microsoft word 90-91-pjac studies on the environmental status of the heavy metals of the road dust in dhaka city contamination of the environment by heavy metals is a phenomenon of global importance today. due to high concentration in the environment, heavy metals may enter the food chain from soils and result in health hazards. accumulation in the street dust is one of the major way through which heavy metals may find their way into soils and subsequently living tissues of plants, animals and human beings [1]. motorized transportation is one of the major sources of urban air pollution [2,3]. bangladesh is a developing country in south asia with a population of 130 million people. it is located between 20º42´ to 26º38´ north latitude and 88º01´ to 92º42´ east longitude. the department of energy in the ministry of environment and forests is responsible for air quality standard has been implemented as part of the environmental conservation rules [4]. dhaka has a population of 12 million and is known for its low air quality. the number of motorized vehicles in the city was relatively low for long time, but dhaka has experienced an estimated yearly increase in motor vehicle traffic by almost 9% between 1992 and 1999 [5]. by 2001, the total number of motor vehicles in dhaka city was 300000. even after this increase ~60% of all journeys in the city were without motorized transport. they were mainly by walking, bicycle or man-drive rickshaws [6]. to tackle the problem of reduced air quality due to vehicle emissions, several steps have been taken. in 1999, leaded gasoline was banned nationally and catalytic converters are now mandatory for new cars. a particulate filter is mandatory on new diesel vehicles. in dhaka city, ‘baby taxis’, which are three-wheel transport powered by twostroke engines, have been banned since 2003 [7]. the aim of the study was to evaluate the present air quality in dhaka city by measuring the trace elements content in the road dusts. ahsan habib*, atiqur rahman and a.m. shafiqul alam department of chemistry, faculty of science, university of dhaka, dhaka 1000, bangladesh. *corresponding author. e-mail: mahabibbit@yahoo.com experimental sampling samples were collected from different roadside of mothijeel, shahabagh and jatrabari areas in dhaka city. they were collected during the dry season. mothijeel is a commercial and very busy traffic area. many motorized vehicles are available in the street. the average speed of the vehicles is very low leading to the extensive emission of air pollutants. jatrabari is one of the most chaotic places and the prime gate of dhaka to communicate with south, southeast and northeast divisions. as the uncounted number of vehicles ply to different destination from here, therefore the gas that is emerged from this vehicles is spreading the limit of heavy metal especially lead in the air of jatrabari in every moment. shahabagh is one of the busiest traffic point of dhaka city. here two famous hospitals (bangladesh institute of research and rehabilitation for diabetes, endocrine and metabolic disorders, birdem) bangobandhu shaikh mujib medical university (bsmmu), national museum, ramna park, national children park, hotel sheraton are situated. maximum vehicles of dhaka and the adjacent cities ply by passing this point. the vehicles exhaust gas, which is significant for environmental pollution. preparation of stock solution. the collected samples were sieved with two different sieves (425 µm and 600µm) to get samples of different sizes that is shown in table 1. then the samples were dried at 105 ºc in an oven for 24 hours. 3.0 g of sj1 sample was taken in a beaker and 40 ml of 5.6 m (40%v/v) nitric acid was added to the sample. the beaker was kept in the water bath and heated on the electric hotplate to boil gently until acid evaporated. the beaker was removed from the hot plate and allowed to cool. then the beaker was rinsed inside with a small volume of water and agitated carefully. finally, the solution was filtered using acid filter paper and the solution was poured in the 100 ml volumetric flask and diluted up to the mark. the prepared solution is known as stock solution and then it was preserved in a issn-1680-9955 pak. j. anal. chem. vol. 6, no. 2, (2005) 89 – 90 report pak. j. anal. chem. vol. 6, no. 2, (2005) 90 polyethylene bottle. similarly, the stock solutions of all the samples were prepared and preserved. table 1. samples of different locations with different sizes in dhaka city location dust size > 600 µm 600 to 425 µm < 425 µm jatrabari sj1 sj2 sj3 mothijeel sm1 sm2 sm3 shahabagh ss1 ss2 ss3 reagents and apparatus. nitric acid, lead nitrate, copper sulphate, mohr’s salt and zinc sulphate were purchased from e. merck, india. the standard solutions of each metal were prepared as follows: lead(ii): 1.0, 2.0, 3.0, 4.0 and 5. 0 ppm; copper(ii): 1.0, 2. 0, 3.0, 4. 0 and 5.0 ppm; ir on(iii); 0.5, 1.0, 2.0, 4.0 and 6 ppm; zinc(ii): 1.0,2. 0,3.0,4. 0 and 5. 0 ppm. de -i oniz ed wa t er wa s used thr ough out the experiment. at omi c absorpti on spectr oph ot om et er (aas) (aa 680, shimadzu, japan) wa s used t o anal yz e all the sampl es. results and discussion the arbitrary concentration of the heavy metals obtained in the dust samples collected at shahbagh, motijheel and jatrabari in dhaka city, bangladesh are summarized in table 2. iron exhibited relatively high level in all the areas. the average estimated value of iron was 63.56 ppm. although lead level was low in all the samples but it has significant effect on the environment as well as on human health. the concentration of copper and zinc lie within the concentration of iron and lead. table 2. amount of metals in the different samples sample no. fe (ppm) cu (ppm) zn (ppm) pb (ppm) ss1 71.52 4.85 2.88 0.78 ss2 65.78 1.17 2.54 0.64 ss3 58.75 6.70 2.46 1.22 sm1 66.60 1.30 2.50 0.95 sm2 55.31 5.86 2.89 0.84 sm3 56.82 2.96 2.84 2.26 sj1 68.15 1.87 2.75 0.66 sj2 55.31 9.29 1.98 0.40 sj3 6 7 .0 9 3 .2 7 2.61 1.10 the variation of iron level was not dependent on the particle size but lead level was varied with particle size. the high level of iron in all the samples is justified. this is due to the high abundance of iron in the nature. on the other hand, lead level was dependent on dust particle size. the results indicated that smaller dust particles contained high level of lead. this phenomenon indicated that leaded gasoline are still used in dhaka city. vehicles still emitting exhaust gases that contained lead compounds. the lead compounds accumulate with particulate matters (pm) in the atmosphere. after that the pm gradually settled down on the earth surface. it is reported that lead level in the rural areas in bangladesh was beyond the detection limit. comparatively high lead level (2.264 ppm) was found at motijheel (sm3). this could be the high traffic density at motijheel area. these results suggest probable common sources of lead and iron .the use of leaded petrol, tyre wears and emission from vehicular and roadside artisans activities may account for some of these metals present in the road dust. this result presented is a part of this study, which at a later time will consider the characteristics and the effects of the road dust on the environment. references 1. s. a. mashi, s. a. yaro and p. n. eyong, manag. envir. qual. 16 (2005) 71. 2. a. faiz and p. j. sturm, atmos. environ. 34, 4745 (2000). 3. j. m. baldasano, e. valera and p. jimenez, sci. total environ. 307, 141 (2003). 4. bangladesh environmental conservation act, 1995; (act 1 of 1995). 1997. s.r.o. no. 197-law/ 97, pp.49. 5. bangladesh: reducing emissions from baby-taxis in dhaka. a joint undp / world bank energy sector management assistance programme (esmap), january (2002); http://www.worldbank.org/html/fpd/esmap/pdfs/25 3.pdf 6. pollution from 2 & 3 wheelers in dhaka. 2001; http://www.adb.org/documents/events/2001/reta 5937/hanoi/documents/08_rab.pdf.accessed 12 february (2004). 7. major policy initiatives. 2004; http//www.doebd.org/policy. html accessed 23 june (2004). issn-1680-9955 pak. j. anal. envir. chem. vol. 7, no. 1, (2006) 62 � 67 lewis acid nature of sncl4 and n-bu2sncl2 determined by adduct formation with 3-methyl-1-indanone muhammad danish1*, c.m. ashraf2, ali mohammad3 and faiz-ur-rehman3 1*department of chemistry, university of sargodha, sargodha, pakistan 2department of chemistry, f.c. college university, lahore, pakistan 3department of chemistry, university of the punjab, lahore, pakistan abstract lewis acid nature of sncl4 and n-bu2sncl2 has been studied using 3-methyl-1-indanone. the equilibrium constant has been calculated for both the tin moieties. it has been found that lewis acid character of sncl4 is fourteen times greater than that of n-bu2sncl2. key words: lewis acid nature, adduct formation, 3-methyl-1-indanone introduction tin is inert and does not react with air or water at room temperature. however, at elevated temperatures, it forms a very thin oxide layer on the surface. tin behaves in an amphoteric way and this nature depends upon concentration and temperature of the medium [1,2]. there are numerous reports on synthesis and applications of organotin compounds [3-8]; however, study of lewis acid nature of tin halides and organotin halides covers the academic interest. in tin halides, organotin halides and most of organotin compounds, the tin center behaves as lewis acid. crystallographic data show, that tin center may show coordination number up to seven [10]. if a donor atom is much away from tin, it does not affect the coordination number and this state is also retained in non coordinating solvents [7,8,12]. due to lewis acid character of tin center, it can be used to study basic properties of compounds containing donor atoms. compounds containing carbonyl group fit in this category as they act as lewis bases. in the present work, 3-methyl-1-indanone has been selected to study acidic properties of sncl4 and n-bu2sncl2. furthermore, it is an important ligand in the synthesis of various tricarbonyl chromium complexes [13-15]. experimental all the chemicals were of analytical reagent grade (purchased from merck) and used without further purification. fresh distilled benzene was used whenever required. preparation of 3-methyl-1-indanone dry benzene (50 cm3) [16] was taken in a two-necked round bottom flask equipped with a water condenser and magnetic stirrer. crotonic acid (6.5 g) was then added followed by portionwise addition of anhydrous alcl3 (31.8 g), under inert atmosphere. the reaction being exothermic started without heating. afterwards, the reaction mixture was refluxed for five hours. the reaction mixture was cooled, extracted in dry benzene, washed with distilled water to remove unreacted aicl3. the organic layer was separated and treated with an aqueous solution of sodium *author for correspondence pak. j. anal. envir. chem. vol. 7, no. 1, (2006) 63 bicarbonate to remove un-reacted crotonic acid and -phenyl butyric acid (by product). the organic layer was separated and washed twice with distilled water. the benzene extract was dried over mgso4 for several hours and filtered. benzene was removed under reduced pressure. the residue was dissolved in dry. ether (100 cm3), stirred with activated charcoal for several hours and filtered through alumina. the filtrate was concentrated to half of its volume and kept overnight. the 3methyl-1-indanone was obtained as a crude mass, which was purified by distillation under reduced pressure [17]. measurement of absorption and equilibrium constant a stock solution of indanone (3.8x10-3 m) was prepared. its molar concentration was kept constant throughout the experiment with both tin halides. the molar concentration of tin halides was varied for each determination. stoichiometric amounts of indanone and tin halide were mixed and absorption was measured at various wavelengths using uv-6000 uv-vis-spectrophotometer, r&m marketing, england. the absorbance was calculated using beer-lambert law. discussion ortho-dichlorobenzene was chosen as the solvent for studying the basicity of the ketone towards tin halides. concentration of ketone was maintained constant while the concentration of snc14 or n-bu2sncl2 was varied as far as experimentally possible. the absorption data are given in tables 1 and 2. the absorption spectra of neat of 3-methyl1-indanone and with tin moieties are shown in figs. 1 and 2. on adding snc14 or nbu2snc12 (as solution in ortho-dichlorobenzene), table 1. absorption data for 3-methyl-1-indanone* with sncl4. concentration of snc14 (m) s. no. wave length (nm) 0 1.86×10-3 3.73×10-3 7.45×10-3 14.91×10-3 1 380 0.065 0.252 0.264 .285 0.325 2 390 0.048 0.237 0.252 0.276 0.320 3 400 0.044 0.235 0.247 0.284 0.336 4 410 0.032 0.223 0.250 0.293 0.354 5 420 0.022 0.210 0.256 0.302 0.376 6 430 0.017 0.202 0.260 0.310 0.392 7 440 0.013 0.190 0.230 0.304 0.380 8 450 0.010 0.174 0.202 0.285 0.356 9 460 0.008 0.163 0.177 0.250 0.315 10 470 0.007 0.149 0.156 0.190 0.248 11 480 0.006 0.133 0.140 0.150 0.192 12 490 0.004 0.116 0.124 0.130 0.162 13 500 0.002 0.103 0.114 0.122 0.146 14 510 0.000 0.096 0.107 0.114 0.130 15 520 0.000 0.089 0.101 0.106 0.118 16 530 0.000 0.087 0.098 0.104 0.109 17 540 0.000 0.084 0.086 0.102 0.104 * concentration of 3-methyl-1-indanone taken each time is 3.84 × 10-3m pak. j. anal. envir. chem. vol. 7, no. 1, (2006) 64 table 2. absorption data for 3-methyl-1-indanone* with n-bu2sncl2. concentration of n-bu2snc12 (m) s no. wave length (nm) 0 1.8×10-2 5.4×10-2 10.8×10-2 16.3×10-2 1 380 0.078 0.085 0.100 0.103 0.108 2 390 0.054 0.060 0.071 0.075 0.084 3 400 0.041 0.050 0.060 0.064 0.068 4 410 0.025 0.029 0.039 0.043 0.048 5 420 0.011 0 .020 0.026 0.029 0.032 6 430 0.005 0.014 0.020 0.022 0.028 7 440 0.001 0.010 0.016 0.018 0.023 8 450 0.000 0.008 0.012 0.014 0.021 9 460 0.000 0.005 0.011 0.013 0.018 10 470 0.000 0.004 0.009 0.011 0.016 11 480 0.000 0.003 0.007 0.009 0.014 12 490 0.000 0.002 0.006 0.008 0.011 13 500 0.000 0.001 0.005 0.006 0.009 14 510 0.000 0.000 0.004 0.005 0.007 15 520 0.000 0 .000 0.002 0.004 0.005 16 530 0.000 0.000 0.003 0.003 0.004 17 540 0.000 0.000 0.000 0.001 0.002 * concentration of 3-methyl-1-indanone taken each time is 3.84 × 10-3m figure 1. absorption spectra of 3-methyl-1-indanone (3.84×10-3 m) in presence of varying amounts of sncl4 using o-dichlorobenzene as solvent. a b sa rb an ce wavelength (nm) pak. j. anal. envir. chem. vol. 7, no. 1, (2006) 65 figure 2. absorption spectra of 3-methyl-1-indanone (3.84x10-3 m) in presence of varying amounts of n-bu2sncl2 using o-dichlorobenzene as solvent. table 3. concentration and absorption data of the ligand with sncl4 conc. of sncl4 absorption at 430 nm d-do [sncl4] / d-do 1/d 1.86 × 10-3m 0.202 0.185 10.05 × 10-3 4.950 3.73 × 10-3m 0.260 0.243 15.35 × 10-3 3.850 7.45 × 10-3m 0.310 0.293 25.43 × 10-3 3.230 14.91 × 10-3m 0.292 0.375 39.76 × 10-3 2.550 concentration of indanone (base) = 3.84×10-3m absorption of pure indanone solution at 430 nm = do = 0.017 relation:[sncl4] / d-do = -1/k × 1/d + 1/kd∞ where do=absorption of pure indanone solution d=absorption at a given concentration of alkyltin halide d∞=absorption for complete aduct formation k=equilibrium constant from graph (fig. 3) k = 69.7 a b sa rb an ce wavelength (nm) pak. j. anal. envir. chem. vol. 7, no. 1, (2006) 66 table 4. concentration and absorption data of the ligand with n-bu2sncl2. conc. of bu2sncl2 absorption at 420 nm d-do [bu2sncl2] / d-do 1/d 1.81 × 10-2m 0.020 0.009 2.640 50.00 5.44 × 10-2m 0.026 0.015 3.630 38.46 10.87 × 10-2m 0.029 0.018 5.040 34.48 16.31 × 10-2m 0.033 0.022 7.410 30.30 concentration of indanone (base) = 3.84×10-3m absorption of pure indanone solution at 420 nm = do = 0.011 relation:[n-bu2sncl2] / d-do = -1/k × 1/d + 1/kd∞ from graph (fig. 4) k = 4.6 figure 3. plot of [sncl4]/d-do vs 1/d for the interaction of 3-methyl-1indanone with sncl4. figure 4. plot of [n-bu2sncl2]/d-do vs 1/d for the interaction of 3-methyl-1-indanone with n-bu2sncl2. [s n c l 4 ]/ d -d 0 1/d [n -b u 2 s n c l 2 ]/ d -d 1/d pak. j. anal. envir. chem. vol. 7, no. 1, (2006) 67 the absorption increased. absorption maxima were observed at 430 and 380 nm for snc14 and nbu2sncl2 respectively. further increase in snc14 or n-bu2sncl2 concentration was not possible experimentally because of weaker interaction between ketone and tin moieties. attempts are made to calculate the equilibrium constant for the aduct formation between snc14 or bu2sncl2 with 3-methyl-1-indanone and the results are shown in tables 3 and 4. a plot of [snc14]/d-do and nbu2snc12/d-do against 1/d gave a straight line (fig. 3 and 4). the equilibrium constant k found from the plot shown in figs. 3 and 4 is 69.7 for snc14 and 4.6 for n-bu2snc12. these results show that 3-methyl-1-indanone interacts with sncl4 or n-bu2snc12 in the aprotic medium and the interaction is reversible. the compound formed in solution has a l:l stoichiometry. the equilibrium constant values show that sncl4 is about fourteen time stronger acid than nbu2sncl2. it can be explained on the bases of replacement of two chlorogroups by butyl groups. this dictates that presence of highly electronegative group on tin increases its lewis acidity. references 1. mc graw hill encyclopedia of science and technology, mc graw hill, new york 18 (1987) 367-369. 2. william benton, encyclopedia britannica, london, 9 (1997) 1019-1021. 3. s. tsunoi, h. shioji and m. tanika, analytical sciences, 20 (2004)101. 4. p. novak, i. gisurora, r. jamber, a. ruzicka & j. holecek, appl. organometal. chem.,18 (2004) 241. 5. w. rehman, m.k. baloch, b. muhammad, a. badshah and k. m. khan, chinese sci. bull., 49 (2004) 119. 6. i. din, m. mazhar, k. m. khan, m.f mohan, k.c. molloy, j. organomet. chem., 689 (2004) 899. 7. m. danish, h. g. alt, a. badhah, s. ali, m. mazhar and n. islam, j. organomet. chem., 486 (1995) 51. 8. m. danish, s. ali, m. mazhar, a. badshah, m. i. chaudhry, h.g. alt and g. kehr, polyhedron, 14 (1995) 3115. 9. m. danish, s. ali, m. mazhar, and a. badshah, main group metal chem., 19 (1996) 121. 10. m. danish, s. ali, m. mazhar, a. badshah and e.r.t. tiekink, main group metal chem., 18 (1995) 617. 11. m. danish, s. ali, m. mazhar, t. masood and e.r.t. tiekink, main group metal chem., 18 (1995) 27. 12. m. danish, s. ali, and m. mazhar, heteroatom chem., 7 (1996) 233. 13. c. m. ashraf, w.r. jackson and d. rosh, aust. j. chem., 28 (1975) 197. 14. c. m. ashraf and w. r. jackson, aust. j. chem., 31 (1978) 1845. 15. c. m. ashraf, arab gulf j. scient. res. math. phys. sci., a5 (1987) 11. 16. d. d. perrin and w. l. f. armergo, �purification of laboratory chemicals� ed 3rd, pergamon, oxford, (1987). 17. f. koelsch. h. hockmann and c.d. claire, j. am. chem. soc., 65 (1943) 59. microsoft word 65-71-pjacissn-1680-9955 pak. j. anal. chem. vol. 6, no. 2, (2005) 65 – 71 studies on the effect of additives on the strength of ordinary portland cement ahsan habib*, anarul islam, dulal solomon tudu and a m shafiqul alam department of chemistry, faculty of science, university of dhaka, dhaka 1000, bangladesh *corresponding author. e-mail: mahabibbit@yahoo.com received: 15-08-2006, revised: 26-09-2006, accepted: 30-09-2006 abstract the effects of additives, such as slag, limestone and fly ash on the strength of ordinary portland cement (opc) have been studied. the percentages of the additives were varied from 9 to 29%. 2.5% gypsum was used in all preparations. the influence of the additives on the strength of opc was monitored by measuring compressive strengths. the results indicated that the strengths of all the composite cements were nicely satisfied the respective american society for testing and materials (astm) values recommended for different times of curing. it was observed that the strengths of the all composite cements gradually increased with time of curing. interestingly, slag composite cements showed higher strengths at all ages (3, 7 and 28 days). on the other hand, limestone composite cements showed comparatively lower strengths but higher than that of astm recommended values. it has also been observed that the strengths are independent of the fineness of the composite cements. keywords: ordinary portland cement (opc), additives, composite cement. introduction portland composite cement results from milling 40-64 parts (by weight) of portland cement clinker together with a corresponding amount (60-36 parts) of pozzolans or other suitable additives [1-5]. composite cements are largely comparable to portland cement in terms of their construction properties and their inclusion in the setting of regulation. portland composite cement has better characteristics than the ordinary portland cement. this kind of cement is suitable to construct buildings, bridges and other commercial structures in coastal area and saline environmental area. the long-term strength is 15 to 20% higher than the ordinary portland cement [6]. the use of portland composite cements enhances the ecological efficiency of concrete construction. the utilization of main constituents other than clinker reduces the co2 emission during cement manufacture in particular as the clinker content of portland composite cements is lower than that of portland cements [7]. composite being slow setting was hot very popular in earlier times but gradually it has become an acceptable building material [8]. slag is a non-metallic product consisting essentially of glass silicates, alumino-silicates of lime and other bases and is obtained as a byproduct in the manufacture of pig-iron in blast furnace. fly ash or pulverized fuel ash is formed as a result of burning pulverized coal. it is a very fine material about 60-70% of which has a size below 0.076 mm. the principal contents of fly ash are normally silica (30-60%), alumina (15-30%), iron oxide and carbon in the form of unburnt fuel up to 20%, lime 7% and small quantities of magnesium oxide and sulphate. limestone occurs in nature and is widely distributed as minerals known by various names as limestone, marble, chalk, iceland spar, coral etc. there are two forms of crystals of calcium carbonate: calcite and aragonite. from the beginning, there was only one cement factory in bangladesh and the production pak. j. anal. chem. vol. 6, no. 2, (2005) 66 of cement was not enough as per demand. at that time cement was imported from thailand, indonesia, malaysia, china etc. to fulfillment the demand, a group of industrialists set up some cement factories in bangladesh. after that cement price was very much controlled in our country. latter, the price of clinker became higher and eventually the price of cement was increased. now the time has come to carry out a research to develop a process to manufacture low cost cement. in the present research, we have attempted to use some additives (slag, limestone and fly ash) with clinker to produce composite cements, which are cost effective compare to ordinary portland cement (opc). we have investigated the effects of additives on the strength of ordinary portland cement. the results are expected to be useful in the manufacturing of composite cements in bangladesh. experimental reagents ethylenediaminetetraacetate (edta), triethanolamine (tea), ammonium chloride, sodium-potassium tartarate, ammonium hydroxide, pentahydrate copper sulphate, methylthymol blue (mtb), mixed indicator (kb), silver nitrate, 1-(2-pyridilazo)-2-naphthol (pan) were purchased from e. merck, india. potassium hydroxide, hydrochloric acid, sulphuric acid, sodium salicylsulfonate were purchased from bdh. standard sand (bs en 196 part i) was purchased from england. preparation and procedure preparation of composite cements all the materials were dried in an oven at 105 0c about 1 hour. the dried materials were taken according to the following composition as shown in table 1 and ground in the mini ball mill (model no. tcl 175, china) for 25-30 minutes. the total weight of composition was 5 kg. another sample ‘d’ was prepared by grinding 97.5% clinker and 2.5% gypsum. physical parameters all the physical properties were measured according to the following procedures. (a) fineness: fineness of the composite cements was measured using air permeability method and sieve tests. air permeability method 5 g of cement was placed in the permeability cell in a standard manner. then air was slowly passed on through the cement bed at a constant velocity. the rate of airflow was adjusted until the flow meter shows a difference in level of 30-50 cm .the difference in level of manometer and the difference in level of the flow meter was recorded. these observations were repeated to ensure that steady conditions had been obtained and then specific surface was calculated [1]. table 1. percentages of clinker, additives and gypsum in the composite cements. cementitious materials (%) sample no. a1 a2 a3 b1 b2 b3 c1 c2 c3 clinker 68.5 78.5 88.5 68.5 78.5 88.5 68.5 78.5 88.5 slag 29 19 9       limestone    29 19 9    flyash       29 19 9 gypsum 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 pak. j. anal. chem. vol. 6, no. 2, (2005) 67 sieve test. 100 g of cement was weighed out and taken it on a standard is sieve no. 9 (90 micron). the air-set lumps in the sample were broken down with spatula. then the sample was continuously sieved, giving circular and vertical motion for a period of 15 minutes. the residue left on the sieve was then weighed and calculated [1]. (b) standard consistency test: 500 g of cement was taken and prepared a paste with a weighed quantity of water (say 24 percent by weight of cement) for the first trial. the paste was made in a standard manner and filled into the vicat mould within 3-5 minutes. after completely filling the mould, the mould was shacked to expel air. a standard plunger, 10 mm diameter, 50 mm long was attached and brought down to touch the surface of the paste in the test lock and quickly released allowing it to sink into the paste by its own weight. then the reading was taken by noting the depth of penetration of the plunger. similarly, more trials were conducted with higher and higher water/cement ratios until such time the plunger penetrates to a depth of 33-35 mm from the top. that particular percentage of water, which allowed the plunger to penetrate only to a depth of 33-35 mm from the top, was the percentage of water required to produce a cement paste of standard consistency [9]. (c) setting time test: 500 g of cement sample was taken in a pot and paste it with requisite amount of water to prepare cement paste of standard consistency. the paste was gauged and filled into the vicat mould in specified manner within 3-5 minutes. a stopwatch (diamond788645, china) was started the moment water is added to the cement. the temperature of water and that of the test room, at the time of gauging was maintained within 27±2 0c. a water bath (modelhhs, china) was used to control the temperature of water at 27±20c. initial setting time: the needle was lowered gently and brought it in contact with the surface of the test block and quickly released and to penetrate into the test block. the period elapsing between the time when water was added to the cement and the time at which the needle penetrates the test block to a depth equal to 33-35 mm from the top was taken as initial setting time. final setting time: the needle of the vicat apparatus was replaced by a circular attachment. the cement was considered as finally set when, upon lowering the attachment gently cover the surface of the test block, the center needle makes an impression, while the circular cutting edge of the attachment fails to do so. this could indicate a hardened state, which the center needle did not pierce through more than 0.5mm [1]. (d) compressive strength test: 1375 g portions of standard sand, 500 g portions of composite cements (i.e. ratio of ~1:3) and 242 ml water were taken on a non-porous enamel tray and each sample was mixed with a trowel for one minute. further mixing with different ingredients was continued until the mixtures were of uniform color. the time of mixing was between 3 to 4 minutes. immediately after mixing, cube moulds of size 7.06 cm were filled with the mortars. the mortars were compacted by taking the moulds on the vibrating table. the compacted cubes were kept in a curing box at a temperature of 20±2 0c with at least 90 percent relative humidity for 24 hours. the cubes were removed from the curing box and immersed in clean fresh water until taken out for testing. three cubes were tested for compressive strength at the periods of 2, 7 and 28 days [1, 10-12]. chemical analysis preparation of stock solution. 0.5 g of clinker was taken in a beaker. 2-3 g of ammonium chloride was added and mixed well. this mixture was treated with 3 drops of conc. hno3 and 3 ml of conc. hcl and heated on sand bath to make paste and it was then dried for digestion. after completion of digestion it was cooled to room temperature. then 125 ml of hcl (97:3) was added to make a solution. the residue was then filtered through whatman paper no. 40 and washed thoroughly with hot distilled water until the filtrate was freed from chloride. a 250 ml volumetric flask was used to collect the filtrate. the filtrate was made upto the mark with distill water. this was the stock solution of clinker. similarly, stock pak. j. anal. chem. vol. 6, no. 2, (2005) 68 solutions of additives, gypsum and finished products were made [13, 14]. apparatus a mixture machine (model no. 160a, china) was used to prepare a cement paste with sand and water. fineness of the prepared cements was monitored using vicat needle permeability apparatus (model no. vn-01, china). setting time was recorded using lea and nurse permeability apparatus (id no.brtc 0604/04/ce, china). compressive strengths of the cement cubes were measured using compression testing machine (model no. 82446/2004, china). the prepared cubes were cured in a standard cement curing cabinet (model no. yh-40b, china). results and discussion chemical composition table 2 indicates the chemical compositions of clinker, additives and gypsum. limestone is rich in cao and fly ash is enriched with sio2. on the other hand, slag contains high amount of both sio2 and cao. the five major compounds such as sio2, cao, mgo, al2o3 and fe2o3 were tested because the relative proportions of these oxides responsible for influencing the various properties of cement. there may have many minor compounds but their influence on the properties of cement or hydrated compounds is not significant. the proportions of these five compounds in clinker were determined in the standard manner. furthermore, the chemical compositions of all the composite cements are shown in table 3. table 2. chemical compositions of clinker, additives and gypsum. chemical composition cementitious materials (%) clinker slag limestone flyash gypsum sio2 22.00 32.20 7.36 44.05 6.00 cao 64.78 37.35 42.91 2.25 31.46 mgo 4.08 6.60 0.96 0.36 0.24 fe2o3 3.00 0.72 1.07 0.84 0.50 al2o3 4.20 0.62 1.50 19.45 0.23 table 3. chemical composition of the composite cements. chemical composition %) sample no a1 a2 a3 b1 b2 b3 c1 c2 c3 d sio2 24.5 23.27 22.52 17.35 18.82 20.28 27.95 25.69 23.52 21.52 cao 55.34 58.24 61.29 57.01 59.32 61.55 45.09 51.87 58.12 63.78 mgo 4.65 4.36 4.15 3.02 3.29 3.60 2.80 3.23 3.69 3.95 fe2o3 2.21 2.45 2.65 2.30 2.51 2.71 2.25 2.28 2.53 2.75 al2o3 3.01 3.42 3.72 3.27 3.55 3.81 8.47 6.95 5.42 4.04 physical parameters fineness table 4 indicates the fineness of all prepared composites. fineness of cements was tested in two ways: 1) by sieve test and 2) by airpermeability method. an average (4.55 ± 0.85) percent of residue was present in each sample. according to the air permeability method, a range of surface area (3200  4800 cm2/g) was observed where the standard surface area according to astm is 2800 cm2/g. pak. j. anal. chem. vol. 6, no. 2, (2005) 69 table 4. percentage of residue, blaine and water consistency of the composite cements. sample no residue (%) blaine (sq.cm/g) water consistency (%) a1 3.6 3872 24.50 a2 4.6 3399 24.00 a3 4.0 3804 23.50 b1 3.8 4840 22.00 b2 5.2 4764 21.50 b3 3.4 3490 22.50 c1 3.7 4753 26.00 c2 3.0 4353 26.25 c3 4.3 3194 24.50 d 5.4 3384 24.00 setting time table 5 indicates the initial and final setting times of all cement composites. the results of both the initial and final setting times satisfied the astm standard (initial setting is not less than 45 minutes and final setting time is not more than 375 minutes). table 5. setting times of the composite cements. sample no. setting time (min) initial final a1 125 285 a2 100 200 a3 65 170 b1 55 160 b2 65 185 b3 90 215 c1 105 235 c2 110 245 c3 95 220 d 75 220 compressive strength table 6 indicates the compressive strengths of the composite cements as well as ordinary portland cement. the results show that the strengths of the composite cements at all ages are higher than those for ordinary portland cement. table 6. compressive strengths of the composite cements. sample no. compressive strength (mpa) age (days) 3 7 28 a1 15.75 33.25 51.75 a2 17.25 34.31 53.78 a3 22.50 43.25 55.78 b1 15.87 27.25 34.96 b2 18.25 31.81 37.03 b3 21.31 34.81 37.68 c1 14.62 29.68 43.70 c2 15.62 29.80 44.06 c3 17.00 30.50 44.87 d 17.93 30.75 39.62 portland composite cement (astm standard) 12.00 19.00 28.00 interestingly, sag cements of all ratios show higher strengths at all ages. the early (3 and 8 days) strength is due to the hydration reaction between portland cement and water, resulting in the formation of calcium-silicate-hydrate (csh) and calcium hydroxide [ca(oh)2]. csh is a gel that is responsible for strength development in portland cement pastes. ca(oh)2 is a by-product of the hydration process that does not significantly contribute to strength development in normal portland cement. silicates in slag cement combine with the calcium hydroxide by-product of hydration and form additional csh. this in turns leads to a denser, harder cementitious paste, which increases ultimate strength as compared to 100% portland cement systems. on the other hand, although fly ash cements show higher strength than that of ordinary portland cement but show lower strength compared to slag cement. in the case of fly ash cement, pak. j. anal. chem. vol. 6, no. 2, (2005) 70 hydration reaction also occur and results in the formation of calcium-silicate-hydrate(csh) and ca(oh)2. eventually, the usual strength of cement is due to the formation of csh gel. the higher strength of fly ash cement is due to the formation of additional csh from the reaction of ca(oh)2 and silica in fly ash. but the lower strength compared to slag cement may due to the inadequate amount of cao needed for the formation of csh. limestone cements show comparative strengths at 3 and 7 days but show lower strength at 28 days compared to any one of the composite cements as well as ordinary portland cement. the early higher strength is due to the formation of calcium-silicate-hydrate during the hydration reaction. but at higher ages (28 days) no further formation of csh is possible since limestone does not contain adequate quantity of sio2. slag cement does not contain carbon and will not cause fluctuation in air content. the percentage of slag cement to get highest flexural strength varies depending on the specific mix design and constituents used. however, slag cement used at replacement rates greater than 25% can cause a dramatic increase in time of set. the lower heat evolution characteristic of slag cement in the summer can be beneficial because it allows more time for placing and finishing concrete. in spring and fall, the delayed set may cause problems with joint sawing, texturing and secondary paving operations. a rule of thumb is that the set time is delayed 3 minutes for every 10% slag replacement of portland cement. slag cements demonstrate improved workability and finishability compared to ordinary portland cement. this is due to the several factors including increased paste cohesiveness, glassy structure of slag cement, and low initial water absorption. fly ash cement also produces less heat of hydration and offers greater resistance to the attack of aggressive waters compared to ordinary portland cement. moreover, it reduces the leaching of calcium hydroxide when used in hydraulic structures [1]. the main problem of utilization of fly ash comes from the unburnt carbon in it as it has no binding force. carbon content variability in fly ash is one of the major causes of fluctuating air contents. the addition of fly ash increases the paste volume, drying shrinkage may be increased slightly if the water content remains constant [11]. at normally specified replacement levels, concrete made with slag cement have lower permeability than concrete made with fly ash. limestone is a plasticizing material. the addition of limestone with portland cement reduces setting time and facilitate workability. it is primarily used for spreading onto walls to make exterior stucco, as portland cement would have poor spreadability. it has also been investigated that the strengths are irrespective of the fineness of the composite cements. conclusion the composite cements of all ratios of slag and fly ash showed better results upto 28 days compared to ordinary portland cement. in addition, slag containing composite cements showed much better strengths compared to ordinary portland cement and other composite cements. on the other hand, limestone containing composite cements showed slightly lower strengths compared to ordinary portland cement. however, this strength is higher than that recommended by astm standard. acknowledgment the authors acknowledge the bose center for advanced study and research in natural sciences, dhaka university, bangladesh for financial support, and mtc cement factory, bangladesh for providing laboratory facilities. references 1. astm c 125, standard terminology relating to concrete and concrete aggregates, 1994 annual book of astm standards. 2. astm c 618, standard specification for coal fly ash and raw or calcined natural pozzolan for use as a mineral admixture in portland cement concrete, 1994 annual book of astm standards. pak. j. anal. chem. vol. 6, no. 2, (2005) 71 3. s. n. ghosh, advances in cement technology, 1st edn. pergamon, oxford, 1983. 4. f. massazza, puzzolanik cimento seminari, (1995) ankara, turkey. 5. p.k. mehta, concrete structure, properties and materials, prentice hall, london, 1986. 6. “heidelberg cement brings in composite cement” mark van kempen, heidelberg cement bangladesh ltd. 9/2/2004, dhaka, bangladesh. 7. s. mueller, “ einsats von cem 11zementen. in: 41 rorschungs kolloquium des dafstb, dusseldorf (2000). 8. k. n. farooque, annual report, 2003-2004, institute of glass and ceramics research and testing, bangladesh council of scientific and industrial research (bcsir). 9. astm c 187, standard test methods for normal consistency of hydraulic cement, 1994. 10. astm c 778, specification for standard sand, 1994 annual book of astm standards. 11. astm c 511, standard specification for moist cabinets, moist rooms and water storage tanks used in the testing, 1994 annual book of astm standards. 12. astm c 109, standard test methods for compressive strength of hydraulic cement, 1994. annual book of astm standards. 13. astm c 114, standard test methods for chemical analysis of hydraulic cement, 1994 annual book of astm standards. 14. j. mendham, r.c. denney, j.d. barnes, m.j.k. thomas, r.c. denney and m.j.k. thomas, vogel’s quantitative chemical analysis (6th edition). microsoft word 122-127-pjaec-02042018-70.doc cross mark issn-1996-918x pak. j. anal. environ. chem. vol. 19, no. 2 (2018) 122 – 127 http://doi.org/10.21743/pjaec/2018.12.13 synthesis of copper nanoparticles via trigonella foenumgraecum seed extract for antibacterial response tahira moeen khan * , amat ul mateen and bushra khan department of chemistry, lahore college for women university, jail road, lahore, pakistan. *corresponding author email: tahira.moeen@lcwu.edu.pk received 02 april 2018, revised 16 october 2018, accepted 20 october 2018 -------------------------------------------------------------------------------------------------------------------------------------------abstract in view of the immense capability of plants this work is planned to employ seed extract as a source for the reduction of cu ions in to cu nanoparticles (cu nps). for this purpose seed extract of trigonella foenum-graecum (fenugreek seeds) was utilized as a substitute of classical methods. this green path for synthesizing cu nps is easy, natural, low cost, sustainable and eco-friendly as compared to conventional methods. in this experiment harmful chemical/physical methods for the production of cu nanoparticles is replaced by using minimum concentration of seed extract. the stepwise characterization was done by using atomic absorption spectroscopy, uv-vis spectroscopy, ftir spectrophotometer and x-ray diffraction (xrd) which have given much valuable information about these materials. antibacterial activity of these nanoparticles is observed at different concentration so their zoi (zone of inhibition) and mic (minimum inhibitory concentration) was also calculated against four human pathogenic strains. keywords: nanoparticles, synthesis, seed extract, ftir, xrd, bacterial culture. -------------------------------------------------------------------------------------------------------------------------------------------introduction nanotechnology has achieved a prominent place in today’s research. with reducing size (1-100 nm) nanoparticles possess a higher surface to volume ratio which is significant for catalytic reactivity and other similar properties such as antimicrobial activity in nanoparticles [1]. nanoparticles can be prepared by either physical methods including pulsed laser ablation [2], vacuum vapor deposition [3], pulsed wire discharge [4], mechanical milling [5] and chemical methods involving chemical reduction [6], micro emulsion techniques [7], sonochemical reduction [8], electrochemical [9], microwave assisted [10] and hydrothermal methods [11]. he usage of lethal chemicals and by products with in these methods make the nanoparticles synthesis more challenging and problematic. moreover, these methods also demand high energy and uneconomical purifications. green synthesis provides advancement over both chemical and physical methods as it is economical, eco-friendly, capable for large scale synthesis and does not require consumption of harmful chemicals, high energy, temperature and elevated pressure [12]. presently green chemistry involve the biosynthesis through microorganism and plant extracts [13, 14]. by using natural extract of aspalathus linearis crystalline perovskite znsno3 nanoclusters, nio, pd & pdo nanoparticles are biosynthesized [15,16]. sageretia thea & moringa oleifera natural extracts are used as chelating agent to prepare zno nanoparticle [17,18 ]. copper nanoparticles exist in three forms i.e. cu metal nanoparticle, cu(i) oxide nanoparticle [cu2o] and cu(ii) oxide nanoparticle [cuo].these have catalytic [19], optical [20] & sensing activities [21]. they are also helpful as resistance materials [22], inorganic-organic nanocomposite [23] & in solar cells [24]. metal pak. j. anal. environ. chem. vol. 19, no. 2 (2018) 123 nanoparticles can show a good antibacterial response. this antibacterial activity of cu may be accredited to their micron range size less than the pore size of the bacteria and thus, they are capable of easily crossing the cell membrane without any hindrance [25]. fenugreek, trigonella foenum-graecum (greek hay) is an annual herb of the pea family (fabaceae) cultivated in north africa, the middle east and asia. it is used mostly as ingredient in traditional medicine in egypt, india and asia. fenugreek helps to heal inflammation and its extracts can be added in manufacturing cosmetics and also in soaps due to its long history as antitumorigenic, antioxidant, antidiabetic, and antimicrobial activities. its seeds extract help control in digestion, arthritis, diabetes, cholesterol, cancer, heart attack, improvement in breast milk, kidney and liver function [26, 27]. his research work aims to throw light on the bio reduction of cu +2 ions into cu 0 nanoparticles by employing aqueous fenugreek seeds extract under varied percentage compositions and there by implementing eco-friendly chemistry for the future research. to the best of our knowledge this green synthesis of copper nanoparticles by using aqueous extract of fenugreek seeds is a novel research and has not been reported earlier. experimental addition of the aqueous plant extract into a solution of the appropriate metal salt initiates a plant extract-facilitated green reduction. the preparation of nps takes place at room temperature and completes within a few minutes. seed extract preparation they were thoroughly washed with distilled water, dried and crushed. the powder was further used for preparation of 1 g/100 ml aqueous seeds extract. this extract was boiled for 30 minutes approx. at 80-90ºc, shaked (5 c/s for 10 minutes) & filtered to obtain homogenized solution which was kept at 4°c in 250 ml erlenmeyer flask. in each and every step of the experiment, sterility conditions were maintained for the efficacy and accuracy in results without any impurity. biosynthesis of nanoparticles for biosynthesis of nanoparticles, 2.0 ml of the seeds extract was mixed with 20 µl of freshly prepared 1×10 -2 m aqueous copper sulphate pentahydrate solution in 250 ml erlenmeyer flask under continuous magnetic stirring. the cu nanoparticles obtained were purified by repeated centrifugation method at 6,000rpm for 40 min followed by dispersion of the pellet in deionized water. later the cu nanoparticles were dried in an oven at 90-100 ºc for 3-4 hours [28]. table 1 shows the preparation of different concentrations by varying the amount of seed extract and keeping the concentration of cuso4.5h2o constant i.e., 1×10 -2 m. table 1. procedure for the preparation of different concentrations concentration procedure 5% 5ml of the seed extract (fenugreek) was mixed with 95ml of the 0.01m cuso4 solution. 15% 15ml of the seed extract was mixed with 85ml of the 0.01m cuso4 solution. 25% 25ml of the seed extract was mixed with 75ml of the 0.01m cuso4 solution. procedure for antibacterial activity for antibacterial activity agar well diffusion method was employed [25]. in each well 50ul of the sample was dispensed . four bacterial strains were selected including pseudomonas aeruginosa (atcc 27853), proteus mirabilis (atcc 7002), agrobacterium tumefaciens (atcc 5577) and bacillus subtilis (atcc 6051). for each strain different concentrations 5%, 15% & 25% was prepared and also measured the zone of inhibition (zoi) of each in mm [29]. biosynthesized nanoparticles characterization 1ml of the colloidal solution of prepared cu nps was diluted with 10 ml of the distilled water and then this diluted solution was aspirated pak. j. anal. environ. chem. vol. 19, no. 2 (2018)124 into the atomic absorption spectrophotometer through capillary tube to confirm the nps synthesis. uv-vis spectral analysis was also done between 200-700 nm. ftir spectral studies was done on shimadzu irtracer-100, fourier transform infrared spectrometer between 4000 and 375 cm -1 with resolution of 4 cm -1 . the powder x-ray diffraction was conducted on a philips analytical xpert diffractometer using a cu kα radiation (λ = 1.540598 °a) with a miniprop detector and operating at 40 kv generator voltage and 40 ma generator current. x-ray diffraction patterns were recorded between 2θ = 5◦ and 79◦ with a step of 0.04◦ and a time of 0.2 s by step. results and discussion atomic absorption spectrometry the presence of copper nanoparticles was confirmed by concentration-absorption linear relationship. fig. 1 shows the increase in absorption with the growing concentration of cu nanoparticles with gradual increase of seed extract concentration. figure 1. effect of the fenugreek seed extract percentage composition on the synthesis of cu nps. visual observation and uv-vis spectroscopy reduction of cu +2 into cu 0 during addition of fenugreek seeds extract is monitored as a consequence of the change in colour and thus can be studied through uv-vis spectroscopy. the change in colour is because of the surface plasmon resonance (spr). the spr band was observed at a maximum absorbance of approximately 560 nm which was confirmed from the literature value [30]. different parameters were optimized including concentration of seeds extract and reaction time. it was identified that the yields of copper nanoparticles were affected by varying these parameters. effect of contact time at room temperature increasing the reaction time results in gradual increase of absorbance which was quantitatively monitored (fig. 2). furthermore, the color intensity also increases with the duration of incubation from faint blue to yellow brown and then to deep brown as shown in (fig. 3). this increase in absorbance and color intensity is attributed to the higher concentration of cu nps. [31]. 1.41 1.58 3.44 4.43 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 2 6 24 48 time (hours) c o n c en tr a ti o n o f c u n p s (p p m ) figure 2. cu nps concentration at different time intervals with 15% seed extract added to 0.01m cuso4 solution a b c d a: 0.01m cuso4 solution b: aqueous seed extract c: addition of seeds extract into 0.01m cuso4 solution d: nanoparticles emulsion after 24 hrs. figure 3. formation of nps with colour change. increase in absorption intensity as the concentration of the fenugreek seeds extract increases, the absorption peak gets more sharpness with slight increase in wavelength. if we increase the extract concentration from 5% to 25%, there is c o n ce n tr a ti o n o f c u n p s pak. j. anal. environ. chem. vol. 19, no. 2 (2018) 125 increase in intensity of absorption as presented in fig. 4. 0 0.05 0.1 0.15 0.2 0.25 0.3 7 0 0 6 7 8 6 5 6 6 3 4 6 1 2 5 9 0 5 6 8 5 4 6 5 2 4 5 0 2 4 8 0 6 8 8 6 6 6 6 4 4 6 2 2 6 0 0 5 7 8 5 5 6 5 3 4 5 1 2 4 9 0 6 9 8 6 7 6 6 5 4 6 3 2 6 1 0 5 8 8 5 6 6 5 4 4 5 2 2 5 0 0 4 7 8 wavelength (nm) 5% 15% 25% a b so rb a n c e figure 4. for 5% seed extract absorbance is observed at 536nm and with 25% at 560 nm fourier transform infrared spectroscopy (ftir) fig. 5 shows ftir spectrum of cu nanoparticles from fenugreek seeds extract. a wide band at 3358 cm -1 is due to the oh group indicating the presence of intermolecular hydrogen bonding. a peak at 2358 cm -1 is observed which can be attributed to c≡c stretching vibrations i.e. alkyne group present in phyto constituents of extract. a band at about 1645 cm -1 is due to c=o stretching vibrations associated with amide i functional group. the amide i band is directly linked to the backbone conformation indicating the presence of proteins in seeds extract [32]. figure 5. ftir spectrum of cu nanoparticles synthesized from fenugreek seeds extract xray diffraction studies the results of xrd pattern analysis revealed three intense peaks in the whole spectrum of 2 theta values ranging from 20 to 80 for the cu nanoparticles. the sample demonstrated a high crystallinity level with diffraction angles of 28.86, 32.14 and 46.55 which correspond to the characteristic of face-centred cubic of copper lines indexed at (210), (111), and (222) fig. 6. peaks for cu nps are compared with jcpds literature. cu nanoparticles with different sizes were obtained. by applying debye–scherrer equation to the obtained xrd pattern of the cu nps, the average nanoparticles size was found to be 14-17 nm, which may indicate a high surface area-tovolume ratio of nanocrystals.cu nanoparticles are easily prone to oxidation and results in the formation of cuo and cu2o nanoparticles as shown below [33]. 2 0 3 0 4 0 5 0 6 0 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 in te n s it y (c o u n ts ) a n g le ( 2 th e ta ) i n t e n s it y d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o d e m o figure 6. xrd plot of cu nps synthesized from fenugreek seeds extract cu +2 + 2e→ cu 0 + air (o2) → cuo/ cu2o but by using fenugreek seed extract as reductant cu nps were not oxidized to cuo nps. thus, from these observations it may be concluded that fenugreek seed extract is not only an excellent reducing agent but also a strong capping agent that it coats the surface of cu nps avoiding its contact with the air and hence no oxidation took place over the surface of synthesized nps. determination of mic (minimum inhibitory concentration) four bacterial cultures including bacillus subtilis (one gram positive), pseudomonas aeruginosa, proteus mirabilis, agrobacterium tumefaciens(three gram negative bacteria) were pak. j. anal. environ. chem. vol. 19, no. 2 (2018)126 used to check the antibacterial potential of cu nanoparticles (5% to 25% concentration) synthesized from fenugreek seed extract. ciprofloxacin was used as control against gram positive bacterium while amoxicillin for gram negative bacteria. 25% (max) concentration give maximum antibacterial potential i.e., 10 mm zone of inhibition for bacillus subtilis, 5 mm for pseudomonas aeruginosa, 9 mm for proteus mirabilis and 10 mm for agrobacterium tumefaciens table 2. with 5% concentration which is minimum inhibitary concentration (mic) 5 mm zoi was observed for bacillus subtilis, 2 mm for pseudomonas aeruginosa, 3 mm for proteus mirabilis and 5 mm for agrobacterium tumefaciens fig. 7. a c b d a = bacillus subtilis b = pseudomonas aeruginosa c = proteus mirabilis d = agrobacterium tumefaciens. figure 7. observed antibacterial activities against four human pathogenic strains conclusion in this research a simple, convenient, cost effective, significant and eco-friendly method for the synthesis of copper nanoparticles through the reduction of copper salts has been demonstrated. aqueous seed extract of fenugreek is used as reducing and capping agent. characterization of cu nanoparticles was done by aas, uv spectroscopy, ftir spectroscopy and xrd analysis. these nanoparticles were evaluated for their antibacterial activity against one gram positive and three gram negative bacterial strains. these biologically synthesized copper nanoparticles using seed extract showed sufficient antibacterial potential. the minimum inhibitory concentration of nanoparticles synthesized from aqueous seed extract of fenugreek was 5%. in the concluding remarks, the copper nanoparticles synthesized using fenugreek seed extract would be a better antimicrobial effective against various bacterial species. acknowledgement all this research is done and supported by lahore college for women university jail road lahore, pakistan. references 1. j. perez, l. bax and c. escolano, willems & van den wildenberg (w&w) (2005) espana sl: barcelona-spain. nanoparticles.org/pdf/perezbaxescolano.pdf 2. m. s. yeh, y. s. yang and y. p. lee, j. phy. chem. b., 103 (1999) 33, 6851. http://pubs.acs.org/doi/abs/10.1021/jp984163 %2b 3. z. liu and y. bando, adv. mater., 15 (2003) 303. doi/10.1002/adma.200390073. 4. k. yatsui, c. grigoriu and h. kubo, appl. phys. lett., 67 (1995) 1214. https://doi.org/10.1063/1.115011 5. d. oleszaka and ph. shingu, j. appl. phys., 79 (1996). https://aip.scitation.org/doi/abs/10.1063/1.36 1294. 6. y. wang, p. chen and m. liu, nanotechnology, 17 (2006) 6000. http://dx.doi.org/10.1590/s151770762014000300002 7. m. p. pileni, j. phys. chem., 97 (1993) 6961. https://pubs.acs.org/doi/abs/10.1021/j100129 a008 8. r. v. kumar, y. mastai and y. diamant, j. mater. chem., 11 (2001) 1209. doi: 10.1039/b005769j pak. j. anal. environ. chem. vol. 19, no. 2 (2018) 127 9. met. molares, v. buschmann , d. dobrev, adv. mater., 13 (2001) 62. aip.scitation.org/doi/abs/10.1063/1.4712536 10. m. hasanpoor, m. aliofkhazraei, h. delavari, procedia materials sci., 11 (2015) 320. https://doi.org/10.1016/j.mspro.2015.11.10 11. l. y. chu, y. zhuo and l. dong, adv. funct. mater., 17 (2007) 933. http://journals.bmsu.ac.ir/jabr/index.php/jabr/ article/view/94/103 12. s. ahmed, m. ahmad, b. l. swami and s. ikram, j. adv. res., 7 (2016) 17. doi: 10.1016/j.jare.2015.02.007. 13. b. t. sone, e. manikandan, a. gurib-fakim, m. maaza, j. alloys and compd., 650 (2015) 357. https://doi.org/10.1016/j.jallcom.2015.07.272. 14. n. jayaprakash, j. judith vijaya, k. kaviyarasu, k. kombaiaha, j. kennedy, r. jothi, ramalingam, murugan a. munusamy , hamad a. al-lohedan, j. photochem. photobiol. b., 169 (2017) 178. https://doi.org/10.1016/j.jphotobiol.2017.03. 013. 15. n. mayedwa, n. mongwaketsi, s.khamlich, k. kasinathan, n. matinise, m. maaza, appl. surf. sci., (2017). https://doi.org/10.1016/j.apsusc.2017.12.161. 16. n. mayedwa, n. mongwaketsi, s. khamlich, k. kasinathan, n. matinise, m. maaza, appl. surf. sci., 446 (2018) 266. https://doi.org/10.1016/j.apsusc.2017.12.116 17. n. mayedwa1, a. t. khalil, n. mongwaketsi, n. matinise, z. k. shinwari , m. maaza , nano res. appl., 3, 2 (2017). doi: 10.21767/2471-9838.100026 18. n. matinise, x. g. fuku, k. kaviyarasu, n. mayedwa, m. maaza, appl. surf. sci., 406 (2017) 339. https://doi.org/10.1016/j.apsusc.2017.01.219 19. s. yang, c. wang, l. chen, s. chen, mater. chem. phys., 120 (2010) 296. https://doi.org/10.1016/j.matchemphys.2009. 11.005 20. t. yu, f. c. cheong, c. h. sow, nanotechnology, 15 (2004) 1732. https://doi.org/10.1088/0957-4484/15/12/005 21. a. umar, m. m. rahman, a. al-hajry and y. b. hahn, electrochem. commun., 11 (2009) 278. https://doi.org/10.1016/j.elecom.2008.11.027 22. x. g. zheng, c. n. xu, y. tomokiyo, e. tanaka, h. yamada and y. soejima , phys. rev. lett., 85 (2000) 5170. https://doi.org/10.1103/physrevlett.85.5170 23. e. alzahrani, anal. chem. insights., 13 (2018). doi: 10.1177/1177390118763361 24. k. iqbal, m. ikram, m. afzal and s. ali, mater. renew. sustain. energy, 7, 4 (2018). https://doi.org/10.1007/s40243-018-0111-2 25. d. prabu, r. parthiban p. s kumar and k. r namasivayam, int. j. pharm. pharm. sci., 7 (2015) 139. https://innovareacademics.in/journals/index.p hp/ijpps/article/view/3548/7675. 26. m. m. al-oqail, n. n. farshori., e. s. alsheddi, j. musarrat, a. a. al-khedhairy, m. a.siddiqui, asian pac. j. cancer p, 14 (2013) 1829. http://dx.doi.org/10.1155/2016/1250387. 27. d. tiran, complementary therapies in nursing and midwifery, 9 (2003) 155. 28. d. vasudev, p. s. kulkarni and s. kulkarni, int. j. chem. stud., 1 (2013) 1. 29. j. p. yadav, s. kumar, l. bhudhwar, a. yadav, m. yadav, j. nanomed. nanotechnol., 7 (2016) 384. doi:10.4172/2157-7439.1000384. 30. n. k. leela and k. m. shafeekh, chemistry of spices fenugreek (editors: v. a. parthasarathy, b. chempakam., and t. j. zachariah., eds., biddles ltd, cab international, king’s lynn, uk) 242 (2008). doi:10.1079/9781845934057.0000. 31. r. k. meena and n. chouhan, res. j. recent sci., 4 (2015) 47. http://www.isca.in/rjrs/archive/v4/iivc2015/10.isca-ivc-2015-04cs-004.pdf 32. m. younas, organic spectroscopy and chromatography, third edition, ilmi kitab khana, pakistan (2014) 58. http://ilmikitabkhana.com/m-younasilmi/organic-spectroscopy-andchromatography-m-younas.html 33. p. sutradhar, m. saha and d. maiti, j. nanostruct. chem., 4 (2014) 86. https://doi.org/10.1007/s40097-014-0086-1. microsoft word 05-pjac-251005-07 issn-1680-9955 pak. j. anal. chem. vol. 6, no. 1, (2005) 22 – 27 conjugated linoleic acid: a mixture of bioactive fatty acids in milk fat of ruminants farah n. talpur* and m. i. bhanger national center of excellence in analytical chemistry, university of sindh, jamshoro, 76080 pakistan. abstract conjugated linoleic acid (cla) present in ruminant milk, have several health benefits including anticarcinogenic, antiatherogenic, immuno-modulating, growth promotion and lean body mass promotion. in the present work we have investigated the content of one dominant (c9, t11-cla), one intermediate (t10, c12-cla) and the three minor cla isomers (t11, c13, t7, c9cla) in ruminant milk from three districts of sindh. a total of 167 milk samples were collected throughout the year from dairy farms in thatta, dadu and hyderabad, sindh. the results show strong variation in cla content, depending on ruminant milk and season. the mean cla concentration was higher in cow’s milk fat ranging from 8.81 -10.99 mg /g followed by sheep 8.39 – 9.10 mg /g, goat 5.90 -6.35 mg /g and buffalo milk fat 5.10 – 6.22 mg /g respectively. summer milk fat contains 7.60 – 24.00 % higher content of total cla as compared to winter milk in all ruminants. the differences in cla contents are possibly due to the different activity of desaturase enzymes among ruminants, while seasonal variations in milk cla concentrations reflect the availability of green pasture and its quality. keywords: conjugated linoleic acid, seasonal variation, ruminants, green pastures introduction conjugated linoleic acid (cla) represent a mixture of positional and geometrical isomers of octadecadienoic acid with conjugated double bounds. cla have been demonstrated to have a range of positive health effects in experimental animals. these include suppression of carcinogenesis [1 – 2], antiobesity effect [3], modulation of the immune system [4] and reduction in atherogenesis [5] and diabetics [6]. lee et al. (1994) [7] and nicolas et al. (1993) [8] have also reported positive effects of cla on cardiovascular risk factors in animal models. cla occurs naturally in food; however, the principal dietary sources are dairy products and other foods derived from ruminant animals [9]. milk and dairy products from ruminants ( cow, goat, sheep etc.) are the opulent sources of c9, t11-cla isomer incorporate 90% of total cla in milk fat, small amounts of t7, c9-, c11, t13and t10, c12-cla isomers may also occur [10]. naturally occurring c9, t11 cla has been shown to reduce the number and incidence of mammary tumours in the rat [11 – 12], suggesting that predominant cla isomer in bovine milk fat is the active component. the evidence became so convincing that the national academy of science advised in 1996 that "conjugated linoleic acid (cla) is the only fatty acid shown unequivocally to inhibit carcinogenesis in experimental animals" [13]. in the rumen c9, t11-cla isomer results primarily as a product of endogenous synthesis via the enzyme δ9-desaturase, with the subtract being t -11 18:1, an intermediate formed in rumen biohydrogenation of linoleic and linolenic acids [10, 14] .conjugated linoleic acid is also an intermediate in the rumen biohydrogenation of linoleic acid, and some escapes complete biohydrogenation and provides the reminder of the cla in milk [15]. pak. j. anal. chem. vol. 6, no. 1, (2005) 23 the content of cla in milk can vary widely, about 3 to 25 mg g-1 fat [16 -18]. the underlying factor resulting in this variation are predominately related to the ruminant diet, including forage to concentrate ratio [19], level of intake [20] and intake of unsaturated fatty acids, especially plant oils that are high in linoleic acid [21]. recently thordottir et al. (2004) [22] investigated the distribution of c9, t11 cla in milk fat from nordic countries with comparatively lower content of cla than european countries [23]. frequency distribution of conjugated linoleic acid and t fatty acids contents in europeans bovine milk fats. these differences are attributed due to shorter summers, hence shorter periods of pasture grazing, as the ruminal outflow of c9, t11-cla is enhanced by pasture intake [24]. there is however, no data available on cla contents of ruminant milk in pakistan. therefore the present study was design to investigate cla content in the milk of ruminant from sindh (pakistan), under varying dietary habitats of ruminants in this region. material and methods milk sampling total 167 samples of milk were collected from dairy cows (n = 46), buffaloes (n = 50), goats (36), and sheep (n=35) during one year, twice in winter (december and march) and twice in summer (june and august). samples were procured from six dairy farms; situated in dadu, thatta and hyderabad region, while milking times at 0500 and 1700 h. milk samples were composted for each ruminant and divided in to three sub samples in order to get triplicate analysis. milk samples were packed in ice and dispatched to laboratory, where stored at –20 0c until analysis. fat extraction fat in milk (1 ml) was extracted using a procedure for total lipids in human milk as described by jensen et al. (1991) [25]. except that 1 ml of an internal standard (1 mg/ml, of methyl nonadecnoic acid [c19: 0]; sigma chemical co., st. louis, mo; dissolved in chloroform/methanol, 2:1, vol/vol) was added to each sample before extraction, and the extracted lipids were evaporated under nitrogen. the lipids were then esterified in capped screw-top tubes (teflon lined) with 6 ml of 0.5 n sodium methoxide heated at 50°c for 10 min [26]. the fatty acid methyl esters (fame) were then cooled to room temperature, and 2 ml of iso-octane and 3 ml of 10% acetic acid were added. the tubes were recapped to prevent evaporation of the short chain fatty acids. the fame were centrifuged (2000 × g) for 10 min, and a portion of the top layer removed and placed in sealed gas chromatography vials and kept at −20°c until analyzed. gas chromatographic analysis the fame were analyzed in a gas chromatograph (perkin elmer 8700) fitted with sp2340 fused silica capillary column (60 m × 0.25 mm i.d. × 0.2-µm film thickness: supelco, inc., bellefonte, pa) using manual injection. nitrogen was the carrier gas, which was set at a 33 psi. the injection volume was 2µl with a split/ splitless ratio (80/20). the column parameters were as follows: initial column temperature was held at 70 °c for 2 min; increased 15 °c/ min to 155 °c (held for 25 min), then increased at 3 °c / min to 215 °c (held for 8 min). the total run time was 61 min. data was collected automatically using the computer program: chromperfect for windows (justice innovations, mountain view, ca). high purity individual c9, t11and t10, c12cla (matreya, inc., pleasant gap, pa) were used to identify the cla isomers of interest. additional cla standard mixture (sigma chemical co.) was used to identify cla isomers in ruminants. statistical analysis statistical analysis was carried out using minitab (minitab, 13.0, pa, usa). differences between seasonal periods were assessed by anova using glm model. differences between periods mean were determined by tukey’s pak. j. anal. chem. vol. 6, no. 1, (2005) 24 studentized procedure and were accepted as significantly different p < 0.05. data for fatty acid concentrations are presented as least square mean with pooled standard error of mean (sem). result and discussion the concentration (mg / g) of conjugated linoleic acid (cla) isomers including c9, t11-, t10, c12and the sum of t7, c9 and t11, c13cla in ruminants (i.e. buffalo, cow, goat and sheep) milk fat is reported in table 1. depending on ruminant milk and season, the cla content was subjected to strong variation (p ≤ 0.05). the concentration of mean cla was found between 5.35 to 10.42 mg / g of total fatty acids, with higher in cow’s milk fat ranging from 8.81 -10.68 mg /g followed by sheep 8.39 – 9.10 mg /g, goat 5.90 -6.35 mg /g and buffalo milk fat 5.10 – 6.22 mg /g respectively. variation in cla content of ruminants has been associated with several factors such as stage of lactation, parity [27], and breed [28 29].however, diet is the most important factor influencing cla concentration [17, 29-30]. in particular, the cla concentration has been found higher in milk from animals fed pasture than those fed dry diets [31]. summer milk produce more elevated concentration 7.60 – 22 % (5.93 – 10.99 mg /g) of total cla in comparison to winter (5.55 – 8.59 mg/g), when less pasture and fresh grass was available and ruminants were also fed dry forages. lawless et al. (1998) [32] and dhiman et al (1999) [33] have also established that animals grazing fresh grass have higher levels of cla in their milk and meat than those consuming conserved forages. pasture is a richer source of linoleic and α -linolenic acids; the latter comprise more than 50% of total fa, whereas the preservation of grass, particularly ensilage, causes the loss of these fatty acids [34]. further more main fatty acid in grass lipids is αlinoleic acid (c18:3) that decreases both in absolute and relative values as grass matures [30, 35]. the majority of cla content was comprised of c 9, t11isomer contributing 8990 % of cla, while t10, c12consisted of 3.2 – 5.90 % of cla in all ruminants investigated in present study. these results are consistent with earlier reports by parodi (1999) [36] and bauman et al., (2003) [37]. the mechanism behind the variation of cla levels among ruminants would primarily be related to two factors, a) rumen production of its t11-18:1 isomer trans vaccenic acid (tva) and cla, and b) the activity of ∆9 desaturase. however rumen production of tva and cla are related to the biohydrogenation of substrates available from the diet, and the type and species of bacteria that biohydrogenate the available substrate to produce cla and tva. considering the fact that rumen out put of cla contributes only marginally to the overall cla content in milk and possibly meat fat, the activity of ∆9desaturase is important to describe at least some of the animal to animal variation [37]. table 2 demonstrates the comparison of total cla content in ruminant with earlier reported data. it is evident from the table that cla concentrations obtained in present investigation are consistent with previous studies in ruminants under varying conditions. however, the mean cla content in milk samples obtained in present work is higher than reported earlier [22] for nordic countries (0.76 vs. 0.58 %). similarly precht and molketin (2000) [23] have also detected higher cla content in european milk in comparison with nordic countries (0.89 vs. 0.58%). according to authors this variation can be explained by the different feeding periods in winter and summer, as it is well known that pasture feeding (summer) increases the content of cla in milk fat compared to high concentrate allocations in winter. this is due to the high concentrations of omega-3 fatty acids in grass, which are a source of cla. shorter summers and longer winters in nordic countries could explain these indicated cla differences between the nordic and pakistan as well as other european countries. pak. j. anal. chem. vol. 6, no. 1, (2005) 25 table – 1. conjugated linoleic acid isomers content mg / g in milk fat of different ruminants conjugated linoleic acid (cla) isomers content in different ruminants (mg / g) summer (may july) ruminant c9,t11-cla t10, c12-cla ∑ (t7,c9-, t11, c13-) cla mean sem buffalo 5.55 0.31 0.36 6.22 0.02 cow 10.00 0.49 0.50 10.99 0.03 goat 5.76 0.27 0.32 6.35 0.02 sheep 8.22 0.38 0.50 9.10 0.01 winter (december – february) ruminant c9, t11-cla t10, c12-cla ∑ (t7, c9-, t11, c13-) cla mean sem buffalo 4.65 0.19 0.26 5.10 0.01 cow 8.01 0.37 0.43 8.81 0.01 goat 5.30 0.25 0.35 5.90 0.02 sheep 7.55 0.38 0.46 8.39 0.01 table 2. comparison of total cla (mg / g) in ruminant milk fat obtained in present study with literature values ruminant present study (mean of both seasons) literature values reference buffalo 5.75 5.00 – 8.40 39, 40 cow 9.43 4.4 0 – 17.00 41, 42 goat 6.23 2.77 – 6.90 43, 44 sheep 8.46 8.00 20.00 44, 45 conclusion present study reveals that the cla content in ruminant’s milk is subjected to variation depending on ruminant and season. the cla content is higher in summer than in winter, all ruminant studied showing the similar changes. the availability of pasture and fresh grass explain the seasonal differences. the results indicate a higher cla content in sindh (pakistan) than in nordic countries ruminant milk in average, which may be related to longer summers and hence, longer periods of pasture feeding in the pakistan. the implications for varying cla concentration on health have to be studied references 1. m.a. mcguire and m.k. mcguire, proc. am. soc. anim. sci. annu. mtg. (2000) 1999. 2. m.a. burely, annu. rev. nutr. 22 (2002) 501. pak. j. anal. chem. vol. 6, no. 1, (2005) 26 3. y. park, k.j. albright, w. liu, j.m. storkson, m.e. cook and m.w. pariza, lipids.32 (1997) 853. 4. m.e. cook, c.c. miller, y. park and m. pariza, poult. sci.71 (1993) 1301. 5. r.j. nicolosi, e.j. rogers, d. kritchevski, j. a. scimeca and p.j. huth, artery. 22 (1997) 266. 6. k.l. houseknecht, j.p. vanden heuvel, s. y. moya-camerana, c. p. portocarrero, l. w. peck, k.p. nickel and m.a. belury. biochem. biophys. res. commun. 244 (1998) 68. 7. k.n. lee, d. kritchevsky and m.w. pariza, atherosclerosis. 108 (1994) 8. r.j. nicolosi, k.v. courtemanche, l. laitinen, j.a. scimeca and p.j. huth, circulation. 88 suppl. (1993) 1 -457. 9. tanaka keiichi, animal science journal. 76 (2005) 291. 10. d.e. bauman, b.a. corl and d.g. peterson. the biology of conjugated linoleic acid in ruminants. aoac press, champaign, il. 2 (2003) 146. 11. y.l. ha, j.m. storkson and m.w. pariza, cancer research. 50 (1990) 1097. 12. c. ip, m. singh, h.j. thompson, j.a. scimeca, cancer research. 54 (1994) 1212. 13. national research council (nrc): carcinogens and anticarcinogens in the human diet. national academy press, washington dc 1996. 14. d.e. bauman, b.a. corl and l.h. baumgard and j.m. griinari, conjugated linoleic acid (cla) and the dairy cow. nottingham university press, nottingham, u.k. (2001) 237. 15. j.k. kay, t.r. mackle, m.j. auldist, n.a. thomson and d.e. bauamn. j. dairy. sci. 87 (2004) 369. 16. j. fritsche and h. steinhart, fetts lipids. 100 (1998) 190. 17. m. collomb, u. butikofer, r. sieber, b. jeangors and j.o. bosset, int. dairy j. 12 (2002) 661. 18. m. collombs, h. sollberger, u. butikofer, r. sieber, w. stoll, and w. schaeren, int. dairy j. 14 (2004) 549. 19. j.m. griinari, d.a. dwyer, m.a. mcguire, d.e. bauman, d.l. palmquist and k.v.v. nurmela, j. dairy sci. 81 (1998) 1251. 20. c.r. stockdale, p.g. walker, j.w. wales, e.d. dalley, a. birkett, z. shen and doyle. j. dairy. res. 70 (2003) 267. 21. a.t. ward, k.m. wittenberg and r. przybylski. j. dairy sci. 85. (2002) 1191. 22. i. hill thorsdottir, j. a. ramel and j. dairy. sci. 87 (2004) 2800. 23. d. precht, j. molkentin milchwissenschaft. 55 (2000) 687. 24. r.c. khanal, t.r. dhiman, d.j. msmahon, r.l. boman and j. dairy. sci. 85 suppl. 1 (2002) 142. 25. r.g. jensen, a.m. ferris and c.j. lammikeefe, j. dairy sci. 74(1991). 3228. 26. j.kg. kramer, v. fellner, m. e. r .dugan, f.d. sauer, m.m. mossoba and m.p. yurawecz. lipids. 32 (1997) 1219. 27. m.l. kelly, j.r. berry, d.a. dwyer, j.m. griinari, p.y. chouinard, m.e. van amburgh and d.e. bauman, j. nutr. 128 (1998) 881. 28. p. secchiari, m. mele, a. serra, a. buccioni, m. antongiovanni, g. farruzzi, f. paoletti and l. andreotti, progr. nutr. 3 (2001) 37. 29. s.l. white, j.a. bertrand, m.r. wade, s.p. washburn, j.t. green, t.c. jenkins and j. dairy sci . 84 (2001) 2295. 30. d.e. bauman, j.m. griinari and livest. prod. sci. 70 (2000) 15. 31. y.a. chilliard, r.m. ferlay and m. doreau, ann. zootech. 49 (2000) 181. 32. a. elgersma, s. tamminga and g. ellen, grassland science in europe. 8(2003) 271. 33. f.lawless, j.j. murphy, d. harrington, r. devery and c. stanton, 1998. j. dairy. sci. 81 (1998) 3259. 34. t.r. dhiman, g.r. arnand, l.d. sattar and m.w. pariza, j. dairy sci. 82 (1999) 2146. 35. c.g. harfoot and g.p. hazlewood. lipid metabolism in the rumen. blackie. prof. london, (1997) 382. 36. a. nudda, m.a. mcguire, g. battacine and g. pulina, j. dairy sci. 88 (2005) 1. 37. p.w. parodi, j. dairy. sci. 82 (1999)1339. 38. d.e. bauman, b.a. corl and g. peterson. the biology of conjugated linoleic acids in ruminants. aocs press, champaign, il, 2 (2003) 146. 39. p.r. aneja, and. murti n.t. ind. j. dairy sci. 43 (1990) 231238. pak. j. anal. chem. vol. 6, no. 1, (2005) 27 40. d. lal, k.m. narayanan, ind. j. dairy sci. 37 (1984) 225. 41. a.j. kelsey, a.b. corl. j.r. collier and e.d. bauman 42. r.c. khanal, t.r. dhiman, d.j. mcmahan and r.l. boman, j. dairy sci. 85 (2002) 142. 43. r.l. wolff, j. am. oil chem. soc.72 (1995) 259. 44. p. parodi, conjugated linoleic acid in food. aoacs press, champaign, il. 2 (2003) 101. 45. a. prandini, d. geromin, f. conti, f. masoera, a. piva and g. piva, j. food. sci.13 (2001) 243. microsoft word 09-270-276-pjaec-15092022-452-c-revised galley pro cross mark issn-1996-918x pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 270 – 276 http://doi.org/10.21743/pjaec/2022.12.09 determination of diclofenac diethylamine levels in emulgel preparations using nir spectroscopy combined with chemometrics lestyo wulandari 1 *, karima pratiwi 1 and yuni retnaningtyas 1 1faculty of pharmacy, university of jember, kalimantan street 37 jember, 68121, indonesia. *corresponding author email: lestyowulandari@unej.ac.id received 15 september 2022, revised 28 december 2022, accepted 29 december 2022 -------------------------------------------------------------------------------------------------------------------------------------------abstract diclofenac is an nsaid-class drug with activity as an analgesic and anti-inflammatory recommended for treating various acute and chronic pain conditions. one of the topical preparations of diclofenac that is often used is emulgel. in this study, diclofenac diethylamine levels in emulgel preparations were determined using nir (near-infrared) spectroscopy and chemo metric methods. simulation sa mples were prepared and divided into 24 training sets and 9 test set samples. nir spectra of training set samples were correlated with the concentration of diclofenac diethyla mine using partial least squares (pls), principal component regression (pcr), and support vector regression (svr). the best model was validated using leave one out cross validation (loocv) and external validation using test set samples. the co mparison method used in this study was the validated tlc densitometry method. the best calibration model was pls, with an r2 value of 0.990 and rmse of 0.171. the results of r2 and rmse of loocv were 0.989 up to 0.990 and 0.167 up to 0.178, respectively. the result of r2 and rmsep external validation were 0.991 and 0.146, respectively. the precision and accuracy method showed rsd of 3.37% and a % recovery of 99.78%. the results of determining the sample levels obtained from nir and tlc densitometry methods tested with the two-paired sample t test and showed that the two methods have no significant differences with a significance value of more than 0.05. keywords: nir spectroscopy, chemo metrics, diclofenac diethylamine, emulgel -------------------------------------------------------------------------------------------------------------------------------------------introduction osteoarthritis (oa) is a disease that often occurs in the elderly. osteoarthritis is characterized by cartilage degeneration, where the damage can cause pain and loss of ability to move [1]. world health organization (who) in 2018 stated that the number of people suffering from osteoarthritis was 343 million worldwide [2]. the initial treatment for mild osteoarthritis is paracetamol, this is because paracetamol is safe, effective, and cheap. however, the u.s. food and drug administration does not recommend taking more than 4,000 mg of paracetamol per day to avoid liver toxicity. when paracetamol cannot reduce symptoms, in cases of moderate to severe osteoarthritis, nsaid treatment is recommended [3]. diclofenac is an nsaid (non-steroidal anti-inflammatory drugs) class drug that has activity as an antiinflammatory, analgesic, and antipyretic. this drug is commonly used to treat acute and chronic pain, rheumatoid, and osteoarthritis. one of the topical preparations of diclofenac that is often used is emulgel. emulgels are pak. j. anal. environ. che m. vol. 23, no. 2 (2022)271 emulsions, either oil in water or water in oil, which are mixed into gel preparations with gelling agents [4]. the use of the topical route can avoid first pass metabolism, direct administration to the target site, the administration may be more acceptable to patients to improve compliance, effective for patients who have difficulty swallowing [5]. several research methods have been conducted to determine the levels of diclofenac diethylamine. these methods were hplc (high-performance liquid chromatography) [6], tlc (thin layer chromatography) [7], uv-vis spectroscopy [8,9], and spectrofluorometry [10]. in this study, the determination of diclofenac diethylamine levels in emulgel preparations was evaluated using nir spectroscopy and chemometric methods. this method was chosen because there has been no analysis of diclofenac diethylamine in emulgel preparations using nir spectroscopy. nir spectroscopy is an effective analytical technique because it does not require solvents, does not cause contamination, does not require chemicals, and has the high analytical capability [11]. however, the nir spectra were complicated and overlapping, so a multivariate analysis was needed. multivariate analysis is a mathematical and statistical method that can separate data from analytical information, such as nir spectrum information called chemometrics [12]. chemometric techniques were used to correlate the spectrum profile and the information contained in the sample [13]. quantitative multivariate analysis techniques were used partial least squares (pls), principal component regression (pcr), and support vector regression (svr) [14]. materials and methods chemical and reagents diclofenac diethylamine used in this study was pharmaceutical grade (aarti drugs ltd, india). all ingredients of emulgel preparation were pharmaceutical grade, i.e., carbopol (cv kimia jaya labora), liquid paraffin, peg 400, nipagin, nipasol (sigmaaldrich), propylene glycol, tea (cv nurra gemilang malang). reagents used were analytical grade, i.e., methanol pro analysis (merck), toluene, ethyl acetate, glacial acetic acid, filter paper (whatman), distilled water, and tlc plates (merck). four commercial samples of diclofenac diethylamine emulgel were purchased from a pharmacy store in east java, indonesia, in august of 2021. instrumentation the tools used in this study were a densitometer scanner (camag), wincats software, nir spectroscopy (brimrose luminar 3070), the unscrambler x 10.4 software (camo), analytical balance (sartorius), ultrasonicator (elmasonic), capillary micro pipette (socorex), mortar and stamper, and glassware. sample simulation preparation the preparation of emulgel simulation samples was based on bhanu et al. with modification. diclofenac diethylamine emulgel simulation samples were made in oilin-water type with the addition of diclofenac diethylamine. simulated emulgel samples were prepared by distinguishing between the oil phase and the liquid phase. in the aqueous phase, carbopol and distilled water were crushed in a mortar, then tea was added. nipagin and nipasol were dissolved in propylene glycol. in the oil phase, liquid paraffin was dissolved, and peg 400 was heated in a cup at 75°c. the oil phase was added gradually to the water phase with continuous stirring until a fine emulsion was formed, and then spiking diclofenac diethylamine to the emulgel gradually until pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 272 varying concentrations were obtained. the simulation samples were divided into a training set and a test set sample. the training set sample consists of 24 samples with a concentration variation range of diclofenac diethylamine of 0% 5.75%, while the test set sample consists of 9 samples with a concentration of 0.6% 5.4%. determination of nir spectra the samples were analyzed with a nir instrument, luminar 3070. before the samples were measured, the instrument was heated for 30 min. the sample was placed on the sample holder plate. each sample was replicated 5 times, and each replication was subjected to 5 shots. the spectra wavelength range was 850 nm 2000 nm. preparation of diclofenac diethylamine standard solution the standard solution of diclofenac diethylamine in methanol was made at a concentration of 100, 200, 400, 600, 800, 1000 and 1200 µg/ml. preparation of sample the emulgel sample was weighed 300 mg in a beaker glass and extracted with methanol, then ultrasonicated for 15 min. the extracted sample was put into a 10 ml volumetric flask and rinsed the beaker glass with the solvent, then added methanol up to 10 ml. the extracted sample was filtered using filter paper and put into a vial. method validation the determination of the levels of the training set, test set, and commercial samples was carried out after this comparison method was validated through the stages of eluent optimization, wavelength optimization, linearity, specificity, detection limit and quantitation limit, precision and accuracy [15]. chemometrics calibration model and validation the chemometrics calibration model for quantitative analysis in this study was formed with pls, pcr, and svr multivariate analysis techniques. the selected calibration model was validated using loocv and external validation. loocv evaluated the model using the training set data by removing a set of data then the remaining data was used to form a new model. the process was repeated until all data was used as a validation set. external validation used an independent sample (test set) to evaluate the model by comparing the predicted value of the test set sample generated from the model with the reference value [16]. the accuracy and precision of the method were evaluated using three levels of concentration of the sample and three replication [17]. the valid calibration model was applied to the determination of diclofenac diethylamine in the commercial sample and then compared with the levels obtained from the comparison method (tlc densitometry). the comparison methods were tested with the two paired samples t-test to determine whether there was a significant difference. results and discussion in this study, the diclofenac diethylamine standard solution concentration range of 0% 5.75% was chosen as the training set because this range already covered the concentration range of diclofenac diethylamine on commercial emulgel. the simulated training set and commercial emulgel sample (fig. 1) have similar spectral patterns. the spectra of the training set simulation pak. j. anal. environ. che m. vol. 23, no. 2 (2022)273 sample and the commercial emulgel sample have different transmittance values. figure 1. nir spectras of diclofenac di ethyl ami ne (ddea), real sample (v), and si mul ation sample 2% (ss 2%) the tlc densitometry results of eluent optimization were toluene: ethyl acetate: glacial acetic acid (v/v/v) = 8:2:0.3 with rf value of 0.48 which is included in the range of optimum rf 0.2-0.8; rs value of 2.214 which has met the resolution requirements of greater than 1.5; the largest n value was 237.037; and the smallest h value was 0.379. the optimum wavelength was 284 nm because it had the highest reflectance value. the method used as a comparison method has been validated with the results of the parameter assessment of each validation stage listed in table 1. table 1. tlc densi tometry method validation resul ts. vali dation parameters results linearity linierity range (n=5) 404 – 3232 ng correlation coefficient (r) 0.998 coefficient of variation (vx0) 3.867% lod 98.179 ng loq 294.54 ng specificity purity and identity test r >0.99 precision (rsd, n=9) 1.227% accuracy (%recovery ± rsd (%) simulation 0.6% 100.33 ± 0.831 simulation 1.2% 100.583 ± 0.911 simulation 1.8% 97.44 ± 1.938 this method fulfilled the linearity requirement, i.e., correlation coefficient (r) ≥ 0.99 and the coefficient of function variation (vxo) < 5%. the purity test was determined based on the r(s,m) value and the r(m,e) value which produces a value of more than 0.99. the identity test was determined based on the r(s,s) value and the r(s,a) value where the r(s,s) value showed the spectral correlation between the two standard tracks. in contrast, r(s,a) showed the correlation between the standard track and the analyte track in the sample. the analyte in the sample was identical to the standard if the correlation value was more than 0.99 [18]. it can be concluded that the analytes in the standard and sample are pure and identical. the assessment of the precision and accuracy fulfilled the acceptance requirement of the rsd value for the precision test of aoac [19]. the results of the calibration model in table 2 showed that the three calibration models formed met the criteria for a good calibration model where the r 2 value was more than 0.91. in this study, the pls calibration model was the best model because it has the highest r 2 value of 0.990 and the smallest rmse value of 0.171. table 2. trai ni ng set sample cali bration model resul ts. no. model rmse r2 1. pls calibration validation 0.171 0.176 0.990 0.989 2. pcr calibration validation 0.492 0.495 0.918 0.917 3. svr calibration validation 0.394 0.399 0.948 0.947 the loocv results are shown in table 3. loocv has r2 >0.91, and the result of the rmse value was small. the pls model was valid in loocv. pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 274 table 3. pls cali bration model loocv validation results. data removed rmse r2 no data removed calibration validation 0.167 0.174 0.989 0.989 training set 1.5% calibration validation 0.173 0.178 0.990 0.989 training set 4.25% calibration validation 0.169 0.177 0.990 0.989 training set 5.75% calibration validation 0.168 0.176 0.989 0.989 the results of external validation shown in fig. 2, which have an r 2 value >0.91 and an rmse value was small, so the pls calibration model has good reliability to be implemented on commercial samples [20]. the precision and accuracy of the method result showed an rsd of 3.37% and a % recovery of 99.78%. the results of the determination of diclofenac diethylamine levels in commercial samples of emulgel by tlc densitometry and nir spectroscopy methods can be seen in table 4. table 4. results of diclofenac diethylami ne level determination in commerci al samples. diclofenac diethyl ami ne content w/w (%) sampl e nir spectroscopy ± sd tlc densi tometry ± sd a 1.129 ± 0.028 1.140 ± 0.027 m 1.150 ± 0.027 1.153 ± 0.031 f 1.152 ± 0.027 1.149 ± 0.016 v 1.148 ± 0.024 1.151 ± 0.031 the results of the determination of diclofenac diethylamine levels showed that the normality test value >0.05, meaning that the data in both methods are normally distributed. the two paired samples t-test has a significant value (2-tailed)>0.05, so it can be concluded that there is no significant difference between the nir spectroscopy and tlc densitometry methods [21]. figure 2. results of external vali dation method using test set samples pak. j. anal. environ. che m. vol. 23, no. 2 (2022)275 conclusion from this research, it can be concluded that the diclofenac diethylamine levels in emulgel can be determined by nir spectroscopy combined with chemometric methods using the best calibration model, namely pls with an r2 value of 0.990 and rmse of 0.171. there is no significant difference in the determination of diclofenac diethylamine levels using tlc densitometry and nir-chemometric evidenced by the results of two paired samples t-test with a significance value (2-tailed) > 0.05. acknowledgments the authors are grateful to the research group of pharmaceutical analysis and chemometrics, faculty of pharmacy, university of jember. conflict of interest the authors declare no conflict of interest of this article. references 1. j. martel-pelletier, osteoarthr. cartil., 12, (2004) 31. https://doi.org/10.1016/j.joca.2003.10.002 2. world health organization, “musculoskeletal conditions,” (2018). https://www.who.int/news-room/factsheets/detail/musculoskeletal-conditions 3. k. sinusas, am. fam. physician, 85 (2012) 49. doi: 10.1136/bmj.1.5222.355-a. https://doi.org/10.1136/bmj.1.5222.355-a 4. p. v. bhanu, v. shanmugam, and p. k. lakshmi, int. j. compr. pharm., 6 (2011) 81. http://www.pharmacie-globale.info 5. m. l. mcpherson and n. m. cimino, pain med. (united states), 14 (2013) 35. https://doi.org/10.1111/pme.12288 6. y. shah, s. joshi, k. c. jindal and s. khanna, drug dev. ind. pharm., 9045 (2016) 1. https://doi.org/10.3109/03639049409038 370 7. w. thongchai, b. liawruangrath, c. thongpoon and t. machan, chiang mai j. sci., 33 (2006) 123. http://www.science.cmu.ac.th/journalscience/josci.html 8. r. bucci, a. d. magrì and a. l. magrì, fresenius j. anal. chem., 362, (1998) 577. https://doi.org/10.1007/s002160051127 9. d. kumble, n. badiadka and s. majal, int. j. pharm. sci. res., 4 (2013) 3635. http://dx.doi.org/10.13040/ijpsr.09758232.4(9).3635-40 10. h. j. patel, int. j. chem. life sci., 6, (2017) 2006. https://doi.org/10.21746/ijcls.2017.2.1 11. a. a. gouda, m. i. k. el-sayed, a. s. amin and r. el sheikh, arab. j. chem., 6 (2013) 145. https://doi.org/10.1016/j.arabjc.2010.12. 006 12. m. otto, chemometrics: statistics and computer application in analytical chemistry (w iley-vch, weinheim, germany) 3rd edition, (2017). https://www.wiley.com/enus/chemometrics%3a+statistics+and+c omputer+application+in+analytical+ch emistry%2c+3rd+edition-p9783527699384 13. h. bin zou, g. s. yang, z. r. qin, w. q. jiang, a. q. du, and h. y. aboulenein, anal. lett., 38 (2005) 1457. https://doi.org/10.1081/al-200062153 14. y. roggo, p. chalus, l. maurer, c. lema-martinez, a. edmond, and n. jent, j. pharm. biomed. anal., 44 (2007) 683. https://doi.org/10.1016/j.jpba.2007.03.023 15. g. indrayanto, m. yuwono and suciati, encyclopedia of chromatography (crc press, florida, us) (3rd edition), vol. 24, pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 276 no. 6. (2010). https://doi.org/10.1108/09504121011067 166 16. l. wulandari, r. idroes, tr. noviandy and g. indrayanto, application of chemometrics using direct spectroscopic methods as a qc tool in pharmaceutical industry and their validation (academic press, cambridge, us) (2021). https://doi.org/10.1016/bs.podrm.2021.1 0.006 17. bp, “ sc iii f. validation of analytical procedures,” be. pharmacopoeia 2, vol. 2022, (2019) 2021. european medicines agency, amsterdam, netherlands https://www-pharmacopoeiacom.queens.ezp1.qub.ac.uk/bp2020/supplementary-chapters/sc-3/sc-iiif--validation-of-analyticalprocedures.html?published-date=201908-01&text=validation. 18. m. yuwono and g. indrayanto, profiles drug subst. excipients relat. methodol., vol. 32 (2005) 241. https://doi.org/10.1016/s00995428(05)32009-0 19. aoac, “appendix f : guidelines for standard method performance requirements,” 2016. 1-18. https://www.aoac.org/resources/guidelin es-for-standard-method-performancerequirements/ 20. q. zhang, q. li and g. zhang, spectrosc. (new york), 27 (2012) 93. https://doi.org/10.1155/2012/276795 21. k. tae kyun, recipes sci., 68 (2015) 540. https://doi.org/10.4097/kjae.2015.68.6.5 40 microsoft word pjaec-100608-15.doc pdfmachine from broadgun software, http://pdfmachine.com, a great pdf writer utility! issn-1996-918x pak. j. anal. environ. chem. vol. 9, no. 2 (2008) phenol removal from aqueous system by jute stick a. i. mustafa1, m. saiful alam2, m. n. amin1, n. m. bahadur2 and a. habib3* 1department of applied chemistry and chemical technology, university of dhaka, dhaka-1000, bangladesh. 2applied chemistry and chemical technology, noakhali science and technology university, noakhali -3802, bangladesh 3*department of chemistry, university of dhaka, dhaka-1000, bangladesh ------------------------------------------------------------------------------------------------------------------------------------------- abstract the adsorption technique using jute sticks has been applied for the removal of phenol from aqueous solutions. the extent of removal was dependent on concentration of phenol, contact time, ph, and quantity of adsorbent. with an initial concentration of 40 ppm phenol in 100 ml and ph 10.0, the removal was found to be about 68% with 3.0 g jute stick. the time to reach equilibrium was found to be 5 hr. the applicability of freundlich isotherm to the adsorption of phenol system was tested at 25 0c to 40 0c at ph 10.0. the spent adsorbent was regenerated by acid treatment. keywords: removal; phenol; jute stick; batch system; freundlich isotherm. ------------------------------------------------------------------------------------------------------------------------------ introduction phenolic compounds which are generated from petroleum and petrochemical, coal conversion, and phenol-producing industries, are common contaminants in wastewater and suspected as toxic and carcinogenic. therefore, phenol and phenolic compounds were designated as priority pollutants by the us epa, which take the 11th place in the list of 129 chemicals [1]. also the european union (eu) has classified several phenols as priority contaminants and the 80/778/ec directive lays down a maximum concentration of 0.5 µg l-1 for total phenols in drinking water [2]. apart from their toxicity and carcinogenity, phenols can cause bad taste and odor, even at low concentration [3]. for this reason it is necessary to eliminate phenols from wastewater before it is discharged. various treatment technologies such as adsorption [4�6, 21, 22], photodegradation [7], flocculation [8], chemical oxidation [9, 23, 24], biological process [10], etc. are available for the removal of phenol from the wastewater. biological process is particularly suited to wastewater containing small amount of phenol. oxidation is used when phenol concentration in wastewater is very high. in coagulation and flocculation process, large amount of sludge is generated which may cause disposal problems. among various physicochemical processes, adsorption is widely used for the removal of phenol from wastewater [4,11]. literature on the adsorption of phenolic compounds onto activated carbon is abundant [12�14]. due to the relatively high cost of activated carbons there have been attempts to utilize low cost, naturally occurring adsorbents include straw, auto mobile tires, fly ash, coal reject, sewage sludge, bagasse, fertilizer waste and saw dust [15�19] to remove organic pollutants. however, the adsorption behavior of phenol on jute stick has not so far been extensively studied. the present study is intended to use of this locally available as a conventional cheap material as a phenol adsorbent. experimental materials and method adsorbent the jute sticks were carefully collected without any contamination from local area. after collection, jute sticks were treated to make them ready for use. the jute sticks were dried initially in an oven at about 70 0c and then ground to fine mesh and the particles size of 17762 µm were separated by sieving through standard test sieves. then these were boiled in distilled water continuously for 30 minutes. the suspension was then left to settle to allow the supernatant to be poured off. this process was repeated several times until the coloured water-soluble components were removed completely. finally the washed adsorbent was dried in an oven at 80 0c, allowed to cool and sieved into 177� 62µm for subsequent use. *corresponding author email: mahabibbit@yahoo.com pdfmachine is a pdf writer that produces quality pdf files with ease! get yours now! “thank you very much! i can use acrobat distiller or the acrobat pdfwriter but i consider your product a lot easier to use and much preferable to adobe's" a.sarras usa mailto:mahabibbit@yahoo.com http://www.pdfmachine.com?cl pak. j. anal. environ. chem. vol. 9, no. 2 (2008) chemicals and apparatus all the reagents and chemicals used were of a. r. grade (bdh). the solutions were prepared in double distilled water. a stock solution of phenol was prepared by dissolving 0.5g phenol in 500ml capacity volumetric flask. this was treated as stock solution of phenol (1000ppm). the phenol estimation was done spectrophotometrically by the 4-aminoantipyrene method using shimadzu uv-160 double beam spectrophotometer [20]. buffer solutions of different ph values procured from e. merk (germany) were used to keep the ph pf the solution fixed at a definite value. ph of the substrate was determined using a microprocessor bench ph meter (hanna ph 300). estimation of phenol by modified method the 4-aminoantipyrine method gave unsatisfactory result [20]. so attempts were made to modify the 4-aminoantipyrine method. preliminary experiments showed that phenol does not give any appreciable colour with 4-aminoantipyrine in presence of potassium ferricyanide in acidic medium. but at ph 8.0 phenol gives a deep brownish red colour whose intensity gradually increases up to ph 10.0 and then sharply decreases. this colour system was made the basis for the development of the modified spectrophotometric method for the determination of trace amount of phenol at a ph 10.0 instead of ph 8.0. absorbance measurements, carried out against reagent blanks, revealed that the absorption maxima is at 497 nm and that the time required for full colour development is 40 minutes. experimental result revealed that 2 ml of 2.0% (w/v) 4-aminoantipyrine solution is sufficient for the development of maximum colour intensity of the system containing 40 ppm phenol solution. results of the effect of potassium ferricyamide indicates that 6.0 ml of 8.0% of (w/v) potassium ferricyanide solution is the optimum amount for the development of the maximum colour intensity of the system containing 40 ppm phenol solution. the colour intensity of system under investigation was unaffected by variation in temperature from 25 to 40 0c. it was also observed that the colour intensity gradually decreases beyond this range. it was found that the modified method was more suitable. removal of phenol from aqueous solution batch adsorption: the batch experiments were conducted using 1.0g of adsorbent in 250ml capacity stopper bottles with 100ml of phenol solution. the adsorbate concentrations were varied in the range of 10� 60ppm. the whole study was carried out at ph 10.0, at higher ph the texture of the adsorbent is changed. the bottles were then shaken at uniform speed at room temperature using an electric shaker. however, experiments at higher temperatures were carried out in beakers in a thermostatically controlled water bath using electrical stirrer. at predetermined time intervals the contents were centrifuged and the remaining concentration of phenol in the supernatant were analyzed spectrophotometrically against respective reagent blank. the bottles containing phenol of different concentration and the specified ph, were shaken for 6 hours to ensure complete saturation. the amount of phenol adsorbed was determined from the difference between the amount of phenol initially added and that left after adsorption. batch experiments were also performed to study the effect of temperature at (25, 30 and 400c) on adsorption of phenol by jute sticks. interestingly, temperature (250�400c) had no appreciable effect on the adsorption of phenol and thus all subsequent experiments were carried out at room temperature. the concentration range of phenol adhering to beer's law under the conditions of investigation for the system was 10�60ppm. results and discussion effect of concentration and contact time batch experiments were carried out to investigate the effect of phenol concentration on the extent of adsorption as a function of time for the initial phenol concentration of 10, 40, 50 and 60 ppm which is shown in fig. 1. 0 2 4 6 8 10 0 100 200 300 400 x /m , m g /g m time , min fig.1: effect of initia l concentra tion a nd conta ct time on phenol remova l 60 ppm 50 ppm 40 ppm 10 ppm the percentage removal of phenol and phenolic compounds as well as the adsorptiondesorption behavior were also varied. the maximum percentage of phenol removal took place within 5 hours. the equilibration time is independent of concentration and hence this period was chosen for further equilibrium batch adsorption studies. figure. 1. effect of initial concentration and contact time on phenol removal pdfmachine is a pdf writer that produces quality pdf files with ease! get yours now! “thank you very much! i can use acrobat distiller or the acrobat pdfwriter but i consider your product a lot easier to use and much preferable to adobe's" a.sarras usa http://www.pdfmachine.com?cl pak. j. anal. environ. chem. vol. 9, no. 2 (2008) adsorption isotherm the adsorption behavior of phenol on jute sticks at all temperatures was in close agreement with the freundlich equation. the constants kf and n obtained from the linear regression analysis of log x/m= log kf + 1/n log ce the intercept log kf is roughly an indicator of sorption capacity and the slope 1/n is adsorption intensity. results of jute sticks are graphically presented in fig. 2. 0 0.5 1 1.5 -5 -3 -1 1 3 5 l o g x /m log ce fig. 2 : freundlich adsorption isotherm 40oc 30oc effect of temperature temperature is an important parameter for any separation process. removal of phenol by jute sticks was studied at four different temperatures: 25, 30, 35 and 40 0c. it was found that the temperature in the range of 25�40 0c had no effect on the phenol removal. therefore all subsequent experiments were carried out at room temperature. effect to ph the removal of phenol from the wastewater is highly dependent on ph of the solution. ph affects the surface charge of the adsorbent and degree of ionization and specification of phenol. the uptake with 40 ppm phenol is small at low ph ranges and gradually increases up to ph 10.0, where maximum removal 68% occurs. thus 10.0 ph was chosen as the optimum and all the subsequent experiments were carried out at this ph. as was expected, the adsorbed amount decreases with increasing the ph value. this can be attributed to the dependency of phenol ionization on the ph value. the ionic fraction of phenolate ion increases as the ph value increased. accordingly, phenol, which is a weak acid (pka=10), will be adsorbed to a lesser extent at higher ph values due to the repulsive forces prevailing at higher ph values. similar behavior has been reported for the adsorption of phenol by activated carbon [13]. regeneration 0.5 g of spent adsorbent after adsorption at ph 10.0 was washed with distilled water to remove any adhering phenol and was shaken with 100ml of 0.1m hcl for regeneration which was completed within 30 minute duration. about 68% of the adsorbed quantity of phenol from the initially present 40ppm was desorbed from all the samples in a single step. it was then washed with distilled water dried in a hot air oven and was reused in subsequent operations. in this way after regeneration samples can be reused in the operation. it reduces the operation cost and thus may be very useful. optimum quantity of adsorbents the amount of jute sticks taken were 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 4.0 g to determine the optimum quantity of jute sticks. the amount of reagents and chemicals added were kept unchanged and ph was also maintained at 10.0. from the adsorption data, it was found that adsorption is completed within 5 hr and the percentage of adsorption increases with the increasing amount of jute stick. the highest percentage of adsorption was obtained by using the 3.0g of jute stick. therefore, optimum quantity was found to be 3.0g which is shown in the table 1. table 1. optimum quantity of jute stick conclusion the present study shows that the jute stick is an effective adsorbent for the removal of phenol from aqueous solutions. moreover, in the context of the present efforts for the diversified uses of jute, a new field will be opened if jute sticks are taken as adsorbents. jute stick can be used as an adsorbent for phenol removal in a wide ph range of 6�12. under amount of jut stick (gm) adsorption (%) 0.5 54.35 1.0 59.78 1.5 61.86 2.0 63.87 2.5 65.96 3.0 67.69 4.0 67.68 figure 2. freundlich adsorption isotherm pdfmachine is a pdf writer that produces quality pdf files with ease! get yours now! “thank you very much! i can use acrobat distiller or the acrobat pdfwriter but i consider your product a lot easier to use and much preferable to adobe's" a.sarras usa http://www.pdfmachine.com?cl pak. j. anal. environ. chem. vol. 9, no. 2 (2008) batch conditions equilibrium was attained in 5 h. maximum removal of phenol was obtained with 3.0 g jute stick at an initial ph of 10.0. the freundlich isotherm show very good fit with the experimental adsorption equilibrium data. references 1. u. s. epa, federal register, washington, dc, 52, 131 (1987) 25861. 2. i. rodriguez, m. p. llompart and r. cela, solidphase extraction of phenols. journal of chromatography 885, (2000) 291. 3. v. k. gupta, s. sharma, i. s. yadav and d. mohan, j. chem. technol. biotechnol. 71 (1998) 180. 4. a. a. m. daifullah and b. s. girgis, water res. 32 (1998) 1169. 5. s. dutta, j. k. basu and r. n. ghar, sep. purif. technol. 21 (2001) 227. 6. s. haydar and m. a. ferro-garcia, j. riverautrilla, j. p. joly, carbon 41 (2003) 387. 7. c. wu, x. liu, d. wei, i. fan and l. wang, water res. 35 (2001) 3927. 8. t. a. ozbelge, o. h. ozbeze and s. z. baskaya, chem. eng. processing 41 (2002) 719. 9. v. kavita, k. palanivelu, chemosphere 55 (2004) 1235. 10. k. karim and s. k. gupta, biores. technol., 80 (2001) 179. 11. p. k. asthana and s. bhatia, ind. j. environ. protection, 14 (1994) 490. 12. g. bercic and a. pintar,. desorption of phenol from activated carbon by hot water regeneration, desorption isotherms. industrial engineering and chemistry research 35, (1996) 4619. 13. halhouli, k. a., darwish, n. a., al-jahmany, y., effects of temperature and inorganic salts on the adsorption of phenol from multicomponent system on a decolorizing carbon. separation science and technology 32, (1997) 3027. 14. a. r. khan, t. a. al-bahri and a. al-haddad, adsorption of phenol based organic pollutants on activated carbon from multicomponent dilute aqueous solutions. water research 31, (1997) 2102. 15. m. streat, j. w. patrick and m. j. camporro perez, water res. 29 (1995) 467. 16. g. q. lu, j. c. f. low, c. y. liu and a. c. lua, fuel 74 (1995) 344. 17. g. q. lu, environ. prog. 15 (1996) 12. 18. s. k. srivastava, v. k. gupta, n. johri and m. dinesh, indian j. chem. technol. 2 (1995) 333. 19. v. k. gupta, s. k. srivastava and t. renu, water res. 34 (2000) 1543. 20. american public health association. standard methods for the examination of water and waste waters, 16th ed. 556 (1985) 558. 21. s. mishra, j. bhattacharya, malaysian journal of chemistry, 9 (2007) 51. 22. p. s. nayak and b. k. sing, disalination 207 (2007) 71. 23. s. ghasempur, s. f. turab, s. o. ranaei-saidat, n. ghaemi and k. khajeh, environ. sci. technol. 41 (2007) 7073. 24. c. díaz,a g. ovejero,a j. l. sotelo, a. rodríguez, and j. garcíaa proceedings of european congress of chemical engineering (ecce-6) copenhagen, 16-20 september (2007). 25. p. ghosh, s. biswas and c. datta, journal of material science, 24 (1) (1989) 205.. pdfmachine is a pdf writer that produces quality pdf files with ease! get yours now! “thank you very much! i can use acrobat distiller or the acrobat pdfwriter but i consider your product a lot easier to use and much preferable to adobe's" a.sarras usa http://www.pdfmachine.com?cl issn-1996-918x pak. j. anal. environ. chem. vol. 8, no. 1 & 2, (2007) short communication voltammetric study of arsenic (iii) and arsenic (v) in ground water of hajigonj and kalkini in bangladesh samir chandra paul, mohammad arifur rahman, nur-e-alam siddique and a. m. shafiqul alam* department of chemistry, university of dhaka, dhaka-1000, bangladesh ------------------------------------------------------------------------------------------------------------------------------------------- abstract the speciation of arsenic in groundwater samples using square wave anodic stripping voltammetry (swasv), differential pulse anodic stripping voltammetry (dpasv) and normal pulse anodic stripping voltammetry (npasv) are described. good resolution of the species, arsenic (iii) and arsenic (v) is achieved using swasv. the reliability of the methods was checked by analyzing the total arsenic content of the samples by hydride generation atomic absorptioion spectrophotometer and by analyzing prepared controlled laboratory standard solution. since this technique is comparatively cheaper than other available techniques it could be a better analytical technique for arsenic speciation from water. in this study, the assessment of inorganic arsenic species in ground water of kalkini (madaripur) and hajigonj (chandpur) is reported. it shows that arsenic content in water in different locations is irregular. most of the locations contain higher level of as(iii) than as(v). the highest concentration of arsenic is found in anayetnagor (554.46 ± 0.07 g/l) of kalkini and raichar (562 ± 0.50 g/l) of hajigonj. however, the level of total arsenic and as(iii) of most of the villages of the study areas are more than the who guide line value (50g/l). therefore a proper monitoring process should be evolved along with the development of methods to keep the water free from arsenic. ------------------------------------------------------------------------------------------------------------------------------ introduction arsenic is ubiquitous in the environment, being naturally present in soil, air, water and food, and concentrations may be increased by anthropogenic contamination [1]. it is present in the environment in a number of different inorganic and organic chemical forms due to its participation in complex biological and chemical process. some of the most important arsenic species from a toxicological perspective include the two oxidation states as(iii), as(v), monomethylarsenic acid (mma), dimethylarsinic acid (dma), arsenobetaine and arsenocholine [2]. in recent years, the presence of high levels of arsenic in ground water, the main source of drinking water in many countries around the world, has drawn attention of the scientific communities. moreover, contamination of food chain by arsenic contaminated water is another threat [3]. numerous recent investigations have demonstrated that arsenic constitutes a serious health risk spot in bangladesh [2-6]. bangladesh, a country in south asia with a population of about 150 million, is one of a number of countries that has arsenic contamination in its groundwater, which results from the underlying mineralogy and geology of the area. arsenic contamination has been reported in groundwater in 41 out of 64 districts in bangladesh [3]. about 61% of the water analysed from tubewells has arsenic content above 0.01 mg/l [4]. the average concentration of arsenic in contaminated water is about 0.26 mg/l with a maximum level of 0.83 mg/l. this is significantly higher than the world health organization (who) maximum permissible limit in drinking water which is 0.05 mg/l [7]. moreover, arsenic shows toxicity and chemical property depending on its oxidation state and physicochemical forms. inorganic arsenic is known to be more toxic than organic ones, and as3+ is reported at least ten times more toxic than as5+ [8]. thus it is *corresponding author e-mail: amsalam2004yahoo.com pak. j. anal. environ. chem. vol. 8, no. 1 & 2, (2007) important to determine each of arsenic species rather than the total amount. rasul et al., and hussam et al., studied the as (iii) and as (v) with the aid of anodic stripping voltammetry (asv) and other metals with aasgf-z (zeeman effect-atomic absorption spectrometer with graphite furnace) from groundwater of kustia, bangladesh [9,10]. but there is no work has been reported with study of the as(iii), as(v) with square wave anodic stripping voltammetry (swasv), differential pulse anodic stripping voltammetry (dpasv) and normal pulse anodic stripping voltammetry (npasv) at hajigonj and kalkini of bangladesh which are most endemic areas. therefore, speciation of arsenic rather than quantification of total arsenic in drinking water present in groundwater are necessary. speciation of arsenic has been performed with different hyphenated techniques, often the coupling of ion chromatography or liquid-chromatography to detection system like: direct uv detection, direct coupling to atomic absorption spectrometry, aas, online hydride generation aas (hg-aas), icp atomic emission spectrometry (icp-aes), icp mass spectrometry [11-13]. these techniques are very expensive. several reports have appeared on electrochemical stripping procedures for the determinations of arsenic [9, 10, 14]. the instrumentation required is relatively simple and generally the cost is far less than that required for the other techniques. another advantage of electrochemical techniques is their ability to distinguish between the different oxidation states of arsenic. the anodic stripping voltammetry is very sensitive [14] and compared with expensive multielement analysis techniques like icp-ms, is an economical procedure for trace determination of arsenic down to the g/l level. in the present work, there are three different electronalytical methods such as square wave anodic stripping voltammetry (swasv), differential pulse anodic stripping voltammetry (dpasv) and normal pulse anodic stripping voltammetry (npasv), were used for the determination of arsenic with speciation to satisfy the lack of proper analytical techniques and implement such analytical methods that can provide accurate and interference free measurements of arsenic at the g/l levels of concentrations in water of hajigonj and kalkini of bangladesh. experimental the study area tubewell water was collected from nine villages of hajigonj and nine villages of kalkini. hajigonj thana at chandpur district is 53 kilometers away from comilla district and kalkini thana at madaripur district is 260 kilometer away from dhaka city. sampling tube well water was collected in polythene containers which were washed before collecting samples with 5% hno3, distilled de-ionized water and finally with the tube well water at the sampling sites for several times. the information about the depth of the tube well, year of installation and also red or green marked were collected from the local inhabitants by supplying the questions-answers sheets. sample preparation the samples collected from different points were filtered with a microfilter paper under vacuum and preserved in acid (1ml conc. hcl per 100ml of water sample). for the measurements of total arsenic, the samples were mixed with concentrated hcl and na2so3 (s) (10ml sample + 10ml conc. hcl + 30-40 mg na2so3(s)) in an acid clean pyrex glass electrolysis cell and heated at 70�80 0 c for 20-30 minutes without stirring, then 15 minutes with stirring and also 15 minutes with purging nitrogen with stirring until all so2 fumes cleared. the sample was then cooled to room temperature and was thus made ready for the measurements of the concentration of total arsenic [14]. preparation of stock standard solutions standard 1000 mg/l stock solutions of as3+ and as5+ were prepared by dissolving as2o3 (merck, germany, analytical grade) and na2haso4.7h2o (merck, germany, analytical grade) in deionized water respectively. these solutions were made acidic by the addition of 2-3 ml conc. hcl acid. fresh standard solutions of lower concentrations were made from the stock solution at the beginning of the everyday experiment. analytical procedure for quantification of as3+ and as5+ at first the gold electrode was made shiny yellow, almost scratch free surface by polishing with fine alumina powder (0.3µm) on wet polishing cloth. then the electrode was cleaned with deionised water and then 1m hcl and also stored in 6m hcl. for measurements of as3+, the stair bar was put into the cell and the cell was filled with 10 ml sample and 10 ml 6m hcl [9]. the solution was purged for 10 minutes with nitrogen. all the electrodes were inserted and tapped off to remove any bubbles from them. a computerized pak. j. anal. environ. chem. vol. 8, no. 1 & 2, (2007) e(mv) vs. ag/agcl (sat. kcl) -200 -100 0 100 200 300 400 500 600 c u rr e n t, i (µ a ) -5 0 5 10 15 20 25 npasv swasv dpasv background current electrochemical system, model hq-2040 by advanced analytics, usa was used for the analysis. the deposition potential (initial potential), accumulation or deposition time (initial delay), quit time delay is 150mv, 120sec. and 30 sec. respectively. the run replication (number of addition) is 3. in these analytical measurement procedures two standard additions were performed and corresponding currents were measured after subtraction of background current. the signal current was then plotted against concentration. the concentration of arsenic was calculated from the slop of the regression line drawn through the points using software sigma plot based on ms-excel. total arsenic was determined by same procedure after treatment of sample with nahso3 as described in sample preparation method. as5+ was evaluated with subtraction of as3+ from total arsenic [14]. results and discussion pulse voltammetric techniques, introduced by barker and jenkin [15], are aimed at lowering the detection limits of voltammetric measurements. by substantially increasing the ratio between the faradaic and nonfaradaic currents, such techniques permit convenient quantification down to the 10-8m concentration level. because of their greatly improved performance, modern pulse techniques have largely supplemented classical polarography in the analytical laboratory. among the voltammetric techniques normal pulse voltametry (npv), differential pulse anodic stripping voltammetry (dpasv), square wave voltametry (swv) was checked to evaluate the sensitivity for the trace arsenic analysis and speciation from groundwater. analytical comparison between swv, dpv and npv the swas, dpas and npas voltammograms of arsenic (as3+) for a 50 ppb standard solution at solid gold electrode (aubutton) are demonstrated in figure1. out of three asv techniques in use, npasv is considered to be least sensitive and selective method due to its highest signal to background ratio. in npv, the nonfaradaic current could not be avoided completely because the current is sampled once at the end of pulse amplitude. this charging current increases the signal current and for this reason npv shows high signal current with respect to swv and dpv where the current is sampled twice, just before the pulse application and late pulse life. although in npv, small amount of charging current is present it is also good sensitive method for as3+ analysis. out of these techniques used for arsenic analysis, the swasv technique is most sensitive and selective electroanalytical method because of its ability to enhance the analytical signal by removing non-faradaic current. so less time is required for arsenic analysis and ultra trace analysis can be easily performed with this method. however, among these methods dpasv shows moderate signal for arsenic but it is also a sensitive method for arsenic analysis. figure 1: comparison between three different electroanalytical methods for as3+(same concentration) at solid gold electrode. speciation of arsenic in ground water it is reported that shallow aquifer layer is contaminated with arsenic in almost all of the districts of bangladesh (dphe-bgs, 2000). in this study, arsenic level is determined in hajigonj (chandpur) and kalkini (madaripur) two contaminated areas of bangladesh. the different species of arsenic (as3+and as5+) content of ground water samples from different locations of the study area was measured with three different electroanlytical methods (swasv, dpasv and npasv) and the data thus obtained are represented in the table 2 and table 3. from the results, it is clear that the distribution of arsenic is not regular rather scattered due to the spatial variation. this is because of the variation in the depth of tube wells, amount of water withdrawn and also the geological phenomenon. in hajigonj, the level of as3+ ranges from 48.19 to 285.28 g/l. among all the water samples collected from the nine different villages of hajigonj, the highest arsenic (iii) concentration was found in raichar (285.28 g/l) and the lowest was in toraghor (48.19/l). the value of as5+ is also the lowest in satbaria (40.05g/l) and the highest in raichar (276.95g/l). however, all of the pak. j. anal. environ. chem. vol. 8, no. 1 & 2, (2007) water samples contain total arsenic above the who guide line value (50 g/l). the level of as3+ ranges from 26.91 to 286.0 g/l in kalkini. among the nine water samples collected from the nine different villages of kalkini, the highest arsenic(iii) concentration was found in anayetnagor (286.0 g/l) and the lowest was in charluxmi (26.91/l). the lowest value of as5+ is in shadipur (26.10g/l) and the highest in anayetnagor (268.46g/l). most of the water samples contain total arsenic above the who guide line value (50 g/l). table 1. concentration of as3+and as5+ in ground water of the study area, hajigonj (chandpur) sample no. sampling location depth of tube wells methods conc. of as3+ conc. of total as (as3++ as5+) conc.of as5+ (feet) (g/lsd) (g/lsd) (g/l) swasv 80.73.0.05 120.78.0.09 40.50 dpasv 72.530.12 119.900.16 47.37 1 satbaria 90 npasv 74.580.70 116.540.30 41.96 swasv 72.710.07 118.670.70 45.96 dpasv 76.560.09 123.090.17 46.53 2 khalpara 100 npasv 78.750.06 115.430.18 36.68 swasv 197.090.21 320.870.01 123.78 dpasv 198.94.0.08 307.270.06 108.33 3 bolakhal 140 npasv 190.060.09 312.120.13 122.06 swasv 80.240.30 129.450.07 49.21 dpasv 70.800.70 115.560.06 44.76 4 bakila 100 npasv 72.760.16 121.490.18 48.73 swasv 285.280.90 562.230.50 276.95 dpasv 288.330.60 594.530.13 306.20 5 raichar 120 npasv 203.430.23 437.600.23 243.17 swasv 276.570.04 380.890.60 104.32 dpasv 296.340.30 389.600.30 93.26 6 uchaa gram 140 npasv 280.980.50 381.260.04 100.28 swasv 83.560.40 140.430.40 56.87 dpasv 85.900.30 145.870.30 59.97 7 dherra 100 npasv 84.370.13 143.590.60 59.22 swasv 48.190.70 94.540.30 46.35 dpasv 46.470.15 91.010.19 44.54 8 toraghor 70 npasv 41.580.50 82.920.50 41.34 swasv 120.270.20 209.760.50 89.49 dpasv 121.680.40 209.00.40 87.32 9 shendra 110 npasv 110.100.14 187.230.16 77.13 no. of analysis (n=3) pak. j. anal. environ. chem. vol. 8, no. 1 & 2, (2007) table 2. concentration of as3+and as5+ in ground water of the study area, kalkini (madaripur) no. of analysis (n=3) the variation of arsenic concentration with depth of the different tube wells is presented in table 1 and table 2. it was found that the concentration of total arsenic was increased with the increase of depth within this range of 70 to 160 ft. this indicates high percentage of water was withdrawn from these aquifers. moreover, it is interesting that the concentration of as (iii) and as (v) varied with the depth of the tube well. this may be due to the change of geology with the variation of depth of soil. hussam et al., 2003 found the concentration of as (iii) and as (v) was present as aso3 3(0.712 mg/l) and aso4 3(0.973 mg/l) in groundwater at kustia respectively [10]. from the table 1 and table 2, it is noticed that most of the locations of kalkini and hajigonj contain higher level of as(iii) than as(v). this observation implies that more inorganic arsenic in ground water present in reduced form. sample no. sampling location depth of tube wells methods concentration of as3+ concentration of total as (as3++ as5+) concentration of as5+ (feet) (g/lsd) (g/lsd) (g/l) swasv 166.290.21 306.510.05 140.22 dpasv 161.170.03 299.590.09 138.42 1 charbivagdi 120 npasv 166.640.08 287.260.13 120.62 swasv 51.970.12 98.200.12 46.23 dpasv 41.550.30 89.670.50 48.12 2 dharichar 90 npasv 42.750.40 72.810.80 30.06 swasv 32.530.08 47.590.07 15.06 dpasv 32.400.10 51.840.14 19.44 3 charluxmi 70 npasv 26.910.14 42.420.30 15.51 swasv 75.960.16 102.060.80 26.10 dpasv 95.160.06 126.770.17 31.61 4 shadipur 80 npasv 82.160.09 102.520.07 20.36 swasv 46.300.70 76.270.15 29.97 dpasv 35.520.12 69.560.19 34.04 5 kashimpur 70 npasv 30.600.15 64.020.10 33.42 swasv 110.710.05 197.310.07 86.60 dpasv 110.110.07 190.610.40 80.50 6 shikar mangol 120 npasv 89.960.40 156.220.08 89.96 swasv 53.800.50 76.240.09 22.44 dpasv 47.520.09 67.760.40 20.24 7 khishnanagor 70 npasv 39.680.08 51.720.05 12.04 swasv 126.180.4 186.200.20 60.02 dpasv 97.440.07 160.510.70 63.07 8 alipur 120 npasv 86.460.23 138.900.04 52.44 9 anayet nagor 160 swasv 286.00.14 554.460.07 268.46 dpasv 275.450.16 539.400.20 263.95 npasv 268.210.02 542.400.09 274.19 pak. j. anal. environ. chem. vol. 8, no. 1 & 2, (2007) table 3. concentration of arsenic in water sample of manikgonj considered as controlled area (swasv method). sample no. locations concentration of as3+ concentration of total as (as3++ as5+) concentration of as5+ (g/lsd) (g/lsd) (g/l) 1 nabagram 14.430.05 24.120.02 9.69 2 khilinda 9.020.6 16.450.13 7.43 3 jagir 10.980.82 19.760.09 8.78 4 macshimul 8.120.7 19.670.41 11.55 no. of analysis (n=3) table 4. validation of results with hg-aas with developed anodic stripping voltammetry methods variation with hg-aas hg-aas swasv dpas npasv swasv dpasv npasv controlled lab. standard (g/l) (%) 125.01.20 119.900.16 116.540.30 120.78.0.09 4.08 6.78 4.22 8.25 95.41.50 98.200.12 89.670.50 72.810.80 2.93 6.02 23.68 9.50 120.52.20 118.670.70 123.090.17 115.430.18 1.51 2.14 4.20 8.35 no. of analysis (n=3) comparison of the endemic areas with the controlled area the water samples of manikgonj which was considered as a controlled area (free from arsenic contamination) were also analysed with swasv method as presented in table 3. the average value of as content in this area was found 20 g/l. on the other hand, the average as contents in different locations of the study areas are about 10 times higher than the average content of the controlled area. validation of the results by hg-aas to validate the results obtained from the electroanalytical method, few water samples were analysed by hg-aas. it was observed that the results obtained by the electroanalytical methods were closed to results obtained by the hg-aas method. this indicates that the results obtained by the developed electroanalytical methods were effective to analyse arsenic from the groundwater where the variation of results between hg-aas and electroanalytical method was about 1.5-9.5%. conclusion from the study of the three electroanlytical methods (swasv, dpasv and npasv), the swasv technique is most selective and suitable electroanalytical method. this method could be a better analytical technique for arsenic speciation from water. moreover this technique is comparatively cheaper than other available methods. the results obtained in this study have been compared with the value of unaffected area (manikgonj).the observations show that arsenic content of the study area in ground water about 10 times higher than the average content of the controlled area. the total arsenic content and arsenic (iii) in water is higher than the who guide line value (50g/l). so, arsenic contamination in ground water of the study areas is in alarming proportions. therefore, a proper monitoring process should be evolved along with development of methods to keep the water free from arsenic. references 1. m. c. villa-logo, e. rodriguez, p. l. mahia, m. s. mnuiategui, d. p. rodringuez, talanta, 57 (2002) 741. 2. s. w. al rmalli, p. i. haris, c. f. harrington, m. ayub, sci. total environ., 337 (2005) 23. pak. j. anal. environ. chem. vol. 8, no. 1 & 2, (2007) 3. m. a. hasan, k. m. ahmed, o. sracek, p. bhattacharya, m. v. bromssen, s. broms, j. fogelstrom, m. l. mazumder, g. jacks, hydrology j. (2007), doi:10 .1007/s10040-0070203-z . 4. m. ali, s. a. tarafdar, j. radional nucl chem. 256(2) (2003) 297. 5. m. g. m. alam, e. t. snow, a. tanaka, total environ., 308 (2003) 83. 6. h. k. das, a. k. mitra, p. k. sengupta, a. hossain, f. islam, g. h. rabbani, enviro int., 30(3) (2004) 383. 7. who. vol. 224, geneva: world health organization; (2001). 8. dcht, arsenicosis in bangladesh, dhaka community hospital trust, february. dhaka, bangladesh, (1998). 9. s. b. rasul, a. k. m. munir, z. a. hossain, a. h. khan, m. alauddin and a. hussam, talanta, 58(1) (2002) 33. 10. a. hussam, m. habibuddowla, m. alauddin, z. a. hossain, a. k. m. munir, a. h. khan, j environ sci health, 38 (2003) 71. 11. m. van holderbeke, y. n. zhao, f. vanhaeck, l. moens l, r. darns, p. sandra, j. anal at spectrom, 14 (1999) 229. 12. j. l. gomez-ariza, d. sanchez-rodas, r. beltran, w. corns, p. stockwell, appl organonet chem, 12 (1988) 439. 13. c. soros, e. t. bodo, p. fodor, r. morabito, anal bioanal chem, 377 (2003) 25. 14. f. t. henry, t. o. kirch, t. m. thorpe, anal. chem, 51(2) (1979) 215. 15. g. c. barker, i. l. jenkin, analyst, 77 (1952) 685. 16. w. h. horwitz, official methods of analysis of aoac international, vol. 1 (2000). microsoft word 4-39-47-pjaec-15092022-454-c-revised galley proof-26-05-2023-4.docx cross mark issn-1996-918x pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 39 – 47 http://doi.org/10.21743/pjaec/2023.06.04 monitoring of tetracycline group antibiotic residues in various food products of animal origin in the turkish market senem şanlı1 , seyfi sardoğan2 and nurullah şanlı3* 1department of chemistry, faculty of science and arts, uşak university, uşak, turkey. 2department of food engineering, faculty of engineering, uşak university, uşak, turkey. 3department of chemistry and physics, roger williams university, one old ferry road, bristol, ri 02809, usa. *corresponding author email: nsanli@rwu.edu received 15 september 2022, revised 15 may 2023, accepted 20 may 2023 ------------------------------------------------------------------------------------------------------------------------------------------- abstract the aim of this study was to detect the presence of antibiotic residues in foods of animal origin, including 42 pieces of chicken gizzard and 46 pieces of bovine kidney and 102 chicken eggs belonging to various brands. these samples were gathered from december 2020 to april 2021 in the aegean province of turkey. a sensitive, simple, rapid, experimentally convenient and cost effective rp-lc method with high recovery output was developed. the method was thoroughly validated for the optimized parameters and produced satisfactory results. the analysis of bovine offal by the developed rp-lc method showed the presence of oxytetracycline, tetracycline, and chlortetracycline residues in 14 (30.43%) kidney samples. chlortetracycline was detected in 7 (16.67%) chicken gizzard samples. in addition, the analysis of chicken eggs revealed the presence of oxytetracycline and tetracycline residues in nine egg samples (8.82%). since, the amount of antibiotic residues in these samples was below the detection limit, quantification could not be carried out. only one (0.98%) of the 102 egg samples exceeded the mrl (267.1 mg/kg) for oxytetracycline concentration. according to the study's overall findings, it is recommended that tetracycline antibiotics should be regularly checked in a variety of foods made from animals because they were found in 32 out of 190 analysed samples. tetracycline residues may pose dangers to human health, so it's important to conduct further research and more information should be given for both producers and consumers. keywords: antibiotics, veterinary drug residues, food safety, tetracyclines, validation, hplc. ------------------------------------------------------------------------------------------------------------------------------------------- introduction antibiotics are actively injected into animal bodies to support their protective systems during the treatment period. antibiotics such as penicillin, gentamicin, tetracycline, danoflaxacin, neomycin, etc. are widely used to prevent and treat diseases, especially mastitis and respiratory system diseases [1]. antibiotic residues in animal foods have become a significant threat to public health and food safety. for this reason, it has become necessary to concentrate on the amount of residue in foods [2]. tetracyclines (tcs) are a broadspectrum cluster of antibiotics, and they've been employed in the treatment of microorganism infections in animals for over fifty years. however, the employment of those medicines has become a significant drawback as a result of antibiotic residues in animal pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 40 food. the adverse effects of this class of antibiotics include organ injury, allergic reactions, gastrointestinal distress, and tooth discoloration. residues of antibiotics in animal feed are not recommended in veterinary applications. the maximum residue levels (mrls) for tcs in several foods have been set by european regulation 2377/90 and its subsequent amendments [3]. despite the occurrence of resistance, tetracyclines are still widely in use in animals owing to their low prices. egg mrls are 200 ng/g for oxytetracycline (otc), tetracycline (tc) and chlortetracycline (ctc), chicken mrls are 100 ng/g for otc, tc, ctc, and doxycycline (dc), but the latter value is temporary and still being investigated. a certain and reliable methodology for detecting tetracycline residues in animal foods is incredibly important. for this purpose, chromatographic techniques, such as high performance liquid chromatography (hplc) with different detection modes like spectrophotometry, fluorescence and mass spectrometry [4-10], and capillary electrophoresis [11-13] have been used for the analysis of tcs. in addition, general descriptions of immunoassay and microbiological test procedures for tc screening in food products have been provided [14–15]. the lack of specificity and the lengthy incubation period necessary for microbiological testing are their biggest drawbacks [16]. due to the very comparable structural similarity of tcs immunoassays, a misleading detection may also occur [17]. as a consequence of this, confirmatory research is necessary in order to quantify the results of screening tests performed on food products. there is little information about the presence of tcs residues, especially in milk samples in turkey [18-20]. however, there is a lack of information about tcs residues in chicken, bovine offal and eggs in turkey. it appears that a useful antimicrobial residue monitoring system should be brought in place. a fast, sensitive, and economical reversed phase liquid chromatography (rp-lc) technique was built up for the analysis of otc, tc, ctc, and dc residues. consequently, the purpose of this study was to detect the presence of antibiotic residues in foods of animal origin, including 42 pieces of chicken gizzard, 46 pieces of bovine kidney, and 102 chicken eggs of various brands, collected in the aegean province of turkey between december 2020 and april 2021. the chemical structures and pka values of studied tcs are listed in table 1 [21]. the developed method has been validated according to ich rules, and recovery values were also calculated. table 1. chemical structures of the tetracycline antibiotics studied. compounds chemical structure oxytetracycline (otc) pka1 = 3.53 [21] pka2 = 7.25 pka3 = 9.58 c22h24n2o9 mw: 460,434 g/mol cas no: 79-57-2 tetracycline (tc) pka1 = 3.35 [21] pka2 = 7.29 pka3 = 9.88 c22h24n2o8 mw: 444,435 g/mol cas no: 60-54-8 chlortetracycline (ctc) pka1 = 3.25 [21] pka2 = 6.72 pka3 = 8.84 c22h23cln2o8 mw: 478,88 g/mol cas no: 57-62-5 doxycycline (otc) pka1 = 3.02 [21] pka2 = 7.97 pka3 = 9.15 c22h24n2o8 mw: 444.44 g/mol cas no:564-25-0 pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 41 materials and methods reagents and chemicals tetracycline, oxytetracyline, chlortetracycline, and doxycycline were bought from sigma (sigma–aldrich, st. louis, mo, usa). methanol (meoh) and acetonitrile (acn) of hplc grade were obtained from fisher scientific (pittsburgh, pa, usa). phosphoric acid (h3po4), hydrochloric acid (hcl), formic acid (hcooh), trichloroacetic acid (cl3ccooh, tca), sodium hydroxide (naoh), and edta were procured from fisher chemical (fairlawn, nj, usa). preparation of solutions in meoh, 100 µg ml-1 stock standard solutions of tc, otc, ctc, and dc were prepared. the mobile phase was used to dilute the working solutions to 10 µg ml-1. after being diluted, these solutions were used to make a series of working standard solutions, which were then used for the daily generation of calibration curves and standard addition spikes. by injecting a solution of uracil [0.01% (v/w), in water], which was found for each combination of mobile phase and ph level, the dead time (to) was measured. instrument description the study was performed using an agilent 1260 series hplc system that includes a ternary solvent pump, an automated injection system, an in-line degasser, column heater, and a multi-wavelength detector. uv identification for the analyzed substances was done at 271 nm. the analysis was performed at a 1.2 ml min-1 flow rate. as the stationary phase, a synergi 4 hydro-rp 80a column (250 x 4.60 mm i.d. 4 µm) was employed at 25 °c. for measuring ph, a mettler toledo, hanna hi 1332 ag/agcl combined ph electrode was used (hanna inst.). the water used was double-distilled and deionized through a millipore direct-q 3uv; 0.22 µm water purification unit. a mixer with 100-300 ml flasks and a vortex (ika ms 3 basic vortex) were used depending on the sample. additional glassware and required equipment include 50 ml conical falcon tubes and graduated cylinders. the used glassware was detergent cleaned and rinsed with 0.01 m hcl and pure water. sample collection from december 2020 to april 2021, a total of 88 offal samples and 102 chicken eggs belonging to various brands were collected in aegean province, turkey. the samples were bought from major supermarkets and local grocery stores and meat galleries, including butchers and chicken stores, and carried to the laboratory after sampling. sample extraction and clean-up meat and chicken tissue samples for meat and chicken tissue samples, 42 pieces of chicken offal (gizzard) and 46 pieces of bovine offal (kidney) were purchased and stored in plastic containers at 4 oc in the dark until used within four days in this study. tissue was cut into small pieces with a side of 1 cm or less. approximately 8 g of offal sample (or larger or smaller as desired) was weighed exactly into a 300 ml mixing glass. the mixture was stired for two minutes or until no visible pieces of tissue remained. after that, it was placed in a falcon tube and 15 ml of methanol was added, and vortex treatment was applied for 5 minutes. thus, protein precipitation was achieved. then, 4 ml of 1% formic acid was added and vortex treatment was applied for 3 minutes. finally, 400 µl of pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 42 0.01 m edta was added and vortex treatment was applied for 2 minutes. the resulting mixture was centrifuged at 2000 rpm for 10 minutes. after the centrifugation process, the supernatant part was removed with the help of an injector and injected into the hplc device by passing it through a 0.22 µm filter. eggs a batch of 6 of the eggs belonging to each brand was taken, blended, and homogenized. 4 grams of the resulting homogeneous mixture was taken and placed in a falcon tube and left in a dark place for 15 minutes. vortex treatment was applied for 10 minutes by adding 8 ml of 5% tca to it. after that, 15 minutes were spent for centrifuging the mixture at 4000 rpm. after centrifugation, a 0.22 µm filter was used to separate the supernatant fraction, which was then injected into the device. results.and discussion. method. development.and validation. the presented hplc technique offers a facile method for the simultaneous determination of tc, otc, ctc, and dc in chicken and beef tissues, as well as in egg samples by diode array detection. the investigated tetracycline compounds were effectively determined simultaneously by using the chosen column as the stationary phase. it is a c-18 bonded phase and endcapped column with non-polar groups to supply extreme retention of polar and hydrophobic compounds in high aqueous mobile phases with 19% carbon loading. the high silica area (475 m2g-1) of the 4 µm surface combined with the coverage of the dense bonded phase permits the high interaction between the analyte and the bonded phases. the obtained results are so persistent in the c-18 phase, they are well suited for separating the compounds studied. to specify the best conditions for the chromatographic separation of tcs, the flow rate, ph of the buffer, temperature, and buffer concentration parameters were investigated. for separation, a 15–30 mm concentration was studied. a 25 mm phosphate buffer concentration was selected because of the best separation of the studied tcs. in terms of the mobile phase ph value, values in the range of ph 2.5–3.0 were tested. the optimum peak resolution was seen at ph 2.8. the applied flow rate was examined in the range of 0.8, 1.0, 1.2, 1.5, and 1.7 ml/min. sharper peaks and shorter retention times were observed with increasing flow rates. since the minimum retention time is necessary for lc, 1.2 ml/min was selected for the optimum results due to the back pressure. the temperature was tested in the range of 25 to 35 oc as the temperature was increased by 10 oc and worked at 35 oc. here, with the increase in temperature, distortions in peak shapes and an in crease in retention time were observed. the column temperature was fixed to 25 oc. the acn concentration in the mobile phase was changed from 30 to 20% (v/v), and 20% (v/v) acn was chosen as the optimum condition due to the peak shape and analysis time. hplc separation was obtained using synergy 4µ hydro-rp 80a (250 × 4.60 mm id × 4 µm) column at 25℃, with a mobile phase acetonitrile-water (20:80, v/v), ph 2.8 (phosphate buffer) and flow rate of 1.2 ml/min. under these circumstances, the analysis time increased to almost nine minutes with symmetrical peaks, and the retention times were 3.16 ± 0.05, 3.92 ± 0.08, 6.44 ± 0.06, and 7.64 ± 0.09 min for otc, tc, ctc, and dc, respectively. a representative chromatogram for the examination of tcs standards is shown in fig. 1 at a wavelength of 271 nm. pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 43 figure 1. the chromatogram of standard mixture of studied compounds 1) otc 5 µg ml−1, 2) tc 5 µg ml−1, 3) ctc 50 µg ml−1, 4) dc 20 µg ml−1 monitored at 271 nm absorbance under optimum conditions. the detector response linearity was tested using otc and tc standard solutions ranging from 0.1 to 20 µg ml-1, chlortetracycline standard solutions ranging from 1 to 80 µg ml-1, and doxycycline standard solutions ranging from 0.5 to 20 µg ml-1, respectively (table 2). plotting concentration versus peak area from the chromatograms of the standard samples allowed for the construction of calibration curves. table 2. statistical evaluation of the calibration data of otc, tc, ctc and dc by rp-lc. compounds otc tc ctc dc linearity range/ µg ml−1 (n = 7) 0.10 – 20.0 0.10 – 20.0 1.0 – 80.0 0.50 – 20.0 slope 238.516 374.857 17.785 215.874 intercept -48.171 -94.127 -27.997 -91.531 correlation coefficient (r2) 0.9994 0.9991 0.9991 0.9994 se of slope 2.580 5.311 0.284 2.713 se of intercept 22.452 46.225 10.688 25.508 limit of detection (lod) / µg/ml 0.0050 0.0041 0.1163 0.0076 limit of quantification (loq) / µg/ml 0.0151 0.0125 0.3525 0.0229 retention time (min) (n = 16) 3.16 3.92 6.44 7.64 rsd% of retention time 1.58 2.04 0.932 1.18 tetracycline standards were prepared every day, and estimations of the concentrations of the analytes in the samples were extrapolated from the graphs. the precision of the approach is demonstrated by the low slope and intercept standard error (se) values. the standard deviation of the response (s) and the slope (m) of the related calibration curve were utilized in the following formulae to calculate the lod and loq values [22, 23]. m s loq m s lod 10 ; 3 .3  (1) recovery studies were determined from egg, chicken, and beef offal samples for the accuracy and precision of this method. the recovery values were determined by spiking the previously studied samples with the appropriate amounts of otc, tc, ctc, and dc at the time of homogenization. the results of the recovery analysis are given in table 3. table 3 suggests that the recovery calculated from these antibiotics ranges from 82.31% to 96.22%. table 3. recovery of studied compounds from egg, chicken and beef offal samples. percent recovered co mp oun ds egg rsd % chicken gizzard rsd % bovine kidney rsd % otc 86.96 2.16 89.06 4.16 92.99 4.25 tc 92.34 1.64 96.22 5.21 89.63 6.55 ctc 90.88 1.97 94.82 2.38 90.90 3.73 dc 82.31 1.05 95.74 2.43 93.74 7.90 as an example, chromatograms of bovine kidney, chicken gizzard, and egg samples were given in fig. 2. pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 44 figure 2. chromatograms of the tc compounds in studied samples. optimum conditions and number of studied compounds are the same as fig. 1. (a) bovine kidney, (b) chicken gizzard, (c) egg (a) (b) (c) pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 45 the proposed method offers better recovery and greater sensitivity. it is clear from the calculated recovery data of all tetracyclines in various samples, which are within the aoac acceptable range for trace analysis; 60-115% [24], and the values of otc, tc, ctc, and dc of meat and egg samples, which were provided in accordance with the codex alimentarius commission and european union regulation 2002/657/ec. additionally, relative standard deviation (rsd) values calculated in this work were less than 10%, which complies with the codex alimentarius commission. according to a statement, if the rsd data are sufficient, the method may be regarded as verified. after rp-lc analysis (table 4), otc and tc were found in nine of the egg samples. according to turkish legislation on veterinary drug residues, oxytetracycline and tetracycline given for maximum residue in chicken eggs (mrl) of 200 µg/kg. a value above the mrl value (267 µg/kg oxytetracycline) was detected in only 1 (0.98%) sample [25]. the levels of tc (lod 0.004 µg/kg) ranged from 89.4 to 122.3 µg/kg was determined in two samples. table 4. levels of antibiotic residues detected by the rp-hplc technique. rp-hplc results number of samples analysed otc tc ctc dc 7d (< 0.004 -0.06; 8.82%) 102a 1 (139.0; 0.98%) 1 (267.1; 0.98%) 1 (122.3; 0.98%) 1 (89.4; 0.98%) 42b 7 (< 0.12; 16.67%) 46c 14 (< 0.004 – 0.12; 30.43%) notes: a number of egg samples collected from various brands. b number of chicken gizzard samples c number of bovine kidney samples d number of positive samples (antibiotic levels, µg kg-1; percentage). other samples had residual levels that were lower than the global standards established by the european union and the turkey-allowed limits. 32 bovine kidney samples, 35 gizzard samples, and the remaining 93 egg samples tested negative for rp-lc detection. in the production of eggs and meat, the massive and incorrect application of tcs and the misguided following of withdrawal periods may result in the presence of their residues in food products. the risks to human health from the maximum residual tc ranges remain even though they were below the limit. conclusion egg, chicken gizzard, and bovine kidney samples collected from supermarkets, local grocery stores, and meat galleries, including butcher shops and poultry shops, in the aegean province contain traces of tc. although the majority of these levels fall below the thresholds established by the european union and turkish law, their presence can still be viewed as posing a threat to consumer health. this is due to their potential to trigger allergic reactions or help to breed bacteria that are resistant to antibiotics, both of which have emerged as major issues in the treatment of infectious diseases that affect humans. therefore, comprehensive surveillance plans for the management of veterinary drug residues in animals and their products must be established by national authorities. in addition, the rp-lc method was used due to its accuracy, straightforward, quick, convenient, cost-effective, and high recovery throughput. the method was completely validated, with satisfactory results for each of the examined method validation parameters. the developed method promises to be applicable to the identification and analysis of frequently found tcs in other offal pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 46 samples such as spleen, liver, and chicken breast as well. acknowledgment the scientific and technological research council of turkey (tubitak) provided financial assistance for the current investigation, which is acknowledged by the authors. declaration of interest the authors declare no conflict of interest. references 1. b. shaikh and w. a. moats, j. chromatogr., 643 (1993) 369. https://doi.org/10.1016/00219673(93)80573-q. 2. i. philips, m. casewell, t. cox, b. de groot, c. friis, r. jones, c. nightingale, r. preston and j. waddell, j. antimicrob. chemother., 53 (2004) 28. https://doi.org/10.1093/jac/dkg483 3. commission regulation no. 2377/90, off. j. eur. commun., 18 august (1990), no. l224. https://eur-lex.europa.eu/legalcontent/en/txt/?uri=celex%3a3199 0r2377 4. b. arabsorkhi and h. sereshti, microchem. j., 140 (2018) 241. https://doi.org/10.1016/j.microc.2018.04. 030 5. j. k. parmar, k. k. chaubey, v. gupta and m. n. bharath, veterinary world, 14 (2021) 1650. www.doi.org/10.14202/vetworld.2021.1 650-1664 6. m. x. feng, g. n. wang, k. yang, h. z. liu and j. p. wang, food control, 69 (2016) 171. https://doi.org/10.1016/j.foodcont.2016. 04.050 7. k. saridal and h. i̇. ulusoy, microchem j., 150 (2019) 104170. https://doi.org/10.1016/j.microc.2019.10 4170 8. f. cetinkaya, a. yibar, g. e. soyutemiz, b. okutan, a. ozcan and m. y. karaca, food addit. contam., part b., 5 (2012) 45. https://doi.org/10.1080/19393210.2012.6 55782 9. c. pizan-aquino, a. wong, l. avilesfelix, s. khan, g. picasso and m. sotomayor, mat. chem. phys., 245 (2020) 122777. https://doi.org/10.1016/j.matchemphys.2 020.122777 10. m. a. gab-allah, y. g. lijalem, h. yu, d. k. lim, s. ahn, k. choi and b. kim, j. chromatogr. a, 1691 (2023) 463818 https://doi.org/10.1016/j.chroma.2023.46 3818 11. j. zhou, g. c. gerhardt and a. r. baranski cassidy, j. chromatogr. a, 839 (1999) 193. https://doi.org/10.1016/s00219673(99)00152-1 12. s. sardoğan, s. şanlı, b. sardoğan and b. atalay, j. pharm. res. int., 32 (2020) 1. https://doi.org/10.9734/jpri/2020/v32i15 30618 13. p. kowalski, j. pharm. biomed. anal., 47 (2008) 487. https://doi.org/10.1016/j.jpba.2008.01.03 6 14. k. bahmani, y. shahbazi and z. nikousefat, food sci. biotechnol., 29 (2020) 441. https://doi.org/10.1007/s10068-01900665-x 15. m. h. ngoc do, t. yamaguchi, m. okihashi, k. harada, y. konishi and k. uchida, food control, 69 (2016) 262. https://doi.org/10.1016/j.foodcont.2016. 05.004 pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 47 16. y. shahbazi, f. ahmadi and n. karami, food agric. immunol., 26 (2015) 821. https://doi.org/10.1080/09540105.2015.1 036357 17. a. barani and a. a. fallah, food agric. immunol., 26 (2015) 420. https://doi.org/10.1080/09540105.2014.9 50199 18. h. aksu, o. cetin, o. arun and o. ergun, medycyna weterynaryjna, 60 (2004) 1171. http://agro.icm.edu.pl/agro/element/bwm eta1.element.agro-article-db383374ce29-4164-95b9fd0ef77cf341?q=bwmeta1.element.agronumber-32d6e6b6-e781-49d2-95cf4416e8da5546;9&qt=childrenstateless 19. n. unusan, int. j. food sci. nutr, 60 (2009) 359. https://doi.org/10.1080/09637480701664 555 20. y. k. das, o. yavuz, e. atmaca and a. aksoy, fresenius environ. bull., 28 (2019) 5982. https://www.researchgate.net/publication /334151750_tetracycline_antib iotics_in_raw_cow%27s_milk_ produced_in_samsun_provin ce_turkey 21. s. şanli, n. şanli and g. alsancak, j. braz. chem. soc., 20 (2009) 939. http://static.sites.sbq.org.br/jbcs.sbq.org. br/pdf/v20n5a20.pdf 22. c. m. riley and t. w. rosanske, development and validation of analytical methods (elsevier, new york) (1996). https://shop.elsevier.com/books/develop ment-and-validation-of-analyticalmethods/riley/978-0-08-042792-8 23. m. e. swartz and i. s. krull, analytical method development and validation (marcel dekker inc, new york) (1997). https://www.tandfonline.com/doi/abs/ 10.1080/10826079808000502 24. u. koesukwiwat, s. jayanta and n. leepipatpiboon, j. chromatogr. a, 1149 (2007) 102. https://doi.org/10.1016/j.chroma.2007.02 .075 25. turkish food codex. march 2017. turkish food codex regulation on the classification and maximum residue limits of pharmacologically active substances that can be found in animal foods. official gazette no: 30000. https://www.resmigazete.gov.tr/eskile r/2017/03/20170307-3.htm issn-1996-918x pak. j. anal. environ. chem. vol. 14, no. 1 (2013) 68 – 74 production of indigenous and enriched khyber pakhtunkhwa coal briquettes: combustion and disintegration strength analysis muddasar habib1, amad ullah khan 1, abdul rehman memon*2 and unsia habib1 1department of chemical engineering, university of engineering and technology, peshawar, pakistan 2department of chemical engineering, mehran university of engineering and technology, jamshoro, pakistan received 15 april 2013, revised 25 june 2013, accepted 30 june 2013 ------------------------------------------------------------------------------------------------------------------------------------------- abstract khyber pakhtun khwa province of pakistan has considerable amounts of low ranked coal. however, due to the absence of any centrally administered power generation system there is a need to explore indigenous methods for effectively using this valuable energy resource. in the present study an indigenous coal briquetting technology has been developed and evaluated in terms of combustion characteristics such as moisture content, volatile matter, ash, fixed carbon and calorific value of the resulting coal briquette and disintegration strength using polyvinyl acetate (pva) in combination with calcium carbonate (sample no 3 with highest disintegration strength value of 2059n). comparison of test samples with the commercially available coal briquettes revealed improved combustion characteristics for the pva bonded (sample no 1 and 5) coal briquettes having higher fixed carbon content and calorific value, lower ash contents as well as lower initial ignition time. keywords: coal briquettes; disintegration strength analysis; volatile matter; ash content; fixed carbon; calorific value. ------------------------------------------------------------------------------------------------------------------------------------------- introduction the developing countries including pakistan are currently facing huge energy crisis due to everincreasing cost of imported fuels. one option to contain the present energy crisis is to harness the power of indigenous coal reserves, which are poorly developed but abundantly available in pakistan. the past two decades have seen the importance of coal being recognised as an alternative source of energy in comparison to the petroleum products. this is mainly triggered by the diminishing oil reserves as well as international and maritime security issues. the use of indigenous coal offers an inexpensive and locally available alternative. an estimated 2.87 million tonnes of coal reserves (equivalent to 700 million tonnes of crude oil) are currently available in the world [1]. however, the handling and burning of raw coal for domestic, semi-commercial and commercial applications poses a challenging problem mainly due to its high ignition temperature, varying coal quality, higher ash, micron-size particle emission and smoke generation. in addition to the above factors and with existing coal profile, coal is prone to disintegration during the course of its mining, transportation, handling and on exposure to weather. the amount of disintegrated coal and/or flakes form a considerable percentage of the mined coal in comparison to the lumped coal and are sold at considerably low rates. the conversion of flaked coal into briquettes upgrades the value and is often sold at higher prices than that for the lumped coal. *corresponding author email: enxarm@gmail.com pak. j. anal. environ. chem. vol. 14, no. 1 (2013) 69 in the absence of any centrally administered large scale power generation system for coal reserves in pakistan, especially in khyber pakhtun khwa (kpk) province, there is a need to develop methods that can effectively utilise the indigenous coal reserves to uplift the local domestic and small scale industrial economy. many solutions exist in this regards such as coal gasification, liquefaction and briquet0ting, which all have their own advantages and disadvantages. due to the lack of any local, technical and/or practical knowhow about coal gasification and liquefaction, the proposed method of coal briquetting technology is aimed at effectively utilizing the indigenous coal reserves and to develop and implement it for the local market. this is in line with the current trend where the use of coal briquettes and improved performance stoves are encouraged in the developing world especially in countries where coal is found in abundance in comparison to other fuel sources [2]. the improved briquetting technology is mainly focused on offsetting the pollution and health impacts that result due to the inefficient burning of low quality raw coal. firewood and charcoal use in pakistan has led to extensive deforestation. the country has rich resources of low ranked indigenous coal which can be utilized in the form of coal briquettes to heat the residential, commercial, and industrial processes [3]. glen (2003) also highlighted the social acceptance of coal briquettes in the pakistani domestic market and suggested that coal briquettes can easily replace the traditional wood burning in the urban areas of punjab and kpk provinces [3]. however, the acceptance of coal briquetting technology in the industrial sector was welcomed with hesitation indicating its slow adoption in these areas. burring characteristics of coal briquettes such as ignition temperature, steady state burning, smoke and ash content along with non-burning characteristics such as mechanical strength, moisture content and moisture resistance will all have an effect in the adoption of this technology by the domestic and industrial market. to address some of these issues altun et al., (2004) has looked into the burning characteristics of coal briquettes from a combustion kinetics point of view and suggested that the coal briquettes ignition and the efficiency and effectiveness of combustion reaction was considerably dependent on the binder type, amount of binder agent and water addition [4]. mechanical disintegration strength of the prepared coal briquettes affects its storage and transportation to the intended markets and hence blesa et al., (2003) looked into the mechanical characteristics of the heat cured coal briquettes made from molasses and suggested that curing brings a uniform morphology in the resulting structures and hence give good disintegration strength for normal use [5]. ellison and stanmore (1981) have referred to the production of high mechanical strength coal briquettes by mechanical compression without the addition of any binder [6]. yildirim and ozbayogh (1997) and rubio et al., (1999) have looked into using different binders for coal briquettes production [7, 8]. coal briquettes can be made in a variety of different ways by adjusting the shape, filler, binder and coal composition. a variation in all these factors affect the calorific value, initial ignition temperature, ash content, moisture, storage and transportation of the resulting coal briquettes [9-10]. the problem of briquetting is not always about how to make the best possible briquettes in a certain locality but the quality of coal flakes and the binder prices influences the production of satisfactory grade for the intended use [11]. no such study has been carried out to look into these factors for the indigenous kpk coal reserves and hence it was the principal purpose of this research to investigate the effects of different binders on the strength, dryness and burning characteristics of the resulting coal briquettes. materials and methods coal briquettes used in the present study were locally prepared in the clean energy engineering laboratory of the department of chemical engineering at the university of engineering and technology peshawar, pakistan. the major experimental tasks involved in this research are given in (fig. 1). pak. j. anal. environ. chem. vol. 14, no. 1 (2013) 70 figure 1. overview of the experimental tasks. in the initial trial experiments different coals were analysed for their moisture, volatile matter, ash, calorific value and fixed carbon contents. although several coal deposits exist in the kpk province but based on the initial trial experiments only three were selected for the coal briquetting experiments reported here. the preliminary selection was based on the amount of fixed carbon content, ash, moisture, volatile matter and calorific value as well as their proximity to the project site. coal sourced from dera adam khel was labelled as coal-1, from doaba as coal-2 and that from hangu was labelled as coal-3. the particle size of coal in this study was limited to anything passing through a 600 micron sieve. the initial tests performed were to find out the moisture level, volatile matter, ash content, fixed carbon and calorific value of the three different types of coal as given in (table 1). table 1. measured moisture content, volatile matter, ash content, fixed carbon, and calorific values of raw coal samples coal sample moisture level / % volatile matter / % ash content / % fixed carbon content / % calorific value / joules dera adam khel, coal-1 1.10 10.66 19.2 69.04 29840 doaba, coal-2 1.28 15.16 20.4 63.16 22468 hangu, coal-3 1.40 12.20 21.3 65.10 25984 a = coal-1 with 1000 ml pva; b = coal-1 with 1000 ml pva and 250gm caco3; c = coal-1 with 1000 ml pva and 500gm caco3; d = coal-2 with 1000 ml pva and 250gm caco3; e = coal-3 with 1000 ml pva; f = available coal briquettes in market (ptc); g = available coal briquettes in market (phoenix) after selecting the coal types, next step involved the mechanical construction of a compression test rig to make the different composition coal briquettes for onward combustion analysis to optimise the optimum composition. a cylindrical shaped pattern was selected based on the ease of briquettes production, storage, use and mechanical strength to make the coal briquettes as shown in (fig. 2). the dimensions of the coal briquette pressing cylinder are shown in (fig. 3). figure 2. cylindrical coal briquettes designed for this study. figure 3. mechanical layout of the coal briquetting cylinder. the actual design of the coal briquetting press was simple and consisted of an iron metal frame upon which a 4 inch diameter and 6 inch trial experiments, construction and commissioning of rig sample preparation for testing fuel combustion property evalualtion by moisture, volatile, fixed carbon and calorific value tests pak. j. anal. environ. chem. vol. 14, no. 1 (2013) 71 length cylinder was welded. the cylinder had removable top and bottom circular plates with 14, 0.25 inch diameter holes that were provided to linearly move on the circular bars inside the cylinder during coal briquettes compression and removal cycles as is shown in (fig. 4 & fig. 5). figure 4. coal briquetting press machine showing the compression and jack removal mechanisms. figure 5. top view of the compression cylinder with top circular steel plate with holes to slide inside the cylinder under the influence of sufficient load. the presence of holes in the resulting briquettes also aided in the ease of combustion and drying due to the circulation of air in the holed structure. to make a briquette, the bottom plate was in-placed and coal, filler and binder mixture was poured into the 10.16cm cylinder to make up the desired height which in this case was limited to 15.24cm in the horizontal plane. once filled, the perforated top steel plate was moved downward by the application of an appropriate load (20 kg concrete block). once compressed for at-least 10 minutes, the cylindrical shaped contents were removed from the cylinder by moving the bottom perforated plate by means of an ordinary car jack which ensured the slow and integrated removal of the resulting coal briquette. the labelled coal briquettes were then placed in sunlight to dry for at-least 10 hours (fig. 6). after drying the coal briquettes were ready for different combustion tests. figure 6. cylindrical coal briquettes drying in the sunlight. moisture content in this study was reported by heating the weighted quantity of coal sample in a laboratory scale muffle furnace (japanese model fm-38) at 105oc for one hour volatile matter was reported by heating the moisture free sample in a covered crucible at 950oc for 7 minutes ash content was reported by burning a known quantity of sample in the muffle furnace until a constant weight of the burned content was achieved. fixed carbon value was found by difference from the moisture, volatile and ash content values. calorific value reported in this work was determined by using a bomb calorimeter (shimadzu model ca4pj). the coal briquettes used in this work were made by mixing the coal samples (table 1) with poly vinyl acetate (pva) and caco3 in varying compositions. the contents were well mixed in a laboratory scaled ribbon mixer. furthermore, the vertical axis disintegration strength of the coal briquettes was measured by the universal testing machine (testometric model-m500-100kn) which gave the maximum disintegration load values in newton (n). pak. j. anal. environ. chem. vol. 14, no. 1 (2013) 72 results and discussion the compositions of coal briquettes made with varying binder type are shown in (table 2). the sole addition of poly vinyl acetate (pva) as binder in coal-1 resulted in somewhat less disintegration strength in comparison to other samples but gave high calorific value, high fixed carbon content, less ash and volatile matter (sample no 1 and 5 in table 2). the amount of moisture in all the investigated samples was nearly the same and stayed below 2% (table 2). the values reported in this work showed good reproducibility with 5% deviation on either side of the mean value obtained by five repeat measurements. the use of pva and calcium carbonate (caco3) as binding material resulted in showing an improved disintegration strength for the resulting coal briquettes but at the cost of reduced calorific value, fixed carbon, and high ash and volatile matter as shown in the case of sample no 2, 3, and 4 in (table 2). the combustion characterisation values of moisture content, volatile matter, ash content and fixed carbon values, shown in table 2, are graphically compared and shown in (fig. 7), which shows the comparison of test samples 1, 2, 3, 4 and 5 with the commercially available samples 6 and 7. the test samples of coal briquettes showed a clear advantage of giving high calorific value, high fixed carbon, and low ash content at a marginal disadvantage of reduction in disintegration strength in comparison to the commercially available coal briquettes. figure 7. measured combustion properties of samples used in this study (sample numbers correspond to the compositions given in table 2. the initial ignition times shown in (fig. 8) for all test and commercially available briquette samples were obtained by burning a unit quantity of fuel in laboratory furnace at 800°c and noting the time when the combustion reaction first started. figure 8. initial ignition time in minutes. the sample numbers correspond to the compositions given in table 2. table 2. measured combustion characteristics of coal briquetts samples used in this study measured values sample number sample name moisture % volatile matter % ash % fixed carbon (% by difference) calorific value, joule/g disintegration strength, newton / n 1 a 1.53 11.55 24 63 30463 1471 2 b 1.30 18.58 32 48.12 23268 1765 3 c 1.7 14.85 47 36.45 22251 2059 4 d 1.44 12.0 18 67.6 25893 1667 5 e 1.51 18.13 26 54.3 28354 1569 6 f 1.51 13.15 58 32.12 16232 1863 7 g 1.41 13.50 43 52.15 24523 1667 a = coal-1 with 1000 ml pva; b = coal-1 with 1000 ml pva and 250gm caco3; c = coal-1 with 1000 ml pva and 500gm caco3; d = coal2 with 1000 ml pva and 250gm caco3; e = coal-3 with 1000 ml pva; f = available coal briquettes in market (ptc); g = available coal briquettes in market (phoenix) pak. j. anal. environ. chem. vol. 14, no. 1 (2013) 73 the measurements of initial ignition time for all the investigated samples varied in the range of 0.5 to 1.1 min for a fixed ignition temperature of 800°c. the initial ignition time for the commercially available samples 6 and 7 was slightly higher as compared to others mainly due to the existence of less volatile matter. the low initial ignition time for all samples reported in this work can be attributed to the presence of low moisture content in all samples. since water (i.e. moisture) has high heat capacity thus the ignition of coal briquettes is delayed until all the moisture is nearly driven off from the coal briquette after which the volatile matter drive off to the atmosphere to make high enough concentration for initial ignition. samples 1, 4, 6 and 7 had the least percentage of volatile matter while samples 2, 3, and 5 had the highest value and thus this would have contributed to their quicker initial ignition. the comparison of calorific values showed that samples 1 and 5 had the highest magnitude followed by sample 4 as compared to commercially available sample 6 and 7 that lagged behind in this respect and were only better than test sample 3. the increased calorific value of the test samples can be attributed to the presence of high fixed carbon content in the test samples except for that in sample 3 where the addition of an increased amount of caco3 led to a decrease in fixed carbon content. the measured calorific values of all samples are shown in (fig. 9). the decrease in calorific value of samples 3, 6 and 7 is justified due to the presence of less fixed carbon content in comparison to other samples as shown in fig. 9 and in table 2, which also show the amount of ash content left after complete combustion. the ash content represents the minerals that form a noncombustible residue after carbon, oxygen, sulphur and moisture has been driven off during the combustion reaction. in addition to other factors the amount of ash content in a sample has an effect on the resulting calorific value, which can be observed table 2 for the different test samples. the disintegration strength comparison among all the samples revealed marginal weaknesses of test samples 1, 2, 4 and 5 in comparison to the commercially available samples 6 and 7. sample 3 was an exception which gave the highest value of vertical axis disintegration strength possibly due to the presence of high caco3 content which would have fused in the presence of moisture and applied stress to give cementation strength to the resulting coal briquette. however, this came at the expense of decreased fixed carbon presence and low calorific value. all the disintegration strength values varied marginally between 2059 and 1471 n, as shown in (fig. 10), and did not show any significant disadvantage for normal combustion testing use in this work. figure 9. caloric values of different coal briquette samples. the sample numbers correspond to the compositions given in table 2. figure 10. disintegration strength of different coal briquette samples. the sample numbers correspond to the compositions given in table 2. the results obtained from this research work suggested that a simple coal briquetting technique can be devised that can make coal briquettes from the locally available cheap sources of disintegrated low ranked coal, polyvinyl acetate (pva) and calcium carbonate. a comparison of combustion characteristics among all the samples revealed that the sole use of pva as a binding agent resulted in disintegration strength that was only marginally inferior to the other combinations pak. j. anal. environ. chem. vol. 14, no. 1 (2013) 74 of binders. the loss of marginal disintegration strength was found to be insignificant for the normal conditions of use. the briquettes samples that contained pva (1 and 5) as a binding agent showed considerable amount of fixed carbon, volatile matter and had less ash. all this resulted in giving these samples a high calorific value in comparison to other samples. the increase in calorific value of samples 1 and 5 briquettes can also be attributed to the presence of pva binder which is combustible and would have aided in the overall calorific value. this is also evident from comparing the calorific values of raw coal in table 1 with that of the coal briquettes test samples with pva binder in table 2. the presence of high ash and less volatile and fixed carbon content in other samples resulted in decreased caloric value and difficulty in attaining a low ignition time for combustion. analysis of three different types of local coals revealed that dera adam khel coal (designated as coal-1 in table 1) contained the least amount of volatile matter among all but had the highest proportion of fixed carbon and hence gave the highest calorific value during bomb calorimeter testing. next in rank was hangu coal (coal-3 in table 1) which had a volatile matter content of 12.2% and the fixed carbon content of 65.1 giving the second highest calorific value. doaba coal (coal-2 in table 1) had the highest volatile matter content but had the least fixed carbon content and thus gave the lowest calorific values. conclusion based on the findings given in this work it can be concluded that, in the absence of any large scale coal utilization plants for power production, the low ranked kpk coal can be effectively utilised by devising the simple indigenous technology for coal briquetting. coal samples tested in this work showed that the use of pva as a binding material can help elevate the calorific value of the resulting briquettes by aiding in combustion (sample no 1 and 5). the use of caco3 in any combination with pva resulted in low calorific value due to an increase in the noncombustible content of the resulting briquettes; however, its presence in one sample resulted in increasing the vertical axis disintegration strength. the entire test briquettes used in this work had considerably low moisture content and thus had low initial ignition time. all test briquettes made in this work had an acceptable level of disintegration strength for normal use. the use of caco3 in combination with pva gave the highest disintegration strength of 2059n. in line with other findings, the coal sample with high volatile matter attained the least initial ignition time, the one with high fixed carbon and less ash content gave the high calorific value. based on the findings reported in this work, it is proposed to use this technique for producing low cost coal briquettes that have low initial ignition time, low ash and high calorific value for the ease of utilisation in the domestic and small scale industry. this work forms a first step in a series of efforts towards devising and assessing the indigenous coal briquetting technology in the kpk region. references 1. p. siritheerasa, c. chomthida and p. sethabunjong, chiang mai j. sci., 35 (2008) 35. 2. g. zhi, c. peng, y. chen, d. liu, g. sheng and j. fu, environ. sci. technol., american chemical society, 43 (2009) 5586. 3. g. s. glenn, energy, 18 (2003) 371. 4. n. altun, c. hicyilmaz and a. s. bagci, fuel process. technol., 85 (2004) 1345. 5. m. j. blesa, j. l. miranda, r. moliner and m. t. izquierdo, fuel, 82 (2003) 1669. 6. g. ellison and b. r. stanmore, fuel process. technol., 4 (1981) 277. 7. m. yildirim and g. ozbayoglu, fuel, 76 (1997) 385. 8. b. rubio, m. t. izquierdo and e. segura, carbon, 37 (1999) 1833. 9. n. e. altun, c. hicyilmaz and a. s. bagci, energy fuels, 17 (2003a) 1266. 10. n. e. altun, c. hicyilmaz and a. s. bagci, energy fuels, 17 (2003b) 1277. 11. a. boyano, m. e. gálvez, m. j. lázaro and r. moliner, carbon, 44 (2006) 2399. characterization of metal exchanged zeolite-a using quantasorb and mercury porosimeter issn-1996-918x pak. j. anal. environ. chem. vol. 11, no. 2 (2010) 30 – 37 utilization of bis(salicylaldehyde)orthophenylenediamine for the separation of gold and chromium by capillary zone electrophoresis rubina soomro1, saima q. memon2, m. j. ahmad3 and najma memon*1 1national center of excellence in analytical chemistry, university of sindh, jamshoro, pakistan, 2institute of advance research in chemical sciences, university of sindh, jamshoro, 3laboratory of analytical chemistry, department of chemistry, university of chittagong chittagong – 4331, bangladesh. ------------------------------------------------------------------------------------------------------------------------------------------ abstract bsopd, bis(salicylaldehyde) orthophenylenediamine) is investigated as complexing agent in capillary electrophoresis for determination of gold and chromium. bsopd was chosen as the uv-visible absorbing chelating ligand because of its ability to form stable complexes with metal ions. both the metal ions can be determined in single run under optimized conditions with run time of 12 minutes including coexisted ions usually present in waste water. separation was achieved at optimized conditions of 50 mm phosphate buffer as a background electrolyte at ph =3.4, at applied voltage of -10 kv and detection wavelength of 231 nm. under above mentioned conditions, limit of quantification (0.5 and 10 µg ml-1) and detection limit (0.1667 and 3.33 µg ml-1) were found for au(iii) and cr(vi), respectively. linear calibration graphs were obtained 0.5 – 50 µg ml-1 for au(iii) and 10 – 60 µg ml-1 for cr(vi) with the correlation coefficient value 0.996 and 0.993, respectively. utility of this method for metal analysis has been investigated by determining gold from wastewater samples of goldsmith factories and chromium in some environmental waters (portable and polluted).the method was validated by comparing results obtained with capillary zone electrophoresis with atomic absorption spectroscopy. keywords: bis(salicylaldehyde)orthophenylenediamine (bsopd), separation of gold and chromium, cze. ------------------------------------------------------------------------------------------------------------------------------------------ introduction capillary electrophoresis is one of the most powerful techniques used to simultaneously separate and determine inorganic ions. it yields highly efficient separation, good repeatability, simple operation and rapid detection, along with very low consumption of electrolyte and sample [1, 2]. one of the problems using ce for the separation of cation has a similar mobility, resulting in poor resolution. however, complexation of cations with a ligand to form charged complexes can be used to modify the mobility of cation as each cation will complex with ligand to a different degree determined by the complex stability [3]. this approach allows for direct uv detection of metal ions after chelating with suitable uv absorbing ligand [3].bsopd which is one of the most popular symmetrical tetradentate ligands, forms complexes with various metal ions and organic compounds [4]. the study of complexation reaction of this ligand in aqueous and nonaqueous matrices could be used as an efficient strategy to design the analytical systems such as, bulk liquid membrane[5], solid-phase extraction [6-9 ], catalyst [10,11], sensors[12-19] and biochemistry [20,21]. bsopd related complexing agents have also found applications as spectrophotmetric derivatizing reagents for metal ions in ce and hplc [22, 23]. selectivity of analysis depends on ph and reaction conditions. bsopd itself is reported to form stable complexes *corresponding author email: najmamemon@gmail.com mailto:najmamemon@gmail.com pak. j. anal. environ. chem. vol. 11, no. 2 (2010) 31 with gold [24], chromium [25] and cobalt [26] have been utilized for their spectrophotometeric determination. gold belongs to the element that occurs on the earth with very low natural contents. it is one of the most important noble metal due to its wide applications in industry and economic activity. for this reason, a simple, sensitive and selective method for determination of trace gold is required strongly [27]. chromium is a major pollutant for environment, cr(vi) is carcinogen because of its unique biochemical role [28]. the separation, peconcentration and determination process of trace metal ions from different matrices especially in water samples are mainly based on the utilization and application of a number of available techniques [29].these includes uv-visible, atomic absorption spectroscopy (aas), high performance liquid chromatography (hplc) and voltammetry [30 – 33]. keeping in view of the simplicity of ce based assay procedures; bsopd has been investigated for its utility as complexing agent for gold and chromium in capillary zone electrophoretic conditions. to the best of our literature survey, there is no previous reported work on the use of bsopd for the separation and determination of these two metal cations; au(iii) and cr(vi) by capillary zone electrophoresis. experimental reagents and instrumentation all reagent used were of analytical or equivalent grade and were obtained from fluka (switzerland) and merck (germany). the reagent bsopd was synthesized according to report [33]. the solution was prepared by dissolving the requisite amount of bsopd in a known volume of double distilled ethanol (merck, darmstadt). more dilute solution of the reagent was prepared as required. appropriate amounts of metal salts gold chloride and potassium dichromate were dissolved to make stock standard solution of 1000 mg l-1. buffer solution ph 1-10 at unit interval were prepared as: hydrochloric acid (0.1m) and potassium chloride (0.1m) ph 1-2; acetic acid (0.1m) and sodium acetate (0.1m) ph 3-6; boric acid (0.1m) and sodium tetraborate (0.1m) ph 8-9; sodium bicarbonate, sodium carbonate ph 10 and phosphoric acid (0.1m) and sodium dihydrogen phosphate(0.1m) ph 2-7.the wastewater samples were obtained from common tannery treatment plant of tannery complex and goldsmith factories (karachi, pakistan). the ce system consists of beckman coulter p/ace mdq, usa equipped with photodiode array detector and mdq 32 karat software was used. uncoated fused silica capillaries of 60 cm total length, 54 cm effective length and 75µm i.d were obtained from beckman instruments. the temperature of capillary and samples were maintained at 25°c. prior to sample run, the capillary was regenerated and conditioned as reported [34].the sample was injected by an auto sampler with a pressure of 0.5 psi. analytical procedure aqueous solution containing 25 µg ml-1 of gold and 50 µg ml-1 amounts of chromium was transferred to 10 ml volumetric flask, and 4 ml of reagent (0.05% w/v solution in ethanol) and running buffer was added. the mixture was diluted to the mark with de-ionized water. finally an aliquot of complex was transferred to the ce tube and electropherogram was recorded as shown in (fig.1). the similar complexation method was used to study spectrophotometeric condition for optimization by uv-visible spectrophotometer. figure 1. separation of au(iii) and cr(vi), 50mm phosphate buffer at ph 3.4 and injection time of 0.4 s, applied voltage of 10kv, and reagent concentration of 4 ml of 1.37mm . determination of gold (iii) in certified reference materials (sh24) pak. j. anal. environ. chem. vol. 11, no. 2 (2010) 32 a 25 g sample was accurately weighed and dissolved in aqua regia and placed in a 250 ml round bottom flask and it was refluxed for 6 h. the contents of the flask were transferred to 25-ml volumetric flask and warmed up to dryness then made up to 25 ml volume with de-ionized water. the quantitation of this solution was carried out with the external calibration curve prepared from the standard solution of au(iii) using the same method described as above. determination of gold in wastewater (goldsmith factories) sample an aliquot (1 – 2 ml) of filtered (with whatman no. 42) wastewater sample was pipetted into a 10-ml calibrated flask, and the gold content was determined with the external calibration curve prepared from the standard solution using the same proposed method. the wastewater sample was collected from goldsmith factory (karachi, pakistan). determination of chromium in wastewater from industrial effluent sample filtered (with whatman no. 42) wastewater sample (100 ml) was evaporated upto 15 ml. the resulting solution was then filtered and volume was made up to 150 ml with de-ionized water. the effluent from the common tannery treatment plant of tannery complex, karachi was taken and diluted as per requirement. an aliquot (1 – 2 ml) of this solution was pipetted into a 10-ml calibrated flask, and the chromium content was determined with the external calibration curve prepared from the standard solution using as describe under procedure. results and discussions pre-column derivatization conditions it was found that au(iii) and cr(vi) could form sensitive color complexes with bsopd in a weak acidic environment. the effect of ph on the chelating reaction, metal to ligand ratio, molar absorptivity and stability of metal ion complexes were investigated by spectrophotometric method (table 1) (fig. 2). it could be found that the absorption maxima of metal complexes in visible region is little different, however another band in uv region at 231 nm can be used for simultaneous detection of gold and chromium in single run. also, shorter wavelength bands are intense and pronounce at low concentrations which are the case in the ce detection, hence detection wavelength of 231 nm was selected for further studies. table 1. quntitative spectrophotometric data of color reaction of bsopd with metal ions. metal ions ph of max absorbance metal: ligand ratio λmax nm ε=104 lmol–1 cm–1 solution stability/ temp. au(iii) 3 1:1 450 32.59 24 hr ( 25 ± 5°c) cr(vi) 4 1:4 440 2.25 24 hr ( 25 ± 5°c) figure 2. a, b and c absorption spectra of au(iii) bsopd, cr(vi) bsopd system and the reagent blank at (λmax = 231nm). selection of separation conditions at the start of the work, certain conditions [35] of buffer concentration (50mm phosphate electrolyte), buffer ph (3.4) and voltage applied (-10 kv) were used for the analytical run of the standards for cations under study keeping the capillary voltage at 25 °c. each of these condition was then systematically adjusted, with the others remaining unchanged, in order to establish variation data for each parameter. the sample was introduced by hydrostatic mode injection. effect of type and ph of background system pak. j. anal. environ. chem. vol. 11, no. 2 (2010) 33 the type of electrolyte played an important role in ce separations. we studied the effects of three electrolytes on the separation, including sodium acetate-acetic acid (naac–hac), boric acidsodium borate and phosphoric acid-sodium phosphate. metal complexes did not show any migration in naac–hac buffer system where as good peaks were observed with borate, but both complexes have similar mobility in borate buffer therefore no separation could be achieved using this buffer along with various surfactants [nonionic {polyoxyethylene -octylphenyl ether (tritonx100)}; cationic {cetyltrimethylammonium bromide (ctab)}; and anionic {sodium dodecyl sulfate (sds)}], and organic modifiers (acetonitril, methanol, butanol, 1-pentanol). phosphate buffer showed preferable separation, better selectivity, high reproducibility, and sharp peaks. therefore, different concentrations of phosphate buffer in range of 12.5 – 80 mm have been optimized and 50 mm phosphate at ph 3.4 was found to have better separation and has been selected as run buffer. effect of electrolyte ph on migration time (tm) it is possible to consider ph as one of the most important separation parameters in cze since it affects the net charge of the cations and also the electro-osmotic flow (eof)[35] consequently movement of analyte species is affected by ph. in the experimental, the effect of ph on the sensitivity and separation was studied in the range of ph 2.00-7.00 (fig. 3.) figure 3. effect of ph on migration time. if the ph was less than 2.5 or more than 6, poor separation between au(iii) and cr(vi) or long run times were observed. as shown in figure both metal ions can be separated including reagent in the range of ph 2.54.00, however, at ph 3.4 good separation was possible with shorter run time hence selected for further studies. influence/effect of voltage on migration time (tm) generally, both applied voltage and temperature affect migration time of ions in electrolyte. increasing the applied voltage, the migration time of ions shortens; the higher temperature could also cause the migration time to be short. that is to say, under higher voltage and temperature the speed of ions would be faster. however, high voltage can cause the joule-heat formed to raise temperature, and higher temperature gives a bad repeatability [36]. from the voltage settings (-5) – (-20) kv; the adopted applied voltage is -10kv at 25oc. (fig. 4) shows baseline separation of both cations au(iii) and cr(vi) within 12 min. figure 4. effect of voltage on migration time. influence/effect of electrolyte concentration on migration time (tm) control of electrolyte concentration is a tool that should be kept in mind as tool in improving efficiency, sensitivity and resolution [37]. different concentrations (12.5 – 80 mm) of phosphate were optimized at ph = 3.4. as seen in (fig. 5) at every instance the tm values are more or less same by increasing concentrations of phosphate buffer but the best resolutions were obtained at the intermediate concentrations within pak. j. anal. environ. chem. vol. 11, no. 2 (2010) 34 the range studied with the data obtained, it was decided the most suitable concentration for our purposes is 50 mm, as it yields the best resolution. all results of optimizations are summarized in (table 2). table 2. ce parameters obtained with optimization experiments. parameters studied range selected value wavelength (λ max ) / nm 231,250,301,310 231 ph of running buffer 50mm (phosphate) 27 3.4 applied pressure for sample injection/psi 0.5 injection time of sample/sec 0.4 10 0.4 length / cm x i.d 50 cm bare silica capillary (id.75m) applied voltage / kv -5 – (-20) -10 concentration of reagent (mm) 0.137 – 0.959 0.548 figure 5. effect buffer concentration on migration time. quantification under optimized as mentioned above, calibration curves of these two metal ions were constructed from five calibration levels by plotting peak heights versus concentrations. detection limits were calculated based on a peak height of thrice the baseline noise while limits of quantification were calculated as 10 times of baseline noise. results for analytical figures of merit are given in (table 3). table 3. quantitative data of metal chelates of bsopd complexes by cze. effect of associated ions effect of some commonly associated metal ions has been investigated at optimized conditions. no significant interferences were found from cr(iii), cu, co, fe, ni, pt, and ag. although bsopd form complexes with co and cr(iii), no peaks were detected, due to insolubility of chromium(iii) and cobalt complexes in aqueous solution during separation. applications the developed method was applied to certified reference samples (sh24) for gold in order to evaluate the quantitative performance. method was also used for the determination of gold in wastewater from goldsmith and tannery wastewater sample from industrial effluent. the assay results are listed in table 4a-4b. good agreement between values obtained by ce method and the certified value for gold were observed. the results of the wastewater analyses by the proposed method were in good agreement with those of by aas. table 4a. determination of gold in crm(sh-24). metal ion calibration range (µgml-1) limit of quntification (µgml-1) limit of detection (lod) (µgml-1) coefficient of determination r 2 linear regression equation au(iii) 0.5-50 0.5 0.167 0.996 y=0.553x + 0.491 cr(vi) 10-60 10 3.333 0.993 y= 0.142x 1.102 pak. j. anal. environ. chem. vol. 11, no. 2 (2010) 35 table 4b. determination of gold and chromium in wastewater. sample amount found by ce with calibration in µgml-1 rsd (%) (n=3) amount found by aas µgml-1 1au(iii) 0.378(2.02) 0.341 2cr(vi) 0.032(0.134) 0.032 conclusions this paper demonstrates the separation and determination of gold and chromium, as their bsopd – complex by ce. bsopd can form water soluble complexes with these two metals ions in mixed system hence the hazardous extraction steps can be avoided. metal ion determination by ce can be proposed as inexpensive alternative at g ml-1 levels. results of the determination of gold from crm sample showed that the method can reliably be applied to real systems. references 1. j. gao, h. fan, w. yang, x. sun, c. li, x. mao, j. wang, r. wang and z. jia, cent. eur. j. chem, 6 (2008) 617. 2. a. v. pirogov and j. havel, j. chromatogr. a, 772 (1997) 347. 3. z. chen, r. naidu and a. subramanian, j. chromatogr. a, 927 (2001) 219. 4. m. joshaghani, m. b. gholivand and f. ahmadi, spectrochim. acta, part a, 70 (2008) 1073. 5. m. b. gholivand, f. ahmadi and e. rafiee, sep. sci. technol., 41 (2006) 315. 6. m. shabany, a. m. haji shabani, s. dadfarnia, a. gorji and s. h. ahmadi, ecletica quimica. sao. paulo., 33 (2008) 61. 7. m. b. gholivand, f. ahmadi and e. rafiee, sep. sci. technol., 42 (2007) 897. 8. e. kim, y. kim and j. choi, bull. korean chem. soc., 29 (2008) 99. 9. y. kim, g. in, m. kim and j. choi, bull. korean chem. soc., 27 (2006) 1757. 10. b. ortiz and s. park, bull. korean chem. soc., 21 (2000) 405. 11. a. n. golikand, j. raoof, m. baghayeri, m. asgari and l. irannejad, russ. j. electrochem., 42 (2009) 192. 12. m. b. gholivand, f. ahmadi and e. rafiee, electroanalysis, 18 (2006) 1620. 13. h. m. abu-shawish, j. hazard mater.,167 (2009) 602 . 14. m. b. gholivand, p. niroomandi, a. yari and m. joshagani, anal. chim. acta, 538 (2005) 225. 15. m. k. amini, j. h. khorasani, s. s. khaloo and s. tangestaninejad, anal. biochem., 320 (2003) 32. 16. m. r. al-saraj, s. m. saadeh, and m. s. abdel-latif, anal. lett., 36 (2003) 2417. 17. h. r. zare, f. memarzadeh, a. gorji and m. m. ardaakani, j. braz. chem. soc.,16 (2005) 571. 18. s. shahrokhian, m. k. amini, r. kia and s. tangestaninejad, anal. chem. 72 (2000) 956. 19. s. shahrokhian and m. amiri, j. solid state electrochem, 11 (2007) 1133. 20. s. kashanian, m. b. gholivand, f. ahmadi, a. taravati and a. hosseinzadeh colagar, spectrochim. acta, part a, 67 (2007) 472. 21. m. cametti, i. piantanida, m. zinic, a. d. cort , l. mandolini, m. marjanovic and m. kralj, j. inorg. biochem. 101 (2007) 1129. 22. a. mallah, s. q. memon, m. y. khuhawar, a. r. solangi and m. i. bhanger, acta chromatogr., 22 (2010) 405. 23. m. y. khuhawar, s. n. lanjwani and t. m. jehangir, anal. sci., 20 (2004) 1193 24. r. soomro, m. j. ahmed, n. memon and h. khan, anal. chem. insights, 3 (2008) 75. 25. r. soomro, m. j. ahmed and n. memon, turk. j. chem., 34 (2010) 1. 26. m. j. ahmed, m. n. uddin, chemoshere, 67 (2007) 2020. certified refernce material (composition) amount present in certified reference material (crm)/µg g-1 % recovery amount found by ce with internal standard addition µg g1rsd (%) (n=3) sh-24 (sio2, al2o3, na2o, k2o, cao, mgo, tio2, mno, p2o5, fe, s) 1.326 91.25% 1.21(0.415) pak. j. anal. environ. chem. vol. 11, no. 2 (2010) 36 27. z. chen, z. huang, j. chen, j. yin, q. su and g. yang, anal. lett., 39 (2006) 579. 28. m. zhang, q. zhang, z. fang and z. lei, talanta , 48 (1999) 369. 29. s. h. babu, k. suvardhan, k. s. kumar, k. m. reddy, d. rekha and p. chiranjeevi, j. hazard. mater, 120 (2005) 213 . 30. a. s. orabi1, a. el marghany, m. a. shaker and a. e. ali, bull. chem. technol. macedonia, 24 (2005) 11. 31. i. narin, m. soylak, l. elci and m. dogan, anal. lett., 34 (11) (2001) 1935. 32. a. padarauskas, a. j. entiene, e. naujalis and v. paliulionyte, j. chromatogr. a, 808 (1998) 193. 33. a. manova, s. humenikova, m. strelec and e. beinrohr, microchim. acta, 159 (2007) 41. 34. m. a. salam and d. a. chowdhury, bull. pure appl. chem. 16 (1997) 45. 35. introduction to capillary electrophoresis (backman coulter), a manual provided with ce model no mdq 32 karat software (http://www. beckmancoulter.com/ literature/ bioresearch/360643-ceprimer1.pdf) 36. e. de paz, b. rabanal and a. negro, j. liq. chromatogr. rel. technol., 21 (1998) 2589. 37. j. gao, x. sun, w. yang, h. fan, c. li and x. mao, j. chil. chem. soc., 53 (2008) 1431. 38. george m. janini, king c. chan, gary m. muschik and haleem j. issaq, j. liq. chromatogr. rel. technol., 16 (1993) 3591. characterization of metal exchanged zeolite-a using quantasorb and mercury porosimeter issn-1996-918x pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 103 – 106 utilization of paper sludge wastes for treatment of wastewater from food processing industries ahmed h.a. dabwan1*, kiyoyuki egusa2, daizo imai2, hideyuki katsumata2, tohru suzuki3 and satoshi kaneco2 1faculty of chemical engineering technology, tati university college, jalan panchor, teluk kalong, 24000 kemaman, terengganu, malaysia 2department of chemistry for materials, graduate school of engineering, mie university, tsu, mie 514-8507, japan 3environmental preservation center, mie university, tsu, mie 514-8507, japan received 18 july 2012, revised 05 october 2012, accepted 11 october 2012 ------------------------------------------------------------------------------------------------------------------------------------------ abstract the food processing industries usually produced large amount of wastewater containing fine and small particles. it takes long time for complete settlement of the fine and small particles in the wastewater. the coagulation method appears to become one of the useful treatments. new inorganic coagulant named “agoclean‒p” has been developed from paper sludge ash. the treatment by coagulation and flocculation were carried out for the wastewater from three different food processing industries namely soup, tofu, and natto. “hi‒biah‒system”, which is an in‒situ solidification system, was used for the continuous treatment of wastewater. the parameters for the water quality were ph, five‒day biochemical oxygen demand (bod5), chemical oxygen demand (cod), total suspended solids (tss), total nitrogen (tn) and total phosphorus (tp). these parameters after the treatment became much lower values relative to those obtained before the treatment. keywords: food processing industries; paper sludge ash; coagulant; wastewater treatment; chemical oxygen demand. ------------------------------------------------------------------------------------------------------------------------------------------ introduction wastewater treatment from the food processing industries needs special treatment method owing to high chemical oxygen demand (cod), high concentration of fats, oils, greases, ammonia, and minerals, and high levels of suspended solids with low settling property. generally, the food treatment processing uses large amount of water at different stages. because large amount of wastewater contains fine and small particles, it takes long time for complete settling. different approaches for wastewater treatment have been investigated in order to develop effective methods for the treatment of wastewaters from food processing before releasing the wastewater into the open environment. therefore, the coagulation method seems to be one of the promising treatments, because it is simple, easy to operate and low cost. paper sludge is generally dried in air, and subjected to incineration. the resulting ash is then disposed by landfilling or is used by mixing in cement. the quantity of paper sludge produced is approximately 1.5 million tons per year in japan, and about 0.6 million tons of incinerated ash is formed thereby [1]. paper sludge ash has been used in many applications such as cement replacement materials [2], raw material for constructional brick [3], and effective adsorbent for arsenic, cadmium *corresponding author email: ahmedmie2000@gmail.com pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 104 and lead ions in the wastewater [4]. in the present work, new inorganic coagulant named “agoclean‒p” has been developed from paper sludge ash. furthermore, the inorganic coagulant has been applied to the treatment of wastewater from food processing industries. materials and methods preparation of inorganic coagulant named “agoclean‒p” waste paper sludge was provided from the paper mill wastewater treatment plant. the paper sludge was dewatered and given to the incinerator for the generation of heat energy. paper sludge ashes were collected from fluidized bed‒reactor in the incinerator. the temperature in the incinerator was 800 oc or higher in order to operate the system without the formation of dioxins. since the paper sludge ashes alone could not become a coagulant, the addition of other inorganic materials was necessary. wastewater from food processing industries the slurry wastewaters were collected from three food processing industries (soup, tofu and natto factories). approximately 5 m3 of each wastewater was applied into the continuous flow wastewater treatment process. jar tester experiment (coagulation and flocculation) the jar‒tester was used to evaluate the optimum time for coagulation and flocculation with inorganic coagulant (agoclean‒p). the tests were performed in one liter plastic jar (rectangular shape) at ambient temperature (about 25oc). continuous wastewater treatment with hi‒biah‒ system (hbs) the in‒situ solidification system named “hi‒biah‒system” was used for the continuous wastewater treatment. the detailed information has been described previously [5,6]. the hbs consists of a main stock tank of sediments, a coagulant chamber, reactors (coagulation and flocculation) and a dewatering section. the treatment capacity was approximately 1~2 m3/hour. the agglomeration of small particles into big flocks, the separation, handling and disposal for the sludge were performed during the continuous treatment. analytical methods chemical oxygen demand (cod), five‒day biochemical oxygen demand (bod5), total nitrogen (tn) and total phosphorus (tp) were measured by japanese industrial standard (jis)‒standard analytical methods. the concentrations of total suspended solid (tss) was determined by a spectrophotometer with a resonance line of 660 nm, based on the japanese industrial standard (jis)‒standard analytical methods (k0101). results and discussion the developed inorganic coagulant was applied into the treatment of wastewater from japanese food processing industries such as soup, tofu and natto factories. soup is used in the instant noodle making. tofu is very popular in japan and japanese people have had the tofu in daily life. tofu is prepared from grained soybeans. natto is produced from the ferment of soybeans. the chemical compositions of paper sludge ash are shown in (table 1). since the paper sludge ashes alone cannot be applied as the coagulant, the inorganic coagulant agoclean‒p was prepared by the addition of other inorganic materials. the chemical compositions for agoclean‒p are shown in (table 2). table 1. chemical compositions for paper sludge ash components content (%) cao 10~35 sio2 25~35 al2o3 23~40 so3 3~8 fe2o3 1.5~5 mgo 1.5~5 tio2 0.5~1.5 pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 105 k2o 0.3~1 table 2. chemical compositions for agoclean-p coagulant components (%) cao 44.2 sio2 26.9 al2o3 12.7 so3 12.2 fe2o3 1.2 mgo 1.2 tio2 0.8 k2o 0.4 others 0.4 although the traditional coagulants such as polyaluminium chloride (pac) and polymer coagulant were tested for the treatment of wastewater from japanese food processing industries, the coagulation could not occur for the wastewater. the reason is not clear. may be, high concentration of fats, oils and greases in the wastewater hinder the coagulation and flocculation processes. first, the jar‒tester was applied to evaluate the optimum time for coagulation and flocculation with agoclean‒p. rapid mixings at 200 rpm for 4 minutes after the addition of agoclean‒p, followed by slow mixing 20 rpm for 1 minute, were selected as the optimum conditions. the purification parameters before and after the treatment are ph, 5‒day biochemical oxygen demand, chemical oxygen demand, total suspended solids, total nitrogen and total phosphorus. the continuous hbs treatment with inorganic coagulant agoclean‒p was applied into the treatment of wastewater from japanese food processing industries. the results are summarized in (table 3). although the initial ph for the three wastewaters was in the acidic range from 5 to 6, ph values were neutral between 8‒8.5 after the treatment. the values for chemical oxygen demand and five‒day biochemical oxygen demand drastically decreased after the treatment, and the removal efficiencies for cod and bod5 were 88‒92 and 85‒97%, respectively. although the total suspended solid before the treatment was very high (>8000 ppm), all of the values became less than 100 ppm after the continuous hbs treatment with inorganic coagulant agoclean‒p. also, the values of total nitrogen and phosphorus after the treatment were much lower compared with those obtained before the treatment. table 3. continuous wastewater treatment with hbs with inorganic coagulant agoclean‒p food factory ph bod5 (ppm) cod (ppm) tss (ppm) tn (ppm) tp (ppm) soup before 4.5 600 1100 >8000 80 39 after 8.2 16 120 <100 18 <1 tofu before 4.5 1100 2100 8000 100 52 after 8.0 92 170 <100 5 <1 natto before 5.5 660 1500 >8000 30 27 after 8.5 100 180 100 19 2.6 flow rate; 1.5 ton/hour. dose of agoclean‒p; 2000 ppm for soup, 1000 ppm for tofu, 2500 ppm for natto. in order to explain possible basic fundamental coagulation mechanisms for the wastewater treatment with agoclean‒p, the micro structure of the surface of the dried products after the coagulation was evaluated by scanning electron microscopy (sem). the needle‒shaped ettringite was observed in the coagulated products (fig. 1). we can conclude that the ettringite formed a three‒dimensional network structure in the coagulated products for the wastewater treatment [7]. therefore, the coagulation owing to the formation of ettringite may play the significant role on the purification of wastewater. pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 106 figure 1. ettringite observed in the dried products after the coagulation. conclusion the wastewater treatment by the coagulation with agoclean‒p inorganic coagulant, which consisted of paper sludge ash wastes, appears to become one of very effective methods for the wastewater from food processing industries. the continuous hbs treatment system with inorganic coagulant agoclean‒p can be applied into 5m3/h of wastewater. the quality of aqueous solution released from different food processing industries after the treatment could be drastically improved by the present methods acknowledgments the main research was partly supported by the ministry of education, culture, sports, science, and technology of japan. all experiments were conducted at mie university, japan and tati university college, malaysia. support was provided to a. h. a. dabwan as the researcher for an invitation fellowship program for research in japan (long‒term) from the japan society for the promotion of science. any opinions, findings, conclusions, or recommendations expressed in this paper are those of the authors and do not necessarily reflect the view of the supporting organizations. references 1. m. koshikawa and a. isogai, j. mater. cy. waste manag., 6 (2004) 64. 2. c. y. yin, s. a. s. abdul kadir, a. s. abdul malik, s. n. syed-ariffin, s. z. mahazer and z. zamzuri, sci. asia, 33 (2007) 473. 3. c. t. liaw, h. l. chang, w. c. hsu and c. r. huang, j. hazard. mater., 58 (1998) 93. 4. b. ahmadi and w. al-khaja, resour. conserv. recycling, 32 (2001) 105. 5. a. h. a. dabwan, d. imai, t. kato, s. kaneco, h. katsumata, t. suzuki and k. ohta, océanis, 34 (2008) 39. 6. d. imai, s. kaneco, a. h. a. dabwan, h. katsumata, t. suzuki, t. kato and k. ohta, int. j. soil. sed. water, 2 (2008) 451. 7. k. egusa, s. kaneco, a. h. a. dabwan, h. katsumata, t. suzuki and k. ohta, seikatsu eisei 54 (2010) 330. ettringite issn-1996-918x pak. j. anal. environ. chem. vol. 14, no. 1 (2013) 47 – 53 determination of heavy metals in eight barley cultivars collected from wheat research station tandojam, sindh, pakistan ghulam qadir shar1, tasneem gul kazi3, wahid bux jatoi1, pirbho mal makhija4, shamroz bano sahito1, abdul hussain shar2 and fateh m. soomro2 1department of chemistry, shah abdul latif university, khairpur (sindh), pakistan. 2department of microbiology, shah abdul latif university, khairpur (sindh), pakistan. 3national centre of excellence in analytical chemistry, university of sindh jamshoro, 76080, pakistan. 4ghulam muhammad mahar medical college, sukkur, sindh, pakistan received 31 january 2013, revised 19 june 2013, accepted 29 june 2013 ------------------------------------------------------------------------------------------------------------------------------------------- abstract barley (hordeum vulgare l.) is one of the most important foods for animals and possesses high nutritional value. in this paper, we have focused our study to find out the chemical parameters, especially metal content of this class of food commodity and its soil, which is not frequently used for human food. wet digestion method was used to destroy the organic matrix to determine the content of eleven metals i.e. iron, zinc, manganese, copper, cobalt, chromium, nickel, lead, cadmium, barium and aluminium, from eight pakistani barley cultivars. the highest level of elements were determined in order; cd < ni < pb < cr < co < cu < ba < al < mn < zn < fe in mg/kg. all analysis was carried out by using airacetylene except al and ba where as both of these metals analysed on air-acetylene and nitrous oxide flame on atomic absorption spectrophotometer. among all these entries, maximum concentration of fe was detected in b6, zn in b5, mn, & cu in b2 & b5, co in b1, ba in b7, al in b8 and rest of the elements i.e. cr, pb, ni and cd were found to be with little difference of concentration among cultivars. keywords: cereals; barley; metals; chemical analysis. ------------------------------------------------------------------------------------------------------------------------------------------- introduction the barley (hordeum vulgare) is one of the oldest cultivated cereal commodities of poaceae family that also include wheat, rice and maize. information about the barley cultivation has been observed in soil tablets from 1700 b.c. [1]. more than 150 million tons of barley was produced in 2009, comprising 54 million hectares of agricultural land worldwide [2]. in united states, barley is ranked third amongst cultivated cereal grains [3, 4]. in most of countries including europe, it is cultivated in large scale and is used as cereal grain for animal as well as birds feed [5]. barley is almost cultivated in cold areas being a cold cultivated plant. however, it also grows at hot areas of the world by producing new varieties of the barley, which can sustain to the temperature of that specific area. the change in climatic conditions was found to change the dietary value in cultivars of barley [6]. barley is used commercially for animal feed, to produce malt, for seed and for human food applications as it is rich in protein, carbohydrates, dietary fibers, minerals, and vitamins. we have studied the amount of essential and trace metals present in barley and its soil using atomic absorption spectroscopy, that is, to our opinion, an important advancement towards the assessment of mineral content present in this important food commodity. *corresponding author email: drgq_khp@yahoo.com pak. j. anal. environ. chem. vol. 14, no. 1 (2013) 48 protein storage in barley is about 50% in mature grains that is the most important nutritional and functional property of grains [7]. different plants follow different paths of uptake and accumulation for various elements [8]. barley accumulates a large amount of starch and is utilized as source of energy [9]. the level of protein in barley cereal was found in the ranges of 7.5 – 17 % at a dry basis where as 75% of the protein is digestible. the protein value in greater amount, about 13.6% on dry matter would not be greatly raising the level of barley to livestock feeders [10]. calcium value was detected at low level in maize, barley and sorghum grains but barley possesses high level of phosphorus as compared to other grains [11]. however, the calcium is a basic macro-element for life, particular for human being for the formation of bones and teeth. low level of minerals in barley cultivars needs supplement in food, which can be fulfilled by taking the supplement from mineral salt. micro-elements deficiency is mainly an environmental crisis that can be overcome with consultation of soil specialists as well as nutritionists of the concerned quarter. the suggestions of experts are essential to resolve this problem by giving their recommendations about supplement and also type of supplement that is needed. most of the elements and vitamins can increase the shell life of the food; therefore, these metals and vitamins are added in barley to preserve for longer time [12]. the element contents vary area-wise on the basis of geographic variations, which badly affect the barley crop than that of seed grown in soil [13]. the demand of barley (hordeum vulgare l.) is going to increase day by day due to the interest of human being as delicious food and its industrial use in addition to animal and bird’s feed [14,15]. some of the barley varieties possess soft husk which can be easily and fairly removed. new industries have been developed around to remove the husk and make it digestible for animal and poultry as their feed. husk less contains fiber and flour and has been used in many edible products [16]. due to rich in fiber contents, glucan and protein, husk less is getting more importance than other grains, such as oats, that is well known for its β-glucan contents [17-19]. the -glucan compound attracts the customers for the reason that it decreases blood cholesterol and glucose levels [20]. the contents of various essential, trace and toxic elements in barley and unpolished rice and its soil of different areas of pakistan were studied using spectroscopic techniques [21, 22]. various analytical methods are used to study the mineral contents and grain yield data of numerous cultivars of barley [23]. heavy metals are essential, needed in small quantities for the health of all living things that may be animals or plants during whole life for their physiological functions, these nutrients cannot be synthesized by the organism itself but are obtained from surrounding [24]. most of the microelements are used for enzyme cofactors, these are easily supplied through the soil to plants in a trace amounts, which is further transferred to animal and human body. human requires less than 100 micrograms per day in diet and is known as dietary minerals. the micro-minerals or trace elements include; iron, cobalt, chromium, copper [25], iodine, manganese, selenium, zinc and molybdenum are not usually necessary in plant fertilizers, because its much quantity may cause harm the growth of a plant. the micronutrients are also included; vitamins that are organic compounds required as nutrients in tiny amounts by an organism but these are not known as microelements but micronutrient [26]. this research was carried out to analyze the variable uptake of concentration of minerals in eight barley lines pedigree (b1-b8) and existence of the concentration of same elements in that specific soil where these cultivars were grown. materials and methods barley sampling wheat research institute tandojam, sindh, pakistan, was selected for the collection of barley samples. eight different genotypes; arar (b1), rihane-03 (b2), beca’s’(b3), beecher (b4), orge (b5), giza 120/5 (b6), albert (b7), and arizon (b8) were grown and harvested at matured stage. the samples were collected on hit or miss basis in the maturation period to make a model sample just after harvesting and samples were de-husked, packed into polyethylene bags without chemical treatment and properly labeled, finally brought into pak. j. anal. environ. chem. vol. 14, no. 1 (2013) 49 laboratory of national centre of excellence in an analytical chemistry, university of sindh, jamshoro for further chemical analysis. the growing period of barley lies in between november to january and harvesting season is by the end of march. all varieties were grown in experimental farms in 2010 at above wheat research station (wri). the clay loam soil was collected from experimental field where all these varieties were grown. the soil was air-dried and sieved (<0.5mm). chemical analysis different barley cultivars and soil samples where these cultivars were grown dried at variable temperatures 80oc to 110oc in oven till invariable weight was attained. two grams of each cultivar and one gram of soil in 5 replicates were weighed in conical flasks (100ml), then were treated with hno3 (5ml). same quantity of hno3 of same concentration was also taken in to empty conical flask that serves as a blank. all barley and soil samples were placed in conical flasks, covered with watch glasses and the samples heated lightly on an electric plate, as content cannot splash. the heating was continued for about 60 minutes till semi dried content obtained in flasks. sample flasks and blank were removed from hot plate for few minutes until flasks were cooled. again 5 ml of hno3 were added to all flasks and placed on an electric plate for further 50-60 minutes to obtain semi-dried content. as semi-dried content obtained, flasks were removed from hot plates, left to cool down for 20 minutes then were added 2 3 ml of 35% h2o2 into the contents of flasks, heating was continued for further 50-60 minutes till semidried contents obtained. after removing watch glasses, 50 ml of deionized water was added to each flask, and was heated until the volumes of contents evaporated up to 25 ml. the contents of the flasks were cooled up to 150c 200c, filtered from first to last by whatman # 42 paper into 25 ml volumetric flasks. the volume of the 25 ml volumetric flasks was made up to mark with deionized water by adding 5% nitric acid to preserve the sample, finally transferred the content to sample bottles, which were previously washed and soaked. besides that an internationally certified reference material was used to maintain analytical quality assurance. the concerned standard reference substances were selected for quality control i.e. bcr crm191 brown bread, nist srm1567a wheat flour, nist rm8433 corn bran and nist rm8436 durum wheat flour. all these sample bottles along with blank were transferred to atomic absorption spectrometry to analyze essential trace elements as well as trace and toxic elements such as: iron, zinc, manganese, copper, cobalt, chromium, nickel, lead, cadmium, barium and aluminium levels. all samples were pre-treated by the wet digestion method to destroy the organic matrix. standard official methods of analysis of the association of official analytical chemists [27] were used for the analysis of above elements. entire unsolvable metals were estimated by using flame atomic absorption spectrophotometer (faas), according to approved methods of the american association of cereal chemists [28]. statistics were accounted on a dehydrated weight origin. results of all above measurements have been mentioned in (table-1). table1. concentration of eleven heavy metals in eight barley cultivars in mg/kg element b1 b2 b3 b4 b5 b6 b7 b8 soil fe 1546.66 1229.84 1181.84 841.01 1537.06 2146.9 1820.28 1316.24 4966.8 zn 48.64 43.22 30.24 37.93 53.05 41.58 44.35 42.09 70.03 mn 21.93 38.41 25.06 22.78 38.69 30.45 33.86 21.93 663.71 cu 9.03 10.31 7.50 7.02 10.46 9.87 8.92 7.17 21.86 co 4.41 3.45 2.73 2.73 3.45 2.26 1.66 3.29 11.15 cr 1.04 0.84 1.03 0.88 1.22 0.83 0.99 1.00 13.09 ni 0.91 0.80 0.70 0.56 1.23 0.92 1.04 0.97 15.69 pb 1.21 1.10 0.78 0.82 0.98 0.55 0.78 1.19 3.49 cd 0.45 0.49 0.33 0.42 0.34 0.23 0.43 0.32 2.94 ba 8.32 6.76 4.43 8.78 3.96 8.32 9.25 6.30 139.41 al 19.06 15.92 10.42 24.57 22.21 19.06 21.42 25.35 42035.7 pak. j. anal. environ. chem. vol. 14, no. 1 (2013) 50 table 2. instrumental conditions for the aas measurement of fe, zn, mn, cu, co, cr, ni, ba, al, pb and cd e le m en ts wave length (nm) slit width (nm) lamp current (ma) fuel flow (acetylene) (l/min) flow rate (air) (l/min) burner height (mm) oxidant (air) kg/cm2 fuel (acetylene) kg/cm2 signal out put fe 248.3 0.2 9.5 2.30 9.4 7.5 1.60 0.3 100% zn 214.0 1.3 9.5 2.0 9.4 7.5 1.60 0.2 100% mn 279.8 0.4 9.5 2.0 9.4 7.5 1.60 0.2 100% cu 325.0 1.3 9.5 2.0 9.4 7.5 1.60 0.2 100% co 250.0 0.2 9.5 2.0 9.4 10.0 1.60 0.35 100% cr 358.2 1.3 6.0 2.0 9.4 7.0 1.60 0.30 100% ni 232.3 0.2 9.5 2.0 9.4 7.0 1.60 0.30 100% ba 553.8 1.3 9.5 5.61 5.91(n2o) 7.5 1.60(n2o) 0.45 100% al 309.5 1.3 9.5 5.61 5.91(n2o) 12.5 1.60(n2o) 0.45 100% pb 232.3 1.3 7.0 2.0 9.4 7.5 1.60 0.2 100% cd 229.0 1.3 7.0 2.30 9.4 7.5 1.60 0.30 100% instrumentation all tabulated results of eleven heavy elements such as, fe, zn, mn, cu, co, cr, ni, ba, al, pb and cd were analyzed by hitachi model 180-50 atomic absorption spectrophotometer. the lamps known as hollow cathode lamps (made by meltorika ) were used as radiation energy source to find out the concentration of given elements. for the fuel, in atomization steps of elements, an air acetylene gas was used. however, for analysis of al and ba, an additional nitrous oxide was used with air-acetylene-nitrous oxide as a source of high hotness. the lamp’s current 9.5, 9.5, 9.5, 9.5, 9.5, 6.0, 9.5, 9.5, 9.5, 7.0, and 7.0ma respectively were maintained by hollow-cathode lamps (mtiorika) for these elements. flow-rate of fuel was 2.21 1/min for fe and cd; 2.0 1/min for zn, mn, cu, co, cr, ni and pb; 5.61 1/min for ba and al respectively. flow-rate for air 9.40 1/min for fe, zn, mn, cu, co, cr, ni, pb, and cd; 5.91 1/min for al and ba were used respectively to get a clear yellow flame. spectrophotometer was coupled to a hitachi recorder 056 with a range of 5mv. the signals calculated were the heights of the absorbance/division peaks. instrumental parameters have been mentioned in (table 2). reagents and calibration pure hno3 (65% w/v), h2o2 (35% w/v) chemicals (merck) and highly purified water / deionized water having zero electrical conductivity was produced with a milli-q system millipore, ma, usa. the calibration curve was got by using the external and internal standards of analyzed elements. for preparation of standard solutions, they were diluted in 1000mg/l multi element solution (icp multi element standard iv, merck, darmstadt, frg) with similar acids, which were used for pre-treatment of sample. all sample solutions were analysed by aspiring through capillary tube into atomic absorption spectrophotometer through absorbance / division’s capacity of each element by maintaining optimum conditions for flame atomization mode. for inter calibration, standard references; 0.5 to 10 ppm was also run along with our own made standards. elemental concentrations were computed on an ibm compatible pc using excel computer program. the statistical calculations for standards are given in (table 3). pak. j. anal. environ. chem. vol. 14, no. 1 (2013) 51 table 3. statistical data for standards of elements statistical calculation y = m x + c elements concentration range ppm (x) absorbance/ division (y) m c r2 fe 0 – 3 0 910 03016 -0.0022 0.9996 zn 0 – 1 0 0.235 0.2349 -0.0027 0.9989 mn 0 – 1 0 – 0.134 0.1324 0.0021 0.9985 cu 0 – 2 0 –0.138 0.0693 -0.0001 0.999 co 0 – 1 0 – 0.053 0.0529 0.0004 0.9997 cr 0 – 1 0 – 0.070 0.0698 0.0002 0.999 ni 0 – 1 0 – 0.111 0.1104 0.0022 0.998 ba 0 – 1 0 – 22 div. 22.057 -0.0001 0.9974 al 0 – 25 0 – 0.0358 0.0014 -0.086 0.9999 pb 0 – 1 0 – 0.063 0.0638 -0.0009 0.9992 cd 0 – 0.125 0 – 25 div. 356.52 0.5074 0.999 absorbance* div. =divisions results and discussion the concentration of the metals in the eight barley hybrids and its soil is given in table 1. among all eight hybrids, the value of fe was found highest in b6 and its lowest level detected in b4 cultivar and the second highest value of iron observed in b7, while b1 and b5 got intermediate position among all cultivars and their values were very close to each other. analytical results of iron of same plot of soil where eight genotypes were grown indicate that the soil accumulated about three times more iron as compared to their barley cultivars. it may be possible that due to high concentration of iron in soil, barley cultivars contained larger value of iron in grains than that of listed metals. zinc was second essential heavy metal that had preserved maximum status in b5 and minimum concentration in b3. meanwhile there was little bit difference in values b2 and b7 as well as b6 and b8 cultivars. as far as its soil was concerned, it possessed round about double concentration of the zn. manganese concentration was found in the range of 21.93 – 38.69 mg/kg in which the minimum and identical value was obtained in b1 & b8 (21.93 mg/kg) and maximum value (38.69 mg/kg) calculated in b5 cultivars. however, there was significant difference level of b2 and b5 barley hybrids (38.41 and 38.69 mg/kg) respectively, wherever soil of that specific area haunted 23 folds higher concentration (663.71 mg/kg) of same element compared to barley hybrids. the high up take of cu was observed in b2 and b5 (10.31 and 10.46 mg/kg) respectively, which were precise and low up take of same element was detected in b3, b4 and b8 variety (7.50, 7.02 and 7.17 mg/kg) in that order with a minute distinction and its soil keeps (21.86 mg/kg) that was 2.5 times higher level of copper as compared to their grains. cobalt and lead are highly toxic in nature for human being and are required at trace level to play active role in metabolism of the body. analytical results of table 1 established that the b1 got uppermost level (4.41 mg/kg) of co and lowest in 1.66 mg/kg in b7, at the same time as two pairs; b2 and b5 (3.45 mg/kg) and b3 and b4 (2.73 mg/kg) had identical values. in close proximity was no significant difference in values of b2 and b8 (3.45 and 3.29 mg/kg) cultivars though soil of same crop of variable genotypes accumulated 11.15 mg of co that was higher than values of all cultivars. the maximum concentration (1.04 mg/kg) of cr was found in b1 and minimum (0.83 mg/kg) in b6, on the other hand, no mark able deviation was observed in pairs of barley: b1 and b3 (1.04 and 1.03 mg/kg); b2 and b6 (0.84 and 0.83 mg/kg); b7 and b8 (0.99 and 1.00 mg/kg) while soil of that pak. j. anal. environ. chem. vol. 14, no. 1 (2013) 52 specific plot contained 13.09 mg/kg. investigated results of ni illustrated so as to chief value was detected in b5 and bottom one in b4 although negligible distinction observed in b1, b6 and b8 varieties. overall, trend of ni absorption was found in decreasing order from b1 to b4 then jumps at peak level in b5 and shows zigzag trend in rest of cultivars, furthermore, randomly collected soil samples of that plot possessed 15.69 mg/kg of nickel. moreover, soils accumulate three fold higher concentration of cobalt and lead 3.5 folds higher that of the lead. as for as cd concentration was concerned, all barley genotypes hold below 0.5 mg/kg of cd and the values of b1, b2, b4 and b7 (0.45, 0.49, 0.42 and 0.43 mg/kg) respectively were high and very close to one another, lowest value of cd was found in b6 (0.23 mg/kg) and its soil have 2.94 mg which is higher (7.82 times) than that of barley level. comparative study of ba in eight cultivars indicates that b7 attained uppermost assessment and b5 lowest one, on the other hand identical values calculated in b1 and b6, at the same time close concentration was detected in pairs: b2 and b8 (6.76, 6.30 mg/kg) and b3 and b5 (4.43 and 3.96 mg/kg) respectively as soil of that plot acquired 139.41 mg/kg. aluminum got exceptional position in capacity of higher concentration among all trace and toxic elements given in table 1, but its maximum concentration (25.35 mg/kg) was observed in last variety (b8) and minimum concentration in b3 (10.42 mg/kg). however, identical data observed in b1 and b6 (19.06 mg/kg), on the other hand, insignificant level was detected in b4 and b8 (24.57 and 25.35 mg/kg) respectively, whereas experimental plot of genotypes keep hold on (42035.7 mg/kg). there was significant difference in the results of al, fe, mn, ba, zn, cu, ni, cr, co and pb in barley given in table 1, which were compared with previous related published work for the same elements [29-34]. the soil was collected from those locations where all these varieties were grown. results of the soil, where these cultivars/hybrids were grown shows that it accumulates the concentration of metals in order; al > fe > mn > ba > zn > cu > ni > cr > co > pb and cd respectively. conclusion it is found that b6 cultivar accumulated highest concentration of iron where as lower concentration of iron, zinc and copper was found in b4. highest level of zinc, manganese, copper, chromium and nickel was detected in b5. maximum level of ba and al was found in b7 and b8 respectively. highest level of pb and cd was observed in b1 and b2 respectively and lowest level of both elements accumulated in b6 variety. the value of trace, essential and toxic elements in soil was detected from double to thousands folds higher than that of barley cultivars, which grown in the same soil. acknowledgement we are grateful to syed iqrar hussain shah, muhammed aslam nushad (seed certification officers), seed certification & registration department and mr. biland rai oad, wheat botanist for their help in collection of the barley samples from wheat research station (wrs), tandojam. author is also thankful to the authorities of shah abdul latif university, khairpur, sindh, pakistan for financial assistance and grant of study leave. references 1. j. r. harland, usda handbook, 338 (1979) 10. 2. takashi matsumoto, jsuyoshi tanaka, kiyosumi hori, kazuhiro sato, plant physiol., 156 (2011) 20. 3. w. lin, s. evans and g. davenport. ag. info. bull., 477 (1984) 40. 4. klaus, f. x. mayer, mihaela martis, pete e. hedley and nils stein, the plant cell, 23 (2011) 1249. 5. ivan t. todorov, padha n. philipova, nikolai, z., zhelev, asenov and hadjolov, biology of the cell, 62 (1988) 105. 6. hoppner, k. h., b. d. owen and f. w. sosulski. can. j. anim. sci., 48 (1968) 135. 7. milan chnapek, eva palencarova, zlenka galova, zelmira balazova, marian tomka, j. microbiol., biotech. & food sci., 1 (2012) 622. pak. j. anal. environ. chem. vol. 14, no. 1 (2013) 53 8. a. aksoy and d. demirezen. polish j. env. studies, 15:1 (2006) 27. 9. natioanal research council (nrc), recommended dietary allowances, 10th ed. national academy press, washington d.c. (1989). 10. c. w. newman, c. f. mcguire and d. c. rasmusson. am. soc. agron., crop sci. soc. and soil sci. soc., ed. (1985) 403. 11. natioanal research council (nrc), nutrient requirements of beef cattle, 7th ed. national academy press, washington d.c (1984). 12. w. e. dinusson, d. o. erickson, c. n. haugse and m. l. buchanan. nd farm res. 26:1 (1968) 3. 13. w. e. dinusson, d. o. erickson, d. w. bolin, m. l. buchanan. nd farm res. 21:7 (1960) 8. 14. m. a. o. oscarson, r. anderson, a. c. salomonsson and p. aman, j. cereal sci., 24 (1996) 161. 15. c. elfverson, a. m. anderson., p. aman. and s. regnér. cer. chem., 76 (1999) 434. 16. r. s. bhatty, cer. chem., 76 (1999) 589. 17. a. lapveteläinen and t. aro. cer. chem., 71 (1994) 133 18. e. marconi, m. graziano. and r. cubadda. cer. chem., 77 (2000) 133. 19. a. de francisco and r. m. de sa. beta glucanas: localização, propriedades utilização. in: lajolo, f. m.; saura-calixto, f. d. e. pena, e. w. de menezes, e. w. fibra dietética en iberoamérica: tecnologia y salud. obtención, caracterización, efecto fisiológico y aplicación en alimentos. são paulo: varela, (2001) 91. 20. r. k. newman, k. c. ore., j. abbott. and c. w. newman. cereal foods world, 43 (1998) 23. 21. g. q. shar, l. a. shar, t. g. kazi, and i. h. afridi, j. res. sci. b. z. univ. 18:2 (2007) 69. 22. g. q. shar, l. a. shar, t. g. kazi, s. sahito and s. a. arain, j. chem. soc. pak. 27:1 (2005) 38. 23. soner yuksel, mevlut akcura, turk. j. agric., 36 (2012) 285. 24. canadian unicef committee, global child survival and health, (2006) 67. 25. http://en.wikipedia.org/wiki/copper in health 26. s. lieberman and n. bruning, the real vitamin & mineral book. ny: avery group, 3 (1990). 27. association of official analytical chemists – aoac. official methods of analysis of the association of official analytical chemists. washington (1995). 28. american association of cereal chemists – aacc. 2000. approved methods of the american association of cereal chemists. 10th ed. st paul (2000). 29. f. madrid and m. b. kirkham, 17th wcss, (2002) 401. 30. j. wieczorek, z. wiezorek and bieniaszewski, polish. j. env. studies., 14 (2005) 535. 31. r. juknys, m. racaite, g. vitkauskaite and j. vencloviene, zemdirbyste-agric., 96 (2009) 111. 32. f. s. maleki, m. r. chaichi, d. mazaheri, a.r. tavakkol and g. savaghebi, j. agr. sci. tech., 13 (2011) 315. 33. r. k. singh and r. p. sharma, int. j. green & herbal chem., 1 (2012) 21. 34. m. samaranda, d. neculai, m. florica, n. j. luminita and p. dumitru, animal sci. biotech., 45 (2012) 237. microsoft word 368-376-pjaec-20012020-221-c.doc cross mark issn-1996-918x pak. j. anal. environ. chem. vol. 21, no. 2 (2020) 368 – 376 http://doi.org/10.21743/pjaec/2020.12.39 the conductivity of emimcl and bmimpf6 ionic liquids for limited range of temperature (25 to 75 °c) under optimal electrolyte combination conditions muhammad tariq sarwar 1 , zhan hanhui 1 and yang jiaxin 2 1 school of environmental sciences and spatial informatics, china university of mining and technology, xuzhou, p.r china 221116. 2 nanjing research institute of environmental science, nanjing, p.r china 210042. *corresponding author email: tariqsarwar98@yahoo.com received 20 january 2020, revised 02 november 2020, accepted 03 november 2020 -------------------------------------------------------------------------------------------------------------------------------------------abstract the purpose of this study was to evaluate the ionic conductivity as a function of temperature, range (25 to 75°c) in the imidazolium-based ionic liquids 1-ethyl-3-methylimidazolium chloride (emimcl) and 1-butyl-3-methyl imidazolium hexafluorophosphate (bmimpf6) of different volume percentages under optimal electrolyte combination conditions. the findings revealed that when 1% emimcl or 1% bmimpf6 was applied, conductivity decreased significantly in relation to the kohlrausch relationship. when 1% emimcl or 2% bmimpf6 was added, the study showed the highest coefficient of alpha (α), while beta (β) was the lowest coefficient for temperature. in conclusion, the influence of the change in volume percentage on the conductivity is weakened by temperature control. keywords: emimcl, bmimpf6, conductivity, temperature coefficient. -------------------------------------------------------------------------------------------------------------------------------------------introduction room temperature ionic liquids (rtils) that can be abbreviated as ionic liquids (ils) present at low temperatures. ils is one of the green solvent systems following water and supercritical carbon dioxide [1]. compared to the commonly used conventional solvents, ils exhibit unique physicochemical properties and their unique functions [2]. usually, the ils consist of organic cations and inorganic or organic anions, which possess the desirable characteristics of a wide range of liquid temperatures, thermal stability, low vapor pressure and no volatilization, reusable, high conductivity, preferably chemical stability and large electrochemical windows, etc. [3-5]. ils are thus considered to be an effective replacement for organic solvent in the reaction-separation coupling system [6]. many ils can be used as green solvents because they do not have the characteristics of volatilization [7]. the solubility of ils is closely related to the properties of their cations and anions. as a new type of green solvent, called ils have been widely used in many fields and rapidly developed into a research and gain popularity. the application fields of ils mainly include organic synthesis [8], catalytic reaction [9], electrochemical extension [10], extraction separation [11], biochemistry [12] pak. j. anal. environ. chem. vol. 21, no. 2 (2020)369 and material engineering [6]. but so far, there have been few studies on the deposition of metals by ils as additives [13]. some research shows that ils considerably amend the properties like density, viscosity, polarity and conductivity by addition of polar solvents [14]. conductivity results for a range of 1alkyl-3-methylimidazol tetrafluoroborates extending to significantly lower temperatures have been reported. the general picture given by reported studies is that the ionic mobilities which are primarily related to the values of electrical conductivity that are highly affected by the exact existence of the anion-cation interaction. the coulombic interaction between the charges of the ions and the van der waals interaction between the induced charges of the ions builds up this interaction. the precise essence of this interaction then relies on characteristics such as the composition of the ions, their polarizability and the hydrogen bonding possibilities [15]. when choosing an ils for an electrochemical application, conductivity (k) is of vital importance. one impediment to the use of ils is the lack of accurate data for conductivity. few or no data has been released for many ils. an example of such a case is 1ethyl-3-methylimidazolium chloride, abbreviated herein as [emim cl] for which the only available data are (25°c ± 2 to 45°c ± 2) conductivity [16]. ils are highly promising in pure form or either in mixtures with proton conductors as electrolytes in electrochemical devices [17]. conductivity is used to describe the extent to which electrons move in a substance. the type of ions in the solution and the ion concentration of the solution all have an effect on the conductivity of the solution [18]. there has been a surge of significance on this topic and a considerable number of researches on the physiochemical properties of il like conductivity. although, physiochemical properties are the apparent sign of infinitesimal stage of interactive features, therefore detail analysis of properties can present the extensive explanation about interactions with the variation in temperature in binary system [19]. similarly, in ils the conductivity is critical and if the il has high conductivity and a high current density, the current efficiency will be high [5,20]. studying the conductivity of ils and their variations in a variety of different situations are critical to introducing ils into the electrolyte to promote electrolysis [20]. in this paper, the conductivity of different volume percentages of ils in solution was determined, and the law of variation was studied. materials and methods reagent 1-(3-aminopropyl) imidazole (≥97%), purchased from aldrich chemicals; methyl isothiocyanate (97%); 1-ethylimidazole chloride (≥97%) and 1-butylimidazole (97%), purchased from suiyuan chemical technology (shanghai) ltd; 1-bromooctane (99%), 1-bromohexane (99%), 1-bromobutane (99%), from shanghai civic chemical technology co., ltd; acetonitrile (hplc grade) from labing chemical; carbon tetrachloride, purchased from aladdin chemical company. multi-parameter conductivity instrument model multi 3320, purchased from xylem analytics germany. copper sulphate, sodium chloride and sulfuric acid was purchased from aladdin chemicals (shanghai). 1-ethyl-3-methylimidazolium chloride (emimcl) ionic liquid purchased from aldrich chemicals and 1butyl-3-methyl imidazolium hexafluorophosphate (bmimpf6) was synthesized. pak. j. anal. environ. chem. vol. 21, no. 2 (2020) 370 structure of ionic liquids 1-butyl-3-methylimidazolium hexafluorophosphate, [bmimpf6] was synthesized using previously published literature [21]. the chemical structure of butyl imidazolium ionic liquid is shown in scheme 1; [bmim] + [br] + kpf6   h24,c40 o [bmim] + [pf6] + kbr scheme 1. 1-butyl-3-methylimidazolium hexafluorophosphate ionic liquid the chemical structure of ethyl chloride ionic liquid is shown in scheme 2; [emim] + + [cl]   h24,c40 o [emimcl] scheme 2. 1-etyl-3-methylimidazolium chloride ionic liquid physical measurements in the range of 25°c~75°c, used the german made multi 3320 conductivity meter to measure the optimal conditions of the electrolyte ratio by respectively adding 0%, 1%, 2%, 3%, 4%, and 5% of emimcl il and the conductivity of the bmimpf6 il with mixed solution fig. 1. in the assessment, cuso4 concentration: 30 g/l; nacl concentration: 40 g/l; h2so4 concentration: 150 g/l of 12 parts, 10 ml each was prepared. 0%, 1%, 2%, 3%, 4%, and 5% of emimcl and bmimpf6 were added to each of 12 solutions. figure 1. multi instrument conductivity meter the specific measurement process is as follows: (1) turned on the conductivity operation switch and preheated it for 30 min to slowly enter the steady-state and selected the best standard solution at a specific electrode constant. (2) connected the conductivity electrode and set the instrument temperature at 25°c. rinsed the conductivity electrode with distilled water, immerse the conductivity electrode in a standard solution, and read the conductivity ka. calculated the electrode constant j=k/ka according to the standard solution conductivity. (3) performed electrode constant setting according to the electrode used and then washed the conductivity electrode with distilled water and then added the emimcl and bmimpf6 ions with volume percentages of 0%, 1%, 2%, 3%, 4%, and 5%, respectively under the optimum conditions of the electrolyte solution. the liquid (mixed solution) rinsed the conductivity electrode and then inserted it into the sample to be tested. pressed the “tds” button and waited for stable the readings on conductivity instrument screen. after the reading is stable then read the test display results. pak. j. anal. environ. chem. vol. 21, no. 2 (2020)371 (4) after the examination, the conductivity electrode is sufficiently cleaned and stored in distilled water. results and discussion effect of volume percentage of emimcl and bmimpf6 on conductivity the relationship between the volume percentage of emimcl and bmimpf6 and the conductivity of the sample is shown in fig. 2. the variation of conductivity of pure ils at different temperatures is shown in table 1 and also compared the data with previously published work. figure 2. relationship between volume fraction of emimcl and bmimpf6 conductivity of the solution it can be seen from fig. 2 that when the temperature was fixed, the conductivity decreases with the increase of the volume percentage of the il. when the volume percentage was more than 1%, the change was stable, which may be due to the conductivity of the solution. the conductivity of mixed solution attained and at the same time obtained high viscosity of il. when the il is added into the solution, the overall viscosity and density of the solution was swiftly increased and when increased the volume percentage of il more than 1% then the conductivity stands weakened. at the same time its effect on the conductivity of the solution that was not obvious. the conductivity is related to the mobility of the load carriers, which can be explained by the higher viscosity caused by stronger van der waals interactions between the longer alkyl chains and the larger cation size. in addition, the longer alkyl chain results in a greater volume fraction of the neutral hydrocarbon component of the organic cation. it can be concluded from table 1 that the conductivity of the two pure ils differs significantly from the conductivity of the electrolyte used in the test. the conductivity of the two pure ils was relatively low and the viscosity of emimcl and bmimpf6 was high. in the large group the anion was inactive and the transition speed was slow, which makes the conductivity of the whole mixed sample system significantly reduced. table1. conductivity of pure emimcl and bmimpf6 at different temperatures. ionic liquids temperature this work literature reference emimcl 2 5°c 3 5°c 4 5°c 5 5°c 6 5°c 7 5°c 1.3 1.9 2.4 3.2 4.1 4.7 0.9 1.5 2.46 3.59 4.54 5.04 [16, 22] [16] [16] na na na bmimpf6 2 5°c 3 5°c 4 5°c 5 5°c 6 5°c 7 5°c 0.7 0.8 0.9 1.3 1.4 1.5 0.304 0.518 0.820 1.222 1.735 1.102 [23-26] [23-26] [23-25] [23, 25] [24, 25] [25] na=not available pak. j. anal. environ. chem. vol. 21, no. 2 (2020) 372 from previous studies (literature) it has been shown that the conductivity κ of the mixed solution confirms to the following formula: jjj j cu zf (1) κ is the conductivity s/m; f is the faraday constant usually 96.5 kc/mol; jz is the charge number; cj is the molar concentration, mol/m 3 ; uj is the twist, m 2 /(s•v). from equation (1) the conductivity of the mixed sample is the sum of the conductance of the individual components in the sample and is related to the concentration change and the twist. the twist was determined by the solution diffusion coefficient, such as the einstein relation (2) f rt d i i i z u  (2) where: di is the diffusion coefficient which is affected by the ui degree. moreover, according to previous studies (literature) there was a corresponding linear relationship between the ui degree ui and the viscosity η and the ui degree ui decreases as the viscosity η increases and the specific relationship was as shown in the formula (3). r.6 ez u i i   (3) where e is the electron charge 1.6022×10 -19 c; ui is the twist, m 2 /(s•v); η represents the viscosity of the medium; r represents the radius of the charged body m; π is the pi. it is known from the formula (3) that ui degree ui is negatively correlated with the charged body radius r and the medium viscosity η. the conductivity of the mixed solution is the result of the interaction of the individual ions contained in the solution and the magnitude of the twist can affect the conductivity. the twist was positively correlated with di. when di was large, the resistance of the ion motion was small and the twist becomes large which resulting in high conductivity. since the cation group radius of emimcl and bmimpf6 is large, the diffusion coefficient of the solution was small resulting in a small degree of twist so that the conductivity of the entire solution was lowered. among them bmimpf6 has a larger ionic radius so its conductivity was smaller. effect of temperature on conductivity the conductivity of the solution is easily affected by temperature. therefore, in the test the law of conductivity changes with temperature under different ils and different addition amounts was determined as shown in fig. 3. figure 3. the influence of temperature on conductivity of electrolyte conductivity of electrolyte as seen in fig. 3, the conductivity of the test sample was positively correlated with pak. j. anal. environ. chem. vol. 21, no. 2 (2020)373 temperature. this is because of ions in the solution increase with the temperature and the diffusion coefficient becomes more substantial. according to the equations (1) and (2) the viscosity of the solution was negatively correlated and the viscosity decreases causing the particles to accelerate migration and become active that was responsible for increasing the conductivity. there are many factors that affect particle motion which can be divided into: (1) ion radius: the motion resistance in the solution was positively correlated with the ionic radius. the larger the radius becomes greater resistance. the positive ion radii of both ils was large so the resistance of the solution was also increased. (2) ion valence state: the valence state was high; the electric field force was obvious and the movement speed was large. (3) the concentration of a mixed solution: the concentration increases the spacing between the ions was reduced and the mutual influence between the ions becomes strong so that the movement was difficult. (4) temperature: group was active by increasing the temperature increases and the reaction transition speeds up which resulting an increase in haste. (5) solution viscosity: positively correlated with motion resistance. when the viscosity enhanced then resistance increased and the swiftness slowed down. for further study the data of the conductivity of the two different ils with temperature in different additions the polynomial fitting of the results and explore the correlation can get fig. 4. figure 4. regression relationship between temperature and conductivity of electrolyte the data is shown in table 2. according to the analysis of data the experimental data was in accordance with the kohlrausch relationship: table 2. correlation parameters of solution conductivity and temperature regression equation. type volume ratio% equation type intercept a b1 b2 0 96.10357 1.00061 0.00313 1 54.46786 1.01839 -0.00734 2 40.52857 0.60871 0.00179 3 39.44857 0.57066 0.0021 4 37.30714 0.78907 -0.00196 emimcl 5 30.56071 0.43104 0.0052 0 99.27143 0.60814 -0.0035 1 45.23214 0.47132 -0.00209 2 29.03214 0.78961 -0.00566 3 27.72143 0.62407 -0.00261 4 25.53214 0.68261 -0.00316 bmimpf6 5 y=a+b1*x +b2*x 2 22.35357 0.28696 -0.00234 pak. j. anal. environ. chem. vol. 21, no. 2 (2020) 374     2000 t-tt-t1kk   (4) where: k0： the conductivity when t=t0, s/m; α, β are temperature coefficients; t is temperature °c; t0=25°c. the calculated and α, β are listed in table 3. table 3. coefficient parameters of solution conductivity and temperature in kohlrausch. temperature coefficient temperature coefficient correlation coefficienttypes volume percenta ge (s•m-1) (α) (104β) r 0% 96.10357 0.010411788 0.32569 0.9917 1% 54.46786 0.018697081 -1.34758 0.9908 2% 40.52857 0.015019281 0.441664 0.9967 3% 39.44857 0.014465924 0.532339 0.9963 4% 37.30714 0.017150643 -0.52537 0.9999 emim cl 5% 30.56071 0.014104384 1.701531 0.9998 0% 99.27143 0.006126032 -0.35257 0.9981 1% 45.23214 0.010420024 -0.46206 0.9931 2% 29.03214 0.027197788 -1.94956 0.9995 3% 27.72143 0.022512186 -0.94151 0.9996 4% 25.53214 0.026735323 -1.23766 0.9988 bmim pf6 5% 22.35357 0.012837323 -1.04681 0.9946 the temperature coefficients α and β was plotted on the ordinate and the volume percentages of emimcl and bmimpf6 was plotted on the abscissa as shown in fig. 5 and 6. as can be seen from the fig. 5 the temperature coefficient α was the largest when the volume fraction of emimcl in the solution was 1%. it indicates that the influence of temperature on the conductivity of the sample system was gradually weakened when the volume percentage of emimcl exceeds 1%. figure 5. relationship between emimcl、 bmimpf6 volume percentage and temperature coefficient (α) with the increase of bmimpf6 volume percentage α reached at maximum when the volume fraction of bmimpf6 was 2% in solution and the temperature coefficient α decreased with the change of bmimpf6 volume percentage. it shows that the increase of the volume percentage of bmimpf6 to 2% that effect the temperature which getting lower and the temperature coefficient α decreases with the volume percentage of bmimpf6. it can be concluded from fig. 6 that when 1% emimcl was added to the solution, the temperature coefficient β reaches the minimum peak and then the volume fraction of emimcl changes. the temperature coefficient β does not fall below to the minimum peak because the effect of the coefficient β was relatively small. similarly, when the volume fraction of bmimpf6 is 2% the temperature coefficient β pak. j. anal. environ. chem. vol. 21, no. 2 (2020)375 was the smallest and then with the change of the volume fraction of bmimpf6 β increases gradually which shows the influence of β on relatively small content. figure 6. relationship between emimcl、 bmimpf6 volume percentage and temperature coefficient (β) conclusions through the experimental study on the conductivity of the best ils and the optimal electrolyte system, it is revealed that the volume percentage of emimcl and bmimpf6 is inversely proportional to the conductivity of the test sample, and the conductivity is significantly reduced when 1% il is added. conductivity is positively correlated with temperature and is in accordance with kohlrausch. when the volume fraction of emimcl is 1% or when the content of bmimpf6 is 2%, the temperature coefficient α is the largest, and β is the smallest. therefore, when more than 1% emimcl or more than 2% bmimpf6 is added, the effect of the change in il content on the conductivity is weakened by temperature control. conflict of interest the authors declare that there is no conflict of interest regarding the publication of this article acknowledgment this research was completed with the support of the national natural science foundation of china (grant no.51574238) and the university student innovation training program of china (grant no.20181030). references 1. h. zhang, m. zhu, w. zhao, s. li and g. feng, green energy environ., 3 (2018) 120. https://doi.org/10.1016/j.gee.2017.11.00 2. 2. r. l. vekariya, j. mol. liq., 227 (2017) 44. https://doi.org/10.1016/j.molliq.2016.11. 123. 3. s. sardar, c. devi, a. mumtaz, z. rashid and j. leveque, j. mol. liq., 271 (2018) 621. https://doi.org/10.1016/j.molliq.2018.09. 024. 4. w. j. li weixiong, chem. prog., 21 (2002) 43. 5. t. zhong, z. le, z. xie, x. cao and x. lu, chinese j. org. chem., 30 (2010) 981 6. w. w. li hongyan and l. lingfei, sci. technol. innov., 14 (2017) 25. 7. y. jiazhen, j. yi and c. yinghua, j. chem. high. sch. chem., 25 (2004) 1733. pak. j. anal. environ. chem. vol. 21, no. 2 (2020) 376 8. c. q. wu jingxuan and s. xinghai, j. phys. chem., 29 (2013) 1705. 9. j. fraga-dubreuil and j. p. bazureau, tetrahedron lett., 42 (2001) 6097. https://doi.org/10.1016/s00404039(01)01190-x 10. m. shuai, z. yiwei and z. xiaoxi, j. nanjing univ. technol., 40 (2018). 11. a. s. amarasekara, chem. rev., 116 (2016) 6133. https://doi.org/10.1021/acs.chemrev.5b0 0763. 12. m. marium, a. auni, m. m. rahman, m. y. a. mollah, a. bin and h. susan, j. mol. liq., 225 (2017) 621. https://doi.org/10.1016/j.molliq.2016.11. 074. 13. a. p. c. ribeiro, s. i. c. vieira, j. m. frança, c. s. queirós, e. langa, m. j. v. lourenço, s. m. s. murshed and c. a. n. de castro, ionic liquids: theory, properties, new approcahes, intech publisher, (2011). https://doi.org/10.5772/13920 14. m. v. fedorov and a. a. kornyshev, chem. rev., 114 (2014) 2978. 15. k. ahmed, a. auni, g. ara, m. m. rahman, m. y. a. mollah, and m. a. b susan, j. bangladesh chem. soc., 25 (2012) 146. 16. m. g. freire, c. m. s. s. neves, i. m. marrucho, j. a. p. coutinho and a. fernandes, j. phys. chem. a, 114 (2010) 3744. 17. j. he, j. yang, m. t. sarwar, c. duan, and y. zhao, j. clean. prod., 256 (2020) 120368. https://doi.org/10.1016/j.jclepro.2020.12 0368. 18. y. geng, c. siliu, w. tengfang, y. dahong, p. changjun, l. honglai, and h. ying, j. mol. liq., 143 (2008) 100. https://doi.org/10.1016/j.molliq.2008.06. 014. 19. j. leys, w. michael, p. m. chirukandath, r. ravindran,t. jan, g. christ, n. peter, t. ben, b. koen and l. stéphane, the jol. chem. phy., 128 (2008) 064509. https://doi.org/10.1063/1.2827462. 20. a. papancea and a. p. s. paţachia, bull. transilvania uni. braşov, 8 (2015) 57. 21. j. picálek and k. jiří, j. mol. liq., 134 (2007) 29. https://doi.org/10.1016/j.molliq.2006.12. 015. 22. k. s. singh and w. s. anthony, j. mol. liq., 297 (2020) 112038. https://doi.org/10.1016/j.molliq.2019.11 2038. 23. j. vila, p. ginés, e. rilo, o. cabeza and l. m. varela, fluid phase equilib., 247 (2006) 32. https://doi.org/10.1016/j.fluid.2006.05.0 28. 24. j. a. widegren, e. m. saurer, k. n. marsh and j. w. magee, the j. chem. thermodyn., 37 (2005) 569. https://doi.org/10.1016/j.jct.2005.04.009. 25. xu, haitao, z. dacheng, x. peng, l. fengqi and g. gao, j. chem. eng. data, 50 (2005) 133. https://doi.org/10.1021/je049787p. 26. zech, oliver, a. stoppa, r. buchner and w. kunz, j. chem. eng. data, 55 (2010) 1774. https://doi.org/10.1021/je900793r. characterization of metal exchanged zeolite-a using quantasorb and mercury porosimeter issn-1996-918x pak. j. anal. environ. chem. vol. 11, no. 1 (2010) 08–15 photoelectrochemical sensors for the rapid detection of dna damage induced by some nanoparticles m. jamaluddin ahmed*, bin-tian zhang and liang-hong guo state key laboratory of environmental chemistry and ecotoxicology, research center for eco-environmental sciences, chinese academy of sciences, p.o. box 2871beijing 100085, china ------------------------------------------------------------------------------------------------------------------------------------------ abstract photoelectrochemcal sensors were developed for the rapid detection of oxidative dna damage induced by titanium dioxide and polystyrene nanoparticles. each sensor is a multilayer film prepared on a tin oxide nanoparticle electrode using layerby-layer self assembly and is composed of separate layer of a photoelectrochemical indicator, dna. the organic compound and heavy metals represent genotoxic chemicals leading two major damaging mechanisms, dna adduct formation and dna oxidation. the dna damage is detected by monitoring the change of photocurrent of the indicator. in one sensor configuration, a dna intercalator, ru(bpy)2 (dppz) 2+ [bpy=2, 2′ -bipyridine, dppz=dipyrido( 3, 2-a: 2′ 3′-c) phenazine], was employed as the photoelectrochemical indicator. the damaged dna on the sensor bound lesser ru(bpy)2 (dppz) 2+ than the intact dna, resulting in a drop in photocurrent. in another configuration, ruthenium tris(bipyridine) was used as the indicator and was immobilized on the electrode underneath the dna layer. after oxidative damage, the dna bases became more accessible to photoelectrochemical oxidation than the intact dna, producing a rise in photocurrent. both sensors displayed substantial photocurrent change after incubation in titanium dioxide / polystyrene solution in a time – dependent manner. according to the data, damage of the dna film was completed in 1h in titanium dioxide / polystyrene solution. in addition, the titanium dioxide induced much more sever damage than polysterene. the results were verified independently by gel electrophoresis and uv-vis absorbance experiments. the photoelectrochemical reaction can be employed as a new and inexpensive screening tool for the rapid assessment of the genotoxicity of existing and new chemicals. keywords: photoelectrochemical sensors; dna damage; nanoparticles. ------------------------------------------------------------------------------------------------------------------------------------------ introduction nanoparticles are small enough to penetrate cell membranes and defenses, yet they are large enough to cause trouble by interfering with normal cell processes as replaced by the researchers at the university of massachusetts. they examined the genotoxicity of silica, titanium dioxide, polystyrine and c60 fullerene nanoparticle suspensions using the alkaline single-cell gel electrophoresis assay (comet assay) to quantify breaks in single and double stranded dna. such nanoparticles are currently in use in electronics, cosmetics, and chemical manufacturing, among others industries. because of their extremely small size, they can be difficult to isolate from the large environment, as they are too small to be removed by conventional filtering techniques. nanoparticles, engineered materials are about a billionth of a meter in size, could damage dna and lead to cancer, according to research presented at the 2007 annual meeting of the american association for cancer research [1]. moreover, there roughly 100,000 chemicals available on the global market, 10,000 of them are hazardous, including about 200-300 confirmed carcinogenic agents [2]. in addition, *corresponding author: present address: department of chemistry, university of chittagong, chittagong-4331, bangladesh, email: pmjahmed55@gmail.com pak. j. anal. environ. chem. vol. 11, no. 1 (2010) 9 thousands of new chemicals are produced and utilized each year. unfortunately, the vast majority of these chemicals do not have sufficient safety and health data, thus posing a great danger to human health and the ecosystem [2, 3]. many chemicals have been found to possess carcinogenic toxicity. some of these carcinogenic materials assert their toxic effect by causing damage in dna, leading to gene mutation. in general, dna damage is produced by one of the two major chemical routes, dna oxidation by reactive oxygen species (ros) and dna adduct formation with exogenous chemicals and their in vivo metabolites [4,5]. according to the international agency for research on cancer, cr(vi), ni(ii), ti(iv), be, cd, and as(iii) compounds have been confirmed to be human carcinogens [6]. a number of studies have shown that metals induce their toxic effects primarily through their ability to produce ros. therefore, there is an urgent demand for rapid detection methods to screen the large number of existing and new chemicals for their genotoxicity. there are currently a number of cell-based assays as well as biochemical and chemical analytical techniques for the detection of dna damage and the assessment of genetic toxicity. dna damage products have been identified and quantified by a wide range of analytical techniques, such as single-cell gel electrophoresis, 32ppostlabeling, immunoassay, gas chromatography/mass spectrometry, high performance liquid chromatography, and electrochemical and electrochemiluminescence sensors [7-9]. as a detection method, photoelectrochemistry is well suited for the rapid and high-throughput screening of genotoxic chemicals [10]. the photoelectrochemistry-based analytical method is potentially very sensitive, as the excitation source(light) is different from the detection signal(current). in addition, the instrument should be simpler and of lower cost than all the optical detection methods due to the use of electronic detection, particularly in an array format. it compares favorably with the optical detection methods such as fluorescence, chemiluminescence, and electrochemiluminescence, which have to use complex and expensive optical imaging devices and sophisticated imagerecognition software. over the years, photoelectrochemistry-based analytical methods have been employed in the quantification of dna[11] and dna hybridization[12]. recently, we reported a photoelectrochemical sensor for the detection of dna damage by fe2+ and styrene oxide[13]. the sensor was assembled by depositing a layer of calf-thymus dna on a tin oxide nanoparticle electrode. a dna intercalator, ru(bpy)2 (dppz) 2+ [bpy=2, 2′ -bipyridine, dppz=dipyrido( 3, 2-a: 2′ 3′-c) phenazine], was employed as the photoelectrochemical signal reporter. when the sensor was exposed to a solution containing 10µm tio2 or 10 µm polystyrene nanoparticle , the dna on the sensor surface was damaged by the nanoparticles, resulting in less binding with ru(bpy)2 (dppz) 2+ and consequently lower signal than the native dna (scheme 1). scheme 1. illustration of experimental procedure: (1) preparation of dna film electrode, (2) dna damage reaction, (3) binding of signal molecule, and (4) photocurrent measurement tin oxide nanoparticle electrode was prepared by the alternate layer-by –layer electrostatic self assembly approach with poly(diallyldimethyl ammonium chloride)(pdda) and ds-dna solution was immobilized on it, which was exposed by titanium dioxide/ polystyrene (as damaging agent) for 1h at 37oc and rotation 200 rpm then dna was damaged. a dna intercalator ru(bpy)2 (dppz) 2+ , was employed as the photoelectrochemical signal reporter. the photocurrent was produced by the conversion of ru2+* to ru3+. thermodynamically, ru3+ can oxidize guanine and adenine bases in dna in the presence of oxalate buffer and get reduced back to ru2+ resulting in the recycling of metal complex an enhance the photocurrent. the photocurrent was measured on a chia electrochemical analyzer using pt flag counter electrode, and ag/agcl reference electrode at 473 nm blue laser light. tio2 pak. j. anal. environ. chem. vol. 11, no. 1 (2010) 10 experimental reagents and solutions poly-(diallyldimethyl ammonium chloride) (pdda) and single and double-stranded calf thymus dna (ss-dna and ds-dna, 13k base pairs), were purchased from sigma-aldrich(st. louis, mo,usa). titanium dioxide nanoparticles (99.9% purity referred to as tio2 40nm) and polystyrene oxide nanoparticles (99.5% purity) were also purchased from sigma-aldrich (st. louis, mo, usa). fifteen percent tin(iv) oxide, as a colloidal dispersion of 15 nm particles, was obtained from alfa aesar (ward hill, ma), and so was hydrogen peroxide. all other chemicals and solvents were of analytical grade. ru-(bpy)2 (dppz) (bf4)2 was synthesized according to the published procedure [14, 15]. all solutions were prepared in high – purity water from a millipore milli-q (biocel water purification system). tin-doped indium oxide conductive glass was supplied by weiguang corp.(shenzhen, people’s republic of china). titanium dioxide nanoparticle standard solution (1000 mg l -1 ) a 100-ml stock solution (1mg ml-1) was prepared by dissolving titanium dioxide (tio2 40nm, purity 99.9%) in 500 µl of 32% h2o2 and 300 µl of 1% w/v na2co3 solutions following the published procedure [16, 16a]. the volume was made up to the mark with milli q water. the resulting solution was ultrasonicated for half an hour until transparent clear aqueous solution was obtained. electron microscopy studies revealed that the actual size of titanium nanoparticle was 2-5 nm. polystyrene nanoparticle standard solution (1000 mg l -1 ) a 100-ml stock solution (1mg ml-1) was prepared by dissolving polystyrene (polystyrene 100nm, purity 99%). a 100mg portion was placed in a 15 ml centrifuge tube fitted with a glass stopper, and 10-ml of diethylbenzene was added. the flask was stoppered and placed in an eberbanch horizontal shaker. the mixture agitated until all polymer had dissolved (within 1hr) following the published procedure [17], and the solvent evaporated thoroughly under vacuum. the residue was redissolved in 10 ml of 10% triton x-100 solution and diluted to 100-ml with highly purified water and the aqueous solution thus obtained was sonicated (1/2 hr) to produce a clear solution. electron microscopy studies revealed that the actual size of polystyrene nanoparticle was 5-7 nm. film assembly sno2 nanoparticle electrodes were prepared by following the previous method [18, 19] (scheme 1). the concentrations of pdda and dna solutions for film deposition were 2 and 0.5 mgml-1, respectively. the dna – modified electrode was denoted as sno2 / pdda / dna. the dna film on the electrode was damaged by exposing to tio2 / polystyrene solution at 37 0c with vortex (200rpm) for 1h for a time period as specified. then the electrode was taken out and rinsed with water. photoelectrochemical measurement the photocurrent was measured on a chi 630a electrochemical analyzer (austin, tx) using a pt flag counter electrode, ag / agcl (3m kcl) reference electrode, and a bias voltage of +0.1v. the area of the working electrode in contact with the electrolyte was 0.25 cm2. the light source of photocurrent measurement was a 473 nm blue laser with 1.5 mw/cm2 power and an illumination area of 0.18 cm2. the light source for action spectrum measurement was a 500w xenon lamp with a light intensity of 0.168 mw/cm2. for sno2 / pdda / tio2 / ds-dna sensor, after dna damage reaction and washing, the electrode was further reacted with 50 µm ru(bpy)2 (dppz) 2+ for 30 min for the intercalation to take place. after the reaction the unbound metal complex was washed off by water. photocurrent was then measured by placing the electrode in 20 mm oxalate buffer ph 5.8. gel electrophoresis the damaged ds-dna sample for gel electrophoresis was prepared by the incubation of 0.1 mg ml-1 ds-dna, 10 mm h2o2 5 mm na2co3 and 100 mg l-1 (2, 1 and 0.5 mgl-1 final concentrations) tio2 or polystyrene at 37 0c with pak. j. anal. environ. chem. vol. 11, no. 1 (2010) 11 vortex (200 rmp) for 1 and 1.5 h, respectively. the incubated dna sample was then electrophoresed on a 1.2% agarose gel in 0.5 ×tbe (45 mm tris, 45 mm boric acid, 1 mm edta, ph 8.0) and 0.5 µg ml-1 ethidium bromide for 30 min at 7.5 v / cm. uv / vis absorption measurment the absorbance intensity was measured on a du 800 double-beam uv – vis spectrophotometer using 250 nm. a solution containing 5 µg ml-1 intact or damaged ds-dna in 20 mm phosphate buffer ph 7.3 and various concentrations of ru(bpy)2 (dppz) 2+ in each well was shaken for 2 min before the measurement. the light intensity from a well containing the buffer alone was used for background(blank) subtraction. the effect of 10 mm h2o2 and 5 mm na2co3 solutions on dna damage was also studied. the damaged ds-dna sample for absorbance measurement was obtained by reacting with 2, 1 and 0.5 mg l-1 tio2 or polystyrene at at 37 0c with vortex (200 rmp) for 1 and 1.5 h, respectively. results and discussion detection methods here we present two photoelectrochemical methods to detect dna damage. one is based on photoelectrochemically catalyzed base oxidation, and the other employs a photoelectrochemical indicator (scheme 1). in the first method, a ruthenium tris(bipyridine)-labeled avidin film and a ds-dna film were assembled successively on a tin oxide nanoparticle film electrode. photocurrent enhancement requires regeneration of the ground state ru2+ complex by a reducing agent. by analogy with previously proposed mechanisms for electrocatalytic oxidation of dna, [20, 21] the photoelectrochemical oxidation reaction in the current system could be represented as in scheme 2. initial excitation of ru2+ after absorbing photon energy gives ru2+* (eq 1). ru2+* injects an electron into the semiconductor (sno2) and produces ru3+ (eq 2), which is then reduced back to ru2+ (eq 3) by guanine and adenine bases in dna, resulting in the recycling of the metal complex and enhanced photocurrent. because the oxidation potential of ru2+*/1+ (0.78 v) is much lower than that of guanine and adenine [10], the excited state does not oxidize the dna bases directly. scheme 2. proposed mechanisms of photoelectrochemical oxidation of dna by ru(bpy)3 2+ ru2+ + hυ → ru2+* ----------------------(1) ru2+* → ru3+ + e ---------------------------(2) ru3+ + g (or a) → ru2+ + gox( or aox) ----(3) in the second method, an unlabeled avidin film and a ds-dna film were assembled on the semiconductor electrode. a dna intercalator, ru(bpy)2(dppz) 2+, was employed as the photoelectronchemical signal reporter. the metal complex binds to the ds-dna film by inserting its dppz ligand into the space between adjacent base pairs with high affinity (binding constant k= 106107 m-1) and selectivity [22]. a steady-state photocurrent was measured in an oxalate buffer which serves as the electron donor to recycle the indicator. after damage, less ru(bpy)2(dppz) 2+ binds to the dna film due to the reduced binding sites, and results in a drop in photocurrent. detection of dna damaged by polystyrene nanoparticle polystyrene is used extensively in the chemical industry and is classified as a carcinogen. in vivo, polystyrene is metabolized by liver enzymes such as cytochrome p450 into styrene 7, 8-oxide, a much more potent carcinogen [23]. the polystyrene reacts in vitro with guanine and adenine nucleotides to form a variety of adducts, leading to dna damage. many other genotoxic organic chemicals follow a similar mechanistic pathway, i.e., from enzyme activation to adduct formation to dna damage [24]. therefore, a rapid method for the detection of dna adducts is valuable to screen organic chemicals for their potential genotoxicity. dna damage induced by polystyrene nanoparticle was first detected by the photoelectrochemically catalyzed base oxidation method. the avidin-ru / ds-dna multilayer film was assembled on the sno2 electrode as described above. the electrode was incubated in 2, 1, 0.5 mg l-1 polystyrene at 37 °c for the time required. after the reaction, photocurrent was measured in a pak. j. anal. environ. chem. vol. 11, no. 1 (2010) 12 phosphate buffer. (fig. 1) shows the photocurrent response for different period of incubation time. the current increased with incubation time and reached its maximum after 1.5 h, at which time the reaction was presumably completed. in the absence of polystyrene, the photocurrent was essentially unchanged, proving the increase was caused by polystyrene nanoparticle. one of the major dna adducts is with the 2-nh2 group of guanine, and involved in the hydrogen bonding interaction with cytosine. adduct formation disrupts the baseparing interaction and changes the local dna structure, thus exposing more bases for photoelectrochemical oxidation. when the damage was complete, the photocurrent was about 2 times higher than that of the control. in a previous report, dna films damaged by styrene oxide were detected by catalytic voltammetry [25]. the chemical reaction was found to be complete within 30 min, accompanied by a 60% increase in the oxidation current. our results indicate the photoelectrochemical method is much more sensitive than catalytic voltammetry. the absolute sensitivity of the photoelectrochemical method cannot be assessed at present due to the lack of information about the amount of damaged dna in the film, which will be estimated in future work by established methods. polystyrene -induced dna damage was also monitored using the ru(bpy)2(dppz) 2+ intercalator. the avidin/ds-dna film on sno2 was treated in polystyrene nanpaticle, reacted with the intercalator, and then measured in 30 mm oxalate buffer. figure 1 shows the photocurrent response as a function of the reaction time in polystyrene nanoparticle. because the number of intercalation sites in the damaged dna is less than that in the intact dna, the photocurrent is reduced. similar to the results obtained in the base oxidation measurement, the current decreased progressively with the reaction time and stabilized after 2h(when adjusted for the control). the control also showed gradual loss of signal over the time the dna film was immersed in the phosphate buffer, probably due to slight desorption of some dna molecules in the film. the de-sorption was also observed in (fig. 1). the small change in the photoelectrochemical response of the indicator after polystyrene nanoparticle reaction is consistent with the structural information. sno2 / pdda / dna figure 1. anodic photocurrent response of ru(bpy)2(dppz) 2+ bound to sno2 / pdda /dsdna electrode after the dna film was exposed to a: 2 mg ml-1 psnp, b: 1 mg ml-1 psnp, c: 0.5 mg ml-1 psnp and d: phosphate buffer. detection of dna damaged by the titanium dioxide nanoparticle among the environmentally polluted metal compounds, cr(vi), ni(ii), cd, ti(iv) and as(iii) have been confirmed to be carcinogenic to human beings. cobalt(ii) and iron(iii) nitrilotriacetate are suspected human carcinogens. these compounds assert their carcinogenic effect either by inducing dna damage or by inhibiting dna repair processes [26]. one of the frequently investigated routes of dna damage is through metal catalyzed generation of reactive oxygen species such as hydroxyl free radical in the presence of h2o2, the so-called fenton reaction. in vitro the fenton reaction causes dna cleavage at almost every nucleotide site, leading to base loss, chain breakage, and base oxidation [27]. many of the base oxidation products are also oxidizable, the most cited of which is 8-oxo-7,8-dihydro-2¢deoxyguanosine (8-oxo-dg). the tio2 nanoparticles were studied in our work as a model for metal-induced oxidative dna damage. the damage was first investigated by the base oxidation detection method described above. in the experiment, the sno2 / pdda / dna sensor was assembled as usual and then exposed to 1, 0.5 and 0.1 mg l-1 ti4+ for the required time. finally, the dna film was allowed to bind to ru(bpy)2(dppz) 2+. the photocurrent was then measured in a phosphate buffer. figure 2 shows the photocurrent change as a function of the reaction time, from which it is obvious that the a d pak. j. anal. environ. chem. vol. 11, no. 1 (2010) 13 damage process proceeded at a much faster rate than polystyrene nanparticle adduct formation and was completed in 1h. as can be seen in figure 2, the measured signal was reduced by one – third as compared with the blank control (buffer only) and also reduced than h2o2 control (h2o2 only), na2co3 control (na2co3 only) and mixture of h2o2 + na2co3 control (h2o2 + na2co3 only).as can be seen in figure 2, there is no any effect of h2o2, na2co3 and mixture of h2o2 and na2co3 on the dna damage response. incubation in either h2o2 or na2co3 or mixture of h2o2 + na2co3 alone did not have any appreciable effect on the response. after 1 h in the tio2 reagents, the current decreased with increase of ti4+ concentration. it was observed that dna was totally damaged with high concentration (1mgl-1) of tio2 nanoparticle. dna damaging tendency was decreased with decreasing concentration of tio2 which is shown in fig. 2. the maximum photocurrent was observed for phosphate buffer which was in good agreement with theoretical concept. sno2 / pdda / dna figure 2. anodic photocurrent response of ru(bpy)2(dppz) 2+ bound to sno2 / pdda / dsdna electrode after the dna film was exposed to a: 1 mg ml-1 of tio2, b: 0.5 mg ml -1 of tio2, c: 0.1 mg ml-1 of tio2, d: 100μm na2co3, e: 50μm na2co3+2.5m h2o2 f: 5 m h2o2 and g: 20 mm sodium phosphate buffer (ph 7.3). the final signal is more than 3 times higher than that of the reaction with polystyrene nanoparticle, see (fig. 1 & 2) suggesting that the metal induced dna damage is much more severe than that induced by the organic compound. detection of the tio2-damaged dna film with the ruthenium intercalator produced results consistent with those of the base oxidation method. the photocurrent dropped immediately after the reaction and became steady after 1 h, at which time the response was only about 15% of the original signal. the concentration of tio2 used in the work is most likely higher than the concentration found in vivo. to validate our findings in the in vivo situation, a concentration range covering the nanogram regime will be investigated. verification of the results by gel electrophoresis and uv-visible absorbance experiments the results were verified independently by gel electrophoresis and uv-visible absorbance experiments. agarose gel electrophoresis of the dna incubated with polystyrene and titanium dioxide nanoparticles. it was clearly found that dna was totally damaged with increase of polystyrene nanoparticle concentration (fig. 3). it was also found that dna was totally damaged by higher concentration of titanium dioxide nanoparticle concentration (fig. 4). the maximum brightness was observed for control buffer. the damaging tendency is gradually decrease with decreasing the concentration of polystyrene or tio2 nanoparticles, respectively (fig. 3 & 4). it can be seen from (fig. 4) that there is no appreciable effect of h2o2, na2co3 or mixture of h2o2 + na2co3 on dna damage. so the results obtained by our photoelectrochemical method were in good agreement with those obtained by agarose gel electrophoresis. the results were also verified by uvvisible spectrophotometry. it was found that dna was damaged by polystyrene except water which gave dna spectra at 255nm. the results are shown in (fig. 5). it can be seen from (fig. 6), that dna was totally damaged by different concentration of tio2 nanoparticle, except only na2co3 and phosphate buffer which gave dna peaks at 255 nm. since h2o2 absorbs uv light and gives high absorbance so all the solutions which contains h2o2 gave high uv absorption spectrums. the results are shown in (fig. 6). so the results obtained by our photoelectrochemical method were in good agreement with those obtained by uv – visible absorbance measurements. it can be found from both the experiments that dna was more severely damaged by tio2 nanoparticle than polysterene. g a pak. j. anal. environ. chem. vol. 11, no. 1 (2010) 14 figure 3. agarose gel electrophoresis of the dna incubated with psnp. a: 2mg ml-1psnp, b：1mg ml-1psnp, c：0.5mg ml-1psnp, d: phosphate buffer and e：marker figure 4. agarose gel electrophoresis of the dna incubated with tio2 a: 1 mg ml-1 of tio2 , b: 0.5 mg ml -1 of tio2, c: 0.1 mg ml-1 of tio2 , d: 100μm na2co3, e: 5 m h2o2 , f: 50μm na2co3+2.5m h2o2, g: 20 mm sodium phosphate buffer (ph 7.3) h: marker figure 5. uv-vis absorption spectrum of the dna incubated with psnp solution. a: 1mg ml-1 psnp, b: 0.5mg ml-1 psnp and c: blank (water) figure 6. uv-vis absorption spectrum of the dna incubated with tio2 solution. a: 1 mg ml-1 of tio2, b: 0.5 mg ml -1 of tio2, c: 0.1 mg ml-1 of tio2, d: 100μm na2co3, e: 5 m h2o2 f: 50μm na2co3+2.5m h2o2, g: 20mm phosphate buffer (ph 7.3) and h: blank (water) conclusions this is a rapid, highly sensitive and inexpensive technique for the detection of dna damage and a powerful tool for the large-scale screening of chemical genotoxicity. this is the first time titanium dioxide was completely dissolved in water using nontoxic h2o2 and na2co3 without strong acid or carcinogenic organic solvents. a b c d e a b c d e f g h a b c e f g d h a c pak. j. anal. environ. chem. vol. 11, no. 1 (2010) 15 the titanium dioxide nanoparticle induced much more sever damage than polystyrene. the detection apparatus is inexpensive and is made of some common electronics and a low –power laser light as compared to other large instruments (e. g. spectrofluorometer, lc-ms etc). the developed dna sensor (induced by tianium dioxide nanoparticle or polystyrene nanoparticle) has the potential to become a powerful tool for the rapid, low cost and large – scale screening of chemical genotoxicity. acknowledgements authors are highly grateful to the authorities of the state key laboratory of environmental chemistry and ecotoxicology, research center for eco-environmental sciences, cas, beijing for providing us all laboratory facilities to complete this project. one of us (m. j. ahmed) is grateful to the authorities of third world academy of sciences and chinese academy of sciences for awarding me castwas visiting scholar fellowship to complete this project. he is also thankful to the university of chittagong, bangladesh for sanctioning sabbatical leave to complete this project. references 1. b. trouiller, r, relience, a westbrook, p, solaimani and r. h. schiesll, cancer research, 69 (2009) 8784. 2. k. christen, environ. sci. technol. 37 (2003) 241a. 3. p. d. thacker, us companies get nervous about eu’s reach, environ. sci. technol. 39 (2005) 171a. 4. k. shosuke, h. yusuke, m. mariko and o. shinji, free radical biol. med. 32 (2002) 822. 5. w. l. xue and d. warshawsky, a review, toxicol. appl. pharmacol. 206 (2005) 73. 6. monographs on the evaluation of carcinogenic risks to humans, international agency for research on cancer: lyon, france, 23 (1980) 1. 7. m. s. cooke, m. d.evans, m. dizdaroglu and j. lunec, faseb j. 17 (2003) 1195. 8. g. eisenbrand, b. pool-zobel, v. baker, m. balls b. j. blaauboer, a. boobis and j. kleiner, food chem. toxicol. 40 (2002) 193. 9. g. p. pfeifer, tecnologies for detection of dna damage and mutations, plenum press, new york, (1996). 10. s. g. weber, d. m. morgan and j. m. elbicki, clin. chem. 29 (1983) 1665. 11. z. gao and n. c. tansil, nucleic acids res. 33 (2005) e123. 12. i. willner, f. patolsky and j. wasserman, angew. chem. int. ed. 40 (2001)1861. 13. m. liang and l. h. guo, environmental science and technology, 41 (2007) 658. 14. k. keyer and j. a. imalay, proc. natl. acad. sci. usa, 93 (1996) 13635. 15. c. c. chen, x. z. li, w. h. ma and j. c. zhao, j. phys. chem. b, 106 (2002) 318. 16. an xu, y. chai, t. nohmi and t. k. hei, part. and fib. toxicol., 6 (2009) 1743. 16a. us pto, 2006, patent no. h0033084 17. e. d. garcia, y. choi, h. moon and b. jung, mater. res. soc., acs nano, 2 (2008) 283. 18. m. liang, s. liu, m. wei and l. h. guo, anal. chem., 78 (2006) 621. 19. m. liang, s. jia, s. zhu and l. h. guo, environ. sci. and technol., 42 (2008) 635. 20. h. h. thorp, tibtech, 16 (1998) 117. 21. h. h. thorp, top. curr. chem. 237 (2004) 159. 22. r. e. holmlin, e. d. a. stemp and j. k. barton, inorg. chem. 37 (1998) 29. 23. j. a. bond, crit. rev. toxicol. 19 (1989) 227 24. s. jia, m. liang and l. h. guo, j. phys. chem. b. 35 (2008) 123. 25. l. p. zhou and j. f. rusling, anal. chem. 73 (2001) 4780 26. e. t. snow, pharmacol. ther. 53 (1992) 31 27. d. dong, d, zheng, f. wang, x, yang, n. wang, y. li, l. h. guo and j. cheng, anal. chem. 76 (2004) 499 microsoft word 152-157-pjaec-08102018-115.galley proof revised.do cross mark issn-1996-918x pak. j. anal. environ. chem. vol. 21, no. 1 (2020) 152 – 157 http://doi.org/10.21743/pjaec/2020.06.18 the studies on water quality for cobalt and manganese content in drinking water of multan area, southern punjab, pakistan ansar mehmood 1 *, muhammad arif bhatti 1 , rashid mahmood 1 , zahid mahmood 1 and zafar iqbal 2 1 mineral processing research center, pcsir laboratories complex, ferozepur road, lahore-54600, pakistan. 2 department of chemistry, comsats, institute of information technology, abbottabad, pakistan. *corresponding author email: miasheikh@hotmail.com received 08 october 2019, revised 30 december 2019, accepted 26 february 2020 -------------------------------------------------------------------------------------------------------------------------------------------abstract the trace elements, cobalt (co) and manganese (mn) were determined in the drinking water of multan city and areas in its vicinity. for this purpose, ten water samples were collected from various points within a circle of one kilometer radius, each time around seven disposal units. water samples were stored in sealed glass flasks at room temperature. the quality of these water samples was compared with reference samples collected from various far off places which were not affected by any disposal unit. analysis for co and mn was carried out using flame-atomic absorption spectrophotometer. the highest concentration of co (0.31 ppm) was found in new multan disposal area, while highest concentration of mn (0.45 ppm) was noted in suraj miani disposal area. the contents of co and mn metals in most of the points are found to be greater than permissible limits of who guide lines for drinking water. keywords: water quality, drinking water, heavy metals, concentration, cobalt, manganese, multan area -------------------------------------------------------------------------------------------------------------------------------------------introduction contamination of surface and ground water has become one of the major environmental issues in big cities. clean drinking water is the basic obligation for all citizens [1]. there is a wide diversity of pollutants like heavy metals, textile dyes, pesticides, pathogens etc. affecting the water bodies [2]. heavy metals gain a particular distress due to their strong toxicity and bioaccumulating nature even at lower concentrations [3]. industrial processes like metal plating units, hydrometallurgical operations, mineral beneficiation and tanneries discharge waste containing multiple types of heavy metal ions [4]. majority of heavy metals are persistent and toxic especially when accumulated above the level of permissible limit [5, 6]. intake of heavy metals causes severe problems related to human health like gastro, dysentry, cholera, typhoid, hepatitus. according to the report published by unesco in 2003, almost 2.3 billion people all over the world are suffering from water borne diseases [7]. in developing countries more that 2.2 million people die each year by drinking contaminated water [8]. in pakistan, condition of water quality is not satisfactory in most of the areas of the country and water pollution is becoming a serious threat due to mismanagement of resources [9]. ground water contamination has been reported in several areas of pakistan [9, 10]. studies have shown that industrial effluents from major cities of pakistan pak. j. anal. environ. chem. vol. 21, no. 1 (2020) 153 have higher concentrations of heavy metals than neqs limits [11-14]. the present study has been carried out purely to asses the quality of drinking water in multan area of southern punjab, pakistan in order to meet the purpose of providing clean and standard quality water. this communication presents the results of contents of co and mn in drinking water samples collected from various points of the city for academic purpose. it is a fact that health of a community depends not only on the availability of health services but also on the better hygienic atmosphere. the provision of health services together with clean drinking water is the surest safeguard against communicable diseases. there is a dire need to provide full information to the population of areas under study using water especially in highly populated and polluted areas. in pakistan people carry many diseases solely because of water as majority of the population in our country is not provided with standard water. materials and methods sample collection drinking water samples were collected from various disposal unit areas and areas away from these units fig. 1. for this purpose, seven sampling points of multan city region were selected as new multan disposal area (tube well), vehari road disposal area (motor pump), kiri jamandan disposal area (motor pump), suraj miani (motor pump), chungi no. 9 (motor pump), old shuja abad road (tube well) & bzu campus (tube well). these samples were kept in sealed glass flasks at room temperature. preparation of standard solutions standard co stock solution (1000 ppm) was prepared by dissolving 4.9322 g of a.r. grade cobalt nitrate [co(no3)2.6h2o] of merck/ bdh in water with the help of 1% nitric acid. the volume in the flask was made up to 1000 ml similarly, standard mn stock solution (1000 ppm) was prepared by dissolving 3.6065 g of a.r. grade manganese chloride [mncl2.4h2o] of merck/ bdh per 1000 ml of water containing 1% hydrochloric acid. working standards were prepared by appropriate dilutions form the stock solutions. determination of trace elements the concentration of co and mn in water samples was determined by atomic absorption spectrophotometer (model: z-8000 hitachi, japan) under optimized conditions. the results obtained were reported in table-1. the concentration ranges of various samples were also found and presented in table-1. statistical analytical data of cobalt and manganese in drinking water samples around various disposal areas (n=10) was calculated by following relation and the data was given in table-2. s.d.= x-x/n-1 analytical results were compared with reference to origin of water (municipal hand pumps and motor pumps) and disposal unit areas. figure 1. location map of main water collection points of multan city area pak. j. anal. environ. chem. vol. 21, no. 1 (2020)154 table 1. concentrations (ppm+sd) and ranges (ppm) of cobalt & manganese in drinking water samples collected from various disposal areas of multan city. sampling area or station cobalt concentration mean (ppm+sd) concentration range of cobalt (ppm) manganese concentration mean (ppm+sd) concentration range of manganese (ppm) b.z.u. campus (tube well) 0.14 ± 0.013 0.01-0.26 0.03 ± 0.006 0.00-0.17 old shuja abad road (tube well) 0.22 ± 0.016 0.16-0.29 0.02 ± 0.003 0.00-0.17 new multan area (tube well) 0.15 ± 0.014 0.07-0.31 0.03 ± 0.006 0.00-0.12 chungi # 9 (motor pump) 0.12 ± 0.008 0.03-0.19 0.14 ± 0.012 0.08-0.26 kiri jamandan (motor pump) 0.07 ± 0.005 0.00-0.22 0.04 ± 0.011 0.00-0.24 vehari road (motor pump) 0.02 ± 0.01 0.00-0.12 0.08 ± 0.011 0.04-0.26 suraj miani (motor pump) 0.08 ± 0.01 0.00-0.13 0.14 ± 0.013 0.02-0.45 table 2. statistical analytical data of cobalt and manganese in drinking water samples around various disposal areas (n=10). sampling area or station cobalt concentration (ppm) manganese concentration (ppm) b.z.u. campus (tube well) 0.15 ± 0.09 0.04 ± 0.04 old shuja abad road (tube well) 0.22 ± 0.03 0.03 ± 0.05 new multan area (tube well) 0.16 ± 0.07 0.04 ± 0.04 chungi # 9 (motor pump) 0.12 ± 0.05 0.15 ± 0.06 kiri jamandan (motor pump) 0.08 ± 0.07 0.05 ± 0.07 vehari road (motor pump) 0.03 ± 0.04 0.09 ± 0.06 suraj miani (motor pump) 0.09 ± 0.04 0.14 ± 0.12 results and discussion the observed range of co concentration in all water samples collected from tube wells and motor pumps was 0.0-0.31 ppm as is evident from table 1. however, higher level of co was found in tube well water samples of new multan disposal area 0.07-0.31 ppm, whereas low concentration range 0.0-0.12 ppm of co was found in water samples of motor pumps obtained from vehari road, disposal area. among the water samples obtained from motor pumps, higher concentration 0.0-0.22 ppm of co was present in samples of kiri-jamandan disposal, while lower concentration 0.0-0.12 ppm was found in vehari road area. whereas, water samples of suraj miani and chungi no. 9 disposal units contained 0.01-0.13 ppm and 0.03-0.19 ppm of co respectively. in municipal water supply, highest concentration 0.07-0.31 ppm of co was found in new multan disposal area as mentioned earlier. the old shuja abad road disposal area samples contain co concentration in the range of 0.16-0.29 ppm of co level slightly lower than new multan disposal area. from these results of co analysis, it is obvious that its higher concentration is found in tube well water samples of three areas i.e. bahauddin zakaria university (bzu), new multan area and old shuja abad road. the level of co is slightly on the higher side than the standard value (co = 0.07 ppm) [15]. it is suggested that the concentration of co in water can be reduced to less than 0.07 ppm by using either ion exchange pak. j. anal. environ. chem. vol. 21, no. 1 (2020) 155 resins [16] or low cost adsorbents [17] in order to provide quality water to local population. the observed range of mn level in various tube well water samples was 0.0-0.45 ppm (table-1). higher concentration (0.02-0.45 ppm) was present in motor pump samples of suraj miani disposal area, while lower concentration (0.000.12 ppm) was found in motor pump water of new multan area. it was noted that new multan area contains lower concentration (0.00-0.12 ppm) than those of municipal water supply (0.00-0.17 ppm). water samples obtained from tube well as source contain mn concentration level of 0.000.17 ppm collected form bzu campus and old shuja abad road disposal area respectively, while new multan areas samples contain 0.00-0.12 ppm of manganese. water samples obtained from motor pumps, contain maximum mn level of 0.020.45 ppm in suraj miani disposal area samples whiles other disposal areas contain comparable concentration 0-0-0.26 ppm of mn. it is recommended that concentration of mn in water can be reduced to less than 0.05 ppm by increasing chlorine doses or increasing ph to make it suitable for drinking purposes [15]. samples of drinking water for comparison were collected from different sources (tube wells, motor pumps) away from these disposal areas. trace elements co and mn were estimated in these samples. the concentration range values of these trace metals are comparable with the concentration ranges of drinking water samples collected from different places around disposal units. the comparison of the concentration ranges of the trace metals (co and mn) with reference to source of water (tube well and motor pump) shows that the upper level of these metals is generally higher in water samples obtained from motor pumps around various disposal unit areas. among all disposal areas studied, the water samples obtained from old shuja abad road and vehari road areas contain relatively lower average concentrations of the both heavy metals (table-1). the british permitted concentration values for co and mn are 0.07 ppm and 0.05 ppm, respectively. a comparison of these levels with the observed concentrations of these metals in water samples reported reveals that co and mn concentration range in water samples from all disposal location are slightly higher than those reported by british water quality act 1989 [15]. it is suggested that population using low quality water must be provided with standard quality water after carrying out complete analysis regarding macro and micro elements. this will lead us towards healthy generations. co and mn occurs naturally in many soils that may erode into surface and ground water sources. however, human activities are also responsible for much of the co and mn contamination in water in some areas. higher levels in drinking waters are usually associated with industrial pollution. the concentration of co and mn in ground water samples of the study area and other studies along with permissible limits are given in table-3. the present study was compared with drinking water quality in the rural areas of the bhongiri region, india. chennaiah et al. [18] studied water quality of ghatkesar, pagidipalli, bibinagar, yamnampeta, annampatla, aushapur and guduru towns. a total of 42 drinking water samples were collected from these areas to assess the status of drinking water. the concentration of mn in the drinking water samples ranged from 0.0049 to 2.046 ppm, while the concentration of co in the drinking water samples ranged from 0.0003 to 0.0028 ppm. the concentration of mn exceeds the permissible limits of who 2004 [19] in the study area (0.100 ppm). co and mn are needed at low levels as catalysts for enzyme activities. drinking water containing high levels of these essential metals may be hazardous to our health. high level of mn in drinking water is associated with neurological damage. the accumulation of mn may cause hepatic encephalopathy [20]. high level of co in drinking water may cause lung cancer [21]. the removal of co and mn, the metallic toxic pollutants from drinking water is very important to safeguard the habitats of the particular area. different processes are being used throughout the world for the removal of heavy and toxic metals from polluted water to make it drinkable. these processes include solvent extraction, membrane separation, micro & ultrafiltration, forward and reverse osmosis, pak. j. anal. environ. chem. vol. 21, no. 1 (2020)156 coagulation, electrolysis, precipitation, phytoremediation, ion exchange and adsorption [22, 23]. the adsorption of co and mn on low cost media such as alumina or bentonite seems to be an economically viable solution to make the polluted water of the multan city areas as drinkable [24, 25]. table 3. the concentration ranges of trace elements (cobalt and manganese) in ground water samples of the study area along with permissible limits and its comparison with some other areas . concentration range of elements (ppm) multan city area, pakistan bhongiri region, india who (2004) british water quality act 1989 cobalt 0.000-0.31 0.000320.0028 not available 0.07 manganese 0.000-0.45 0.00492.046 0.100 0.05 conclusion it is concluded that the concentration ranges of cobalt and manganese are higher in water samples obtained from motor pumps as compared to the tube well samples. the concentration levels of cobalt and manganese in water samples obtained from various disposal areas are comparable with the water samples obtained from areas away from disposal units. the concentration of both metals is slightly higher than those of permitted values. the concentration of these metals can be reduced to permissible limits by adsorption on suitable media. references 1. s. t. trudgill, d. e. walling and b. w. webb, water quality process and policy (john wiley and sons, ltd, england) (2000) 3. isbn: 978-0-471-98547-1 2. m. faisal and s. husnain, afr. j. biotechnol., 3 (2004) 610. issn 1684–5315 © 2004 3. j. m. harsfall and a. i. spiff, electron. j. biotechnol., 8 (2005) 4. doi: 10.2225/vol8-issue2-fulltext-4 4. s. e. bailey, t. j. olin, r. m. bricka and d. d. adrian, water res., 33 (1999) 2469. doi: 10.12691/jaem-2-1-5 5. j. c. igwe, e. c. nwokennaya and a. a. abia, afr. j. biotechnol., 4 (2005) 1109. 6. w. viessman, j. mark and j. hammer, water supply and pollution, 7/e (suarabh printers pvt. ltd india) (2005) 475. isbn-10: 1292026073 7. unesco. 2003. water for people, water for life. the united nations world water development report, united nations educational, scientific and cultural organization and berghahn books. 8. who, unicef. 2000, global water supply and sanitation assessment report. isbn 92 4 156202 1 9. m. sajjad, s. rahim and s. s. tahir, pcrwr 2005 national water quality monitoring programme, water quality report 2005, chemical quality of ground water in rawalpindi-islamabad water engineering and development, 24 (1998) 271-274. 10. r. ullah, r. n. malik and a. qadir, afr. j. sci. tech., 3 (2009) 429. isbn978-0-12-818965-8 11. m. tariq, m. ali and z. shah, environ., 25 (2006) 64. doi:10 (11), 1823-1828, 2007 12. s. i. hussain, a. ghafoor, s. ahmad, g. murtaza and m. sabir, pak. j. agr. sci., 43 (2006) 97. 13. m. j. khan, a. u. bhatti, s. hussain and wasiullah, soil environ., 26 (2007) 139. doi: 10.1016/s1002-0160(09)60279-4 14. g. murtaza, a. ghafoor and m. j. qadir, j. sci. food. agric., 88 (2008) 100. doi: 10.17957/ijab/15.0026 15. c. e. gilbert and e. j. calabrese, british water quality act, 1989. section. 20 (5) (b), department of environments, regulating drinking water quality (lewis publishers, london, u.k) (1992) 186. 16. s. rengaraj and s. h moon, water res., 36 (2002) 1783. doi: 10.1016/s0043-1354(01)00380-3 17. j. c moreno, r. gómez and l. giraldo, materials, 3 (2010) 452. doi.org/10.3390/ma3010452 18. j. b. chennaiah, m. a. rasheed and d. j. patil, int. j. plant animal environ. sci., 2 (2014) 2231. pak. j. anal. environ. chem. vol. 21, no. 1 (2020) 157 19. guidelines for drinking-water quality, 2004. 3rd ed. vol. 1. world health organization, geneva. isbn: 978 92 4 154761 1 20. g. p. layrargues, c. rose, l. spahr, j. zayed, l. normandin and r. f. butterworth, metabol. brain dis., 13 (1998) 311. doi: 10.1023/a: 1020636809063 21. department of health and human services, public health service. 2004. agency for toxic substances and disease registry. url:http://www.atsdr.cdc.gov/csem/csem.h tml. 22. z. mahmood, s. zahra, s. iqbal, m.a. raza and s. nasir, appl. water sci., 7 (2017) 3469. doi: 10.1007/s13201-017-0624-3 23. y. c. sharma, e. jalilnejad and s. yarusova, int. j. environ. sci. dev., 8 (2017) 195. 24. world health organization (who), ministry of health, the govt. of pakistan, quality drinking water: guidelines and standards for pakistan (2005) 4. https://doi.org/10.1155/2017/7908183 25. u. zafar, m. a. bhatti and a. akram, pak. j. anal. environ. chem., 19 (2018) 53. doi: http://dx.doi.org/10.21743/pjaec/2018.0 6.05 issn-1996-918x pak. j. anal. environ. chem. vol. 9, no. 1 (2008) short communication voltammetric study of arsenic (iii) and arsenic (v) in ground water of hajigonj and kalkini in bangladesh samir chandra paul, mohammad arifur rahman, nur-e-alam siddique and a. m. shafiqul alam* department of chemistry, university of dhaka, dhaka-1000, bangladesh ------------------------------------------------------------------------------------------------------------------------------------------- abstract the speciation of arsenic in groundwater samples using square wave anodic stripping voltammetry (swasv), differential pulse anodic stripping voltammetry (dpasv) and normal pulse anodic stripping voltammetry (npasv) are described. good resolution of the species, arsenic (iii) and arsenic (v) is achieved using swasv. the reliability of the methods was checked by analyzing the total arsenic content of the samples by hydride generation atomic absorptioion spectrophotometer and by analyzing prepared controlled laboratory standard solution. since this technique is comparatively cheaper than other available techniques it could be a better analytical technique for arsenic speciation from water. in this study, the assessment of inorganic arsenic species in ground water of kalkini (madaripur) and hajigonj (chandpur) is reported. it shows that arsenic content in water in different locations is irregular. most of the locations contain higher level of as(iii) than as(v). the highest concentration of arsenic is found in anayetnagor (554.46 ± 0.07 g/l) of kalkini and raichar (562 ± 0.50 g/l) of hajigonj. however, the level of total arsenic and as(iii) of most of the villages of the study areas are more than the who guideline value (50g/l). therefore a proper monitoring process should be evolved along with the development of methods to keep the water free from arsenic. ------------------------------------------------------------------------------------------------------------------------------ introduction arsenic is ubiquitous in the environment, being naturally present in soil, air, water and food, and concentrations may be increased by anthropogenic contamination [1]. it is present in the environment in a number of different inorganic and organic chemical forms due to its participation in complex biological and chemical process. some of the most important arsenic species from a toxicological perspective include the two oxidation states as(iii), as(v), monomethylarsenic acid (mma), dimethylarsinic acid (dma), arsenobetaine and arsenocholine [2]. in recent years, the presence of high levels of arsenic in ground water, the main source of drinking water in many countries around the world, has drawn attention of the scientific communities. moreover, contamination of food chain by arsenic contaminated water is another threat [3]. numerous recent investigations have demonstrated that arsenic constitutes a serious health risk spot in bangladesh [2-6]. bangladesh, a country in south asia with a population of about 150 million, is one of a number of countries that has arsenic contamination in its groundwater, which results from the underlying mineralogy and geology of the area. arsenic contamination has been reported in groundwater in 41 out of 64 districts in bangladesh [3]. about 61% of the water analysed from tubewells has arsenic content above 0.01 mg/l [4]. the average concentration of arsenic in contaminated water is about 0.26 mg/l with a maximum level of 0.83 mg/l. this is significantly higher than the world health organization (who) maximum permissible limit in drinking water which is 0.05 mg/l [7]. moreover, arsenic shows toxicity and chemical property depending on its oxidation state and physicochemical forms. inorganic arsenic is known to be more toxic than organic ones, and as3+ is reported at least ten times more toxic than as5+ [8]. thus it is important to determine each of arsenic species rather *corresponding author e-mail: amsalam2004@yahoo.com mailto:amsalam2004@yahoo.com pak. j. anal. environ. chem. vol. 9, no. 1 (2008) 2 than the total amount. rasul et al., and hussam et al., studied the as (iii) and as (v) with the aid of anodic stripping voltammetry (asv) and other metals with aasgf-z (zeeman effect-atomic absorption spectrometer with graphite furnace) from groundwater of kustia, bangladesh [9,10]. but there is no work has been reported with study of the as(iii), as(v) with square wave anodic stripping voltammetry (swasv), differential pulse anodic stripping voltammetry (dpasv) and normal pulse anodic stripping voltammetry (npasv) at hajigonj and kalkini of bangladesh which are most endemic areas. therefore, speciation of arsenic rather than quantification of total arsenic in drinking water present in groundwater are necessary. speciation of arsenic has been performed with different hyphenated techniques, often the coupling of ion chromatography or liquid-chromatography to detection system like: direct uv detection, direct coupling to atomic absorption spectrometry, aas, online hydride generation aas (hg-aas), icp atomic emission spectrometry (icp-aes), icp mass spectrometry [11-13]. these techniques are very expensive. several reports have appeared on electrochemical stripping procedures for the determinations of arsenic [9, 10, 14]. the instrumentation required is relatively simple and generally the cost is far less than that required for the other techniques. another advantage of electrochemical techniques is their ability to distinguish between the different oxidation states of arsenic. the anodic stripping voltammetry is very sensitive [14] and compared with expensive multielement analysis techniques like icp-ms, is an economical procedure for trace determination of arsenic down to the g/l level. in the present work, there are three different electronalytical methods such as square wave anodic stripping voltammetry (swasv), differential pulse anodic stripping voltammetry (dpasv) and normal pulse anodic stripping voltammetry (npasv), were used for the determination of arsenic with speciation to satisfy the lack of proper analytical techniques and implement such analytical methods that can provide accurate and interference free measurements of arsenic at the g/l levels of concentrations in water of hajigonj and kalkini of bangladesh. experimental the study area tubewell water was collected from nine villages of hajigonj and nine villages of kalkini. hajigonj thana at chandpur district is 53 kilometers away from comilla district and kalkini thana at madaripur district is 260 kilometer away from dhaka city. sampling tube well water was collected in polythene containers which were washed before collecting samples with 5% hno3, distilled de-ionized water and finally with the tube well water at the sampling sites for several times. the information about the depth of the tube well, year of installation and also red or green marked were collected from the local inhabitants by supplying the questions-answers sheets. sample preparation the samples collected from different points were filtered with a microfilter paper under vacuum and preserved in acid (1ml conc. hcl per 100ml of water sample). for the measurements of total arsenic, the samples were mixed with concentrated hcl and na2so3 (s) (10ml sample + 10ml conc. hcl + 30-40 mg na2so3(s)) in an acid clean pyrex glass electrolysis cell and heated at 70�80 0 c for 20-30 minutes without stirring, then 15 minutes with stirring and also 15 minutes with purging nitrogen with stirring until all so2 fumes cleared. the sample was then cooled to room temperature and was thus made ready for the measurements of the concentration of total arsenic [14]. preparation of stock standard solutions standard 1000 mg/l stock solutions of as3+ and as5+ were prepared by dissolving as2o3 (merck, germany, analytical grade) and na2haso4.7h2o (merck, germany, analytical grade) in deionized water respectively. these solutions were made acidic by the addition of 2-3 ml conc. hcl acid. fresh standard solutions of lower concentrations were made from the stock solution at the beginning of the everyday experiment. analytical procedure for quantification of as3+ and as5+ at first the gold electrode was made shiny yellow, almost scratch free surface by polishing with fine alumina powder (0.3µm) on wet polishing cloth. then the electrode was cleaned with deionised water and then 1m hcl and also stored in 6m hcl. for measurements of as3+, the stair bar was put into the cell and the cell was filled with 10 ml sample and 10 ml 6m hcl [9]. the solution was purged for 10 minutes with nitrogen. all the electrodes were inserted and tapped off to remove any bubbles from them. a computerized electrochemical system, model hq-2040 by advanced pak. j. anal. environ. chem. vol. 9, no. 1 (2008) 3 e(mv) vs. ag/agcl (sat. kcl) -200 -100 0 100 200 300 400 500 600 c u rr e n t, i (µ a ) -5 0 5 10 15 20 25 npasv swasv dpasv background current analytics, usa was used for the analysis. the deposition potential (initial potential), accumulation or deposition time (initial delay), quit time delay is 150mv, 120sec. and 30 sec. respectively. the run replication (number of addition) is 3. in these analytical measurement procedures two standard additions were performed and corresponding currents were measured after subtraction of background current. the signal current was then plotted against concentration. the concentration of arsenic was calculated from the slop of the regression line drawn through the points using software sigma plot based on ms-excel. total arsenic was determined by same procedure after treatment of sample with nahso3 as described in sample preparation method. as5+ was evaluated with subtraction of as3+ from total arsenic [14]. results and discussion pulse voltammetric techniques, introduced by barker and jenkin [15], are aimed at lowering the detection limits of voltammetric measurements. by substantially increasing the ratio between the faradaic and nonfaradaic currents, such techniques permit convenient quantification down to the 10-8m concentration level. because of their greatly improved performance, modern pulse techniques have largely supplemented classical polarography in the analytical laboratory. among the voltammetric techniques normal pulse voltametry (npv), differential pulse anodic stripping voltammetry (dpasv), square wave voltametry (swv) was checked to evaluate the sensitivity for the trace arsenic analysis and speciation from groundwater. analytical comparison between swv, dpv and npv the swas, dpas and npas voltammograms of arsenic (as3+) for a 50 ppb standard solution at solid gold electrode (aubutton) are demonstrated in figure1. out of three asv techniques in use, npasv is considered to be least sensitive and selective method due to its highest signal to background ratio. in npv, the nonfaradaic current could not be avoided completely because the current is sampled once at the end of pulse amplitude. this charging current increases the signal current and for this reason npv shows high signal current with respect to swv and dpv where the current is sampled twice, just before the pulse application and late pulse life. although in npv, small amount of charging current is present it is also good sensitive method for as3+ analysis. out of these techniques used for arsenic analysis, the swasv technique is most sensitive and selective electroanalytical method because of its ability to enhance the analytical signal by removing non-faradaic current. so less time is required for arsenic analysis and ultra trace analysis can be easily performed with this method. however, among these methods dpasv shows moderate signal for arsenic but it is also a sensitive method for arsenic analysis. figure 1: comparison between three different electroanalytical methods for as3+(same concentration) at solid gold electrode. speciation of arsenic in ground water it is reported that shallow aquifer layer is contaminated with arsenic in almost all of the districts of bangladesh (dphe-bgs, 2000). in this study, arsenic level is determined in hajigonj (chandpur) and kalkini (madaripur) two contaminated areas of bangladesh. the different species of arsenic (as3+and as5+) content of ground water samples from different locations of the study area was measured with three different electroanlytical methods (swasv, dpasv and npasv) and the data thus obtained are represented in the table 2 and table 3. from the results, it is clear that the distribution of arsenic is not regular rather scattered due to the spatial variation. this is because of the variation in the depth of tube wells, amount of water withdrawn and also the geological phenomenon. in hajigonj, the level of as3+ ranges from 48.19 to 285.28 g/l. among all the water samples collected from the nine different villages of hajigonj, the highest arsenic (iii) concentration was found in raichar (285.28 g/l) and the lowest was in toraghor (48.19g/l). the value of as5+ is also the lowest in satbaria (40.05g/l) and the highest in raichar (276.95g/l). however, all of the pak. j. anal. environ. chem. vol. 9, no. 1 (2008) 4 water samples contain total arsenic above the who guide line value (50 g/l). the level of as3+ ranges from 26.91 to 286.0 g/l in kalkini. among the nine water samples collected from the nine different villages of kalkini, the highest arsenic(iii) concentration was found in anayetnagor (286.0g/l) and the lowest was in charluxmi (26.91g/l). the lowest value of as5+ is in shadipur (26.10g/l) and the highest in anayetnagor (268.46g/l). most of the water samples contain total arsenic above the who guide line value (50 g/l). table 1. concentration of as3+and as5+ in ground water of the study area, hajigonj (chandpur) sample no. sampling location depth of tube wells methods conc. of as3+ conc. of total as (as3++ as5+) conc.of as5+ (feet) (g/lsd) (g/lsd) (g/l) swasv 80.73.0.05 120.78.0.09 40.50 dpasv 72.530.12 119.900.16 47.37 1 satbaria 90 npasv 74.580.70 116.540.30 41.96 swasv 72.710.07 118.670.70 45.96 dpasv 76.560.09 123.090.17 46.53 2 khalpara 100 npasv 78.750.06 115.430.18 36.68 swasv 197.090.21 320.870.01 123.78 dpasv 198.94.0.08 307.270.06 108.33 3 bolakhal 140 npasv 190.060.09 312.120.13 122.06 swasv 80.240.30 129.450.07 49.21 dpasv 70.800.70 115.560.06 44.76 4 bakila 100 npasv 72.760.16 121.490.18 48.73 swasv 285.280.90 562.230.50 276.95 dpasv 288.330.60 594.530.13 306.20 5 raichar 120 npasv 203.430.23 437.600.23 243.17 swasv 276.570.04 380.890.60 104.32 dpasv 296.340.30 389.600.30 93.26 6 uchaa gram 140 npasv 280.980.50 381.260.04 100.28 swasv 83.560.40 140.430.40 56.87 dpasv 85.900.30 145.870.30 59.97 7 dherra 100 npasv 84.370.13 143.590.60 59.22 swasv 48.190.70 94.540.30 46.35 dpasv 46.470.15 91.010.19 44.54 8 toraghor 70 npasv 41.580.50 82.920.50 41.34 swasv 120.270.20 209.760.50 89.49 dpasv 121.680.40 209.00.40 87.32 9 shendra 110 npasv 110.100.14 187.230.16 77.13 no. of analysis (n=3) pak. j. anal. environ. chem. vol. 9, no. 1 (2008) 5 table 2. concentration of as3+and as5+ in ground water of the study area, kalkini (madaripur) no. of analysis (n=3) the variation of arsenic concentration with depth of the different tube wells is presented in table 1 and table 2. it was found that the concentration of total arsenic was increased with the increase of depth within this range of 70 to 160 ft. this indicates high percentage of water was withdrawn from these aquifers. moreover, it is interesting that the concentration of as (iii) and as (v) varied with the depth of the tube well. this may be due to the change of geology with the variation of depth of soil. hussam et al., 2003 found the concentration of as (iii) and as (v) was present as aso3 3(0.712 mg/l) and aso4 3(0.973 mg/l) in groundwater at kustia respectively [10]. from the table 1 and table 2, it is noticed that most of the locations of kalkini and hajigonj contain higher level of as(iii) than as(v). this observation implies that more inorganic arsenic in ground water present in reduced form. sample no. sampling location depth of tube wells methods concentration of as3+ concentration of total as (as3++ as5+) concentration of as5+ (feet) (g/lsd) (g/lsd) (g/l) swasv 166.290.21 306.510.05 140.22 dpasv 161.170.03 299.590.09 138.42 1 charbivagdi 120 npasv 166.640.08 287.260.13 120.62 swasv 51.970.12 98.200.12 46.23 dpasv 41.550.30 89.670.50 48.12 2 dharichar 90 npasv 42.750.40 72.810.80 30.06 swasv 32.530.08 47.590.07 15.06 dpasv 32.400.10 51.840.14 19.44 3 charluxmi 70 npasv 26.910.14 42.420.30 15.51 swasv 75.960.16 102.060.80 26.10 dpasv 95.160.06 126.770.17 31.61 4 shadipur 80 npasv 82.160.09 102.520.07 20.36 swasv 46.300.70 76.270.15 29.97 dpasv 35.520.12 69.560.19 34.04 5 kashimpur 70 npasv 30.600.15 64.020.10 33.42 swasv 110.710.05 197.310.07 86.60 dpasv 110.110.07 190.610.40 80.50 6 shikar mangol 120 npasv 89.960.40 156.220.08 89.96 swasv 53.800.50 76.240.09 22.44 dpasv 47.520.09 67.760.40 20.24 7 khishnanagor 70 npasv 39.680.08 51.720.05 12.04 swasv 126.180.4 186.200.20 60.02 dpasv 97.440.07 160.510.70 63.07 8 alipur 120 npasv 86.460.23 138.900.04 52.44 9 anayet nagor 160 swasv 286.00.14 554.460.07 268.46 dpasv 275.450.16 539.400.20 263.95 npasv 268.210.02 542.400.09 274.19 pak. j. anal. environ. chem. vol. 9, no. 1 (2008) 6 table 3. concentration of arsenic in water sample of manikgonj considered as controlled area (swasv method). sample no. locations concentration of as3+ concentration of total as (as3++ as5+) concentration of as5+ (g/lsd) (g/lsd) (g/l) 1 nabagram 14.43  0.05 24.12  0.02 9.69 2 khilinda 9.02  0.6 16.45  0.13 7.43 3 jagir 10.98  0.82 19.76  0.09 8.78 4 macshimul 8.12  0.7 19.67  0.41 11.55 no. of analysis (n=3) table 4. validation of results with hg-aas with developed anodic stripping voltammetry methods variation with hg-aas hg-aas swasv dpasv npasv swasv dpasv npasv controlled lab. standard (g/l) (%) 125.0  1.20 119.900.16 116.54  0.30 120.78  .0.09 4.08 6.78 4.22 8.25 95.4  1.50 98.200.12 89.67  0.50 72.81  0.80 2.93 6.02 23.68 9.50 120.5  2.20 118.670.70 123.09  0.17 115.43  0.18 1.51 2.14 4.20 8.35 no. of analysis (n=3) comparison of the endemic areas with the controlled area the water samples of manikgonj which was considered as a controlled area (free from arsenic contamination) were also analysed with swasv method as presented in table 3. the average value of as content in this area was found 20 g/l. on the other hand, the average as contents in different locations of the study areas are about 10 times higher than the average content of the controlled area. validation of the results by hg-aas to validate the results obtained from the electroanalytical method, few water samples were analysed by hg-aas. it was observed that the results obtained by the electroanalytical methods were closed to results obtained by the hg-aas method. this indicates that the results obtained by the developed electroanalytical methods were effective to analyse arsenic from the groundwater where the variation of results between hg-aas and electroanalytical method was about 1.5-9.5%. conclusion from the study of the three electroanlytical methods (swasv, dpasv and npasv), the swasv technique is most selective and suitable electroanalytical method. this method could be a better analytical technique for arsenic speciation from water. moreover this technique is comparatively cheaper than other available methods. the results obtained in this study have been compared with the value of unaffected area (manikgonj).the observations show that arsenic content of the study area in ground water about 10 times higher than the average content of the controlled area. the total arsenic content and arsenic (iii) in water is higher than the who guide line value (50gl-1). so, arsenic contamination in ground water of the study areas is in alarming proportions. therefore, a proper monitoring process should be evolved along with development of methods to keep the water free from arsenic. references 1. m. c. villa-logo, e. rodriguez, p. l. mahia, m. s. mnuiategui and d. p. rodringuez, talanta, 57 (2002) 741. pak. j. anal. environ. chem. vol. 9, no. 1 (2008) 7 2. s. w. al rmalli, p. i. haris, c. f. harrington and m. ayub, sci. total environ., 337 (2005) 23. 3. m. a. hasan, k. m. ahmed, o. sracek, p. bhattacharya, m. v. bromssen, s. broms, j. fogelstrom, m. l. mazumder and g. jacks, hydrology j. (2007), doi:10 .1007/s10040-0070203-z . 4. m. ali and s. a. tarafdar, j. radional nucl chem., 256 (2003) 297. 5. m. g. m. alam, e. t. snow and a. tanaka, total environ., 308 (2003) 83. 6. h. k. das, a. k. mitra, p. k. sengupta, a. hossain, f. islam and g. h. rabbani, enviro int., 30 (2004) 383. 7. who. vol. 224, geneva: world health organization; (2001). 8. dcht, arsenicosis in bangladesh, dhaka community hospital trust, february. dhaka, bangladesh., (1998). 9. s. b. rasul, a. k. m. munir, z. a. hossain, a. h. khan, m. alauddin and a. hussam, talanta., 58 (2002) 33. 10. a. hussam, m. habibuddowla, m. alauddin, z. a. hossain, a. k. m. munir and a. h. khan, j environ sci health, 38 (2003) 71. 11. m. van holderbeke, y. n. zhao, f. vanhaeck, l. moens l, r. darns and p. sandra, j. anal at spectrom., 14 (1999) 229. 12. j. l. gomez-ariza, d. sanchez-rodas, r. beltran, w. corns and p. stockwell, appl organonet chem., 12 (1988) 439. 13. c. soros, e. t. bodo, p. fodor and r. morabito, anal. bioanal chem., 377 (2003) 25. 14. f. t. henry, t. o. kirch and t. m. thorpe, anal. chem., 51 (1979) 215. 15. g. c. barker, i. l. jenkin, analyst., 77 (1952) 685. 16. w. h. horwitz, official methods of analysis of aoac international., vol. 1 (2000). microsoft word 243-251-pjaec-01112020-303-c-received galley proof cross mark issn-1996-918x pak. j. anal. environ. che m. vol. 22, no. 2 (2021) 243 – 251 http://doi.org/10.21743/pjaec/2021.12.03 histamine detection in mackerel (scomberomorus sp.) and its products derivatized with 9-flourenilmethylchloroformate muhammad abdurrahman munir 1,2 , lee yook heng 1 , edison eukun sage 1 , muhammad mukram mohamed mackeen 1 and khairiah haji badri 1* 1department of chemical sciences, faculty of science and technology, universiti kebangsaan malaysia, bangi, selangor. 2department of pharmacy, faculty of health science, universitas alma ata, bantul, yogyakarta. *corresponding author email: kaybadri@ukm.edu.my received 02 november 2020, revised 23 june 2021, accepted 13 september 2021 -------------------------------------------------------------------------------------------------------------------------------------------abstract histamine is commonly present in food containing proteins, like in mackerel. consuming fish is imperative for the improvement of human muscles. nevertheless, so me studies reported ingesting fish containing histamine more than 50 mg·kg-1 can cause toxicity. this study analyzed and determined the composition of histamine in mackerel and its products co mmonly consumed in malaysia, especially on the east coast of malaysia. these included processed mackerel such as canned products, satay (skewed fish) and keropok lekor (fish cake/ cracker). histamine analysis was performed using high performance liquid chro matography (hplc) equipped with a fluorescence detector. a derivatizing reaction was applied to increase the sensitivity of hplc to hista mine using 9-flourenilmethylchloroformate (fmoc-cl). the chromatographic separation was achieved in 15 min. method validation was in accordance to co mmission decision 657/2002/ce. the linear range was at 0.16 – 5.00 µg· ml-1 (histamine) with the lod at 0.10 µg·ml-1 and loq at 0.30 µg· ml-1. method applicability was checked on seven real samples involving raw, cooked, and dry products, yielding acceptable recovery. keywords: histamine, mackerel, hplc, validation, lod, loq -------------------------------------------------------------------------------------------------------------------------------------------introduction an increase in the number of food types and their products are produced and distributed for human consumption. nevertheless, several problems still exist for many years in relation to consumers' health, such as heavy metals, usage of pesticides, and traces of toxic compounds such as biogenic amines that are commonly detected in fish [1] and fish products. biogenic amines are simple nitrogen compounds that generally can be found in food and beverages containing protein such as fish, meat, cheese, milk, and others. the main biogenic amines present in these products are putrescine, tyramine, cadaverine, and histamine [2]. they are produced by amination and/or transamination of aldehydes and ketones. they can also be formed by decarboxylation of free amino acids (fig. 1) during the degradation process of proteinbased food and beverages involving specific bacteria such as lactobacillus, pediococcus, and leuconostoc species. the determination of biogenic amines in fish is of great interest simply because of their toxicity besides being used as an indicator for quality [3] or freshness or even spoilage level of fish [4]. pak. j. anal. environ. che m. vol. 22, no. 2 (2021)244 n h n c h2 h2 c nh2 + co2n h n c h2 ch nh2 cooh histi dine hi stami ne figure 1. production of hi stami ne from i ts correspondi ng decarboxyl ation of ami no acids among all biogenic amines found in food, histamine is considered toxic and easily present in food and beverages, as informed by the european food safety authority [5-6]. according to food and drug administration (fda), the acceptable amount of histamine that can be consumed is below 50 mg·kg -1 . it becomes toxic and leads to several symptoms such as nausea, palpitation, and headache [78] when it goes beyond this level. histamine fish poisoning, called scombroid poisoning, is the most general foodborne ailment related to fish consumption [6, 9-10]. therefore, the demand for a safer approach to consuming fish and its products has promoted more studies to tackle the histamine issues, especially in detecting its existence. several chromatography techniques have been studied in order to determine histamine in fish. these include usage of thin– layer chromatography (tlc), high performance liquid chromatography (hplc) and gas chromatography (gc). some researchers use hplc and gc equipped with mass spectrometry (ms) since ms has some advantages in the selectivity and anti-jamming capability [5, 10-11]. tlc is simple and requires no sophisticated instruments but several studies reported several issues from the longer time needed for analysis, inaccurate acquired results, and more for qualitative demand. whereas gc is not often applied due to inherent tailing problems. histamine is a polar and non-vaporized compound, thus the use of derivatization reagent prior to gc analysis is compulsory in order to detect histamine [12-13]. among these methods, hplc is the most popular technique used in order to detect histamine in food and beverages, owing to its high selectivity property and simpler than any other method [14-17]. generally, histamine separation is performed in the c8 or c18 column, in gradient elution with the mobile phase consisting of methanol, acetonitrile, or water [18]. extraction is the most critical step in the histamine detection procedure. according to some studies, the extraction method can influence analytical recovery. the extraction of histamine from a solid sample such as fish and meat is usually carried out using an acid such as hydrochloric acid (hcl), perchloric acid (hclo4), or trichloroacetic acid (tca) [19]. biogenic amines are strong organic bases, thus it is important to take advantage of this feature for their separation from the sample matrix, where tca represented a better choice for fish [12, 20]. due to the structural characteristic of histamine, the derivatization step is an imperative step, and several reagents are applied such as ophthaldialdehyde (opa), dansyl chloride, dansyl chloride and 9-flourenilmethylchloroformate (fmoc-cl) [21]. fish mackerel is widely consumed all over the world due to its nutritional values. furthermore, monitoring fish freshness is imperative to ensure the safety of human consumption [22]. of particular interest are the products of mackerel such as satay, fish ball, or even the popular malaysia product prepared from mackerel (known locally as “keropok lekor”) that is consumed in several countries in south east asia such as thailand and indonesia. there is a lacking of reporting on histamine analysis. therefore, the aim of the present work was to establish a method for determining histamine in mackerel and its products using hplc analysis and pak. j. anal. environ. che m. vol. 22, no. 2 (2021) 245 fluorescence as a detector. in this study, fmoc-cl was used as a derivatizing agent in order to increase the sensitivity and selectivity of histamine (fig. 2). the proposed method was validated in terms of linearity, accuracy, precision, detection, and quantification limit. moreover, the developed and validated method was applied to the histamine analysis in mackerel samples. o o cl + n h n c h2 h2 c nh2 n n c h2 h2 c nh2 + hcl o o fmoc-cl histamine histamine fm oc figure 2: deri vati zation of histamine with fmoc-cl materials and methods materials histamine dihydrochloride (his) was purchased (≥99% purity) from sigma– aldrich. fmoc-cl, tca, hplc-grade acetonitrile (acn) and hplc-grade methanol (meoh) were also purchased from sigmaaldrich. hydrochloric acid (hcl), acetone, potassium borate buffer, and glycine were obtained from ukm laboratory (purchased from sigma-aldrich). standard (stock) solution of histamine was prepared at a concentration of 1000 mg·l-1 in 0.1 m hcl. all chemicals and reagents used were of analytical grade. preparation of standard solution a stock solution of histamine was prepared with 0.1 m hcl in a volumetric flask. the solution was stored at 4oc. standard solutions were prepared by diluting the stock solution in order to construct the calibration curve (0.16–5 µg·ml -1 ). sample preparation about 5 g of fresh fish sample in wet condition was placed in a beaker and homogenized with 10 ml of 10% tca for 20 min. the slurry was then centrifuged for 5 min at 2000 rpm. the supernatant was filtered through a filter paper and rinsed with 10% tca. the sample was derivatized using 5 mm fmoc-cl. the homogenate was stored at 4 o c before further analysis using hplc-fd. derivatization procedure 10 µl of the sample was mixed with 20 µl of fmoc-cl (5 mm) and 10 µl of potassium borate buffer in a vial. the mixture was incubated at 10oc for 20 min. the supernatant was then mixed with 10 µl of 0.1 m glycine. finally, 950 µl of 50% acetonitrile was added to the mixture before further analysis using hplc-fd. hplc – fd detection of histamine the hplc analysis was carried out on a waters 2475 – c18 column (150 mm × 2.1 mm id, 3 µm particle size), equipped with a fluorescence detector (ex: 267/ em: 314). chromatographic separation was carried out using an isocratic elution. the mobile phase was acetonitrile: deionized water (63:37, v/v), and the flow rate was kept at 0.34 ml·min -1 with the injection of 0.5 µl. the mixture was filtered using a membrane filter (0.45 nm) and degassed in an ultrasonic bath for 10 min before being analyzed. the target compound was identified according to the retention time of its corresponding standard. quantification was measured using the standard's calibration curves that underwent a similar derivatization step of sample preparation. validation study all analytes injected were performed in triplicate to ensure accuracy, precision, pak. j. anal. environ. che m. vol. 22, no. 2 (2021)246 recovery, detection, and quantification threshold limit. the correlation coefficient and linear regression studies, repeatability assessment, mean measurement, standard, and relative standard deviation were calculated using microsoft excel 2019 software. results and discussions histamine extracted from fish sample extraction of histamine from fish samples is an imperative step before analysis using hplc histamine is one of the biogenic amines and has a diverse chemical structure, and occur at a wide range of concentration in food and beverage samples [11]. most of the extraction methods reported in the literature for histamine detection were generally using the acidic extraction method, starting with a solid matrix. organic solvents such as methanol and ethanol are rarely used. the choice of acid must be related to the sample characteristics. several compounds other than histamine might be disturbances and can be eliminated in this step. hcl becomes a good choice for cheese and fruits analysis but is not suitable for meat and fish owing to the difficulties related to occasional sample turbidity [12]. in this study, histamine in fish samples extracted with the use of tca is the best choice due to its efficacy to precipitate amino acids from the fish sample. method validation the analytical method was optimized by determining the linear range, precision, recovery detection and quantification threshold limit. results are presented in table 1. linearity of the calibration curves was established by injecting six standard mixtures with concentrations ranging from 0.16 to 5 µg·ml-1. a series of standard working solutions at 0.16, 0.31, 0.63, 1.25, 2.50 and 5 µg·ml -1 were obtained from optimized conditions. satisfactory linearity was acquired between the peak area and concentration of the analyte (fig. 3). the detection limit was validated from the lowest concentration of histamine required to give a signal to noise ratio of three (s/n = 3), while the quantification limit was validated with a signal to noise ratio of 10 (s/n = 10). both of them were reflected by the limit of detection (lod) and limit of quantification (loq) values [10, 23]. table 1. li near range, cali bration curve, correl ation coefficient (r), detection and quanti fication li mit of histami ne. parameters vali dated results linear range (µg·ml-1) 0.16 – 5 calibration curve y = 4e + 06x + 175075 r2 0.9998 lod ( µg·ml-1) 0.10 loq ( µg·ml-1) 0.30 intraday (% rsd) 1.73 interday (% rsd) 7.38 recovery (%) 103 r2: square of regression coefficient; tr: retention time; lod: limit of detection; loq: limit of quantification; rsd: relative standard deviation. figure 3. cali bration curve of hi stami ne – fmoc, peak area versus concentration the repeatability and reproducibility of the method were measured by injecting histamine standard at six replicates on the same day (intraday) and over ten days (interday), respectively. good reproducibility of both the peak area (rsd ≤ 1.73 %) for pak. j. anal. environ. che m. vol. 22, no. 2 (2021) 247 intraday and (rsd ≤ 7.38 %) for inter-day, they are presented in table 1. various studies have reported the application of other derivatizing reagents such as opa and dansyl chloride. both reagents are generally applied to derivatize biogenic amines before analyzing with hplc, nevertheless, compared to fmoc – cl, they have several drawbacks. opa cannot derivatize primary amine, and derivatization to primary and secondary amines are unstable, while dansyl chloride upon application is time – consuming and needs to be heated to high temperature in order to complete the derivatization process [24, 25]. the selectivity of fmoc – cl was reported by ramachandra et al., after derivatized using fmoc, the analyte can be detected using hplc equipped with uv and fl detectors at the picomole level. the stability of fmoc – cl is better than opa [26]. in terms of reactivity toward nucleophiles, fmoc – cl is also better than opa. moreover, the application of acid solvents such as tca and hcl in this research to dilute histamine is satisfactory. the products have a better collision-induced dissociation (cid) energy, which will provide a straightforward way to identify the reactive amines by applying ms/ms analysis [27]. after ten days, histamine derivatized with fmoc-cl has degraded owing to several factors such as temperature and the condition of the environment. fmoc-cl composition inside histamine is possible to reduce if exceed more than ten days. a mackerel sample was selected to conduct the precision and accuracy assay. the intraday precision was measured from the result of six replicated analytes prepared at 5 µg·ml -1 of histamine standard to fish sample in a single day, whereas the inter-day precision was determined from the analytes for ten consecutive days. the mixture was derivatized prior to hplc analysis. satisfactory recovery for histamine was acquired (103%) (table 1). it can be concluded that (tca) can be applied to extract histamine from fish and its products. analysis of mackerel the established and validated method was adopted to determine the histamine concentration in fish samples. in histamine analysis of fish mackerel and its products, peak identification of each sample was identified on the basis of retention time by comparing with standard solutions. it was also confirmed by spiking standards to the fish samples. the retention time of histamine was predetermined and detected to be stable and consistently reproducible. quantification was based on the external standard method using calibration curves fitted by linear regression analysis. during the experimental conditions, baseline separation of histamine was obtained in less than 15 min. no interference peaks were presented at the retention times of the analyte (fig. 4). this method was applied for histamine detection in 7 different products of mackerel. the samples tested and the concentration found of the histamine are shown in table 2. table 2. histami ne concentration i n mackerel and i ts products (n = 6). sampl e type histami ne (µg·kg-1) mackerel fish 1.16 canned mackerel 0.28 fish ball mackerel 0.53 fish satay mackerel 0.19 keropok lekor (mackerel cracker) 0.84 fish chip mackerel 0.17 salted mackerel 0.03 several studies have been carried out in order to verify the presence of histamine derivatized with fmoc-cl. according to (fig. 4a), three components appeared at a 2.080, 3.510 and 3.839 min retention time. pak. j. anal. environ. che m. vol. 22, no. 2 (2021)248 the peak for the derivatized histamine standard appeared at a retention time of 2.080 min. the peak for fmoc-cl was detected at 5.380 min, glycine appeared at 4.310 min, and tca appeared at 3.344 min. analysis of fmoc-cl and hcl has also been studied where the retention time of hcl and tca is almost similar. the fish samples have successfully been derivatized using fmoc-cl, where tca was used to extract histamine in the fish prior to derivatization. the detected histamine appeared at 2.188 min, and it was similar to the histamine standard, as shown in (fig. 4b). 2 .0 8 0 3 .5 1 0 3 .8 3 9 e u 0 . 0 0 2 0 . 00 4 0 . 00 6 0 . 00 8 0 . 00 1 0 0 .0 0 0. 0 0 5 .0 0 1 0 .0 0 1 5 .00 2 .1 8 8 2 .9 6 5 3 .3 1 3 e u 0.00 20.00 40.00 60.00 80.00 100.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 figure 4. (a) the chromatogram of histami ne standard deri vati zed by fmoc-cl (ex: 267/ em: 314) and (b) the chromatogram of mackerel deri vati zed with fmoc-cl (ex: 267/ em: 314) fish and fish products contain histamine at various concentrations. the concentration of histamine is influenced by several factors, such as the presence of bacteria. bacterial growth in fish and fish products results in a shelf life reduction of the fish and fish products, furthermore an increase in the risk of fish–borne diseases [28]. yusoff et al. also reported that the content of histamine is influenced by the storage time due to microbial activity [29]. some other studies related to histamine detection in food samples are on different approaches to analysis. zhai et al. detected histamine in canned sardines at 7.5 mg·kg -1 [30], while parchami et al. acquired histamine at 0.104 mg·kg -1 in fish samples [31]. francisco et al. studied histamine detection using uv and fluorescence detectors and found histamine below the fda regulation limit and detection limit at 0.17 and 1.6 mg·kg -1, respectively [32]. kounnoun et al. used opa as a reagent to derivatize histamine and later analysed using hplc equipped with uv and fl detectors. histamine in fish samples was detected at below 25 mg·kg -1 and detection limit of 1.8 mg·kg-1 [33]. zhu et al. reported the application of hplc to analyse histamine after derivatization with a fluorogenic compound with a detection limit at 1.3 nmol·l-1 [34]. chong et al. reported that sardine was the only fish where histamine still could be analyzed under 4 o c during the degradation process [35]. therefore, a reliable method is needed in order to detect histamine in food to prevent fish-borne intoxication, maintain good control of fish production and check safety quality. in this study, hplc is reliable to analyze histamine in the fish samples [36]. according to the findings in this study, the concentration of histamine in mackerel and its products can be categorized safe and can be consumed since the histamine concentrations were at a lower level following the compliance policy guideline. this guideline suggests that if histamine level is at 10 mg·kg -1 , it is considered not toxic, nevertheless, the concentration at 30 mg·kg -1 is considered as decay, whereas above 30 mg· kg-1 indicates the fish is totally decomposed re tention time (min) (a) (b) re tention time (min) pak. j. anal. environ. che m. vol. 22, no. 2 (2021) 249 [37]. histamine poisoning generally occurred upon improper consumption of scombroid fish species such as tuna, bonito, saury and mackerel. they have a high level of histidine in their flesh [38]. however, fish and fish products are necessary for humans. nevertheless, the quality of fish and fish products is more difficult to control than meat owing to the quality of them influenced by several parameters such as species, age, habitats and enzymes activity [39]. various concentrations of histamine are regulated in different countries. fda has issued an approved level of 50 mg·kg -1 , while a higher concentration of histamine is allowed by the european community, south africa, and italy at 100 mg·kg-1. australia and germany suggested a much higher histamine level at 200 mg·kg -1 [40]. conclusion this study indicated that the developed derivatization method was suitable for histamine determination in mackerel and its by-products using hplc-fd chromatographic technique. the chromatographic separation was achieved within 15 min, and satisfactory peak resolution was acquired. the fmoc-cl reaction was carried out at ambient temperature. the method was reproducible and accurate, with recovery at 103%. using this method, we were able to detect and monitor histamine in the fish sample. though there are zero cases of histamine poisoning reported during the consumption of mackerel and its by-products in malaysia, and although histamine concentration is low in these fish products, this technique is handy in offering a trustable quantified result. acknowledgments the authors would like to extend their gratitude to universiti kebangsaan malaysia and malaysia ministry of education for providing the necessary funding for this research through the research incentive grant (ggp-2019-021). conflict of interest the authors declare that they have no conflict of interest in any parts of this article to any concerned party. references 1. m. papageorgiou, d. lambropoulou, c. morrison, e. klodzinska, j. namiesnik and j. plotka – wasylka, trac – trend anal. chem., 98 (2017) 128. doi: 10.1016/j.trac.2017.11.001 2. m. a. sahudin, m. s. su’ait, l. l. tan, l. h. heng and n. h. a. karim, anal. bioanal. chem., 411 (2019) 6449. doi.org/10.1007/s00216-019-02025-4 3. l. pester, food addit. contam. a, 28 (2011) 1547. doi: 10.1080/19440049.2011.600728 4. y. q. wang, d. q. ye, b. q. zhu, g. f. wu and c. q. duan, food chem., 163 (2014) 6. doi.org/10.1016/j.foodchem.2014.04.064 5. c. i. g. tuberoso, f. congiu, g. serreli, s. mameli, food chem., 175 (2015) 29. doi: 10.1016/j.foodchem.2014.11.120 6. r. parchami, m. kamalabadi and n. alizadeh, j. chromatogr. a, 1481 (2017) 37. doi: 10.1016/j.chroma.2016.12.046 7. food and drug administration, scombrotoxin (histamine) formation. in fish and fishery products hazards and controls guide, 4 th ed. office of seafood, washington dc (2011). 8. c. proestos, p. loukatos and m. komaitis, food chem., 106 (2008) 1218. doi.org/10.1016/j.foodchem.2007.06.048 9. m. trevisani, r. mancusi, r, m. cecchini, c. costanza and m. prearo, vet. sci., 4 (2017) 31. pak. j. anal. environ. che m. vol. 22, no. 2 (2021)250 doi: 10.3390/vetsci4020031 10. y. fu, z. zhou, y. li, x. lu, c. zhao and g. xu, j. chromatogr. a, 1465 (2016) 30. doi.org/10.1016/j.chroma.2016.08.067 11. a. jain, m. gupta and k. k. verma, j. chromatogr. a, 1442 (2015) 60. doi.org/10.1016/j.chroma.2015.10.036 12. e. dadakova, m. krizek and t. pelikanova, food chem., 116 (2009) 365. doi.org/10.1016/j.foodchem.2009.02.018 13. s. bomke, b. seiwert, l. dudek, s. effkemann and u. karst, anal. bioanal. chem., 393 (2009) 246. doi: 10.1007/s00216-008-2420-2 14. h. dong and k. xiao, food chem., 229 (2017) 502. doi.org/10.1016/j.foodchem.2017.02.120 15. j. j. zhong, n. liao, t. ding, x. ye and d. h. liu, j. chromatogr. a, 1406 (2015) 331. doi.org/10.1016/j.chroma.2015.06.048 16. a. m. piaste, a. jastrzebska, m. p. krzeminski, t. m. muziol and e. szlyk, anal. chim. acta, 834 (2014) 58. doi.org/10.1016/j.aca.2014.05.028 17. r. mandrioli, l. mercolini and m. a. raggi, anal. bioanal. chem., 405 (2013) 7941. doi: 10.1007/s00216-013-7025-8 18. c. lanfranco, s. moret and g. purcaro, hplc in food analysis, crc press, boca raton (2008). 19. m. a. munir and k. h. badri, j. anal. methods chem., 2020 (2020) 1. doi.org/10.1155/2020/5814389 20. c. a. lazaro and c. a. conte – junior, bra. j. vet. res. animal sci., 50 (2013) 430. doi.org/10.11606/issn.16784456.v50i6p430-446 21. y. y. guo, y. p. yang, q. peng and y. han, food sci. technol. int., 50 (2015) 1523. doi.org/10.1111/ijfs.12833 22. f. f. faizal, l. l. tan and s. i. zubairi, sensor actuat. b – chem., 269 (2018) 36. doi.org/10.1016/j.snb.2018.04.141 23. s. jia, y. ryu, s. w. kwon and j. lee, j. chromatogr. a, 1282 (2013) 1. doi.org/10.1016/j.chroma.2013.01.041 24. y. gao, j. tan, j. lu, z. sun, z. li, z. ji, s. zhang and j. you, anal. bioanal. chem., 412 (2020) 8339. doi.org/10.1007/s00216-020-02969-y 25. x. huang, q. zhang, x. li, x. ao and x. wang, chromatographia, 84 (2021) 463. doi.org/10.1007/s10337-021-04020-3 26. r. a. ramachandra, v. murugan and k. b. premakumari, j. appl. pharm. sci., 10 (2020) 31. doi.org/10.7324/japs.2020.10505 27. a. lkhagva, c. c. shen, y. s. leung and h. c. tai, j. chromatogr. a, 1610 (2020) 60536. doi.org/10.1016/j.chroma.2019.460536 28. b. kuswandi, a. j. restyana, a. abdullah, l. y. heng and m. ahmad, food control., 25 (2012) 184. doi.org/10.1016/j.foodcont.2011.10.008 29. m. n. m. yusoff, m. h. jaffri and s. azhari, malays. j. sci. health tech., 7 (2020) 53. doi.org/10.33102/mjosht.v7i.108 30. h. zhai, x. yang and l. li, food control, 25 (2012) 303. doi.org/10.1016/j.foodcont.2011.10.057 31. r. parchami, m. kamalabadi and n. alizadeh, j. chromatogr. a, 1481 (2017) 37. doi.org/10.1016/j.chroma.2016.12.046 32. k. c. a. francisco, p. f. brandao, r. m. ramos, l. m. goncalves, a. a. cardoso and j. a. rodrigues, int. j. food sci. technol., 55 (2019) 1. doi.org/10.1111/ijfs.14300 33. a. kounnoun, m. el maadoudi, f. cacciola, l. mondello, h. bougtaib, n. alahlah, n. amajoud, a. el baaboua and a. louajri, chromatographia, 83 (2020) 893. pak. j. anal. environ. che m. vol. 22, no. 2 (2021) 251 doi.org/10.1007/s10337-020-03909-9 34. f. zhu, h. liu, w. zhang, c. du, h. zhu, x. du, x. hu and y. lin, rsc adv., 11 (2021) 19541. doi.org/10.1039/d1ra01436f 35. c. y. chong, f. a. bakar, r. a. rahman, j. bakar and m. z. zaman, j. food sci. technol., 51 (2014) 1118. doi.org/10.1007/s13197-012-0621-3 36. efsa, efsa j., 9 (2011) 2393. doi.org/10.2903/j.efsa.2011.2393 37. s. oguri, m. enami and n. soga, j. chromatogr. a, 1139 (2007) 70. doi.org/10.1016/j.chroma.2006.10.077 38. n. richard, l. pivarnik, p. c. ellis and c. lee, j. aoac int., 91 (2008) 768. doi.org/10.1093/jaoac/91.4.768 39. c. m. keow, f. a. bakar, a. b. salleh, l. y. heng, r. wagiran and s. siddiquee, int. j. electrochem. sci., 7 (2012) 4702. 40. d. carelli, d. centonze, c. palermo, m. quinto and t. rotunno, bionsen. bioelectron., 23 (2007) 640. doi.org/10.1016/j.bios.2007.07.008 issn-1680-9955 pak. j. anal. & envir. chem. vol. 7, no. 2, (2006) 107 � 111 cathodic stripping voltammetric determination of cefadroxil in pharmaceutical preparations and in blood serum amber r. solangi*1, arfana mallah2, m. y. khuhawar2 and m. i. bhanger1 1national center of excellence in analytical chemistry, university of sindh, jamshoro, pakistan. 2m.a. kazi institute of chemistry, university of sindh, jamshoro, pakistan. email: amber@boston.usa.com abstract an analytical method has been developed using hanging mercury drop electrode (hmde) for the quantitative determination of antibacterial drug cefadroxil (cfl) from pharmaceutical preparations and blood serum. cathodic adsorptive stripping voltammetry was carried out in hydrochloric acid (0.1m): methanol (80: 20 v/v) and potassium chloride (0.1m) as supporting electrolyte. the reduction wave was obtained within -700 to -800 mv. linear calibration curve was within 1-50µg/ml with detection limit of 0.1µg/ml of cefadroxil. relative standard deviation for inter and intra day analysis of cfl was within 1-2%. the number of additives present in pharmaceutical preparations did not interfere the determination of cefadroxil. the analysis of pharmaceutical preparations and blood serum after chemotherapy with cefadroxil indicated relative standard deviation (rsd) within 0.8-1.2% and 2.6-3.8% respectively. the satisfactory results were obtained for quality control of cefadroxil in pharmaceutical preparations and in blood serum. keywords: cathodic stripping voltammetry, phenolic beta lactam, cefadroxil, pharamaceuticals, hmde. introduction the phenolic -lactam antibiotic cefadroxil (cfl) is a broad spectrum first generation cephalosporin and is used for the treatment of urinary tract infections. it has significant activity against both gram-positive and gram-negative bacteria [1]. it contains a -lactam ring fused with a six membered dihydrothiazine ring bearing substitutes r1 and r2 in the side chain at c3 and c7. various analytical procedures are described for the determination of cfl mainly, spectrophotometry [2-3], derivative spectrophotometry [4-5], spectrofluorometry [6-7], liquid chromatography [8-11], atomic absorption [12], capillary zone electrophoresis [13], chemiluminescence [14-15] and electroanalytical technique [16-18]. the british pharmacopeias, 1998, describes a liquid chromatographic method for the analysis of bulk drug [19]. electroanalytical methods are sensitive, selective and suitable for biological active compounds. they involve less expensive equipment. ivaska et al [16] reported a differential pulse polarographic method for cfl at ph 1-2 as a distinct peak at -1.25 to 1.30 v (vs ag/agcl) and a linear scan voltammetric method at glassy carbon electrode as anodic oxidation wave at +0.8v (vs ag/agcl) at ph 7.3. ozkan et al [17] reported electro oxidation of cfl using glassy carbon electrode by cyclic voltammetry and differential pulse voltammetry. the method was applied for determination from pharmaceutical preparations. gaber et al [18] examined differential pulse polarography and cyclic voltammetry to investigate the coordination of cfl with cd(ii), pd(ii) and zn(ii). however these methods have not been applied for the determination of cfl from biological fluids. pd fm ac hi ne tr ia l v er sio n mailto:amber@boston.usa.com pak. j. anal. & envir. chem. vol. 7, no. 2, (2006) 108 the procedure which could be used for the analysis of cfl from biological fluids, without extensive sample preparations or derivatization in the presence of excipients could be of analytical interest. hanging mercury drop electrode (hmde) can be examined, because glassy carbon electrode requires surface polishing during each run and may be time consuming. the precision and accuracy may also be affected. the present work examines simple and sensitive method for the determination of cfl from pharmaceutical preparation and blood serum using differential pulse cathodic stripping voltammetry at hmde. the determination of cfl from pharmaceutical preparations could be of interest for quality control and analytical procedure used for the analysis of cfl in biological fluids may be of value in evaluation of bioavailability and pharmacokinetic properties. experimental chemicals and reagents gr grade methanol, hydrochloric acid (37%), potassium chloride, potassium nitrate, lithium chloride, sodium acetate, potassium dihydrogen phosphate (e. merck, germany) and cefadroxil (cfl) (sigma, switzerland) were used. freshly prepared doubly distilled deionized water was used throughout the study. a stock solution of cfl (1mg/ml) was prepared by dissolving 100 mg of cfl in methanol (20 ml) and adjusting the volume with hydrochloric acid (0.1n) to 100ml. this solution (5ml) was further diluted to 100ml with methanol: hcl (0.1m) (20:80v/v) on each working day. potassium chloride (0.1m) as base electrolyte prepared in deionized water was used. buffer solutions within ph 1-10 at unit interval were prepared from the following: hydrochloric acid (0.1m), potassium chloride (0.1m), acetic acid (0.1m), sodium acetate (0.1m), ammonium acetate (0.1m) sodium bicarbonate (0.1m), sodium carbonate (0.1m) ammonium chloride (0.1m) and ammonia (0.1m). instrumentation the ph measurements were made with ph meter (wtw � inolab, germany) with glass electrode and internal reference electrode. voltammetric measurements were performed with metrohm automatic 746-va trace analyzer equipped with 747-va stand (metrohm, switzerland). the 747 stand includes a three electrode system, an ag/agcl (3m kcl), reference electrode, a platinum wire as auxiliary electrode and hanging mercury drop electrode as working electrode. a printer epson lx-300 was used for printing purposes. ptfe coated string bar, rotated by a magnetic stirrer was used during preconcentration step. analytical procedure determination of cefadroxil solution of cefadroxil (in methanol: (0.1m) hcl 1:4 v/v) (10ml) containing 10-500 µg and 1ml of potassium chloride (0.1m) was transferred into polarographic vessel. the ph was adjusted to 4 by adding 1ml of (0.1m hcl:kcl) buffer. the sample was purged for 200 seconds with oxygen-free nitrogen (british oxygen company, boc, karachi). the preconcentration potential (-700 mv) measured against ag/agcl reference electrode was applied to the fresh mercury drop for 60 seconds (tacc= 60 sec) while the solution was stirred. the stirring was then stopped for a period of 10 seconds (equilibration time=10 sec). the voltammogram was then recorded by applying a cathodic differential pulse scan with pulse amplitude of -50 mv. the voltammograms were recorded in triplicate for each run automatically by the instrument. the average peak heights (n=3) were assessed on the basis of the difference between peak height of the analyte and that of base electrolyte alone recorded under the same conditions. the quantitation was carried out by calibration curve and standard addition technique. determination of cefadroxil in pharmaceutical preparations eight tablets of each helicef (helix pharma (pvt.) ltd. karachi, pakistan), neucef (sami pharmaceutical (pvt.) ltd. karachi, pakistan), evacef (highnoon laboratories, lahore, pakistan) were separately ground to fine powder. a quantity equivalent to one tablet (500mg of cefadroxil) of each was weighed, dissolved in 20ml methanol, transferred to a 100 ml volumetric flask and diluted to the mark with pd fm ac hi ne tr ia l v er sio n pak. j. anal. & envir. chem. vol. 7, no. 2, (2006) 109 (0.1m) hcl. this solution was slightly turbid but no further treatment was made. further dilution was made with 20% methanol in hcl (0.1m). 10 ml of this solution and 1 ml potassium chloride (0.1m) were transferred to polarographic cell and analysis was carried out by analytical procedure 2.3.1. determination of cefadroxil in blood serum the blood sample (5ml) of volunteers was collected after 2hrs of taking a tablet helicef containing 500 mg of cefadroxil. the blood sample was collected by vein puncture in a clean screw capped vial and centrifuged at 3000 rpm for 15 minutes. supernatant was separated and added acetonitrile (1ml) and contains were mixed for two minutes. the sample was centrifuged for 15minutes at 4000 rpm and supernatant was collected and organic solvent was evaporated at 45°c under nitrogen stream. subsequently the sample was transferred to volumetric flask, volume was adjusted to 10ml with methanol: hcl (0.1m) (1:4 v/v) and was analyzed using analytical procedure 2.3.1. a blank determination was carried out following the same procedure with blood sample (5ml) from the volunteer who had not taken any medicine at least for one week. results and discussion the electrochemical reduction of cfl by differential pulse cathodic stripping voltammetry (csv) was examined by hanging mercury drop electrode (hmde). the effect of ph and supporting electrolyte was examined on electrochemical reduction of cfl. the influence of ph was examined within ph 2�10. it was observed that peak potential shifted linearly to more negative side with an increase in ph indicating that the mechanism of electrode reaction is ph dependent. the highest peak current was obtained at ph 4 at -0.75 v (figure 1) and was selected. the effects of different electrolyte on peak potential (mv) and peak current (na) were examined. the solution (1ml) of potassium chloride, lithium chloride, sodium oxalate, potassium citrate, sodium carbonate, sodium acetate of 0.1m concentration were added and determination were carried out at ph 4. the peak potential was measured at -0.75v. average response of at least (n=3) determination was calculated. the maximum peak current was obtained when potassium chloride was used. at optimized conditions the peak current was observed to be concentration dependent and linear calibration curve was obtained by recording average peak current (n=6) against concentration of cfl and was obtained with 1-50 g/ml (figure 2). 10 13 16 19 0 2 4 6 8 10 12 ph c u rr e n t (n a ) fig. 1. effect of ph on current (na) for cefadroxil in 20% methanol plus 80% 0.1m hcl plus 0.1 m kcl. -600 -700 -800 -900 mv i / na 2.5 65.5 97.0 128.5 160.0 34.0 fig. 2. cathodic stripping voltammogram for cfl indicating potential (mv) verses current (na) in 20% methanol plus 80% 0.1m hcl plus 0.1 m kcl, ph 4.0, concentrations 1, 10, 20, 30, 40, 50 g/ ml, peak potential -0.75 v. the coefficient of determination, r2 for a six points calibration was obtained 0.9822 with y= 4.8542x (figure 3). the analysis of test solutions pd fm ac hi ne tr ia l v er sio n pak. j. anal. & envir. chem. vol. 7, no. 2, (2006) 110 of cfl (n=4) was carried out within the calibration range and relative % error for the analysis was obtained within ± 0.5%. the reproducibility of the measurements in terms of peak potential and peak current for cfl with 10g/ml (n=5) was calculated and relative standard deviations (rsd) were obtained with 0.5% and 0.6% respectively. the detection limit measured as signal to noise ratio (3:1) as compared to the blank was obtained 0.1g/ml. the inter and intra day variation in the response for 10g/ml was examined for five days and each day analysis was carried out in triplicate (n=3). the rsd for pooled data was obtained 1.2%. for the determination of cfl in pharmaceutical preparations, the interfering effects of common additives glucose, lactose, sorbitol, gum arabic were examined at the concentration five times the cfl (10g/ml). the average variation (n=3) in the response was observed less than 5% and did not interfere the determinations. the results obtained were compared with a spectrophotometric method [20] by measuring the absorbance at 455nm and a good agreement with observed results with csv was obtained (shown in table 1). the method was applied for the determination of cfl in the pharmaceutical preparations helicef, neucef and evacef. the results of analysis are summarized in table 1 and the results agreed with the labeled values with relative % deviation within 1.2 to 5.2%. the rsd for replicate analysis (n=3) was obtained within 0.8 � 1.2%. y = 4.854x r2 = 0.9822 0 50 100 150 200 250 300 0 10 20 30 40 50 60 concentration (g/ml) c u rr e n t ( n a ) fig. 3. calibration plot for cfl from figure 2. the method was examined for the analysis of cfl from human serum. the blood samples of healthy volunteers were collected after 2hrs from a single dose of 500mg of cefadroxil. the serum after de-proteinization with acetonitrile was analyzed. the results of analysis are summarized in table 2 and are within 15 to 20 g/ml with rsd 2.6-3.8%. the peak potential was however observed at slightly shifted position towards negative but stable signals were observed including after spiking the sample with 100 g of cfl. table 1. results of analysis for cefadroxil in pharmaceutical preparations *amount found by spectrophotometric method [20] s. no. name of drug labeled amount (mg/ tablet) amount found (mg/ tablet) ± c.l at 95% *amount found (mg/ tablet) ± c.l at 95% rsd % (n=3) 1 helicef 500 497 ± 4 492±2 0.8 2 neucef 500 479 ± 6 472±5 1.2 3 evacef 500 493 ± 4 488±4 0.8 pd fm ac hi ne tr ia l v er sio n pak. j. anal. & envir. chem. vol. 7, no. 2, (2006) 111 table 2. results of analysis for cefadroxil in blood serum conclusion simple cathodic stripping voltammetric method has been developed with hmde for quantitative determination of cefadroxil at ph-4. the csv methodology is simple, sensitive and rapid for the determination of the drug in pharmaceuticals and in blood serum. the sample preparation procedure is simple without need to eliminate the excipients. the satisfactory results obtained with rsd 0.6% and allow recommending the procedure for quality control of cefadroxil in concentration range 1-50 µg/ml in pharmaceutical preparations and blood serum. references 1. w. a. ramers, j. n. delagado (eds), wilson and gisfold�s text book on organic medicinal pharmaceutical chemistry, 10th ed. lippincott-raven publishers, philadelphia, new york, (1998) 274 2. h. salem, anal. chim. acta. 515, (2004) 333 3. g. a. saleh, h. f. askal, m. f. radwan, and m. a. omar, talanta, 54, (2001)1205. 4. a. el-gindy, a. f. m. el walily, m. f. bedair,. j. pharm. biomed. anal. 23, (2000) 341. 5. b. morelli, j. pharm. biomed. anal., 32: 2, (2003) 257. 6. m. hefnawy, , y. el-shabrawy and f. belal, j. pharm. biomed. anal. 21: issue 4, (1999) 703-707. 7. a. f. m. el walily, a. a. gazy, s. f. belal and e. f. khamis, j. pharm. biomed. anal. 20, (1999) 643. 8. v. f. samanidou, e. a. hapeshi and i. n. papadoyannis, j. chromatogr. b, 788 issue 1-5, (2003) 147. 9. n. snippe, n. c. van de merbel, f. p. m. ruiter, o. m. steijger, h. lingeman and u. a. j. chromatogr. b: biomedical sciences and applications, 662: issue 1-2, (1994) 61. 10. c. hendrix, yongxin zhu, c. wijsen, e. roets and j. hoogmartens, j. chromatogr. a, 634 issue 2, (1993) 257. 11. k. lindgren, j. chromatogr.: biomedical applications, 413, (1987) 347. 12. h. salem and h. askal, j. pharm. biomed. anal. 29, (2002) 347. 13. y. mrestani, r. h. h. neubert, a. härt and j. wohlrab, anal. chim. acta. 349, issues 1-3 29, (1997) 207. 14. yuanyuan sun, yuhai tang, hong yao and xiaohui zheng, talanta, 64 issue 1, (2004)156. 15. i. n tomita, l. o. s bulhoes, anal. chim. acta. 442, (2001) 201. 16. a. ivaska and f. nordström, anal. chim. acta. 146, (1983) 87. 17. sibel. a. ozkan, n. erk, b. uslu, n. yilmaz, i. biryol, j. pharm. biomed. anal. 23: (2000) 263. 18. a. a. abdel gaber, m. a. ghandour and h. s. el-said, anal. lett. 36: (6), (2003) 1245. 19. the british pharmacopoeia, hmso, london, (1998). 20. s. m. el-ashry. f. belal, m. m. elkerdawy, d. r. elwasseef, microchim. acta, 135, 3-4, (2000) 191. blood samples no. amount taken helicef (mg) amount found (g/ml)  c.l at 95% % rsd 1 500 15.2±0.5 3.8 2 500 20.4±0.5 2.7 3 500 17.6±0.4 2.6 pd fm ac hi ne tr ia l v er sio n microsoft word 3-31-38-pjaec-15092022-460-c-revised galley proof-19-05-2023-3.docx cross mark issn-1996-918x pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 31 – 38 http://doi.org/10.21743/pjaec/2023.06.03 aroma component analysis using hs/spme-fid gas chromatograph in basmati rice varieties of punjab, pakistan farah shamim*, mohsin ali raza, syed sultan ali, samina sarfraz and misbah riaz rice research institute, kala shah kaku, punjab-39018, pakistan. *corresponding author email: farah_tirmazi@yahoo.com received 15september 2022, revised 16february2023, accepted 20february 2023 ------------------------------------------------------------------------------------------------------------------------------------------- abstract aroma is a promising quality factor for rice grain that impacts consumer acceptability. the principal volatile compound that adds basmati rice fragrance is 2-acetyl-1-pyrroline (2ap). milled white rice of 04 promising varieties i.e., super basmati, basmati-515, basmati 2000and basmati 370 were evaluated for volatile compounds by gas chromatography (gc) coupled with solid phase micro extraction unit (spme) using flame ionizing detector (fid). six volatile compounds (nonanal, decanal, and alcohols such as benzyl alcohol, indole) were identified in the tested varieties, among them 2-ap is only present in aromatic rice varieties. this study confirmed the occurrence of 2-ap in all studied varieties with highest concentration in super basmati followed by basmati-515, basmati 2000 and basmati 370. keywords: basmati rice, 2-acetyl-1-pyrroline, aroma, pakistan, gas chromatograph, spme. ------------------------------------------------------------------------------------------------------------------------------------------- introduction fragrant/ aromatic rice varieties demand has amplified in this decade worldwide. scent is supreme feature in rice grain, especially where consumer approval is taken a benchmark. currently, buyer are more conscious of the eminence of the rice consumed [1,2]. recent world consumption data and scientific studies showed that in european countries demand for aromatic rice varieties increased, particularly basmati due to emergent attention in eastern ethnic cuisine. premium quality basmati rice only originated in pakistan and india, and jasmine rice grown in thailand get good market price. the tropical countries have their own scented rice varieties that are popularly consumed locally besides basmati [3, 4]. compounds responsible for aroma in rice have been under study since previous 33 years. several compounds were recognized in cooked and un-cooked rice. preliminary rice selection decisions were made by eating individual grains or breathing the aroma of leaf tissue or grains either heating in water or retorting with koh. this practice is not quantifiable and it allows classification of progeny only as scented, moderately scented, and non-scented. presence of 2-ap has been an indicator of aroma in the selection of fragrant rice lines [5]. in 1965, ammonia, hydrogen sulfide along with acetaldehyde were found in the volatiles of heated rice. the biochemical pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 32 nature of the aroma complex remained unidentified till 1982, once ron buttery with his team of scientists from the us department of agriculture (albany, ca) successfully recognized 2-acetyl-1-pyrroline (2ap) as the prime compound apt for the pleasing aroma in rice [6]. 2-ap [iupac tag: 5-acetyl-3,4dihydro-2h-pyrrole, density 1090 kgm−3, molecular weight 0.111145 kg mol−1] has minute odor verge (0.1 µgkg−1) so nominally detected by the human nose. it contributes popcorn like aroma in rice. in non-scented rice varieties, it is present at negligible levels reportedly 10 times lower than aromatic rice [4, 7, 8]. rice aroma is interaction of numerous complex volatile compounds affected by pre and post-harvest operations. the rapid advancement in the instrumentation and sampling methods for the isolation have made it possible to analyze compounds even at trivial concentrations (ppb levels). extraction methods can be divided into traditional and modern. traditional methods include purge and trap, steam distillation–solvent extraction, direct distillation and solvent extraction. recent methods contain static headspace (shs) followed by separation of aroma compounds using gas chromatography (gc) with flame ionization detector (fid) or mass spectroscopy (ms) and headspace–solid phase micro extraction (hs-spme) [9, 10]. spme is newest effective aroma analytical equipment combines sampling, extraction unified with liquid or gas chromatography. it has excellent repeatability and detection as compared to previous methods [11, 12]. newly harvested aromatic rice varieties have robust smell but later successive operations i.e., milling, drying and storage decreases aroma. lesser the milling degree, the greater the aroma absorbed in the milled rice [13]. rice aroma component analysis is important to understand the aromatic nature of rice. pakistan has quite a lot of traditional basmati rice cultivars retaining the fragrance. nevertheless, their volatile profiles and the extent of impulsive compounds have thus far studied. this study may help to identity the variety and interpret the cooking quality of rice to relate it to further analysis for future rice breeding. methods and materials rice varieties (super basmati, basmati-515, basmati-2000, basmati-370) were grown at experimental farm area of rice research institute, kala shah kaku, punjab, pakistan under standard agronomic conditions. paddy samples were harvested at 20% moisture content, dried to 11% moisture, dehulled, polished with lab polisher (satake trg058, japan) from milling lab to obtain white rice [14]. freshly milled white rice samples were grinded using udy cyclone mill to make fine powder. 4g of flour sample was immediately placed in 20ml glass vial. prior analysis empty vials were heated in hot air oven at 150oc for 1 h to remove any unintended compound. after placing rice flour, vials were then instantly sealed with silicon septum/ptfe and crimp cap made with aluminum. vial was well shaken at ambient temperature prior injection in gc [15]. the volatile compounds of rice were recognized by their different retention times from standard. following standards were acquired from authentic manufacturer. i.e., nonanal (sigma aldrich, usa), decanal (sigma aldrich, usa), vanillin (daejung, korea), benzyl alcohol (sigma aldrich, usa), 2-acetylpyroline (sigma aldrich, usa), indole (supelco, spain). different dilutions of standards (0.1, 1.0, 2.0, 3.0, 4.0, 5.0 ppm and 10 ppm) were made in hplc grade toluene (sigma aldrich, usa). pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 33 separation of volatiles mixture was achieved by gas chromatograph (master gc with complete automation, dani instruments s.p.a, italy) united by flame ionization detector (fid), capillary column dn-wax (30m×0.25mm×0.25µm) was used. nitrogen gas used as carrier with flow rate of 1 ml/min. initially 40°c column temperature was employed and then heated to 165°c @ 10°c/ min and then elevated to 250°c @ 15°c/min for 3min. the spme fiber (57341u) supelco, bellefonte, pa, usa) was desorbed in gc injector for 5 min having 250°c temperature in splitless mode. gc oven programme was hold for 1 min at 50°c to reach 100°c @ 4°c/min in increments then ramped to 240°c @ 50°c/min with ultimate hold of 2min. identification of 2-ap and other selected volatiles was done by comparing the sample peak with standard solution peak. the vials were incubated in the auto sampler oven at 80°c, after 20 min the spme fiber assembly was exposed to the headspace overhead the surface of the solid sample for 30 min at the same temperature. extraction temperature set at 80°c, pre-incubation time at 10 min with adsorption period at 10 min was used for complete recovery. area computation was used as extent of the comparing quantity during optimization. calibration graph for quantification of volatile compounds viz. 2-ap, decanal, nonanal, vanillin, benzyl alcohol and indole was plotted. volatile compounds were calculated by the fraction values got from the chromatogram. chromatogram values were subtracted from the blank solvent reading. clarity chromatographic station software was used in this study. results and discussion the concentration of aromatic compounds evaluated by gc analysis varied among tested aromatic rice varieties. identification of individual volatile constituents were based on comparison with standard. six volatile compounds were separated from all tested varieties. the chromatograms are presented as fig.1. a, b, c, d. component detail with data is summarized in table 1-4, as peak number, name of volatile compounds (benzyl alcohol, vanillin, nonanal, decanal, indole and 2-ap) retention time (12.8-19.5 min), percentage peak area (548.38 mv.s) and concentration (ppm) of the identified compounds. aroma in rice may be created by assimilated expression of many volatile compounds. the predominant compounds identified in this study were benzyl alcohol, vanillin, nonanal, decanal, indole and 2-ap respectively in different range as discussed below. nonanal in food and feed is responsible for nutty flavor. its concentration was highest in super basmati (14.06ppm) followed by basmati 370 (12.52ppm) and basmati 515. indole exhibits musty,jasmine like flavor in food and feed.the highest concentration of indole was present in super basmati and basmati-370. minimal traces of indole were detected in basmati 2000. though benzyl alcohol, responsible for the sweet floralodour, was identified in all varieties ranging 5.66 to10.67 ppm. 2-ap is the principal aromatic compound responsible for basmati aroma, which was recognized in all studied varieties. the highest 2-aplevel was detected in super basmati, followed by basmati 370 as depicted by fig. 1. a, b, c, d. it is apparent from table 5, that super basmati contained highest quantity of all volatiles except indole and vanillin. basmati -515 is leading aromatic cultivar of punjab, pakistan exhibiting notable content of 2-ap. likewise basmati-370, an old variety still grown in pakistan, showed aroma volatiles in prominent quantity. basmati-2000 contained all the scented contents with the exception of indole content which might be a varietal character. pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 34 figure 1. a, b, c, d. chromatogram of basmati-515, super basmati, basmati-370, basmati-2000 (a) (b) (c) (d) pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 35 table 1. basmati-515 aroma component analysis. retention time (min) area (m v.s) concentration (ppm) peak type compound name 1 12.835 20.962 12.33 ordnr nonanal 2 14.688 82.167 5.33 ordnr decanal 3 16.576 60.325 9.25 ordnr benzyl alcohol 4 16.875 1.549 0.46 ordnr 2ap 5 18.619 62.848 9.9 ordnr indole 6 19.528 38.052 18.4 ordnr vanillin total 265.902 45.67 table 2. super basmati aroma component analysis. retention time (min) area (m v.s) concentration (ppm) peak type compound name 1 12.827 56.378 14.06 ordnr nonanal 2 14.691 194.241 14.26 ordnr decanal 3 16.576 150.125 16.04 ordnr benzyl alcohol 4 16.875 2.204 0.68 ordnr 2ap 5 18.619 183.386 18.46 ordnr indole 6 19.536 34.573 11.67 ordnr vanillin total 620.907 75.17 table 3. basmati 370 aroma component analysis. retention time (min) area (m v.s) concentration (ppm) peak type compound name 1 12.827 112.504 12.52 ordnr nonanal 2 14.691 404.245 17.92 ordnr decanal 3 16.576 301.525 10.67 ordnr benzyl alcohol 4 16.875 37.305 0.59 ordnr 2ap 5 18.619 305.783 19.27 ordnr indole 6 19.517 59.244 21.71 ordnr vanillin total 1240.608 132.14 table4. basmati 2000 aroma component analysis. retention time (min) area (m v.s) concentration (ppm) peak type compound name 1 12.824 27.839 12.33 ordnr nonanal 2 14.693 11.077 5.33 ordnr decanal 3 16.6 3.723 9.25 ordnr benzyl alcohol 4 16.84 3.816 0.46 ordnr 2ap 5 18.619 0.987 9.9 ordnr indole 6 19.523 18.69 18.4 ordnr vanillin total 66.144 28.75 pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 36 table 5. aroma component analysis of basmati varieties of punjab, pakistan. sr. no. compound name basmati 515 super basmati basmati 2000 basmati 370 1 nonanal 12.38b 14.06a 11.60c 12.52b 2 decanal 5.33d 14.26b 11.37c 17.92a 3 benzyl alcohol 9.25c 16.04a 5.66d 10.67b 4 2-ap 0.46c 0.68a 0.43c 0.59b 5 indole 9.90b 18.46a 0.00d 19.27c 6 vanillin 18.40b 11.67d 14.69c 21.17a results are in line with research findings of many indian scientists. bryantetal. 2011 [16] documented hexanal (0.787 ppm), decanal (0.065 ppm), nonanal (0.242 ppm), benzyl alcohol (0.052 ppm), guaiacol (0.033 ppm) and vanillin (0.324 ppm) in indian basmati 370. scented cultivars basmati, della khao dawk and mali were evaluated for flavor quantification and 2-ap concentration in these varieties varied from 0.05 to 0.34 ppm [17]. in another study, [15] jezussek et al. 2011 reported 0.122 to 0.411 ppm 2-ap concentration in 11 basmati varieties of india. furthermore, 0.214 ppm was reported in basmati 370 collected from market of punjab, india in the same study. in a latest study, 152 volatile compounds were separated among the tested 12 rice cultivars using solvent extraction technique [18]. besides about 65 volatiles identified in rice bran previously [19]. another study [15] revealed that basmati grown in parts of karnataka state and maharashtra excel in 2ap (0.122 to 0.411 ppm) on other area’s basmati. in another study slight 2ap contents were reported in black rice [20]. the majority of the volatile (heterocyclic) are prevalent in freshly harvested rice than stored [21]. the present study confirmed the prior findings that the aroma strength in rice is affected by variety selection and growing conditions mainly. aromatic rice cultivars cannot adequately characterize just by 2-ap because other distinctive odors (e.g., earthy, nutty, roasty and green) are also demonstrating the expression [9, 22, 23]. in scented rice genotypes, 2ap can be spotted in all parts of the plant excluding roots [6] whereas in nonaromatic rice, 2ap is also found at a much lower concentration (0.0015 mg/kg) that cannot be professed easily [13]. conclusion aroma profile analysis and quantification in pakistani rice varieties was accomplished first time regarding the cultivars which was not reported earlier. we can apply it as targeted breeding approach in aromatic rice variety development as it is timeconsuming and laborious to cultivate new varieties without aroma profiling. this study can be used for identification of aroma compounds of other non-basmati varieties for adulteration detection in export consignments. acknowledgment authors are thankful to all rri scientists for execution of analysis workand field trial. conflict of interest there is no conflict of interest. pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 37 references 1. k. sakthive, r. m. sundaram, r. n. shobha, s. m. balachandran and c. n. neeraja, biotechnol. adv., 27 (2009) 468. https://doi.org/10.1016/j.biotechadv.200 9.04.001 2. f.s.g. hashemi, m. y. rafii, m. r. ismail, t. m. m. mahmud, h. a. rahim, r. asfaliza, m. a. malek and m. a. latif, crit. rev. plant sci., 32 (2013) 445. https://doi.org/10.1080/07352689.2013.8 07716 3. m. akram, pak. j. agric. res., 22 (2009) 154. http://agris.fao/agris-search/search/ search.do?recordid=pk2010000303 4. m. a. u. baradi and a. r. elepaño, philippine agric. ssc.n, 95 (2012) 260. https://agris.fao.org/agris-search/ search.do?recordid=ph2013000782 5. d. s. yang, r. l. shewfelt, k. lee and s. j. kays, j. agric. food chem., 56 (2008) 2780. https://doi.org/10.1021/jf072685t 6. r. g. buttery, l. c. ling and t. r mon, j. agric. food chem., 34 (1986) 112. https://doi.org/10.1021/jf00067a031 7. v. hinge, p. hemant and a. nadaf,appl.biochem. biotechnol., 178 (2015) 1. https://doi.org/10.1007/s12010-015-1898-2 8. k. wakte, r. zanan, v. hinge, k. khandagale, a. nadaf and r. henry,j. sci. food agric., 97 (2016) 384. https://doi.org/10.1002/jsfa.7875 9. c. j. bergman, j. t. delgado, r. bryant and k. r. cadwallader, cereal chem., 77 (2000) 454. https://doi.org/10.1094/cchem.2000.7 7.4.454l 10. m. b. mariani, e. testani and v. giannett, j. commodity sci. technol. quality, 50 (2011) 185. https://doi.org/10.1016/j.foodcont.2 017.02.036 11. n. s. kottearachchi, e. g. d.priyangani, and d. p. s. t. g attanayaka. j. nat. sci. foundation of sri lanka, 38 (2010) 139. https://doi.org/10.4038/jnsfsr.v38i2.2040 12. z. zeng, h. zhang, t. zhang, s. tamogami and j. y. chen,j. food compos. anal., 22 (2009) 347. https://doi.org/10.1016/j.jfca.2008.11.020 13. r. widjaja, j. d. craske and m. wootton, j. sci. food agric., 70 (1996) 151. https://doi.org/10.12691/jfnr-3-2-7 14. s. v. mathure, k. v. wakte, n. jawali and a. b. nadaf, food analysis methods, 4 (2011) 326. http://doi.org/10.1007/s12161-010-91713 15. m. jezussek, b. o. juliano and p. schieberle, j. agric. food chem., 50 (2002) 1101. https://doi.org/10.1021/jf0108720 16. r. j. bryant and a. m. mcclung, food chem., 124 (2011) 501. https://doi.org/10.1016/j.foodchem.2010. 06.061 17. d. k. verma and p. p. srivastav, food rev. int., 38 (2020) 111. https://doi.org/10.1080/87559129.2020.1 720231 18. k. ashokkumar, m. govindaraj, s. vellaikumar, v. g. shobhana, a. karthikeyan and m. akilan, j.sathishkumar. front nutr., 7 (2020) 1. https://doi.org/10.3389/fnut.2020.599119 19. v. r. hinge, h. b. patil and a. b. nadaf,rice, 178 (2016) 619. https://doi.org/10.1007/s12010-0151898-2 20. l. g. dias, g. h. b. duarte, l. r. b mariutti and n. bragagnolo,food res. int., 123 (2019) 550. https://doi.org/10.1016/j.foodres.2019.05 .025 pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 38 21. y. m. a. m. wijerathna, n. s. kottearachchi and d. p. s. t. g. attanayaka, trop.l agric., 160 (2012) 123. https://doi.org/10.4172/23298863.1000187 22. v. ramtekey, s. cherukuri, k. g. modha, a. kumar, u. b. kethineni, g. pal, a. n. singh and s. kumar, rev. anal. chem., 40 (2021) 272. https://doi.org/10.1515/revac-2021-0137 23. z. h. prodhan and s. h. u. qingyao, rice. sci., 27 (2020) 86. https://doi.org/10.1016/j.rsci.2020.01.001 microsoft word pjaec-010708-23.doc pdfmachine from broadgun software, http://pdfmachine.com, a great pdf writer utility! issn-1996-918x pak. j. anal. environ. chem. vol. 9, no. 2 (2008) analytical studies on the quality and environmental impact of commercial motor gasoline available in multan region of pakistan ghulam yasin*1, t. m. ansari1, s. m. s. r. naqvi2 and f. naz talpur3 1*department of chemistry, bahauddin zakariya university, multan 60800, pakistan 2hdip petroleum testing laboratory, multan, pakistan 3national centre of excellence in analytical chemistry, university of sindh, jamshoro 76080, pakistan ------------------------------------------------------------------------------------------------------------------------------------------- abstract physico-chemical characteristics such as specific gravity, reid vapour pressure, copper corrosion, distillation (i.b.p., f.b.p., total recovery & residue) and hydrocarbon contents (saturates, aromatics and polars) of gasoline of different oil marketing companies collected from retail outlets in district multan have been analysed using standard astm procedures. results have been compared with the pakistani, indian and european specifications to assess the quality of pakistani gasoline (petrol). the environmental impact of gasoline has also been assessed. keywords: quality, aromatics, ps specification, environmental impact. ------------------------------------------------------------------------------------------------------------------------------ introduction the term gasoline, an american term, is used widely in usa and the word petrol is used in britain for motor fuels. this product is usually associated with fuel available at car service station. gasoline [1] consists of organic compounds containing carbon and hydrogen (hydrocarbon). substances derived from crude oil have great commercial value. the customary processing of crude oil does not involve the separation and handling of pure hydrocarbons. the products derived from crude oil are always mixtures, occasionally simple, but more often very complex. gasoline is a complex mixture of hydrocarbons that normally boils below 355°f (180°c) or at the most, below 390°f (200°c). the hydrocarbon constituents in this range are those that have 4 to 12 carbon atoms in their molecular structure. these hydrocarbons fall into three categories such as paraffins, olefins and aromatics [2]. automotive gasoline has been classified into two grades, premium and regular on the basis of octane number. gasoline with higher octane number has numerous benefits including reduced exhaust emissions [3] and engine noise, improved cold starting and engine durability. quality [4,5] of motor gasoline is generally determined by measuring its various physico-chemical parameters such as specific gravity, octane number, distillation range, residue, copper corrosion and sediments, etc.employing standard test methods [6,7]. due to speedy mechanization in this era, there has been a tremendous increase in the number of light and heavy vehicles in pakistan that has resulted in a very high demand for motor gasoline. the petroleum [8, 9] products have gained prime importance in our daily life. it has been noticed that some greedy petroleum [10] dealers exploit this essential need of the transport system and mix cheaper oils with these expensive products to earn more profit. there is a general complaint about the poor engine performance and increased exhaust emission by a considerable number of consumers from time to time. consumers suspect the adulteration of different commercially available motor gasoline products. keeping in view, the consumers complaints about poor quality of these commercial products, we thought it worthwhile to test the quality of different locally marketed brands of motor gasoline products. the trend today is towards making gasoline more environment and human friendly or making *corresponding author e-mail: gyasinmalik@hotmail.com id7923921 pdfmachine by broadgun software a great pdf writer! a great pdf creator! http://www.pdfmachine.com http://www.broadgun.com mailto:gyasinmalik@hotmail.com pak. j. anal. environ. chem. vol. 9, no. 2 (2008) gasoline a really clean fuel. most petroleum refineries are facing the challenge of producing motor gasoline having all the desirable properties and also complying with the ever-increasing environmental regulations and health restrictions on automotive emissions. the environmental regulations were created to guard against high levels of lead, aromatics, olefins and sulfur in gasoline, reduction in volatile organic compounds, toxic nitrogen oxides (nox) in automotive tailpipe emissions, and emissions at retail out lets. among the techniques, high-resolution capillary gc equipped with flame-ionization detection (fid) [11] and capillary gc-ms are the most important and most widely used techniques for separation, characterization, and identification of hydrocarbons present in gasoline. in the present study gasoline was fractionated into aliphatic, aromatic and polar hydrocarbons. characterization and identification of individual aliphatic, aromatic, and polar was accomplished by using capillary gc with fid detector [1217]. fifty compounds were identified based on comparison of gc retention data with individual authentic standards. petroleum products contain toxic components and produce acute toxic effects, chronic toxicity and carcinogenity. they can foul shorelines and interfere with water treatment. petroleum oils can cause devastating physical effects such as smothering, causing oxygen depletion, suffocation, egg contamination, destruction of existing and future food supply, affecting breeding animals and habitat. petroleum oil spills can have a severe impact on drinking water resources. oil pollution seriously damages the terrestrial and aquatic environment. it does not take a spill of catastrophic magnitude to have a serious impact on an aquatic habitat. the complex food chain or web, from microorganisms and plants to shellfish, mammals, and birds is affected by even small spills experimental commercially available motor gasoline samples were collected randomly from retail outlets of different marketing companies available in district multan. samples were tested using standard astm procedures. distillation measurements, final boiling points (°c) and residual (% vol.) of motor gasoline samples were determined using astm d-86 method. instrument used for distillation was manufactured by gallenkamp, england. specific gravity 60/60of was measured by glass hydrometer (poulten selfe&lee, england) following the astm method d-1298. reid vapour pressure (rvp) @37.8of was measured using astm method d-323. copper strip corrosion @ 50oc was measured by using copper strip and copper strip corrosion bomb & bath (koehler, usa) following the astm test method d-130. hydrocarbons were measured by perkin elmer model (8700) gas chromatograph. analysis of hydrocarbons by gas chromatography a 0.2-µl of each gasoline sample was injected without prior treatment to a perkin elmer (pe) 8700 gas chromatograph equipped with a flame-ionization detector (fid) with a 1-min purge-off. a polar capillary column sp-2340 (60 m × 0.25mm) having stationary phase of methyl lignocerate was used. the carrier gas, nitrogen (3.5 ml/min) transported the vaporized sample through the column in which it partitioned into individual components, which were sensed by fid detector as they eluted from the end of the column. the detector signal was recorded with integrating computer. each eluting component of each gasoline sample was identified by comparing its retention time with the retention time of reference standard under identical conditions (table-1). following temperature programme was used: initial temperature70°c, ramp rate 4°c per minute, second temperature 120°c, ramp rate 10°c per minute, final temperature 220°c, stay time 10 minute at 220°c.the concentration of each component in wt% was determined by measuring the peak area. the components were sum up as saturates, aromatics and polars. motor gasoline (petrol) is an important fuel in human life but it is also responsible for deteriorating ambient air quality (aaq) through fugitive and exhaust emissions. oxygenates help to reduce co emission and they are excellent octane improvers. oxygen limit at a maximum of 2-7 wt/wt % in the fuel is put by the international specifications. the us clean air act of 1990 had specified that oxygenates must be added in the gasoline sold in 41 cities of us which were under non-attainment limit of co. in pakistan oxygenates are not used in gasoline and ministry of petroleum has not specified about oxygenates in petrol while european gasoline specification for oxygenates is 2-3 wt/wt % (table-2) and indian specification is 2-5 wt/wt % (max.) (table-2). it means that oxygenates are not present in petrol. lower aromatics reduce the reactivity of emissions; produce less engine deposits and contribute to lower benzene emissions. presently there are no limits for aromatics and olefins in pakistani gasoline. european specification for olefins is 18 %v/v (max.) and for aromatics is 42 % v/v (max.) (table-2) while indian specifications for olefins are 10 %v/v (max.) and 35 %v/v aromatics (max) (table-2). in future pakistan mailto:@37.8 pak. j. anal. environ. chem. vol. 9, no. 2 (2008) may have to limit the aromatics up to a maximum of 3540 % v/v or wt/wt %. similarly, the limits for olefins at around 15-20 % v/v or wt/wt % may have to be implemented. in our study we have determined aromatics 43.19 wt %( table-3) in pso gasoline, 42.35 wt % (table-4) in shell gasoline and 32.98 wt % (table-5) in caltex gasoline. these quantities of aromatics in pakistani gasoline are high. pso gasoline has been found paraffins (saturates) 32.34 wt % (table3), 39.74 wt % (table-4) in shell gasoline and 41.68 wt % (table-5) in caltex gasoline. the concentration of polars 7.830 wt %(table-3) in pso gasoline, 6.810 wt %(table-4) in shell gasoline and 4.170 wt % (table-5) in caltex gasoline while indian standards have maximum 10 %v/v olefins and 35 % v/v aromatics. pakistani gasoline has very high concentration of aromatics as compared to indian gasoline and is not environmentally favorable. caltex gasoline has low concentration of aromatics (32.98 wt %) as compared to pso and shell gasoline. european and indian standards have 1 % v/v benzene limit. the benzene (carcinogenic) concentration in pakistani petrol sample is high which must be reduced and its limit should be specified by the psqca. the benzene concentration in pso gasoline is 3.515-5.073 wt % (table-3), 4.930-6.640 wt % (table4) in shell gasoline and 3.890-7.540 wt % in caltex gasoline (table-5). these concentrations of benzene in petrol are very high. as discussed earlier, the concentration of total aromatic compounds must be decreased. the concentration of benzene and total aromatics show that pakistani petrol is of not a good quality as compared to european gasoline. if aromatics concentration is reduced in gasoline then there will be problems that octane number of petrol will be reduced, that affects the performance of the engine. aromatics are octane number boosting agents, and lower aromatics concentrations lower the octane number, and result in poor quality of petrol. octane boosting compounds are not environmentally friendly because aromatics produce more smoke and smog. olefins form engine fouling gums, more smoke and smog. short-term effects of benzene and other aromatics include: anemia, nervous system disorders, depressed immune system and death when exposed to extremely high concentrations, dizziness, drowsiness, rapid heart beat, disorientation, unconsciousness, headaches, giddiness, loss of muscular control are other symptoms. long-term effects include: possible reproductive damage (sex depression), damage to chromosomes, cancer, especially a type of leukemia known as acute myeloid leukemia (aml), dermatitis. ground level pollution includes: ozone depletion, greenhouse effect and acid rain. specific gravity range of pso petrol is 0.7510.767 (table-3), shell petrol is 0.754-0.760 (table4), caltex petrol is 0.755-0.762 (table-5) and ps limit is 0.76(max.) (table-2). on the basis of specific gravity all petrol samples are according to the prevalent pakistan standard limits. low specific gravity shows good quality of gasoline. lower rvp reduces vehicle evaporation emissions from fuel tanks, carburetor, running losses, and refueling emissions. ried vapour pressure range of pso petrol is 9.500-9.800 psi, shell petrol range is 9.700-10.00 psi, caltex petrol range is 9.500-9.900 psi and ps limit is 10 psi (max.) (table-2). this data shows that shell petrol is of good quality. high reid vapour pressure shows that there are high concentrations of light components. these light components give adequate vaporization of fuel air mix for easy engine cold start. too many heavy components contribute to chamber deposits and spark plug fouling causing release of unburnt hydrocarbons into the atmosphere. copper strip corrosion of all these petrol samples is according to the ps limit (1 max.). it means that all the patrol samples are neutral. in distillation, the final boiling point range of pso petrol samples is 182.0-190.0oc, shell petrol range is 180.0-182.0oc, caltex petrol range is 185.0188.0oc and ps limit is 205oc(table-2).lower value of final boiling point indicates good quality. it means that shell petrol is of good quality. indian standard limit for final boiling point is195oc (table-2) while ps limit is 205oc (table-2) which is very high and must be improved. lowering of 90 % point helps to reduce hydrocarbon contents and co emissions and engine deposits during cold start and warm-up. the gas chromatographic results, reveal the presence of c14 & c15 in all petrol samples which show that there is a mixing of some heavy fraction to earn more profit. this practice has bad impact on the petrol quality and on the environment. pak. j. anal. environ. chem. vol. 9, no. 2 (2008) table-1. retention times of hydrocarbon standards by gas chromatography _______________________________________________________________ standard retention time (min.) standard retention time (min.) (saturates) pentane 3.40 hexane 3.43 heptane 3.48 nonane 3.63 decane 3.89 c14h30 8.04 c15h32 9.50 (aromatics) toluene 3.79 benzene 4.1 ethylbenzene 5.28 xylene 5.34 cumene 5.60 ter. butyl benzene 6.34 polars pentanol 7.17 alpha naphthol 6.99 table 2. standard specification of european, indian and pakistani gasoline. _____________________________________________________________________________________ property european* aiamcharter** pakistani standard*** parliament 2005(indian) specification 2005 colour red (ulp) pinkish sp. gravity 60/60of**** (max.) 0.76 reid vapour pressure (psi)@37.8of max. 50 kpa 10 copper strip corrosion @ 50oc max. 1a unleaded (%) 100 distillation initial boiling point (oc) report 10% recovery (oc) max. 70 10-40 % recovery (oc) max. 70 46% recovery (oc) max 10 50% recovery (oc) max. 125 65%recovery (oc) max. 100 75% recovery (oc) max. 100 90% recovery (oc) max. 180 180 final boiling point (oc) max 195 205 total recovery (vol %.) report residue (% v/v) maximum 2.0 .olefins (% v/v) max. 18 10 aromatics (%v/v) max 42 35 benzene (% v/v) max. 1.0 1.0 oxygen (wt/wt %) 2-3 2-5 sulphur (%wt/wt) max. 0.005 0.003 *data reproduced from cleaning the air �better vehicle, better fuels; published by tata energy research institute (teri) india, p.245, 2002. ** data source. aiam (1999) and bis (1995 b), clearing the air �better vehicle, better fuels; published by tata energy research institute (teri) india, p-231, (2003). ***standard test limits are produced from pakistan standard 1430:1999 (udc: 665.733.5) ****ministry of petroleum & natural resources of has directed �to report� the sp. gravity at 60/60of. mailto:(psi)@37.8 pak. j. anal. environ. chem. vol. 9, no. 2 (2008) table-3. analytical results of physical parameters and hydrocarbon contents in gasoline of pso _____________________________________________________________________________________ parameter astm s-1 s-2 s-3 s-4 range mean stdev. method colour pink pink pink pink 0.000-0.000 0.000 0.000 sp. gravity 60/60of d-1298 0.761 0.754 0.767 0.761 0.751-0.767 0.761 0.005 reid vapour pressure (psi) @37.8of d-323 9.800 9.600 9.500 9.700 9.500-9.800 9.650 0.129 copper strip corrosion @ 50oc d-130 1a 1a 1a 1a 0.000-0.000 1a 0.000 residue/loss 1/1 1/1 1/1 1/1 0.000-0.000 1/1 0.000 distillation d-86 i.b.p. (oc) 45.00 42.00 42.00 42.00 42.00-45.00 42.75 1.500 10%recovery (oc) 58.00 58.00 58.00 58.00 0.000-0.000 58.00 0.000 50% recovery (oc) 98.00 95.00 100.0 98.00 95.00-100.0 97.75 2.061 90% recovery (oc) 145.0 142.0 148.0 145.0 142.0-148.0 145.0 2.450 f.b.p. (oc) 185.0 182.0 190.0 185.0 182.0-190.0 184.0 3.317 total recovery (vol %) 98.00 98.00 98.00 98.00 0.000-0.000 98.00 0.000 pentane (wt %) 12.71 7.220 12.11 -7.220-12.71 10.68 3.011 hexane (wt %) 13.96 7.540 11.77 -7.540-13.96 11.09 3.264 heptane (wt %) 2.249 2.500 1.990 -1.990-2.500 2.246 0.255 octane (wt %) 2.247 3.220 2.970 -2.247-3.220 2.812 0.505 nonane (wt %) 0.000 1.630 1.530 -1.530-1.630 1.580 0.071 decane (wt %) 0.000 0.525 0.640 -0.525-0.640 0.583 0.081 c14h30 (wt %) 2.741 2.555 1.911 -1.911-2.741 2.402 0.436 c15h32 (wt %) 0.425 0.637 0.406 -0.406-0.637 0.489 0.128 toluene (wt %) 0.930 0.000 0.420 -0.420-0.930 0.675 0.361 benzene (wt %) 5.037 3.515 5.073 -3.515-5.073 4.542 0.889 ethylbenzene (wt %) 11.16 9.126 18.20 -9.126-18.20 12.83 4.761 xylene (wt %) 19.27 13.13 11.56 -11.56-19.27 14.65 4.074 cumene (wt %) 11.16 7.620 7.890 -7.620-11.16 8.890 1.971 ter.butylbenzene (wt %) 2.269 1.880 1.340 -1.340-2.269 1.830 0.500 alpha naphthol (wt %) 9.447 7.863 6.180 -6.180-9.447 7.830 1.630 total saturates(wt %) 34.34 29.34 33.33 -29.34-34.34 32.34 2.640 total aromatics (wt %) 49.83 35.27 44.48 -35.27-49.83 43.19 7.360 total polars (wt %) 9.450 7.860 6.180 -6.180-9.450 7.830 1.640 ____________________________________________________________________________________ mailto:@37.8 pak. j. anal. environ. chem. vol. 9, no. 2 (2008) table 4. analytical results of physical parameters and hydrocarbon contents in gasoline of shell _____________________________________________________________________________________ parameter astm s-1 s-2 s-3 s-4 range mean stdev. method colour pink pink pink pink 0.000-0.000 pink 0.000 sp. gravity 60/60of d-1298 0.760 0.756 0.754 0.759 0.754-0.760 0.757 0.003 reid vapour pressure (psi) @37.8of d-323 10.00 9.900 9.700 9.800 9.700-10.00 9.850 0.129 copper strip corrosion @ 50oc d-130 1a 1a 1a 1a 0.000-0.000 1a 0.000 residue/loss 1/1 1/1 1/1 1/1 0.000-0.000 1/1 0.000 distillation d-86 i.b.p. (oc) 45.00 45.00 45.00 42.00 42.00-45.00 44.25 1.500 10%recovery (oc) 58.00 56.00 56.00 58.00 56.00-58.00 57.00 1.155 50% recovery (oc) 98.00 95.00 95.00 98.00 95.00-98.00 96.50 1.732 90% recovery (oc) 148.0 145.0 148.0 145.0 145.0-148.0 146.5 1.732 f.b.p. (oc) 182.0 182.0 182.0 180.0 180.0-182.0 181.5 1.000 total recovery (vol %) 98.00 98.00 98.00 98.00 0.000-0.000 98.00 0.00 pentane (wt %) 3.910 8.020 18.93 13.92 3.910-19.92 11.21 6.585 hexane (wt %) 11.25 15.28 22.70 14.97 11.25-22.70 16.05 4.797 heptane (wt %) 2.250 7.520 7.500 7.000 2.250-7.520 6.068 2.556 octane (wt %) 2.170 3.132 3.100 2.960 2.170-3.132 2.841 0.453 nonane (wt %) 2.100 2.220 2.200 0.973 0.973-2.220 1.873 0.603 decane (wt %) 0.250 0.092 0.092 0.109 0.092-0.250 0.136 0.077 c14h30 (wt %) 2.599 0.225 0.529 1.493 0.225-2.599 1.212 1.071 c15h32 (wt %) 0.376 0.229 0.595 0.238 0.229-0.595 0.360 0.171 toluene (wt %) 1.707 0.101 0.000 0.139 0.101-1.707 0.649 0.916 benzene (wt %) 4.950 6.640 5.257 4.930 4.930-6.640 5.444 0.811 ethylbenzene (wt %) 5.600 7.030 6.600 10.00 5.600-10.00 7.308 1.892 xylene (wt %) 32.15 11.00 17.30 9.650 9.650-32.15 17.53 10.30 cumene (wt %) 11.38 11.38 11.19 4.480 4.480-11.38 9.608 3.420 ter.butylbenzene (wt %) 2.280 1.320 3.040 1.260 1.260-3.040 1.975 0.850 pentanol (wt %) 5.762 0.206 0.445 0.179 0.179-5.762 1.648 2.745 alpha naphthol (wt %) 0.000 5.148 10.37 5.136 5.136-10.37 6.886 3.020 total saturates(wt %) 24.91 36.72 55.65 41.66 24.91-55.65 39.74 12.73 total aromatics (wt %) 58.07 37.47 43.39 30.46 30.46-58.07 42.35 11.74 total polars (wt %) 5.760 5.350 10.82 5.320 5.320-10.82 6.810 2.680 ___________________________________________________________________________________ mailto:@37.8 pak. j. anal. environ. chem. vol. 9, no. 2 (2008) table-5. analytical results of physical parameters and hydrocarbon contents in gasoline of caltex _____________________________________________________________________________________ parameter astm s-1 s-2 s-3 s-4 range mean stdev. method colour pink pink pink pink 0.000-0.000 pink 0.000 sp. gravity 60/60of d-1298 0.756 0.762 0.755 0.760 0.755-0.762 0.758 0.003 reid vapour pressure (psi) @37.8of d-323 9.600 9.900 9.500 9.700 9.500-9.900 9.670 0.171 copper strip corrosion @ 50oc d-130 1a 1a 1a 1a 0.000-0.000 1a 0.000 residue/loss 1/1 1/1 1/1 1/1 0.000-0.000 1/1 0.000 distillation d-86 i.b.p. (oc) 42.00 42.00 45.00 42.00 42.00-45.00 42.75 1.500 10%recovery (oc) 55.00 58.00 55.00 58.00 55.00-58.00 56.50 1.732 50% recovery (oc) 98.00 100.0 95.00 98.00 95.00-100.0 97.75 2.061 90% recovery (oc) 148.0 148.0 145.0 148.0 145.0-148.0 147.3 1.500 f.b.p. (oc) 188.0 188.0 185.0 185.0 185.0-188.0 186.5 1.732 total recovery (vol %) 98.00 98.00 98.00 98.00 0.000-0.000 98.00 0.000 pentane (wt %) 13.88 14.28 15.80 15.16 13.88-15.80 14.78 0.865 hexane (wt %) 8.710 30.82 14.70 9.180 8.710-30.82 15.86 10.34 heptane (wt %) 6.860 7.240 8.020 2.310 2.310-8.020 6.108 2.577 octane (wt %) 2.180 2.070 2.350 2.430 2.070-2.430 2.258 0.163 nonane (wt %) 0.839 0.227 2.130 1.050 0.227-2.130 1.062 0.793 decane (wt %) 0.357 0.113 0.256 0.289 0.113-0.357 0.254 0.103 c14h30 (wt %) 1.667 1.286 0.239 1.600 0.239-1.667 1.198 0.661 c15h32 (wt %) 0.306 0.196 0.000 0.175 0.175-0.306 0.226 0.070 toluene (wt %) 0.263 0.125 0.238 0.263 0.125-0.263 0.222 0.066 benzene (wt %) 4.724 3.890 4.853 7.540 3.890-7.540 5.252 1.584 ethylbenzene (wt %) 8.271 9.722 9.882 9.630 8.271-9.882 9.376 0.744 xylene (wt %) 10.90 9.720 9.342 12.70 9.342-12.70 10.67 1.510 cumene (wt %) 0.431 6.050 6.890 7.870 0.431-7.870 5.310 3.337 ter.butylbenzene (wt %) 5.104 1.132 1.040 1.340 1.04-5.1040 2.154 1.974 pentanol (wt %) 0.233 0.166 0.280 0.172 0.166-0.233 0.213 0.054 alpha naphthol (wt %) 0.000 4.684 5.370 5.753 4.684-5.753 5.269 0.542 total saturates(wt %) 34.80 56.23 43.50 32.19 32.19-56.23 41.68 10.84 total aromatics (wt %) 29.69 30.64 32.25 39.34 30.64-39.34 32.98 4.370 total polars (wt %) 0.023 4.850 5.650 5.930 0.230-5.930 4.170 2.660 ___________________________________________________________________________________ conclusion the motor gasoline available in multan region does not conform to the european or indian standard specifications. these gasoline samples are according to the ps specification. we must improve its quality according to the international standards to save the environment. references 1. g. a. schoonveld and w.f. marshall, �the total effect of a reformulated gasoline on vehicle mailto:@37.8 pak. j. anal. environ. chem. vol. 9, no. 2 (2008) emission by technology.� warrendale: society of automobile engineers [sae paper no. 990380], (1999). 2. bis (bureau of india standards), �motor gasoline � specifications.� (second revision), [is 2796:1995a], (1995). 3. s. s. brown, s. nomoto and f. w. sunderman, �physics and chemistry of petroleum products.� ranjan k. bose (ed.), tata energy research institute, darbari seth block, new delhi, india (1999). 4. j. m. john and s. prakash �clean fuels: an essential need for automotive emission control.� proceedings 20th world petroleum congress, engelhard corporation, 101, wood avenue, nj, usa (2000). 5. t. g. skryabyna, l. i. fedotova, t. i. chekmasova, and v. a. vorotnykova, new methods for monitoring the quality of petroleum and petroleum products, j. khim. tekhnol. topl. masel. (1993) 26. 6. f. k. gad, �a basic programme to estimate basic properties of petroleum oils�, j. trans. egypt. soc. chem., 12(1991) 1-3. 7. roussel, j. and boulet, r., �characterization of crude oils and petroleum fractions�, j. pet. refin.,1 (1995) 453. 8. guibet, jean-claude, �characteristics of petroleum products for energy use�, j. pet. refin. 1 (1995) 453. 9. thiault, b., �standards and specifications of petroleum products�, j. pet. refin., 1 (1995) 453. 10. i. a. l, rhodes, r. z. olvera and j. a. leon, j. hydrocarbon contam. soils. 1, (1991) 273. 11. z wang, and m. fingas, j. gas chromatogr. a., 774 (1&2) (1997) 51. 12. r. hua, y. li, w. liu, j. zheng, h. wei, j. wang, x. lu, h. kong, g. xu, j. chromatogr. a 1019 (2003) 101. 13. j. m. d. dimandja, g. c. clouden, i. colon, j. f. focant, w. v. cabey, r.c. parry, j. chromatogr. a 1019261 (2003). 14. c. vendeuvre, f. bertoncini, l. duval, j.l. duplan, d. thiebaut, m. c. hennion, j. chromatogr. a 1056 (2004) 155. 15. j. beens, m. adahchour, r.j.j. vreuls, k. van altena, u. a. th. brinkman, j. chromatogr. a 919 (2001) 127. 16. f. c. y. wang, w. k. robbins, f. p. di sanzo, f. c. mcelroy, j. chromatogr. sci. 41(2003) 519. 17. colombe vendeuvre, rosario ruiz-guerrero, fabrice bertoncini, laurent duval, didier thiebaut, marie-claire hennion, j. chromatogr. a 1086 (2005) 21. microsoft word 115-126-ok-pjaec-10112020-306-c-received galley pr cross mark issn-1996-918x pak. j. anal. environ. chem. vol. 22, no. 1 (2021) 115 – 126 http://doi.org/10.21743/pjaec/2021.06.12 determination of lisinopril in pure and tablet form by using 2-hydroxynaphthaldehyde as derivatizing reagent zahid ali zounr 1 *, ayaz ali memon 2 , abdul ghani memon 1 , f. m. a. rind 1 , m. y. khuhawar 3 , ghulam quadir khaskheli 1 , mazhar iqbal khaskheli 4 , nazir ahmed brohi 5 and saeed akhtar abro 6 1 dr. m. a. kazi institute of chemistry, university of sindh, jamshoro, pakistan. 2 national center of excellence in analytical chemistry, university of sindh, jamshoro, pakistan. 3 institute of advanced research studies in chemical sciences, university of sindh, jamshoro, pakistan. 4 department of chemistry, government college university, kali mori hyderabad, sindh, pakistan. 5 department of microbiology, university of sindh, jamshoro, pakistan. 6 institute of plant science, university of sindh, jamshoro, pakistan. *corresponding author email: zahid_zounr100@yahoo.com received 10 november 2020, revised 05 march 2021, accepted 10 march 2021 -------------------------------------------------------------------------------------------------------------------------------------------abstract an easy, sensitive and accurate spectrophotometric method has been developed for the determination of lisinopril (lnp) in pure and tablet formulations based on derivatization reaction with 2-hydroxynaphthaldehyde (2hna). the derivatization reaction was carried out in methanol solvent at ph-5.5 at 95±2c for 15 min. the linear calibration curve was obtained that obeyed the beer’s law within the concentration range 5-50 µgml -1 of lnp at 433 nm with a coefficient of determination r²=0.996. the recovery was in the range from 98.25-101.82 with molar absorptivity of drug 9×10 3 mole -1 cm -1 . the method was accurate and precise (intra-day variation 0.05-0.97% and inter-day 0.07-1.6%), with limit of detection (lod) and limit of quantification (loq) 0.264 µgml -1 and 0.8 µgml -1 , respectively. no interferences from the excipients were detected. the method was applied for the rapid analysis of lnp in pharmaceutical products. keywords: lisinopril, spectrophotometry, 2-hydroxynaphthaldehyde, derivatization -------------------------------------------------------------------------------------------------------------------------------------------introduction lisinopril 1-[6-amino-2-(1-carboxy-3-phenylpropylamino)-hexanoyl]-pyrrolidine-2 carboxylic acid. it has a molecular formula of c21h31n3o5 and a molecular weight of 441.52 gmol -1 [1-3]. the structure of drug lnp & reagent 2hna are given below (fig.1). lnp, is the third ace inhibitor permitted for use in the united states; lnp itself is active, unlike enalaprilat (ena). lnp is a significantly more potent inhibitor of ace than enalaprilat in vitro. both are used for heart failure and hypertension treatment and diuretic medications. lnp is an angiotensin converting enzyme inhibitor used in the medication of hypertension and heart failure in prophylactic treatment following myocardial infarction and diabetic nephropathy. lnp are amongst the key therapeutic developments of modern medicine due to their histrionic impact in the treatment of congestive cardiac failure and arterial hypertension. the renin-angiotensin system is instantaneously stimulated as a reflex response in order to conserve blood volume. pak. j. anal. environ. chem. vol. 22, no. 1 (2021)116 o ho 2hydroxynaphthaldehyde o oh n nh2 n h ho o o lisinopril figure 1. structure of drug lisinopril & reagent 2-hydroxynaphthaldehyde the lnp causes blood pressure reduction by 5-6 mm hg, due to which 40% hazard of stroke and 15-20% coronary heart disease can be decreased. various classes of allopathic drugs such as diuretics, antagonists of adrenergic receptors, adrenergic agonists, blockers of calcium channels, ace inhibitors, antagonists of angiotensin ii receptors, antagonists of aldosterone, vasodilators and centrally acting adrenergic drugs are used in the maintenance and treatment of all types of hypertension [4]. it has been observed that in some cases, sub-standard medicinal drugs which do not contain the amount of active ingredient stated on the label are sold in the market. this has prompted the quality control labs to do random sampling of the marketed drugs and determine the active ingredient content for quality control purpose. in developing countries, the quality control labs do not have expensive, sophisticated equipment to embark upon the drug quality control task. the literature reveals various analytical methods were described for the determination of lnp such as titrimetric [5, 6], spectrophotometric [7-15], spectrofluorometric [16-20], chromatographic [21-38], derivative uv-spectrophotometric, [39-40] polarography [41-42], radioimmunoassay [43] and fluoroimmunoassay [44]. spectroscopy is still the most widely used analytical tool for major qualitative and quantitative analysis of pharmaceutical formulations. it delivers key financial and experimental advantages over other techniques. for example, many derivative spectrophotometric methods were established using different reagents, one of them paraskevas and co-workers developed a spectrophotometric method [12] in single and multi-component tablets also containing hydrochlorothiazide (hct), based on the derivatization reaction with 1-fluoro-2,4dinitrobenzene (fdnb, sanger reagent). the active ingredient contents of drug in pure and dosage form were determined, using acetonitrile solvent, at ph 8.2 (borate buffer) in the dark at 60 °c for 45 min. the lnp complex was measured at λmax 356.5 or 405.5 nm (only at 405.5 nm if hct is present). another method was proposed by sbârcea [45] and her team for the quantitative determination of lnp in bulk and pharmaceutical formulations based on the reaction with ninhydrin in the presence of potassium hydroxide. the reaction quantitatively proceeds at a temperature of 95 ± 2ºc, in 10 min and the end product, purple colored, exhibits maximum absorption at 567 nm. pak. j. anal. environ. chem. vol. 22, no. 1 (2021) 117 c o r r h a r n h h . 1 r oh n h r rc b h a b.. 2 r r n r c + h2o schiff's base scheme 1. principle of reaction (schiff’s base reaction) finally, the latest method was developed by shraitah [46] based on the reaction of alizarin with primary amine present in the lnp in the presence of 80% ethyl alcohol. the reaction produced a complex red colored product that absorbs maximally at 434 nm. the above reported methods were not only time consuming but also involve expensive solvents, and thus hard to apply in routine analysis. this prompted us to develop a simple, economical and rapid spectrophotometric procedure to determine lnp from pure and drug formulations. the process is based on schiff base reaction by an aldehyde or ketones reacting with a primary amino group under acid or base catalysis or heat. in the same way, amino group of lnp reacts with the aldehyde group of 2hna reagent. the following general reaction is shown in scheme 1. materials and methods reagents and chemicals all of the materials and reagents used were of analytical grade. lnp (100.21%) was obtained from bosch pharmaceutical (pvt.) ltd. the reagent 2hna (100.1%) was purchased from emd chemicals (gibbstown, nj, usa), acetic acid, potassium chloride, hydrochloric acid, sodium carbonate, sodium bicarbonate, methanol, ethanol were from merck, germany. sodium acetate was from fluka, switzerland. de-ionized water was used throughout the study. commercial tablets tablets corace (bosch pharmaceutical pvt. ltd.), zestril (ici pakistan ltd.), trupril (getz pharma pakistan), which obtained labeled amount of 10 mg/tablet, while lisna (zafa pharmaceutical lab. pakistan) contained 20 mg/tablet lnp and purchased from local market hyderabad, sindh, pakistan. stock solutions standard solutions the stock solution (0.02% w/v) of drug lnp was prepared by dissolving exact weighed 20 mg in 10 ml volumetric flask in methanol. then 1ml above solution diluted up to 10 ml calibrated volumetric flask in the same solvent. the stock solution (1% w/v) of reagent 2hna was freshly prepared by dissolving 0.1 gm in sufficient methanol and the volume was adjusted to 10 ml, achieving 10 mgml -1 concentrations. buffer solutions the buffer solutions were prepared of ph 1-2 by utilizing (0.1m) hydrochloric acid and (0.1m) potassium chloride, ph 3-5.7 pak. j. anal. environ. chem. vol. 22, no. 1 (2021)118 (0.1m) acetic acid and (0.1m) sodium acetate, ph 5.8-8 sodium phosphate monobasic and sodium phosphate dibasic, ph 8-10 sodium carbonate and sodium bicarbonate. instrumentation all measurements were performed using the perkin elmer double beam spectrophotometer,lambda 35 uv/visible spe ctrometer (usa), connected to dell computer, using 1 cm quartz cuvette, and the ph meter was used by model orion 420a ph meter fitted with a glass electrode and reference electrode (orion research inc. boston, usa). method-1 analysis of standard drug lnp the lnp drug solutions 1-5 ml were dissolved in methanol at concentrations 5-50 µgml -1 , and 1 ml of each drug solution was transferred to 10 ml calibrated volumetric flask, followed by the addition of 2 ml of reagent 2hna (1% w/v) prepared in methanol and 1 ml alcoholic acetate buffer ph-5.5. the mixture was heated for 15 min at 95 c ± 1c on a water bath. then flask contents were allowed at room temperature and the volume was adjusted with methanol up to the mark. the absorbance was finally measured at 433 nm against the blank. method-2 application on analysis of commercial tablets an accurately weighed mass of 20 tablets was crushed. the powder of tablets equivalent to 20 mg lnp was weighed and transferred into a 100 ml volumetric flask containing a sufficient amount of solvent methanol. the suspension was stirred for 10 min. the solution was filtered through a filter paper (whatman no. 1), after rinsing with methanol the volume was adjusted to 100 ml. then 1 ml of the resulting solution was shifted to 10 ml stoppered volumetric flask, and 2 ml of reagent 2hna was added, followed by 1 ml acetate buffer ph-5.5. the contents were heated up to 15 min at 95⁰c ± 1⁰c on a water bath, cooled at room temperature (25⁰c), the volume was adjusted and absorbance recorded at 433 nm, against the blank. validation of method for derivatization a newly developed spectrophotometric method for the determination of imine derivative of lnp was validated for linearity, accuracy, % recovery, sensitivity, precision and stability of solutions. linearity for calibration and linearity, five different concentrations of the imine derivative were used in the range of 5-50 µgml -1 . the linearity of the method was determined by plotting the absorbance versus concentration of drug lnp derivative. the slope (m), intercept (b), and the correlation coefficient (r 2 ) were determined from the regression analysis. percent recovery measurement the % recovery was calculated by added pure drug lnp with 2hna derivative solution as % recovery = [(dt – ds) / da] × 100 where dt is the total drug concentration after standard addition; ds is the drug concentration in the imine derivative mixture and da is the drug concentration added. sensitivity the sensitivity of the proposed method was calculated by a limit of detection (lod) and lower limit of quantification (loq) of imine derivative using signal to noise ratio (σ/s) of 3.3 σ /s and 10 σ /s, respectively; pak. j. anal. environ. chem. vol. 22, no. 1 (2021) 119 where σ is the standard deviation of the signal and s is the slope of a corresponding calibration curve. precision the imine derivative solution was analyzed at three intervals a day at 08:00, 16:00, 24:00, h for repeatability and for three consecutive days for reproducibility in order to assess the intermediate precision (intra-day and inter-day). the outcome was expressed as the mean ± sd and percent relative standard deviation (%rsd). results and discussion the motive of this research work was to develop a simple approach for the determination of lnp in pure and pharmaceutical formulations. scheme 2 illustrates the reaction mechanism of preparation of new imine derivative lnp2hna by primary amino group of lnp drug with derivatizing reagent 2hna. o oh n nh2 n h ho o o + o ho 2hydroxynaphthaldehyde (s)-1-((s)-6-amino-2-((s)-1-carboxy-3-phenylpropylamino)hexanoyl)pyrrolidine-2carboxylic acid o oh n n n h ho o o ho (s)-1-((s)-2-((s)-1-carboxy-3-phenylpropylamino)-6-((e)-(2-hydroxynaphthalen-1-yl)methyleneamino)hexanoyl)pyrrolidine-2carboxylic acid -h2o scheme 2. proposed reaction pathway for derivatization of drug to complex formation (lnp-2hna) pak. j. anal. environ. chem. vol. 22, no. 1 (2021)120 the 2hna was used as a derivatizing reagent for the colorimetric analysis of lnp. the reagent 2hna reacted with the drug lnp to produce imine derivative lnp-2hna having a light yellow color. this reaction took place in the acid medium at ph-5.5 with maximum absorbance at (λmax) 433 nm, molar absorptivity of 9×10 3 mole -1 cm -1 . the specific parameters were optimized, which effect the preparation of the 2hna-lnp derivative similarly effect of reagent quantity, ph, heating time and temperature. analytical parameters optimization selection of wavelength the wavelength of maximum absorbance shows a vital role for quantitative determinations. it is crucial to choose the wavelength where the derivative gives optimal absorbance. the absorbance of 20 μgml -1 of lnp and 2hna derivative was measured within the range of 350-500 nm. the (λmax) is optimized in the visible range at 433 nm against a reference. selection of optimal temperature and heating time for the preparation of derivative initially, it was observed that the rate of reaction was very slow at room temperature, therefore the mixture contents were heated and the derivatization reaction was monitored on the optimal wavelength (λmax) 433 nm for 0-30 min with an interval of 5 min at 95 c. figure 2. effect of temperature to the yield of reaction it was observed that the best derivatization occurred by heating the reaction mixture for 15 min at 95 c ± 1c (fig. 2). optimization volume and concentration of reagent the effect of adding different quantities of reagent 2hna solution to 1ml of drug lnp (0.02% w/v) was also studied. the reagent concentration of (1% w/v) 2hna was varied between 0.5-3.0 ml in the 10 ml volumetric flask containing 1ml of drug lnp. there was no change in rising absorbance noticed after the addition of 2 ml reagent. therefore, the best absorbance was measured by adding 2 ml of reagent 2hna as shown in fig. 3. figure 3. effect of reagent 2hna concentration on color development ph effect on derivative at the most optimal conditions, the effect of adding 1 ml of 0.1 m different buffer solutions at ph ranges 2-10 was studied on the derivative. the consistent increase in absorbance was examined from ph 4-6. further ph was specified by using buffer solution at a difference of 0.5 like ph 5.0, 5.5, 6.0, etc. the best maximum absorbance was obtained utilizing acetate buffer solution at ph 5.5 (fig. 4). the addition of other buffers ph 8-10 revealed precipitation. thus acetate buffer ph 5.5 was considered as optimal. pak. j. anal. environ. chem. vol. 22, no. 1 (2021) 121 figure 4. effect of ph on derivative color intensity effect of solvent the effect of solvents on derivative was investigated by the addition of 2 ml of mentioned solvent and compared with 2 ml methanol. the procedure of determining solvent effect explained that the mixture contents containing 1 ml of drug lnp (0.02% w/v), 2 ml reagent 2hna (1% w/v) and 1 ml acetate buffer ph-5.5, were heated for 15 minutes, then cooled same at room temperature 25 c, then 2 ml of following mentioned solvents were added in 10 ml volumetric flask and in the blank. it was observed that none of the following solvents interfered in the lnp-2hna derivative (table 1). table 1. effect of solvents on derivative in terms of maximum absorbance. abs. with methanol abs. with other solvents solvent volume (ml) added lnp-2hna derivative lnp-2hna derivative thf 2 0.422 0.425 acetone 2 0.424 0.426 n-hexane 2 0.421 0.418 ethyl acetate 2 0.423 0.420 isopropanol 2 0.418 0.418 acetonitrile 2 0.425 0.421 propanol 2 0.423 0.420 butanol 2 0.422 0.425 effect of mixing order of reagents various mixing orders in the current work were applied. the absorbance decreased when mixed 1 ml acetate buffer ph-5.5 in drug lnp (0.02% w/v), then reagent 2hna (1% w/v). altering the sequence of mixing by adding 2hna first, then buffer followed by lnp solution also has revealed little amount of absorbance. it was confirmed that the addition of 1ml of drug lnp drug first, then 2 ml reagent 2hna followed by 1 ml buffer ph-5.5 solution provided maximum absorbance of derivative. effect of additives the effect of the possible presence of additives like calcium hydrogen phosphate, maize starch, mannitol, pregelantised maize starch, magnesium stearate on absorbance in the determination of drug lnp was studied. two concentration levels, first at an equal concentration of the drug lnp (0.02% w/v), and second at 10 times the concentration of drug, did not change the absorbance significantly. not more than ±2% change in absorbance was calculated and no any additive interfered in the derivative lnp-2hna during the determination of lnp drug (table 2). table 2. effect of additives on absorption of derivative. abs. with additives (lnp-2hna) derivative additive abs. without additives (lnp-2hna) derivative equal conc. to drug 10x conc. to drug calcium hydrogen phosphate 0.420 0.422 0.423 maize starch 0.421 0.424 0.419 mannitol 0.422 0.418 0.421 pregelantised maize starch 0.423 0.420 0.418 magnesium stearate 0.418 0.416 0.421 percent recovery from dosage form table 3 shows the percentage recovery of lnp-2hna derivative from four different commercial drugs by above pak. j. anal. environ. chem. vol. 22, no. 1 (2021)122 mentioned method (2). the percentage recovery was found more than 98 % in all particular formulations. table 3. application of proposed method on commercial drugs. drug brands labeled amount per tablet (mg) amount found per tablet % recovery corace 10 9.81 98.1 zestril 10 10.06 100.6 trupil 10 9.93 99.3 lisna 20 19.87 99.3 stability of derivative the stability of lnp-2hna derivative was analyzed in terms of absorbance at the concentration of 20 µgml -1 lnp. there was no significant change in absorbance was evaluated within 48 h. calibration graph (beer’s law) a linear calibration curve (fig. 5) regarding the correlation between absorbance and different concentrations of the drug lnp was depicting linearity within the concentration range 5-50 µgml -1 of lnp with 2hna, and correlation coefficient of 99.96% (r²=0.9996). figure 5. linearity curve of spectrophotometric determination reproducibility /repeatability for the stability of derivative, the assessment of interday and intraday repeatability of the procedure is an important parameter. the methanolic solution of lnp 20 µgml -1 was taken in three separate (10 ml) calibrated flasks and the method was applied as mentioned method (1). the method was repeated for three days (n=3). the average mean absorbance of intraday and interday reproducibility for imine derivative was seen as 0.264 µgml -1 and 0.8 µgml -1 with (rsd) values 0.97% and 1.6%, respectively (table 4). table 4. sensitivity comparison of proposed method for imine derivative with ab. parameters imine derivative lnp drug precision (n=3) inter-day intra-day 0.264 0.801 _ limit of detection (lod) 0.97 1.77 sensitivity (μgml-1) limit of quantification (loq) 1.60 3.97 validation of the proposed method statistical evaluations for linearity, sensitivity, percentage recovery, precision, lod and loq of the proposed method were given in (table 5). the comparative study of our developed method with previous reported spectrophotometric methods given in (table 6), that reveals the lod and loq values were smaller over other mentioned reported methods. table 5. statistical evaluations of the developed method. parameters observation derivative color absorption maxima (nm) linearity range (µg/ml) molar absorptivity (l mol-1cm-1) sandell sensitivity (μg/cm2/0.001 abs unit) correlation coefficient (r2) rsd (%) slope (b) intercept (a) percentage of recovery (%) limit of detection (lod) µg/ml limit of quantification (loq) µg/ml intra-day variation (%) inter-day variation (%) light yellow 433 5.0-50.0 0.9104 5.9×10-2 0.996 0.775 0.01 0.0014 98.74 -99.52 0.264 0.8 0.05 -0.97 0.07-1.60 pak. j. anal. environ. chem. vol. 22, no. 1 (2021) 123 table 6. comparison of proposed methods with existing spectrophotometric methods for the assay of lnp in pharmaceutical formulations. precision four drug formulations were being repeatedly analyzed in three successive days in order to evaluate intra-day and interday reproducibility for imine derivative was seen at 0.07% and 1.6%, respectively. the %rsd values lower than 2% were obtained in our studies witness that the developed method was precise (table 5). accuracy the accuracy of the proposed method has been evaluated by applying the developed method for the determination of lnp in pharmaceutical formulations. the concentration of each drug was determined from the corresponding regression equations. the obtained percentage recoveries indicate the appropriate accuracy of the proposed method. the standard addition method was also carried out to analyze the accuracy of the method. method accuracy was assessed for the determination of the commercial tablets by adding varying amounts of the standard lnp to a certain concentration of filtrate tablet solution. the findings showed good recoveries with low rsd. commercial formulations have been successfully analyzed for the proposed lisinopril method and the results were compared with the reference method [8], (table 4). the proposed method produces good results in both raw and pharmaceutical formulations (table 5). specificity the proposed method was determined successfully for lnp without any interference from tablet excipients, as depicted in table 2. conclusion a rapid, simple and economical spectrophotometric method using inexpensive reagents was developed for determination of lisinopril in pure and tablet form. our method is robust in terms of reproducibility and high sensitivity. the novelty of this proposed method is to utilize first time 2hna reagent for derivatization of lnp drug. the lod and loq values are smaller over other spectrophotometric methods reported in the literature. moreover, the synthesized lnp imine derivative is highly stable. acknowledgement authors are thankful to alkemy pharmaceutical laboratories (pvt.) ltd. hyderabad for kindly provide us the drug derivatizing reagent λmax (nm) reaction time (min/temp) lod (µg ml–1) loq (µg ml–1) linear range (µg ml–1) references 1,2-naphthoquinone-4sulfonic (nqs) 481 5/ 25°c 1.16 3.53 5-50 [9] alizarin 434 7/40°c 2.08 6.94 4-300 [46] ninhydrin 600 5/80 °c 5.587 18.437 10-150 [47] phenylhydraizine 362 20/85°c 40-200 [48] ascorbic acid 530 15/100°c 0.349 1.152 5-50 [49] chloranil 346 4-20 [50] 2-hydroxynaphthaldehyde (2hna) 433 15/95⁰c 0.264 0.8 5-50 current work pak. j. anal. environ. chem. vol. 22, no. 1 (2021)124 samples. we express our gratitude to prof. dr. shahabuddin memon, head of national centre of excellence in analytical chemistry, for providing laboratory facilities. conflict of interest the authors declare that there is no conflict of interest. references 1. british pharmacopoeia commission, british pharmaco-poeia 2012, the stationery office, norwich, (2012). 2. united states pharmacopeia and national formulary (usp 36–nf 31), united states pharmacopeia convention, rockville, (2012). 3. society of japanese pharmacopoeia, japanese pharmacopoeia, 16th ed., tokyo maruzen company ltd., (2012). 4. l. l. brunton, j. s. lazo, & k. l. parker, (2006). goodman & gilman. as bases farmacológicas da terapêutica. 11ª ed. rio de janeiro: mc gwaw-hill suteramericana do brasil. 5. a. w. olalowo, o. m. adegbolagun, and o. a. bamiro, african j. pharm. pharmacol., 9 (2015) 165. https://doi.org/10.5897/ajpp2014. 4031 6. k. basavaiah, k. tharpa and k. b. vinay, eclética química, 35 (2010) 7. https://doi.org/10.1590/s010046702010000200001 7. a. g. memon, pak. j. anal. environ. chem., 21 (2020) 27. http://doi.org/10.21743/pjaec/2020.06 .04 8. v. m. sarma, n. v. s. venugopal, and l. giribabu, am. j. anal. chem., 11 (2020) 289. http://doi.org/10.4236/ajac.2020.118023 9. a. a. a. ali and a. a. elbashir, ame. acad. schol. res. j., 5 (2013) 106. 10. c. m. jamakhandi, c. javali, i. j. disouza, s. u. chougule and k. a. mullani, int. j. pharm. sci., 3 (2011) 185. 11. s.naveed, mod. chem. appl., 2 (2014) 2 http://dx.doi.org/10.4172/23296798.1000137 12. g. paraskevas j. pharm. biomed. anal., 29 (2002) 865. https://doi.org/10.1016/s07317085(02)00207-8 13. s. a. shama, a. s. amin, h. omara, j. chil. chem. soc., 56 (2011) 566. http://dx.doi.org/10.4067/s071797072011000100009 14. f. f. mohammed, k. badr el-din and s. m. derayea, j. adv. biomed. pharm., sci., 2 (2019) 47. http://doi.org/10.21608/jabps.2019.676 6.1031 15. z. zaheer, s. khan, m. sadeque, m. s. baig and j. n. sangshetti, j. indian assoc. pediatr. surg.,1 (2016) 12. 16. s. m. derayea, k. m. badr eldin, f. f. mohammed. luminescence, 32 (2017) 1482. https://doi.org/10.1002/bio.3348 17. s. m. derayea, k. m. b. el-din and f. f. mohammed, spectrochim. acta part a, 188 (2018) 318. https://doi.org/10.1016/j.saa.2017.07.021 18. m. sobhy, m. e. h. el-sadek and n. m. saeed, j. pharm. pharm. res., 2 (2018) 1. http://doi.org/10.26502/jppr.0005 19. c. m. jamakhandi, c. javali, s. kumar, s. kumar and s. d. s. kumar, int. j. pharm. sci. drug res., 2 (2010) 182. https://innovareacademics.in/journal/ijpp s/vol2suppl4/899. pak. j. anal. environ. chem. vol. 22, no. 1 (2021) 125 20. c. k. zacharis, p. d. tzanavaras, d. g. themelis, g. a. theodoridis, a. economou and p. g. rigas, anal. bioanal. chem., 379 (2004) 759. http://doi.org/10.1007/s00216-004-2530-4 21. k. m. fahelelbom, m. m. al-tabakha, n. a. eissa, d. e. e. obaid and s. sayed, res. j. pharm. technol., 13 (2020) 2647. http://doi.org/10.5958/0974360x.2020.00470.9 22. m. s. arayne, n. sultana, a. tabassum, s. n. ali and s. naveed, med. chem. res., 21 (2012) 4542. http://doi.org/10.1002/cjoc.201190226 23. f. elsebaei and y. zhu, talanta, 85 (2011) 123. http://doi.org/10.1016/j.talanta.2011.03.037 24. y. z. fawzi elsebaeia. talanta, 85 (2011) 123. http://doi.org/10.1016/j.talanta.2011.03.037 25. n. s. goud, g. achaiah, v. sivaramakrishna and p. mayuri, int. j. pharm. tech. res., 8 (2015) 448. https://www.scholarsresearchlibrary.com /articles 26. v. kumar, r. p. shah and s. singh, j. pharm. biom. anal., 47 (2008) 508. http://doi.org/10.1016/j.jpba.2008.01.041 27. y. latha and d. g. sankar, asian j. res. chem., 8 (2015) 27. http://doi.org/10.59558/09744150.2015.00006.1 28. y. x. liu, d. shou, m. l. chen, z. d. chen, p. m. zhang and y. zhu, chin. chem. lett., 23 (2012) 335. https://doi.org/10.1016/j.cclet.2011.11.024 29. v. maslarska and j. tencheva, int. j. pharm. bio. sci., 4 (2013) 163. 30. j. v. odovic, b. d. markovic, r. d. injac, s. m. vladimirov and k. d. karljikovic-rajic, j. chromatogr. a, 1258 (2012) 94. https://doi.org/10.1016/j.chroma.2012.08 .038 31. j. j. pandya, m. sanyal and p. s. shrivastav, j. liq. chromatogr. rel. technol., 19 (2017) 12. https://doi.org/10.1080/10826076.2017.1 324482 32. n. m. rao and d. gowrisankar indian j. pharm. sci., 78 (2016) 217. http://doi.org/10.4172/pharmaceuticalsciences.1000106 33. n. rastkari and r. ahmadkhaniha, biomed. chromatogr., 1 (2017) 10. http://doi.org/10.1002/bmc.4120 34. a. sana, s. naveed, f. qamar, s. shakeel pak. j. pharm. sci., 30 (2017) 635. http://doi.org/10.12991/marupj.300842 35. s. şenkardeş, t. özaydın, t. uğurlu and ş. g. küçükgüzel, marmara pharm. j. 21 (2017) 338. http://doi.org/10.12991/marupj.300842 36. n. shafi, f. a. siddiqui, n. sultana and m. s. arayne. j. liq. chromatogr. rel. tech., 38 (2015) 1466. https://doi.org/10.1080/10826076.2015.1 050503 37. t. b. stoimenova, m. piponski, g. t. serafimovska and m. stefova, maced. j. chem. chem. eng., 36 (2017) 201. http://dx.doi.org/10.20450/mjcce.2017.1 210 38. n. sultana, s. naveed and m. arayne, j. chromatogr. separ. techn., 4 (2013) 1. http://doi.org/doi: 10.4172/jbb.1000171 39. c. v. n. prasad, r. n. saha and p. parimoo, pharm. pharmacol. commun., 5 (1999) 383. pak. j. anal. environ. chem. vol. 22, no. 1 (2021)126 https://doi.org/10.1211/14608089912873 5027 40. n. erk, spectroscopy letters, 31 (1998) 633. https://doi.org/10.1080/00387019808002756 41. v. k. redasani, p. r. patel, d. y. marathe, s. r. chaudhari, a. shirkhedkar and s. j. surana, j. chil. chem. soc., 63 (2018) 4126. http://dx.doi.org/10.4067/s071797072018000304126 42. w. gul, z. augustine, s. khan, k. saeed and h. raees, j. bioequiv. availab., 9, (2017) 31. http://doi.org/10.4172/jbb.1000320 43. p. j. worland and b. jarrott, j. pharm. sci., 75 (1986) 512 https://doi.org/10.1002/jps.2600750518 44. a. s. yuan and j. d. gilbert, j. pharm. biomed. anal., 14 (1996) 773. https://doi.org/10.1016/07317085(95)01718-6 45. l. sbarcea, l. udrescu, l. drăgan, c. trandafirescu, z. szabadai and m. bojiţă, farmacia, 62 (2014) 107. 46. m. shraitah and m. m. okdeh, mod. chem. appl., 4 (2016) 2. doi: 10.4172/2329-6798.1000172 47. a. raza and t. m. ansari, j. chin. chem. soc., 52 (2005) 1055. https://doi.org/10.1002/jccs.200500149 48. a. el-gindy, j. pharm. biomed. anal., 25 (2001) 913. https://doi.org/10.1016/s07317085(01)00376-4 49. n. rahman, j. braz. chem. soc., 16 (2005) 1001. https://doi.org/10.1590/s010350532005000600018 50. a. f. el-yazbi, h. h. abdine, j. pharm. biomed. anal., 19 (1999) 8. https://doi.org/10.1016/s07317085(98)00110-1 microsoft word 03-205-214-pjaec-17022022-431-c-revised-02-01-2023 cross mark issn-1996-918x pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 205 – 214 http://doi.org/10.21743/pjaec/2022.12.03 impact of heavy metals toxicity on children and adults from local rice varieties of northern nigeria abdul a. olaleye *1 . habibat o. adubiaro 2 , aminu dauda 1 and yahaya a. adamu 1 1 department of chemistry, faculty of science, federal university dutse, nigeria. 2 department of industrial chemistry, federal university oye-ekiti, nigeria. *corresponding author email: lebdul@fud.edu.com received 17 february 2022, revised 07 december 2022, accepted 14 dece mber 2022 -------------------------------------------------------------------------------------------------------------------------------------------abstract the current study aimed to investigate the levels of heavy metals [cadmium (cd), zinc (zn), arsenic (as), lead (pb), and mercury (hg)] in local rice varieties named jamila (jm), santana (stn), kwandala (kw), and sipi (sp) collected from danbatta town of kano state, northwestern nigeria. the sa mples of local rice varieties were digested using hno3 and hcl as digestion acids in a ratio of 2:1 (v/v). the digested samples were later analyzed for heavy metals using atomic absorption spectrophotometer. moreover, the health risk assessment of heavy metals by the consumption of local rice varieties among local children and adults was also estimated based on estimated daily intake (edi), target hazard quotient (thq), hazard index (hi), and target cancer risk (tcr). the concentrations of cd, zn, and pb dry weight basis observed in the range of 0.0020.06, 0.02-20.0, and 1.16-14.2 mg/kg, respectively. hg was detected only in the sp rice variety with a concentration of 0.022 mg/kg. whereas, as was detected in stn (0.086 mg/kg), kw (0.006 mg/kg), and sp (0.028 mg/kg). the resulting data showed that cd, zn, hg, and as were within the maximum permissible limits set by regulatory bodies. the edi values ranged from 1.21e-5 -1.21e-1 and 5.0e-6 – 5.0e-2 for children (24 kg body weight) and adults (70 kg body weight), respectively. the data of the non-carcinogenic risk assessment indicated that the thq values of cd, hg, and as were less than the maximum permissible limit of 1.0 for both children and adults. the hi data showed the potentially high possible health risk of the heavy metals by the consumption of the studied local rice varieties, with pb being the major contributor. similarly, resulting data of tcr for cd and pb showed high cancer risk upon the consumption of the studied local rice varieties over a long time. keywords: local rice, heavy metals, target hazard quotient, hazard index -------------------------------------------------------------------------------------------------------------------------------------------introduction food safety is a significant challenge in most developing countries, especially in africa. most foods consumed in these countries are contaminated with a series of pollutants. feeding on unwholesome food materials has severe health and economic impacts on the populace [1, 2]. nigeria ranks second among the nations across the globe in rice importation, purchasing about two million metric tons in one year from countries like thailand and china [3]. due to their public health implications, exposure to heavy metals by humans has been the main focus of attention among researchers and health and nutrition experts [4]. these contaminants could occur naturally as constituents of the earth's crust and could also be distributed through various activities of human beings. pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 206 pollution of the environment by heavy metals, even at low concentrations, and their long– term cumulative health effects are among the leading global health concerns. heavy metals are non-biodegradable and remain in aquatic and terrestrial environments for long periods. they could be transported from soil to water bodies or absorbed by cereal crops and eventually gets to humans through their consumption. undoubtedly, high traffic and industrial activities are the major contributors to high levels of heavy metals in the environment. plants growing around these areas are likely to be contaminated by these heavy metals by the absorption from the soil, or the leaves may absorb atmospheric contaminants. soil contamination by heavy metals can transfer to food and accumulate in consumers. for instance, plants may be accumulating these heavy metals from contaminated soil without physical changes or visible indication, which could pose a potential risk for humans, and animals. most of these heavy metals may have long last harmful effects due to their non-biodegradable nature, and long biological half-lives lead to accumulation in different body parts. most of them are very toxic because they are soluble in water. these heavy metals may harm humans and animals even at low concentrations because the body finds it difficult to get rid of them [5, 6]. for example, continuous cd consumption leads to respiratory system damage and lung cancer [7]. hg is a potent neurotoxin found in a variety of products. it affects the brain, liver, and kidneys and can cause developmental disorders in children. methyl mercury is an organic contaminant capable of damaging the central nervous system [8]. heavy metals generally affect the physiological functions of plants and retard nitrogen fixation and plant growth [9]. heavy metals usually combine with the thiol, amino, and imino groups of protein and form a metal complex. this makes the proteins lose their biological functions and cause the breakdown of the cells [9]. rice is a common staple food produced in northern nigeria. zamfara, jigawa, and borno states are the major contributors to rice production because of the application of specialized processing equipment [10]. statistical data indicated that the annual rice consumption in nigeria is about 5.5 million tons, while the local production of rice is about 1.8 million tons [11]. rice farmers in nigeria account for nearly three million out of about sixty million rural dwellers, and approximately five million hectares of arable land are suitable for rice production [10]. rice is an important cereal food that fulfilled the potential nutrition effectively for the poor population in remote areas of emerging countries [12]. orisakwe et al. [13] and otitoju et al. [4] reported high levels of pb and hg in pumpkin leaves obtained from a construction site in uyo in the eastern part of nigeria. consumption of local rice is increasing in nigeria because rice is one of the major staple foods available in the country. local rice is currently being eaten by more than one million people daily. therefore, there is a need to investigate local rice varieties being consumed for their toxic effects. uddin et al. [14] reported that many countries have assessed and monitored heavy metals in foods and vegetables. however, information about the toxicity and health risk parameters of the heavy metals in locally grown rice in nigeria is scarce. therefore, this study investigates the levels of heavy metals in locally produced rice varieties in northern nigeria, followed by the heavy metals' possible carcinogenic and non-carcinogenic pak. j. anal. environ. che m. vol. 23, no. 2 (2022)207 risks assessment based on their consumption by children and adults. materials and methods location of the rice cultivation the local rice varieties were grown at danbarta town in danbarta local government area of kano state, nigeria. danbarta is located about forty-nine miles north of kano city at the northern border of kano state, northwestern nigeria. it has an area of 932 square kilometers with a latitude of 12 o 25 ' 12 '' and a longitude of 8o38'4''. danbatta is bordered north, east, south, and west by kazaure, babura, minjibir, and makoda local government areas, respectively. the average temperature is 33oc with a humidity level of 14%. the zonal office of kano state agricultural and rural development agency is located in this area, where agriculture is a major economic activity. residents of this region are recognised for their farming endeavours. because they adhere to the philosophy of eating what they create, they consume a lot of local cuisines, particularly rice. samples collection and preparation the local rice varieties were purchased in the shuwarin market in dutse, jigawa state, from retailers who had earlier obtained them from the danbarta local government area of kano state, nigeria. the samples were washed with distilled water in the department of chemistry, federal university dutse, and spread on a clean stainless steel tray to allow the water to drain off. the samples were separately packaged inside labeled envelopes and dried in the gallenhkamp oven at 65 o c for two days, pulverized into a fine powder using electric stainless steel excella mixer grinder. the pulverized samples were sieved by a 2 mm sieve to obtain fine particles. figure 1. map of the study area, danbarta area of kano state, nigeri a materials, chemicals and reagents all the materials and chemicals used for the analysis were of analytical grade. they include ceramic mortar and pestle, analytical balance (mettler toledo, switzerland), digital timer hotplate (thermo scientific, usa), vortex shaker, different sizes of volumetric flasks (pyrex, usa), different sizes of the beaker (pyrex, usa), whatman filter paper (150 mm diameter, ge healthcare, usa), hno3 and hcl (sigma aldrich product of united states of america), distilled water and stock solutions of the metals obtained from physical and chemical laboratories, n.i.m.g, jos, nigeria. samples digestion and analysis five grams of each dried sample of local rice varieties was weighed into digestion flasks, and add 8.0 ml of hno3 and 4.0 ml hcl (2:1; v/v). the digestion flasks were placed on a hot plate and heated at 80 oc. the temperature was gradually increased up to 120 o c until there was complete digestion. the digested samples were diluted with distilled water and made up to 100 ml, as reported elsewhere [15]. heavy metal concentrations in the samples (pb, as, zn, cd and hg) were determined using a perkin elmer as 3100 flame atomic absorption spectrophotometer (faas) facility from physical and chemical laboratories, n.i.m.g, jos, nigeria. pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 208 quality control and assurance the instrument used for the analysis was well calibrated to check the accuracy of the instrument and traceability of the measurement with certified reference standard solutions covering the desired concentration of the analytes in the samples. the reference standard stock solution (1000 mg/l) of each heavy metal was obtained from n.i.m.g, jos, nigeria. the stock solution was later diluted for calibration by aas. purification of the reagents blanks (acids) was carried out by subboiling distillation to remove the trace amounts of the metals in the acids, thereby removing background contamination of heavy metals. the spike recovery technique was used for the analytical procedure. this was achieved by introducing a known standard of the metals into already analyzed samples and re-analyzing it again. there was more than 96% recovery for all the metals. the relative standard deviation, a measure of the precision of results, was less than 5.0%. the limit of quantification (loq) for zn and cd was 0.005 mg/l, whereas that of as, pb, and hg was 0.007 mg/l. health risk assessment the following parameters were used to assess the non-carcinogenic and carcinogenic health risk, such as estimated daily intake, target hazard quotient, and chronic hazard index. estimated daily intake (edi) of the heavy metals edi of heavy metals was calculated using the equation [14]: where cm is the concentration of heavy metals (mg kg-1 dry weight), rf represents the daily intake of food in kg per person per day, and bw is the average body weight in kg (24 kg for children; 70 kg for adults). non-carcinogenic risk target hazard quotient (th q) thq was calculated using the following formula [14]: where thq denotes non-cancer risks, ef represents the exposure frequency (365 days/year), de represents exposure duration (65 years), and df denotes reference dose. df of zn, pb, cd, hg, and as are 0.03, 0.0035, 0.003, 0.0001, and 0.0003 mg/kg/day, respectively [16, 14], and tavncar represents the average time for non-carcinogens is 365 days/year x de [17]. chronic hazard index (hi) the chronic hazard index is the sum of more than one hazard quotient for multiple toxicants or multiple exposure pathways [18]. this was calculated using the equation: hi = ∑ thq (3) carcinogenic risk target cancer risk (tcr) tcr was estimated by using the formula [16]: tcr = thq x sepo (4) se po = carcinogenic potency slope. the reference values for cd and pb are 6.1 and 0.0085 mg/kg bw/day, respectively [16]. pak. j. anal. environ. che m. vol. 23, no. 2 (2022)209 data analysis the data analysis was carried out using the microsoft excel package. results and discussion the concentrations of rice samples in mg/kg are presented in table 1. this study showed that cd was present in very low concentrations in all the local rice varieties, with stn (0.064 mg/kg) having the highest concentration and kw, and sp having the lowest concentration. the levels of cd in this study were lower than 0.07 mg/kg, as reported by wang et al. [19] for irrigated rice samples and rezaei et al. [20] for rice samples from various countries (mg/kg): korea (0.08), brazil (1.60), saudi arabia (6.16) and china (0.23). the samples cd were also lower than the 0.2 mg/kg permissible limit set by european union [12], the chinese regulatory agency [21] and who/fao [22]. also, it was reported that entering the food chain could cause damage to the lungs and bones, leading to anemia and sometimes high blood pressure [23]. hg was not detected in jm, stn, and kw. this shows that the environment where they were grown was not contaminated with hg. however, hg was in sp with a value of 0.022 mg/kg. the permissible limit set european union commission, chinese regulation and who/fao is 0.02 mg/kg. the result of this study compares well with 0.022 mg/kg for irrigated rice, as reported by wang et al. [19] but is comparatively lower than 0.041-0.798 mg/kg local and imported rice samples available in iraq [24]. hg is toxic and inimical to human health, even at low concentrations. zn had concentrations ranging from 0.02 – 20.0 mg/kg. zn is one of the trace metals that are needed in our diets in small amounts. the maximum permissible limit for zn is 6.0 mg/kg [22]. two of the samples under consideration stn (20.0 mg/kg) and kw (8.06 mg/kg), were higher than this critical level; others (0.02 – 0.028 mk/kg) were comparatively lower. however, the present finding showed that the samples' zn levels were all lower than the 50 mg/kg and 60 mg/kg permissible limits by the chinese regulatory agency and w ho/fao, respectively. excess zn interacts with cu and fe, decreasing their absorption. excess zn also decreases the functioning of the immune system [25]. this study revealed that levels of as in the local rice samples ranged from 0.006-0.086 mg/kg. it was, however, not detected in jm. this finding indicated that the local rice varieties are low in as as compared with chinese standard (0.15 mg/kg) [21], eu commission standard (0.10 mg/kg) [12] and who/fao standard (0.15 mg/kg) [22]. according to mousavi et al. [26], negative effects of as include general weakness in the muscle, loss of appetite, nausea, diarrhea, vomiting, inflammation of the mucous membrane of the eyes, skin lesions, anemia and reduced white blood cells and malignant tumors. this study shows that consumers of the local rice varieties may not be vulnerable to these diseases following their consumption. table 1. levels of heavy metals (mg/kg) i n four varieties of l ocall y consumed rice i n ni geri a. metals jm stn kw sp mean sd cv% cd 0.038 0.064 0.002 0.002 0.027 0.030 112 hg nd nd nd 0.022 nc nc nc zn 0.020 20.0 8.06 0.028 7.03 9.44 134 as nd 0.086 0.006 0.028 nc nc nc pb 2.30 6.84 1.16 14.2 6.13 5.92 96.5 jm = jamila local rice; stn = santana local rice; kw = kwandala local rice; sp = sipi local rice; sd = standard de viation; cv% = coefficient of variation percent; nd = not detected; nc = not computed the concentration of pb in the samples followed the sequence sp > stn > jm > kw with pb values of 14.2, 6.80, 2.30 and 1.16 mg/kg, respectively. the pb concentrations in this study were higher than 0.3 mg/kg which is pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 210 the maximum limit set by fao/who [22] and 0.2 mg/kg limit set by chinese department of preventive medicine [21]. these findings should arouse the attention of researchers and the government; since, on average, it has been revealed that locally produced rice from the northern region is contaminated with high levels of heavy metals and that rice is one of the commonest staples produced in the northern region [27]. a comparison of the findings of this work with research carried out on local rice grown in kaduna by umar and wunzani [28] revealed the pb content found in the literature (0.183 mg/kg) was lower than the pb content of the present study. the variation could be due to variations in the species under consideration, total heavy metal contents of the soil, and physical and chemical properties, which could affect heavy metals' bioavailability [29, 30]. the pb content of rice in this research was higher than 0.01 mg/kg of pb in rice from taiwan [31] but well below 61.17 mg/kg of local rice from owerri, imo state [13]. acute effects of heavy metals may not be common like the chronic effects of accumulated metallic elements in tissues which could pose health issues. research has indicated that contaminated foods happened to be the major sources of heavy metals for humans, and rice was the major route of cd and pb accumulation for asians [32]. otitoju et al. [4] reported high levels of heavy metals in vegetables planted along a construction site in uyo. this could be due to the waste coming from the various industries in the area. consumers of locally produced rice from the northern region are at greater risk of lead toxicity. otitoju et al. [4] reported that it is important to monitor and gather information systemically on the level of heavy metals in the environment to have effective pollution control and mitigate the contamination of food stuffs by heavy metals. table 2. comparison of sample resul ts with standard permissi ble limits. pb jm d (%d) stn d (%d) kw d (%d) sp d (%d) who/fao (5.00) 2.30 +2.70 (54.0) 6.84 -1.84 (36.8) 1.16 +3.84 (76.8) 14.2 -9.20 (184) eu standard (0.20) 2.30 -2.10 (1050) 6.84 -6.64 (3320) 1.16 -0.96 (480) 14.2 -14.0 (7000) chinese standard (0.20) 2.30 -2.10 (1050) 6.84 -6.64 (3320) 1.16 -0.96 (480) 14.2 -14.0 (7000) cd jm d (%d) stn d (%d) kw d (%d) sp d (%d) who/fao (0.20) 0.038 +0.162 (81.0) 0.064 +0.136 (68.0) 0.002 +0.198 (99.0) 0.002 +0.198 (99.0) eu standard (0.20) 0.038 +0.162 (81.0) 0.064 +0.136 (68.0) 0.002 +0.198 (99.0) 0.002 +0.198 (99.0) chinese standard (0.20) 0.038 +0.162 (81.0) 0.064 +0.136 (68.0) 0.002 +0.198 (99.0) 0.002 +0.198 (99.0) zn jm d (%d) stn d (%d) kw d (%d) sp d (%d) who/fao (60.0) 0.02 +59.98 (99.9) 20.0 +40.0 (66.7) 8.06 +51.9 (86.6) 0.028 +59.9 (99.9) eu standard (na) 0.02 nc 20.0 nc 8.06 nc 0.028 nc chinese standard (50.0) 0.02 +49.98 (99.9 20.0 +30.0 (60.0) 8.06 +41.94 (83.9) 0.028 +49.97 (99.9) hg jm d (%d) stn d (%d) kw d (%d) sp d (%d) who/fao (0.02) nd nc nd nc nd nc 0.022 -0.002 (10.0) eu standard (0.02) nd nc nd nc nd nc 0.022 -0.002 (10.0) chinese standard (0.02) nd nc nd nc nd nc 0.022 -0.002 (10.0) as jm d (%d) stn d (%d) kw d (%d) sp d (%d) who/fao (0.15) nd nc 0.086 +0.064 (42.7) 0.006 +0.144 (96.0) 0.028 +0.122 (81.3) eu standard (0.10) nd nc 0.086 +0.014 (14.0) 0.006 +0.094 (94.0) 0.028 +0.072 (72.0) chinese standard (0.15) nd nc 0.086 +0.064 (42.7) 0.006 +0.144 (96.0) +0.144 (96.0) +0.122 (81.3) pak. j. anal. environ. che m. vol. 23, no. 2 (2022)211 table 3 showed the estimated daily intake of the selected heavy metals by the consumption of local rice varieties by children (24 kg bw) and adults (70 kg bw). the edi for children were (mg/kg/day): cd (1.21e-53.87 e-4); zn (1.21 e-4 – 1.21 e-1); pb (7.01 e-3 – 8.58 e-2); as (3.63 e-5 – 5.20e-4) and hg (1.33 e-4 for sp only). for adults (70 kg bw), edi levels were cd (5.0e-61.60 e-6); zn (5.0 e-5 – 5.0 e-2); pb (2.90 e-3 – 3.55 e-2); as (1.50 e-5 – 2.150e-4) and hg (5.50 e-5 for sp rice variety only). the highest edi values of cd, zn, and as were observed by the consumption of stn rice variety in the children and adults, whilst the high edi of pb and hg was calculated by the consumption of sp rice variety. awareness about dietary intake is fundamental to assess the risk of heavy metals to human health. the target hazard quotient, hazard index and target cancer risk of heavy metals for the consumption of the local rice varieties in children and adults are presented in table 4. for the children category, the thq values of cd, zn, pb, and as ranged from 0.004 – 0.129, 0.004 – 4.03, 2.00 – 16.6, and 0.12 – 1.73, respectively. whereas thq value of hg was found to be 1.33 for sp rice variety only. in the adult's category, thq values ranged from cd (0.002 – 0.053); zn (0.002 – 1.67); pb (0.830 – 10.1); as (0.05 – 0.72); hg (0.55 for sp only). thq is a measure of the noncarcinogenic risk of heavy metals. it is a measure of developing non-carcinogenic health challenges. the maximum limit for thq is 1. if the thq is less than 1, it is considered safe for the exposed consumers but if it is equal or greater than 1, it could be dangerous to the health of the consumers [33]. results of this study showed that thq for cd, as and hg ha values less than 1. this suggests that consumption of the samples would pose no health risk due to these metals in all the samples for both children and adults. however, for zn, the thq was greater than 1.0 by the consumption of stn rice variety by children and adults, respectively. the thq for pb were less than 1 in kw (0.830) for childern and greater than 1 (2.00 – 16.6) by the consumption of local rice varieties by adults. this implies that long term exposure to the rice samples could pose health risk as a result of pb poisoning. the levels of hazard index ranged from 3.74 – 18.5 (for 24kg bw) and 1.69 – 10.9 (for 70kg bw). hazard index is the addition of all thq values in any given sample. a value of hi less than 1 in any food sample indicates that the exposed consumers would not have any adverse lifetime health effects due to consumption of that sample. hi above 1.0 implies that the combined effects of heavy metals could cause long-term noncarcinogenic health problems to the consumers. hazard index reported for root, fruit, and leafy vegetables were 11.24, 8.35, and 13.16, respectively [14]. in this study, hi levels were higher than 1.0 in children and adults, and pb is the major contributor to these high levels of hi. table 3. estimated dail y i ntake of the rice vari eties for 24 kg and 70 kg body weight. chil dren (24 kg body weight) adul ts (70 kg body weight) samples cd hg zn as pb cd hg zn as pb jm 2.30 e-4 nd 1.21e-4 nd 1.39e-2 9.5e-5 nd 5.00e-5 nd 5.75 e-3 stn 3.87 e-4 nd 1.21 e-1 5.20 e-4 4.13 e-4 1.60 e-4 nd 5.0 e-5 2.15 e-4 1.71 e-2 kw 1.21 e-5 nd 4.87 e-2 3.63 e-5 7.01 e-3 5.0 e-6 nd 2.02 e-2 1.50 e-5 2.90 e-3 sp 1.21 e-5 1.33 e-4 1.69 e-4 1.69 e-4 8.58 e-2 6.0 e-6 5.50 e-5 7.00 e-5 7.0 e-5 3.55 e-2 mean 1.60 e-4 nc 4.25 e-2 nc 3.70 e-2 6.63 e-6 nc 1.80 e-2 nc 1.50 e-2 sd 1.83 e-4 nc 5.71 e-2 nc 3.57 e-2 7.55 e-5 nc 2.40 e-2 nc 1.50 e-2 cv% 114 nc 134 nc 96.6 114 nc 131 nc 100 jm = jamila local rice; stn = santana local rice; kw = kwandala local rice; sp = sipi local rice; sd = standard deviation; cv% = coefficient of variation percent; nd = not detected; nc = not computed pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 212 table 4. target hazard quoti ent, hazard i ndex and target cancer risk of the sampl es for chil dren and adul ts. jm stn kw sp mean sd cv% jm stn kw sp mean sd cv% metals children (24 kg body weight) adults (70 kg body weight) cd 0.077 0.129 0.004 0.004 0.063 0.053 85.3 0.032 0.053 0.002 0.002 0.022 0.025 102 hg nd nd nd 1.33 nc nc nc nd nd nd 0.550 nc nc nc zn 0.004 4.03 1.62 0.006 1.41 1.90 135 0.002 1.67 0.670 0.002 0.586 0.788 135 as nd 1.73 0.120 0.560 nc nc nc nd 0.720 0.05 0.230 nc nc nc pb 3.97 11.8 2.00 16.6 8.59 6.81 9.3 1.64 4.89 0.830 10.1 4.37 4.21 96.3 hi 4.05 15.0 3.74 18.5 10.3 7.56 73.4 1.69 7.33 1.54 10.9 5.37 4.57 85.1 tcr (cd) 0.468 0.787 0.025 0.025 0.326 0.372 114 0.193 0.325 0.014 0.014 0.137 0.151 110 tcr (pb) 0.034 0.100 0.017 0.141 0.073 0.058 79.2 0.014 0.042 0.007 0.086 0.037 0.036 96.6 jm = jamila local rice; stn = santana local rice; kw = kwandala local rice; sp = sipi local rice; sd = standard deviation; cv% = coefficient of variation percent; nd = not detected; nc = not computed the levels of tcr of cd in children (24 kg bw) and adults (70 kg bw) were 0.025 – 0.787 and 0.014 – 0.325, respectively whilst the levels of tcr of pb were 0.017 – 0.141 and 0.007 – 0.086, respectively. fao/who [22] has shown that prolonged exposure to a specific carcinogen might lead to cancer, and the probability may increase with prolonged consumption contact time. the tcr levels of cd and pb by the consumption of the studied local rice varieties were within the range of 0.042 – 0.330 in vegetables in satkhira, bangladesh [14] but higher than 4.48e-6 – 1.74e-3 in fourteen leafy vegetables sold in ekiti state, nigeria [34]. the level of carcinogenic risk is linked to the tcr level of heavy metals in any food sample [34]. if the tcr value is less than 10 -6 , the carcinogenic risk is low. but, the tcr values between 10 -5 and 10 -3 showed a moderate risk. similarly, the tcr value between 10-3 and 10-1, and equal or greater than 10-1, indicated the high and very high risk, respectively. the levels of tcr in this study for cd and pb indicate a high risk of carcinogen upon consumption of the rice samples over a long period. conclusion this study examined the toxicity levels of heavy metals in four varieties of local rice consumed in the northwestern region of nigeria. the results revealed that the studied heavy metals were found in very trace amounts in the samples of the local rice varieties, while some were not detected. the concentrations of pb in the samples of the local rice varieties were more than the permissible limits set by most of the regulatory agencies. this is a significant concern as this might be due to the onset of environmental pollution. the target hazard quotient of cd, as, and hg showed that the consumption of the local rice varieties would not pose any health risk in children and adults. however, the zn levels in local rice varieties were above the permissible limit in only one sample. whereas, pb was greater than the permissible limit in most of the samples of local rice varieties. the combined effects of heavy metals in the samples of the local rice varieties can pose a health risk for the consumers over a long period of time. the carcinogenic risk is high for the exposed populations over time due to high tcr values. high levels of heavy metallic elements in the local rice varieties, especially pb, are undesirable. more research on soil and water samples from the region is recommended to determine whether the contamination is coming from irrigation water or the soil to the rice variety. generally, there were high variations in the levels of the local rice pak. j. anal. environ. che m. vol. 23, no. 2 (2022)213 varieties, as evident in the values of the coefficient of variation percent. conflict of interest authors declare that there is no conflict of interest. references 1. h. cao j. chen, j. zhang, h. zhang, l. qiao and y. men, j. environ. sci., 22 (2010) 1792. https://doi.org/10.1016/s10010742(09)60321-1 2. s. r. smith, environ int., 35 (2009) 142. https://doi.org/10.1016/j.envint.2008.06. 009 3. usaid, increasing competitiveness and food security in nigeria. (2012). https://www.usaid.gov 4. o. otitoju, m. i. akpanabiatu, g. t. o. otitoju, j. i., ndem, a. f. uwah, e. o. akpanyung and j. t. ekanem, res. j. environ. earth sci., 4(2012) 371. https://www.researchgate.net/publication /235987925 5. y. chen, c. wang and z. wang, environ. int., 31 (2005) 778. https://doi.org/10.1016/j.envint.2005.05. 024 6. k. p. singh, d. mohan, s. sinha and r. dalwani, chemosphere, 55 (2004) 227. https://doi.org/10.1016/j.chemosphere.20 03.10.050 7. k. ganguly, b. levanen, l. palmberg, a. akesson and a. linden, eur. respr. rev., 27 (2018). article id: 170122 8. p. w. davidson, g. j. myers and b. weiss, pediatrics, 113 (2004) 1023. available at: publications.aap.org 9. a. kumar and seema, j. environ. sci. toxicol. food technol., 10 (2016) 8. available at: www.iosrjournals.org 10. o. oluwatomi, about rice production and processing, 2011. http://tribune.com.ng/index.php/wealthcreationthru-agric/26645-about-riceproductionand-processing. 11. a. femi, smuggling as bane of local rice production. the guardian nigeria newspaper (mon, jan 7 th ), (2013). 12. european commission. commission regulation (ec) no. 1881/2006 of 19 december 2006 setting maximum levels for certain contaminants in foodstuffs. off. j. eur. union., l364 (2006) 5. 13. o. e. orisakwe, j. k. nduka, c. n. amadi, d. o. dike and o. bede, chem. central j., 6 (2012) 77. http://journal.chemistrycentral.com/cont ent/6/1/77 14. m. n. uddin, m. k. hasan and p. k. dhar, j. mat. environ. sci., 10 (2019) 543. http://www.jmaterenvironsci.com 15. unep/fao/who, assessment of dietary intake of chemical contaminants. united nations environmental program, nairobi, (1992). 16. usepa, risk based screening table. composite table: summary tab 0615, (2015). www.nigeriamarket.org. 17. usepa, usepa regional screening level summary table, (2011). https://www.epa-prgs.ornl.gov 18. u. c. ekhator, n. a. udowelle, s. igbiri, r. n. asomugha, z. n. igweze and o. e. orisakwe, j. environ. pub. health, (2017) 1. https://doi.org/10.1155/2017/8458057. 19. z. wang, x. zeng, m. geng, c. chen, j, cai, x. yu, y. hou and h. zhang, pol. j. environ. stud., 24 (2015) 1379. http://www.pjoes.com 20. l. rezaei, v. alipour, p. sharafi, h. ghaffari, a. nematollahi, v. pesarakloo and y. fakhri, environ. health eng. manag., 8 (2021) 67. https://doi.org/10.34172/ehem.2021.10 21. chinese department of preventive medicine (cdpm), thresholds for food pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 214 hygiene. china standard press, beijing, (1994). 22. fao/who, technical reports “guidelines for the safe use of waste water and food stuff”. report of the joint who/fao, vol 2 no 1, world health organisation (w ho) and food and agriculture organisation (fao), geneva, switzerland (2013). 23. s. himeno and k. aoshima, cadmium toxicity: new aspects in human diseases, rice contamination and cytotoxicity. springer, singapore (2019). available at: www.springer.com 24. r. a. abu-almaaly, i. f. ali karm and n. m. a. alsaffar, pharm. sci. res., 10 (2018) 1761 www.jpsr.pharmainfo.in 25. e. i. adeyeye and f. j. faleye, j. appl. environ. sci., 3 (2007) 150. www.researchgate.net 26. k. a. mousavi, y. fakhri, a. nematollahi and m. pirhadi, trends food sci. technol., 96 (2020) 30. https://doi.org/10.1016/j.tifs.2019.12.007 27. o. oluwatomi, about rice production and processing, 2011. http://tribune.com.ng/index.php/wealthcreationthru-agric/26645-about-riceproductionand-processing 28. m. a. umar and d. k. wunzani, international j. sci. technol. res., 2 (2013) 2277. corpus id: 15540124. www.ijstr.org 29. w. cheng, g. zhang, h. yao, w. wu and m. zu, j. zhejiang univ. sci. b, 7 (2006) 565. https://doi.org/10.1631/jzus.2006.b0565 30. m. i. rubio, i. escrig, c. martinezcortina, f.j. lopezbenet and a. sanz, plant growth regul., 14 (1994) 151. https://doi.org/10.1007/bf00025217 31. l. haw-tarn, m. c. wang and g. li, chemosphere, 56 (2004) 1105. https://doi.org/10.1016/j.chemosphere.20 04.05.018 32. c. z. fangmin, h. x. ningchum, l. yi, z. wenfang, z. zhiwei and c. mingxue, sci. total environ., 359 (2006) 156. https://doi.org/10.1016/j.scitotenv.2005. 05.005 33. t. adefa and m. tefera, chem. africa, 3 (2020) 1073. https://doi.org/10.1007/s42250-02000181-0 34. a. j. adesina, o. a. ibigbami, a. a. olaleye, a.m. olatunya, s.d. oyeyemi, o. k. popoola, a. f. akinsola, s. a. olagboye, a.y. gbolagade and o. ayodele, curr. topics toxicol., 16 (2020) 157. www.researchtrends.net microsoft word 92-101-pjaec-24022020-232-c-galley proof revised.d cross mark issn-1996-918x pak. j. anal. environ. chem. vol. 21, no. 1 (2020) 92 – 101 http://doi.org/10.21743/pjaec/2020.06.11 effect of wastewater irrigation on trace metal accumulation in spinach (spinacia oleracea l.) and human health risk ilker ugulu 1* , zafar iqbal khan 2 , sidrah rehman 2 , kafeel ahmad 2 , mudasra munir 2 and humayun bashir 2 1 faculty of education, usak university, usak, turkey. 2 department of botany, university of sargodha, sargodha, pakistan. *corresponding author email: ilkerugulu@gmail.com received 24 february 2020, revised 11 may 2020, accepted 21 may 2020 -------------------------------------------------------------------------------------------------------------------------------------------abstract the present research determines the effect of wastewater for irrigation on heavy metal accumulation in vegetables in the example of spinach (spinacia oleracea l.) and to evaluate human health risk from consumption. trace metal values of cd, cu, cr, fe, zn, ni and mn, were determined in the water, soil and plant samples by atomic absorption spectrophotometer. trace metal concentrations in spinach samples ranged from 0.29 to 0.37, 0.14 to 1.25, 0.07 to 0.67, 1.12 to 2.48, 0.33 to 0.38, 1.92 to 2.90 and 0.51 to 0.63 mg/kg for cd, cr, cu, fe, ni, zn and mn, respectively. these values of trace metals were lower than the permissible limits except for cd. all health risk index (hri) values except for cd were less than 1. however, the hri values related to spinach samples irrigated with canal water and sugar mill water were generally higher than the values of the samples irrigated with groundwater. the hri value of cd was higher than 1 and consumers of such vegetables in which hri of metal was greater than 1 will be at risk. keywords: biomonitoring, spinach, vegetable, heavy metal -------------------------------------------------------------------------------------------------------------------------------------------introduction wastewater irrigation is an important source of agricultural irrigation especially in countries that have difficulties in terms of freshwater resources [1-4]. one of the most prominent problems faced by cities and factories is the discharge and disposal of wastewater. if the industrial and sewage wastewaters are not disposed of within the scope of appropriate solutions, they pose significant problems for the environment [5-11]. so, irrigation of agricultural soil by wastewater for a long period may lead to the accretion of trace metals in soils and vegetables [12-13]. also, these metals can pass to animals and humans through the food chain and cause ecosystem-wide contamination. potential health risks and food safety problems make this one of the most alarming environmental aspect [14-18]. the term heavy metal is used for metals with a density higher than 5 g/cm 3 in terms of physical properties. some metals are indispensable for living beings, while others are highly toxic. however, indispensable metals are also known to be toxic after a certain amount for living beings. many studies on the subject have shown that the use of industrial and sewage wastewater in agricultural irrigation causes heavy metal pollution in soil and plants [19-22]. high level of trace metals is present in the upper layer of soil due to wastewater irrigation. this depth zone is located in the root area of many crops [23]. for this reason, pak. j. anal. environ. chem. vol. 21, no. 1 (2020) 93 this layer has an important place in the uptake of plants from the soil. vegetables are one of the most important elements of human and animal nutrition. necessary components of daily diet like protein, calcium, vitamins and other nutrients are supplied by vegetables. vegetables may also store trace metals in their edible and non-edible parts [4]. trace elements are essential for normal metabolic functions in plants, but at higher concentrations, these metals are toxic and can seriously interfere with physiological and biochemical functions [13]. cd, cu, cr, fe, zn, ni and mn are the type of heavy metals that react as micronutrients at minor concentrations, they act as toxic compounds at higher concentrations. health issues produced by contaminated soil and vegetables have been widely reported throughout the world [24-28]. for this reason, determination of heavy metal accumulation values, especially in plants consumed as food, has an important place in the researches on environmental pollution. spinach (spinacia oleracea l., amaranthaceae) is grown in pakistan and all over the world. s. oleracea is used in traditional medicine to treat constipation, alleviate stomach acidity, treat anaemia and as diuretic and carminative. with all these features, spinach is an important plant in terms of nutrition, protection against diseases and alternative medicine practices. pakistan is an important agricultural country and the most important economic activity of the country is agriculture. however, there is a shortage of clean water in the country and wastewaters such as industrial wastewater and sewage water are widely used in agricultural irrigation. studies showed that wastewater irrigation is effective in heavy metal accumulation for spinach, as in other agricultural products [22]. also, the literature studies on the subject showed that the studies on the irrigation of spinach with wastewater, heavy metal accumulation and its effect on health are not sufficient. in this direction, the present research was aimed to determine the effect of using wastewater for irrigation on heavy metal accumulation in vegetables in the example of spinach (spinacia oleracea l.) and to evaluate human health risk from the consumption. materials and methods study area this study was performed in khushab district of punjab, pakistan (fig. 1). the maximum temperature measured in the region in the summer is about 50 °c, and the minimum temperature recorded in the winter is about 12 °c. due to this temperate feature, the city of khushab offers a favourable environment for agricultural applications. figure 1. the map of study area plant cultivation and sample preparation spinach (spinacia oleracea l.) samples were grown at the end of october 2016 in 60 small plastic pots. approximately 2.5 kg of soil was filled to each plastic pot and a different treatment was applied in every 20 plastic pots. ten seeds were sown in each plastic pot, and each pot was irrigated twice a week with a litre of groundwater (ti: gwi), canal water (tii: cwi) and sugar mill water (tiii: mwi). after the plant samples in the pots matured, only four plants were left in each pot and 210 kg ha -1 urea fertilizer was applied to each pot. the samples of water used in the irrigation of the pots were also taken as examples in the metal analysis. soil samples were taken from the pots from a depth of 5 cm with the help of an auger. at the end of april 2017, spinach leaves were collected for analysis, dried outdoors and pak. j. anal. environ. chem. vol. 21, no. 1 (2020)94 ground by pounding in a mortar. ground powdered plant samples were dried in an oven for 3 days at 75 0 c. after it was completely dry, the samples were prepared for metal analysis using the wet digestion method. metal analysis cd, cu, cr, fe, zn, ni and mn amounts were determined in the water, soil and plant samples with the help of atomic absorption spectrophotometer (shimadzu model aa-6300). table 1 shows the operating conditions used for each heavy metal in the analysis process. table 1. operating conditions for the analysis of metals using atomic absorption spectrometry. elements parameters cd cr cu fe ni zn pb wavelength (nm) 228.8 422.7 324.8 248.3 232.0 213.9 283.3 slit width (nm) 0.7 0.7 0.7 0.2 0.2 0.7 0.7 lamp current (ma) 8 10 6 12 12 8 10 air flow rate (l/min) 15 15 15 15 15 15 15 acetylene flow rate (l/min) 1.8 2.8 1.8 2.2 1.6 2 2.0 burner height (mm) 7 9 7 9 7 7 7 statistical analysis the variance of the metal values for water, soil and vegetables were analysed by one-way anova by spss 23. in evaluating the differences in metal concentrations in the samples, 0.001, 0.01 and 0.05 values were determined as the level of significance. bioconcentration factor (bcf) bioconcentration factor refers to metal accumulation in the plant as a result of the heavy metal transition from soil to plant. the following formula is used to calculate the bioconcentration factor: bcf = cveg / csoil while cveg refers to the metal accumulation value in plant tissues (mg/kg, fresh weight), csoil refers to the metal concentration in the soil (mg/kg, dry weight) [12]. daily intake of metals (dim) one of the certain methods considered to detect consumer-based health risks is the daily intake of metals. dim was measured using the following formula: daily intake of metal s= cmetal × cfood intake / baverage weight while cmetal denotes metal concentration in plant samples, cfood intake indicates daily food intake and baverage weight indicates average body weight. in this study, the daily food intake of a person was taken as 0.345 mg/kg and an average bodyweight of 60 kg as a standard. health risk index (hri) the hri indicates a health threat to people who consume contaminated food. in this study, it was used to calculate the heavy metal exposure that can occur if spinach samples are consumed by humans. hri is described as the ratio of dim in food crops to the oral reference dose [9]. hri = dim / oral reference dose pollution load index (pli) according to each metal value in the soil, pli provides an estimation to the metal accumulation status. pli was calculated for each treatment using the following formula [29]: pli = determined metal value of researched soil / reference metal value of soil. the reference trace metal values of soil for cd (1.49 mg/kg), cr (9.07 mg/kg), cu (8.39 mg/kg), ni (9.06 mg/kg), zn (44.19 mg/kg), and mn (46.75 mg/kg) were taken according to khan et al. [9] and fe (56.90 mg/kg) was taken according to ahmad et al. [1]. results and discussion trace metal concentration in water samples in the current study, the recorded fe and zn concentrations in the water samples used for pak. j. anal. environ. chem. vol. 21, no. 1 (2020) 95 irrigation were greater than other metal values (fig. 2). however, it was observed that heavy metal accumulation values in canal and sugar mill water were higher than groundwater accumulation values. the anova results indicated that there were no significant differences (p>0.05) between the metal concentrations for cr, cd, cu, ni and mn while the significant differences for fe and zn in the water samples (table 2). the maximum permissible limits of the cd, cr, cu, fe, ni, zn and mn in water were reported by standard guidelines in europe as 0.01, 0.5, 0.2, 5, 0.2, 2 and 0.2 mg/l, respectively [30]. trace metal values in water samples except mn were above the maximum limits reported for water. in line with these results, it can be said that there is pollution in the waters used for irrigation in the study area. in the study conducted in khushab, khan et al. [9] noticed the metal values in groundwater, canal water and industrial water samples from the region as 0.01-0.02-0.03 mg/l for cu, 1.69-1.76-1.88 mg/l for cd, 0.640.72-0.83 mg/l for fe, 0.54-0.57-0.65 mg/l for cr, 0.08-0.10-0.14 mg/l for ni, 0.07-0.08-0.12 mg/l for mn and 0.57-0.61-0.66 mg/l for zn, respectively. the metal values obtained from this study were found above the maximum permissible limits by usepa [31]. figure 2. trace metal concentrations in irrigation water table 2. analysis of variance for heavy metals and metalloids in soil and spinach. mean squares sample source of variation (sov) degree of freedom (df) cd cr cu fe ni zn mn treatments 4 .215ns .002ns .364ns 6.189* .750ns .006*** .186ns water error 10 .052 .001 .074 .813 .307 .001 .039 treatments 4 .010* .030** .026ns .534ns .001ns 33.458*** .050** soil error 10 .002 .003 .023 32.603 .003 2.259 .006 treatments 4 .006ns 1.510ns .396ns 204.0** .002ns 1.151* .013ns spinach error 10 .004 .623 .219 16.956 .001 .281 .006 *, **, *** significant at 0.05, 0.01, and 0.001 levels; ns, non-significant water trace metal concentration in soil samples in the present study, the determined mean metal values in soil samples were 0.35, 0.155, 0.348, 6.52, 0.38, 6.64 and 5.20 mg/kg for cd, cr, cu, fe, ni, zn and mn, respectively. the mean fe, zn and mn concentrations were higher, and the mean cd and cr concentrations were lesser among the three treatments (fig. 3). these values also clearly showed that heavy metal accumulation values in soil samples irrigated with sugar mill water were higher than the metal accumulation values of soil samples irrigated with other waters. according to the statistical analysis, while different irrigation regimes produced a statistically significant difference in the accumulation of cd, cr, zn and mn in the soils where spinach was grown, it did not make a significant difference in terms of cu, fe and ni accumulation (p>0.05) (table 2). usepa [31] reported the maximum permissible limits in the soil for the accumulation of cd, cr, cu, fe, ni, zn and mn as 3, 100, 50, 21000, 50, 200 and 2000 mg/kg, respectively. all metal values in the present research identified below permissible limits for all treatments. findings of alrawiq et al. [32] showed a higher amount of metals than the presented values in this study except for cd. many studies performed in pakistan reported on the high concentration of trace metals in vegetables irrigated with industrial water or sewage sludge. ahmad et al. [33] examined the heavy metal accumulation in the soil samples irrigated with wastewater and tap water in their study in khushab, pakistan, and found that the cobalt accumulation in the soil irrigated with sewage water (20.2 mg/kg) was more than irrigated with tap water (13.5 mg/kg). as mentioned in this study, it was concluded that heavy metal accumulation was higher as a result of irrigation with the sewage water. the reason for these results may be low adsorption property of this metal in soil [9]. however, many factors such as the geological characteristics of the soil of the region, industrial establishments in the environment, climate and precipitation can be shown among the factors affecting the heavy metal level in the soil. figure 3. fluctuation in metals in soil of spinach soil pak. j. anal. environ. chem. vol. 21, no. 1 (2020) 97 trace metal concentration in vegetable samples trace metal concentrations in spinach samples ranged from 0.29 to 0.37, 0.14 to 1.25, 0.07 to 0.67, 1.12 to 2.48, 0.33 to 0.38, 1.92 to 2.90 and 0.51 to 0.63 mg/kg for cd, cr, cu, fe, ni, zn and mn, respectively. among the three treatments, the mean fe and zn concentrations were higher in all treatments and the mean cr and cu concentrations were lesser in treatment i and ii (fig. 4). these values showed that heavy metal accumulation values in spinach samples irrigated with sugar mill water were higher than the metal accumulation values of spinach samples irrigated with other waters except cd, mn and ni. according to the statistical analysis, while different irrigation regimes produced a statistically significant difference in the accumulation of cd, cr, cu, ni and mn in the spinach samples, it did not make a significant difference in terms of fe and zn accumulation (p>0.05) (table 2). usepa [31] reported the maximum permissible limits in the plants for the accumulation of cd, cr, cu, fe, ni, zn and mn as 0.1, 5, 73, 425, 67, 100 and 500 mg/kg, respectively. the range values of trace metals in spinach samples were lower than these permissible limits except for cd. also, the cd concentration was considerably higher than the values (0.0020.08 mg/kg) in egypt reported by dogheim et al. [34]. however, the present cd values were lesser than the vegetables studied by gupta et al. [35] in india (10.37-17.79 mg/kg) and within the range (0.03-0.73 mg/kg) noted by liu et al. [36] in china. demirezen and aksoy [37] examined various vegetables and determined that zn contents were in the range of 3.56-4.59 mg/kg which was higher than the present study as in the range from 1.92 to 2.90 mg/kg. ahmad et al. [33] observed a higher range of cobalt in the root samples (1.07–1.26 mg/kg) of the plants irrigated with the sewage water. as mentioned in this study, it was concluded that heavy metal accumulation was higher as a result of irrigation with the sewage water. the main factors that affect the heavy metal intake of plants from the soil are factors such as ph, temperature, cation exchange capacity of the soil, the rate of other metals in the soil, chemical selectivity, oil value and species of the plant [29]. in line with the findings obtained from this study, it can be said that the use of wastewater for irrigation increases the heavy metal accumulation in the soil and maybe the reason for the high heavy metal level in other factors mentioned above. figure 4. fluctuation of metals in spinach spinach bioconcentration factor (bcf) analysis of various metals in three irrigations, zn showed the maximum and cu showed the minimum value in groundwater treatment. in treatment-i (gwi), transfer factor for cr, fe and cu was lower than cd, mn, ni and zn. in treatment-ii (cwi), transfer factor for zn, fe, cu and cr was lower as compared to cd, mn and ni. finally, in treatment-iii (mwi), fe, zn and ni values were lower than the values of cr, cu, cd and mn (table 3). bioconcentration factor values for cd, cr, cu and zn in spinach samples irrigated with sugar mill water and mn and zn values in samples irrigated with groundwater were found higher than 1. the bcf is the best way to know the availability of important metals transferred from soil to grow vegetable. the bcf values of cd, cr, cu and zn in sugar mill irrigated samples, and the bcf values of mn and zn in groundwater irrigated samples were higher than 1, which shows that these metals were easily available to vegetables and diffused more in them [1]. the mean values for bcf reported by ahmad et al. [33] were 0.036 and 0.038 for co in brassica rapa grown at tap water and sewage water irrigated sites, respectively. although it was close to each other, it was observed that bcf value in wastewater irrigation area is higher like as in the present study. table 3. bioconcentration factor for spinach. heavy metals irrigations cd cr cu fe ni zn mn i 0.9300 0.699 0.273 0.42 0.934 2.22 1.613 ii 0.954 0.767 0.511 0.187 0.939 0.809 0.907 iii 1.147 8.116 1.759 0.254 0.878 1.836 0.995 pollution load index (pli) pli values for spinach grown with three different irrigations was in the following sequence. order of pli in treatment-i (gwi), treatment-ii (cwi) and treatment-iii (mwi) were cd>fe> ni>cu>cr>zn>mn, cd>fe>zn>cu>ni>cr>mn and cd>fe>zn>cu>ni>cr>mn, respectively. the highest pli was observed for cd and the lowest pli for cr and mn in all three irrigations (table 4). it was observed that the pli values of the sugar mill water and canal water irrigated samples were higher than the groundwater irrigated samples. however, the measured values were lower than the values found by khan et al. [16]. higher pli value (1.493) was noticed by ahmad et al. [33] when the sewage source was used to irrigate the soil instead of tap water. in the study conducted in the region, ahmad et al. [1] found the highest pli value for cd. in this direction, the results of the present study were parallel to the values reported by ahmad et al. [1]. a pli value greater than 1 indicates that the food is contaminated with metal, and less than 1 indicates that it is not contaminated [15]. the fact that the pli values obtained as a result of the study were less than 1 indicates that the spinach samples are consumable. daily intake of metals (dim) dim values for fe and zn were higher and cr was the lowest value in sugar mill water and canal water treatments. the order of dim values in in treatment-i (gwi), treatment-ii (cwi) and treatment-iii (mwi) were fe>zn>mn>ni>cd> cr>cu, zn>fe>mn>ni>cd>cu>cr and zn>fe> cu>mn>cd>ni>cr, respectively (table 5). according to who/fao, dim values for cd, cr, cu, ni and zn were 0.06, 0.05–0.2, 3, 1.4, 60 mg/day, respectively. the dim values for all metals presented in this study are below the standard values. mahmood and malik [38] pointed out that, daily intake of metal was higher for zn and less for cr and cd in foodstuff grown at wastewater. in the present study, the consequences of the dim value of vegetables irrigated with sugar mill wastewater showed resemblance to that given by mahmood and malik [38]. health risk index (hri) in the present study, the order of hri values of the metals in treatment-i (gwi), treatment-ii (cwi) and treatment-iii (mwi) were cd>ni>mn>zn>fe>cu>cr, cd>ni>mn>zn>cu> fe>cr and cd>ni>cu>mn>zn>fe>cr, respectively (table 6). the metal with the lowest health risk index for spinach samples in all pak. j. anal. environ. chem. vol. 21, no. 1 (2020) 99 irrigation environments was chromium. the fact that the calculated hri value was greater than 1 indicates that the consumption of this food carries health risks and that less than 1 indicates that consumption is not a problem in terms of health [7]. in the present study, cd value was higher than 1 and consumers of such vegetables in which hri of metal was greater than 1 will be at risk [15]. on the other hand, although the hri values were less than 1, it was seen that the values related to spinach samples irrigated with canal water and sugar mill water were generally higher than the values of the samples irrigated with groundwater. a similar finding was reached by ahmad et al. [33] and it was reported that hri values were higher in products that were irrigated with wastewater. the hri indicates a health threat to people who consume contaminated food [3940]. table 5. daily intake of metal for spinach. heavy metals irrigations cd cr cu fe ni zn mn i 0.0017 0.00083 0.00042 0.01430 0.00210 0.01106 0.00362 ii 0.00216 0.00143 0.00125 0.00649 0.00222 0.0161 0.00319 iii 0.00198 0.00123 0.00388 0.00956 0.00194 0.01667 0.00297 table 6. health risk index values. heavy metals irrigations cd cr cu fe ni zn mn i 1.721 0.002 0.0106 0.0204 0.1051 0.0368 0.0885 ii 2.167 0.0009 0.0312 0.0092 0.111 0.0536 0.0780 iii 1.980 0.0048 0.097 0.0136 0.0973 0.0555 0.072 table 7. metal correlation between soil-vegetable. correlation metals soil-vegetable cd .669 cr -.680 cu .476 fe -.405 ni .928 zn .883 mn -.721 correlation the results presented positive nonsignificant correlation of cd, cu, zn and ni and negative non-significant correlation of cr, fe and mn (table 7). bibi et al. [17] reported that the correlation between soil and vegetable was positive and non-significant for cd and ni. results of correlation in the current study were similar to that study. conclusion in the present study, cd, cr, cu, fe, ni, zn and mn values in spinacia oleracea samples pak. j. anal. environ. chem. vol. 21, no. 1 (2020)100 irrigated with groundwater, canal water and sugar mill water were examined. the values of the trace metals in spinach samples except cd were below the maximum permissible limits. also, these values showed that heavy metal accumulation values in spinach samples irrigated with sugar mill water were higher than the metal accumulation values of spinach samples irrigated with other waters except cd, mn and ni. according to the findings of the study, health risk index value of cd was higher than 1 and consumers of such vegetables in which hri of metal was greater than 1 will be at risk. even so, it can be said that legal measures should be taken and implemented sensitively by local governments and appropriate authorities. acknowledgement laboratory facilities are provided by high tech lab university of sargodha. the authors also thank all the supporters for suggestions and comments for the improvement of this manuscript. references 1. k. ahmad, k. nawaz, z. i. khan, m. nadeem, k. wajid, a. ashfaq, b. munir, h. memoona, m. sana, f. shaheen, r. kokab, s. rehman, m. f. ullah, n. mehmood, h. muqaddas, z. aslam, m. shezadi, i. r. noorka, h. bashir, h. a. shad, f. batool, s. iqbal, m. munir, m. sohail, m. sher, s. ullah, i. ugulu, y. dogan, fresen. environ. bull., 27 (2018) 846. https://www.researchgate.net/publication/32 3144653 2. m. nadeem, t. m. qureshi, i. ugulu, m. n. riaz, q. u. an, z. i. khan, k. ahmad, a. ashfaq, h. bashir and y. dogan, pak. j. bot., 51 (2019) 171. http://doi.org/10.30848/pjb2019-1(14) 3. z. i. khan, i. ugulu, s. sahira, k. ahmad, a. ashfaq, n. mehmood and y. dogan, int. j. environ. res., 12 (2018) 503. https://doi.org/10.1007/s41742-018-0110-2 4. i. ugulu, appl. spectrosc. rev., 50(2) (2015) 113. https://doi.org/10.1080/05704928.2014.9359 81 5. i. ugulu, m. c. unver and y. dogan, oxid. commun., 39 (2016) 765. http://scibulcom.net/ocr.php?gd=2016&bk= 1 6. m. c. unver, i. ugulu, n. durkan, s. baslar and y. dogan, ekoloji, 24 (2015) 13. https://doi.org/10.5053/ekoloji.2015.01 7. y. dogan, i. ugulu and n. durkan, pak. j. bot., 45 (2013) 177. https://www.pakbs.org/pjbot/pdfs/45(si)/2 4.pdf 8. i. ugulu and s. baslar, j. altern. complem. med., 16 (2010) 313. http://doi.org/10.1089=acm.2009.0040 9. z. i. khan, i. ugulu, s. umar, k. ahmad, n. mehmood, a. ashfaq, h. bashir and m. sohail, bull. environ. contam. toxicol., 101 (2018) 235. https://doi.org/10.1007/s00128-018-2353-1 10. y. dogan, i. ugulu and s. baslar, ekoloji, 19 (2010) 88. http://doi.org/10.5053/ekoloji.2010.7512 11. n. durkan, i. ugulu, m. c. unver, y. dogan and s. baslar, trace elem. electroly., 28 (2011) 242. http://doi.org/10.5414/tex01198 12. i. ugulu, y. dogan, s. baslar and o. varol, int. j. environ. sci. technol., 9 (2012) 527. https://doi.org/10.1007/s13762-012-0056-4 13. y. dogan, s. baslar and i. ugulu, appl. ecol. environ. res., 12 (2014) 627. http://doi.org/10.15666/aeer/1203_627636 14. y. dogan, m.c. unver, i. ugulu, m. calis and n. durkan, biotech. biotechnol. equip. 28 (2014) 643. http://doi.org/10.1080/13102818.2014.9470 76 15. i. ugulu, int. j. educ. sci., 26 (2019) 49. https://doi.org/10.31901/24566322.2019/26. 1-3.1086 16. z. i. khan, k. ahmad, h. safdar, i. ugulu, k. wajid, h. bashir and y. dogan, res. j. pharm. biol. chem. sci., 9 (2018) 759. https://www.rjpbcs.com/pdf/2018_9(5)/[94]. pdf 17. z. i. khan, i. ugulu, s. umar, k. ahmad, n. mehmood, a. ashfaq, h. bashir and m. sohail, bull. environ. contam. toxicol., 101 (2018) 235. https://doi.org/10.1007/s00128-018-2353-1 pak. j. anal. environ. chem. vol. 21, no. 1 (2020) 101 18. i. ugulu, biotech. biotechnol. equip., 29 (2015) 20. http://doi.org/10.1080/13102818.2015.1047 168 19. n. yorek, i. ugulu and h. aydin, comput. intel. neurosci., article id 2476256, (2016) 1. http://dx.doi.org/10.1155/2016/2476256 20. i. ugulu, biotech. biotechnol. equip., 23 (2009) 14. 21. z. i. khan, i. ugulu, k. ahmad, s. yasmeen, i. r. noorka, n. mehmood and m. sher, bull. environ. contam. toxicol., 101 (2018) 787. https://doi.org/10.1007/s00128-018-2448-8 22. z. i. khan, k. ahmad, s. rehman, a. ashfaq, n. mehmood, i. ugulu and y. dogan, pak. j. anal. environ. chem., 20 (2019) 60. http://doi.org/10.21743/pjaec/2019.06.08 23. z. i. khan, h. safdar, k. ahmad, k. wajid, h. bashir, i. ugulu and y. dogan, pak. j. bot., 52 (2020) 111. http://dx.doi.org/10.30848/pjb2020-1(12) 24. i. ugulu, z. i. khan, s. rehman, k. ahmad, m. munir, h. bashir and k. nawaz, bull. environ. contam. toxicol., 103 (2019) 468. https://doi.org/10.1007/s00128-019-02673-3 25. k. ahmad, k. wajid, z. i. khan, i. ugulu, h. memoona, m. sana, k. nawaz, i. s. malik, h. bashir and m. sher, bull. environ. contam. toxicol., 102 (2019) 822. https://doi.org/10.1007/s00128-019-02605-1 26. z. i. khan, h. safdar, k. ahmad, k. wajid, h. bashir, i. ugulu and y. dogan, environ. sci. pollut. res., 26 (2019) 14277. https://doi.org/10.1007/s11356-019-04721-1 27. z. i. khan, n. arshad, k. ahmad, m. nadeem, a. ashfaq, k. wajid, h. bashir, m. munir, b. huma, h. memoona, m. sana, k. nawaz, m. sher, t. abbas and i. ugulu, environ. sci. pollut. res., 26 (2019) 15381. https://doi.org/10.1007/s11356-019-04959-9 28. i. ugulu, int. j. educ. sci., 28 (2020) 7. https://doi.org/10.31901/24566322.2020/28. 1-3.1088 29. s. farooq, a. barki, m.y. khan and h. fazal, pak. j. weed sci. res., 18 (2012) 277. https://www.wssp.org.pk/weed/resources/im ages/paper/18wq1450364169.pdf 30. t. m. chiroma, r. o. ebewele and f. k. hymore, int. refereed j. eng. sci., 3 (2014) 1. http://dx.doi.org/10.183x/a03020109 31. usepa. preliminary remediation goals, region 9. united states environmental protection agency, washington, dc. (2002). 32. n. alrawiq, j. khairiah, m.l. talib, b.s. ismail and s. anizan, int. j. chem. tech. res., 6 (2014) 2347. http://www.sphinxsai.com/2014/vol6pt4/2/( 2347-2356)jul-aug14.pdf 33. k. ahmad, z.i. khan, s. yasmin, m. ashraf and a. ishfaq, pak. j. bot., 46 (2014) 1805. https://www.pakbs.org/pjbot/pdfs/46(5)/35. pdf 34. s. m. dogheim, e. m. m. ashraf, s. a. g. alla, m. a. korshid and s. m. fahmy, food add. contam., 21 (2004) 323. https://doi.org/10.1080/02652030310001656 361 35. s. gupta, v. jena, s. jena, n. davic, n. matic, d. radojevic and j. s. solanki, croatian j. food sci. tech. 5 (2013) 53. https://bib.irb.hr/datoteka/1016071.manus cript_gupta_5.pdf 36. w.h. liu, j.z. zhao, z.y. ouyang, l. soderlund and g.h. liu, environ. int., 31 (2005) 805. https://doi.org/10.1016/j.envint.2005.05.042 37. d. demirezen and a. aksoy, j. food qual., 29 (2006) 252. https://doi.org/10.1111/j.17454557.2006.000 72.x 38. a. mahmood and r. n. malik, arab. j. chem., 7 (2014) 91. https://doi.org/10.1016/j.arabjc.2013.07.002 39. k. wajid, k. ahmad, z.i. khan, m. nadeem, h. bashir, f. chen and i. ugulu, bull. environ. contam. toxicol., 104 (2020) 649. https://doi.org/10.1007/s00128-020-02841w 40. i. ugulu, m.c. unver and y. dogan, euromediterr. j. environ. integr., 4 (2019) 36. http://dx.doi.org/10.1007/s41207-019-01287 microsoft word 64-68-galley proof pjaec-18032016-36.doc issn-1996-918x pak. j. anal. environ. chem. vol. 18, no. 1 (2017) 64 – 68 http://doi.org/10.21743/pjaec/2017.06.06 detection of zinc in nail samples of iron welders hina chaudhry* 1 , maryam ijaz 2 , gul e fatima 3 , aisha masood 4 and numrah nisar 5 department of environmental science, lahore college for women university, lahore, pakistan *corresponding author email: hinachaudhry.env@hotmail.com received 18 march 2016, revised 29 may 2017, accepted 30 may 2017 -------------------------------------------------------------------------------------------------------------------------------------------abstract the current study was conducted to quantify zinc in nail samples of iron welders from different areas of lahore, pakistan. this study intended to assess nutritional deficiencies of zinc in welders. as nails serve a beneficial biomarker of concentrations of trace elements, hence in the present study the nails of welders were used for monitoring. the total number of nail samples collected from workers of iron welding shops, were 40. in a standardized washing procedure the nail samples were scrapped and cleaned of dust particles with nonionic detergent (triton x-100) and then nail samples were digested afterwards in acid mixture. the concentration of zinc was evaluated by atomic absorption spectrophotometer. the results revealed that concentration of zinc in nail samples ranged 0.297 – 1.718 mg/kg and averaged at 0.88±0.39 mg/kg which is below the ideal zinc levels in nail samples. correlation of zinc (mg/kg) was significant with age (0.214< 0.5), weight (0.320< 0.5) and body mass index (0.268< 0.5) of the welders, while a weak correlation of zinc (mg/kg) was found with height (0.042< 0.5) of the welders. keywords: zinc, nail samples, iron welders, atomic absorption spectrophotometer, pearson’s correlation. -------------------------------------------------------------------------------------------------------------------------------------------introduction zinc significantly contributes in constancy of human metabolism. participation of this trace metal in all major biochemical pathways and formulation of genetic material such as dna transcription, rna translation and cell division is well documented [1]. zinc content ranges from 1.5 to 2.5 grams in normal adult humans. average content is higher in males and lesser in females comparatively. human organs, fluids, tissues and major secretions contain zinc in them [2]. zinc is found in a wide array of foods. highest zinc per serving is offered by oysters while huge quantities are provided by poultry and red meat too. dairy products, nuts, cereals, beans, whole grains and different types of sea foods as in lobsters and crabs are rich in zinc content. institute of medicine of the national academies (formerly national academy of sciences) supports food and nutrition board (fnb) for the formulation of dietary reference intakes (dris) for zinc [3]. impairment of immune function, retardation in the growth and loss of appetite are some of the serious aftermath of zinc deficiency in human body. severity of zinc deficiency is characterized by skin and eye lesions, hair loss, male hypogonadism, impotence and delayed sexual maturation (prasad, 2004). reduced intake of zinc leads to lowered absorption of zinc in the body which is not compensated by reduced zinc excretion. ultimately, zinc is lowered in the body as reserved mobile zinc is depleted [1]. both acute and chronic types of zinc toxicities are common. when zinc is administered in high quantities it can lead to acute effects such as headaches, nausea, and diarrhea, loss of appetite, vomiting and cramps in abdomen [4]. as galvanized materials are welded, zinc oxide is formed which leads to metal fume fever on inhalation [5]. trace elements found in human nails have been thoroughly studied using variety of pak. j. anal. environ. chem. vol. 18, no. 1 (2017) 65 techniques. elemental analysis of trace metals is achieved through atomic fluorescence spectrometry, atomic absorption spectrometry, inductively coupled plasma atomic emission spectrometry (icp-als) [6]. fibrous proteins that make up tissue of nail are richly loaded with keratins appearing as residues of cysteine. healthy cells influence their roots and transient concentrations are offered by fluids and blood therefore nails serve as a beneficial biomarker of concentrations of trace elements [7]. the present study focuses on the importance of zinc in human body. zinc concentration in the body directly impacts immune system; development and growth of human body thereby influence the body directly. materials and methods sampling area and sample collection: for quantifying zinc in nail samples iron welders were selected from different welding shops found in lahore, pakistan. male subjects from welding shops were chosen for nail sample collection. hand of the respondents were washed using distilled water and metal free medicated soap. drying was done by clean towel or tissue papers in order to get rid of external contamination. clean scissors were used for collection of fingernails of male subjects between age ranges of 14-66 years. nail samples were stored in air tight plastic jars at room temperatures. analysis was carried out at environmental science research laboratory of lahore college for women university, lahore, pakistan. the general health status of the welders was assessed by means of a questionnaire related to their personal profile, general health, dietary habits, life style, age and gender, source of drinking water, smoking habits, health condition and medication. instrumentation: atomic absorption spectrophotometer (aas thermo scientific m series gf95z zeeman furnace) was used. washing of nail samples: nonionic detergent triton x-100 was used to wash nail samples in a standardized procedure [8]. nail samples were cleaned off any dust particles and then soaked in acetone which cleared off contamination. afterwards the nail samples were washed with deionized water five times. oven was used for drying and then samples were stored in desiccator. wet-acid digestion and preparation of waterclear solution: wet acid digestion was employed, whereby 10 ml of mixture of concentrated nitric acid and perchloric in ratio of 6:1 was used to digest dried nail samples. it was kept overnight at room temperature and excessive foaming was prevented. then it was heated at 160-180°c to get water clear mixture. heating reduced the volume of solution to 1 ml. wet acid digestion destroys the organic matter and in this way solution is obtained that contains metal in its elemental form. then each sample was diluted using 0.1 m nitric acid [9]. analytical procedure: flame mode of atomic absorption spectroscopy was used for analysis of nail samples. instrument calibration was achieved by running three different standards at the start of the analytical procedure. sample injection was done using a small capillary. for analyzing zinc in nail samples, hollow cathode lamp with the atoms of zinc was used (table 1). table 1. concentration of zinc in nail samples of iron welders. sample id zinc concentration mg/kg sample id zinc concentration mg/kg 1 0.821 21 0.469 2 1.350 22 0.395 3 0.869 23 1.395 4 0.572 24 1.199 5 0.746 25 1.083 6 0.735 26 1.602 7 0.464 27 1.319 8 0.483 28 1.178 9 0.541 29 1.014 10 0.420 30 1.180 11 0.297 31 1.388 12 1.219 32 1.118 13 0.470 33 0.448 14 0.457 34 1.239 15 0.454 35 1.108 16 0.515 36 1.192 17 0.661 37 1.718 18 0.583 38 1.333 19 0.482 39 1.115 20 0.553 40 1.145 pak. j. anal. environ. chem. vol. 18, no. 1 (2017)66 data interpretation and analysis: after analysis, the concentration of zinc in nail samples was depicted using statistical methods of averages, ranges, standard errors and pearson’s correlation. spss-17 and microsoft excel 2010 were utilized for statistical calculations and data interpretations. results and discussion the present study revealed low concentrations of zinc in nails of iron welders (table 2). as per reports of world health organization (who), concentration range of zinc in body must be between 2-3 g/kg. study of lawrence wilson in 2012 showed that zinc levels in nails ranging between 0.8-2.8 mg/dl or 8-28 mg/kg is considered ideal. comparison of average zinc concentration in the samples was made with allowable limits. the results revealed that the zinc concentration was well below the prescribed limit. according to the present study, in almost all of the workers deficiency of the zinc was observed. the zinc concentration studied in welders’ nails increases significantly with age. the younger group showed least levels when compared with older group (fig. 1). table 2. mean and standard deviation of the variables. n minimum maximum mean std. deviation age (years) 40 14 66 28.95 13.27 weight (kg) 40 40 106 64.57 12.68 height (meters) 40 1.52 2.00 1.66 0.1016 body mass index kg/m3 40 18.6 31.6 23.61 3.4 concentrati on (ppm) 40 0.2970 1.7183 0.8836 0.3944 figure 1. comparison of average value of zinc (mg/kg) in nails of welders from different age groups data which was gathered during survey revealed that 57.5% of welders have largest proportion of vegetable in their daily diet (fig. 2). diets rich in zinc were missing from their meals. zinc is critically important for healthy skin, men’s reproductive health and for mood and brain health. zinc deficiency may be associated with certain nutritional reasons. deficiencies appear in the consumers, if zinc content in the consumed food is low or if the forms of zinc are unavailable. other causes include deficiency associated with some ailments and genetic disorders which effect absorption of zinc in the intestine. in some severe cases, zinc levels fall due to loss of zinc from intestines [10]. it can be inferred from the studies that the lack of balanced diet and poor nutritional status of food are the most evident causes of zinc deficiency. assessment of variety of food consumed by the workers showed that there was over dependence on food items such as vegetables and pulses while meat, fruits and rice were consumed comparatively less frequently by these workers. such nutritional imbalances have led to a low mineral diet deficient in zinc. similar studies with use of nails as biomarkers for quantifying heavy metals in a wide variety of age groups of workers from iron welder workshops found in maiduguri metropolis, borno state, nigeria were depictive of strong links between nutritional deficiencies and low concentration of zinc in nails. poor socioeconomic conditions, haphazard outdoor works, unhygienic food intake and rapid loss of zinc from body were the main reasons for lower levels of zinc in respondents [6]. figure 2. graph showing preference of food items by welders pak. j. anal. environ. chem. vol. 18, no. 1 (2017) 67 descriptive statistic study variables were analyzed using descriptive statistics and averages and standard deviations were interpreted. ranges were calculated. table 3 of the study showed mean age (28.95 0±13.27yrs), mean weight (64.57±12.68kg), mean height (1.66±0.101m) and average body mass index bmi (23.61±3.4). the mean concentration of zinc in nail samples of iron welding workers was 0.883±0.394. ranges calculated in the present study are shown in the table. table 3. correlation of concentration of zinc with age, weight, height and bmi. age weight height bmi pearson’s correlation 0.214 0.093 0.042 0.454 sig.2 (tailed) 0.294 0.32 0.341 0.268 concentration zinc n 40 40 40 40 analysis of relationship the present study also utilized pearson's correlation (level of significance set at 0.05) for determining the relationship between variables. correlation determined the strength of relationship between variables. 1 means the relationship is strong. 0.5 or above means relationship is positive strong. less than 0.5 means there is weak relationship. negative means inverse relationship. table 3 showed strength of correlation (0.320 < 0.5) between weight and zinc concentration. similar strength was depicted between zinc concentration and bmi (0.268 < 0.5). rest of the variables determined in the study such as age (0.214 < 0.5) and height (0.042 < 0.5) showed a comparatively weaker correlation with concentration of zinc in mg/kg table 4. thus the data analysis indicated that there was a significant but weak relationship of zinc concentration with age, height, weight and bmi of the workers. table 4. faas specifications for zinc analysis. lamp current (m ao) 5 wavelength (nm) 213.9 linear range (mg/litre) 0.4–1.5 slit width (nm) 0.5 integration time (seconds) 2.0 flame air acetylene body weight and metabolism of zinc have shown a strong interrelationship in a variety of clinical and experimental studies conducted on the subject. when a person becomes underweight either due to insufficient diets, regular attacks of illnesses and infections then it causes low intake of protein diet, vitamins and minerals particularly zinc. if the workers are obese or overweight, then deficiency of zinc is critically harmful, this was concluded in world health organization (who) studies. this is further complicated by prevalence of decreased resistance to infections and behavioral and learning issues. the respondents which fall in 1.52 m category of height showed least levels of zinc concentration when compared with the, 2.0 m categories. it was evident from the finding of this study that respondents with low bmi had low zinc concentration as compared to normal and high bmi. this result can be related to other study in which nail mineral analysis was performed on over three-hundred males with bmi ranging between low, normal and high. significant differences were noted in zinc levels between men with a low compared to those with a high bmi [11]. institute of medicine (2001) supported the fact that zinc deficiency has become a leading risk factor for a number of diseases and disorders prevailing worldwide. occupants involved in iron welding are at a higher risk of developing such zinc deficiencies due to risky operations they are involved in make them more vulnerable to such deficiencies. developing countries depict higher zinc deficiencies which hampers the health as well as productivity [12]. the influence of certain factors (age, sex, health, occupation, etc.) causing the change in zinc levels is obvious, whereas the influence of other factors (structure of nail, height of the subject, etc.) is obscure. it is very important to consider all the factors at the time of investigation for effective interpretation, validity, and application of results of nail analysis [13]. further organized feeding programs for industrial workers in public sector undertakings is gaining importance to promote better nutritional status, since well-fed labor force and productivity are closely related. pak. j. anal. environ. chem. vol. 18, no. 1 (2017)68 conclusion zinc deficiency is one of the leading risk factors for disability and death worldwide affecting an estimated billion people. nutritional zinc deficiency arises when physiological requirements cannot meet by zinc absorption from diet. the high prevalence of zinc deficiency in the developing world has substantial health and economic costs including decreased productivity. zinc is an important component of the human body and its level greatly influence the body by means of different ways as it plays an important role in immune system, growth and development of the human body. present study utilized nails as a biomarker for quantifying zinc levels in welders. the prevailing situation of zinc that appeared in alarmingly low concentration (0.297 – 1.718 mg/kg) in all samples of nail of the welders is quite concerning. main cause of the zinc deficiency in the body is the lack of balanced diet and poor nutrition. zinc concentration showed positive correlation with age, weight, bmi and height. the poor health conditions of these workers have direct relationship with their socioeconomic status and nutritional deficiencies. references 1. r. a. colvin, w. r. holmes, c. p. fontaine, and w. maret, metallomics, 2 (2010) 306. https://doi.org/10.1039/b926662c 2. m. hambidge, human zinc deficiency. j. nutrition, 130 (2000) 1344s. http://jn.nutrition.org/content/130/5/1344s.fu ll 3. imfnb. dietary reference intakes for vitamin a, vitamin k, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. washington dc: (2001). national academy press. https://www.ncbi.nlm.nih.gov/books/nbk22 2310/ 4. l. m. plum, l. rink and h. haase, int. j. environ., 7 (2010) 1342. https://doi.org/10.1007/bf02703706 5. g. j. li, l. l. zhang, l. lu, p. wu and & w. zheng. j. occup. environ. med., 46 (2004) 241. https://doi.org/10.1097/01.jom. 0000116900.49159.03 6. f. i. abdulrahman, j. c. akan, z. m. chellube and m. waziri, world environ., 2 (2012) 81. https://doi.org/10.5923/j.env.20120204.05 7. p. xun, k. liu and j. s. morris, in. am. j. epidemiol., (2010). https://doi.org/10.1093/aje/kwq001 8. r. mehra and m. juneja, j. biosci., 30 (2005) 253. https://doi.org/10.1007/bf02703706 9. c. hotz and k. h. brown, food nutr. bull., 25 (2004) 99s. https://www.ncbi.nlm.nih.gov/pubmed/18046 856 10. s. bozalioglu, y. ozkan, m. turan and b. simsek, j. trace elem. med. biol., 18 (2005) 243. https://doi.org/10.1016/j.jtemb.2005.01.003 11. d. wang, x. du and w. zheng, toxicol. lett, 176 (2008) 40. https://doi.org/10.1016/j.toxlet.2007.10.003 12. a. s. prasad, bmj. 326 (2003) 409. https://doi.org/10.1136/bmj. 326.7386.409 13. s. sukumar, r. colah and d. mohanty. br. j. haematol., 116 (2002) 671. https://doi.org/10.1046/j.00071048.2001.03328.x microsoft word 303-313-pjaec-22042019-162.doc cross mark issn-1996-918x pak. j. anal. environ. chem. vol. 21, no. 2 (2020) 303 – 313 http://doi.org/10.21743/pjaec/2020.12.32 monitoring of zinc profile of forages irrigated with city effluent zafar iqbal khan 1 , kafeel ahmad 1 , hareem safdar 1 , ilker ugulu 2 , kinza wajid 1 , muhammad nadeem 3 , mudasra munir 1 and yunus dogan* 4 1 department of botany, university of sargodha, sargodha, pakistan. 2 faculty of education, usak university, usak, turkey. 3 institute of food science and nutrition, university of sargodha, sargodha, pakistan. 4 buca faculty of education, dokuz eylul university, izmir, turkey. *corresponding author email: yunus.dogan@deu.edu.tr received 16 september 2019, revised 16 september 2020, accepted 20 october 2020 -------------------------------------------------------------------------------------------------------------------------------------------abstract wastewater contains a surplus amount of trace metals that contaminate the soil and crops. a pot trial was performed to determine the impact of wastewater on the zinc accumulation in forages and their associated health risk. forages both of summer (zea mays, echinochloa colona, pennisetum typhoideum, sorghum vulgare, sorghum bicolor, sesbania rostrata, and cyamopsis tetragonoloba) and winter (trifolium alexandrinum, medicago sativa, brassica campestris, trifolium resupinatum, brassica juncea, and brassica napus) were grown with sewage water and tap water treatment. the experiment was laid down in a completely randomized design with five replicates. the concentration of zinc in water, root and forage samples were analysed by atomic absorption spectrophotometer. in tap water, the zinc value was 0.498 mg/l and in wastewater 0.509 mg/l, respectively. the maximum level of zinc in the forages leaves was 3.582 mg/kg found in brassica napus grown in the winter season. the maximum observed value for zinc bioconcentration factor in brassica juncea was (2.88) grown in winter. the values of pollution load index for zinc were found less than 1. the values of daily intake of metal and health risk index for zinc in all forages were less than 1 indicated that consumption of these forages was free of risk. keywords: bioaccumulation, pollution load index, forage, health risk index, zinc. -------------------------------------------------------------------------------------------------------------------------------------------introduction the shortage of water is a major problem all over the world, and many parts of the world are facing this problem day by day [1]. this problem of water shortage is solved by alternate sources of irrigation [2]. the wastewater is a source of some nutrients essential for soil fertility, but it also contains toxic metals that contaminate the soil and crops [3]. the metals ni, pb, zn, cd, cu, cr, and mn from wastewater contaminate the agricultural land and crops grown there and become the part of the food chain and cause various health hazards in human [4, 5]. the wastewater irrigation is beneficial if it imparts no negative impact on crops as well as human health [6]. however, heavy metals due to their residing natures cause pollution in the environment and ultimately in humans [7]. the water sites such as sewage, canal water and tube-well water used for fields having different food crops. the root apices of pak. j. anal. environ. chem. vol. 21, no. 2 (2020)304 plants are impassable with heavy metals due to their immature cells and low-density cell walls. metals are taken up by plant from contaminated soil and then transfer to the upper parts of the plants [8]. zinc (zn) is considered a vital element for metabolism in animals and plants, but if it exceeds the level severe losses to life occur [9]. zn has great importance as a catalytic element for over 300 enzymes, such as carbonic anhydrase, alcohol dehydrogenase, alkaline phosphatase, cu-zn superoxide dismutase, and dna-rna polymerase [10]. also, mitosis division of a cell is distressed due to zn activity [11]. the activity and permeability of membranes are decreased by the zn attack because it affects the movement of ions and enzymes there [12]. the necrosis of shoots caused by zn and it also can destroy the plant cell finally [13]. zn interrupts the root function [14]. additionally, cd, pb, and zn decrease plant uptake level of necessary elements like mn, but a greater amount of zn can cause a lack of development and reproduction [15]. the current research was conducted to determine the impact of zn on pollution severity and transfer of zn in forages and humans through soils. materials and methods study area the current research (pot trial) was performed at the department of botany, the university of sargodha, pakistan at coordinates 32.0740° n, 72.6861° e. plant cultivation summer cultivation: 4 types of forages bajra (pennisetum typhoideum rich.), sanwak (echinochloa colona l. link), jowar (hybrid) (sorghum bicolor l. moench), jantar (sesbania rostrata bremek & oberm.), maize (zea mays l.), local jowar (sorghum vulgare pers.), gawara (cyamopsis tetragonoloba l. taub.). were planted in 70 pots (35 control and 35 experimental) below 4-5 cm of soil. the physicochemical parameters of soil are given in table 1. the experiment was laid down in a completely randomized design (crd) with 5 replicates. the chemical composition of canal and sewage water is given in table 2. pots were irrigated twice a week. winter cultivation: six winter forages were sown; berseem (trifolium alexandrinum l.), sarsoon (brassica campestris l.), luscern (medicago sativa l.), indian mustard (brassica juncea l. czern.), chatala (trifolium resupinatum l.), and canola (brassica napus l.). forages were planted in 60 including 30 control (tap water irrigated) and 30 experimental pots (sewage water). the plants were harvested on 6-10-2016. table 1. physicochemical properties of water. properties of water tap water sewage water electrical conductivity (µs/cm) 1890 7750 calcium+ magnesium (ca2++mg2+) (meq/l) 5.2 18.5 sodium (na+) (meq/l) 13.7 59.0 carbonate (co23-) (meq/l) 0.4 0.8 bicarbonate (hco3) (meq/l) 8.2 9.6 chloride (cl-) (meq/l) 6.4 51.7 sodium adsorption ratio (sar) 8.5 19.4 residual sodium carbonate (rsc) 3.4 nil table 2. physicochemical properties of soil. properties of soil s-c* s-e** wc*** we**** depth 0-15 0-15 0-15 0-15 ph 7.7 8.1 7.9 8.1 electrical conductivity (ms/cm) 5.64 8.42 3.01 4.51 organic matter (%) 0.90 0.83 0.96 0.76 available phosphorus (mg/kg) 8.8 7.0 8.6 7.4 available potassium (mg/kg) 240 160 200 170 saturation (%) 36 38 40 38 texture loamy loamy loamy loamy *s-c: summer control, **s-e: summer experimental, ***w-c: winter control, ****w-e: winter experimental pak. j. anal. environ. chem. vol. 21, no. 2 (2020) 305 samples collection plastic bottles were washed with distilled water and samples of sewage and tap water (100 ml each) were taken in plastic bottles. conc. hno3 (1 ml) was added in water to prevent the activity of microorganisms. samples (130) were stored in a refrigerator before the digestion. soil samples were sun dried and then oven-dried for 3 days at 75ºc to removes excess moisture. after drying and grinding, these samples were digested. zinc analysis zn contents were analysed by running samples in atomic absorption spectrophotometer (aas-6300 shimadzu japan). statistical analysis zn values for water, soil and forage samples were analysed by statistical package of social sciences (spss 23). independent samples t-test was used to determine whether tap water and sewage water irrigation made a statistically significant difference in the samples. bioconcentration factor bioconcentration factor (bcf) was used to determine the transfer of metals from soil to the edible part of the plant [16]. soilinmetalheavyof kg mg ionconcentrat plantinmetalheavyof kg mg ionconcentrat bcf                  pollution load index pollution severity of soil can be well analysed by using the following formula [17]. soilinmetaltheofvalueferencere soiledinvestigatinionconcentratmetal pli the reference value of zn was (44.19 mg/kg). daily intake of metals daily intake of metals (dim) was computed according to the following formula [18]. weightbodyaverage forageofakeintdailymetalofionconcentrat dim   average body weight was taken as 550 kg. health risk index health risk index (hri) was calculated by the following formula [19]. dosereferenceoral metalofakeintdaily hri rfd values for zn was 0.3 mg/kg/day [20]. results and discussion zinc content in water according to independent samples ttest results, the difference between heavy metal values in tap and sewage water samples was statistically significant (p<0.01). the determined zn value for tap water and sewage water was 0.498 and 0.509 mg/l, respectively (table 3). the zn content in the present findings was found within the permissible limit of 2.0 mg/l given by pescod [21]. the zn values in the present findings were higher than the findings of tariq et al. [22] (0.1 mg/l) in tap water and by murtaza et al. [23] (0.210 mg/l) for sewage water. salawu et al. pak. j. anal. environ. chem. vol. 21, no. 2 (2020)306 [24] found a higher zn value (4.236 mg/l) in sewage water. the present zn values in water were lower than the findings of kumar and chopra [25] (2.17-8.80 mg/l) for borewell and industry effluent. khaskhoussy et al. [26] reported a similar range (0.20-0.55 mg/l) for zn in freshwater and treated wastewater. kumar and chopra [25] analyzed that the higher level of various metals in the wastewater might be due to the application of various chemicals used in the industry. among the household products, the medicated (anti-dandruff) shampoos contain zn pyrithione and the high zn concentrations will thus raise the zn inputs to the sewage waters. also, the differences in the zn values determined in the various studies can be potentially originated from the study areas of the studies. table 3. zinc content in water (mg/l). tap water sewage water p 0.498±0.1274 0.509±0.0506 0.001** permissible maximum limita 2.0 mg/l **: significant at 0.01 level, source: apescod [21] zinc in soil independent sample t-test showed that the zn content in the soil samples of c. tetragonoloba, s. vulgare, b. juncea, and t. alexandrinum were statistically different (p<0.01). the order as a result of tap water irrigation (twi) was: p. typhoideum> z. mays> b. napus> b. campestris> s. bicolor> e. colona> t. resupinatum> b. juncea> t. alexandrinum> s. vulgare> c. tetragonoloba>m. sativa> s. rostrata. the sequence was as: m. sativa> c. tetragonoloba> z. mays> b. campestris> b. napus> p. typhoideum> t. alexandrinum> s. bicolor> e. colona> b. juncea> s. rostrata> t. resupinatum> s. vulgare for sewage water irrigation (swi). the maximum values of zn were found in the soil of m. sativa (2.871 mg/kg) and the minimum was found in the soil of s. rostrate (0.129 mg/kg) (table 4). the values of zn were found within the permissible maximum limits of 200 mg/kg established by usepa [27]. these zn values were contradicted as reported by some researchers (12.13 mg/kg) as in october and 8.47 mg/kg in june [28]. however, kumar and chopra [25] noticed a higher range of zn in soil (3.75-4.15 mg/kg). table 4. zinc content (mg/kg) in soil grown with different forages. *, **: significant at 0.05 and 0.01 levels, ns: non-significant, source: ausepa [27] khaskhoussy et al. [26] found a higher range for zn (59.5-74.5 mg/kg) in soil irrigated with freshwater and treated wastewater. zn accumulation in the soil might be due to various factors metals in water, biological processes, soil and water properties. the activities of soil microflora are affected adversely due to the binding of zn ions with soil particles when irrigation is applied [29] as shown in fig. 1. tap water sewage water p forage summer z. mays 1.546±0.0026 1.824±0.0172 0.193ns p. typhoideum 1.594±0.0498 1.798±0.0021 0.105ns c. tetragonoloba 0.153±0.0089 2.850±0.0379 18.198ns e. colona 0.462±0.0364 0.480±0.0536 0.001** s. rostrata 0.129±0.0317 0.372±0.0187 0.148ns s. bicolor 0.522±0.0292 0.708±0.0232 0.086ns s. vulgare 0.294±0.0028 0.298±0.0137 0.001** winter b. campestris 1.278±0.0018 1.822±0.0169 0.740ns b. napus 1.544±0.0192 1.805±0.0043 0.069ns b. juncea 0.468±0.0347 0.488±0.0520 0.001** m. sativa 0.156±0.0084 2.871±0.0310 18.435ns t. resupinatum 0.154±0.0379 0.370±0.0188 0.117ns t. alexandrinum 0.298±0.0043 0.301±0.0160 0.001** df 24 t -1.108 permissible maximum limita 200 mg/kg pak. j. anal. environ. chem. vol. 21, no. 2 (2020) 307 figure 1. zinc contents in soil zinc in root according to the results of the independent samples t-test, the difference between the heavy metal values of the plant root samples as a result of tap and sewage irrigation was statistically significant except for c. tetragonoloba s. bicolor and m. sativa plants (p<0.01 and p<0.05). the order of zn values as a result of twi was: t. alexandrinum>b. juncea> e. colona> s. bicolor> b. napus> s. vulgare> b. campestris> c. tetragonoloba> p. typhoideum> z. mays> s. bicolor> t. resupinatum> s. rostrata. while as a result of swi was: t. alexandrinum> m. sativa> c. tetragonoloba> s. bicolor> b. jucea> e. colona> b. napus> s. vulgare> b. campestris> z. mays> p. typhoideum> s. rostrata> t. resupinatum. the highest zn content was in the root was 0.390 mg/kg in t. alexandrinum grown in winter and the lowest 0.075 mg/kg in s. rostrata grown in summer (table 5). asdeo [30] and masona et al. [31] found a higher zn range as 6.32-8.92 mg/kg and 24-120 mg/kg, respectively. hassan et al. [32] reported a greater value of zn in plants (35.3 mg/kg). khaskhoussy et al. [26] found a higher trend of zn root than present study and keller et al. [33] observed that various plants with different root systems had diverse reactions and tolerances to heavy metals and minimum heavy metal concentrations in tissues could promote plant growth (fig. 2). table 5. zinc concentration (mg/kg) in roots of forage samples irrigated with tap and sewage water. tap water sewage water p forage summer z. mays 0.120±0.0021 0.190±0.0016 0.012* p. typhoideum 0.123±0.0018 0.183±0.0018 0.009** c. tetragonoloba 0.133±0.0017 0.310±0.0019 0.088ns e. colona 0.235±0.0016 0.258±0.0014 0.001** s. rostrate 0.075±0.0015 0.138±0.0017 0.010* s. bicolor 0.118 ±0.0018 0.305±0.0019 0.088ns s. vulgare 0.147±0.0146 0.238±0.0017 0.021* winter b. campestris 0.138±0.0063 0.193±0.0028 0.008** b. napus 0.185±0.0017 0.216±0.0034 0.002** b. juncea 0.236±0.0021 0.266±0.0021 0.002** m. sativa 0.130±0.0017 0.325±0.0046 0.095ns t. resupinatum 0.105±0.0016 0.155±0.0017 0.006** t. alexandrinum 0.367±0.0023 0.390±0.0017 0.001** df 24 t -1.138 permissible maximum limita 50 mg/kg *, **: significant at 0.05 and 0.01 levels, source: awho [6] figure 2. zinc contents in root irrigated with tap and sewage water zinc in leaves according to the results of the independent samples t-test, the difference between the heavy metal values of the plant leaf samples as a result of tap and sewage irrigation was statistically significant except for b. napus and b. juncea plants (p<0.01 and p<0.05). the level of zn in leaves of forages at twi was found in following order: b. napus> b. juncea> t. resupinatum> z. mays> c. tetragonoloba> m. sativa> p. typhoideum> e. colona> s. rostrata> s. pak. j. anal. environ. chem. vol. 21, no. 2 (2020)308 bicolor> s. vulgare> t. alexandrinum> b. campestris. while as a result of swi was: b. napus> b. juncea> s. bicolor> t. resupinatum> p. typhoideum> z. mays> s. rostrata> t. alexandrinum> s. vulgare> e. colona> z. mays> m. sativa> b. campestris. the highest zn content in the forages leaves was 3.582 mg/kg occurred in b. napus grown in the winter season and the lowest was 0.073 mg/kg in b. campestris also grown in winter (table 6). the current zn values were found within the permissible limit of 50 mg/kg established by who [6]. according to this finding, it seems like no risk for metal toxicity. khan et al. [34] reported higher zn concentrations varied from (25.88 to 42.24 mg/kg) with the lowest values during october and the highest during january. however, kumar and chopra [25] observed a lower range of zn (8.28-11.60 mg/kg) in crops. kansal et al. [35] found a higher range of zn in different plant parts in maize (38-53 mg/kg) and berseem (25-46 mg/kg) irrigated with tube-well and sewage water. the lowest zn prerequisite of livestock varies with the chemical form or combination of the diet [36] (fig. 3). table 6. zinc contents (mg/kg) in leaves of forages. tap water sewage water p forage summer z. mays 0.084±0.0023 0.126±0.0024 0.004** p. typhoideum 0.085±0.0017 0.259±0.0023 0.075* c. tetragonoloba 0.125±0.0176 0.199±0.0025 0.014* e. colona 0.196±0.0023 0.257±0.0025 0.009** s. rostrate 0.189±0.0017 0.240±0.0017 0.007** s. bicolor 0.143±0.0627 0.286±0.0018 0.051* s. vulgare 0.187±0.0017 0.213±0.0019 0.002** winter b. campestris 0.073±0.0019 0.123±0.0018 0.006** b. napus 1.275±0.0017 3.582±0.0026 13.300ns b. juncea 0.265±0.0019 1.350 ±0.0177 2.943ns m. sativa 0.086±0.0018 0.125±0.0021 0.004** t. resupinatum 0.214±0.0222 0.285±0.0017 0.013* t. alexandrinum 0.188±0.0015 0.223±0.0016 0.003** df 24 t -1.257 permissible maximum limita 50 mg/kg ns: non-significant, *, **: significant at 0.05 and 0.01 levels, source: awho [6] figure 3. zinc contents in leaves of forages bioconcentration factor the values of bcf in plants due to twi was found in the following descending sequence: b. napus> c. tetragonoloba> t. resupinatum> s. rostrata> t. alexandrinum> b. juncea> e. colona> b. campestris> z. mays> p. typhoideum> s. vulgare> m. sativa> s. bicolor. as a result of swi was: b. juncea> s. bicolor> b. napus> s. rostrata> t. resupinatum> t. alexandrinum> e. colona> s. vulgare> c. tetragonoloba> b. campestris> p. typhoideum> z. mays> m. sativa. bcf value was higher in b. juncea (2.88) and the minimum in m. sativa (0.0433) (table 7). lu et al. [37] found lower zn bcf value (0.26 mg/kg) in maize shoots as compared to the present study. alrawiq et al. [38] observed a lower range (0.296-0.196) for zn bcf after irrigation with different treatments. asdeo [30] also reported a lower value (0.4049) for bcf in millet. it was reported by pawan et al. [29] that the ions of zn associated with metal pollution caused by the property of zn ions to bind with the soil particles and they also get dissolved in the water found in soil. pak. j. anal. environ. chem. vol. 21, no. 2 (2020) 309 table 7. bioconcentration factor of zinc in forages. pollution load index the order of pli due to twi was: p. typhoideum> z. mays> b. napus> b. campestris> s. bicolor> e. colona> t. alexandrinum> s. vulgar> t. resupinatum> c. tetragonoloba> s. rostrata> b. campestris. the order of soil pli value according to the plant due to swi was: m. sativa> c. tetragonoloba> b. campestris> b. napus> s. bicolor> z. mays> p. typhoideum> e. colona> b. juncea> s. rostrata> t. resupinatum> t. alexandrinum> s. vulgare. the highest pli was noticed in m. sativa (0.0649) and the lowest value showed by s. vulgare (0.0066) (table 8). bao et al. [39] found higher pli for zn in soil (1.04, 1.14, 1.03) in three different zones irrigated with the long-term sewage water. ahmad et al. [40] also noticed higher values of pli for zn (1.528) in soil treated with sewage and canal water. the higher pli suggests that there was more contamination of heavy metals in the area. table 8. pollution load index for zinc in soil. daily intake of metal and health risk index the values of dim for zn due to twi was found in the following sequence: b. napus> b. juncea> e. colona> t. resupinatum> s. rostrata> t. alexandrinum> s. vulgare> s. bicolor> p. typhoideum> c. tetragonoloba> z. mays> m. sativa> b. campestris. while due to swi was found in following descending sequence: b. napus> b. juncea> e. colona> t. resupinatum> s. vulgare> m. sativa> s. bicolor> s. rostrata> b. juncea> t. alexandrinum> c. tetragonoloba> b. campestris> z. mays. the maximum dim value calculated for zn in b. napus (0.0813) and the minimum in b. campestris (0.00164) (table 9). roggeman et al. [41] noticed higher mean dim value (7368-4216 mg/kg) in winter and summer value (3698-2110 mg/kg) in herds of cows as compared to the present study. lawal et al. [42] earlier found similar dim zn values (0.0068-0.0062) in spinach leaves grown around kubanni river in two farmlands. in the present results, the values of dim were bcf irrigation water tap sewage forage summer z. mays 0.046 0.082 p. typhoideum 0.047 0.162 c. tetragonoloba 0.819 0.869 e. colona 0.516 0.598 s. rostrata 0.742 1.543 s. bicolor 0.211 2.446 s. vulgare 0.457 0.475 winter b. campestris 0.052 0.672 b. napus 0.826 1.984 b. juncea 0.552 2.888 m. sativa 0.043 0.547 t. resupinatum 0.770 1.386 t. alexandrinum 0.628 0.739 pli irrigation water tap sewage forage summer z. mays 0.033 0.0402 p. typhoideum 0.036 0.0406 c. tetragonoloba 0.0039 0.0645 e. colona 0.0104 0.0108 s. rostrate 0.0029 0.0084 s. bicolor 0.0118 0.0160 s. vulgare 0.0064 0.0065 winter b. campestris 0.029 0.0412 b. napus 0.035 0.0408 b. juncea 0.0105 0.0109 m. sativa 0.0037 0.0649 t. resupinatum 0.0034 0.0083 t. alexandrinum 0.0067 0.0068 pak. j. anal. environ. chem. vol. 21, no. 2 (2020)310 lower than 1 and it suggests that health risk was linked with the use of such contaminated forages. the maximum hri observed value showed by b. napus (0.965) and the minimum value by b. campestris (0.0054 mg/kg). khan et al. [43] gave higher hri zn value (0.5370.609) and lawal et al. [42] observed lower hri zn value (0.040-0.021) in spinach leaves grown around kubanni river in two farmlands. khan et al. [44] gave similar mean hri value (0.09-0.10) in wastewater irrigated sites. health risk index depends on the physico-chemical characteristics of the soil, type of forage being consumed and the rate of the consumption of forages. table 9. daily intake of metals and health risk index of zinc in forages. dim hri irrigation water irrigation water tap sewage tap sewage forage summer z. mays 0.0020 0.0029 0.0065 0.0093 p. typhoideum 0.0029 0.0058 0.0064 0.0195 c. tetragonoloba 0.0028 0.0045 0.0094 0.0150 e. colona 0.0058 0.0068 0.0144 0.0198 s. rostrata 0.0042 0.0054 0.0142 0.0181 s. bicolor 0.0032 0.0064 0.0107 0.0215 s. vulgare 0.0040 0.0048 0.0142 0.0160 winter b. campestris 0.0016 0.0027 0.0054 0.0092 b. napus 0.0289 0.0813 0.0271 0.965 b. juncea 0.0060 0.0306 0.0200 0.202 m. sativa 0.0019 0.0028 0.0064 0.0094 t. resupinatum 0.0048 0.0064 0.0161 0.022 t. alexandrinum 0.0041 0.0050 0.0142 0.0168 conclusion wastewater irrigation readily contaminates the soil and agricultural land. in the present research work, the level of zn was high in different parts of forages that were irrigated with the sewage water. the concentration of zn in all parts of forages treated with sewage water were higher than those treated with tap water. the values of zn in both treatments were found within the permissible limit. the values of health risk index in all forages were less than 1. thus, it was concluded that forages treated with tap and sewage water were safe for human consumption. conflicts of interest the authors declare that there is no conflict of interest in this paper. references 1. z. i. khan, i. ugulu, s. sahira, k. ahmad, a. ashfaq, n. mehmood and y. dogan, int. j. environ. res., 12 (2018) 503. https://doi.org/10.1007/s41742-0180110-2 2. k. ahmad, k. wajid, z. i. khan, i. ugulu, h. memoona, m. sana, k. nawaz, i. s. malik, h. bashir and m. sher, bull. environ. contam. toxicol., 102 (2019) 822. https://doi.org/10.1007/s00128-01902605-1 3. z. i. khan, i. ugulu, s. umar, k. ahmad, n. mehmood, a. ashfaq, h. bashir and m. sohail, bull. environ. contam. toxicol., 101 (2018) 235. https://doi.org/10.1007/s00128-0182353-1 4. z. i. khan, h. safdar, k. ahmad, k. wajid, h. bashir, i. ugulu and y. dogan, environ. sci. pollut. res., 26 (2019) 14277. https://doi.org/10.1007/s11356-01904721-1 5. s. erkol and i. ugulu, proc. soc. behav. sci., 116 (2014) 4742. pak. j. anal. environ. chem. vol. 21, no. 2 (2020) 311 http://dx.doi.org/10.1016/j.sbspro.2014.0 1.1019 6. z. i. khan, i. ugulu, k. ahmad, s. yasmeen, i. r. noorka, n. mehmood and m. sher, bull. environ. contam. toxicol., 101 (2018) 787. https://doi.org/10.1007/s00128-0182448-8 7. i. ugulu, int. j. educ. sci., 26 (2019) 49. https://doi.org/10.31901/24566322.2019/ 26.1-3.1086 8. i. ugulu and s. baslar, j. alternative compl. med., 16 (2010) 313. http://doi.org/10.1089=acm.2009.0040 9. i. ugulu, biotech. biotechnol. equip., 29 (2015) 20. http://doi.org/10.1080/13102818.2015.1 047168 10. m. nadeem, t. m. qureshi, i. ugulu, m. n. riaz, q. u. an, z. i. khan, k. ahmad, a. ashfaq, h. bashir and y. dogan, pak. j. bot., 51 (2019) 171. http://doi.org/10.30848/pjb2019-1(14) 11. z. i. khan, h. safdar, k. ahmad, k. wajid, h. bashir, i. ugulu and y. dogan, pak. j. bot., 52 (2020) 111. http://dx.doi.org/10.30848/pjb20201(12) 12. i. ugulu, z. i. khan, s. rehman, k. ahmad, m. munir, h. bashir and k. nawaz, bull. environ. contam. toxicol., 103 (2019) 468. https://doi.org/10.1007/s00128-01902673-3 13. z. i. khan, k. ahmad, s. rehman, a. ashfaq, n. mehmood, i. ugulu and y. dogan, pak. j. anal. environ. chem. 20 (2019) 60. http://doi.org/10.21743/pjaec/2019.06.08 14. z. i. khan, n. arshad, k. ahmad, m. nadeem, a. ashfaq, k. wajid, h. bashir, m. munir, b. huma, h. memoona, m. sana, k. nawaz, m. sher, t. abbas and i. ugulu, environ. sci. pollut. res., 26 (2019) 15381. https://doi.org/10.1007/s11356-01904959-9 15. i. ugulu, m.c. unver and y. dogan, euro-mediterr. j. environ. integr., 4 (2019) 36. http://dx.doi.org/10.1007/s41207-0190128-7 16. i. ugulu, environ. educ. res., 21 (2015) 916. http://doi.org/10.1080/13504622.2014.9 23381 17. k. wajid, k. ahmad, z. i. khan, m. nadeem, h. bashir, f. chen and i. ugulu, bull. environ. contam. toxicol., 104 (2020) 649. https://doi.org/10.1007/s00128-02002841-w 18. z. i. khan, k. ahmad, s. siddique, t. ahmed, h. bashir, m. munir, s. mahpara, i. s. malik, k. wajid, i. ugulu, m. nadeem, i. r. noorka and f. chen f, environ. sci. pollut. res., 27 (2020) 26694. https://doi.org/10.1007/s11356-02009062-y 19. z. i. khan, i. ugulu, s. sahira, n. mehmood, k. ahmad, h. bashir and y. dogan, j. water sanit. hyg. dev., 10 (2020) 249. https://doi.org/10.2166/washdev.2020.13 2 20. n. yorek, i. ugulu and h. aydin, comput. intell. neurosci., article id 2476256, (2016) 1. http://dx.doi.org/10.1155/2016/2476256 21. m. d. pescod, wastewater treatment and use in agriculture. in: fao irrigation and drainage paper. 47. food and agricultural organization, rome. (1992). http://www.fao.org/3/t0551e/t0551e00. htm 22. m. tariq, m. ali and z. shah, soil environ., 25 (2006) 64. http://www.se.org.pk/papers.aspx?issuei d=5 pak. j. anal. environ. chem. vol. 21, no. 2 (2020)312 23. g. murtaza, a. ghafoor, m. qadir, g. owens, m. a. aziz, m. h. zia and saifullah, pedosphere, 20 (2010) 23. https://doi.org/10.1016/s10020160(09)60279-4 24. k. salawu, m. m. barau, d. mohammed, d. a. mikailu, b. h. abdullahi and r. i. uroko, j. toxicol, environ. health sci., 7 (2015) 76. https://doi.org/10.5897/jtehs2015.0339 25. v. kumar and a. k. chopra, adv. crop. sci. tech., 4 (2015) 203. https://doi.org/10.4172/23298863.1000203 26. k. khaskhoussy, m. hachicha, b. kahlaoui, b. messoudi-nefzi, a. rejeb, o. jouzdan and a. arselan, j. appl. sci. res., 9 (2013) 132. http://www.aensiweb.com/old/jasr/jasr/2 013/132-140.pdf 27. i. ugulu, int. j. educ. sci., 28 (2020) 7. https://doi.org/10.31901/24566322.2020/ 28.1-3.1088 28. i. ugulu, z. i. khan, s. rehman, k. ahmad, m. munir and h. bashir, pak. j. anal. environ. chem., 21 (2020) 92. http://doi.org/10.21743/pjaec/2020.06.11 29. r. s. pawan, s. pratima, t. chirika and k. b. pradeep, pak. j. anal. environ. chem., 7 (2006) 70. http://www.pjaec.pk/index.php/pjaec/arti cle/view/52/42 30. a. asdeo, inter. refereed j. eng. sci., 3 (2014) 8. http://www.irjes.com/papers/vol3issue4/vesion%201/b340818.pdf 31. c. masona, l. mapfaire, s. mapurazi and r. makanda, j. sust. dev., 4 (2011) 132. https://doi.org/10.5539/jsd.v4n6p132 32. n. u. hassan, q. mahmood, a. waseem, m. irshad and a. pervez, pol. j. environ. stud., 22 (2013) 115. http://www.pjoes.com/assessment-ofheavy-metals-in-wheat-plants-rnirrigated-with-contaminatedwastewater,88959,0,2.html 33. c. keller, d. hammer, a. kayser, w. richner, m. brodbeck and m. sennhauser, plant soil, 249 (2003) 67. https://doi.org/10.1023/a:10225906090 34. z. i. khan, m. ashraf, n. ahmad., k. ahmad and e. e. valeem, pak. j. bot., 41 (2009) 1603. http://www.pakbs.org/pjbot/pdfs/41(4)/ pjb41(4)1603.pdf 35. b. d. kansal, r. kumar and r. sokka. the influence of municipal wastes and soil properties on the accumulation of heavy metals in plants. heavy metals in the environment. cec consultants ltd., edinburg, (1996). 36. z. i. khan, i. ugulu, a. zafar, n. mehmood, h. bashir, k. ahmad and m. sana, pak. j. bot., 53 (2021) in press. http://dx.doi.org/10.30848/pjb20211(14) 37. y. lu, h. yao, d. shan, y. jiang, s. zhang, and j. yang, j. chem., article id 628280, (2015) 1. https://doi.org/10.1155/2015/628280 38. n. alrawiq, j. khairiah, j. talib, m. l. ismail and b. s. anizan, int. j. chem. tech. res., 6 (2014) 2347. http://www.sphinxsai.com/2014/vol6pt4/ 2/(2347-2356)jul-aug14.pdf 39. z. bao, w. wu, h. liu, h. chen and s. yin, pol. j. environ. stud., 23(2013) 309. http://www.pjoes.com/impact-of-longterm-irrigation-with-sewage-r-nonheavy-metals-in-soils-cropsand,89196,0,2.html 40. k. ahmad, z. i. khan, a. ashfaq, m. ashraf and s. yasmin, pak. j. bot., 46 (2014) 1805. http://www.pakbs.org/pjbot/pdfs/46(5)/ 35.pdf 41. s. roggeman, d. v. n. brink, v. n. praet, r. blust and l. bervoets. environ. pollut., 172 (2013) 186. https://doi.org/10.1016/j.envpol.2012.09. 006 pak. j. anal. environ. chem. vol. 21, no. 2 (2020) 313 42. s. n. lawal, o. agbo and a. usman, j. phys. sci., 28 (2017) 49. https://doi.org/10.21315/jps2017.28.1.4 43. z. i. khan, k. ahmad, n.a. akram, n. mehmood and s. yasmeen, pak. j. bot., 49 (2017) 547. https://www.pakbs.org/pjbot/pdfs/49(2) /22.pdf 44. z. i. khan, k. ahmad, h. safdar, i. ugulu, k. wajid, h. bashir and y. dogan, res. j. pharmaceut. biol. chem. sci., 9 (2018) 759. microsoft word 160-167-pjaec-11062021-381-c.doc cross mark issn-1996-918x pak. j. anal. environ. che m. vol. 23, no. 1 (2022) 160 – 167 http://doi.org/10.21743/pjaec/2022.06.16 beta carotene determination in different vegetables by high performance liquid chromatography naseem zahra*, muhammad khalid saeed, khurram shahzad, shamma firdous, ijaz ahmad, muhammad ashraf, bukhtawar tariq and rabea yaseen food and biotechnology research centre, pcsir laboratories complex, ferozepur road, lahore-54600, pakistan. *corresponding author email: drnaseemzahra@gmail.com received 11 june 2021, revised 10 february 2022, accepted 05 april 2022 -------------------------------------------------------------------------------------------------------------------------------------------abstract β-carotene is an enriched source of vitamin a and is frequently present in vegetables. the vegetables rich in β-carotene should be used on the daily menu due to their dietary importance. the deficiency of vitamin a may cause severe health issues like premature deaths of children. so, the present study was conducted for the evaluation of vegetables containing high amounts of β-carotene. six vegetables were selected from the local market of lahore, pakistan, and beta carotene was analyzed by high performance liquid chromatography. it was concluded that carrot was rich in beta carotene contents, i.e., 12950±5.0 µg/100g. the sequence of beta carotene amount in the selected vegetables was carrot>spinach>brinjal>to mato>bitter gourd>cabbage. it is suggested that vegetables like carrot, and spinach should be used on a daily basis for good health maintenance. keywords: beta carotene, vitamin a, hplc, vegetables -------------------------------------------------------------------------------------------------------------------------------------------introduction β-carotene, a member of the carotenoids family, has been known as an essential dietary source of vitamin a for many years [1]. vitamin a dearth has unsympathetic effects on reproduction, growth, and resistance to infection [2]. vitamin a has an important role in many biological processes like cellular differentiation, cartilage and bone development, and growth [3]. carotenoids are commonly produced by photosynthetic organisms like plants, animals, and some species of bacteria, where they are involved in metabolic and biosynthetic processes. the yellow, orange and red colors of different fruits, vegetables, fishes, birds, and flowers are due to carotenoids, therefore, they are also called as colorants [4]. in the food industry, they are used as a natural coloring agent. some carotenoids are a part of the western diet, e.g., lycopene, lutein, zeaxanthin, and cryptoxanthin [5]. the importance of carotenoids lies in their beneficial properties regarding human health. for instance, the antioxidant property of carotenoids is proven to be significant for human health in various ways [6]. research on carotenoids has shown that it is involved in the regulation of cells, gene expression system, boosting up the immune response, and enzymes of drug metabolism [7]. nowadays living habits and lifestyle are bit changed. prevalent use of cigarettes and irregular diet has led to the production of free radicals that can cause serious damage to macromolecules like dna, proteins, fatty acids of low density lipoproteins and cholesterol, etc. [8]. the short communication pak. j. anal. environ. che m. vol. 23, no. 1 (2022) 161 increase in the production of free radicals elevates oxidative stress, which increases the risk of lethal diseases, e.g., cardiovascular diseases, cancer, rheumatoid arthritis, diabetes, and chronic inflammation [9]. β-carotene is an important carotenoid known for its pro-vitamin activity, which is then metabolized into active vitamin a by the human body [10]. β-carotenoid present in fruits and vegetables is regarded as a rich source of antioxidants. it is proven effective for all chronic diseases as it reduces oxidative stress by eradicating the production of free radicals [1, 11]. studies and investigations on carotenoids, especially β-carotene, showed that they combat the effect of certain diseases, most commonly cancer [12], light sensitivity [13] disorders, problems regarding age [14], and cardiovascular diseases. it is broadly assumed that β-carotene in leafy vegetables and orange-colored plants is a rich source of antioxidants and helps to eliminate oxidative damage [15, 16]. based on the antioxidant property and pro-vitamin a activity of βcarotene, it is naturally considered to extend the human life span. experimental in vivo animals showed that β-carotene reduces free radicals [17], thus preventing oxidative damage that is considered the main cause of chronic diseases. observations showed that a high intake of beta carotene through diet reduces the mortality rate [18], and results may vary [19]. moreover, unsatisfactory results were found with the β-carotene supplementation [20]. the reason behind it is that different sources of β-carotene have different effects on metabolism rate [21]. for instance, β-carotene is naturally in food or supplements, and both have an impact on human health. advancement in the methods for the determination of carotenoids in food is for two main reasons. firstly, it was assumed that previously reported values of carotenoids in different vegetables and fruits were inaccurate because methodologies were unreliable and insufficient to report and discriminate the carotenoids and vitamin a of supreme importance for human health [22, 23]. secondly, carotenoids without vitamin a activity are assumed to play other important roles than nutrition and eyesight. due to conjugated double bonds, carotenoids may act as a trap or antioxidant and play a significant role in cancer causatives and elimination [24]. subsequently, from the 1970’s hplc (high performance liquid chromatography) has become the common source of carotenoid determination in various vegetables and fruits because of its rapid separation technique moreover it achieves better resolution and hence better results [25]. some of the studies deal with characterizing many carotenoids, but the processes were difficult and not suitable for routine examination. developments are being made as the determination of carotenoids is a complicated procedure. the present study entails the determination of β-carotene in different vegetables. materials and methods different vegetables collected from the local market of allama iqbal town, lahore were selected for the determination of βcarotene by using hplc. by taking edible parts of the collected samples, a composite sample was made by mixing each vegetable in a minute quantity. a 100 g of composite sample was taken, and a subsample of 10 g was separated for the extraction procedure. after that, the pre-analyzed sample was simply washed with tap water and placed in inert condition at a temperature of -4 ͦ c to avoid any spoilage [26]. the further analysis was conducted at the food and biotechnology research centre, pcsir laboratories complex lahore. pak. j. anal. environ. che m. vol. 23, no. 1 (2022)162 beta carotene extraction beta carotene was extracted from the collected samples and was analyzed by using the high performance liquid chromatography technique [27]. 10 g of inert sample (kept at -4°c) was mixed with 30 ml of acetone, a standard solvent for the extraction of carotene from vegetables. the resulting mixture was then filtered through whatman filter paper. the residue was washed twice with acetone to obtain a colorless extract. after it, the residue was disposed of, and the remaining filtrate was mixed with 20 g of anhydrous sodium sulphate. the removal of anhydrous sodium sulphate was done by filtration and the volume of extract was lessened by a rotary evaporator that works on the principle of evaporation. the extract was then transferred to a 100 ml volumetric flask and the volume was made up to the mark by adding in it acetone and water, making the final extract with 80% of acetone. standard preparation of beta carotene 1 g standard of beta carotene enclosed in a vial (kept at -4°c) was brought from merck. 10 mg was dissolved in 100 ml nhexane to prepare a stock solution of beta carotene. the concentration of standard was made equal to 10 ppm. the stock solution of beta carotene was dissolved in different known concentrations [26]. for instance, 2, 4, 6, and 8 ppm dilutions were attained in 5 ml of each n-hexane solution. every working standard solution was introduced into the hplc system present at the laboratory of pcsir lahore. chromatographic conditions were according to the perkin elmer hplc software containing series 200 hplc pump (isocratic gradient), having a c-18 column coupled with c series 200 detector was used. “tcw s 4.0 software” was used for the quantification and peak identification in the hplc system. analysis was made by running the mobile phase (acetonitrile, dichloromethane, and methanol by the ratio of 70:20:10, respectively) at a flow rate of 1 ml per minute. the wavelength was set at 452 nm, and the pressure of the column was maintained at 1000-1200 psi. the standard solution of beta carotene (20 µl) was introduced when the injector was in the load phase. sample evaluation a sample of beta carotene in 80% acetone was used as a standard for hplc analysis. 20 µl of each vegetable sample was taken by using a microsyringe. peak identification and quantification were obtained by comparing the sample retention time with the standard retention time of βcarotene. results and discussion beta carotene is of peak interest because of its antioxidant properties and various health benefits like protecting vision, improving respiratory health, promote brain health, help to remove dandruff due to its hydrating property, and curing diabetes [28]. the amount of beta carotene varied from trace amounts in sweet potatoes, onion, mushrooms, and mint to thousands µg/100g in cabbage, bitter gourd, tomato, brinjal, spinach, and carrot. from the present data, it was observed that dark green vegetables are considered to have more beta carotene content in comparison with other vegetables, e.g., spinach contained 7824±0.5 µg/100g, followed by cabbage containing 1050±2.0 µg/100g and bitter gourd 1232±2.0 µg/100g were all vegetables pak. j. anal. environ. che m. vol. 23, no. 1 (2022) 163 that tend to have more carotenoids, apart from these vegetable carrots contained maximum amount of β-carotene 12950 ±5.0 µg/100g and the name β-carotene is also due to this reason [29]. some of the vegetables were selected from the local market of lahore specifically to determine the amount of β-carotene. in this research, six vegetables were chosen, and their results are shown in table 1. table 1. amount of β-carotene (µg/100g) i n di fferent vegetables. vegetable local name botani cal name beta carotene (µg/100g) ±sd bitter gourd karaila momordica charantia 1232±2.0 brinjal baingan solanum melongena 2450±1.0 cabbage gobi brasicca capitate 1050± 2.0 carrot gajar daucasa carota 12950±5.0 spinach palak spanacia oleracea 7824±0.5 tomato timater lycopersicum esculentum 1590±1.0 this data was in correspondence with earlier data and proved that among root vegetables, carrots tend to have the highest content of carotenoids and β-carotene in carrots is beneficial for improved night vision [30]. the comparison table 2 for carotene in other vegetables reported worldwide is given below. the comparison showed that the amount of beta carotene is highest in carrot followed by spinach and paprika (table 2). chromatograms and β-carotene content in different vegetables are shown in fig. 1. whereas, fig. 2 represents the β-carotene in standard solution. fig. 3 shows that the concentration of beta carotene was highest in carrot followed by spinach, brinjal, tomato, bitter gourd, and cabbage. the concentration of beta carotene in green leafy vegetables was given in the range of 80-9204 µg/100g [36]. table 2. amount of β-carotene i n di fferent vegetables/food items reported worl dwi de. vegetable amount of β-carotene reference cabbage 2441± 9.8 µg/100g [1] tomato 508071.6 µg/100g [1] spinach 12850± 3.4 µg/100g [1] carrot 6400±10.05 µg/100g [31] paprika 8000± 12.02µg/100g [31] pumpkin 3168 ±10.08µg/100g [31] corn 8460± 5.01µg/100g [31] carrot 20300± 2.0 µg/100g [31] green pepper 125.87 ±10.98 µg/g [32] red pepper 1060.24 ±15.67 µg/g [32] yellow pepper 611.54 ±09.45 µg/g [32] white rice 68.00±0.33 µg/g [33] fried rice 379.00±0.01 µg/g [33] beans porridge 4257.00±0.98 µg/g [33] green pepper 116.08±0.21mg/100g [34] red pepper 126.05±0.15 mg/100g [34] tomato 54.12±0.08 mg/100g [34] bitter gourd 1078±9.64 µg/100g [35] brinjal 2100±11.35 µg/100g [35] cabbage 910±10.81 µg/100g [35] tomato 1610±8.66 µg/100g [35] spinach 9940±23.06 µg/100g [35] red chilli 3290±8.54 µg/100g [35] lady finger 3220±29.81 µg/100g [35] cucumber 280±11.68 µg/100g [35] carrot 11210±72.62 µg/100g [35] bottle guard 140±4.58 µg/100g [35] pak. j. anal. environ. che m. vol. 23, no. 1 (2022)164 a b c d e f figure 1. the chromatogram of beta carotene i n bri njal, bi tter gourd, cabbage, carrot, spi nach and tomato (a-f) pak. j. anal. environ. che m. vol. 23, no. 1 (2022) 165 figure 2. the chromatogram of beta carotene i n standard solution in fig. 3, it was observed that the cabbage has the lowest concentration of βcarotene while the carrot has the highest concentration of β-carotene. figure 3. concentration of beta carotene (µg/100g) i n di fferent vegetables among leafy vegetables, spinach has the highest amount of carotenoids and is closed to the reported value [36]. tomatoes have a mean value of 1590 µg/100g. however, this value is lower than that reported [37]. the carotenoid content varying in different vegetables [38-39] may be due to the reason that carotenoids are highly sensitive to air, temperature [40], and other climatic conditions, so their determination needs much proficiency and experimental processes such as extraction, temperature variation or solvent used in the mobile phase of hplc must be carefully handled to avoid inaccuracies and variation in results. conclusion hplc is considered the most efficient and sensitive technique for carotenoid analysis. vegetables, especially dark green vegetables, are an excellent source of carotenoids and other vitamins, i.e., vitamin a. present research has shown that vegetables can fulfill 50% of our daily need for micro and macronutrients. vegetables are a good source that justifies daily nutrient requirements and helps to induce the immune response against several diseases. β-carotene in vegetables is popular for its antioxidant activity and helps to fight chronic diseases. to alleviate vitamin a deficiency, vegetables are cost effective and a better source to replace expensive animal food resources, which are not affordable for the low income population of any country. more vegetables containing β-carotene must be explored to completely eliminate the vitamin a deficiency in our population. conflict of interest there is no conflict of interest in this research. references 1. d. m. sahabi, r. a. shehu, y. saidu and a. s. abdullahi, j. basic appl. sci., 20 (2012) 225. http://www.ajol.info/index.php/njbas/ind ex 2. n. ullah, a. khan, f. ali khan, m. khurram, m. hussan, s. m. u. khayam, m. amin and j. hussain, middle-east j. sci. res., 9 (2011) 496. https://www.idosi.org/mejsr/mejsr9(4)11 /12.pdf 3. l. m. de luca, the faseb j., 5 (1991) 2924. https://pubmed.ncbi.nlm.nih.gov/166124 5/ pak. j. anal. environ. che m. vol. 23, no. 1 (2022)166 4. h. jackson, c. l. braun and h. ernst, am. j. cardiol., 101(2008) 50. https://doi:10.1016/j.amjcard.2008.02.00 8. 5. n. i. krinsky and e. j. johnson, mol. aspects med., 26 (2005) 459. https://doi: 10.1016/j.mam.2005.10.001. 6. a. v. rao and l. g. rao, pharmacol. res., 55 (2007) 207. https://doi: 10.1016/j.phrs.2007.01.012 7. o. kucuk, f. h. sarkar, z. djuric, w. sakr, m. n. pollak, f. khachik, m. banerjee, j. s. bertram, and d. p. wood jr, exp. biol. med., 227 (2002) 881. https://doi:10.1177/15353702022270100 7. 8. h. tapiero, d. m. townsend and k. d. tew, biomed.pharmacother., 58 (2004) 100. https://doi:10.1016/j.biopha.2003.12.006 9. b. n. ames, l. s. gold and w. c. willett, proc. natl. acad. sci., 92 (1995) 5258. https://doi: 10.1073/pnas.92.12.5258. t. grune, g. lietz, a. palou, a. c. ross, w. stahl, g. tang, d. thurnham, s. a. yin and h. k. biesalski, j. nutr., 140 (2010) 2268s. https://doi: 10.3945/jn.109.119024. 10. s. a. stanner, j. hughes, c. n. m. kelly, and j. buttriss, pub.health nutr., 407 (2004) 407. https://doi: 10.1079/phn2003543. 11. e. giovannucci, a. ascherio, e. b. rimm, m. j. stampfer, g. a. colditz, and w. c. willett, jnci j. natl. canc. int., 87 (1995) 1767. https://doi: 10.1093/jnci/87.23.1767. 12. w. stahl, and h. sies, mol. biotech., 37 (2007) 26. https://doi: 10.1007/s12033-007-0051-z. 13. r. d. semba, f. lauretani, and l. ferrucci, arch. biochem. biophys., 458 (2007) 141. https://doi: 10.1016/j.abb.2006.11.025. 14. j. fiedor, and k. burda, nutrients, 6 (2014) 466. https://doi: 10.3390/nu6020466. 15. d. weber, and t. grune, mol. nutr. food res., 56 (2012) 251. http://dx.doi.org/10.1002/mnfr. 16. a. el-agamey, g. m. lowe, d. j. mcgarvey, a. mortensen, d. m. phillip, t. g. truscott, and a. j. young, arch. biochem. biophys., 430 (2004) 37. https://doi: 10.1007/978-3-7643-749907. 17. a. agudo, l. cabrera, p. amiano, e. ardanaz, a. barricarte, t. berenguer, m. d. chirlaque, m. dorronsoro, p. jakszyn, n. larranaga, and c. martínez, am. j. clin. nutr., 85 (2007) 1634. https://doi: 10.1093/ajcn/85.6.1634. 18. p. henriquez-sanchez, a. sanchezvillegas, c. ruano-rodriguez, a. gea, r. m. lamuela-raventós, r. estruch, j. salas-salvadó, m. i. covas, d.corella, h. schröder, and m. gutiérrez-bedmar, eur. j. nutr., 55 (2016) 227. https://doi: 10.1007/s00394-015-0840-2. 19. l. g. zhao, q. l. zhang, j. l. zheng, h. l. li, w. zhang, w. g. tang and y. b. xiang, sci. rep., 6 (2016) 26983. https://doi: 10.1038/srep26983. 20. e. g. donhowe and f. kong, food bioproc. tech., 7 (2014) 338. https://doi.org/10.1007/s11947-0131244-z. 21. b. a. underwood, retinoids, 1 (1984) 281. https://academic.oup.com/jn/articlepdf/134/1/231s/24004114/z4w0010400s 231.pdf. 22. j. l. bureau and r. j. bushway, j. food sci., 51 (1986) 128. https://eurekamag.com/research/005/571 /005571558.php. 23. g. a. lozano, carotenoids, parasites, and sexual selection: oikos, (1994) 309. https://doi.org/10.2307/3545643. pak. j. anal. environ. che m. vol. 23, no. 1 (2022) 167 24. r. f. taylor, adv. chromatogr., 22 (1983) 157. https://www.ncbi.nlm.nih.gov/pmc/articl es/pmc373008/pdf/microrev000610005.pdf 25. i. a. khalil and f. r. varananis, sarhad j. agric., 105 (1996) 15. https://www.researchgate.net/publication /256703013_determination_of_beta_car otene_content_in_fresh_vegetables_usin g_high_performance_liquid_chromatogr aphy 26. i. a. khalil and f. r. durrani, trop. agric., 67 (1990) 313. https://www.cabdirect.org/cabdirect/abst ract/19906774019. 27. a. bendich and g. s. higdon, am. j. nutr., 32 (2004) 225. https://doi.org/10.1093/jn/134.1.225s. 28. b. borenstein and r. h. bunnell, adv. food res., 15 (1966) 195. https://doi.org/10.1016/s00652628(08)60081-6. 29. c. l. rock, j. l. lovalvo, c. emenhiser, m. t. ruffin, s. w. flatt and s. j. schwartz, j. nutr., 128 (1998) 913. https://doi.org/10.1093/jn/128.5.913. 30. s. javeria, t. masud, s. sammi, s. tariq, a. sohail, s. j. butt, k. s. abbasi, k. s. and s. ali, j. nutri., 12 (2013) 983. 31. r. k. shaha, s. rahman and a. asrul, a., annals biol. res., 4 (2013) 27. https://scialert.net/abstract/?doi=pjn.201 3.983.989. 32. r. a. sanusi and a. e. adebiyi, pak. j. nutr., 8 (2009)1512. https://www.researchgate.net/profile/ras akisanusi/publication/42973236_beta_c arotene_content_of_commonly_consu medfoods_and_soups_in_nigeria/links/ 56a8b4dd08ae997e22bdf675/betacarotene-content-of-commonlyconsumed-foods-and-soups-innigeria.pdf 33. g. e. igbokwe and c. o. anagonye, the biosci. j., 1 (2013) 89. https://bioscientistjournal.com/index.php /the_bioscientist/article/view/51. 34. m.n. ahamad, m. saleemullah, h.u. shah, i.a. khalil and a.u.r. saljoqi, sarhad j. agric., 23 (2007) 767. https://www.aup.edu.pk/sj_pdf/deter mination%20of%20beta%20car otene%20content.pdf. 35. v. v. agte, k. v. tarwadi, s. mengale, and s. a. chiplonkar, j. food comp. anal., 13 (2000) 885. https://nph.onlinelibrary.wiley.com/doi/p df/10.1002/jsfa.1427. 36. j. e. romanchik, e. h. harrison and d. w. morel, j. nutr. biochem., 8 (1997) 681. https://agris.fao.org/agrissearch/search.do?recordid=us1997078 131. 37. e. giovannucci, j. cancer inst., 91 (1999) 317. https://doi.org/10.1093/jnci/91.4.317. 38. k. s. jayaraman and d. d. gupta, drying of fruits and vegetables. in handbook of industrial drying, crc press (2020) 643. https://www.taylorfrancis.com/chapters/ edit/10.1201/9780429289774-21/dryingfruits-vegetables-jayaraman-das-gupta. 39. m. sharma, z. usmani, v. k. gupta and r. bhat, crit. rev. biotech., 41 (2021) 535. https://doi.org/10.1080/07388551.2021.1 873240. 40. s. singh, b. singh, a. k. singh and v. k. pandey, int. j. curr. microbiol. appl. sci., 9 (2020) 1144. https://www.ijcmas.com/jun2020issue.p hp microsoft word 05-d-pjaec-02092021-389-c-revised galley proof-18cross mark issn-1996-918x pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 225 – 236 http://doi.org/10.21743/pjaec/2022.12.05 heavy metal accumulation in goosefoot (chenopodium album l.) irrigated with wastewater ilker ugulu 1* , zafar iqbal khan 2 , sidrah rehman 2 , kafeel ahmad 2 , khalid nawaz 2 , mudasra munir 2 and humayun bashir 2 1faculty of education, usak university, usak, turkey. 2 department of botany, university of sargodha, sargodha, pakistan. *corresponding author email: ilkerugulu@gmail.com received 02 november 2021, revised 30 may 2022, accepted 11 november 2022 -------------------------------------------------------------------------------------------------------------------------------------------abstract wastewater sources contain enormous a mounts of nutrients for plant growth. this study aimed to define the metal accumulation in the goosefoot plant (chenopodium album l.) of wastewater use in agricultural irrigation and to evaluate the risks of this accumulation to human health. the present research was perfor med in field conditions in khushab, pakistan. the cd, cu, cr, fe, zn, ni, and mn concentrations were determined with the analysis perfor med using atomic absorption spectrophotometer-aas. heavy metal concentrations in goosefoot sa mples irrigated with groundwater (gwi), canal water (cwi) and sugar mill water (mwi) ranged fro m 0.84 to 1.08, 0.55 to 0.78, 0.23 to 0.70, 2.09 to 5.56, 2.84 to 13.53, 0.53 to 1.13 and 0.32 to 0.39 mg/kg for cd, cr, cu, fe, ni, zn, and mn, respectively. according to the statistical analyses, wastewater applications had a non-significant effect on cr, cu, and zn concentrations in c. album samples collected from three sites, and a significant effect on cd, fe, mn, and ni concentrations (p>0.05). the results also showed that the health risk index value of cadmium was higher than 1. according to these results, long-term consumption of c. album samples grown in the study area may cause an accumulation of cd in the human body and diseases in many tissues and organs. keywords: biomonitoring, goosefoot, vegetable, trace metal -------------------------------------------------------------------------------------------------------------------------------------------introduction wastewater sources contain enormous amounts of nutrients for plant growth. for this reason, wastewater is used in many parts of the world to irrigate crops in urban and suburban lands [1-3]. another reason for using wastewater in agricultural irrigation is that many countries do not have facilities for the non-toxic disposal of wastewater. for this reason, vegetables in urban areas of many countries are mostly irrigated with industrial and urban wastewater [4-7]. these wastewaters contain a variety of contaminants, including both inorganic and organic hazardous chemicals, medical wastes, and nutrients [8-13]. industrial and domestic wastewater enters the soil and water system through agricultural irrigation and introduces potentially toxic metals [14-15]. therefore, the concentrations of heavy metals increase in the soil due to the intensive usage of wastewater. these metals can affect plant growth and the health of living things as well as negatively affect nutritional value [16-20]. the accumulation of these toxic compounds in pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 226 the food chain may cause various problems in the health of all living things in the ecosystem, including humans and animals. the intake of hazardous substances such as trace metals causes serious diseases in humans [21-22]. heavy metal toxicity can have acute or chronic effects [23-25]. irrigation of agricultural soil by wastewater for a long period may lead to the accretion of metals in agricultural soils and plants. potential health risks and food safety problems make this one of the most alarming environmental aspects [26]. consumption of food crops grown in soils irrigated with contaminated water having a high amount of heavy metals can exert a direct impact on human health [27]. crops of rice contaminated with cd on a farm in asia provide a strong indication that dysfunction of the human renal tubule is due to cd as a heavy metal [13]. it has been indicated that crops have different capacities to translocate and accumulate harmful material and heavy metals in their different parts. it is also described that there is an extensive discrepancy in the uptake and absorbance of metals between different species of plants and even between cultivars of identical species [28]. many chenopodium species are traditionally used in indigenous systems of medicine for the treatment of numerous ailments. chenopodium album, a plant widely distributed in asia, north america, and europe, is grown as a food crop in parts of asia and africa [22]. many studies have been reported on a wide variety of chemical components such as aldehydes, alkaloids, apocarotenoids, and flavonoids in the structure of the plant, and the antifungal and antioxidant properties of the plant. examples of the reported benefits of the herb on its use for phytotherapeutic purposes: appetite enhancer, anthelmintic, laxative, diuretic, biliary, and beneficial against abdominal pain and eye diseases [29]. agricultural activities in pakistan stand out as one of the most important economic activities of the country [16]. on the other hand, pakistan, which can be described as an agricultural country, has a great problem in terms of clean water resources [19]. for this reason, a significant part of the water required for agricultural irrigation is obtained by mixing wastewater such as industrial wastewater and sewage water with groundwater. studies have shown that irrigation with wastewater is effective in metal accumulation in agricultural products, since the water used for agricultural irrigation is not made clean by advanced treatment methods [22]. literature studies on the subject have shown that studies on heavy metal accumulation in c. album samples irrigated with wastewater and the effects of these plants on health are not sufficient. for this reason, the main aim of this study is to define the metal accumulation in the c. album plant of wastewater use in agricultural irrigation and to evaluate the risks of this accumulation for human health. materials and methods study area the present research was performed in field conditions in khushab, pakistan (fig. 1). khushab district of the province of punjab is placed in pakistan country (gps coordinates: 31° 52' 42.4956'' n and 71° 53' 55.0896'' e). the maximum temperature measured in the region in the summer is about 50 °c, and the lowest in the winter is about 12 °c. due to this temperate feature, the city of khushab offers a favourable environment for agricultural applications. pak. j. anal. environ. che m. vol. 23, no. 2 (2022)227 figure 1. the map of the study area plant cultivation goosefoot (chenopodium album l.) samples were grown at the end of october 2016 in 60 small plastic pots. approximately 2.5 kg of soil was filled in each plastic pot and a different treatment was applied to every 20 plastic pots. ten seeds were sown in each plastic pot, and each pot was irrigated twice a week with a litre of groundwater (ti: gwi), canal water (tii: cwi), and sugar mill water (tiii: mwi). after the plant samples in the pots matured, only four plants were left in each pot and 210 kg ha-1 urea fertilizer was applied to each pot. the samples of water used in the irrigation of the pots were also taken as examples in the metal analysis. soil samples were taken from the pots at a depth of 5 cm with the help of an auger. at the end of april 2017, goosefoot leaves were collected for analysis, dried outdoors, and ground by pounding in a mortar. ground powdered plant samples were dried in an oven for 3 days at 75 o c. after it was completely dry, the samples were prepared for metal analysis using the wet digestion method [17]. sample preparation and metal analysis during the preparation of soil and goosefoot samples for analysis by wet digestion method, 1 g of soil and goosefoot sample was digested. soil samples were digested with hno3 using a standard protocol. plant samples were digested with a mixture of hno3, h2 so4, and hclo4 (5:1:1) at 80°c until a transparent solution was obtained. after the solution had cooled, it was filtered through whatman filter paper #42 and the final volume of the solution was made up to 50 ml. the final solution was stored in plastic bottles for metal analysis. the cd, cu, cr, fe, zn, ni, and mn concentrations were determined with the analysis performed using atomic absorption spectrophotometer-aas (shimadzu model aa-6300). the operating conditions for the respective potentially toxic metals are given in table 1. table 1. operati ng condi tions for the anal ysis of metals usi ng atomic absorption spectrometry. element cd cr cu fe ni zn pb wavelength (nm) 228.8 422.7 324.8 248.3 232.0 213.9 283.3 slit width (nm) 0.7 0.7 0.7 0.2 0.2 0.7 0.7 lamp current (ma) 8 10 6 12 12 8 10 air flow rate (l/min) 15 15 15 15 15 15 15 acetylene flow rate (l/min) 1.8 2.8 1.8 2.2 1.6 2 2.0 burner height (mm) 7 9 7 9 7 7 7 pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 228 statistical analysis the variance of the metal values for water, soil, and vegetables was analysed by one-way anova by spss 23 at 0.001, 0.01, and 0.05 significance levels. correlations between vegetables and soil were calculated with pearson’s correlation coefficient. bioconcentration factor (bcf) the following formula was used to calculate the bcf, which shows the metal accumulation in the plant as a result of heavy metal transfer from the soil to the plant: bcf = cveg / csoil cveg: metal value in plant (mg/kg), csoil: metal value in soil (mg/kg) [12]. daily intake of metals (dim) dim, one of the methods used to assess whether the consumed products pose a health risk, was measured using the following formula: dim = cmetal × cfood intake / baverage weight cmetal: metal value in plant, cfood intake: daily food intake, baverage weight: average body weight. the daily food intake of a person was taken as 0.345 mg/kg and an average body weight of 60 kg as a standard [9]. health risk index (hri) in this study, the hri used to evaluate the health risk status that may occur in the case of consumption of c. album samples by humans was calculated using the following formula [9]. hri = dim / oral reference dose pollution load index (pli) the pli for each irrigation application was calculated using the formula below [9]: pli = metal value of soil / reference metal value of soil the reference soil values (mg/kg) of cd (1.49), cr (9.07), cu (8.39), fe (56.90), ni (9.06), zn (44.19), and mn (46.75) were taken according to ugulu et al. [3]. results and discussions the use of wastewater in agricultural irrigation saves water in areas with clean water shortages, but it can positively or negatively affect the development of plants grown with organic and inorganic substances in their content [11]. in addition, harmful chemicals such as heavy metals in the composition of wastewater can seriously threaten human health by participating in the food chain as well as affecting the development of agricultural products [13]. in the present study, it was aimed to determine the influence of using wastewater in agricultural irrigation on the metal contamination in vegetables in the c. album sample in pakistan, an agricultural country where there is a significant deficiency of fresh water, and to evaluate the risks posed by this accumulation for human health. first of all, in this experimental study, when the metal values in the samples used in irrigation for the cultivation of c. album samples were examined, it was determined that the fe and zn values in all irrigation water samples were higher than the other metal values (fig. 2). however, it was determined that heavy metal values in canal water (tii: cwi) and sugar mill water (tiii: mwi) samples were higher than groundwater (ti: gwi) values. the anova results pak. j. anal. environ. che m. vol. 23, no. 2 (2022)229 indicated that there are no significant differences (p>0.05) between the metal concentrations for the cr, cd, cu, ni, and mn while the significant differences for fe and zn in the water samples (table 2). figure 2. fl uctuation of heavy metal s i n irrigation water the limits for the heavy metal content of water suitable for use in agricultural irrigation have been determined by institutions such as who, fao, usepa, and european standard guidelines [27]. many countries and agricultural organizations try to prevent the risks that may arise in terms of environmental health by carrying out their inspections within these limits [8]. accordingly, the maximum metal limits allowed for the water to be used in agricultural irrigation are determined by the european standard guidelines as 0.01, 0.5, 0.2, 5, 0.2, 2, and 0.2 mg/l for cd, cr, cu, fe, ni, zn, and mn, respectively [30]. when these limits are compared with the data obtained in this study, it is seen that the metal values in the irrigation waters are higher than the limit values, except for mn. as stated in the introduction of the study, many countries use wastewater in agricultural irrigation, but they do not or cannot do enough filtration in this process [22]. this situation can be shown as one of the main reasons why the heavy metal values determined in the irrigation water samples are above the reported limit values. in the study conducted in khushab, khan et al. [9] noticed the metal values in groundwater (gwi), canal water (cwi), and industrial water (iw) samples from the region as 0.01-0.02-0.03 mg/l for cu, 1.69-1.76-1.88 mg/l for cd, 0.64-0.72-0.83 mg/l for fe, 0.54-0.57-0.65 mg/l for cr, 0.08-0.10-0.14 mg/l for ni, 0.07-0.08-0.12 mg/l for mn and 0.57-0.61-0.66 mg/l for zn, respectively. ugulu et al. [31] researched the effect of the use of various water sources in agricultural irrigation on metal contamination in vegetables in the ginger plant sample, and found that the cd, co, cr, cu, fe, ni, pb, zn, and mn values in the wastewater samples were between 0.84-1.67, 0.08–0.22, 0.42-0.72, 0.45-0.85, 2.51-9.99, 1.21-1.92, 0.02–0.15, 1.82-9.98, and 0.64-0.91 mg/kg, respectively. as a result of the study, ugulu et al. [31] determined that fe and zn values in wastewater samples were higher than other metals, similar to the findings of this study. likewise, the metal values in the studies mentioned are above the maximum allowable limit values reported by the usepa [32]. in the experimental study, after the metal values in the wastewater samples (gwi, cwi, and mwi) used for irrigation were determined, the heavy metal values of the soil samples taken from the pots where the goosefoot samples were grown were measured. the mean metal values in soil samples ranged from 0.83-1.033, 0.18-0.26, 0.071-0.124, 2.27-6.19, 0.38-0.40, 2.32-7.11, and 0.39-0.61 mg/kg for cd, cr, cu, fe, ni, zn, and mn, respectively. the fe and zn concentrations for all soil samples were higher than other metal values (fig. 3). these values also clearly showed that heavy metal accumulation values in soil samples irrigated with sugar mill water (mwi) were higher than the metal accumulation values of soil samples irrigated with other waters (gwi and cwi). the analysis of the variance of the data showed that the three treatments had a nonsignificant effect (p>0.05) on cr and ni while significant effects on cd, cu, fe, zn, and mn concentrations were observed in the soil used to grow chenopodium album (table 2). pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 230 figure 3. fl uctuation of heavy metal s i n soil of c. album maximum permissible limits (mpl) can be defined as the highest tolerable values for heavy metals or other contaminants [24]. usepa (1997) reported the mpls of cd, cr, cu, fe, ni, zn, and mn for soil as 3, 100, 50, 21000, 50, 200, and 2000 mg/kg, respectively. the metal values determined for soil samples in this study did not exceed these limits. a similar study was performed by jamali et al. [33] who reported that the zn value (208.6 mg/kg) was the highest and the cd value (4.2 mg/kg) was the lowest in the studied soil samples. the results reported by jamali et al. [33] were much higher than the present study. on the other hand, heavy metal concentrations in the soil of the present study were much lower than those reported by chao et al. [34]. according to chao et al. [34], zn, pb, cu, and ni concentrations in soils were 21.17-79.50, 24.36-40.69, 21.67-46.85, and 13.72-24.00 mg/kg (in dry matter), respectively. concentrations of cr and fe noted by balkhair and ashraf [35] were 0.87-1.00 and 0.36-0.75 mg/kg, respectively, and the cr value (0.871.00) was higher and the fe value (0.36-0.75) was lesser than the present study. many studies performed in pakistan reported on the high concentration of heavy metals in vegetables irrigated with industrial water or sewage sludge. ahmad et al. [36] examined the heavy metal accumulation in the soil samples irrigated with wastewater and tap water in their study in khushab, pakistan, and found that the cobalt accumulation in the soil irrigated with sewage water (20.2 mg/kg) was more than irrigated with tap water (13.5 mg/kg). as mentioned in this study, it was concluded that heavy metal accumulation was higher as a result of irrigation with the sewage water. metal concentrations in goosefoot samples ranged from 0.84 to 1.08, 0.55 to 0.78, 0.23 to 0.70, 2.09 to 5.56, 2.84 to 13.53, 0.53 to 1.13 and 0.32 to 0.39 mg/kg for cd, cr, cu, fe, ni, zn, and mn, respectively. the fe and zn concentrations for all soil samples were higher than other metal values (fig. 4). these values showed that heavy metal accumulation values in goosefoot samples irrigated with sugar mill water were higher than the metal accumulation values of goosefoot samples irrigated with other waters except cu, mn, and ni. the analysis of the variance of the data showed that the three treatments had a non-significant effect (p>0.05) on cr, cu, and zn but significant effects were observed on cd, fe, mn, and ni concentrations in collected c. album samples (table 2). table 2. anal ysi s of variance for heavy metals and metalloi ds i n soil and c. album. mean squares sampl e source of vari ation sov degree of freedom df cd cr cu fe ni zn mn treatments 4 .215ns .002ns .364ns 6.189* .750ns .006*** .186ns water error 10 .052 .001 .074 .813 .307 .001 .039 treatments 4 .048* .007 .139*** 15.819* .001 29.093* .049** soil error 10 .010 .005 .009 2.036 .001 6.721 .005 treatments 4 .057** .006 .468 440.392** .005** 54.826 .412** goosefoot error 10 .009 .008 .301 39.272 .001 31.330 .053 *, **, *** significant at 0.05, 0.01, and 0.001 levels; ns, non-significant pak. j. anal. environ. che m. vol. 23, no. 2 (2022)231 figure 4. fl uctuation of heavy metal s i n c. album usepa [31] reported the maximum permissible limits in plants as 0.1, 5, 73, 425, 67, 100, and 500 mg/kg for the cd, cr, cu, fe, ni, zn, and mn, respectively. the determined metal values in c. album specimens did not exceed these mpls except the cd (0.84 to 1.08 mg/kg). ugulu et al. [31] investigated the effect of the use of various water sources in agricultural irrigation on heavy metal accumulation in vegetables in the ginger plant sample and found that cadmium accumulation (1.710 to 1.810 mg/kg) was high in the sample plants, especially as a result of the use of industrial wastewater. ahmad et al. [36] observed a higher range of cobalt in the root samples (1.07–1.26 mg/kg) of the plants irrigated with the sewage water. as mentioned in this study, it was concluded that heavy metal accumulation was higher as a result of irrigation with the sewage water. this result may be due to the cultivation of vegetables grown in soil irrigated by sewage water. sharma et al. [37] reported that the highest value of zn (0.23 mg/kg) was present as compared to other heavy metals studied in the vegetable palak, radish, tomato, cabbage, and carrot of varanasi city, india. this variation of metals' amounts in vegetables may be due to the different metals' absorption capacities in food [17, 22]. many studies were conducted in different cities of pakistan, using different plants, investigating the effect of wastewater use in agricultural irrigation on heavy metal pollution. khan et al. [1] and khan et al. [16] investigated the effect of wastewater irrigation on trace metal/metalloid accumulation in vegetables in jhang and bhakkar cities of pakistan's punjab province and used okra (abelmoschus esculentus) samples as study material. khan et al. [1] determined as, se, cd, fe, zn, cu, and co values between 9.15011.200, 0.540-0.550, 0.270-0.480, 39.39042.940, 55.710-60.270, 20.550-23.510 and 0.480-0.490 mg/kg, respectively, in okra in jhang sample. khan et al. [16] defined mo, as, se, fe, cu, zn, ni, pb, cd, and co values between 7.01-9.29, 2.80-3.58, 0.37-0.48, 39.71-44.04, 11.79-19.84, 28.20-37.05, 6.089.33, 4.88-7.06, 3.37-4.19, and 0.12-0.32 mg/kg, respectively, in okra in the bhakkar sample. khan et al. [22] used luffa (luffa cylindrica) samples in a study on the effects of using untreated wastewater in agricultural irrigation on metal pollution and associated health risks. accordingly, metal/metalloid values in luffa samples watered with municipal wastewater, groundwater, and canal water were defined as follows: 7.9-9.0, 3.74.2, 0.5-0.6, 39.1-43.2, 15.7-20.8, 29.0-42.4, 6.9-8.2, 5.8-7.7, 4.0-4.3 and 0.1-0.4 mg/kg for mo, as, se, fe, cu, zn, ni, pb, cd and co, respectively. the metal values identified by these researchers in the okra and luffa samples in both jhang and bhakkar cities were higher than the values recorded in khushab city for goosefoot in this study. this situation also reveals the diversity of factors affecting heavy metal accumulation. the differences between the studies whose findings are presented may be due to the study areas, the geological characteristics of the regions, and the accumulation characteristics of the plants used in the studies [15]. analysis of various metals in three irrigations, mn and fe showed the highest bcf values in sugar mill water treatment (treatment-iii). in treatment-i, the bioconcentration factor for mn and cu was pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 232 higher than fe and ni. in treatment ii, the bioconcentration factor for mn was higher than cu and in treatment iii, the bcf value of fe was higher than ni (table 3). bioconcentration factor values for cd, cu, and mn for goosefoot samples irrigated with groundwater, cd, cr, and mn values for canal water irrigated samples, and cd, cu, fe, zn, and mn values for sugar mill water irrigated samples were found to be higher than 1. the order of bcf values for treatment i: mn>cu>cd>cr>zn>ni>fe, treatment ii: mn>cr>cd>ni>zn>fe>cu, and treatment iii: fe>mn>cu>zn>cd>cr>ni. the bcf is one of the best methods used in the evaluation of metals transferred from soil to plants in the agricultural production process or other chemicals to be determined [22]. okereke et al. [38] observed higher bcf values for cr (0.14), cu (1.07), and ni (0.38) in seven green leafy vegetables than the bcf values in the present study for cr (1.10), cu (0.4-1.3) and ni (0.9). the mean values for bcf reported by ahmad et al. [36] were 0.036 and 0.038 for co in brassica rapa grown at tap water and sewage water irrigated sites, respectively. although it was close to each other, it was observed that the bcf value in the wastewater irrigation area is higher than in the present study. ugulu et al. [24] determined the highest bcf value for fe (8.009) in trigonella foenicum samples irrigated with municipal wastewater as a result of their study investigating the effect of wastewater irrigation on heavy metal accumulation in vegetables in khushab, another city in pakistan. regional differences may be the reason for the differences between the findings of the studies mentioned, or it may be due to the accumulation characteristics of the plants [7]. the daily intake of metal (dim) analysis values showed that the daily intake of fe was the highest but those of cu and cr were the lowest with all treatments (table 3). according to who/fao (1996), dim values for cd, cr, cu, ni and zn were 0.06, 0.05–0.2, 3, 1.4, and 60 mg/day, respectively. the dim values for all metals presented in this study are below the standard values. mahmood and malik [39] pointed out that, the daily intake of metal was higher for zn and less for cr and cd in foodstuff grown in wastewater. in the present study, the consequences of the dim value of vegetables irrigated with sugar mill wastewater showed a resemblance to that given by mahmood and malik [39]. table 3. bioconcentration factor, dail y intake of metal, pollution load index and health risk index val ues for c. album. heavy metal sirrigation treatment cd cr cu fe ni zn mn i 1.000 0.98 1.320 0.778 0.839 0.900 1.613 ii 1.016 1.196 0.485 0.63 0.919 0.86 1.85bcf iii 1.045 1 1.228 10.34 0.976 1.204 1.310 i 0.00583 0.00148 0.00134 0.01636 0.00184 0.01204 0.00362 ii 0.00488 0.00131 0.00058 0.03419 0.00205 0.01274 0.00650dim iii 0.00621 0.00105 0.00363 0.13530 0.00225 0.04925 0.00306 i 0.681 0.0287 0.0211 0.0642 0.0420 0.0526 0.0083 ii 0.5604 0.0210 0.0248 0.108 0.0429 0.058 0.0130pli iii 0.693 0.020 0.061 0.039 0.044 0.161 0.011 i 5.8398 0.00098 0.03369 0.0233 0.092 0.0401 0.08852 ii 4.8803 0.00087 0.01455 0.08313 0.10278 0.0424 0.1586hri iii 6.21 0.00070 0.0909 0.1932 0.11266 0.16419 0.0747 pak. j. anal. environ. che m. vol. 23, no. 2 (2022)233 the highest pollution load index (pli) value was observed for cd and the lowest pli value was defined for mn in all three irrigations (table 3). it was observed that the pli values of the sugar mill water and canal water irrigated samples were higher than the groundwater irrigated samples. however, the measured values were lower than the values found by khan et al. [16]. a higher pli value (1.493) was noticed by ahmad et al. [38] when the sewage source was used to irrigate the soil instead of tap water. in the study conducted in the region, ahmad et al. [1] found the highest pli value for cd. in this direction, the results of the present study were parallel to the values reported by ahmad et al. [1]. ashfaq et al. [40] investigated the accumulation of cr, mn, fe, mo, pb, and cd in pumpkin (cucurbita maxima) samples grown by wastewater irrigation in the urban area of sargodha city, and the pli values for these metals were 0.4, 0.01-0.03, 0.01-0.009, and 0.01-0.02 for cd, fe, mn, and cr, respectively. all of the pli values found as a result of this study are below one. one (1) is considered the critical value for the pollution load index (pli). accordingly, a pli value higher than this value is interpreted as the food is contaminated with metal, and if it is less than this value, it is interpreted as not contaminated [15]. all of the pli values determined for heavy metal values in c.album samples in this study were below the value of 1. therefore, it can be concluded that these plants are not contaminated with heavy metals. in addition, it can be said that irrigation with wastewater does not cause heavy metal contamination to a certain extent for the c.album plant. in the present study, the metal with the lowest health risk index (hri) for goosefoot samples in all irrigation environments was cr, and the metal with the highest risk was cd (table 3). the fact that the calculated hri value is higher than 1 indicates that the consumption of this food carries health risks and that less than 1 indicates that consumption is not a problem in terms of health [41]. among the hri values calculated in this study, a value greater than 1 was determined only for cd. due to the high hri value determined for cadmium, it can be said that c. album plants consumed in the region pose a risk to human health [15]. on the other hand, although hri values less than 1 were determined for c. album samples irrigated with canal water and sugar mill water, the heavy metal values detected in these samples were generally higher than those irrigated with groundwater. ahmet et al. [36] found higher hri values for vegetables irrigated with wastewater as a result of their study in khushab, pakistan, where they examined heavy metal accumulation in soil samples irrigated with wastewater and tap water. ugulu et al. [31] investigated the effect of agricultural irrigation with wastewater on heavy metal accumulation in ginger plants in sheikhupura (pakistan), a region where industrial settlements are located, and determined a high hri value for the lead as a result of irrigation with wastewater. correlation analysis is one of the most effective methods used to find the relationships between various variables in environmental sciences and environmental pollution studies, as it is in many fields related to science [42-44]. in this study, correlation analysis was used to determine whether the determined heavy metal values were related to each other. according to the results of the analysis, an insignificant positive correlation was found between cd, cu, cr, mn, and ni, and an insignificant negative correlation was found for fe (table 4). the reasons for the positive correlation between heavy metals can be cited as having common sources, the interdependence of their chemical properties, or having similar behaviours during transportation [45-46]. pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 234 table 4. metal correlation between soil-vegetable. corre lation metals soil-vegetable cd .981 cr .847 cu .947 fe -.500 ni .986 zn 1.000* mn .685 * pearson correlation coefficient is significant at the 0.05 level (2tailed). conclusion the research findings showed that the metal contents in the irrigation water samples used in the study were in the order of sugar mill water>canal water>ground water. when the values in the plant samples were evaluated, it was observed that the cd accumulation exceeded the maximum permissible limits. the results also showed that the health risk index value of cadmium was higher than 1.0. the fact that the calculated hri value is greater than 1.0 indicates that the consumption of this food carries a risk in terms of health. according to these results, it can be said that if c. album samples grown in the study area are consumed continuously, cd may accumulate in the human body through food and this may cause diseases in many tissues and organs. conflict of interest the authors declare that there are no conflicts of interest regarding the publication of this paper. references 1. k. salawu, m. m. barau, d. mohammed, d. a. mikailu, b. h. abdullahi and r. i. uroko, j. toxicol, environ. health sci., 7 (2015) 76. https://doi.org/10.5897/jtehs2015.0339 2. a. n. m. al-razee, n. abser, a. mottalib, a. nargis, a. k. jhumur, m. u. thakur, w. liu, s. poddar, s. i. sarker and a. habib, pak. j. anal. environ. chem., 22 (2021) 84. http://dx.doi.org/10.21743/pjaec/2021.06.10 3. i. ugulu, z. i. khan, s. rehman, k. ahmad, m. munir and h. bashir, pak. j. anal. environ. chem., 21 (2020) 92. http://doi.org/10.21743/pjaec/2020.06.11 4. k. khaskhoussy, m. hachicha, b. kahlaoui, b. messoudi-nefzi, a. rejeb, o. jouzdan and a. arselan, j. appl. sci. res., 9 (2013) 132. http://www.aensiweb.com/old/jasr/jasr/2 013/132-140.pdf 5. r. s. pawan, s. pratima, t. chirika and k. b. pradeep, pak. j. anal. environ. chem., 7 (2006) 70. http://www.pjaec.pk/index.php/pjaec/arti cle/view/52/42 6. c. masona, l. mapfaire, s. mapurazi and r. makanda, j. sust. dev., 4 (2011) 132. https://doi.org/10.5539/jsd.v4n6p132 7. n. u. hassan, q. mahmood, a. waseem, m. irshad and a. pervez, pol. j. environ. stud., 22 (2013) 115. http://www.pjoes.com/assessment-ofheavy-metals-in-wheat-plants-rnirrigated-with-contaminatedwastewater,88959,0,2.html 8. i. ugulu, s. baslar, y. dogan and h. aydin, biotech. biotechnol. equip., 23 (se) (2009) 410. https://doi.org/10.1080/13102818.2009.1 0818451 9. c. keller, d. hammer, a. kayser, w. richner, m. brodbeck and m. sennhauser, plant soil, 249 (2003) 67. https://doi.org/10.1023/a:10225906090 10. y. lu, h. yao, d. shan, y. jiang, s. zhang and j. yang, j. chem., 2015 (2015) article id 628280. https://doi.org/10.1155/2015/628280 pak. j. anal. environ. che m. vol. 23, no. 2 (2022)235 11. n. alrawiq, j. khairiah, j. talib, m. l. ismail and b. s. anizan, int. j. chem. tech. res., 6 (2014) 2347. http://www.sphinxsai.com/2014/vol6pt4/ 2/(2347-2356)jul-aug14.pdf 12. s. roggeman, d. v. n. brink, v. n. praet, r. blust and l. bervoets. environ. pollut., 172 (2013) 186. https://doi.org/10.1016/j.envpol.2012.09.006 13. z. i. khan, i. s. malik, k. ahmad, k. wajid, m. munir, i. ugulu and y. dogan, catrina – int. j. environ. sci., 20 (2019) 31. https://cat.journals.ekb.eg/article_85765_83 99f58323f082c77eff657c7c15495a.pdf 14. e. e. santos, d. c. lauria and c. l. porto da silveira, sci. total environ., 327 (2004) 69. http://dx.doi.org/10.1016/j.scitotenv.200 4.01.016 15. i. ugulu, int. j. educ. sci., 26 (2019) 49. https://doi.org/10.31901/24566322.2019/ 26.1-3.1086 16. z. i. khan, k. ahmad, h. safdar, i. ugulu, k. wajid, h. bashir and y. dogan, res. j. pharmaceut. biol. chem. sci., 9 (2018) 759. https://www.rjpbcs.com/pdf/2018_9(5)/[ 94].pdf 17. i. ugulu, z. i. khan, s. rehman, k. ahmad, m. munir, h. bashir and k. nawaz, pak. j. anal. environ. chem., 20 (2019) 107. http://doi.org/10.21743/pjaec/2019.12.14 18. i. ugulu, biotech. biotechnol. equip., 29 (2015) 20. http://doi.org/10.1080/13102818.2015.1 047168 19. n. yorek, i. ugulu, m. sahin and y. dogan, natura montenegrina, 9 (2010) 973. https://www.researchgate.net/profile/yu nus-dogan-8/publication/271498684 20. i. ugulu and s. erkol, nwsa-educ. sci., 8 (2013) 79. https://www.researchgate.net/publication /288825959 21. z. bao, w. wu, h. liu, h. chen and s. yin, pol. j. environ. stud., 23 (2013) 309. http://www.pjoes.com/impact-of-longterm-irrigation-with-sewage-r-nonheavy-metals-in-soils-cropsand,89196,0,2.html 22. z. i. khan, k. ahmad, s. rehman, a. ashfaq, n. mehmood, i. ugulu and y. dogan, pak. j. anal. environ. chem., 20 (2019) 60. http://doi.org/10.21743/pjaec/2019.06.08 23. z. i. khan, h. safdar, k. ahmad, k. wajid, h. bashir, i. ugulu and y. dogan, pak. j. bot., 52 (2020) 111. http://dx.doi.org/10.30848/pjb2020-1(12) 24. i. ugulu, z. i. khan, s. rehman, k. ahmad, m. munir, h. bashir and k. nawaz, bull. environ. contam. toxicol., 103 (2019) 468. https://doi.org/10.1007/s00128-01902673-3 25. m. i. latif, m. i. lone and k. s. khan, soil environ., 27 (2008) 29. https://www.researchgate.net/publication /221718684 26. k. wajid, k. ahmad, z. i. khan, m. nadeem, h. bashir, f. chen and i. ugulu, bull. environ. contam. toxicol., 104 (2020) 649. https://doi.org/10.1007/s00128-02002841-w 27. z. i. khan, n. arshad, k. ahmad, m. nadeem, a. ashfaq, k. wajid, h. bashir, m. munir, b. huma, h. memoona, m. sana, k. nawaz, m. sher, t. abbas and i. ugulu, environ. sci. pollut. res., 26 (2019) 15381. https://doi.org/10.1007/s11356-01904959-9 28. i. ugulu, environ. educ. res., 21 (2015) 916. http://doi.org/10.1080/13504622.2014.9 23381 29. n. yadav, n. vasudeva, s. singh and s. k. sharma, indian j. nat. prod. res., 6 (2007) 131. pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 236 https://www.researchgate.net/publication /288617783 30. fao/who, codex alimentarius commission (2001) food additives and contaminants. joint fao/who food standards programme, alinorm 01/12a:1-289. https://www.fao.org/fao-whocodexalimentarius/thematicareas/contaminants/en/ 31. i. ugulu, z. i. khan, z. sheik, k. ahmad, h. bashir and a. ashfaq, arab. j. geosci., 14 (2021) 702. https://doi.org/10.1007/s12517-02107073-8 32. usepa (2002) preliminary remediation goals, region 9. united states environmental protection agency, washington, dc. https://archive.epa.gov/region9/superfun d/web/pdf/04prgtable.pdf 33. m. k. jamali, t. g. kazi, m. b. arain, h. i. afridi, n. jalbani and a. r. memon, j. agron. crop sci., 193 (2007) 218. http://dx.doi.org/10.1111/j.1439037x.2007.00261.x 34. w. chao, l. xiao-chen, z. li-min, w. pei-fang and g. zhi-yong, polish j. environ. stud., 16 (2007) 199. http://www.pjoes.com/pdf-8797621835?filename=pb_%20cu_%20zn%2 0and%20ni.pdf 35. k. s. balkhair and m. a. ashraf, saudi j. biol. sci., 23 (2016) 32. https://doi.org/10.1016/j.sjbs.2015.09.023 36. k. ahmad, z. i. khan, a. ishfaq, m. ashraf and s. yasmin, pak. j. bot., 46 (2014) 511. http://www.pakbs.org/pjbot/pdfs/46(5)/ 35.pdf 37. r. k. sharma, m. agrawal and f. marshall, ecotoxicol. environ. saf., 66 (2007) 258. https://doi.org/10.1016/j.ecoenv.2005.11 .007 38. c. j. okereke, e. b. essien and m. o. wegwu, j. anal. pharm. res., 3 (2016) 77. https://doi.org/10.15406/japlr.2016.03.0 0077 39. a. mahmood and r. n. malik, arab. j. chem., 7 (2014) 91. https://doi.org/10.1016/j.arabjc.2013.07.002 40. a. ashfaq, z. i. khan, z. bibi, k. ahmad, m. ashraf, i. mustafa, n. a. akram, r. perveen and s. yasmeen, pak. j. zool., 47 (2015) 1051. https://www.zsp.com.pk/pdf47/10511058%20(19)%20qpjz-00662015%209-4-15%20.doccorrected-9%20april.pdf 41. z. i. khan, i. ugulu, a. zafar, n. mehmood, h. bashir, k. ahmad and m. sana, pak. j. bot., 53 (2021) 247. http://dx.doi.org/10.30848/pjb2021-1(14) 42. i. ugulu, m. c. unver and y. dogan, euro-mediterr. j. environ. integr., 4 (2019) 36. http://dx.doi.org/10.1007/s41207-0190128-7 43. i. ugulu, h. aydin, n. yorek, and y. dogan, natura montenegrina, 7 (2008) 165. https://d1wqtxts1xzle7.cloudfront.net/45 852810 44. z. i. khan, t. ahmad, h. safdar, i. ugulu, k. wajid, m. nadeem, m. munir and y. dogan, pak. j. anal. environ. chem., 21 (2020) 303. http://doi.org/10.21743/pjaec/2020.12.32 45. z. i. khan, a. nisar, i. ugulu, k. ahmad, k. wajid, h. bashir and y. dogan, egypt. j. bot., 59 (2019) 753. https://doi.org/10.21608/ejbo.2019.9969. 1296 46. y. dogan, i. ugulu, n. durkan, m. c. unver, h. h. mert, eurasia. j. biosci., 5 (2011) 10. http://dx.doi.org/10.5053/ejobios.2011.5. 0.2 microsoft word 07-d-pjaec-28012022-422-c-revised galley proof-29cross mark issn-1996-918x pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 247 – 258 http://doi.org/10.21743/pjaec/2022.12.07 spectrophotometric determination of catecholamine containing drugs using calcon dye ikram k. othman and theia’a n. al-sabha* chemistry department, college of education for pure science, mosul university, mosul, iraq. *corresponding author email: dr_theiaa@yahoo.co.uk received 28 january 2022, revised 03 july 2022, accepted 27 september 2022 -------------------------------------------------------------------------------------------------------------------------------------------abstract calcon dye has been used for spectrophotometric determination of catecholamine-containing drugs, namely, adrenaline, methyldopa and dopamine in their pure forms and pharmaceutical formulations. the method is based on the oxidation of the above drugs with an excess of nbromosuccini mide (nbs) in an acidic medium. the residual oxidizing agent bleaches the blackish-brown color of calcon measured at 510 nm. the decolorization of the dye is proportional to the residual amount of nbs, which is proportional to the concentration of the drug. linear calibration graphs were obtained in the concentration range 0.5-16.0, 2.0-40.0 and 1.036.0 μgml-1 with molar absorptivity values 1.10×104, 3.2×103 and 4.3×103 lmol-1cm-1 for above drugs, respectively. the method is simple, sensitive, accurate, precise and free from excipients. the developed method was successfully applied to determine the drugs in their pharmaceutical formulations. keywords: catecholamine drugs, calcon, oxidation, spectrophotometry -------------------------------------------------------------------------------------------------------------------------------------------introduction catecholamines are a class of monoamines synthesized from tyrosine, which contain a catechol group and a side chain with an amino group in their structure [1]. catecholaminescontaining drugs are widely used to treat different disorders, such as playing a key role in the mechanisms of emotions, learning, memorization, and sleep, as well as in psychomotor activity and neural regulation [2]. adrenaline (epinephrine) is a hormone that is involved in regulating visceral functions (such as respiration) [3,4]. it is chemically known as 4-[(1r)-1-hydroxy-2(methylamino)ethyl]benzene-1,2-diol (i). it is a neurotransmitter for the mammalian central nervous system. many diseases are associated with a change in catecholamine concentration. it is an active principle of adrenal gland balm and is a drug used to treat nasal congestion, asthma, heart block, hypotension and cardiac arrest. a change in the concentration of adrenaline in biological fluids (blood, urine, cerebrospinal fluid) can serve as a reliable indicator of a violation of homeostasis [5,6]. methyldopa, is in the family of alpha-2-adrenergic receptor agonists. chemically it is named s-2-amino-3-(3,4dihydroxyphenyl)-2-methyl-propanoic acid (ii). it is an antihypertensive drug used to lower blood pressure and treat some hypertension and gestational hypertension cases in renal failure and complicated pregnancies [7,8]. dopamine constitutes approximately 80% of the catecholamine content material pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 248 with inside the brain [9]. chemically known as 2-(3, 4-dihydroxyphenyl) ethylamine (iii), it is classified among the heart-stimulating drugs [10], which is a hormone secreted by the adrenal gland. it contributes to the fight or flight response processes [11] and the circulatory system (shock) due to myocardial infarction and trauma [12]. (i) (ii) (iii) different techniques have been described for determination of catecholamine drugs such as voltammetry [13-16], chromatography [17-21], amperometry [22], capillary electrophoresis [23,24] and fluorometry [25]. however, these techniques are expensive, need experience and are not available in all laboratories. the spectrophotometric technique is still the preferred method due to its simplicity. various spectrophotometric methods using different reagents have been reported for the determination of catecholamine drugs. these methods include ion association using eosin y and application of cloud point extraction technique [26], charge transfer complexes using bromanil [27], oxidation-reduction using ferric ion [28-30], diazotisation using pnitroaniline [31], 4-aminoantipyrine in the basic medium [32], complexation reaction using 4-aminoantipyrine and copper ion, ionpair complex using triiodide ion [33] and using ferrous ion as a complexing agent in alkaline medium [34], oxidative coupling using p-toluidine in the presence of sodium periodide [35], 2, 6-diaminopyridine in the presence of potassium periodate [36] and 3methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate in the presence of potassium ferricyanide [37]. the aim of this study is to develop an accurate, simple, sensitive and cost-effective method for the spectrophotometric determination of catecholamine containing drugs. the method is based on the oxidation of drugs by n-bromosuccinimide (nbs) in an acidic medium, and the residual oxidant, which is proportional to the drug concentration, bleach the color of calcon dye and reduction in absorbance is measured at 510 nm, which forms the basis of determination method. materials and methods instruments: uv-visible spectrophotometer type shimadzu uv-1650 pc equipped with a 1.0-cm path length silica cell, ph-meter with a combined glass electrode type philips pw (9421) was used for ph measurements. all calculations in the computing process were performed in microsoft excel format. reagents: calcon reagent was prepared in a concentration of 500 μgml-1 by dissolving 0.05 g in distilled water in a 100 ml volumetric flask. oxidizing agent (nbs) was prepared in a concentration of 5×10 -3 m by dissolving 0.0890 g in 100 ml distilled water. hydrochloric acid was prepared in a concentration of 1 m by diluting an appropriate volume of conc. hcl. pak. j. anal. environ. che m. vol. 23, no. 2 (2022)249 stock solutions of drugs: stock solutions of adrenaline, methyldopa and dopamine were prepared in a concentration of 100 μgml -1 by dissolving 0.01 g of each drug in 100 ml distilled water in volumetric flasks. the solutions were kept in the refrigerator. sample preparation aliquots containing 0.5-16.0, 2.0-40.0 and 1.0-36.0 μgml -1 of adrenaline, methyl dopa and dopamine standard solutions were added respectively, and separated into three series of 10 ml volumetric flasks, followed by adding 1 ml of 1 m hcl and 1.5 ml of 5×10 3 m nbs. the solutions were left for 10 min in a water bath adjusted at 30ᵒc for completion of the oxidation process, then 2.6 ml of 130 μgml -1 calcon was added. the solutions were diluted to the mark, and the absorbance was measured at 510 nm after 20 min for dopamine and 15 min for adrenaline and methyldopa against respective reagent blank. procedure for drug formulations injection adrenaline: the content of five ampoules of adrenaline (each ampoule contains 1.0 mg/1 ml adrenaline) or adrenaline darnitsa (each ampoule contains 1.8 mg/1 ml adrenaline) were mixed and then diluted to 50 ml with distilled water to obtain 100 or 180 μgml -1 respectively. different volumes of these solutions were taken to obtain concentrations 1.0, 4.0, 6.0 and 10.0 µgml -1, and they were treated according to the general procedure. the concentration of adrenaline per ampoule was found using the standard calibration curve for the drug in its pure form. dopamine hcl: the content of three dopadren ampoules (each ampoule contains 200 mg/5 ml of dopamine. hcl). 2 ml were mixed and diluted to 100 ml with distilled water to obtain a solution of 800 µgml -1 . then, a solution was prepared by dilution with a concentration of 100 μgml -1 . different volumes of the solution were taken to obtain concentrations 4.0, 10.0, 16.0 and 24.0 μgml-1. they were treated according to the general procedure. the dopamine concentration was found in the ampoule using the calibration curve of the drug in its pure form. tablet: ten tablets of aldosam or aldomac (each tablet contains 250 mg methyldopa) were carefully weighed, crushed and mixed well. the weight of the powder equivalent to one tablet was dissolved in 20 ml of distilled water and heated in a water bath for a few minutes to increase the solubility. the solution was allowed to cool and filtered in a 100 ml volumetric flask, then completed to the mark with distilled water to obtain 2500 µgml -1 . the solution was diluted to 100 µgml-1, and 4.0, 10.0, 16.0 and 24.0 µgml-1 treated according to the general procedure. results and discussion calcon, called eriochrome blue black r or mordant black 17. it is chemically known as 2-hydroxy-1-(2-hydroxy-1naphthylazo)naphthalene-4-sulfonic acid sodium salt (i v) that was proposed by hildebrand and reilley in 1958 to be used as an indicator for calcium ion [38, 39]. (iv) in the preliminary investigation, it was found that calcon dye has maximum absorption at 510 nm. experimentally, a quantitative oxidation of calcon dye was noticed in the presence of the oxidizing agent nbs in the acidic medium. the absorbance of the dye decreased by increasing the pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 250 concentration of the oxidizing agent by bleaching its color. based on this feature, the possibility of indirect spectrophotometric determination of the catechol amine drugs (adrenaline, methyldopa and sopamine hydrochloride) has been studied. the increasing concentration of drugs leads to a decrease in the concentration of oxidizing agents for bleaching calcon color. it also leads to an increase of the absorbance at 510 nm, which is proportional to the drug concentration (fig.1). however, the optimum conditions for the quantitative determination of the above drugs have been considered. figure 1. absorption spectra of 130 μgml-1 calcon i n aci dic medi um (a) i n the presence of nbs and 10 μgml-1 adrenaline (b), 20 μgml-1 methyldopa (c) and 15 μgml-1 dopami ne (d) agai nst reagent bl ank (e) optimization of calcon dye concentration: to select the optimum amount of calcon to be used for the determination of catecholamine drugs, increasing volumes (milliliters) of a dye solution at a concentration of 500 µgml-1 were added to a 10 ml volumetric flasks containing 1.0 ml of 1 m hcl. the volume was supplemented with distilled water to the mark, and the absorption was measured at 510 nm. it was found that the linear concentration range of calcon dye was 1-130 µgml -1 . therefore, the concentration was fixed at 130 µgml-1 for the estimation of the drug. optimization of oxidizing agent, concentration and volume: the effect of the oxidizing agents, including nbs, potassium dichromate, chloramine-t and cerium (iv) sulfate with a concentration of 5×10 -3 m on the bleaching of the dye in the medium of hcl was considered. the results, cited in fig. 2, indicated that nbs was the most suitable in the oxidation of the dye, as it gave the lowest absorbance; therefore, it was adopted in the present method. the effect of nbs concentration and volume was studied. it was found that 1.5 ml of 5×10 -3 m was the best for this method. figure 2. effect of oxidi zi ng agent on the bleachi ng of 130 µgml-1 cal con effect of acid experimental results demonstrated that the oxidation of calcon dye and the studied drugs by nbs were present in an acidic medium and at room temperature (23°c) for adrenaline but at 30°c for methyldopa and dopamine. therefore, the effect of different acids of 1 m was examined for determination of the above drugs in the presence of 130 µgml -1 calcon dye separately. the results in table 1 indicate that hcl was suitable acid. however, the concentration and volume of hcl were studied. it was found that 1 ml of 1 m gave maximum absorbance, table 2 & fig. 3. the conducted procedure was recommended in subsequent experiments. figure 3. effect of volume of 1 m hcl on the absorbance of drugs pak. j. anal. environ. che m. vol. 23, no. 2 (2022)251 table 1. effect of 1 ml of 1 m aci d on the absorbance of drugs. absorbance ch3coohhno3h3po 4h2so 4hcl μgml-1drug 0.2510.2840.3170.3690.39310 adrenaline 0.1060.1170.1360.1770.19220 methyldopa 0.1310.1470.1850.2260.28515 dopamine. hcl table 2. effect of mol ar concentration of hcl on the absorbance of drugs. mol ari ty of hcl /absorbance 3.02.52.01.51.00.5 μgml-1drug 0.2210.2520.2960.3490.3850.33110adrenalin 0.0910.1120.1340.1770.1960.15220methyldopa 0.1270.1610.1910.2330.2850.17415dopamine. hcl effect of time on the oxidation and bleaching: this study was conducted to determine the time period required for the oxidation of the pharmaceutical compounds and the dye with nbs in hydrochloric acid at different times with shaking. the results in fig. 4 showed that 10 min was sufficient time for the oxidation of drugs before adding a calcon dye, 15 min for bleaching of a dye for adrenaline and methyldopa, and 20 min for dopamine hydrochloride. the reaction remained stable for at least 24 h for all drugs. figure 4. effect of ti me on the oxi dation of drugs and bleaching of calcon for (a) adrenali ne, (b) methyldopa and (c) dopamine. hcl pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 252 lod =,loq = effect of temperature on oxidation reaction and stability: the effect of the temperature and time on the oxidation of catecholamine drugs was studied in the range 23°c (room temperature; rt) 50°c using the optimum conditions and concentrations of the reagents as mentioned above. the absorbance of the dye was measured after 5 min intervals at 510 nm. the results showed in fig. 5, a, b, and c indicated that high absorbance was reached after 30 min for dopamine and 20 min for adrenaline and methyldopa at 30°c and remained constant for more than 3 h. whereas higher temperature decreases the absorbance. effect of addition sequence: the addition sequence must follow the general procedure; otherwise, a decrease in absorbance was noticed. linearity and calibration graph: under the optimum conditions mentioned above, standard calibration graphs for adrenaline, methyldopa and dopamine. hcl with calcon dye was created by plotting the absorbance versus concentration fig. 6. the proposed methods obeyed beer’s law in the ranges 0.516.0, 2.0-40.0 and 1.0-36.0 µgml -1 with low intercept and good correlation coefficients for above drugs respectively. the molar absorptivity values were estimated, and they indicated that the method was sensitive (table 3). accuracy (average recovery %) and the relative standard deviation (rsd) for the analysis of three replicates of different concentrations for each of the above drugs explain that the method is accurate and precise. the detection limit (lod) and quantitation limit (loq) [40] were calculated by the subsequent equations: where: σ = standard deviation of the blank and b = slope of the calibration curve. the results, cited in table 3, are below the lower limit of beer's law range. figure 5. effect of temperature on the absorbance and stability of the oxi di zation of adrenaline (a), methyl dopa (b) and dopami ne (c) pak. j. anal. environ. che m. vol. 23, no. 2 (2022)253 figure 6. calibration graphs of adrenaline, methyldopa and dopami ne. hcl table 3. quantitative parameters and statistical data for assay of adrenali ne, methyl dopa and dopami ne. average٭ for six determinations suggested mechanism: nbs is an oxidizing agent. it behaves as a bromonation agent in the acidic medium for aliphatic and aromatic organic compounds [41-43]. this method assumed that nbs oxidizes the catecholamine drugs (adrenaline, methyldopa and dopamine hydrochloride) in an acidic medium. then, the unreacted nbs oxidizes the known amount of calcon and bleach its color. the decrease of nbs concentration upon oxidation of known concentration of drugs leads to an increase in the absorbance of the dye at 510 nm, which is linearly proportional to the concentrations of the studied drug compounds. the suggested mechanism is explained in scheme 1. analysis of catecholamine medicines in commercial formulations: the present method has been successfully applied for the determination of catecholamine drugs (injection and tablet). the results mentioned in table 4 indicate that the proposed method is accurate and reproducible. validation of the method: for the purpose of demonstrating the efficiency of the developed method, its success in estimating catecholamine drugs, and its freedom from the effect of excipients in its pharmaceutical preparations, the standard addition technique has been applied to pharmaceutical dopami ne. hclmethyldopaadrenali neparameters 1.0-36.02.0-40.00.5-16.0linearity range (µgml-1) 0.43×1040.32×1041.10×104molar absorptivity (lmol-1cm-1) 44.4474.0716.67sandell’s sensitivity (µgcm-2) average٭100.0699.83101.42 recovery (%) 0.1410.2340.053lod ( µgml-1) 0.4680.7810.176loq ( µgml-1) rsd٭0.350.850.38 0.02160.02390.0423intercept 0.02250.01350.0600slope 0.99700.99630.9968correlation coefficient (r2) pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 254 preparations for the above drugs. the method was summarized by adding increasing amounts of a standard solution of pure drug to a known quantity of the pharmaceutical preparation. by following the described method, the absorption was measured at the wavelength of 510 nm, and the obtained results were included in table 5, which indicates that the method has good selectivity. scheme 1. suggested mechani sm for oxi dation of catechol ami ne drugs and calcon by nbs r= , , 1-naphthol pak. j. anal. environ. che m. vol. 23, no. 2 (2022)255 table 4. anal ysi s of the catechol ami ne medicines i n commerci al formul ations. average recovery (mg) average recovery (%) recoverya (%) drug content found* (mg) amount present (µgml-1) certi fi ed val ue pharmaceutical preparation 100.501.011 98.040.984 99.811.006 0.99599.52 99.721.0010 1 mg adrenali ne ampouleb 98.830.991 100.541.014 98.690.996 0.99299.20 98.720.9910 1 mg adrenali ne ampoulec 100.501.811 99.291.794 100.081.806 1.79999.98 100.051.8010 1.8 mg adrenali ne ampouled 97.96244.904 99.93249.8310 99.49248.7316 243.35099.34 99.97249.9324 250 mg al dosam tablete 99.81249.534 100.67251.6710 100.42251.0416 250.285100.14 99.66249.1524 250 mg al domac tabletf 99.56199.114 100.26200.5210 99.89199.7816 199.2699.63 98.81197.6224 200 mgdopadren ampoul eg aaverage of four determinations manufactured by blincoln pharmaceuticals, india, cosel-turkey, ddarnitsa pharmaceuticals, firm-ukraine,esdi-iraq,fmacleods pharmaceuticals ltd-india and g vem pharmaceuticals-turkey. table 5. effect of exci pient i nterferences by the standard addi tion procedure. drug content found (mg) average recovery (%) recovery (%) amount present (µgml-1) certi fi ed val uepharmaceutical preparation 97.494 0.98798.78 100.076 1 mgadrenaline ampoule (india) 97.054 0.98398.36 99.686 1 mgadrenaline ampoule (turkey) 96.144 1.7597.21 98.286 1.8 mgadrenaline ampoule (ukraine) 97.154 244.8797.95 98.7510 250 mgaldosam tablet (iraq) 99.004 249.3299.73 100.4710 250 mgaldomac tablet (india) 199.3499.6798.374200 mgdopadren ampoule (turkey) pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 256 table 6. comparison between the proposed method and other methods. literature methods present method [44] [36] [29] anal ytical parameter adrenaline methyldopa dopamine adrenaline methyldopa dopamine reagent calcon v+5 diaminopyridinekio4 fe iii-k3 fe(cn)6 method oxidation chelating complex oxidative coupling prussian blue formation λmax (nm ) 510 488 478 735 linearity (µgml-1) 1.0-36.0 2.0-40.0 0.5-16.0 0.5–140 2-24 0.05–6.00 molar absorptivity lmol-1cm-1 1.10×104 0.32×104 0.43×104 2.015×103 1.0481 x 104 3.2 x 104 temp. (cᵒ) 30 70 25 r.t. rsd 0.35 0.85 0.38 ≤ 0.88 ≤ 0.86 0.65 applications injection tablet injection injection tablet injection comparison between the proposed method and other spectrophotometric methods: the suggested method for the determination of catecholamine drugs has been compared with other described spectrophotometric methods in the literature. the current method was distinguished by its sensitivity over some of the spectrophotometric methods listed in table 6. this method is simple and could be applied to all catecholamine-containing drugs in their pure form and pharmaceutical preparations. conclusion a new spectrophotometric method has been suggested for the determination of catecholamine-containing drugs. the method is dependent on the oxidation of drugs by nbs and subsequently reacted with calcon dye. the decolorization of the dye, measured at 510 nm, is proportional to the residual amount of nbs, which is proportional to the concentration of the drug. the method is simple, sensitive and accurate. the linearity range for calibration curves were 0.5-16.0, 2.0-40.0 and 1.0-36.0 μgml-1 with molar absorptivity values 1.10×10 4 , 3.2×10 3 and 4.3×10 3 lmol -1 cm -1 for adrenaline, methyldopa and dopamine respectively. the method was applied successfully for the determination of the intended drugs in their pharmaceutical formulations as tablets and injections with good recovery values. conflict of interest there is no conflict of interest in this research. references 1. p. nagaraja, a. k. shrestha, a. shivakumar, n. g. s. al-tayar and a. k. gowda, quim. nova, 34 (2011) 373. http://dx.doi.org/10.1590/s010040422011000300002 2. m. lieberman, a. marks and a. peet. marks' basic medical biochemistry: a clinical approach (4th ed.). philadelphia: wolters kluwer health/lippincott williams & wilkins, (2013) 175. isbn 9781608315727. 3. r. c. malenka, e. j. nestler and s.e. hyman, "chapter 6: widely projecting systems: monoamines, acetylcholine, and orexin". in a. sydor, r.y. brown (eds.). molecular neuropharmacology: a foundation for clinical neuroscience (2nd ed.). new york, usa: mcgrawhill medical, (2009) 157. isbn 9780071481274 pak. j. anal. environ. che m. vol. 23, no. 2 (2022)257 5. n. djelić, m. radaković, s. borozan, v. dimirijević-srećković, n. pajović, b. vejnović, n. borozan, e. bankoglu, h. stopper and z. stanimirović, mutat. res. genet. toxicol. environ. mutagen., 843 (2019) 8. doi: 10.1016/j.mrgentox.2019.01.013 6. s. y. hassan, cardiovasc. revasc. med., 20 (2019) 907. doi:10.1016/j.carrev.2018.10.026 7. t. easterling, s. mundle, h.bracken, s. parvekar, s. mool, l. a. magee, p. dadelszen, t. shochet and b. winikoff, lancet, 394 (2019) 1011. doi: 10.1016/s0140-6736(19)31282-6. 8. s. braunthal and a. brateanu, sage open medicine, 7(2019)1. doi: 10.1177/2050312119843700 9. k. c. berridge, psychopharmacology, 191 (2007) 391. doi:10.1007/s00213-006-0578-x 10. p. sokoloff1 and b. l. fol, eur. j. neurosci., 45 (2017) 2. doi.org/10.1111/ejn.13390 11. a. bjorklunda and o. lindvallb, j. parkinson’s dis., 7 (2017) s21. doi.org/10.3233/jpd-179002 12. s. a. a. touroutogloua, t. b. rudya, s. salcedoa, r. feldmanc, d. j. m. hookera, b. c. dickersona, c. catanaa and l. f. barrett, proc. natl. acad. sci., 114 (2017) 2361. doi.org/10.1073/pnas.1612233114 13. a. kutluay, s. karabulut and s. abbasoglu, j. chin. chem. soc., 55 (2008)794. doi: 10.1002/jccs.200800119 14. x. zhu, x. ge and c. jiang, j. fluorescence, 17 (2007) 655. http://dx.doi.org/10.1007/s10895-0070206-0 15. b. perez-mella, a. alvarez-lueje, electroanalysis, 25 (2013) 2193. doi.org/10.1002/elan.201300241 16. n. f. atta, a. galal and r. a. ahmed, j. electrochem. soc., 158 (2011) f52. http://dx.doi.org/10.1149/1.3551579 17. m. dolezalova and m. tkaczykova, j. pharm. biomed. anal., 19 (1999) 555. doi.org/10.1016/s0731-7085(98)00257-x 18. j. le, t. sun, r. peng , . yuan, y. feng, s. wang and y. li, mikrochim. acta, 187 (2020) 165. doi: 10.1007/s00604-020-4145-7 19. k. murtada, f. andrés, i. galván, á. ríos and m. zougagh, j. chromatogr. b, 1136, (2020) 121878. https://doi.org/10.1016/j.jchromb.2019.121878 20. j. britto-júnior , n. j. antunes, r. campos , m. sucupira , g. d. mendes, f. fernandes, m. o. moraes, m. e. a moraes and g. d. nucci, biomed. chromatigr., 35 (2021) e4978. doi: 10.1002/bmc.4978. 21. f. chen, b. fang and s. wang, j. anal. meth. chem., id 8821126 (2021)1. doi.org/10.1155/2021/8821126 22. m. p. dossantos, a. rahim, n. fattori, l. t. kubota and y. gushikem, sensors actuators b, 171-172 (2012) 712. doi.org/10.1016/j.snb.2012.05.061 23. s. xiong, h. han, r. zhao, y. chen and g. liu, biomed. chromatogr., 15 (2001) 83. https://doi.org/10.1002/bmc.37 24. l. a. kartsova, e. a. bessonova, a. a. sidorova, v. a. kazakov, i. a. tver'yanovich and l. i. velikanova, russ. j. appl. chem., 77 (2004) 1150. http://dx.doi.org/10.1023/b:rjac.00000 44165.62665.85 25. a. moslehipour, j. chem. rev., 2 (2020) 130. doi: 10.33945/sami/jcr.2020.3.1 26 i. a. shihab and t. n. al-sabha, pak. j. anal. environ. chem., 21 (2020) 10. http://doi.org/10.21743/pjaec/2020.06.02 27. m. m. al-sharook, j. edu. sci., 19(2), (2007) 1. doi.org/10.33899/edusj.2007.5781 28. t. s. al-ghabsha, m. s. al-enizzi and z. z. al-abdaly, j. edu. sci.,19(3), (2007) 1. doi.org/10.33899/edusj.2007.51320 pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 258 29. l. g., y. zhang, q. li., anal. sci., 25 (2009) 145. doi.org/10.2116/analsci.25.1451 30. b. s. nagaralli, j. seetharamappa and m. b. melwanki, j. aoac int., 85 (2002) 1288. doi.org/10.1093/jaoac/85.6.1288 31. p. nagaraja, r. a. vasantha and k. r. sunitha, j. pharm. biomed. anal., 25 (2001) 417. doi.org/10.1016/s0731-7085(00)00504-5 32. a. j. m. al-ogaidi, j. eng. stud. res., 22 (2016) 7. http://dx.doi.org/10.29081/jesr.v22i4.250 33. f. a. nour el-dien, m. a. zayed, g. g. mohamed and r. g. el-nahas, j. biomed. biotechn., (2005) 1. doi.org/10.1155/jbb.2005.1 34. n. j. alaallah, s. a. dhahir and h. h. ali, iop conf. series: mater. sci. eng., 871 (2020) 012033. doi:10.1088/1757-899x/871/1/012033 35. l. rahman, a. m. al-abachi and m. h. al-qaissy, anal. chim. acta, 538 (2005) 331. doi:10.1016/j.aca.2005.02.045 36. i. t. humeidy, j. eng. sci. techn., 15 (2020) 1824. 37. s. ashour, drug anal. res., 5 (2021) 51. doi.org/10.22456/2527-2616.113058 38. g. p. hildebrand and c. n. reilley, anal. chem., 29 (1957) 258. doi.org/10.1021/ac60122a025 39. r. belcher, r. a. close and t. s. west, talanta, 1 (1958) 238. doi.org/10.1016/0039-9140(58)80039-9 40. m. valcarcel, “principles of analytical chemistry: a textbook”, springer (2000) 67. berlin, germany. doi: 10.1007/978-3-642-57157-2 41. k. basavaiah, b. c. somashekar and v. ramakrishna, acta pharm., 57 (2007) 87. doi:10.2478/v10007-007-0007-7 42. zahraa j. al-gubouri and theia’a n. alsabha, j. edu. sci., 29 (2020) 246. doi: 10.33899/edusj.2020.126846.1060 43. k. b. vinay, h. d. revanasiddappa, o. z. devi, p. j. ramesh and k. basavaiah, braz. j. pharm. sci., 47 (2011) 251. doi.org/10.1590/s198482502011000200006 44. a. s. al-ayash, j. al-nahr. univ., 11 (2008) 54. doi.org/10.22401/jnus.11.3.05 microsoft word 10-277-287-pjaec-03082021-388-c-revised galley pro cross mark issn-1996-918x pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 277 – 287 http://doi.org/10.21743/pjaec/2022.12.10 valuation of groundwater contaminated with nitrates and human health risks (hhr) between villages of sinjar and tal afar districts, iraq mohammed hazim sabry al-mashhadany college of education for pure sciences, university of mosul, mosul, iraq. *corresponding author email: mohammedhazemm@uomosul.edu.iq received 03 august 2021, revised 28 october 2022, accepted 01 november 2022 -------------------------------------------------------------------------------------------------------------------------------------------abstract nitrates are one of the most common pollutants in groundwater. to assess the risk of exposure to nitrates in drinking water for age groups, we monitored the concentration of nitrates in the drinking water of the al-jazeera region. the climate of this region is characterized as dry and semi-arid, and its inhabitants depended on the water in the aquifers as a source of drinking for many years, without monitoring, treatment, or filtration system, as there is no public drinking water network. a model was also used to assess the risks of nitrate pollution in groundwater to human health. samples were taken from 30 wells distributed equally over the three villages to collect water samples and measure the concentration of nitrate ions in groundwater. the concentration of nitrate ions in well water is less than 50 mgl-1 and ranged between (4.2 – 48.1) mgl-1. the mathematical model results showed that the ages under 11 years and pregnant wo men have a higher hazard quotient of nitrate value (hq) than one except for wells no. 2 and 9, which are higher than the permissible limits for drinking. as for age groups above 11, well water was suitable for drinking, and the hq value was mainly less than one. the reason for this age group's lower chronic daily intake (cdi). in other words, the groundwater was suitable for adults and not for children under 11 years and pregnant women. keywords: hq, cdi, nitrate, cancer, drinking water. -------------------------------------------------------------------------------------------------------------------------------------------introduction the issue of the environment and environmental pollution has received the attention of specialists and global public opinion, and there have been many studies that dealt with groundwater pollution after it was contaminated with chemical, and biological pollutants, which has contributed greatly to the increase in diseases and the deterioration of environmental components [1]. the use of well water regardless of the degree of pollution and the severity of their use, leads to diseases that may be fatal, and these diseases may not appear when using water until some time has passed; pollution of groundwater with nitrates is an important problem for the rural population of the world, as there are hundreds of wells and tens of thousands of hectares of agricultural land whose groundwater cannot be used for drinking purposes due to nitrate pollution, as in morocco [2], germany [3], and france [4, 5]. therefore cannot use its water to exceed the nitrate concentrations permissible limit [6]. from the above, we note the magnitude of the problem, considering the negative effects of pollution on general human health and infants in particular. the health effects of nitrates in humans are most closely related to pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 278 infants, as their consumption of water containing nitrates with milk leads to the transformation in the stomach that can combine with oxygen molecules in red blood cells leading to oxygen depletion and the possibility of suffocating the child. as for adults, drinking water containing higher concentrations of nitrates than infants may not pose a health risk [7, 8]. however, some studies indicate the possibility of bleeding in the spleen due to the ingestion of water containing large amounts of nitrates. world health organization (who) standards state that nitrate concentrations exceeding 50 mgl-1 can be dangerous for adults and children; also, a low nitrate content can become hazardous when the water containing it boils because nitrites and nitrates are not evaporable [9, 10]. the concentration of nitrates in groundwater and surface water is normally low. still, it may reach high levels due to various nitrogen sources, whether (agricultural or animal) on the earth's surface, in the soil layer, or the shallow layers under the soil, which is transmitted by the filtration process, surface runoff, or others [11, 12]. when the added quantities of chemical fertilizers exceed certain proportions, this often happens through repeated, unexamined, and random additions in many countries, leading to many negative effects, directly or indirectly, on the biological system in particular and the environment in general. the direct repercussions of chemical fertilizers are direct damage to the living components of the ecosystem, including human, animal, and plant health. as for the indirect effects, they negatively affect the vital parts of the ecosystem (water, air, and soil). they occur in a defect in the composition of these natural components and the natural balance between them. on the other hand, the leakage of nitrates into the groundwater is one of the most important risks of pollution with nitrogen fertilizers [13, 14]. in some countries where groundwater is the main source of drinking. some reports indicate groundwater pollution increases the risk of cancer of the pancreas, brain, large intestine, bladder, and thyroid [15, 16]. geospatial technology, such as satellite remote sensing, geographic information systems (gis), and satellite navigation system, are widely used in groundwater research. the most common applications of geospatial technology in groundwater research include identifying and mapping groundwater exploration areas and producing spatial and sensitive groundwater quality for pollution maps using gis [17]. among the studies that were conducted was to measure the concentration of nitrates in groundwater, which are dangerous to human health: noor and others studied the nitrate ion concentration in the groundwater of several wells in the city of mosul, whose concentration ranged between (0.39-10.88 mgl-1) attributed this to pollution with wastewater [18], and the study of al-saffawi and awad of the village of abuwajnah village in the zammar sub-district in nineveh governorate indicated that there is no risk of drinking water by rural consumers due to its low concentrations, as its hq value ranged between (0.0228 0.1125) [19]. the study aims to find out the suitability of drinking water for different age groups by applying the nitrate model. material and methods description of the study area the study was conducted on groundwater in the northwestern part of nineveh governorate (al-jazirah region). it includes the kakhirta village, the village of ein al-hussan and the village of shoueira, where various agricultural and animal activities are spread that depend on the groundwater sources in the area for drinking and various uses [1, 20]. pak. j. anal. environ. che m. vol. 23, no. 2 (2022)279 sample collection samples were collected regularly for five months to cover part of the area, and gps determined the coordinates of each well. from there, they were projected onto the map as identified 30 wells. table 1 shows the number of wells and samples collected from the sites according to geographical division. the total number of samples taken was 150 during the study period, with a sample from each well for each month. table 1. coordi nates (e, n) and al titude of the studied wells. sample preparation at 220 nm, the nitrate ion absorbs uv light, but not at 275 nm. because dissolved organic stuff absorbs light at 220 nm, this is achieved by measuring the absorbance of a water sample at 275 nanometers, a wavelength at which organic matter can absorb electromagnetic radiation but not by nitrates. once known, an experimental correction factor at 220 nm can discriminate between nitrate and organic matter. there are two phases to sample preparation. the sample will first be filtered to prevent uv light from being scattered by suspended particles in the water sample. to avoid interferences caused by the absorption of oh or co3 2, both of which may absorb at 220 nm, the samples were acidified with 1 n hcl. up to 1000 caco3 mgl -1 , acidification should preclude interference from these ions. hydrochloric acid is employed because cldoes not absorb light in the spectrum's 250 – 290 nm region. samples were collected for five replicates of each sample in pyrex glass containers of 250 ml capacity and routinely followed the standard method for taking samples from the source as they filled the sample. the air was expelled inside the package, sealed after washing the container twice or three from the same source, and transferred to the industrial chemistry lab at the university of mosul. the measurement was carried out according to the (ultraviolet screening method) by taking a known volume of the well-filtered sample, then adding to it (1 ml) of hcl (1 n) acid and measured at wavelengths of 220 and 275 nm using a uv spectrophotometer [21]. assessment of the human health risk of nitrates (hhr) by drinking water for the studied wells well water was evaluated for the studied area hhr, according to the united states environmental protection agency well e n al titude (m) 1 42̊ 34ʹ88ʹʹ 36˚55ʹ90ʹʹ 406 2 42̊ 34ʹ71ʹʹ 36˚55ʹ87ʹʹ 406 3 42̊ 34ʹ93ʹʹ 36˚55ʹ73ʹʹ 407 4 42̊ 34ʹ45ʹʹ 36˚55ʹ68ʹʹ 406 5 42̊ 34ʹ59ʹʹ 36˚55ʹ45ʹʹ 407 6 42̊ 34ʹ95ʹʹ 36˚55ʹ60ʹʹ 407 7 42̊ 35ʹ01ʹʹ 36˚55ʹ34ʹʹ 407 8 42̊ 34ʹ68ʹʹ 36˚55ʹ31ʹʹ 407 9 42̊ 34ʹ70ʹʹ 36˚55ʹ12ʹʹ 407 k ak h ir ta v il la g e 10 42̊ 35ʹ00ʹʹ 36˚55ʹ06ʹʹ 407 11 42̊ 21ʹ79ʹʹ 36˚31ʹ16ʹʹ 335 21 42̊ 21ʹ53ʹʹ 36˚31ʹ43ʹʹ 335 13 42̊ 21ʹ42ʹʹ 36˚31ʹ64ʹʹ 335 14 42̊ 21ʹ47ʹʹ 36˚30ʹ26ʹʹ 332 51 42̊ 21ʹ26ʹʹ 36˚31ʹ26ʹʹ 332 61 42̊ 21ʹ11ʹʹ 36˚31ʹ11ʹʹ 342 71 42̊ 20ʹ87ʹʹ 36˚32ʹ02ʹʹ 348 81 42̊ 21ʹ13ʹʹ 36˚32ʹ08ʹʹ 348 91 42̊ 22ʹ19ʹʹ 36˚30ʹ54ʹʹ 327 e in a lh us sa n vi ll ag e 20 42̊ 22ʹ18ʹʹ 36˚31ʹ02ʹʹ 335 12 42˚22'86'' 36˚28'61'' 324 22 42˚24'04'' 36˚29'34'' 313 23 42˚24'24'' 36˚28'75'' 317 24 42˚24'26'' 36˚27'66'' 318 25 42˚24'94'' 36˚26'57'' 310 26 42˚25'01'' 36˚25'01'' 308 27 42˚22'80'' 36˚26'08'' 320 28 42˚25'59'' 36˚30'07'' 325 29 42˚26'53'' 36˚29'59'' 319 s h o u ei ra v il la g e 30 42˚26'32'' 36˚26'80'' 304 pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 280 (usepa), which is widely used to determine the risks of nitrates to human health. a special model was used to calculate the nitrate concentration in well water. this model calculates the cdi and the hq or the following equations: cdi = sw × is × es × de / bz × zt hq = cdi / dn cdi stands for chronic daily intake (mg/kg day), and sw stands for nitrate content in drinking water (mgl -1 ). de indicates the exposure period in years and is represents the average daily intake of water (liters) for different ages of adults, children, and babies. the local population in the study region relies on groundwater for drinking. therefore, the frequency of exposure (es) is 365 days/year. bz: average body weight in kg, meantime values (zt) in days, dn represents the reference dose of nitrates (1.6 mg/kg/d), and these data are obtained from risk information systems. if the hq values are more than one, then it is considered hazardous to human health, and water is not suitable for drinking, but when it is equal to or less than one, drinking water is not dangerous and can be used for drinking [22, 23]. inverse distance weighting used the (idw) method to predict the spatial distribution of nitrates in the groundwater of 30 wells, one of the geostatistical methods and one of the most advanced techniques. fig 2, 3 and 4 show the distribution of nitrates in groundwater over the area of each village [24]. figure 1. village sites i n the study area in ni neveh governorate [23, 24] kakhirta village ein alhussan village shawira village tal afar district pak. j. anal. environ. che m. vol. 23, no. 2 (2022)281 figure 2. spati al distri bution of nitrates i n the kakhi rta vill age figure 3. spati al distri bution of nitrates i n the ei n al -hussan vill age pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 282 figure 4. spati al di stri bution of nitrates i n the shoueira vill age results and discussion nitrogen fertilization is one of the most important agricultural applications that contribute to the pollution of groundwater, which causes health risks, including the carcinogenic effect. so, the amount of nitrates that a person takes in a day should not exceed 200 mg, since nitrates in the body are transformed into nitrites and are toxic through the formation of amines (nitrosamines) [25], which in turn cause liver cancer or esophageal cancer, nitroso compounds are formed in the human body due to the intake of nitrates [26]. after drinking water containing nitrates, about 20% is converted to nitrite by bacteria in the digestive system. the nitrates in the acid conditions of the stomach turn into nitrosoacid (hno2), which reacts with the amines to form n-nitroso compounds (nocs) that may cause cancer when present in high concentrations [27]. the results in this study showed values of nitrate concentration in well water, which ranged between (4.2 48.1) mgl -1 , and their means do not exceed 47 mgl-1, meaning it is less than 50 mgl -1 table 2, the rise is due to the intrusion of animal and agricultural waste into the groundwater. as a result of the pak. j. anal. environ. che m. vol. 23, no. 2 (2022)283 biodegradation processes by microorganisms, the amino acids are transformed into ammonia and then into nitrates by the nitrification process, as in the following equation:  42 nhrnh   324 nononh table 2. ni trate ion val ues for the studied water wells (mgl-1). wells mi n max mean ± sd 1 19.3 48.0 40.7±12.0 2 4.2 31.0 24.5±11.4 3 20.0 47.0 41.1±11.8 4 18.9 45.2 37.4±10.5 5 20.2 48.0 41.5±11.9 6 20.0 48.0 41.4±11.9 7 17.6 45.2 38.9±11.9 8 20.0 45.8 36.9±12.1 9 6.5 34.8 26.8±12.0 k ak h ir ta v il la g e 10 19.9 46.5 40.7±11.6 11 45.5 47.7 46.6±0.8 12 45.5 47.6 46.7±0.8 13 45.7 47.1 46.4±0.6 14 46.1 47.4 46.7±0.6 15 43.0 45.4 44.4±1 16 39.6 45.8 43.4±2.9 17 43.8 46.8 45.1±1.2 18 44.2 46.2 45.1±0.9 19 45.5 47.2 46.3±0.7 e in a lh u ss an v il la g e 20 45.8 47.0 46.4±0.6 21 43.2 46.3 44.8±1.3 22 43.3 46.5 45.2±1.3 23 46.1 47.6 46.8±0.6 24 45.6 47.4 46.6±0.7 25 45.7 47.6 46.5±0.8 26 45.0 47.7 46.5±1 27 46.1 48.1 47.0±0.8 28 45.3 47.6 46.7±0.9 29 45.3 47.7 46.6±0.9 s h o u ei ra v il la g e 30 44.2 45.9 44.9±0.8 these values are considered permissible for drinking according to the standards of the world health organization (who). the harmful effect of the nitrate ion appears through the presence of methemoglobinemia in the blood of infants. thus nitrates are reduced to nitrite by the reductase enzyme, both inside and outside the human body; the formed nitrite binds with hemoglobin to form methemoglobinemia (methb), which cannot transport oxygen to various body tissues as a result of the oxidation of iron (fe +2 to fe +3 ), fig. 5. this creates a health problem known as a blue baby syndrome. children over three months old are more likely to have this disease, as they get large amounts of nitrates by consuming drinking water through artificial feeding; the effect of nitrates on this group of children appears more than on adults because of low concentrations of nitrates cause them disease. the study showed that all water samples from wells for ages under six years are not suitable for drinking due to exceeding the hq value of one, which ranged between (1.0910 2.0939). the hq value of more than one exceeded 80%. for ages between 6-11 years, it ranged between (0.6894 1.3242), which poses a threat to health safety, whereas, for ages above 11 years, the hq value was less than one and varied between (0.5026 1.0145) and thus be suitable for drinking, shown in table 3 [28-32]. figure 5. hemoglobi n converts to methemoglobi nemi a i n the blood pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 284 table 3. cdi and hq val ues for well water. age groups wells < 1.0 6 11 11-16 16-18 18-21 21 -65 > 65 cdi 2.8976 1.8326 1.3359 1.0919 1.4039 1.3525 1.3882 1 hq 1.8110 1.1454 0.8349 0.6825 0.8775 0.8453 0.8676 cdi 1.7442 1.1031 0.8041 0.6573 0.8451 0.8141 0.8356 2 hq 1.0901 0.6894 0.5026 0.4108 0.5282 0.5088 0.5223 cdi 2.9271 1.8512 1.3495 1.1031 1.4182 1.3663 1.4023 3 hq 1.8294 1.1570 0.8434 0.6894 0.8864 0.8539 0.8765 cdi 2.6650 1.6854 1.2286 1.0043 1.2912 1.2439 1.2767 4 hq 1.6656 1.0534 0.7679 0.6277 0.8070 0.7774 0.7980 cdi 2.9541 1.8683 1.3619 1.1132 1.4313 1.3788 1.4153 5 hq 1.8463 1.1677 0.8512 0.6958 0.8945 0.8618 0.8845 cdi 2.9492 1.8652 1.3597 1.1114 1.4289 1.3766 1.4129 6 hq 1.8432 1.1657 0.8498 0.6946 0.8931 0.8604 0.8831 cdi 2.7708 1.7524 1.2774 1.0442 1.3425 1.2933 1.3275 7 hq 1.7318 1.0952 0.7984 0.6526 0.8391 0.8083 0.8297 cdi 2.6252 1.6603 1.2103 0.9893 1.2719 1.2253 1.2577 8 hq 1.6407 1.0377 0.7564 0.6183 0.7949 0.7658 0.7860 cdi 1.9057 1.2052 0.8786 0.7181 0.9233 0.8895 0.9130 9 hq 1.1911 0.7533 0.5491 0.4488 0.5771 0.5559 0.5706 cdi 2.8999 1.8340 1.3370 1.0928 1.4050 1.3536 1.3893 k ak hi rt a v il la g e 10 hq 1.8125 1.1463 0.8356 0.6830 0.8782 0.8460 0.8683 cdi 3.3210 2.1003 1.5311 1.2515 1.6091 1.5501 1.5910 11 hq 2.0756 1.3127 0.9569 0.7822 1.0057 0.9688 0.9944 cdi 3.3289 2.1053 1.5347 1.2545 1.6129 1.5538 1.5948 12 hq 2.0805 1.3158 0.9592 0.7840 1.0080 0.9711 0.9968 cdi 3.3056 2.0906 1.5240 1.2457 1.6016 1.5429 1.5837 13 hq 2.0660 1.3066 0.9525 0.7786 1.0010 0.9643 0.9898 cdi 3.3243 2.1024 1.5326 1.2527 1.6107 1.5517 1.5926 14 hq 2.0777 1.3140 0.9579 0.7830 1.0067 0.9698 0.9954 cdi 3.1653 2.0019 1.4593 1.1928 1.5336 1.4774 1.5165 15 hq 1.9783 1.2512 0.9121 0.7455 0.9585 0.9234 0.9478 cdi 3.0881 1.9530 1.4237 1.1637 1.4962 1.4414 1.4794 16 hq 1.9300 1.2206 0.8898 0.7273 0.9351 0.9009 0.9247 cdi 3.2099 2.0301 1.4799 1.2096 1.5552 1.4983 1.5378 17 hq 2.0062 1.2688 0.9249 0.7560 0.9720 0.9364 0.9611 cdi 3.2156 2.0337 1.4825 1.2118 1.5580 1.5009 1.5405 18 hq 2.0097 1.2710 0.9266 0.7573 0.9737 0.9381 0.9628 cdi 3.2992 2.0866 1.5210 1.2433 1.5985 1.5399 1.5806 19 hq 2.0620 1.3041 0.9507 0.7770 0.9991 0.9625 0.9879 cdi 3.3030 2.0889 1.5228 1.2447 1.6003 1.5417 1.5824 e in a lh us sa n v il la ge 20 hq 2.0643 1.3056 0.9517 0.7779 1.0002 0.9636 0.9890 cdi 3.1908 2.0180 1.4711 1.2024 1.5460 1.4894 1.528721 hq 1.9943 1.2613 0.9194 0.7515 0.9662 0.9309 0.9554 cdi 3.2202 2.0366 1.4846 1.2135 1.5602 1.5030 1.5427 22 hq 2.0126 1.2729 0.9279 0.7584 0.9751 0.9394 0.9642 cdi 3.3331 2.1080 1.5367 1.2560 1.6149 1.5557 1.5968 23 hq 2.0832 1.3175 0.9604 0.7850 1.0093 0.9723 0.9980 cdi 3.3179 2.0984 1.5297 1.2503 1.6076 1.5487 1.5896 24 hq 2.0737 1.3115 0.9560 0.7815 1.0047 0.9679 0.9935 cdi 3.3133 2.0955 1.5275 1.2486 1.6053 1.5465 1.5873 25 hq 2.0708 1.3097 0.9547 0.7804 1.0033 0.9666 0.9921 cdi 3.3152 2.0967 1.5284 1.2493 1.6063 1.5465 1.5883 26 hq 2.0720 1.3104 0.9553 0.7808 1.0039 0.9666 0.9927 cdi 3.3502 2.1188 1.5445 1.2625 1.6232 1.5637 1.6050 27 hq 2.0939 1.3242 0.9653 0.7891 1.0145 0.9773 1.0031 cdi 3.3265 2.1038 1.5336 1.2536 1.6117 1.5527 1.5937 28 hq 2.0790 1.3149 0.9585 0.7835 1.0073 0.9704 0.9960 cdi 3.3174 2.0981 1.5294 1.2501 1.6073 1.5484 1.5893 29 hq 2.0734 1.3113 0.9559 0.7813 1.0046 0.9678 0.9933 cdi 3.1991 2.0233 1.4749 1.2056 1.5500 1.4932 1.5327 s h o u ei ra v il la g e 30 hq 1.9995 1.2646 0.9218 0.7535 0.9688 0.9333 0.9579 pak. j. anal. environ. che m. vol. 23, no. 2 (2022)285 finally, the use of chemical fertilizers is increasing, resulting from the increase in the proportion of nitrogen and the lack of vegetation during the winter to the disruption of the nitrogen cycle. this led to groundwater supplies with high concentrations of nitrates, which slowly seep nitrates into the soil at a rate of 1 meter annually until it reaches the underground water level. however, a large amount of nitrogen enters groundwater through surface runoff and seepage every year [33, 34]. good agricultural practices should be encouraged to avoid excessive nitrogenous fertilizers. large quantities of nitrogen fertilizers will increase nitrate concentrations in groundwater by losing a large proportion of the fertilizer used by leaching into the soil [35, 36]. conclusion nitrate concentrations for all well water in the study area were within the permissible limits, less than 50 mgl -1 according to who standards. the study relied on the nitrate ion model, which requires age groups, body weight, exposure rate, and human consumption of drinking water containing nitrate concentrations. the study showed that the main hq values were inappropriate for ages under 11 years and therefore posed a health risk to children, while older ages were good and safe to drink and did not pose a health risk in cancerous diseases. it also showed that the main reason for the high concentration of nitrates in groundwater is farmers' excessive use of nitrogen fertilizers, animal waste, and wastewater. we must try to keep the current nitrate levels from rising. by reducing agricultural fertilizers, future generations will pay the price for the current bad practices in agriculture. international water quality guidelines allow a maximum of 25 mgl -1 for infants and pregnant women and 50 mgl -1 for adults. individuals with weaker immune systems, such as children and the elderly, are more vulnerable to the harmful consequences of nitrate pollution. acknowledgement i thank the university of mosul and the college of education for pure sciences for providing the facilities and completing the paper. conflict of interest the author declares no conflict of interest. references 1. m. h. s. al-mashhadany, materials today, 42 (2021) 2756. https://doi.org/10.1016/j.matpr.2020.12.717. 2. m. lahmar, n. el khodrani, s. omrania, h. dakak, r. moussadek, a. douaik, in e3s web of conferences, (2020) 01001. https://doi.org/10.1051/e3sconf/2020150 0100. 3. g. sundermann, n. wägner, a. cullmann, c. r. von hirschhausen and c. kemfert, diw weekly report, 10 (2020) 61. http://dx.doi.org/10.18723/diw_dwr:202 0-8-1. 4. m. parvizishad, a. dalvand, a. h. mahvi and f. goodarzi, health scope, 6 (2017) 43. doi: 10.5812/jhealthscope.14164. 5 j.-c. roux, sci. total environ., 171 (1995) 3. https://doi.org/10.1016/00489697(95)04695-x. 6. a.-a. y. al-saffawi, pak. j. anal. environ. chem., 20 (2019) 75. http://dx.doi.org/10.21743/pjaec/2019.06.10 pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 286 7. n. adimalla, human ecol. risk assess. j., 26 (2020) 310. https://doi.org/10.1080/10807039.2018.1 508329. 8. m. yousefi, s. ghalehaskar, f. b. asghari, a. ghaderpoury, m. h. dehghani and m. ghaderpoori, regul. toxicol. pharmacol., 107 (2019) 104408. https://doi.org/10.1016/j.yrtph.2019.104 408. 9. s. k. gupta, r. gupta, a. gupta, a. seth, j. bassin and a. gupta, environ. health perspect. j., 108 (2000) 363. https://dx.doi.org/10.1289%2fehp.00108 363. 10. m. h. ward, r. r. jones, j. d. brender, t. m. de kok, p. j. weyer and b. t. nolan, int. j. environ. res. public health, 15 (2018) 1557. https://dx.doi.org/10.3390%2fijerph150 71557. 11. n. adimalla, p. li and h. asakura, human ecol. risk assess., 25 (2018) 1107. https://doi.org/10.1080/10807039.2018.1 460579. 12. k. nakagawa, h. amano, h. asakura and r. berndtsson, environ. earth sci., 75 (2016) 234. http://dx.doi.org/10.1007/s12665-0154971-9. 13. f. edition, who chronicle, 38 (2011) 104. https://www.who.int/publications/i/item/ 9789241549950 14. a. javid, a. a. roudbari, a. mashayekh -salehi, m. ghanbarian and m. ghanbarian, int. j. water, 14 (2020) 1. http://doi.org/10.5812/jhealthscope.14164. 15. n. espejo-herrera, k. p. cantor, n. malats, d. t. si lve ma n , a. tardón, r . cía-closas, c. se r r a, m. kogevinas and c. m. villanueva. environ. res., 137 (2015) 299. http://dx.doi.org/10.1016/j.envres.2014.1 0.034 16. m. h. ward, b. a. kilfoy, p. j. weyer, k. e. anderson, a. r. folsom and j. r. cerhan, epidemiology, 21 (2010) 389. http://doi:10.1097/ede.0b013e3181d6201d. 17. a. a. elnazer, e.-m. m. seleem, s. a. zeid, i. s. ismail, h. a. bahlol and s. a. salman, environ. geochem. health, 12 (2021) 100517. http://doi:10.1016/j.gsd.2020.100517. 18. n. a. al-hamdany, y. m. al-shaker and a. y. al-saffawi, biochem. cell. arch., 2 (2020) 6063. https://connectjournals.com/03896.2020. 20.6063. 19. a. al-hamdani, a. kaplan and a. alsaffawi, j. physics: conf. ser., 1999 (2021) 012028. http://doi:10.1088/17426596/1999/1/012028. 20. a. y. t. al-saffawi, b. s. u. ibn abubakar, l. y. abbass and a. k. monguno, nigerian j. technol., 39 (2020) 632. http://dx.doi.org/10.4314/njt.v39i1.35. 21. apha. (2017). standard method for examination of water and waste water. 23rd ed., washington, dc, usa. https://doi.org/10.2105/smww.2882.089. 22. q. zhang, p. xu and h. qian, int. j. environ. res. public health, 16 (2019) 4246. http://doi:10.3390/ijerph16214246. 23. n. adimalla and p. li, human ecol. risk assess., 25 (2019) 81. https://doi.org/10.1080/10807039.2018.1 480353. 24. k. sathees , b. sangeetha, groundw. sustain. dev., 10 (2020) 100297. https://doi.org/10.1016/j.gsd.2019.100297. 25. a. rahman, n. mondal and k. tiwari, sci. rep., 11 (2021) 1. https://doi.org/10.1038/s41598-02188600-1. 26. i. d. buller, d. m. patel, p. j. weyer, a. prizment, r. r. jones and m. h. ward, int. j. environ. res. public health, 18 (2021) 68221. pak. j. anal. environ. che m. vol. 23, no. 2 (2022)287 https://doi.org/10.3390/ijerph18136822 27. r. alex, a. kitalika, e. mogusu and k. njau, j. geofluids, 2021 (2021) 1. https://doi.org/10.1155/2021/6673013. 28. m. h. s. al-mashhadany, earth environ. sci., 722 (2021) 012029. htttp://doi:10.1088/17551315/722/1/012029. 29. s. s. herrmann, k. granby and l. duedahl-olesen, food chem., 174 (2015) 516. http://doi: 10.1016/j.foodchem.2014.11.101 30. d. karunanidhia, p. aravinthasamya, t. j. subramanib and k. srinivasamoorthy, j. human ecol., 25 (2019) 21. https://doi:org/10.1080/10807039.2019.1 568859. 31. a. j. a. jaafer and a. y. t. al-saffawi, j. plant arch., 20 (2020) 3221. 32. m. nujiae, and m. h. stanae, sci. j. nutr. diet, 6 (2017) 63. 33. m. h. s. al-mashhadany, j. a. a. mamaree and a. y. t. al-saffawi, j. plant arch., 20 (2020) 3928. 34. l. huljek, d. dario perkoviæ and z. kovaè, j. maps, 15(2) (2019) 570. http://doi.org/10.1080/17445647.2019.1 642248. 35. y. guimei, w. jiu, l. lei, l. yun, z. yi and w. songsong, j. bmc public health, 20 (2020) 3. https://doi.org/10.1186/s12889-02008583-y. 36. r. a. a. al-shanona, n. m. sadeq and a. y. t. al-saffawi, j. environ. stud., 18 (2018) 14. https://dx.doi.org/10.21608/jesj.2018.20 4149. microsoft word 70-78-pjaec-14092021-392-c-.docx cross mark issn-1996-918x pak. j. anal. environ. che m. vol. 23, no. 1 (2022) 70 – 78 http://doi.org/10.21743/pjaec/2021.06.07 effect of aloe vera extract as green corrosion inhibitor on medium carbon steel in sulphuric acid environment suhail mashooque 1 , mukesh kumar 1 * and imran nazir unar 2 1department of metallurgy and materials engineering, mehran university of engineering and technology , jamshoro, sindh, pakistan. 2department of chemical engineering, mehran university of engineering and technology, jamshoro, sindh, pakistan. *corresponding author email:mukesh.kumar@faculty.muet.edu.pk received 14 september 2021, revised 20 december 2021, accepted 22 december 2021 -------------------------------------------------------------------------------------------------------------------------------------------abstract medium carbon steel is widely consumed by various industrial sectors due to its attractive set of mechanical properties and low cost, but it experiences deterioration when exposed to a corrosive environment. in the present study, aloe vera plant extract was studied as a green corrosion inhibitor for medium carbon steel in an acidic medium. the presence of inhibitive compounds in aloe vera plant extract was deter mined by ftir. moreover, the inhibition efficiency was determined through gravimetric analysis and electrochemical analysis. the results show that the aloe vera plant extract provided inhibition efficiency of more than 90% in both gravimetric and electrochemical analyses. furthermore, the shift in polarization curves depicts that this plant extract is a mixed type inhibitor acting as an anodic and cathodic inhibitor. overall, aloe vera plant extract provides excellent corrosion inhibition to medium carbon steel in the h2so4 environment and can be used as a green corrosion inhibitor for mitigating inter nal corrosion of pipelines and storage tanks. keywords: aloe vera, h2so4 environment, internal corrosion, medium carbon steel, eco-friendly inhibitor -------------------------------------------------------------------------------------------------------------------------------------------introduction medium carbon steel is commonly known as mild steel. due to its availability in the market with low price and good mechanical properties like strength, ductility, and malleability [1]. it has been used in almost every industrial sector. but the main problem with medium carbon steel is its susceptibility towards corrosion, especially in acidic mediums [2]. different industrial sectors such as manufacturing, automobile, chemical processing, oil, and gas use medium carbon steel extensively in their operations. furthermore, in these industries almost in all, an acidic environment is present in various operations, as a result, such materials like medium carbon steel tend to be prone to corrosion [3]. it is obvious that when medium carbon steel comes in contact with the acidic medium, it experiences degradation and deterioration by corrosion. in industrial operations, corrosion leads to serious consequences, including leakage of hazardous fluids from containers and pipelines. which causes product contamination and catastrophic incidents, which ultimately becomes the reason behind the shutdown of the plants and huge loss of economy [4]. in 2002, the usa, civil administration, and national association of corrosion pak. j. anal. environ. che m. vol. 23, no. 1 (2022) 71 engineers (nace) collectively researched corrosion cost, and they estimated that around $276 billion are being spent on over corrosion prevention techniques every year in the usa, which is almost 3.1 per cent of the us gross domestic product (gdp) [5]. thereby, it can be said that corrosion affects different areas of life directly or indirectly. for suppose sudden failure of giant oil and gas pipelines or collapse of industrial operations results in the release of toxic substances and raises serious technical and environmental concerns. statistically, corrosion has a significant impact on the economy, therefore it is necessary to mitigate corrosion by adopting suitable prevention techniques [6]. corrosion is broadly categorized as internal and external corrosion. particularly, internal corrosion is observed in pipelines and storage tanks and controlled by injecting corrosion inhibitors. because corrosion inhibitors provide better inhibition action, that’s why they are far better and more effective than other prevention techniques when it comes to the internal protection of pipelines [7]. conventionally, synthetic inhibitors are used by industries. since, the last few decades, researchers have greatly emphasized introducing green (natural) inhibitors due to the adverse effects of synthetic compounds on workers and the environment. natural extracts are found to be friendly with nature while performing anticorrosive actions. meanwhile, the green inhibitors occur naturally, are readily available, biodegradable in nature, cost-effective, and do not have any momentous effect on health and the environment [8]. so, for the corrosion inhibition of various metals, multiple green inhibitors have been studied in different environmental conditions. like gum arabic, oil palm frond, henna extract, and aloe vera plant extract are studied in different environments for different metals. and to support such type of eco-friendly inhibitors environmental legislation have also preferred the use of green inhibitors in the industrial sector [9]. furthermore, aloe vera extract as a green inhibitor has been studied by many researchers in recent times, such as abiola and james studied aloe vera in 2 m hcl for zinc in which the efficiency of aloe vera was increasing as concentration was increasing [10]. singh et al. studied the corrosion inhibition of aloe vera extract on mild steel in 1 m hcl medium and observed that its efficiency was 90% at 200 ppm concentration [11], likewise these researchers mehdipour et al. also studied aloe vera for stainless steel in 1 m h2 so4 in which electrochemical study showed that as the concentration increased the effectiveness of inhibitor also increased [12]. meanwhile, the efficiency of the inhibitor can be observed by its adsorption behaviour because adsorption is the key property of any inhibitor. it tends to protect metal through adsorbing mechanism, which acts as a barrier between the metal and the environment [13]. moreover, it is reported that the adsorption effect of green corrosion inhibitor is a function of the presence of some functional groups, which includes such as (a) anthraquinone, (b) p-coumaric acid, (c) caffeic acid, (d) ferulic acid, which is shown in fig.1 [14]. figure 1. phenolic compound structures: (a) anthraqui none (b) p-coumaric aci d, (c) caffei c acid, (d) ferulic aci d in the present study, the inhibition efficiency of aloe vera plant extract is pak. j. anal. environ. che m. vol. 23, no. 1 (2022)72 determined in an acidic environment for medium carbon steel. it is reported that aloe vera contains several free anthraquinones and phenolic compounds, which offer good adsorption characteristics on metallic surfaces [15]. moreover, the aloe vera plant extract possesses such aromatic structures and aliphatic compounds within which the presence of electron-rich oxygen, pi-electrons, and double bonds are significant, and which can easily form bonds with the ions of the metal surface in order to create a barrier between the metal and acidic environment [16]. materials and methods preparation of aloe vera extract the fresh succulent aloe vera plant was taken from the garden of metallurgy & materials engineering department, muet, jamshoro, pakistan. the colourless extract of clean-fleshy aloe vera was collected into a beaker at 25°c room temperature by peeling it off with a knife and pressing it with hand. furthermore, the obtained pulpy extract of aloe vera was filtered to get a more clean and pure extract. fourier transform infrared spectroscopy (model: perkin elmer spectrum) was used to confirm the presence of phenolic compounds in the aloe vera plant extract. environment preparation sulphuric acid (h2 so4) of analytical grade (98.5% pure) was used to prepare the 1 m solution in deionized water. then the aloe vera plant extract was used as an inhibitor at various concentrations (200 ppm, 400 ppm, 600 ppm, and 800 ppm). coupons preparation medium carbon steel coupons used for experimental work were cut from a round bar with 1x1 inch as per the reported size of coupons [17]. table 1 shows the chemical composition of medium carbon steel and for test spark spectrometer (model: bruker q2 ion) were used. furthermore, initially, coupons were ground with the emery papers and polished using alumina paste to remove rust and dust from the metallic surface. after that, coupons were rinsed with deionized water and acetone ((ch₃)₂co) to remove the dirt or grease from the surface of the coupons. table 1. chemi cal composi tion of medi um carbon steel (wt.%). elements phosphorus (p) manganese (mn) silicon (si) iron (fe) sulphur (s) carbon (c) composi tion 0.0020 0.581 0.488 98.7 0.0030 0.387 gravimetric analysis gravimetric analysis (weight loss measurements) was performed at room temperature 25°c [18]. the coupons were precleaned and weighed by using a weight balance machine(shimadzu balance, model ay62), then placed into the 100 ml beakers containing 1 m h2 so4 and various concentrations of inhibitor 200 ppm, 40 ppm, 600 ppm, 800 ppm. the test was performed in an open atmosphere. weight loss of coupons was determined after an immersion time of 24 h, 48 h, and 72 h. the weight loss of the coupons was calculated by the difference between initial weight (wi) and final weight (wf) using equation (1) [19]. ∆w = wi -wf (1) corrosion rates (cr) were determined using weight loss data in given equation (2) [20]. at w cr   (2) whereas w is the weight loss of coupon in mg, a indicates the surface area of pak. j. anal. environ. che m. vol. 23, no. 1 (2022) 73 the specimen, and t determines the time of each experiment in hours. from the obtained corrosion rate at different concentrations of inhibitor, the inhibition efficiency (ie) of the inhibitor was determined by using equation (3) [21]. 100 cr crcr (%)ie blank inhblank    (3) crblank (absence of inhibitor) and crinh (presence of inhibitor) represent the corrosion rates. electrochemical analysis the electrochemical analysis was carried out using versastat4 potentiostat using corrosion kit, which contained 300 ml of respective test solution. three electrode setup was used containing saturated calomel electrode (sce), medium carbon steel coupon, and platinum mesh electrode as reference electrode, working electrode, and auxiliaryelectrode, respectively. the analysis was conducted at room temperature 25°c. for each test, samples were properly ground using emery papers of various sizes, i.e., 400, 600, 800, 1000, and 1200–polished and cleaned with acetone. potentiodynamic polarization was performed in ranges from -0.4v to +0.4v at open circuit potential with a scan rate of 1 mv sec -1 . an electrochemical impedance spectroscopy (eis) study was performed in the frequency range from 100000 hz to 0.004 hz [22]. results and discussion fourier transform infrared spectroscopy (ftir) fig. 2 depicts the results of the ftir spectrum of aloe vera extract between transmittance and wavelength. ranges from 500 cm -1 to 4000 cm -1 were obtained. peaks in the graph represent the evidence of phenolic compounds in aloe vera plant extract, the peaks at 572.8 cm-1, 1251 cm-1, and 1402 cm-1 correspond to phenolic-oh, c=c bond, coc. furthermore, the presence of -no2 group and weak bonds justifies the absorption bands at 1865 cm -1 2351 cm -1 and 3451 cm -1 . and the polymeric compounds indicate the presence of c-h bonding in the extract [23]. figure 2. ftir of al oe vera pl ant extract gravimetric analysis weight loss analysis was carried out to study the effect of aloe vera plant extract as an eco-friendly corrosion inhibitor in a 1m h2 so4 environment on medium carbon steel. the test was conducted at room temperature 25°c. samples were immersed in test solution at various concentrations of aloe vera plant extract 0 ppm, 200 ppm, 400 ppm, 600 ppm and 800 ppm for 24 h, 48 h, and 72 h. weight loss measurement at different concentrations and immersion times were calculated using eq: 01, 02, and 03. table 2 shows the results of weight loss, corrosion rate (cr), and obtained inhibition efficiency values (ie%). the obtained data shows that weight loss of medium carbon steel samples significantly decreases with an increase in inhibitor concentration at different exposure times, as shown in fig. 3. table 2 indicates the ie% of aloe vera plant extract as a function of adsorption inhibitor – as the concentration of inhibitor is increasing results the substantial increase in surface adsorption of anthraquinone molecules pak. j. anal. environ. che m. vol. 23, no. 1 (2022)74 which increase inhibition efficiency of aloe vera plant extract. moreover, the maximum efficacy was obtained at 800 ppm, approximately 97%, 96%, and 92% for 24 h, 48 h, and 72 h exposure times, respectively. it is reported that inhibitors are adsorbed on the metallic surface and provide a barrier between the metal and the environment [24]. figure 3. results of weight loss of medi um carbon steel at various concentrations of al oe vera pl ant extract i n 1m h2so4 potentiodynamic polarization (tafel curves) electrochemical polarization measureements were conducted on medium carbon steel in 1 m h2 so4 solution with and without the addition of a green inhibitor. five concentrations of 0 ppm, 200 ppm, 400 ppm, 600 ppm, and 800 ppm were added in the corrosive solution, and the obtained tafel curves are depicted in fig. 4. moreover, table 3 presents the computed potentiodynamic parameters in the presence and absence of inhibitors. it can be observed from fig. 4 that the addition of aloe vera plant extract changes the anodic (βa) and cathodic (βc) slopes by reducing anodic dissolution and slowing down the hydrogen evolution process, respectively. it is cited that the classification of corrosion inhibitors as cathodic, anodic, and mixed type is based on the variations observed in polarization tafel curves after the addition of inhibitor in it [25]. however, the considerable change in tafel curves (anodic and cathodic region) after the addition of green inhibitor specifies that aloe vera plant extract is a mixed-type inhibitor. it is reported that the addition of natural inhibitors slowdowns the anodic and cathodic reactions through adsorbing on active sites as a result, corrosion reactions are retarded [26]. furthermore, the addition of inhibitor offers better surface coverage to the metallic surface in an acidic environment and shifts the corrosion behaviour towards a more inhibition region [27]. table 2. gravi metric data of al oe vera pl ant extract at various concentrations. time (h) inhi bitor concentration (ppm) weight loss (g) corrosion rate (mpy) inhi bition efficiency (ie%) surface coverage (ө) 24 0 200 400 600 800 0.7289 0. 5340 0.4571 0.3962 0.3687 164.9128 119.7312 103.9177 90.3637 83.5859 36.34 59.38 83.77 97.69 0.3634 0.5938 0.8377 0.9769 48 0 200 400 600 800 1.9544 1.5517 1.2567 1.0406 0.9987 220.2603 175.0787 142.3220 117.4721 112.9540 25.95 87.81 93.39 95.96 0.2595 0.8781 0.9339 0.9596 72 0 200 400 600 800 2.7439 2.2831 1.9217 1.7933 1.4327 206.3293 171.6901 144.5811 134.7918 107.6828 20.18 42.73 62.04 91.51 0.2018 0.4273 0.6204 0.9151 pak. j. anal. environ. che m. vol. 23, no. 1 (2022) 75 figure 4. tafel plot of aloe vera pl ant extract at di fferent concentrations on medi um carbon steel i n 1m h2so4 results also show that corrosion current density (jcorr) values are continuously decreasing with increasing the inhibitor concentration. it confirms that increased corrosion inhibitor concentration hindered the corrosion reaction by developing a resistive layer of adsorbed molecules. in fact, a gradual decrease in corrosion rate is the function of inhibitor concentration, as the inhibitor concentration increases the corrosion rate decreases [28]. table 3. electrochemical polari zation parameters of al oe vera pl ant extract at various concentrations on medi um carbon steel i n 1m h2so4. inhibitor conc. (ppm) eco rr (mv) ico rr (µa) βa (mv) βc (mv) jco rr (µa/cm2) corrosion rate (mm/yr) 0 -491.31 7.24 309.7 261.1 14.49 3.75 x 10-05 200 -516.08 3.84 200.8 188.5 7.68 1.98 x 10-05 400 -509.26 3.26 155.0 171.5 6.52 1.68 x 10-05 600 -508.59 2.97 150.3 168.4 5.95 1.54 x 10-05 800 -507.76 2.79 82.3 89.1 5.58 1.44 x 10-05 electrochemical impedance spectroscopy (eis) the eis study was conducted to understand the effect of inhibitor concentration on the impedance behaviour of steel in a 1 m h2so4 environment. semicircles in fig. 5 show impedance change in the presence and absence of inhibitor. these results were also fitted by using the equivalent circuit (ec), and the extracted data is displayed in table. 4. the lower values of double-layer capacitance (cdl) and higher values of charge transfer resistance (rct) justified that inhibitor creates a strong protective barrier to resist the corrosion reaction by adsorbing on the metallic surface [29]. figure 5. eis (nyqui st) plots at various concentrations of aloe vera pl ant extract on medium carbon steel i n 1m h2so4 table 4. eis parameters at various concentrations of al oe vera pl ant extract on medium carbon steel i n 1m h2so4. inhi bitor concentration (ppm) double-layer capaci tance cdl(µf) charge transfer resistance rct (ω) 0 2.2507 2.777 200 3.0576 2.926 400 2.8853 3.125 600 3.2327 4.878 800 4.6641 5.377 adsorption isotherm an adsorption study was carried out to understand the interaction of inhibitor molecules with medium carbon steel substrate during corrosion test. various methods (weight loss, eis, and potentiodynamic polarization) can be adapted to calculate the degree of surface coverage (θ) for adsorption. because surface coverage (θ) helps to evaluate the isotherms. in this study, surface coverage pak. j. anal. environ. che m. vol. 23, no. 1 (2022)76 (θ) is determined from weight loss data shown in table 2 and fitted to temkin, langmuir, frumkin, flory-huggins, bockris-swinkels, and dhar-flory-huggins model [30]. but the best fit was observed with the langmuir model, then it was considered for the present research. this model illustrates the interaction between the metal surface and inhibitor molecules. langmuir adsorption model is expressed in given equation (4): ink ads inh c k 1c   (4) where (cinh) is the concentration of the inhibitor (aloe vera plant extract), (θ) is the surface coverage, and (kads) is the adsorption equilibrium constant. the graph between inhibitor concentration (cinh) versus (c/θ) showed a straight line as shown in fig. 6. this confirms that the langmuir model is reliable to explain the interaction between inhibitor molecules and medium carbon steel surface in 1m h2 so4 solution. figure 6. langmuir adsorption i sotherm of al oe vera pl ant extract on medi um carbon steel conclusion the results conclude that aloe vera plant extract can be used commercially as a green corrosion inhibitor to prevent medium carbon steel from h2 so4 attack. it is ecofriendly and readily available at a low cost. both gravimetric and electrochemical analysis validate its corrosion inhibition performance in an acidic medium. the maximum inhibition efficiency of 97% was achieved by gravimetric analysis for 24 h immersion time. whereas the effectiveness of inhibitor was also confirmed from nyquist plots. the study also reveals that aloe vera plant extract acts as a mixed-type inhibitor that follows the chemical and physical adsorption on medium carbon steel surface followed by the langmuir adsorption isotherm model. acknowledgment the authors acknowledge the department of metallurgy and materials engineering and chemical engineering, mehran university of engineering and technology jamshoro, pakistan, for providing lab facilities and space for conducting necessary experiment work. conflict of interests there are no conflicts of interest to disclose. references 1 g. t. galo, a. de a. morandimgiannetti, f. cotting, i. v. aoki and i. p. aquino, met. mater. int., 48 (2020) 85. http://doi: 10.1007/s12540-020-00679-9. 2. r. rodrigues, s. gaboreau and j. gance, const. build. mater.,269 (2021) 121240. http://doi.10.1016/j.conbuildmat.2020.12 1240 3. t. h. ibrahim, y. chehade and m. a. zour, int. j. electrochem. sci., 6 (2011) 15. http://doi: 10.17675/2305-6894-2011-91-15. 4. c. a. loto and r. t. loto, der pharma chemica,12 (2016) 63. http://doi.org/10.1016/dr.dpc.2016.07.00 4. pak. j. anal. environ. che m. vol. 23, no. 1 (2022) 77 5. a. ayoola, o. s. i. fayomi and i. g. akande. j. biotribo-corros., 67 (2020) 6. http://doi: 10.1007/s40735-020-00361-y. 6. j. panchal, d. shah, r. patel, s. shah, m. prajapati and m. shah, j. biotribocorros., 7 (2021) 107. http://doi:10.1007/s40735-021-00540-5 7. c. verma, e. e. ebseno, i. bahadur and m.a. qureshi, j. mol. liq., 255 (2018) 577. http://doi:10.1016/j.molliq.2018.06.110. 8. b. tan, j. he, s. zhang, c. xu, s. chen, h. liu and w. li, j. colloid interf. sci., 585 (2021) 287. http://doi:10.1016/j.jcis.2020.11.059. 9. l. t. popoola, corros. rev., 37 (2019) 71. http://doi:10.1515/corrrev-2018-0058. 10. o. k. abiola and a. o. james, corros. sci.,52 (2010) 661. http://doi:10.1016/j.corsci.2009.10.026. 11. a.k. singh, s. mohapatra and b. pani, j. ind. eng. chem., 33 (2016) 288. http://doi:10.1016/j.jiec.2015.10.014. 12. m. mehdipour, b. ramezanzadeh and s.y. arman, j. ind. eng. chem., 21 (2015) 318. http://doi: 10.1016/j.jiec.2014.02.041. 13. s. dahiya, s. lata, p. kumar and r. kumar, corros. rev., 34 (2016) 241. http://doi: 10.1515/corrrev-2016-0015. 14. n. o. eddy and s. a. odoemelam, pigment resin technol., 38 (2009) 111. http://doi: 10.1108/03699420910940617. 15. f. suedile, f. robert, c. roos and m. lebrini, electrochim. acta,133 (2014) 631. http://doi:10.1016/j.electacta.2013.12.07 0. 16. a. m. abdel-gaber, b. a. abd-elnabey, i. m. sidahmed, a. m. elzayady and m. saadawy, corros. sci., 48 (2006) 2765. http://doi: 10.1016/j.corsci.2005.09.017. 17. a. zaher, a. chaouiki, r. salghi, a. boukhraz, b. bourkhiss and m. ouhssine, int. j. corros., 2020 (2020) 1. http://doi: 10.1155/2020/9764206. 18. m. abdallah, port. electrochim. acta, 22, (2004) 161. http://doi: 10.4152/pea.200402161. 19. a. ostovari, s. m. hoseinieh, m. peikari, s. r. shadizadeh and s. j. hashemi, corros. sci., 51 (2009) 1935. http://doi: 10.1016/j.corsci.2009.05.024. 20. a. m. samsudin, a. s. pamungkas and r. e. nugraheni, the 24th regional symposium on chemical engineering (rsce 2017), matec web conf., 156 (2018) 1. http://doi:10.1051/matecconf/201815603 050 21. t. bellezze, g. giuliani, a. vicere and g. roventi, corros. sci., 130 (2018) 12. http://doi:10.1016/j.corsci.2017.10.010. 22. a. o. michael, “corrosion inhibition of mild steel in 15 wt% hcl solution by synthetic and green compounds” university of nottingham, doctor of philosophy, nov. (2018). http://eprints.nottingham.ac.uk/id/eprint/ 56647. 23. o. s. i. fayomi, a. a. ayodeji, e. b. omoniyi and s. t. okolie, int. j. adv. manuf. tech., 99 (2018) 2579. https://doi.org/10.1007/s00170-0182655-9. 24. r. t. vashi and h. g. chaudhari, int. j. innov. res. sci. eng. tech., 11 (2017) 22081. http://www.ijirset.com/upload/2017/nov ember/96_11_ijirset_%20paper%20ij 61111674.pdf 25. m. pais and p. rao, j. biotribocorros., 5 (2019) 92. http://doi: 10.1007/s40735-019-0286-9. 26. h. j. habeeb, h. m. luaibi, r. m. dakhil and a. a. h. kadhum, results phys., 8 (2018) 1260. http://doi:10.1016/j.rinp.2018.02.015. pak. j. anal. environ. che m. vol. 23, no. 1 (2022)78 27. a. s. fouda, m. m. hegazi and a. elazaly, int. j. electrochem. sci., 14 (2019) 4668. http://doi: 10.20964/2019.05.47. 28. y. yaocheng, y. caihong, a. singh and y. lin, new j. chem., 43 (2019) 16058. http://doi:10.1039/c9nj03378e. 29. g. t. galo, a. a. morandim-giannetti, f. cotting, i. v. aoki and i. p. aquino, metals mater. int., 27 (2021) 3238. http://doi:10.1007/s12540-020-00679-9. 30. m. h. shahini, m. keramatinia, m. ramezanzadeh, b. ramezanzadeh and g. bahlakeh, j. mol. liq., 342 (2021) 117570. http://doi:10.1016/j.molliq.2021.117570 microsoft word 01-10-pjaec-02102018-111-p.doc cross mark issn-1996-918x pak. j. anal. environ. chem. vol. 20, no. 1 (2019) 01 – 10 http://doi.org/10.21743/pjaec/2019.06.01 development of gas chromatographic method with electron capture detector for determination of some pcdds in wheat and rice grain matrix iffat abdul tawab khan 1 , qurrat-ul-ain *2 and zahida tasneem maqsood 2 1 department of chemistry, federal urdu university of arts, science and technology, gulshan-e-iqbal campus, karachi-75300, pakistan. 2 department of chemistry, university of karachi, karachi-75270, pakistan. *corresponding author email: qurrat_chem@uok.edu.pk received 02 september 2018, revised 06 may 2019, accepted 13 may 2019 -------------------------------------------------------------------------------------------------------------------------------------------abstract this study develops a gas chromatographic method coupled to micro-electron capture detector to determine four basic polychlorinated dibenzo-p-dioxins (pcdds) congeners: 1, 1,2,3,7,8pentachlorodibenzo-p-dioxin; 2, 2,3,7,8-tetrachlorodibenzo-p-dioxin; 3, 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin; and 4, 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin in wheat and rice. the pcdds were extracted using 1:1 acetone:n-hexane mixture followed by cleaning with acidic aluminium oxide in polypropylene mini columns and eluted with dichloromethane. in quantitative determinations, the limit of detection for congener 1 of pcdds was 0.4 ng ml -1 while for other congeners (2–4) it was found to be 1.0 ng ml -1 . the congener 1 was checked at spiking levels of 0.02, 0.05 and 0.1 ng g -1 , and its recovery was 85.96–120.74% and 95.32–116.88% from wheat and rice, respectively. wheat and rice were also spiked by congers 2–4 at spiking levels of 0.05, 0.1 and 0.5 ng g -1 ; the recovery ranges from wheat were 87.70–115.54%, 85.64–117.88% and 88.40–119.32% for congener 2, 3 and 4, respectively, while from rice the recovery was 77.67– 115.68%, 83.18–119.68% and 79.76–131.15% for congener 2, 3 and 4, respectively. the limit of quantification was determined as 0.1 ng g -1 for congener 1 and 0.5 ng g -1 for other three pcdds (2–4). the intra-day and inter-day rsds of peak areas (n = 3) for four congeners (2 ng ml -1 ) were ranged at 2.5–8.1% and 3.1–10.6%, respectively. this study provides a simple and cost-effective gas chromatographic-electron capture detector method to study some basic pcdds in wheat and rice grains first time in pakistan with fair precision and accuracy when expensive high resolution gas chromatographic-mass spectrometry method is not accessible. keywords: gc-µ-ecd, congeners of pcdds, limit of detection (lod), spiking levels, recovery percent, limit of quantification (loq) -------------------------------------------------------------------------------------------------------------------------------------------introduction wheat and rice are the two most important cereal grains, constituting our main diet. polychlorinated dibenzo-p-dioxins (pcdds) can contaminate food grains and other food products through their entrance in food chain. the pccds mainly accumulate in high fat food due to their high lipophilicity. about 90% human exposure to pcdds is ascribed to consumption of contaminated food [1-4]. the pccds are released in the environment in small amounts mainly as unintentional byproducts of anthropogenic activities (steel manufacturing, iron sintering, waste incineration, etc.) or processes such as forest fires, which specifically involve organic chlorine or inorganic chlorides [5, 6]. the pcdds belong to highly harmful class of organic compounds called ‘persistent organic pollutants (pops)’, extremely resistant to biological, chemical, and photolytic degradation. pak. j. anal. environ. chem. vol. 20, no. 1 (2019)2 the pcdds-induced toxicity includes skin lesions (e.g., chloracne), endometriosis, teratogenic effects, reproductive effects and carcinogenicity to living organisms [1, 7]. out of two hundred ten possible congeners of pcdds, at least 17 have been identified with broad range toxicity, and 2,3,7,8-tetrachlorodibenzo-p-dioxin is recognized with highest toxicity among them [5]. twelve different pops including pcdds have been known to be removed and controlled by 150 countries under stockholm convention treaty [3]. the recommended tolerable/allowable daily intake (tdi/adi) value for pcdds is 4 pg-teq/kg/day [7, 8], while tolerable weekly intake (twi) from the european community (ec) is 14 pg whoteq/kg/week. teq value represents total toxic equivalence for pcdds mixtures present in various matrices. the teq is the sum of products of the individual congener concentrations and tefs (toxic equivalence factors). the insight and control of biochemical and chemical risks of dioxins in food has been comprehensively reviewed by various researchers [1, 7, 9, 10]. the impact of cooking processes on total concentrations of pcdds has been assessed by perello et al. in various common food stuffs in spain [11]. pcdds have been detected in different food items under several monitoring programs carried out in several countries such as china [5], japan [12], italy [13], kuwait [14], united states [15], germany [16], canada [17], france [18], egypt [19], spain [20] and greece [21]. huang et al. estimated potential influence of dietary patterns changes (i.e., decreasing vegetable and total grain consumption and increasing animal-derived food intake) on chinese population health risk (increasing cancer risk) to dioxins [22]. mccrady et al. [23] and uegaki et al. [24] examined the pathways of the pcdds in food chain through grains. otani et al. also investigated the dioxins levels in food grains and some beans [8]. therefore, it is necessary to acquire information on residual levels of pccds in food and feed to evaluate their health importance. various extraction, clean-up and detection techniques for pcdds in different matrixes have been developed and reported so far [25-28]. some efficient quantification techniques to determine toxicity levels of pcdds include high resolution gas chromatography-high resolution mass spectrometry (hrgc-hrms), gc-tandem mass spectrometry (gc-qitms/ms), gc-triple quadrupole mass spectrometry (gc-qqqms/ms) and gc-fourier transform ion cyclortron resonance mass spectrometry (gc-fticrms) [1]. the hrgc-hrms is recognized as the confirmatory tool for pcdds analysis due to its excellent sensitivity and selectivity. however, the very high cost of hrgc-hrms operation, maintenance and instrument itself demand researchers to develop alternative approaches with reduced analysis costs for dioxins [5]. particularly in pakistan, it is very difficult at present to conduct monitoring of pcdds due to the unavailability of up-to-date and highly sensitive techniques intended for their clean-up and quantification. thus, only few studies have been reported for pakistan on dioxins monitoring, including dioxins level studies in chicken eggs [29], human milk [30], animal milk [31] and river sediments [32]. the study of international pops elimination network (2005) observed exceeding dioxins levels in the eggs from peshawar compared to newly proposed european union (eu) action level, supporting the need for further monitoring in all environment compartments, particularly in food and long-term changes to reduce pcdds releases into the pakistani environment [29]. for proper monitoring of pcdds in pakistan, first there is a prerequisite of developing multiple detection system for all dioxins congeners. therefore, the aim of current study is to develop a systematic method to analyze few basic pcdds in food samples. in this regards, wheat and rice grains have been selected to study four different congeners of pcdds, 1,2,3,7,8pentachlorodd (1), 2,3,7,8-tetrachlorodd (2), 1,2,3,6,7,8-hexachlorodd (3) and 1,2,3,4,6,7,8heptachlorodd (4). to our knowledge, no systematic data has been reported so far on using gas chromatographic-micro-electron capture detector (gc-µ-ecd) method to determine pcdds in food grains in particular in pakistan. therefore, samples of wheat and rice were monitored for a mixture of selected pcdds congeners using gc-µecd to develop a method of their analysis in food pak. j. anal. environ. chem. vol. 20, no. 1 (2019) 3 grains, and the performance and feasibility of method was evaluated and discussed. materials and methods chemicals solvents (n-hexane, dichloromethane and acetone) were obtained from tedia company (usa), and they were of chromatographic grade. analytical grade alumina (al2o3, ph 4.5, activity 1) and anhydrous sodium sulphate (na2so4) were procured from merck (darmstadt, germany). acidic alumina was heated to 450 °c for 3 h to activate prior to analysis, and anhydrous sodium sulphate was heated to 150 °c for 3 h to dry and stored in a desiccator before use. pcdds congeners 1–4 (50 µgml -1 ) were obtained from wellington laboratories (canada). n-hexane was used to prepare separate stock standard pcdds solutions (10 µgml -1 ) in 5 ml. an intermediate mixed standard solution of four congeners (0.2 µgml -1 each) and the calibration working standard mixtures (containing four congeners) in the equivalent concentration range of 0.1–160 ngml -1 were prepared by further dilutions in 100 ml to determine linearity (table 1). the working standard mixtures of pcdds congeners 1–4 for spiking were separately prepared in n-hexane from the stock standard solutions as per requirement (table 2) to determine % recoveries of congeners. the stock and working standards were prevented from light and stored at 4°c. under prescribed storage conditions, all the solutions were stable for at least one month. instruments and gc-µ-ecd conditions an agilent technologies 6890 n (usa) gas chromatograph was coupled to ni 63 µ-ecd. an hp-5ms capillary column (30 m, 0.25 µm film thickness, 0.25 mm id) and 7683 series auto injector were equipped to gc system. the injection mode was splitless. for data collection, the system was provided with a software enhanced data analysis. the operating temperature conditions were adjusted as follows: injector 280 oc, column oven 250 oc and detector 300 °c. nitrogen (purity 99.9995%) with a flow rate of 0.5 ml min -1 was operated as a carrier gas, while 60 ml min -1 of nitrogen was adjusted as makeup flow. a buchi v-512 model (switzerland) rotary evaporator with chiller and dynac (usa) centrifuge machine were also used. sample collection and preparation cereal grain (wheat and rice) samples were investigated for pcdds to verify gc-µ-ecd instrument performance using previously reported methods of pesticide analysis in grains with some modifications [33]. all procedures were applied to wheat and rice grains separately. one kg of rice and wheat samples were acquired from a local market of gulshan-e-iqbal town (karachi), initially cultivated and refined in larkana city before transport to karachi. each sample was ground in a laboratory mill, sieved using a 40mesh size sieve, and used as a control. each experiment was performed in triplicate. to 5 g of ground grain sample, a mixture of pcdds congeners (1–4) was added in specified amount (table 2) and agitated for 5 minutes. this spiked sample was kept on standing for three hours for complete absorption of the congeners. the spiked samples and control (the congener free sample) were then allowed to pass through the following extraction and clean-up procedures before gc-µecd instrumental analysis. extraction process for extraction, the spiked or control grain sample was transferred to a centrifuge tube of 100 ml, and acetone:n-hexane (1:1, v/v) extraction mixture (50 ml) was added to it. the tube contents were thoroughly mixed/ stirred with a glass-rod for three minutes. the resultant mixture was centrifuged at a speed of 2,500 rpm for three minutes, and the supernatant was directly decanted into a 1 liter capacity separatory funnel. then, 40 ml acetone:n-hexane (1:1) solvent mixture was added again to the same centrifuge tube, homogenized, centrifuged and decanted as previously in the same separatory funnel. afterward, about 200 ml aqueous na2so4 (2.5 g per 100 ml) and dichloromethane (25 ml) were sequentially added to the above separatory funnel followed by vigorous shaking for two minutes. after separating the phases, the lower dichloromethane layer was collected in a 250 ml pak. j. anal. environ. chem. vol. 20, no. 1 (2019)4 conical flask. using dichloromethane in two more 25 ml portions, the aqueous layer partitioning was repeated. the combined extract of dichloromethane was then passed through a glass column (25 mm id x 450 mm) over 25 g of anhydrous sodium sulphate to collect dry extract while the aqueous extract was discarded. finally, 10 ml dichloromethane was used to wash the sodium sulphate column, and the combined extracts along with washings were concentrated upto 1 ml on rotary evaporator before clean-up. clean-up process for clean-up, a 21 cm long polypropylene column (1.2 cm id) was plugged up with cottonwool (pre-washed with dichloromethane), about 1 g anhydrous na2so4 was transferred over it, and then activated acidic alumina (about 4 g) was added to column with continuous tapping to achieve compact packing. about 1 g anhydrous na2so4 was added again to the column forming a bed on the column top. the column was pre-washed with 5 ml dichloromethane with subsequent transfer of 1 ml dried dichloromethane extract to the column. about 15 ml of eluate was obtained using dichloromethane flow rate of 1 ml min -1 , evaporated to dryness, and resulting residue was dissolved in n-hexane (0.5 ml) prior to instrumental analysis. quantitative analysis auto injector was used to inject 5.0 μl of dissolved residue to gas chromatograph. the areas under different peaks in the resultant chromatogram were compared with those obtained from respective standards injections, and instrument performance was evaluated based on linearity, percent recovery, repeatability and limits of detection and quantification. results and discussion dioxins are ‘persistent organic pollutants’ that are extremely toxic, and humans are exposed to them mainly through food ingestion [1, 7, 22]. the present study develops a method to determine four different polychlorinated dibenzo-p-dioxins (pcdds) congeners in wheat and rice samples. the selected congeners were 1,2,3,7,8pentachlorodibenzo-p-dioxin (congener 1), 2,3,7,8tetrachlorodibenzo-p-dioxin (congener 2), 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin (congener 3) and 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (congener 4). to determine pcdds in rice and wheat grain matrix, the gas chromatographic method described by khan et al. (2007) for pesticides analysis in grains was followed with some modifications [33]. for the quantitative analysis, the gc column was equipped with µecd. initially, the pcdds from spiked grain samples were extracted through 1:1 nhexane:acetone mixture. the propylene mini columns containing acidic aluminium oxide and anhydrous sodium sulphate were then utilized for clean-up to eliminate the possible residual fat and co-extractives from the sample extracts. otherwise, inadequate clean-up of sample can cause fast deterioration of gas chromatographic system particularly ecd, thus ruling out reliable results because of resulting inaccuracies and decreased precision [34]. pcdds generally show low water solubilities, and highly chlorinated pccds exhibit high hydrophobicity [35]. thus, they usually accumulate in lipid fraction of biological samples, and a medium polarity solvent system (consisting of either one solvent or a binary solvent mixture) is often recommended for the extraction of pcdds [36]. a study by kooke et al. evaluated soxhelt extraction efficiency of seven different solvent systems for pcdds in fly ash and found benzene or toluene as most efficient extractant [37]. literature shows the use of different solvents for the pcdds extraction from various matrices prior to quantitative gc analysis, for example, nhexane:acetone (80:20, v/v) for fruits and vegetables [13], dichloromethane:n-hexane (1:1, v/v) for fish, beef and corn silage [5], diethyl ether:n-hexane (7:10, v/v) followed by n-hexane for milk [38] and toluene for chicken eggs [29]. most commonly applied solvent system is dichloromethane/n-hexane; however, due to carcinogenicity and high cost of various solvents, and possibility of deterioration of detector (µecd), we recommend n-hexane/acetone as a better choice in the extraction of pcdds. sample extracts must be concentrated before clean-up or analysis, therefore the boiling point of solvent is important. after extraction, the sample extract is often pak. j. anal. environ. chem. vol. 20, no. 1 (2019) 5 a 8.88 b 12.97 c 19.22 d 29.37 s time response partitioned with either one solvent or by using two separate solvents. anhydrous salt (sodium sulphate or sodium chloride) can be added to absorb water so that the crude extract can be purified before subsequent partitioning step with a water immiscible solvent to avoid stable emulsion formation. removal of any water from extract is also necessary prior to concentration. the control and blank (solvent) were also run in order to assess the efficiency and purity of materials. no contamination peak was observed apart from two peaks emerged just after peak of solvent; however, those might not be characteristic of the congeners studied and might have appeared due to some impurity in the blank or matrix. the external standardization method was used for quantitative determinations. accuracy is affected mostly by the capability of injecting exact sample amounts through a syringe. in this study a previously calibrated auto sampler was operated. limit of detection (lod) shows instrument sensitivity for selected congeners. based on signalto-noise-ratio (s/n) ≥3, the lod value was determined as 0.002 and 0.005 ng/injection for congener 1 and other three congeners (2–4), respectively. under applied experimental conditions with constant 5 μl injection volume, x ng/injection ≈ 200x ngml -1 . the gc-µ-ecd chromatograms of mixture of standard congeners (1–4) are illustrated in (fig. 1 and 2) at 0.02 ng and 0.10 ng per 5 μl injection of each standard congener, respectively. the figures show sufficient gas-chromatographic separation of four pcdds with retention times of congeners 1, 2, 3 and 4 observed at 13.0, 8.9, 19.2 and 29.4 min, respectively. it is also evident from chromatograms that response of congener 1 is about double as compare to response of other three congeners, which is in agreement with its lower lod. the sensitivity results suggest that the presented gc-µ-ecd method has the ability to detect trace amounts of dioxins. linear calibration curve was constructed for each selected congener as a plot of respective peak areas versus analyte concentrations under the proposed chromatographic conditions. (table 1) presents derived calibration parameters for linearity test (linear range, correlation equation and correlation coefficient) for all congeners. congener 1 shows linearity in the range of 0.0020– 0.1000 ng/injection i.e., 0.4–20 ngml -1 with correlation coefficient (r 2 ) value of 0.9989. the other three congeners (2–4) reveal linearity within 0.0050–0.5000 ng/injection (1.0–100 ngml-1) and r 2 in the range of 0.9976–0.9991. figure 1. gc-µ-ecd chromatogram for standard mixture of four pcdds congeners (0.02 ng/5 μl injection). s: peak of solvent; a: congener 2; b: congener 1; c: congener 3; d: congener 4 pak. j. anal. environ. chem. vol. 20, no. 1 (2019)6 a 8.87 b 12.96 c 19.21 d 29.35 s time response figure 2. gc-µ-ecd chromatogram for standard mixture of four pcdds congeners (0.10 ng/5 μl injection). s: peak of solvent; a: congener 2; b: congener 1; c: congener 3; d: congener 4 table 1. statistical parameters for calibration of selected congeners. pcdds congeners linear range (ng/injectiona) correlation equation correlation coefficient (r2) 1 0.0020-0.1000 y = 733.3 + 58882431 x 0.9989 2 0.0050-0.5000 y = 808.2 + 16265950 x 0.9991 3 0.0050-0.5000 y = 8765.9 +23045998 x 0.9988 4 0.0050-0.5000 y = 457.7 + 14346032 x 0.9976 based on correlation coefficient values from five point calibration curves, a reasonable linearity was demonstrated by all tested congeners. from the precision analysis of calibration data, the intra-day rsds of peak areas (5 μl injection, 3 injections per day) for standard pcdds solutions (2 ngml -1 ) were 8.1, 2.5, 3.3 and 3.2% for congeners 1, 2, 3 and 4, respectively. similar concentration (2 ngml -1 ) standard solutions of four congeners yielded inter-day rsds of peak areas (5 μl injection, 3 injections per day x 3 days) in the range of 3.1–10.6%. the peak areas rsds ˂ 15% indicate good repeatability of gc-μ-ecd method. the instrument performance was also evaluated based on retention time shifts in repeated analysis. the percent rsds of retention times for 10 successive 5 μl injections of four congeners (2 ng ml -1 ) were ranged at 0.09–0.18. during the whole study, the repeated analysis (5 μl injection, 10 injections per month x 13 months) of standard congeners (2 ng ml-1) gave shifts in the retention times as follows: 12.28–12.95 min (0.06–0.19 % rsd) for congener 1, 8.39–8.74 min (0.09–0.51 % rsd) for congener 2, 18.21–19.20 min (0.03–0.12 % rsd) for congener 3, and 27.85–29.35 min (0.02–0.07 % rsd) for congener 4. this data shows excellent repeatability of instrument and column (hp-5ms) performance. the recovery percents in wheat and rice grains were examined at applied spiking levels of 0.02, 0.05 and 0.1 ng g -1 for congener 1, while 0.05, 0.1 and 0.5 ng g -1 levels of spiking were set for congeners 2, 3 and 4. limit of quantification (loq) of used method for both wheat and rice samples was observed as 0.1 ng g -1 in case of congener 1 and 0.5 ng g-1 in case of congeners 2–4, based on s/n ratio ≥10. the reproducibility of spiking data was checked by three injections of each extract. the percent recovery and precision found at applied spiking levels for wheat and rice grains are listed in (table 2 and 3), respectively. pak. j. anal. environ. chem. vol. 20, no. 1 (2019) 7 the gc-µ-ecd chromatograms of spiked samples of wheat and rice are presented in (fig. 3 and 4), respectively, at 0.1 ngg -1 congener 1 and 0.5 ngg -1 congeners 2, 3 and 4. over applied spiking range, the average recovery of congener 1 was in the range of 85.96–120.74% and 95.32–116.88% from wheat and rice, respectively, with rsd range of 8.31–13.95%. in contrast, the congeners 2, 3 and 4 were recovered from wheat at their applied spiking levels in the average range of 87.70–115.54%, 85.64–117.87% and 88.40–119.32%, respectively, with rsd 3.99–10.82%. the average recovery of congener 2, 3 and 4 from rice at applied spiking levels was in the range of 77.67–115.68%, 83.18– 119.68% and 79.76–131.15%, respectively, with their rsd values in the range of 5.57–14.75%. interestingly, at lower spiked levels in all cases (i.e., 0.02 ngg -1 for congener 1 and 0.05 ngg -1 for other three congeners) the percent recovery values are greater than 100%. it is because of the reason that the chance of error is often enhanced at lower residual concentration, but the stated results are well within the european union (eu) permissible error range, i.e., within 15% rsd [5, 39]. at loq, the average percent recovery of congeners 1–4 for wheat was 85.64–93.59% with rsd 3.99–8.31%, while for rice the average recovery of congeners 1– 4 was 77.67–95.32% with rsd 6.29–9.88%. the difference in recoveries and %rsds of pcdds congeners in wheat and rice is due to different matrix effect. the given results reveal that the recovery of selected congeners by presented method is quite satisfactory with reasonably good reproducibility. hence, the developed method can satisfy the requirement of monitoring pcdds in wheat and rice samples. table 2. applied spiking levels, percent recoveries and percent rsd values for selected congeners in wheat grains (n = 3). pcdds congeners applied spiking levels (ngg-1) mean % recovery rsd % 1 0.02 0.05 0.10 120.74 85.96 91.33 9.35 10.82 8.31 2 0.05 0.10 0.50 115.54 87.70 88.79 10.79 6.57 5.07 3 0.05 0.10 0.50 117.88 85.83 85.64 9.93 4.79 3.99 4 0.05 0.10 0.50 119.32 88.40 93.59 10.71 7.40 4.50 table 3. applied spiking levels, percent recoveries and percent rsd values for selected congeners in rice grains (n = 3). pcdds congeners applied spiking levels (ngg-1) mean % recovery rsd % 1 0.02 0.05 0.10 116.88 104.01 95.32 13.95 13.79 9.88 2 0.05 0.10 0.50 115.68 80.74 77.67 14.75 14.71 9.11 3 0.05 0.10 0.50 119.68 83.18 88.18 10.75 11.16 8.30 4 0.05 0.10 0.50 131.15 104.73 79.76 8.14 5.57 6.29 ainjection volume = 5 μl (x ng/injection ≈ 200x ngml-1) pak. j. anal. environ. chem. vol. 20, no. 1 (2019)8 a 8.87 b 12.96 c 19.21 d 29.35 s time response c2 c1 a 8.87 b 12.96 c 19.21 d 29.35 s time response c2 c1 figure 3. gc-µ-ecd chromatogram for spiked wheat grains. applied spiking level: congener 1 = 0.10 ngg-1; congener 2, 3, 4 = 0.50 ngg-1. s: peak of solvent; c: contamination peak; a: congener 2; b: congener 1; c: congener 3; d: congener 4 figure 4. gc-µ-ecd chromatogram for spiked rice grains. applied spiking level: congener 1 = 0.10 ngg-1; congener 2, 3, 4 = 0.50 ngg-1. s: peak of solvent; c: contamination peak; a: congener 2; b: congener 1; c: congener 3; d: congener 4 pak. j. anal. environ. chem. vol. 20, no. 1 (2019) 9 conclusion the current study has developed an inexpensive method first time in pakistan to determine few basic congeners of pcdds in wheat and rice grains. present study focused on a simple operation, presenting a method with fair sensitivity and repeatability which is affordable by many researchers who cannot presently afford high resolution mass spectroscopy. however, this method, which implied simple bench-top gc-µecd, is not suitable for studies requiring a limit of detection ˂ 5pg. the recommended tolerable daily intake for pcdds (4 pg-teq/kg/day) is beyond the quantification limit of this method; however, the target can be attained by exploiting either automated and more effective clean up processes or more sensitive quantification, such as by 2dimentional gc-µ-ecd. therefore, the developed method can efficiently provide screening and identification of four tested pcdds with accurate quantification in samples, particularly the matrices heavily contaminated with dioxins, in routine analysis. acknowledgments we greatly thank pakistan agricultural research council (parc), southern zone agricultural research centre, for essential scientific assistance on gc-µ-ecd. references 1. s. kanan and f. samara, trends environ. anal. chem., 17 (2018) 1. https://doi.org/10.1016/j.teac.2017.12.001 2. world health organization, dioxins and their effects on human health (2016). http://www.who.int/mediacentre/factsheets/f s225/en/ 3. united states environmental protection agency (usepa). learn about dioxin (2016). https://www.epa.gov/dioxin/learn-about-dioxin 4. g. c. cakirogulları, d. kilic and y. ucar, food control, 21 (2010) 1245. https://doi.org/10.1016/j.foodcont.2010.02.010 5. h. sun, p. wang, h. li, y. li, s. zheng, j. matsiko, y. hao, w. zhang, d. wang and q. zhang, sci. china chem., 60 (2017) 670. https://doi.org/10.1007/s11426-016-9017-9 6. j. d. urban, d. s. wikoff, a. t. bunch, m. a. harris and l. c. haws, sci. total environ., 466–467 (2014) 586. https://doi.org/10.1016/j.scitotenv.2013.07.0 65 7. z. lali, j. pollut. eff. cont., 6 (2018) 1. https://doi.org/10.4172/2375-4397.1000215 8. t. otani, n. seike and t. miwa, j. food hyg. soc. japan (shokuhin eiseigaku zasshi), 47 (2006) 182. https://doi.org/10.3358/shokueishi.47.182 9. g. a. kleter, m. j. groot, m. poelman, e. j. kok and h. j. p. marvin, food chem. toxicol., 47 (2009) 992. https://doi.org/10.1016/j.fct.2008.08.021 10. l. r. bordajandi, g. gomez, e. abad, j. rivera, m. d. m. fernandez-baston, j. blasco and m. j. gonzález, j. agric. food chem., 52 (2004) 992. https://doi.org/10.1021/jf030453y 11. g. perello, r. marti-cid, v. castell, j. m. llobet and j. l. domingo, food cont., 21 (2010) 178. https://doi.org/10.1016/j.foodcont.2009.05.003 12. t. tsutsumi, s. takatsuki, r. teshima, r. matsuda, t. watanabe and h. akiyama, chemosphere, 191 (2018) 514. https://doi.org/10.1016/j.chemosphere.2017. 10.046 13. m. esposito, a. d. roma, s. cavallo, g. diletti, l. baldi and g. scortichini, toxics, 5 (2017) 1. https://doi.org/10.3390/toxics5040033 14. a. husain, b. gevao, b. dashti, a. brouwer, a. p. behnisch, m. al-wadi and m. alfoudari, ecotoxicol. environ. saf., 100 (2014) 27. https://doi.org/10.1016/j.ecoenv.2013.12.002 15. y. wan, p. jones, r. holem, j. khim, h. chang, d. kay, s. roark, j. newsted, w. patterson and j. giesy, sci. total environ., 408 (2010) 2394. https://doi.org/10.1016/j.scitotenv.2010.02.003 16. e. bruns-weller, a. knoll and t. heberer, chemosphere, 78 (2010) 653. https://doi.org/10.1016/j.chemosphere.2009. 12.014 17. k. srogi, environ. chem. lett., 6 (2008) 1. https://doi.org/10.1007/s10311-007-0105-2 pak. j. anal. environ. chem. vol. 20, no. 1 (2019)10 18. a. tard, s. gallotti, j. leblanc and j. volatier, food addit. contam., 24 (2007) 1007. https://doi.org/10.1080/02652030701317293 19. n. loufty, m. furhacker, p. tundo, s. raccanelli and m. a. tawfic, chemosphere, 66 (2007) 1962. https://doi.org/10.1016/j.chemosphere.2006. 07.081 20. a. bocio and j. l. domingo, environ. res., 97 (2005) 1. https://doi.org/10.1016/j.envres.2004.01.012 21. a. papadopoulos, i. vassiliadou, d. costopoulou, c. papanicolaou and l. leondiadis, chemosphere, 57 (2004) 413. https://doi.org/10.1016/j.chemosphere.2004. 07.006 22. t. huang, w. jiang, z. ling, y. zhao, h. gao and j. ma, sci. rep., 6 (2016) 1. https://doi.org/10.1038/srep21997 23. j. k. mccrady, c. mcfarlane and l. k. gander, chemosphere, 21 (1990) 359. https://doi.org/10.1016/00456535(90)90006-f 24. r. uegaki, n. seike and t. otani, chemosphere, 65 (2006) 1537. https://doi.org/10.1016/j.chemosphere.2006. 04.009 25. g. suzuki, m. someya, h. matsukami, n. m. tue, n. uchida, l. h. tuyen, p. h. viet, s. takahashi, s. tanabe, a. brouwer and h. takigami, emerg. contam., 30 (2016) 98. https://doi.org/10.1016/j.emcon.2016.03.001 26. s. li, g. liu, m. zheng, m. wang, k. xiao, c. li and y. wang, chemosphere, 126 (2015) 73. https://doi.org/10.1016/j.chemosphere.2015. 02.014 27. l. rodenburg, s. du, h. lui, j. guo, n. oseagulu and d. fennell, environ. sci. technol., 12 (2012) 6612. https://pubs.acs.org/doi/10.1021/es300560q 28. l. zhang, j. li, y. wu and y. zhao, chin. j. chromatogr., 25 (2007) 887. https://doi.org/10.1016/s18722059(08)60007-3 29. international pops elimination network (ipen) secretariat, sustainable development policy institute-sdpi (pakistan) and arnika association (czech republic) islamabad-prague, contamination of chicken eggs near the dump site on the edge of peshawar, pakistan by dioxins, pcbs and hexachlorobenzene (2005). https://www.researchgate.net/publication/27 7328134 30. a. scheeter, j. r. startin, m. rose, c. wright, i. parker, d. woods and h. hansen, chemosphere, 20 (1990) 919. https://doi.org/10.1016/00456535(90)90201-4 31. i. a. t. khan and z. t. maqsood, bull. environ. contam. toxicol., 82 (2009) 722. https://doi.org/10.1007/s00128-009-9683-y 32. n. khan, a. inam, j. f. mueller, t. herrmann and o. paepke, organohalogen compd., 66 (2004) 1420. https://espace.library.uq.edu.au/view/uq:10 1225 33. i. a. t. khan, riazuddin, p. zahida and a. mubarik, bull. environ. contam. toxicol., 79 (2007) 454. https://doi.org/10.1007/s00128-007-9266-8 34. y. zhang, j. yang, r. shi, q. su, l. yao and p. li, j. sep. sci., 34 (2011) 1675. https://doi.org/10.1002/jssc.201100058 35. a. aberg, m. macleod and k. wiberg, j. phys. chem. ref. data, 37 (2008) 1997. https://doi.org/10.1063/1.3005673 36. f. bruner, j. chromatogr. libr., 20 (1981) 241. https://doi.org/10.1016/s03014770(08)60135-9 37. r. m. m. kooke, j. w. a. lustenhouwer, k. olie and o. hutzinger, anal. chem., 53 (1981) 461. https://doi.org/doi:10.1021/ac00226a017 38. p. korytar, c. danielsson, p. e. leonards, p. haglund, j. de boer and u. a. t. brinkman, j. chromatogr. a, 1038 (2004) 189. doi:10.1016/j.chroma.2004.03.026 39. p. haglund, p. korytar, c. danielsson, j. diaz, k. wiberg, p. leonards, u. a. t. brinkman and j. de boer, anal. bioanal. chem., 390 (2008) 1815. https://doi.org/10.1007/s00216-008-1896-0 microsoft word 9-pdf-94-105-pjaec-03052023-514-c-revised galley proof-22-06-2023-9.docx cross mark issn-1996-918x pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 94 – 105 http://doi.org/10.21743/pjaec/2023.06.09 assessment of heavy metal contents and their risks in vegetables collected from district ghotki, sindh liaquat ali shar*, shafique ahmed arain and ghulam qadir shar institute of chemistry, shah abdul latif university, khairpur, sindh, pakistan. *corresponding author email: liaquatalishar1978@gmail.com received 30 may 2023, revised 10 june 2023, accepted 16 june 2023 ------------------------------------------------------------------------------------------------------------------------------------------- abstract vegetables are renowned for their nutritional value, as they are rich sources of dietary fiber, minerals, and vitamins offering a wide range of health benefits. they also possess antioxidative properties that contribute to overall well-being. the aim of this study was to evaluate the contamination of heavy metals like arsenic (as), cadmium (cd), chromium (cr), copper (cu), iron (fe), manganese (mn), nickel (ni), and zinc (zn) in the agriculture soil and most frequently consumed vegetables including; sweet potato (ipomoea batatas), turnip (brassica rapa), onion (allium cepa), carrot (daucus carota), garlic (allium sativum), radish (raphanus sativus), lotus (nelumbo nucifera), potato (solanum tuberosum), beetroot (beta vulgaris), and ginger (zingiber officinale) collected from various sites of district ghotki, sindh. the agriculture soil and vegetable samples were digested, and heavy metal levels were determined using atomic absorption spectrophotometer (aas). the zn content was found higher in all vegetable samples. cu, fe, mn, and zn concentrations were found within the permissible levels of the food and agriculture organization and world health organization (who/fao). however, the concentrations of as, cd, cr, and ni were found higher than the permissible limit suggested by who/fao. hazard index (hi), target hazard quotient (thq), daily intake of metals (dim), and estimated daily exposure to heavy metals (edem) were also measure. the hazard index (hi) values of as for all the vegetable samples were greater than 1, indicating potential health risks to those consuming these vegetables. keywords: arsenic, heavy metals, vegetables, target hazard quotient, estimated daily exposure of heavy metals. ------------------------------------------------------------------------------------------------------------------------------------------- introduction in recent years, the issue of heavy metals (hms) contamination has become a global concern due to their non-biodegradable nature, bioaccumulation behavior, and the contamination of agricultural soils. the high levels of toxic metals in agricultural soil can negatively impact crop quality and reduce overall product yield. this contamination can also upset the structure and function of soil, leading to adverse environmental impacts. furthermore, the transportation of these metals through the food chain can have serious implications for human health. the sources of hms contamination in agricultural soils can be attributed to either anthropogenic or natural factors [1-4]. the concentrations of naturally occurring hms in unpolluted agricultural soils are primarily governed by the geological composition of parent material, which influences their release and incorporation into the soil matrix [5]. human activities can significantly impact the levels of hms in soils, leading to potential environmental and health consequences. the pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 95 use of chemical fertilizers and pesticides in agriculture, along with emissions from vehicles and industrial activities, can introduce high levels of toxic metals into the soil, altering its natural balance and contaminating crops and groundwater [6]. furthermore, mining operations can release heavy metals into the soil and water, affecting nearby ecosystems and posing a risk to human health [7-10]. vegetables are a vital component of a healthy diet for individuals of all ages, offering a rich source of minerals, fiber, and vitamins necessary for optimal bodily function and well-being [11-13]. however, it should be noted that vegetables can also contain high levels of hms. this is because they can absorb these metals from polluted water and soil through their roots and subsequently transfer them to the edible portions of the plant [14, 15]. the presence of toxic metals in vegetables is a concern for human health as ingestion of these metals can have adverse effects on various bodily functions, which may be including cancer, developmental anomalies, hematological and reproductive effects, kidney and liver damage, cardiovascular diseases and nervous system disorders [16,17]. in recent years, numerous studies conducted worldwide by researchers have focused on investigating the potential risks of hms on human health through the consumption of vegetables [18-23]. these studies analyze the levels of toxic metals in vegetables, such as lead, cadmium, arsenic, and mercury, and assess the potential health implications for individuals who consume them. the aim of this study is to determine the concentrations of arsenic and hms in the common vegetables grown in the ghotki district, sindh province, and to compare the levels of hms among the vegetables. moreover, the health risks associated with hms exposure through vegetable consumption will be assessed to create crucial data for public health protection. materials and methods study area the headquarters of the ghotki district is located in the city of mirpur mathelo in the sindh province of pakistan. the district was established in 1993, having previously been a part of the sukkur district. the total area of the ghotki district is 6975 square kilometers or 1,555,528 acres. a significant portion of this area, approximately 25,000 acres, is classified as deserted land, while 402,578 acres are flooded. nevertheless, the cultivated area spans the district's desert and flooded regions. the ghotki district includes the white desert, also known as achhro thar, which is characterized by windblown hills. additionally, a flooded area or kacha stretches along the indus river for approximately 87 kilometers and contains forests (fig. 1). according to the 2017 census, the population of the ghotki district was recorded as 1,648,708, with 21.89% residing in urban areas. the majority, 93.67% of the total population, follows islam, while 6.19% practice hinduism, including the scheduled castes. the district is home to people of different languages, including 1.05% saraiki, 1.64% punjabi, 2.49% urdu, and 93.37% sindhi. notably, the historic hindu temple, shadani darbar, is situated in this district. map of study area map of district ghotki map of sindh province figure 1. sampling area of district ghotki sample collection pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 96 five sites were selected for sample collection. at each site, one kg of each type of sample was collected depending on the ripeness of the vegetables in the field during sampling. in total ten types of vegetable samples were collected which included, sweet potato (ipomoea batatas), turnip (brassica rapa), onion (allium cepa), carrot (daucus carota), garlic (allium sativum), radish (raphanus sativus), lotus (nelumbo nucifera), potato (solanum tuberosum), beetroot (beta vulgaris), and ginger (zingiber officinale). for soil sample collection five spots were selected from where vegetable samples were collected. collections of vegetable samples were made in triplicates. samples were wrapped in neat and clean bags, properly labeled and shifted to the laboratory, institute of chemistry, shah abdul latif university, khairpur, where samples were initially washed using tap water to eliminate dirt particles. double distilled water was used to rinse vegetable samples, kitchen knife was used to cut into smaller pieces for drying purposes. sliced vegetable samples were dried in an oven at temperatures below 100 °c for 5 to 7 h. dried vegetable samples were properly labeled and shifted to the national center of excellence in analytical chemistry, university of sindh, jamshoro for heavy metal and as analyses. sample preparation and analyses 2 g of each vegetable sample was taken in a beaker containing nitric acid and perchloric acid in a 4:1 ratio. contents were kept for digestion purposes overnight. samples were heated using electric hot plates at different temperatures until the formation of a clear solution. then samples were kept cooling and filtered using whatman #42 filter paper. double distilled water was used to dilute the samples to make up a volume of up to 50 ml in measuring flasks. samples were kept at room temperature for further analysis. to evaluate the reproducibility of measurement, all analyses were repeated three times. the 2% hno3 solution was used to prepare 1000 ppm stock solutions of heavy metals from their salts. double distilled water was used for successive dilution from stock solutions. a hydride generation absorption spectrophotometer was used to detect arsenic from the edible roots of vegetable samples. the quantity of arsenic and heavy metals was measured from the calibration curve of the respective hms. certified reference material obtained from international atomic energy agency (iaea) was used to analyze the accuracy of the analysis. a similar method was followed for the analysis of blank and calibration standard solutions. statistical analysis for the analysis of statistics the spss (statistical package for the social science) version 18.0 was used. descriptive statistics such as average, minimum, maximum, standard deviation, and correlation co-efficient were determined at a 5% level of confidence. risk assessment daily intake of heavy metals (dim) and estimated daily exposure (edem) the ingestion of heavy metals in the samples depicted as daily intake of metals (dim) was calculated using the following equation. cm, dintake, and bw stand for the concentration of hms, daily ingestion of vegetables (166 g/person/day), and body weight (60 kg), respectively [24]. pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 97 edem was determined using the following formula: where dim and bw show daily intake of hms and body weight, respectively. target hazard quotient the target hazard quotient (thq) depends on the consumption of common vegetables by the people of the study area. this procedure of evaluating risk using thq is given in the usepa region iii risk dependent concentration and its equation is as follows [25]: where, mc, ir, ef, ed, cf rfd, bw and atn are metal content, ingestion rate, exposure frequency (365 days/year), duration exposure (60 years), conversion factor (0.085), reference daily dose, body weight (60 kg) and average time exposure, respectively. hazard index (hi) the hazard index from thqs is denoted as the total of the hazard quotients as given in the following equation [24]. hi=thq(cr)+thq(cu)+thq(cd)+thq( cr)+thq(as)+thq(crfe)+thq(mn)+t hq(ni)+thq(zn) results and discussion daily intake of heavy metals the dim range of heavy metals under study in vegetable samples was determined as cr (0.069 0.270), cu (0.163 0.866), cd (0.044 0.087), as (0.013 0.023), fe (0.047 0.135), mn (0.070 0.140), ni (0.052 0.079) and zn (1.001 1.575) mg/kg/day (table 1 & 2). table 1. daily intake (dim) and estimated daily exposure of trace metals (edem) in different vegetables collected from various locations of district ghotki. daily intake of heavy metals (dim) (mg/day, fresh weight) ginger beet root potato lotus radish garlic carrot onion turnip sweet potato cr 0.076 0.114 0.072 0.069 0.103 0.106 0.075 0.075 0.270 0.101 cu 0.866 0.340 0.504 0.332 0.334 0.466 0.353 0.279 0.367 0.163 cd 0.087 0.085 0.068 0.055 0.068 0.058 0.074 0.044 0.049 0.064 as 0.023 0.022 0.022 0.021 0.023 0.017 0.013 0.014 0.019 0.019 fe 0.047 0.059 0.050 0.063 0.065 0.047 0.056 0.053 0.116 0.135 mn 0.114 0.124 0.140 0.135 0.076 0.093 0.083 0.072 0.070 0.083 ni 0.060 0.052 0.068 0.071 0.054 0.066 0.070 0.058 0.061 0.079 zn 1.226 1.537 1.575 1.520 1.165 1.001 1.281 1.390 1.394 1.170 estimated daily exposure of heavy metals (edem) (mg/kg bw/day) cr 0.0011 0.0016 0.0010 0.0010 0.0015 0.0015 0.0011 0.0011 0.0039 0.0014 cu 0.0124 0.0049 0.0072 0.0047 0.0048 0.0067 0.0050 0.0040 0.0052 0.0023 cd 0.0012 0.0012 0.0010 0.0008 0.0010 0.0008 0.0011 0.0006 0.0007 0.0009 as 0.0003 0.0003 0.0003 0.0003 0.0003 0.0002 0.0002 0.0002 0.0003 0.0003 fe 0.0007 0.0008 0.0007 0.0009 0.0009 0.0007 0.0008 0.0008 0.0017 0.0019 mn 0.0016 0.0018 0.0020 0.0019 0.0011 0.0013 0.0012 0.0010 0.0010 0.0012 ni 0.0009 0.0007 0.0010 0.0010 0.0008 0.0009 0.0010 0.0008 0.0009 0.0011 zn 0.0175 0.0220 0.0225 0.0217 0.0166 0.0143 0.0183 0.0199 0.0199 0.0167 pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 98 table 2. descriptive statistics of daily intake (dim) and estimated daily exposure of heavy metals (edem) in vegetables collected from different locations of district ghotki. dim ((mg/day, fresh weight) edem (mg/kg bw/day) max min mean sd max min mean sd cr 0.270 0.069 0.106 0.060 0.004 0.001 0.002 0.001 cu 0.866 0.163 0.400 0.188 0.012 0.002 0.007 0.003 cd 0.087 0.044 0.065 0.014 0.001 0.001 0.001 0.000 as 0.023 0.013 0.019 0.004 0.000 0.000 0.000 0.000 fe 0.135 0.047 0.069 0.031 0.002 0.001 0.001 0.000 mn 0.140 0.070 0.099 0.027 0.002 0.001 0.002 0.000 ni 0.079 0.052 0.064 0.008 0.001 0.001 0.001 0.000 zn 1.575 1.001 1.326 0.189 0.023 0.014 0.018 0.003 estimated daily exposure of heavy metals (edem) however, edem values of heavy metals from same samples were measured as cr (0.001 0.0039), cu (0.0023 0.0124), cd (0.0006 0.0012), as (0.0002 0.0003), fe (0.0007 0.0019), mn (0.001 0.002), ni (0.0007 0.0011) and zn (0.0143 0.0225) mg/kg/day (table 1 & 2). target hazard quotient the maximum value of thq of cr was found in turnip samples, whereas minimum thq was found in lotus samples. the hazard index of cr was measured as 0.33 which is less than unity. similarly, thq values were found in the range of 0.0115 0.0613 unit minimum in sweet potato and in ginger samples (table 3). health indexes the hi value greater than one shows possible health hazard which was displayed only by arsenic. hi greater than 1 increases the level of distress to the population consuming the vegetables from the study area; while hi values less than 1 concludes that the population is protected. since hi value greater than one (1.83) was found for arsenic. all other metals displayed hi values of less than one (table 3). the order of hi for heavy metals was found as follows; as > cd > cr > cu > mn > zn > fe (table 3). table 3. target hazard quotient (thq) for different heavy metals, their hazard index (hi) from consumption of various types of vegetables collected from different locations of district ghotki. ginger beet root potato lotus radish garlic carrot onion turnip sweet potato hi cr 0.0240 0.0360 0.0227 0.0217 0.0324 0.0334 0.0235 0.0237 0.0849 0.0317 0.33 cu 0.0613 0.0241 0.0357 0.0235 0.0236 0.0330 0.0250 0.0197 0.0260 0.0115 0.28 cd 0.0493 0.0480 0.0387 0.0309 0.0385 0.0331 0.0419 0.0248 0.0277 0.0364 0.37 as 0.2211 0.2117 0.2085 0.1975 0.2179 0.1583 0.1207 0.1364 0.1819 0.1772 1.83 fe 0.0002 0.0002 0.0002 0.0003 0.0003 0.0002 0.0002 0.0002 0.0005 0.0005 0.00 mn 0.0230 0.0250 0.0283 0.0272 0.0154 0.0188 0.0169 0.0146 0.0141 0.0168 0.20 ni 0.0086 0.0074 0.0096 0.0100 0.0077 0.0094 0.0099 0.0083 0.0087 0.0111 0.09 zn 0.0116 0.0145 0.0149 0.0144 0.0110 0.0095 0.0121 0.0131 0.0132 0.0111 0.13 pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 99 heavy metal concentration in studied vegetables the cr content range in various vegetable samples was found as 0.416 and 1.625 mg/kg in turnips and lotus respectively, while the who/fao limit is 1.5 mg/kg. vegetable samples of turnips showed alarming levels of cr, whereas all other vegetable samples declared chromium within allowable levels (table 4 & 5) & (fig. 2). cr is extremely significant for dna transcription as well as insulin activity. although an intake of less than 0.02 mg/day may reduce the cellular responses to insulin [26]. the highest and lowest amount of 5.216 and 0.981 mg/kg of cu was found in vegetables of ginger and sweet potato, respectively. the safe level of cu was found in all vegetable samples than the allowable level of 10 mg/kg (table 4 & 5). the interaction of copper with the atmosphere is complicated; it may be observed from the research that a major part of cu introduced into the atmosphere may readily become stable which cannot pose a health problem. cu is very important for the development of animals and plants and therefore is considered a micronutrient element. it helps in the production of human blood haemoglobin, while in plants cu is extremely significant in water regulation, disease resistance, and seed production. high levels of cu may cause intestinal and stomach irritation, kidney and liver damage and anaemia as well. increased oxidative damage may be caused to dna, lipids and proteins by copper. tubular necrosis in kidney and liver cirrhosis may also be caused due to chronic cu toxicity [27, 28]. table 4. mean content of heavy metals (mg/kg) in different vegetables collected from various locations of district ghotki. ginger (n = 5) beet root (n = 5) potato (n = 5) lotus (n = 5) radish (n = 5) garlic (n = 5) carrot (n = 5) onion (n = 5) turnip (n = 5) sweet potato (n = 5) cr 0.46 0.688 0.434 0.416 0.62 0.639 0.449 0.454 1.625 0.606 cu 5.216 2.046 3.036 2.002 2.01 2.808 2.128 1.678 2.211 0.981 cd 0.524 0.51 0.411 0.329 0.409 0.352 0.445 0.264 0.295 0.387 as 0.141 0.135 0.133 0.126 0.139 0.101 0.077 0.087 0.116 0.113 fe 0.286 0.356 0.303 0.382 0.393 0.285 0.338 0.322 0.700 0.816 mn 0.684 0.744 0.843 0.811 0.457 0.559 0.502 0.436 0.421 0.501 ni 0.364 0.315 0.408 0.426 0.326 0.399 0.423 0.351 0.37 0.473 zn 7.386 9.259 9.485 9.159 7.017 6.028 7.718 8.373 8.397 7.050 table 5. descriptive statistics, maximum contaminant level and reference dose (rfd) of heavy metals for vegetables collected from different locations of district ghotki. hm max: (mg/kg) min: (mg/kg) mean (mg/kg) sd. who/fao limit rfd (mg/kg/day) cr 1.625 0.416 0.639 0.361 1.5 1.5000 cu 5.216 0.981 2.412 1.134 10 0.0400 cd 0.524 0.264 0.393 0.086 0.1 0.0050 as 0.141 0.077 0.117 0.022 0.1 0.0003 fe 0.816 0.285 0.418 0.185 150 0.7000 mn 0.843 0.421 0.596 0.160 2.5 0.0140 ni 0.473 0.315 0.386 0.049 0.10 0.0200 zn 9.485 6.028 7.987 1.137 20 0.3000 pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 100 figure 2. heavy metals (mg/kg) in different vegetables collected from various locations of district ghotki the higher and lower concentration of cd was measured from ginger (0.524 mg/kg) and carrot (0.264 mg/kg) greater than permissible limit of 0.1 mg/kg of the who guideline. the mean cd content (0.393 mg/kg) was also greater than the allowable limit (table 4 & 5). contaminated water and food are the main cd exposure for humans, while cd may be inhaled through cigarette smoke. cd toxicity is because of its buildup in animals and plants for about 25 to 30 years. cd may be removed from food by one of the effective methods like microbial fermentation [29, 30]. phosphate fertilizers and waste incineration procedure is another main source of cd in the atmosphere. there may be a large difference in blood cd levels between nonsmokers and smokers of cigarettes. toxic effects may be found on the gastric system and may lead to lung cancer, breast cancer, gastric cancer, as well as renal cancer. the reference daily dose for cd as established by epa is 0.001 mg/kg/day for food and 0.0005 mg/kg/day for water [31]. the range of as content was determined as 0.077 to 0.141 mg/kg in vegetables of carrot and ginger, respectively. the allowable as level is mentioned as 0.1 mg/kg, although average arsenic concentration of 0.117 mg/kg was greater than the permissible limit. only carrot and onion displayed safe as level. the all remaining vegetables showed as content at an alarming level (table 4 & 5). as is not a metal but a metalloid but because of its carcinogenic and toxic nature, it is therefore presented along with heavy metal toxicity. various researchers have discussed the health effects of arsenic disclosure to humans. foods, groundwater, and atmospheric air are responsible for as exposure for majority of the population [32]. as damages reproductive, renal, endocrine, and cardiovascular systems. but in numerous parts of the globe such as bangladesh, india and pakistan it was observed that groundwater is the main source of as exposure. according to the reports of the researchers, the major source of as exposure in pakistan and bangladesh is groundwater [33, 34]. groundwater contamination of as in bangladesh may be due to the intensive use of agrochemicals for agricultural purposes. chronic exposure of inorganic as may cause “black-foot disease” which is illustrated by a constant failure of circulation in feet and hands, causing eventually gangrene and necrosis. the who/fao allowed fe level given as 150 mg/kg, while all samples collected from the study area showed a safe limit of fe. the lowest and the highest level of 0.285 and 0.816 mg/kg of fe were determined from garlic and sweet potato, respectively. the mean fe content was measured as 0.418 mg/kg from vegetable samples of the study area (table 4 & 5). fe sufficiency may enhance the rate of estrogen–induced kidney tumors in syrian hamsters; it also enhances the carcinogen–induced mammary cancer in mice. fe adequacy also caused a variety of estrogen–induced cancers in humans. for an extensive range of metabolic purposes, fe plays a function as a catalytic center [35]. however, fe deficiency also causes anaemia. numerous signals of fe deficiency are observed like, spoiled cognitive role, less ability of hearing, exhaustion, reduced physical fitness, decreased work efficiency, pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 101 enhanced distractibility, itching, reduced coordination and reactivity, failure to normalize body temperature and ingestion pica [36, 37]. who/fao maximum contaminant point of mn for vegetables is suggested as 2.5 mg/kg, while data shows a safe limit of mn in all vegetables in the study area. the utmost and smallest mn content was determined as 0.843 and 0.421 mg/kg in samples potato and turnip, respectively. the average mn concentration in samples collected from ghotki district was found as 0.596 mg/kg (table 4 & 5). mn is toxic at a high level while it is an essential metal for the nutrition of animals as well as plants. mn is present in various foods of the human diet like oysters, green beans, olive oil, nuts, eggs, soya beans, rice, grains, herbs, tea and spinach. the brain and respiratory tract in human is mostly affected due to mn toxicity, indications include nerve damage, figment of the imagination and absentmindedness. bronchitis, lung embolism, and parkinson’s disease are also caused by mn [38]. deficiency of mn may cause impaired growth and reproduction. its symptoms include neurological symptoms, birth effects, skeleton disorders, skin problems, lowered cholesterol levels, blood clotting, weight gain and glucose intolerance. the ni content range was found as 0.315 and 0.473 mg/kg in vegetables onion and sweet potato, respectively. the average ni content of 0.386 mg/kg is greater than the allowable level of 0.1 mg/kg suggested by who/fao. all vegetable samples displayed alarming levels of ni in the study area of district ghotki (table 4 & 5). no specific function of ni has been observed in humans, but for some microbial intestine enzymes, it acts as a co – factor. higher levels of ni may cause damage to cell and dna structures, which should be less than 0.1 mg/day [39, 40]. the maximum, minimum and mean values of zn in vegetables were determined as 9.485, 6.028 and 7.987 mg/kg. all samples declared zn content below the allowable level of 20 mg/kg as suggested by who/fao (table 4 & 5) & (fig. 2). zn insufficiency in humans’ diet may be more harmful to health because it is an essential element. health effects related to zn deficiency include dermatitis, immune puzzlements, deferred wound healing, growth retardation, damaged neuropsychological functions, oligospermia and neurosensory changes. for men and women the recommended dietary allowance (rda) for zn is 11 and 8 mg/day, respectively, while for women during pregnancy and lactation higher rdas are recommended [41, 42]. the concentration of hms in vegetables reported in the literature in pakistan and worldwide is provided in table 6. table 6. the concentration of hms in vegetables reported in the literature in pakistan and worldwide. ginger beet root potato lotus radish garlic carrot onion turnip sweet potato cr 3.17 [43] 7.61 [44] 5.87 [45] 17.27 [46] 10.11 [45] 7.89 [45] 16.32 [45] 22.18 [45] 2.70 [47] 16.7 [48] cu 65.14 [43] 10.94 [44] 14.35 [45] 32.18 [46] 24.85 [45] 18.76 [45] 28.40 [45] 6.25 [45] 8.10 [47] 3.3 [48] cd 4.60 [43] 0.00 [44] 0.280 [45] 1.52 [46] 0.77 [45] 0.00 [45] 0.96 [45] 0.13 [45] 0.10 [47] 18.8 [48] as ---------0.073 [44] 4.82 [46] 0.073 [44] ----0.148 [44] ---------16.6 [48] fe 78.64 [43] 80.52 [44] 66.78 [45] ----59.81 [45] 65.21[45] 80.51[45] 182.4 [45] 93.53 [47] 20.5 [48] mn 16.22 [44] 15.87 [45] --------13.65 [45] 14.76 [45] 20.15 [45] 11.10 [47] 8.9 [48] ni 7.01 [43] 2.16 [44] 3.90 [45] ---3.41[45] 8.21 [45] 3.37 [45] 0.54 [45] 0.00 [47] 10.3 [48] zn 16.74 [43] 34.18 [44] 26.52 [45] 88.4 [46] 39.48 [45] 24.83 [45] 29.20 [45] 23.94 [43] 50.95 [45] 15.9 [48] correlation coefficient the correlation coefficient was determined with the help of spss software version 18. this shows that only fe – cr declared a strong positive correlation. none of the other pair displayed either a positive or negative strong or weak correlation coefficient among hms of the studied area (table 7). table 7. correlation coefficient among heavy metals determined from study area. correlations cr cu cd as fe mn ni zn cr 1 cu -.090 1 cd .026 .274 1 as .173 .058 -.080 1 fe .799** -.423 -.184 .189 1 mn -.389 .205 -.268 .429 -.283 1 ni -.066 -.216 -.326 -.235 .424 .000 1 zn -.050 -.338 -.505 .167 .087 .567 .015 1 **. correlation is significant at the 0.01 level (2-tailed). conclusion heavy metals were determined from the vegetable samples collected from district ghotki. the average concentration of chromium, cadmium, arsenic and nickel showed alarming levels in collected vegetable samples of district ghotki. the average content of copper, iron, manganese and zinc were found within the allowable limit of fao/who guidelines in root vegetables of the study area. food plants especially root vegetables are the major dietary source being consumed all over the world. they play a significant role in nutritious commitment to the customers. target hazard quotient (thq) associated with the assessed heavy metals exposure via consumption of root samples for adults were below 1 in all samples. however, health indices of all heavy metals were below 1 except arsenic which was above 1. to avoid risks to human health, strict enforcement must be followed for the maximum allowable ingestion of heavy metals. the nutritional value of vegetables is decreased due to heavy metal contamination. it is recommended that the intake of vegetables by animals and humans must be avoided from those sites which are contaminated by arsenic and heavy metals. order of the average concentration of heavy metals was found as, ginger zn > cu > mn > cd > cr > ni > fe > as, beet root as, zn > cu > mn > cr > cd > fe > ni > as, potato as, zn > cu > mn > cr > cd > ni > fe > as, lotus as, zn > cu > mn > cr > ni > fe > cd> as, radish as, zn > cu > cr > mn > cd > fe > ni > as, garlic as, zn > cu > cr > mn > ni > cd > fe > as, carrot as, zn > cu > mn > cr > cd > ni > fe > as, onion as, zn > cu > cr > mn > ni > fe > cd > as, turnip as, zn > cu > cr > fe > mn > ni > cd > as and sweet potato as, zn > cu > fe > cr > mn > ni > cd > as. it is therefore suggested that heavy metals and arsenic must be regularly monitored in common vegetables in the study area. recommendations it is recommended to establish a robust monitoring system to regularly assess the levels of hms in vegetables across sindh, promote safe agricultural practices among farmers to minimize contamination, enforce strict standards and regulations for hms concentrations in vegetables, enhance consumer awareness of risks and encourage informed choices, and support research and collaboration among stakeholders to develop innovative solutions for reducing hms levels in crops. by implementing these pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 103 recommendations, sindh can ensure food safety, protect public health, and minimize the risks associated with heavy metals in vegetables. acknowledgement i am thankful to shah abdul latif university khairpur to provide facilities and completing the article. conflict of interest the authors declare that there is no conflict of interest. references 1. m. varol, k. gündüz and m.r. sünbül, environ. res., 202 (2021) 111806. https://doi.org/10.1016/j.envres.2021.11 1806 2. c. tokatlı and m. varol, environ. res., 201 (2021) 111571. https://doi.org/10.1016/j.envres.2021.11 1571 3. g. qin, z. niu, j. yu, z. li, j. ma and p. xiang, chemosphere., 267 (2021) 129205. https://doi.org/10.1016/j.chemosphere.20 20.129205 4. a. alengebawy, s.t.abdelkhalek, s.r. qureshi and m.q. wang, toxics, 9 (2021) 42. https://doi.org/10.3390/toxics9030042 5. j. k. saha, r. selladurai, m. v. coumar, m. l. dotaniya, s. kundu, a. k. patra, j. k. saha, r. selladurai, m. v. coumar, m. l. dotaniya and s. kundu, soil poll emer thre agri, 10 (2017) 155. https://doi.org/10.1007/978-981-104274-4_7 6. c. chaza, s. rayane, n. sopheak, b. moomen and o. baghdad, int. j. environ. res., 12 (2018) 631. https://doi.org/10.1007/s41742-0180120-0 7. l. joanne slavin, beate lloyd, adv nutr, 3 (2012) 506. https://doi.org/10.3945/an.112.002154 8. c. luo, c. liu, y. wang, x. liu, f. li, g. zhang, and x. li, j. hazard. mat., 186 (2011) 481. https://doi.org/10.1016/j.jhazmat.2010.1 1.024 9. n. munir, m. jahangeer, a. bouyahya, n. el omari, r. ghchime, a. balahbib, s. aboulaghras, z. mahmood, m. akram, s.m. ali shah and i.n. mikolaychik,. sustainability, 14 (2022) 161. https://doi.org/10.3390/su14010161 10. z. xu, m. shi, x. yu and m. liu, int j environ res public health, 19 (2022) 16, 9835. doi: 10.3390/ijerph19169835 11. f. noli and p. tsamos, sci. total environ., 563 (2016) 377. https://doi.org/10.1016/j.scitotenv.2016. 04.098 12. n. shaheen, n. m. irfan, i. n. khan, s. islam, m. s. islam and m. k. ahmed, chemosphere, 152 (2016) 431. https://doi.org/10.1016/j.chemosphere.20 16.02.060 13. v. antoniadis, s. m. shaheen, j. boersch, t. frohne, g. du laing and j. rinklebe, j. environ. manag., 186 (2017) 192. https://doi.org/10.1016/j.jenvman.2016.0 4.036 14. k. ur rehman, s. m. bukhari, s. andleeb, a. mahmood, k. o. erinle, m. m. naeem and q. imran, agri water manag, 226 (2019) 105816. https://doi.org/10.1016/j.agwat.2019.105 816 15. f. a. rutigliano, r. marzaioli, s. de crescenzo and m. trifuoggi, sci. total environ., 691 (2019) 195. https://doi.org/10.1016/j.scitotenv.2019. 07.037 pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 104 16. a. guadie, a. yesigat, s. gatew, a. worku, w. liu, f. o. ajibade and a. wang,. scie tot environ., 761 (2021) 143302. https://doi.org/10.1016/j.scitotenv.2020. 143302 17. n. gupta, k. k. yadav, v. kumar, s. krishnan, s. kumar, z. d. nejad, m. m. khan, and j. alam, environ. toxicol. pharmacol., 82 (2021) 103563. https://doi.org/10.1016/j.etap.2020.1035 63 18. j. wang, l. wang, y. wang, d. c. tsang, x. yang, j. beiyuan, m. yin, t. xiao, y. jiang, w. lin and y. zhou, environ. inter., 146 (2021) 106207. https://doi.org/10.1016/j.envint.2020.106 207 19. s. park, l. zhao, s. h. lee, h. c. hamner, l. v. moore, d. a. galuska and h. m. blanck, nutrients, 15 (2023) 274. https://doi.org/10.3390/nu15020274 20. d. romero-estévez, g. s. yánezjácome, k. simbaña-farinango and h. navarrete, foods, 8 (2019) 330. https://doi.org/10.3390/foods8080330 21. m. harmanescu, l. m. alda, d. m. bordean, i. gogoasa and i. gergen, chem. cent. j., 5 (2011). 1. https://doi.org/10.1186/1752-153x-5-64 22. a. margenat, v. matamoros, s. díez, n. cañameras, j. comas and j. m. bayona, environ. int., 124 (2019) 49. https://doi.org/10.1016/j.envint.2018.12. 013 23. g. mustatea, e. l. ungureanu, s. c. iorga, d. ciotea and m. e. popa, foods, 10 (2021) 3. 581. https://doi.org/10.3390/foods10030581 24. f. a. o. who. joint expert committee on food additives and world health organization, (2011). world health organization. organization. https://apps.who.int/iris/bitstream/handle /10665/44520/?sequence=1 25. m. bamuwamye, p. ogwok and v. tumuhairwe, j. environ. pollut. human health, 3 (2015) 24. doi:10.12691/jephh-3-2-1 26. f. guerra, a. r. trevizam, t. muraoka, n. c. marcante and s. g. canniattibrazaca, scie agri, 69 (2012). 54. https://doi.org/10.1590/s010390162012000100008 27. m. rehman, l. liu, q. wang, m. h. saleem, s. bashir, s. ullah and d. peng,. environ. sci. poll. res., 26 (2019). 18003. https://doi.org/10.1007/s11356-01905073-6 28. w. hermann, annals trans med, 7 (2019) 1. doi: 10.21037/atm.2019.02.07 29. g. genchi, m. s. sinicropi, g. lauria, a. carocci and a. catalano, inter. j. environ. res. pub. health, 17 (2020) 3782. https://doi.org/10.3390/ijerph17113782 30. m. ebrahimi, n. khalili, s. razi, m. keshavarz-fathi, n. khalili and n. rezaei,. j. environ. health sci. eng., 18 (2020) 335. https://doi.org/10.1007/s40201-02000455-2 31. k. fatema, s. s. shoily, t. ahsan, z. haidar, a. f. sumit and a. a. sajib, toxicol rep, 8 (2021) 1109. https://doi.org/10.1016/j.toxrep.2021.05. 015 32. c. w. chang, c. h. ou, c. c. yu, c. w. lo, c. y. tsai, p. y. cheng, y. t. chen, h. c. huang, c. c. wu, c. c. li and h. y. lee, bmc cancer, 21 (2021) 1. https://doi.org/10.1186/s12885-02107799-4 33. h. ur rehman, s. ahmed, m. ur rahman and m.s. mehmood, environ sci pollut res 29, (2022) 33, 49796– 49807 (2022). https://doi.org/10.1007/s11356-02219405-6 pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 105 doi: 10.1016/b978-0-323-484350.00039-3 34. f. karahan, i.i. ozyigit, i.a. saracoglu, i.e. yalcin, a.h. ozyigit and a. ilcim, biol trace elem res, 197 (2020), 316– 329. https://doi.org/10.1007/s12011-01901974-2 35. a. atamaleki, a. yazdanbakhsh, y. fakhri, f. mahdipour, s. khodakarim and a. m. khaneghah, food res. inter., 125 (2019) 108518. https://doi.org/10.1016/j.scitotenv.2020. 143302. 36. c. wong, paedi child heal, 27 (2017) 527. https://doi.org/10.1016/j.paed.2017.08.004 37. k.m. erikson, m. aschner, met ions life scie. 19 (2019) 253-66. https://doi.org/10.1515/9783110527872016 38. r. c. balachandran, s. mukhopadhyay, d. mcbride, j. veevers, f. e. harrison, m. aschner, e. n. haynes and a.b. bowman, j. biol. chem., 295 (2020) 19. 6312. https://doi.org/10.1074/jbc.rev119.009453 39. f. karahan, i. i. ozyigit, i. a. saracoglu, i. e. yalcin, a. h. ozyigit and a. ilcim, biol. trace elem. res., 197 (2020) 3169. https://doi.org/10.1007/s12011-01901974-2 40. k. shams tabrez and a. malik, eds., sprin nat (2021). https://doi.org/10.1007/978-3-03076609-2 41. c. t. chasapis, p. s. a. ntoupa, c. a. spiliopoulou and m. e. stefanidou, arch toxicol, 94 (2020) 1443. https://doi.org/10.1007/s00204-02002702-9 42. n. n. f. sheet, national food policy plan of action and country investment plan, government of the people’s republic of bangladesh, (2011) 1. https://faolex.fao.org/docs/pdf/bgd16302 9.pdf 43. f. ali, m. israr, s. ur rehman, a. azizullah, h. gulab, m. idrees, r. iqbal, a. khattak, m. hussain and f. m. alzuaibr, plos one, 16 (2021) e0255853. https://doi.org/10.1371/journal.pone.025 5853. 44. n. hussain, shafiq, m. ahmed and s.m hussain, saudi j. biol. sci., 29 (2022) 1813. https://doi.org/10.1016/j.sjbs.2021.10.043 45. m. a. fazili, n. ahmad, j. m. war, r. nazir and m. akram, res squa preprint (version 1) (2022) 1-18. https://doi.org/10.21203/rs.3.rs1814291/v1 1-18 46. i. ashraf, f. ahmad, a. sharif, a. r. altaf and h. teng, appl scie, 3 (2021) 1. https://doi.org/10.1007/s42452-02104547-y 47. n. gupta, k. k. yadav, v. kumar, s. prasad, m. cabral-pinto, b. h. jeon, s. kumar, m. h. abdel latif and a. k. d alsukaibia, front. environ. sci., (2022) 40. doi: 10.3389/fenvs.2022.791052 48. r. sultana, r. u. tanvir, k. a. hussain, a. s. chamon and m. n. mondol, environ. syst. res., 11 (2022) 15. https://doi.org/10.1186/s40068-02200261-9 microsoft word 1-12-pjaec-08082014-04.doc issn-1996-918x pak. j. anal. environ. chem. vol. 15, no. 2 (2014) review use of composite sorbents for the removal of copper (ii) ions from aqueous solution rebecca oyedoyin adeeyo, olugbenga solomon bello* department of pure and applied chemistry, ladoke akintola university of technology, p. m. b. 4000. ogbomoso, oyo state, nigeria. received 08 august 2014, revised 19 november 2014, accepted 19 november 2014 ------------------------------------------------------------------------------------------------------------------------------------------- abstract composite adsorbents are synthesized from two or more materials with different chemical and physical properties in order to increase their selectivity and the reusability. researchers have developed and applied several novel composite materials for enhancing the removal of heavy metal. in this review, extensive list of composites developed via advanced technologies with specified characteristics for the removal of cu (ii) ion are discussed. emphases on their adsorption capacities, reusability, desorption and regeneration with improved mechanical strengths are presented. conclusively, prospects and other challenges to be checked and addressed in future are outlined. keywords: composite; adsorption; cu (ii) ions; desorption; aqueous. ------------------------------------------------------------------------------------------------------------------------------------------- introduction heavy metal pollution produced in industrial wastewater is a major issue throughout the world. in particular, cu (ii) ions, whose sources are metal plating, mining, tanneries, painting, car radiator manufacturing, as well as agricultural sources where fertilizers and fungicidal spray are used intensively [1]. the effect on human health and aquatic life are regarded as harmful, and of course are not biodegradable therefore, the removal in an effective manner from water and wastewater is, thus, ecologically very important. several researchers have reported and established technologies for the recovery of metals from wastewater, which include chemical precipitation [2], flotation [3], biosorption [4-6]. among the various methods described, adsorption is the most promising method that has been successfully applied for the purification and recovery of cu (ii) ions from effluents, due to its high efficiency, easy handling and availability [7]. however, due to their low mechanic intensity, limited reusability and relative low adsorption capacity, there is a shift from expensive commercial activated carbon to low cost ecofriendly and readily available adsorbent [8]. the adsorption capacity of various adsorbents for cu (ii) ions removal has been reported, some of them are kaolinite, montmorrillonite, palm ash, zeolite, activated carbon fiber glycidyl methacrylate chelating resin containing fe2o3 particles and alginate-activated carbon [9-12]. these adsorbents were effective, however, inability to separate the adsorbate from the adsorbent effectively after wastewater treatment has paved way for advanced technologies. composites both inorganic and organic materials may be used as binding materials for effective removal of heavy metals [13]. composites can be defined as natural or synthesized materials made from two or more materials with significantly different physical and chemical properties which remain separate and *corresponding author email: osbello06@gmail.com pak. j. anal. environ. chem. vol. 15, no. 2 (2014) 2 distinct at the microscopic or macroscopic scale within the material. moreover, their high reactivity and excellent selectivity towards specific pollutant makes it a promising and attractive alternative adsorbent [14]. composites are synthesized to combine the desired properties of the materials. in nano composite, nano particles (such as clay, metal, carbon nanotubes, e. t. c.) act as fillers in a matrix. polymer–clay composites may offer combined properties; represent an improvement upon thermal, mechanical and porosity properties compared with the homogenous characteristics of the bare individual clay and polymer components [14]. in this review, the uses of different composites adsorbents obtained through various techniques by several researchers are reviewed; shortcomings and future challenges are discussed. ca-alg2/go gel beads composite alginate materials have biocompatibility and relatively low cost compared to polymer materials [15]. alginate is a binary heteropolymer containing varying proportions of 1, 4-linked α –l guluronic acid (g-blocks) and β–d–mannuronic acid (m-blocks) units in a pyranose form, arranged in linear blocks. alginates are porous in structure with high affinity for various metals. papageogiou et. al., investigated the ability of calcium alginate (ca-alg2) to adsorb cu2+ ions from aqueous solutions and discovered the maximum adsorption capacity of cu2+ ions was 88.95 mg/g, due to the high m/g block ratio. also, gotoh et. al., prepared alginate chitosan hybrid gel beads and found the adsorption of cu2+ ions on the beads very rapid, reaching equilibrium within 10 min with a maximum adsorption capacity of 70 mg/g [16]. moreover, li and ba investigated caalg2/cnts composite. it was found to have a high cu2+ ion adsorption capacity of 84.9 mg/g in wastewater treatment applications [17]. algothmi et al., studied ca-alg2/go (prepared using sol gel chemistry technique) as a novel composite for the removal of cu (ii) ion in waste water [18]. graphene oxide (go) is the main structural element of graphite connected together via van der waals forces. go contains hydroxyl, epoxy, diol, ketone and carboxyl functional groups which are randomly assembled at edges and basal planes. therefore, these functional groups make go sheets strongly hydrophilic and compatible with biopolymers [19]. naphthalene was also used as a drying agent as it causes less damage to the beads than other higher temperature dehydration methods and does not cause the dried beads to shrink [20]. the adsorption isotherm data was fitted using langmuir isotherm. the maximum adsorption capacity is 60.2 mg/g at adsorption equilibrium of 45min. the adsorption kinetic data were described by pseudo second order kinetic equation showing that the ca-alg2/go has higher kinetic adsorption rate at 0.0179 g/mg. fig. 1(a) and (b) shows the sem images of ca-alg2 and ca-alg2/go, respectively. in (fig. 1a), there are limited porous structures observed in ca-alg2 dehydrated beads while in (fig 1b) more defined porous structures can be observed in caalg2/go gel beads (fig. 1b), circled have pore sizes ranging from 1 to 5 µm. figure 1. fib/sem images of (a) ca-alg2 and (b) ca-alg2/go gel beads after drying in molten naphthalene. circled image in (b) indicates more defined porous structures in ca-alg2/go gel beads (b), circled have pore sizes ranging from 1 to 5 µm. [18] reproduced with permission from publisher. source. pak. j. anal. environ. chem. vol. 15, no. 2 (2014) 3 diethylenetriamine-grafted poly(glycidyl methacrylate) diethylenetriamine-grafted poly (glycidyl methacrylate) adsorbent is an amine functionalized absorbent derived from diethylenetriamine (deta) grafted onto poly (glycidyl methacrylate) (pgma) micro-granules. the high content amine (amine groups) have been found to be one of the most efficient functional groups [17, 21] hence, positive effects on the effectiveness, capacity, selectivity and reusability of this adsorbent [22]. different authors have reported amine-containing compounds being immobilized onto various substrates including polyacrylonitrile fiber, activated carbon [22], mcm[22, 23], cellulose [24] for the removal of heavy metal. paredes et al., reported that polyglycidyl methacrylate has good mechanical strength and high reactivity of the epoxy groups for surface grafting, it is also used in column chromatography in industries. liu et. al., reported that the grafting was achieved through a reaction between an amine group of deta with an epoxy group of pgma and the adsorbent showed excellent adsorption performance for copper ions at solution ph >3. formation of surface complex with the neutral amine groups on the adsorbent and higher solution ph values resulted in better adsorption performance of cu (ii) ions on the adsorbent in the ph range of 1–5. ionic strength improved maximum adsorption capacity was found to be 1.5 mmol/g at the solution ph value of 5, resulting into faster adsorption kinetics. the adsorption kinetics closely followed the pseudo-second-order kinetic model, indicating the importance of chemical adsorption in the process. adsorption isotherm data were best fitted with the langmuir–freundlich isotherm model. different hno3 solutions were used in desorption which showed that the acidic conditions greatly affected the desorption performance and 0.1 m hno3 solution achieved the best desorption efficiency, with 80 % of the desorption efficiency being completed in the first 1 min. pgma-deta adsorbent can be effectively reused five times for copper ion adsorption without any significant loss in the adsorption capacity [25]. humic acid-immobilized polymer/bentonite composite humic acid-immobilized polymer/ bentonite composite, a novel product prepared by direct intercalation polymerization technique by immobilizing humic acid (ha) onto aminemodified polyacrylamide/bentonite. anirudhan and suchithra prepared amine-modified polyacrylamide/ bentonite. the multifunctional character is due to the presence of various phenolic, carboxyl and hydroxyl groups in addition to skeleton of aliphatic or aromatic units in weakly acidic to basic media. the adsorption of metal ion was enhanced by these carboxylic and phenolic groups through electrostatic interactions and surface complexation. the percentage loading increased to about 95 % with maximum monolayer adsorption capacity as high as 108.08 mmol /g, with a ph of 6.0 at 30oc. the adsorption followed pseudo second order equation and rate constant decreases with increase in concentration of cu (ii) ions. thermodynamic parameters showed that adsorption process is endothermic therefore influence of enthalpy change is prominent than entropy change. large desorption and regeneration efficiency with 0.1 m hcl was recorded. possibility of reuse for four cycles consecutively was reported [26]. magnetic cu (ii) ion imprinted composite adsorbent a novel method was studied to prepare magnetic cu (ii) ion imprinted composite (cu (ii) – mica) to remove cu (ii) in wastewater treatment. this was achieved by synthesizing waste fungal mycelium from industry, chitosan (cs) and fe3o4 nanoparticles combining with metal imprinting technology. chitosan conjugated fe3o4 nanoparticles for the removal of cu (ii) has been reported with a disadvantage of being nonselective [20]. berger et. al., reported fe3o4 magnetic nanoparticles preparation and cu (ii) – mica preparation, based on metal imprinting technique [11]. the character analyses indicated that the cu (ii) – mica had a diameter of about 40 µm, with many tiny interspaces structure on the surface and possess amido and hydroxide groups which could chelate with cu (ii). the spinel structure of fe3o4 did not change during the synthesis process. cu (ii)–mica was nearly super pak. j. anal. environ. chem. vol. 15, no. 2 (2014) 4 paramagnetic while ms and mr values were 91.0 % and 92.4 % lower than that of fe3o4. (fig. 2) shows the preparation of cu (ii) mica, with regeneration carried out by washing the adsorbent with 0.2m naoh for 1.0 h. figure 2. preparation schematic of cu (ii)-mica composite [27]. reproduced with permission from publisher. source: ren et al., 2008. the langmuir adsorption isotherm fit the adsorption equilibrium data most. the maximum adsorption capacity is 71.36 mg /g at 25 ec and ph 5.5. it also showed a metal ion affinity in the competitive conditions for cu (ii)> zn (ii)> ni (ii). magnetic separation results showed that settling time was shortened by 180s. it can be used five times with an adsorption capacity loss of about 14–15 %. it is a costeffective, easy separation adsorbent and has good practical prospects in the wastewater treatment industry. kinetic studies showed that the adsorption process obeyed pseudo-second-order rate mechanism with an initial adsorption rate of 253.2 g/mg. (table 1) reviews kd, k and k values of zn (ii) and ni (ii) with respect to cu (ii). a comparison of the kd values for the cu (ii) imprinted adsorbent with the control samples showed an increase in kd for cu (ii) while kd decrease for zn (ii) and ni (ii). this shows that imprinted beads indicate selectivity for the target molecule (i.e. cu (ii) ions) due to molecular geometry the cu (ii) sorption capacity of the cu(ii)-mica was much higher than that for other metal ions and can be determined in the presence of zn (ii) and ni (ii) interference. table 1. kd, k and k values of zn (ii) and ni (ii) with respect to cu (ii) [27] reproduced with permission from publisher. mnica cu(ii)-mica metal ion kd(ml/g) k kd(ml/g) k ki cu(ii) 124 210 zn(ii) 116 1.07 85 2.47 2.31 ni(ii) 58 2.13 37 5.67 2.6 sewage sludge/industrial sludge-based adsorbent sewage sludge/industrial sludge-based is a complex adsorbent derived from industrial sludge consisting of sewage sludge and waste oil sludge was achieved by pyrolysis technique. the reason for the effectiveness of this composite was as a result of new chemical entities present on the surface such as basic ph, presence of catalytic centers based on iron and other transition metal forming solid state reaction, surface chemistry, porosity and high volume of pores. the condition of pyrolysis was adjusted in an inert atmosphere at 650 or 9500c, either as single components or as 50:50 mixtures [28, 29]. the high removal ability of materials obtained at 6500c is as a result of cation exchange reactions between calcium and magnesium in aluminosilicates, formed on their surface during heat treatment, and copper. on the other hand, robertson and leckie, 1998 indicate that the high degree of mineralization of the surface of the materials obtained at 9500c promotes copper complexation and its surface precipitation as hydroxides or hydroxylcarbonate entities [30]. this is responsible for change in porosity [29]. bentonite–polyacrylamide composite bentonite is effective due to its high swelling ability and cation exchange capacity. bentonite is classified as a smectic soil composed of an expandable 2:1 type of alumino silicate clay mineral and also consists of flat particles with characteristic size of about 102 to 103 nm with negatively charged surface ions and sodium counter ions [31]. bentonite was embedded in the polyacrylamide (paam) gel to form bentonite polycrylamide composite for the removal of cu (ii) ion. bent paam performs effectively in the sorption of cu (ii) ion be due to its strong pak. j. anal. environ. chem. vol. 15, no. 2 (2014) 5 coordination between cu(ii) and the nitrogen on the surface of bent–paam. the removal of cu (ii) ion increased from about 9 % to 97 % at ph ranging from 2.4 to 7, increased with increasing temperature, and decreasing ionic strength. the sorption of cu (ii) of bent–paam was an endothermic and irreversible reaction. this composite showed higher adsorption capacity with increase from 29 to 33 mg/g at ph 6.2 and follows langmuir isotherm model. ozcan et. al, revealed that e value is in the range of 8 – 16 kj/mol [32], indicating that sorption is governed by chemical ion-exchange according to the theory of d–r mode. both enthalpy and entropy change for bent-paam is higher due to large activation energy [33]. amine-functionalized silica magnetite composite amine-functionalized silica magnetite, nh2/sio2/fe3o4, was synthesized by surface modification of fe3o4 with sio2 and n-[3(trimethoxysilyl)propyl]-ethylenediamine (tped) containing amino functional groups with a cationic charge that can adsorb anionic pollutants by means of electrostatic attraction [19] tped behaved as an anionic or cationic adsorbent. the ph value of the aqueous solution was adjusted to make amino groups protonic or neutral. the most efficient functional group for the removal of cu (ii) ion is neutral nitrogen of amine group with a lone pair electron [34, 35, 36]. adsorption kinetic is best described by a pseudosecond order model and the derived activation energy for cu (ii) ions is 26.92 kj and decreasing ionic strength mol-1). in adsorption–desorption cycles, nh2/sio2/fe3o4, were repeated three times using the same amine magnetic adsorbent resulting in a loss of approximately 13.6 %. the optimum conditions to desorb cationic and anionic adsorbates from nh2/sio2/fe3o4 were provided by a solution with 0.1 m hno3 for cu (ii) ions [23]. pectin iron oxide magnetic nanocomposite adsorbent pectin, a component of higher plant cell walls is a structural polysaccharide with partially esterified polygalacturonic acid (pga) [37]. therefore, pectin can be used as bio-sorbent to remove cu (ii) ions from aqueous solutions. pectin coated iron oxide magnetic nano composite (piomn) adsorbent is prepared by iron salt coprecipitation method, followed by the direct encapsulation without cross linking with calcium ions. this method is promising due to complexation and decreasing ionic strength. when polyglacturonic acid (pga) binds with cu (ii) it strengthens cu-pectin interactions [38]. mata et. al., studied adsorption behavior of cu (ii) ions from waste water by sugar-beet pulp pectin [39]. they use sugar beet-pulp pectin xerogels for copper removal in a fixed bed column [40]. sahu et al., also studied pectin coated iron oxide magnetic which was prepared by co-precipitation method, encapsulation and cross linking with calcium iron [41]. a binding method using glutatradehyde and adapic acid has been used to synthesize pectin iron oxide magnetic nano composites [42]. these nano composites have the advantages of pectin and magnetic properties. gong et. al., studied piomn adsorbent. the sorption kinetic data fitted best to pseudo second order model. sorption isotherms were described by both langmuir and freundlich equation with maximum adsorption capacity of 48.99 mg/g. scanning electron microscope studies shows the diameter of piomn has 77 ± 5nm with a spherical shape. this adsorbent increases with increasing ph, confirming ion exchange and electrostatic force mechanism during adsorption process. furthermore, desorption studies were investigated with 0.1 m hno3, 0.01 m edta and 0.1 m naoh respectively. the adsorbent regenerated using 0.01 m edta is the most efficient, releasing 93.70 % of its original capacity after the first regeneration cycle, it reached 58.66 % of the original capacity after the fifth cycle [36]. carboxymethyl cyclodextrin conjugated magnetic nanoparticles cyclodextrin is a cyclic oligosaccharide consisting of seven -d-glucose units connected through (1,4) linkages with a toroidal structure, truncated cones containing a polar cavity with primary hydroxyl groups lying outside and the secondary hydroxyl groups inside [43, 44]. cyclodextrins can form inclusion complexes with a wide variety of organic and inorganic compounds in its hydrophobic cavity [44]. pak. j. anal. environ. chem. vol. 15, no. 2 (2014) 6 the novel nano adsorbent caboxylmethyl cyclodextrin conjugated magnetic nanoparticles (cmcd-mnps) are prepared by grafting carboxymethyl cyclodextrin onto the magnetite surface via carbodiimide method. aqueous solubility and metal complexation potential can be altered by substituting functional groups on the outside of the cyclodextrin [45]. moreover, metal ion complexes with cyclodextrins could have a wide range of applications in catalysis and molecular identification [46]. hu et. al., showed that the grafted cd on the mwcnt/iron oxides showed enhancement of the adsorption capacity because of the strong complexation abilities of the multiple hydroxyl groups in cd with the lead ions [47]. furthermore, anirudhan and suchithra studied cmcd-mnps, the kinetics of cu (ii) adsorption follows the pseudo second order model. the equilibrium data are fitted well by langmuir model, adsorption of cu (ii) ions reached his equilibrium within 30 minutes and 90 % was adsorbed in 15 minutes. the adsorption of cu (ii) onto cmcd-mnps was found to be temperature dependent. the maximum adsorption capacity for cu2+ ions is estimated to be 47.2 mg/g at 25◦c. the analyses of ftir and xps reveal that the oxygen atoms on cmcd-mnps are the main binding sites in the formation of complexes. treatment with citric acid and na2 edta solution for desorption of cu (ii) on cmcd-mnps was effective [26]. β-cyclodextrin (β-cd) is a cyclic oligosaccharide formed from seven glucose molecules by -1-4-glucosidic linkages, giving a micro-environment of a chiral, hydrophilic outside and a hydrophobic interior cavity [48, 49]. more over, renewable, biodegradable characters and large number of active hydroxyl groups selectively makes cyclodextrin a promising adsorbent for cu (ii) ion removal from waste waters [50]. zhao et. al., investigated chemical modification of cyclodextrin through esterification and oxidation reactions and cross-linking of hydroxyl outside the interior cavity to produce the enhanced adsorption functions on heavy metal ions [3]. cyclodextrin adsorbent cda is a co-polymer resin formed from acrylic acid (aa) and acrylamide (am) by inverse suspension and redox titrations to remove cu (ii) ion from waste water. cda hydrogel exhibited typically three-dimensional cross-link network structure. the significant increase in the adsorption capacity when the ph of the solution and the ionic strength increased was 107.37mg/g at 80 mg/l concentration of cu (ii) ion. experimental data fitted best with the freundlich equation model. the kinetic model results indicate that adsorption of cu (ii) ions onto the cdaa fits the quasi-secondorder and elovich equations [5]. high strength hydrogel hydrogel-based adsorbents are easy loading; it captures cations with simple chemicals in most cases, reusable and has the possibility of semi-continuous operation [51]. moreover, they are functionalized with amino, hydroxyl, carboxyl, imidazole and hydrazine groups to demonstrate high capacities in removal of metal ions from aqueous solutions due to their complexing abilities [52]. the thermal, hydrolytic and chemical stabilities of hydrogels make them very promising in the field of water purification [53]. katime et. al., and trakulsujarichok et. al., reported that wettability and high swelling can facilitate adsorption of target metals because the swelling of three dimensional networks likely give specific surface area and more functional group exposed are readily approachable; while limitation in the mobility of functional group occurs by coordination resin which are poorly swollen in water [54, 55]. poly (vinylpyrrolidone/ acrylic acid (pvp/aac) co-polymer hydrogels was prepared to remove cu (ii) ions from aqueous solution with maximum adsorption capacity of 0.36 mmol/g [56]. also, cavus studied non-competitive removal of metal ions by poly (acrylic acid-comethacrylamide) gels and found that the maximum adsorption capacity of the hydrogels was 0.64 mmol cu2+ /g dry hydrogel. lack of being reusable coupled with poor mechanical strength caused by highly swollen network is one of the serious drawbacks hindering the actual applications of hydrogels in metal ion removal. wang et. al., investigated mechanically strong hydrogel prepared by photo initiated polymerization of oligo (ethylene glycol) methacrylate (oegma), 2-vinyl-4,6-diamino1,3,5-triazine (vdt) and cross-linker n,n – methylene bisacrylamide (mbaa). a series of pak. j. anal. environ. chem. vol. 15, no. 2 (2014) 7 p(oegma-co-vdt) hydrogels whose schematic molecular structure is depicted in (fig. 3). mechanical properties of the hydrogel are strengthened by the introduction of monomer vdt through self-bonding of diaminotriazine, therefore enhancing the adsorption of copper ion onto the hydrogel by chelation between amino group and the metal ion. the experimental adsorption data fits langmuir isotherm most, the maximum adsorption capacity was calculated to be 1.1 mmol/g. 1 m hcl aqueous solution will desorbed cu (ii) ion from the hydrogel with the desorption efficiency of 99 %. moreover, desorption capacity is maintained above 90 % with no significant loss in mechanical properties after six adsorption – desorption cycles [57]. figure 3. schematic molecular structure of p(oegma-co-vdt) hydrogel [57]. reproduced with permission from publisher. macroporous bead adsorbents based on poly(vinyl alcohol) / chitosan (pva/cs) poly(vinyl alcohol) (pva) is a watersoluble material containing large amounts of hydroxyl groups. pva has many advantages such as low cost, non-toxicity, biocompatibility, high durability and chemical stability [58, 59]. the macroporous pva-based beads also showed a good removal for heavy metal from aqueous solution [60]. a lot of poly(vinyl alcohol) synthesized with different material has been investigated by different researchers. yanfeng et al., studied pva/cs bead composites adsorbents, which were prepared by the interpenetrating polymer network (ipn) and cross linking process. it was discovered that the amino group and the hydroxyl group present in the pva / cs bead adsorbent exhibits a synergistic effect on removal of heavy metals from wastewaters. the equilibrium data of cu (ii) ion were best fitted the freundlich isotherm best while adsorption kinetics followed the pseudo-second-order kinetic model. in thermodynamics studies, adsorption process was found to be feasible and endothermic in nature. initial metal concentration, contact time, ph, temperature and ionic strength were some of the factors that affect the adsorption process. after five cycles of adsorption–desorption operations, the readsorption capacities attained 76 – 88 % of initial adsorption values [61]. iron oxide-coated zeolite natural zeolite, an alumino silicate mineral, possesses characteristics of large surface area, strong capability of ion exchange and adsorption due to their particular tetrahedral pore framework. also, they are one of low-cost and easily obtainable materials which have been used as adsorbent for removal of heavy metals [62, 63]. in order to enhance the sorption capacity of natural zeolite for heavy metal ions, some metal oxides such as manganese, aluminum oxides having higher affinity towards metal were used [64]. manganese oxide coated zeolite (mocz) used to adsorb cu (ii) ion [4]. thomas model was found to be suitable for describing the adsorption process of the dynamic behavior of the mocz column. the total adsorbed quantities, equilibrium uptake and total percentage of cu (ii) ions removal related to the effluent volumes were determined by evaluating the breakthrough curves obtained at different conditions. the removal of copper and lead ion by mocz columns was in descending order: pb (ii) > cu (ii). the adsorbed copper and lead ions were easily desorbed from mocz with 0.5 mol-1 hno3 solution. han studied iron oxide coated on the surface of zeolite which was a promising adsorbent for the removal of cu (ii) ion. the initial region of breakthrough curve was defined by adams –bohart model while thomas described the whole breakthrough process. adsorption process strongly depends on the flow of rate, the initial cu (ii) concentration and depth. the mass transfer model provides a good agreement with experimental data. the saturated column was regenerated by 1 mol-1 hydrogen pak. j. anal. environ. chem. vol. 15, no. 2 (2014) 8 chloride solution and iocz could be reused in cu (ii) ion removal [4]. chemically functionalized silica gel silica gel has large surface area and is easily modified, which makes it an effective carrier. amps have amide, sulfonic, and carbonyl groups that could bind metal ions thereby making amps a functional polymer. also, others such as amine group have been reported by haung et. al., glerup et. al. and zoubolis et. al., as one of the most efficient functional group in removing metal ions [5, 33, 65]. deng et. al., investigated on poly acrylonitrile as effective function al group for the adsorption of cu (ii) ion [21]. torress also studied on cellulose an effectual material for the removal of cu (ii) ion [24]. moreover, wang et al., investigated the grafting of amps(2 – acrylamido2-methylpropanesulfonic acid) on silica gel to obtain adsorbent which functions with methacryloxypropyl trimethoxy silane reagent. the adsorption capacity increased with increasing ph while the adsorption kinetics showed that the data fitted well, the pseudo second order kinetic model. the adsorption obeyed both freundlich and langmuir isotherms. the thermo gravimetric analysis shows that surface modification reaction introduced some organic functional groups onto the surface of silica. the maximum copper (ii) ion capacity was 19.9 mg/g. hno3 solution was used as the desorbing solution and the adsorption capacities remain stable after three adsorptions and desorption respectively [57]. magnetite polyvinyl acetate coupled with ligands of imiodiacetic acid (m-pvac-ida) recently, adsorption materials with large specific external surface area, easy recycling, and high reusability has been of great interest [66, 67]. mpa is essentially non-porous, thus preventing the clog problem and having good regeneration ability. yoshitake et. al., synthesized magnetic polymer adsorbent (mpa) of micro-size to recover cu (ii) ions from the aged pickling solution in the pcb plant [68]. dauer and dunlop investigated superparamagnetic fe3o4 (magnetite) prepared by the chemical co-precipitation method coated with polyvinyl acetate (pvac) to form mpa of magnetitepvac (m-pvac) via the suspension polymerization with vinyl acetate (vac) [69]. tseng et. al., studied the synthesis of micro-size magnetic polymer adsorbent (mpa) coupled with metal chelating ligands of iminodiacetic acid (ida) for the removal of cu (ii) ion. superparamagnetic fe3o4 called magnetite was prepared via chemical co-precipitation method, and then coated with polyvinyl acetate (pvac) through suspension polymerization with vinyl acetate (vac), yielding magnetite-pvac (denoted as mpvac). introduction of functional groups on the surface of super-paramagnetite particles of mpvac, without demolishing the magnetite within the particles through several sequential procedures such as alcoholysis, epoxide activation and coupling were subsequently employed [70]. also, the micro-size m-pvac coupling with chelating ligands of ida (denoted as mpvac-ida) was prepared with the desired chemical properties. micro-size of about 1µm and specified functional groups of metal chelating ligands of m-pvac-ida can provide large specific area of external surface and absorbability of metal ions of adsorbent, which are essential to adsorption. moreover, after the use, m-pvac-ida was separated from the solution via applied magnetic force [70]. the summary of the maximum capacity of the different composite adsorbents are shown below in (table 2). table 2. adsorption capacities of composite for the removal of cu (ii) ion using different adsorbents. adsorbents maximum capacity references deta-pega ga-mnp cu(ii)-mica bentpaa m nh2/sio2/fe3so4 cmcd-mnps ha-am paa-b sewage /industrial sludge β-cyclodextrin ca-alg2/go gel beads oegma/vdt m-pvac-ida amps-silica pva/cs piomn 1.5mol/g 38.5mg/g 71.36mg/g 33mg/g at ph 6.2 10.41mg/g 47.2mg/g 108.08mg/g 50mg/g 18.93-107.37mg/g 60.2mg/g 1.1mmol/g 0.12mmol/g 19.9mg/g 238.45mg/g 48.99mg/g 25 7 24 60 23 70 29 13 5 18 57 70 14 17 36 pak. j. anal. environ. chem. vol. 15, no. 2 (2014) 9 diboron trioxide / titanium dioxide titanium dioxide has been widely used as photocatalytic material for removing toxic chemicals from waters. however, certain limitations exist in using bare tio2 in photocatalytic reactors. as a result of small size (about 4–30 nm) tio2 aggregates rapidly in a suspension losing its effective surface area as well as catalytic activity [14]. several researchers have made attempt to immobilize fine tio2 on porous adsorbent materials such as silica, activated carbon, alumina clay and zeolites etc. using sol-gel route to produce composites adsorbent [71, 72, 73]. yoneyama and torimoto also dispersed tio2 on the surface area vermiculite to prepare composite adsorbent for the decomposition of textile dye [74]. al-rashidi et al., synthesized tio2 with diborontrioxide at the nano-size used to remove cu (ii) ions from water by adsorption. nanoparticle materials of interest are of sizes below 50 nm. the adsorption kinetics was well explained by pseudo second order kinetic model. the sorption particle was best fitted to freudlich model therefore suggesting that the copper uptake is a chemi-sorption process. an intra particle diffusion based weber–morris model was applied to evaluate the rate-limiting steps which suggested that pore diffusion controlled the overall sorption process [75]. polyamine functionalized copper high content of amine (amine groups) was discovered to be one of the most efficient functional groups [17] therefore, effects on the effectiveness, capacity, selectivity and reusability of this adsorbent has been positive [22, 76]. different authors have reported amine-containing compounds being immobilized onto various substrates including polyacrylonitrile fiber, activated carbon [22]. yu et. al., investigated immobilizing copper chelating agent: triethylenetetramine and tetraethylenepentamine onto macroporous poly (gma-co-tmptma) microspheres. the adsorbent is of good specificity to cu (ii) ion but have weak adsorption towards other metal ions such as fe2+, mg2+, zn2+ and ca2+[77]. prospects and future challenges although the use of these composite reported in the literature have increasingly improved the pace of cu (ii) ion removal in waste water, there are still some gaps to be filled. some of these are suggested below: (1) the potential of composite adsorbents under multi-component pollutants needs to be assessed. this would make a significant impact on the potential commercial application of composite adsorbents to industrial systems. (2) diversity in the use of each sorbent for considerable removal of other contaminants (heavy metals) not only selective detoxification of copper ion, should be investigated to further contribute to environmental protection. (3) further feasibility studies in utilizing these composites on commercial scale need to be checked, avoiding limitation to laboratory scale batch studies. (4) great concern for environmental friendly disposal of pollutant laden adsorbent should not be ignored. (5) further investigation of these adsorbent with real industrial effluents is required to bring about improvement in the field of adsorption. conclusion this review focus on recent developments of wide varieties of composite adsorbents (clay particles and polymers forming complexes with metal ion and amine groups) in the application of wastewater treatment for copper ion removal. it should be noted that the maximum adsorption capacities reported identifies a promising effect of these composites materials in cu (ii) ion detoxification. the developed sorbents are robust enough to withstand multiple handling and can release the metal ion in response to external stimuli (separation) and then re-adsorbed repeatedly with little or no loss of mechanical strength therefore contributing to economic feasibility. pak. j. anal. environ. chem. vol. 15, no. 2 (2014) 10 acknowledgements the corresponding author acknowledges the support obtained from third world academy of science (twas) in form of grant; research grant number: 11-249 rg/che/af/ ac_1_unesco fr: 3240262674. references 1. e-s. el-astoukhy z, n. k. amin and o. abdelwahab, hydrometallur., 89 (2007) 224. 2. o. j. esalah, m. e. weber and j. h. vera, j. chem. eng., 78 (2000) 948. 3. g. x. zhao, h. x. zhang, q. h. fan, x. m. ren, j. x .li and y. x. chen, j. hazard mater., 173 (2010) 661. 4. r. han, l. zoua, k. zhao, x. yanfang, f. xu, y. li and y. wang, chem. eng. j., 149 (2009) 123. 5. z. haung, s. liu, b. zhang, l. xu and x. hu, carbohydrate polym, 88 (2012) 608. 6. y. sag, b. akcael and t. kutsal, sep. sci. technol., 37 (2002) 279. 7. s. s. banerjee and d. chan, j. hazard. mater, 147 (2007) 792. 8. g. y. yan and t. viraraghavan, biores. technol, 78 (2001) 243. 9. a. m. donia, a. a. atia, h. e. boraey, and d. h. mabrouk, sep. purf. technol., 49 (2006) 64. 10. k. c. kang, s. s. kim, j. w. choi, and s. h. kwon, j. indus. eng. chem., 14, (2008) 131. 11. t. y. kim, j. y. jin, s. s. park, s.j. kim, and s. y. cho, j. indus. eng. chem., 14 (2008) 714. 12. m. s. lee, a. g. ahn and j. w. ahn, hydrom., 70 (2003) 23. 13. m. solener, s. tunali, a. s. ozcan, a.ozcan and t. gedikbey, desalina, 223 (2008) 308. 14. qujiuhui, j. environ. sci., 20 (2008)1. 15. k. i. draget, g. s. braek o. smidsrd o., carbohyd polym., 25 (1994) 31. 16. t. gotoh, k. matsushima and k. i. kikuchi, chemosp., 55 (2004) 135. 17. n. s. li and r. b. bai, sep. puf. technol., 45 (2005) 237. 18. w. m. algothmi, n. badaru, y. yu and j. g. shapter, j. coll. int. sci., 397 (2013) 32 19. t. kuilla, s. bhadra, d. yao, n. k. kim, s. bose and j. h. lee, prog. polym. sci., 35 (2010) 1350. 20. c. f. chang, c. y. chang, and t. l. hsu, coll. surf., 327 (2008) 64. 21. s. deng, r. b. bai and j. p. chen, lang., 19 (2003) 5058. 22. w. yantasee, y. lin, y., g. e. fryxell, k. l alford, b. j. busche and j. d. johnson, indus. eng. chem. res., 43 (2004) 2759. 23. y. lin, h. khan, p. chien, l. chiou c. liu, j. hazard. mater., 185 (2011) 11. 24. y. ren m. zhang, d. zhan desalin., 228 (2008) 135. 25. j. d. torres, e. a. faria and a. g. s. prado j. hazard. mater. 129 (2006) 239. 26. c. liu, r. bai and l. hong, j. coll. int. sci., 303 (2006) 99. 27. t. s. anirudhan and p. s. suchithra j. indust. eng. chem., 16 (2010) 130. 28. t. j. bandosz and k. block, appl. catal, 142 (2008) 168. 29. m. seredych and t. bandosz j. coll. interf. sci., 302 (2006) 379. 30. a. p. robertson and j. o. leckie, environ. sci. technol., 32 (1998) 2519. 31. s. t. yang, j. l. li, d. d. shao, j. hu and x. k. wang, j. hazard. mater., 166 (2009) 109. 32. a. ozcan, e. m. oncu and a. s. ozcan, j. coll. interf. sci., 277 (2006) 90. 33. a. i. zouboulis, k. a. matis, b. g. lanara and c. l. neskovic, sep. sci. technol., 32 (1997) 1755. 34. m. chanda, g. l. rempel, react polym., 25 (1995) 25. 35. m. ghoul, m. bacquet and m. morcellet, water resourc., 37 (2003) 729. pak. j. anal. environ. chem. vol. 15, no. 2 (2014) 11 36. gong j. wang x., zeng g., chen l., j. deng j., x, zhang x. and qiu-ya ni., chem. eng. j., 185– 186 (2012) 100. 37. c. vilhena, m. l. goncalves and a. m. mota, electroanalyt., 16 (2004) 2065. 38. a. m. khvan and k. a. abduazimov, chem. nat. compd. 37 (2001) 388. 39. y. n. mata, m. l. blázquez, a. ballester, f. gonzález and j. a. munoz, chem. eng. j., 150 (2009) 289. 40. y. n. mata, m. l. blazquez, a. ballester, f. gonzalez and j. a. munoz, j. env. mgt., 90 (2009) 1737. 41. s. sahu and r. k. duttan, j. hazard. mater, 323 (2011) 980. 42. r. rakhshaee and m. panahandeh, j. hazard. mater., 189 (2011)158. 43. r. v. rekharsky and y. inoue, chemistry rev., 98 (1998) 1875. 44. j. szejtli, chem. rev., 98 (1998) 1743. 45. e. norkus, j. inclu. phen. macrocy. chem., 65 (2009) 237. 46. m. e. skold, g. d. thyne, j. w. drexler, and j. e. mc cray, j. contami. hydrol., 107 (2009) 108. 47. j. hu, d. shao, c. chen, g. sheng, j. li, x. wang and m. nagatsu, m., j. phy. chem., 114 (2010) 6779. 48. d. k. balta, e. bagdatli, n. arsu, n. ocal and y. yagci, j. photochem. photobio. chem., 196 (2008) 33. 49. e. m. del valle, pro. biochem., 39 (2004) 1033. 50. d. m. xie and w. x. sun, j. mater. sci. and eng., 24(2006) 623. 51. h. kasgoz, s. ozgumus, m. orbay, polym., 44 (2003) 1785. 52. v. bekiari, m. sotiropoulou, g. bokias and p. lianos, colloid surf., 312 (2008) 214. 53. h. s. hoffman, rev., 43 (2002) 3. 54. i. katime and e. rodriguez, j. macromol. sci., a 38 (2001) 543. 55. t. trakulsujaritchok, n. noiphom, n. tangtreamjitmun and r. saeeng, j. mat. sci., 46 (2011) 5350. 56. a. e. ali, h. e. shawky, h. a. e. rehim and e. a. hegazy, european polym. j., 39 (2000) 2337. 57. n. wang, han, liu., t. bai, h. gao, p. zhang,w. wang and w. liu, j hazard mater., 213 (2012) 258. 58. w. c. kao, j. y. wu, c. c. chang and j. s. chang j. hazard mater., 169 (2009) 651. 59. k. m. khoo and t. p. ting, biochem. eng. j., 8 (2001) 51. 60. y. zhang, y. li, l. yang, x. ma, l. wang and z. ye, j. hazard. mater., 178 (2010) 1046. 61. l. yanfeng, x. li and y. zhengfang, chem. eng., j 178 (2011) 60. 62. s. kundu and a. k. gupta, j. coll. int. sci., 290 (2005) 52. 63. m. r. panuccio, f. crea a. sorgonà and g. cacco g., j. environ. manage., 88 (2008) 890. 64. y. al-degs and a. m. khraisheh, sep. sci. technol., 35 (2000) 2299. 65. j. glerup, p. a. goodson, d. j. m. hodgson, k. michelsen, k. m. nielsen and h. weihe, inorg. chem., 31 (1992) 4611. 66. y. c. chang and d. h. chen, coll. int. sci., 283 (2005) 446. 67. v. k. gupta and a. rastogi, coll. surf., 64 (2008) 170. 68. h. yoshitake, t. yokoi and t. tatsumi, chem. mater., 14 (2002) 4603. 69. r. r. dauer and e. h. dunlop, biotechnol. bioeng., 37 (1991) 1021. 70. a. z. m. badruddoza, a. s. h. tay, p. y. tan, k. hidajat and m. s. uddin j. hazard .mater., 185 (2011) 117. 71. j. y. tseng, c. y. changa, y. h. chenb, c. f. chang, and p. c. chiang, physico chem. eng., 295 (2007) 209. 72. p. berger, n. b. adelman and k. j. bechman, j. chem., edu., 76 (1999) 943. 73. g. p. lepore, l. persaud and c. h. langford, j. photochem. photobiol. chem., 98 (1996) 103. pak. j. anal. environ. chem. vol. 15, no. 2 (2014) 12 74. y. xu, w. zheng and w. liu, j. photochem. photobiol. chem., 122 (1999) 57. 75. h. yoneyama and t. torimoto, cata. tod., 58 (2000) 133. 76. b. al-rashdi, c. tizaoui and n. hilal, chem. eng. j. 183 (2012) 294. 77. m. prasad and s. saxena, indus. eng. chem. res., 43 (2004) 1512. 78. z. yu, r. wu, m. wu, l. zhao, r. li and h. zou, b: biointerfaces, 78 (2010) 222. microsoft word 08-259-269-pjaec-15032022-433-c-revised galley pro cross mark issn-1996-918x pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 259 – 269 http://doi.org/10.21743/pjaec/2022.12.08 groundwater contamination study of faisalabad and sargodha metropolitan cities abdullah 1 , islam ud din 2 , uzma rashid 1, aijaz panhwar 3 *, tahseen aslam 1 , fozia hussain 1 , razia kulsoom 1 and muhammad afzal 1 1pcsir laboratories, islamabad, pakistan. 2department of environmental sciences, international islamic university islamabad (iiui), pakistan. 3pcsir laboratories complex, off university road, karachi, sindh, pakistan. *corresponding author email: aijazap@yahoo.com received 15 march 2022, revised 12 december 2022, accepted 25 december 2022 -------------------------------------------------------------------------------------------------------------------------------------------abstract the groundwater is one of the most important sources for fulfilling daily needs. groundwater for drinking purposes is the biggest source in pakistan, but due to population explosion, the rapid development of industrialization, deforestation, urbanization and unplanned housing sche mes on agricultural lands are the major reasons for groundwater contamination and deterioration. the work emphasized evaluating the physicochemical characteristics of the groundwater of the two cities of punjab, faisalabad and sargodha for drinking purposes. overall thirty samples were collected in triplicate, fifteen from each city, faisalabad and sargodha, respectively. physicochemical parameters, trace elements and microbiological analysis were conducted. the results revealed that the quantities of magnesium (mg), calcium (ca), sodium (na), chloride (cl-), and total dissolved solids (tds) were significantly higher than the permissible limits of who in the majority of the samples from sargodha, while tds, total hardness (th), were higher in most samples of faisalabad. the studied trace elements aluminum (al), chro mium (cr), arsenic (as), manganese (mn), iron (fe), cobalt (co), nickel (ni), copper (cu), zinc (zn), selenium (se), cadmium (cd), and lead (pb) were found under safe limits of who except cr, cd, se and mn in sargodha city and in faisalabad se and cd were found to be crossing who levels in few locations. the sa mples from faisalabad were found microbiologically unsafe as compared to sargodha. principal component analysis (pca) revealed that the area's most dominant anion was chloride. many processes are involved in changing water chemistry, and the water quality was controlled by rock water interaction and evaporation procedures. the study concluded that the area's water was brackish; due to this, the water was found unsuitable for drinking purposes. therefore, the supply of safe water and water treatment plant installations are highly recommended in these areas. keywords: groundwater, industrial wastewater, physicochemical, pollution, urbanization, water quality. -------------------------------------------------------------------------------------------------------------------------------------------introduction clean water is a basic human need and should be free from chemicals & microbes. pakistan is blessed by nature with adequate surface and groundwater resources, including rivers, lakes and groundwater in the country [1]. the water is a vital part of our every field of life [2]. safe and hygienic water is important for life as well as for the sustainable development of a nation [3]. however, over 844 million humans living on the earth have no availability of pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 260 clean water for drinking [4]. faisalabad city is one of the largest industrial estates and 3 rd with a population of 3,203,846, while sargodha is the 12 th largest city with 659,862 population in pakistan. the groundwater is the major source for drinking purposes in pakistan, but due to rapid explosion in population, development of industrialization, deforestation, and unplanned housing schemes on agricultural lands are the major reasons for groundwater contamination and deterioration. water contamination is key to diarrhea disease, with around 2.5 million causalities yearly, otherside polluted water is responsible for 5 million deaths in developing countries [5,6]. substandard or low-quality drinking water is responsible for 30% and 40% of disease and deaths in pakistan, while contaminated water affecting 1/5 of the population with diseases [7, 8]. in sargodha city, around 38% of 96% groundwater was found unfit, with a major role of turbidity, tds, fluoride, fe, pb and microorganism [9]. in faisalabad, the major sources of drinking water are ground and surface water, 92% and 8%, respectively; while only twenty four percent samples were found fit for drinking purpose. the key contaminants found were turbidity, hardness, tds, chloride, as, fe, and microorganisms [9, 10]. the groundwater level of faisalabad city is gradually decreasing due to the demand of the public and industries [11]. the water table has dropped upto ten meters in some parts of the city [12]. while the groundwater quality of sargodha city is not much suitable for drinking purposes [13, 14]. in pakistan, the maximum number of populations have no reach to clean and healthy drinking water and merely 40-60% of people have a good water supply [15]. water pollution is the main issue for human health all over the globe [16]. natural water bodies receiving pollution load without treatment are critically depleted of dissolve oxygen levels, disturbing the natural balance of aquatic eco-system while being contaminated with a variety of toxic wastes containing heavy metals (such as as, cd, cr etc.) [17]. according to the pakistan council of research in water resources, the human community is increasing very speedily. fresh water is becoming limited because of poor water management, lack of awareness and professionalism. shortage of freshwater due to overpopulation and industrialization leads to water pollution issues globally. groundwater is the single drinking source for most global communities [18]. major contaminants found in water come from natural and anthropogenic sources [19]. in pakistan, the unsustainable use and regular extraction of water for drinking and farming disturb water quality [19, 20]. therefore, the groundwater status of sargodha and faisalabad districts, punjab, pakistan, was evaluated to know the concentration of different physicochemical parameters and the water's suitability for drinking purposes. the main reason behind the shortage and pollution of water, a resource is the wastage of water, unequal distribution, poor drainage system, and contaminated water supply system is responsible for less access to clean water in cities [21]. due to the huge quantity of industrial sectors and almost running without proper treatment plants, faisalabad is considered one of the most polluted groundwater in the country [22]. the people’s perception of rural areas in a tehsil samundri, district faisalabad, was that the water quality of different sources, that is, hand and electric pumps were poor [23, 24]. the physicochemical analysis of different samples collected from urban areas of faisalabad showed that the overall groundwater used for drinking in urban areas of faisalabad was intensively polluted with sewerage water [25]. pakistan has the largest irrigation system, but due to a poor water distribution system and excess use of fertilizers and pesticides, country is facing around 50% losses and is equally responsible for low groundwater quality. due to the increasing population of pak. j. anal. environ. che m. vol. 23, no. 2 (2022)261 faisalabad, contaminated water is the most alarming problem. in 1999, the requirement of faisalabad city was around 64.7 million gallons of drinking water supply to meet the needs; nearly 03 million gallons of this water came from domestic pumps that come out from subsoil water and tube well [26]. the huge quantity of salt is also the biggest issue of pakistan’s groundwater; the use of salt water for irrigation, dissolution of salts, and intrusion of seawater are also key responsible for the deterioration of the groundwater quality [21,27]. the current work is persistent with the assessment of the drinking water quality assessment of faisalabad and sargodha cities in prevailing climate changes. materials and methods reagents and instrumentation a.r grade chemicals were used in the preparation of reagents and standards. the chemicals used were sodium salt of edta, silver nitrate, sulfuric acid, ammonium chloride, sodium hydroxide, sodium chloride, potassium chloride, ph buffer, eriochrome indicator, calcone indicator, potassium chromate, bromocresol green etc. all necessary precautions were taken while sampling, transportation, and storage [28]. trace metals al, cr, as, mn, fe, co, ni, cu, zn, se, cd, and pb were analyzed by inductively coupled plasma mass spectrometer (icp-ms) agilent 7800 icpms, while microbiological tests were conducted by following standard procedures. analysis was carried out for various water quality parameters in triplicate. physicochemical analysis was performed for each sample in triplicate, and the average values were recorded. the temperature, ph, and electrical conductivity (ec) of all collected water samples were measured by a digital ph meter (jenway-3510) and ec meter (hach hq 14d), respectively. chloride, ca and mg (titrimetric method), na and k were measured on flame photometer (afp−100, sedico), and total dissolved solid (tds) and total suspended solids (tss) were measured gravimetrically after recommended filtration. all the analytical estimations were performed within 48 h of sampling [17]. study area the two cities, faisalabad and sargodha of punjab province of pakistan, were selected for this study. sampling total of thirty groundwater samples (fig 1 & 2), fifteen from different locations of each city, were obtained from (sargodha city: new satellite town, ali town, jamia masjid bilal bypass faisalabad road, shell petrol pump bypass faisalabad road, chak # 50, chak # 47, government institute of commerce for women chak # 47, murad colony, farooq colony, university road, kilyari town, jafria colony chowk, usmania colony, old civil line, essa nagar, civil hospital. faisalabad city: nishtabad, manawala, jameel abad, wapda city, manawala, haji abad, allied hospital, samanabad, azamabad, iqbal stadium, bus station, civic centre, madina town, saifabad, sandhu town) bore wells and community tube wells in pre-cleaned 1.5l polyethylene screw capped container in triplicate. groundwater samples were collected from the aquifers of faisalabad city at a depth of 31 to 40 feet; while the aquifers of sargodha city were at a depth of 50 to 60 feet. overall, more than a hundred samples were collected. but thirty were a representative average of 3 to 4 replicates per location. for metal determination, the samples were acidified with nitric acid. the samples for microbiology were collected in sterilized bottles. all samples were preserved and transported in the ice box and immediately analyzed. pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 262 figure 1. sampl e location map of sargodha ci ty. figure 2. sampl e location map of faisal abad ci ty. icp analytical conditions agilent inductively coupled plasma mass spectrometer 7800 icp-ms with hmi for aerosol dilution was used for analysis. analytical conditions are shown in table 1. table 1. icp-ms operati ng parameters. parameter val ue rf power (w) 1600 sampling depth (mm) 10 carrier gas (l/min) 0.68 dilution gas (l/min) 0.27 helium cell gas (ml/min) 5.0 energy discrimination (v) 5.0 isis 3 loop size (µl) 300 sample preparation a water sample (100 ml) was placed in a pre-cleaned beaker, to which high purity nitric acid (1 ml) was added. the beaker was heated for approximately two hours on a hot plate covered with a watch glass at a temperature just below boiling. after leaving it to cool to room temperature, it was transferred to a volumetric flask, made up to 50 ml with ultrapure water, and used for analysis. for elements present in high concentration, dilution test solutions were prepared by diluting 10-fold with 1% nitric acid solution. the calibration curve samples were prepared by diluting and mixing appropriate amounts of mixed standard solution and single element standard solution (1000 mg/l) from merck. microbiological analysis e. coli, thermo tolerant coliform and total coliform bacteria were evaluated by using standard methods. briefly, media was poured into clean petri dishes; 100 ml of the drinking water was separated through a membrane filter using vacuum filtration assembly. membrane filters were placed in clean petri dishes and incubated at 35 °c for 24 h. after 24 h, visible colonies of coliform bacteria turned pink or dark red with a lustrous shine. triplicate samples were used for each sample. pak. j. anal. environ. che m. vol. 23, no. 2 (2022)263 statistical analysis statistical calculations were performed using the computer program minitab (version 13.2) and excel. results and discussion physico-chemical analyses of both cities are presented in table 2. the results were compared with the world health organization (who) recommended levels, showing that the ph was within the who recommended levels in all water samples from faisalabad and sargodha. the analytical findings of water samples of faisalabad suggest that tds and ec of sample nos. 7, 8, 9, 10 and 15 from faisalabad were within the control limits of who standards. whereas, tds in all samples of sargodha city was beyond the set limits of who. the results were found consistent with the previous studies indicating that the chemical analysis of underground water of faisalabad city from areas along narwala and sargodha road were inadequate for drinking. high levels of tds are objectionable to consumers owing to excessive scaling in water pipes, heaters, boilers and household appliances [29]. the transition in tds and ec values in faisalabad suggests the non-uniform quality of the ground-water [30]. according to the tds levels, all the water was brackish (tds greater than 1,000 mg/l), apart from a small number of samples, which were freshwaters (tds less than 1,000 mg/l) [31]. chloride concentrations were also higher than the who levels except for a few samples. table 2. physicochemical anal ysi s of groundwater samples of sargodha and faisal abad city. city parameters s 1 s 2 s 3 s 4 s 5 s 6 s 7 s 8 s 9 s 10 s 11 s 12 s 13 s 14 s 15 sargodha 4931 4931 7806 2296 9643 4066 2068 3058 8813 2072 7289 3619 1984 2270 2709 faisalabad tds 2727 1221 1632 2306 1587 1548 774 589 1009 403 1218 3182 1956 2823 650 sargodha 96 96 48 46 168 106 46 22 160 12 132 30 16 54 58 faisalabad tss 8 6 4 10 2 4 0 0 2 0 4 26 14 28 0 sargodha 1662 1662 1463 842 1186 2194 1042 953 2793 665 2903 1740 1163 1197 886 faisalabad total hardness as caco3 443 787 399 432 299 321 354 465 990 379 654 621 562 432 366 sargodha 653 653 749 576 845 557 691 672 480 653 634 653 634 557 422 faisalabad alkalinity as caco3 440 509 490 758 557 451 298 461 518 307 499 691 595 662 211 sargodha 2729 2729 727 1142 4526 1752 776 1362 4877 874 4162 1606 898 1079 1113 faisalabad chloride 190 46 149 381 105 125 44 56 64 17 71 290 161 225 198 sargodha 301 301 168 177 106 266 186 195 328 133 319 319 160 2216 151 faisalabad calcium 53 75 49 53 58 93 27 67 100 98 133 71 84 71 62 sargodha 218 218 250 96 221 367 138 112 473 80 505 23 184 154 122 faisalabad magnesium 75 144 67 72 37 21 61 72 40 35 77 61 77 61 3 sargodha 60 3520 2570 750 3230 498 28 42 2900 30 3550 44 47 26 35 faisalabad sodium 643 30 20 25 780 40 29 12 25 4 40 31 28 40 5 sargodha 19 19 18 53 53 16 12 11 50 6 27 14 9 31 44 faisalabad potassium 61 41 7 10 28 8 7 5 13 2 7 14 11 15 3 sargodha nil nil nil nil nil nil nil nil nil nil nil nil nil nil nil faisalabad carbonate nil nil nil nil nil nil nil nil nil nil nil nil nil nil nil sargodha 535 535 614 472 693 457 567 551 394 535 520 535 520 457 346 faisalabad bicarbonate 361 417 401 622 457 370 244 378 425 252 409 567 488 543 173 sargodha 12 124 98 78 197 10 6 8 208 4 168 12 16 8 6 faisalabad sulphate 14 6 7 9 10 8 7 8 6 4 7 6 5 7 10 pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 264 the concentrations of all cations were almost above the who levels, except na. in all water samples, the cations order was ca>mg>na>k. the finding of heavy and trace metals have been presented in table 3, observing the presence of heavy metals in sargodha city. the al concentration was found between the ranges of 0.28-0.56. however, the permissible limit of al is 0.2 mg/l, hence all samples were found unfit for drinking water purpose. a huge variation was found in cr, in sample-8, which was 16.580 mg/l, while other results for cr were 0.010.13, the permissible limit for the parameter is 0.05 mg/l, and hence only one sample was beyond the permissible limit. the highest result of mn was found in all samples between 0.02-12.09 mg/l, while the permissible limit is 0.04 mg/l. the results ranges for fe were 0.25-0.4 mg/l. there are no cobalt guidelines, and results were found within the ranges of 0.01-0.6. the results of ni, cu, zn, se, cd, and pb were 0.02-0.21, 0.02-0.1, 0.5-3.56, 0.02-0.29, 0.01-0.7, and 0.01-0.02, respectively. while the results of heavy metals in groundwater samples of faisalabad were within the ranges of 0.32-0.81, 0.01-0.13, 0.02-0.58, 0.01-0.08, 0.27-0.42, 0.01-0.06, 0.01-0.37, 0.02-0.37, 0.24-0.84, 0.02-0.76, for al, cr, as, mg, fe, co, ni, cu, zn, and se, respectively. table 3. trace el ement anal ysis of water sampl es of sargodha and faisal abad ci ty. city parameters s 1 s 2 s 3 s 4 s 5 s 6 s 7 s 8 s 9 s 10 s 11 s 12 s 13 s 14 s 15 sargodha 0.510 0.470 0.500 0.400 0.440 0.480 0.560 0.410 0.300 0.280 0.350 0.340 0.330 0.370 0.390 faisalabad aluminum 0.480 0.420 0.750 0.750 0.800 0.610 0.500 0.530 0.430 0.390 0.320 0.520 0.640 0.580 0.810 sargodha 0.010 0.020 0.010 0.010 0.010 0.030 0.020 16.580 0.090 0.010 0.010 0.010 0.010 0.130 0.050 faisalabad chromium 0.110 0.060 0.060 0.050 0.130 0.040 0.010 0.040 0.020 0.020 0.020 0.020 0.020 0.010 0.010 sargodha 0.020 0.010 0.020 0.020 0.010 0.020 0.020 0.010 0.020 0.030 0.010 0.010 0.010 0.010 0.020 faisalabad arsenic 0.040 0.020 0.080 0.050 0.050 0.020 0.050 0.070 0.020 0.020 0.020 0.580 0.020 0.050 0.050 sargodha 4.330 12.090 5.880 0.410 2.640 0.400 0.110 0.020 0.020 1.740 6.650 1.030 0.070 4.350 0.800 faisalabad manganese 0.080 0.030 0.020 0.020 0.030 0.020 0.020 0.010 0.020 0.010 0.010 0.020 0.020 0.010 0.020 sargodha 0.400 0.370 0.370 0.310 0.330 0.360 0.410 0.290 0.280 0.250 0.320 0.350 0.330 0.320 0.290 faisalabad iron 0.390 0.280 0.330 0.380 0.390 0.420 0.270 0.270 0.330 0.310 0.320 0.310 0.350 0.420 0.330 sargodha 0.020 0.030 0.020 0.010 0.010 0.060 0.010 0.010 0.000 0.020 0.020 0.010 0.330 0.020 0.010 faisalabad cobalt 0.010 0.000 0.010 0.010 0.010 0.010 0.010 0.030 0.010 0.010 0.010 0.060 0.020 0.010 0.010 sargodha 0.170 0.070 0.160 0.170 0.090 0.050 0.020 0.040 0.020 0.200 0.210 0.080 0.080 0.030 0.080 faisalabad nickel 0.030 0.010 0.320 0.080 0.030 0.060 0.010 0.010 0.010 0.100 0.020 0.090 0.080 0.020 0.370 sargodha 0.040 0.120 0.050 0.040 0.030 0.040 0.030 0.030 0.020 0.040 0.040 0.080 4.630 0.030 0.030 faisalabad copper 0.030 0.020 0.100 0.070 0.050 0.040 0.040 0.050 0.030 0.020 0.020 0.240 0.120 0.050 0.030 sargodha 1.960 1.300 6.060 2.510 0.500 1.060 0.930 1.360 1.390 3.560 2.540 6.200 0.010 1.450 0.850 faisalabad zinc 0.580 0.400 0.480 0.360 0.840 0.620 0.560 0.520 0.630 0.410 0.420 0.420 0.430 0.240 0.260 sargodha 0.080 0.030 0.080 0.030 0.020 0.100 0.080 0.100 0.280 0.180 0.080 0.060 0.030 0.010 0.290 faisalabad selenium 0.110 0.060 0.080 0.080 0.110 0.040 0.030 0.420 0.050 0.050 0.050 0.760 0.060 0.020 0.030 sargodha 0.020 0.020 0.010 0.010 0.010 0.010 0.020 0.010 0.010 0.060 0.040 0.010 0.010 0.010 0.070 faisalabad cadmium 0.040 0.010 0.020 0.010 0.010 0.010 0.010 0.020 0.040 0.010 0.010 0.080 0.070 0.010 0.010 sargodha 0.010 0.020 0.010 0.010 0.010 0.010 0.010 0.010 0.000 0.010 0.010 0.010 0.010 0.010 0.010 faisalabad lead 0.010 0.000 0.020 0.010 0.110 0.010 0.010 0.010 0.020 0.010 0.010 0.030 0.030 0.010 0.010 pak. j. anal. environ. che m. vol. 23, no. 2 (2022)265 while all samples from both cities were found microbiologically unfit for drinking purposes. in the same type of study, 90% of samples from the area of samundri faisalabad were higher than the who limits [32] due to higher results of ec and tds in faisalabad. the outcomes of the microbiological analysis are shown in table 4. total plate count was not detected in all the samples of both cities, whereas faecal and total count was observed in all the samples. in a study, the groundwater from sargodha was reported unsafe due to high tds, na and microbiologically [33]. samples 3, 7 and 12 from faisalabad were found to be highly polluted with faecal colonies suggesting that these water reservoirs are highly polluted by sewage water [34]. collectively, a pollution load of approximately 300 and 260 tons of bod/day from faisalabad's residential, commercial and industrial sectors is added to chenab and ravi rivers, respectively [35]. correlation coefficient the correlation matrix was studied to understand the associations among the studied water variables at a 0.05 significance value in table 5 [36]. tds possesses a strong correlation with cl , mg and k, while a negative correlation existed with ph and ec. among the other parameters, a good correlation of clwas observed with k, mg, th and ta. the na shows no important correlation with cl , suggesting that halite dissolution may not be the main process affecting the water quality. mg was found in a strong correlation with th and tss, cland k, representing the identical origin and/ or same hydro-geochemical behavior during different reactions [37]. table 4. microbiological analysis of water sampl es of sargodha and fai sal abad city. city parameters s 1 s 2 s 3 s 4 s 5 s 6 s 7 s 8 s 9 s 10 s 11 s 12 s 13 s 14 s 15 sargodha nil nil nil nil nil nil nil 0 nil nil nil nil nil nil nil faisalabad tpc nil nil nil nil nil nil nil 0 nil nil nil nil nil nil nil sargodha <2 <2 <2 <2 <2 <2 nil <2 <2 <2 <2 <2 <2 <2 <2 faisalabad t. coliforms <2 <2 23 <2 <2 <2 2 <2 <2 <2 <2 7 <2 <2 <2 sargodha <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 faisalabad f. coliforms <2 <2 1.000 <2 <2 <2 2.000 <2 <2 <2 <2 8.000 <2 <2 <2 sargodha -ve -ve -ve -ve -ve -ve <2 -ve -ve -ve -ve -ve -ve -ve -ve faisalabad e.coli -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve table 5. correl ation among vari ous variabl es of groundwater samples (n=30). vari ables ph ec tds cl ta na k ca mg th tss ph 1 ec -0.092 1 tds -0.241 -0.398 1 cl -0.070 -0.349 0.831 1 ta -0.242 -0.407 0.471 0.301 1 na -0.204 -0.315 0.134 0.290 0.079 1 k 0.070 -0.189 0.433 0.569 0.002 -0.176 1 ca -0.155 -0.171 0.138 0.184 0.019 0.002 0.313 1 mg -0.377 -0.228 0.810 0.744 0.189 0.144 0.453 0.243 1 th -0.470 -0.276 0.794 0.737 0.193 0.224 0.440 0.315 0.933 1 tss -0.244 -0.359 0.902 0.903 0.276 0.189 0.562 0.260 0.869 0.848 1 bold figures are different from 0 with a significance value alpha = 0.05 pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 266 principal component analysis (pca) from pca analysis, three factors (f1, f2 and f3) were derived (table 6), which shows three major eigen values. f1 was loaded with tds, cl , mg, th, tss, ca and k, which shows the dominancy of rock and water contact and weathering of minerals [38]. the high loading of cl− was may be from domestic sewage, industrial discharges, and chemical manures [39, 40]. f2 was loaded with na and ta and shows that natural processes have major involvement, mainly affecting groundwater chemistry. in f3, high loading of ec, mg and th was recorded. three-factor variabilities with different factor loadings recommend the non-uniform hydrogeochemistry of groundwater in the area. mg occurrence may be due to water-rocks interaction because of its moderate loadings. table 6. correl ation between vari ables and factor composi tion i n samples of water. f1 f2 f3 ph -0.337 -0.379 -0.757 ec -0.443 -0.349 0.628 tds 0.919 0.057 -0.089 cl 0.894 -0.069 -0.185 ta 0.384 0.528 -0.270 na 0.249 0.646 -0.001 k 0.570 -0.620 -0.207 ca 0.327 -0.274 0.126 mg 0.905 -0.091 0.235 th 0.914 -0.021 0.279 tss 0.959 -0.095 -0.017 eigen value 5.168 1.448 1.274 variability (%) 46.982 13.164 11.586 cumulative % 46.982 60.147 71.733 gibbs diagram gibbs's diagram [41] illustrated that water chemistry in the investigated area was mainly controlled by evaporation and rock water interaction processes (fig. 2). figure 3. gibbs diagram representing water chemistry in the area conclusions the rapid increase in population, unplanned growth of industrialization, and unplanned housing schemes on agricultural lands and deforestation are the major reasons for groundwater quality worsening. suitable cleaning steps should be followed before using groundwater for drinking purposes. the physicochemical results showed that the majority of the parameters were above the who limit except ph. thus, the study concluded that the water was mostly of pak. j. anal. environ. che m. vol. 23, no. 2 (2022)267 brackish nature, the heavy metals were almost found within the permissible limit of w ho, while chemically and microbiologically, the samples were found unfit for drinking purposes. from the principal component analysis results, it was also concluded that natural processes were mainly responsible for the non-uniform chemistry of the water, which was mainly controlled by evaporation and rock-water interaction mechanisms. therefore, the supply of safe and hygienic drinking water and the establishment of water treatment plants are highly recommended to solve the water shortages by using improved and new technologies in irrigation systems. it is concluded that parallel sewage lines and drinking water pipelines are the leading cause of contamination, while the chemical substance is another major source. discharging chemical substances and effluent into the surface drain, groundwater should be considered a threat to the environment and health. using cost-effective treatment methods can remove the contaminations, but identification of contamination is essential. while overall, drinking water quality improvement has been observed in punjab province from 35 to 49% in 2015 and 2020, respectively. as per data, the drinking water in punjab province has improved, but to achieve sustainable development goals target by 2030, there is a need for more attention and implementation of a safe drinking water policy in pakistan for achieving the target of sustainable development goals. conflict of interest the authors declare no conflict of interest. reference 1. z. a. soomro, m. i. a. khokhar, w. hussain and m. hussain, bio. med. res. int., (2017) 1. https://doi.org/10.1155/2017/7908183. 2. s. l. postel, g. c. daily and p. r. ehrlich, science, 271(1996) 785. doi: 10.1126/science.271.5250.785. 3. r. saha, n. c. dey, s. rahman, l. galagedara and p. bhattacharya, groundw. sust. dev., 7 (2018) 91. https://doi.org/10.1016/j.gsd.2018.03.002. 4. who, progress on drinking water, sanitation and hygiene: (2017) update and sdg baselines. switzerland: who. 5. m. kosek, c. bern and r. l. guerrant, bull. who, 81:3 (2003) 197. doi:10.1590/s004296862003000300010. 6. g. holgate, ewm, 3 (2000) 105. doi: 10.1155/2017/7908183. 7. r. p. country report, pakistan, global water partnership, draft south asia water vision,” vol. 2025, (2000). 8. m. a. kahlown, m. a. tahir, h. rasheed and k. p. bhatti. water quality status, national water quality monitoring programme, fourth technical report pcrwr 5, (2006). https://doi.org/10.1155/2017/7908183. 9. dr. m. aslam tahir, m. akram kahlown, engr. faizan ul hassan, muhammad farooq. report on technical assessment of water supply schemes, northern & central punjab (volume ii). pcrwr (2021) 92 & 326. 10. r. hifza, a. fauzia, a. kiran and m. ashraf. drinking water quality in pakistan: current status and challenges. pcrwr (2021) 21 & 27. 11. nasir a, muhammad s. n, imran s, shafiq a, iqra a. adv. environ. biol., 10 (2016) 155. 12. s. asma, c. arslan, a .nasir and a. khan, pak. j. agric. sci., 49 (2012) 541. doi:10.26480/pjg.02.2018.11.17 13. f. rahman, t. cheema, t. azeem, a. a. naseem, f. rehman, o. riaz, t. abbas, m. f. ullah, s. rehman, fresenius environ. bull., 28:11 (2019) 7695. pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 268 14. o. riaz, t. abbas, m. nasar-uminallah, s. rehman and f. ullah, sci. int., 28:5 (2016) 4715. 15. a. hannan, s. shan and u. arshad, biomedical, 26 (2010) 152. 16. m. m. rahman, m. f. howladar, m. a. hossain, a.s.h. muzemder and m.a. al numanbakth, groundw. sust. dev., 10 (2020) 100310. 17. asian development bank. prefeasibility study, integrated industrial waste management services in faisalabad, cities development initiative for asia, asian dev, (2010) 49. doi.org/10.22617/tcs220086-2. 18. a. panhwar, k. faryal, a. kandhro, s. bhutto, u. rashid, n. jalbani, r. sultana, a. solangi, m. ahmed, s. qaisar, z. solangi, m. gorar and e. sargani, environ. challenges, 6 (2022) 100447. doi.org/10.1016/j.envc.2022.100447 19. c. reimann, d. banks, sci. total environ., 332:1-3 (2004) 13. doi.org/10.1016/j.envpol.2020.116227. 20. a. rashid, d.-x. guan, a. farooqi, s. khan, s. zahir, s. jehan, s.a. khattak, m.s. khan and r. khan, sci. total environ., 635 (2018) 203. doi: 10.1016/j.scitotenv.2018.04.064 21. a. azizullah, m.n.k. khattak, p. richter and d.-p. haider, environ. int., 37:2 (2011) 479. doi: 10.1016/j.envint.2010.10.007 22. m. yamin, a. nasir, m. sultan, w. i. w. ismail, r. shamshiri, f. n. akbar, adv. environ. biol., 9 (2015) 53. 23. h. ali and m. s. akhtar, int. j. sci. res., 4 (2015) 523. 24. h. zulfiqar, q. abbas, a. raza and a. ali, j. global innov. agric. social sci., 4:1 (2016) 40. 25. s. farid, m. k. baloch, and s. a. ahmad, int. j. water res. environ. eng., 4 (2012) 55. doi: 10.5897/ijwree11.086 26. m. k. daud, m. nafees, s. ali, m. rizwan, r. a. bajwa, m. b. shakoor, m. u. arshad, s. a. s. chatha, f. deeba, w. murad, i. malook, s. j. zhu, bio. med. res. int., 3 (2017) 790. doi.org/10.1155/2017/7908183 27. r. bashir, h. nawaz and m. khurshid, pak. j. biol. sci., 2 (1999) 715. doi: 10.3923/pjbs.1999.715.719. 28. s. k. agarwal, pollution management, water pollution (a.p.h. publishing corporation, new delhi, india) (2002). doi: 10.4236/lce.2016.74014 29. nasir, m.s., nasir, a., rashid, h. appl. water sci., 7 (2017) 3197. https://doi.org/10.1007/s13201-0160467-3. 30. american public health association (apha). standard methods for the examination of water and wastewater, 23rd edition (2017). doi: 10.4236/psych.2013.411117 31. r. nagarajan, n. r. mohan, u. mahendran and s. s. kumar, environ. monit. assess., 171:1-4 (2010) 289. doi: 10.1007/s10661-009-1279-9 32. m. a. zia, k.u. rehman, f. anjum and r. latif, j. res. (sciences), 16 (2005) 11. doi: 10.1155/2017/7908183 33. m. mobeen, a. moin and m. naseer, j. biol. environ. sci., 11:5 (2017) 138. 34. s. farid, m. k. baloch and s. a. ahmad, int. j. water resour. environ. eng., 4 (2012) 55. doi: 10.5897/ijwree11.086 35. water and sanitation agency. water supply, sewerage and drainage master plan of faisalabad, interim report, (2017) 49. doi.org/10.3390/w12010166 36. r., freeze, j. cherry, groundwater (prentice-hall, englewood cliffs, nj), (1979) 604. pak. j. anal. environ. che m. vol. 23, no. 2 (2022)269 37. j. wu, p. li, h. qian, z. duan and x. zhang, arab. j. geosci., 7:10 (2014) 3973. doi 10.1007/s12517-013-1057-4 38. a. k. tiwari, a. k. singh, a. k. singh and m. singh, appl. water sci., 7:4 (2017) 1609. doi: 10.12691/aees-8-5-4 39. d. purushotham, m. prakash and a. n. rao, environ. earth sci., 62:8 (2011) 1707. 40. m. bodrud-doza, m. a. h. bhuiyan, s. d.-u. islam, m. s. rahman, m. m. haque, k. j. fatema, n. ahmed, m. rakib and m. a. rahman, groundw. sustain. dev., 8 (2019) 226. doi: 10.1016/j.gsd.2018.11.008 41. r. gibbs, j. sci., 170:3962 (1970) 1088. doi: 10.1126/science.170.3962.1088 microsoft word 02-194-204-pjaec-26042022-442-c-ree revised galley cross mark issn-1996-918x pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 194 – 204 http://doi.org/10.21743/pjaec/2022.12.02 determination of the antiprotozoal and antibacterial drug metronidazole in blood and dosage forms using a simple spectrophotometric method ann h. mahmood and hana sh. mahmood * department of chemistry, college of science, university of mosul, mosul, iraq. *corresponding author email: hanashukermahmood@uomosul.edu.iq received 26 april 2022, revised 01 september 2022, accepted 14 october 2022 -------------------------------------------------------------------------------------------------------------------------------------------abstract a new, precise, and sensitive method for the determination of metronidazole has been created. metronidazole has been determined in both blood and dosage forms. the reaction is based on the reduction of the nitro group of metronidazole followed by coupling with the diazotized paminodiphenylamine to produce a soluble, colored complex measured at 515 nm. beer-lambert law is obeyed over the concentration range from 20 to 100 µg/ml, the molar absorptivity is 1397.88 l/ mol.cm.the method has been used for the determination of metronidazole in the blood of the volunteer after 4, 6, 8, and 12 h from the administration of 500 mg tablet showing that the higher level of the drug was found after 6 h from swallowing the drug orally with excellent precision range (rsd% 0.0122-0.0387). the method has been also applied for the estimation of drug content in injection and tablet dosage forms with accuracy ranging from -4.1to +4.0. keyword: metronidazole, p-aminodiphenylamine, blood, dosage for ms -------------------------------------------------------------------------------------------------------------------------------------------introduction metronidazole (mndz) it is an efficient antiprotozoal and antibacterial agent used to treat trichomoniasis, amoebiasis, and giardiasis [1]. it decreases the cytokines levels produced during the treatment of covid-19 infection [2,3]. mndz is usually modified in the liver by oxidation of the side-chain to form hydrophilic products, the main two oxidative metabolites of metronidazole are hydroxy and acetic acid derivatives [4,5]. mndz binds to the deoxyribonucleic acid of the anaerobic bacteria and protozoa and blocks its replication [6,7]. chemically mndz (fig. 1) is 2-(2-methyl-5-nitro-1h-imidazol-1yl) ethan-1-ol)with chemical formula c6h9n3 o3 and molecular weight 171.16 g/mole) [8]. mndz is known as flagyl; metizol; metro; metrogelv and other brand names [9]. the ultraviolet spectrum of mndz in aqueous acid exhibits a peak at 277 nm and in aqueous alkali exhibits a peak at 319 nm [9,10]. figure 1. metroni dazol e (mndz) mndz reacts with a silver(i)to form the coordination complex [ag (mndz)2 no3] and [(ag (mndz)2)2]so4, which exhibits significant antibacterial activity [11]. pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 195 mndz has been estimated in pharmaceutical dosage forms by hyphenated techniques lc-ms/ms chromatographic methods [12-14], electrochemical methods [15-18], and chromatographic techniques [19, 20]. mndz has been estimated in pharmaceutical formulations by two flow injection methods using metol (n-methyl-paminophenol sulfate) as electron acceptor and the reduced form of metronidazole as an electron donor by two lines manifold procedure in the presence of naio4 in the first method and by reverse flow injection manifold in the second [21]. mndz has been determined by spectrophotometric methods which are based on the reduction of mndz with zinc/hcl, followed by the formation of schiff base with pdimethylaminobenzaldehyde [22] and with vanillin [23]. other spectrophotometric procedures are based on the reduction of the nitro group in mndz followed by oxidation with alkaline potassium permanganate [24], also reduction of the nitro group of mndz, followed by diazotization of it, and coupling with β-naphthol [25], n-(1-naphthyl) ethylenediamine [26], para-hydroxyl benzaldehyde [27], and with α-naphthol in another literature [28]. mndz reacts with chloranilic acid according to the chargetransfer principle, the reaction is taken place in acetone-cetylpyridinium chloride and the formed mndz complex was measured at 513 nm. [29], reduced mndz reacts with p-benzoquinone to form a purple color complex in a methanolic medium measured at 526 nm [30]. spectral changes of metronidazole upon changing the ph of the medium have been followed at 326 nm. the calibration curve has been expressed by the difference in absorbances (∆a) against concentration [31]. some techniques for determination of mndz are complected and require elaborate, expensive, and may not be available. mndz in all spectrophotometric methods acted as a diazotized compound, in this article the reduced mndz acts as a coupling agent, and the proposed method exhibits good applicability for the determination of mndz in blood as well as in dosage forms. material and methods instruments absorption spectra were measured on a double beam jasco v630spectrophotometer with 1.0 cm matched glass cells. chemicals all chemicals used were of analytical grade: prepared solution metronidazole (100 μg/ml), sodium nitrite (nano2) (1%), sulphamic acid (3%), hydrochloric acid (1 m), sodium hydroxide (4 m), p-amino diphenylamine (1x10-3 m): 0.0184 g of pure reagent has been dissolved in 10 ml of ethanol followed by dilution to100 ml. metronidazole tablet/200 mg (ajanta pharma limited indian): the content of five tablets has been mixed, pulverized, and weighed. the mean weight of one tablet was 0.3286 g 0.08215 g (equivalent to 0.05 g active component metronidazole) was reduced according to the reduction procedure, then diluted to prepare 100 µg/ml. metronidazole tablet/500 mg (microlab limited): the content of five tablets has been mixed, pulverized, and weighed. the mean weight of one tablet was 0.65402 g. so, 0.065402 g of tablet powder is equivalent to 0.05 g active component metronidazole) was reduced according to the reduction procedure, then diluted to prepare 100 µg/ml. pak. j. anal. environ. che m. vol. 23, no. 2 (2022)196 metronidazole intravenous injection 500 mg/100 ml (pioneer company iraqsulaymaniyah): 10 ml of the intravenous injection liquid was reduced according to the reduction procedure, then diluted to prepare 100 µg/ml of the drug sample. extraction of mndz from human blood 2 ml of human blood samples (of healthy voluntaries) have been collected after 4, 6, 8, and 12 h after oral administration of a single dose 500 mg tablet mndz tablet/500 mg (microlab limited)in heparinized tubes during non-alternative days, 0.5 ml of sodium citrate kit has been added, separation was carried out by centrifugation 5000 periods per second at room temperature, the decantated supernatant was reduced by 0.4 g zinc powder in acidic medium (5 ml of concentrated hcl), mixed with 10 ml of hot distilled water and boiled for 5 min, then cooled in ice, filtrate and finally diluted to make 25 ml with distilled water [33,34]. reduction step using zinc in acidic medium this step involves the addition of 0.4 g zinc powder to 0.05 g mndz pure powder (provided by the state company of drugs industry and medical appliances), followed by the addition of 5 ml of concentrated hcl, then 10 ml of hot distilled water, after cooling period, the solution was diluted to make 100 ml final volume and 500 μg/ml final concentration. the final solution was diluted to prepare a 100 μg/ml mndz working solution. the reaction was expressed by the chemical equation as in scheme 1 [26]. scheme 1. reduction reaction of mndz starting conditions of the reaction 1 ml of sodium nitrite and 0.5 ml of hcl were added to 2 ml of the reagent paminodiphenyl amine, followed by the addition of 1.5 ml sulphamic acid after two min, another two min then 1 ml of the reduced metronidazole is added, finally 2 ml sodium hydroxide has been added and the solution is diluted to make 10 ml in a volumetric flask. the formed color was orangish-red measured at 515 nm to give a 0.1679 absorbance value against a blank solution. the effluence of many types and amounts of chemicals on the reaction efficiency indicated by absorbance values have been studied and explained below: results and discussion optimization of the reaction conditions the influence of sodium nitrite the reagent p-aminodiphenyl amine is a primary aromatic amine that can be easily diazotized by the addition of sodium nitrite in an acidic medium to form nitrous oxide. nitrous oxide reacts with p-aminodiphenyl amine to form diazonium salt pulling a water molecule according to the equations in scheme 2: nano2 +hcl hno2+nacl p-amino diphenyl amine diazonium salt of p-amino diphenyl amine scheme 2. formation reaction of di azoni um salt pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 197 practically 0.2, 0.4, 0.8, 1, 1.5, and 2 ml of sodium nitrite (1%) have been added in an acidic solution to the organic reagent (1x10 -3 m). fig. 2 shows a maximum at 1.5 ml of sodium nitrite. figure 2. the i nfl uence of the quantity of sodium nitrite the influence of acids the presence of many acids (h3po4, hno3, h2 so4, hcl, ch3cooh) with many quantities on the sensitivity of the colored product exhibits a maximum at 0.5 ml of 1 m hcl, while both nitric acid and phosphoric acids exhibit negative effect fig. 3 summarize the results. figure 3. the i nfl uence of the aci ds adjustment the amount of sulphamic acid an excess of nitrous oxide may cause an undesirable further reaction, so, sulphamic acid is used to adjust the amount of nitrous oxide according to the below reaction equation: fig. 4 shows that 1.5 ml of 3% sulphamic acid is a convenient amount to remove the excess amount of nitrous oxide. figure 4. the i nfl uence of the quantity of sul phamic aci d select the type and adjustment the amount of base the diazonium formation requires an acidic medium while the coupling step requires a basic medium to enhance and increase chromophore area. 0.5 ml of 4 m of bases naoh, koh, and na2co3 as basic salts are used for the last requirements. fig. 5a showed that naoh is the best choice. fig. 5b showed that exactly one milliliter is preferred. figure 5. a: sel ection of base b: selection of naoh amount a b so rb a n ce a b so rb a n ce a b so rb a n ce a pak. j. anal. environ. che m. vol. 23, no. 2 (2022)198 adjustment the amount of organic reagent agent 1.5, 2, and 2.5 ml of p-aminodiphenyl amine agent (1x10-3 m) against 10, 20, 30, 40, 50, 75, 100, and 125 µg of mndz in final volume 10 ml under the above-selected conditions have been followed. table 1 indicates that 1.5 ml of the reagent produces a higher correlation and it is selected. table 1. infl uence of p-ami nodi phenyl ami ne on the absorbance of increasi ng concentrations of mndz. absorbance/µg of mndz vol ume of pami nodi phenyl ami ne (ml) 10 30 50 75 100 125 r 2 1.5 0.011 0.0503 0.325 0.3801 0.6110 0.953 0.974 2 0.022 0.0460 0.3218 0.3620 0.661 1.0532 0.952 2.5 0.039 0.073 0.1965 0.335 0.629 1.066 0.928 effect of standing time table 2 explains the effect of standing time after two steps: table 2. effect of standi ng ti me ti me (mi n.) i mm edi at el y 1 2 3 5 7 after reaction of reagent with nitrous oxide 0.24 47 0.25 47 0.32 80 0.32 10 0.32 00 0.310 0 after release the excess of nitrous oxide -----0.32 1 0.33 41 0.34 00 0.33 5 0.330 1after the diazonium step (addition of sodium nitrite), in which the measurements have been taken after 0, 1, 2, 3, 5, and 7 min of the addition of sodium nitrite. from table, 2 min is sufficient. 2after the release the excess of nitrous oxide step (addition of sulphamic acid), in which the same above periods have been followed as shown in table 2, 3 min is sufficient. effect of surfactant and sequence of addition as table 3 show, the cationic surfactant cetylpyridiniumchloride (cpc) relatively enhance the value of absorbance when 2 ml of the surfactant is added, while fig. 5a exhibits that 2 ml of cpc produces the maximum enhancements following sequence 4 as explained in fig. 5a. table 3. infl uence of surfactant. surfactant sol ution (1 × 10-3 m) absorbance/ order of addi tion* sodium dodecyl sulphate (sds) 0.328 cetyltrimethylammonium bromide (ctab) 0.319 cetylpyridinium chloride (cpc) 0.1% 0.345 * p-aminodiphenyl amine (padpa) + surfactant (s) + hydrochloric acid (h)+sodium nitrite (n) + sulphamic acid (f) + metrazol (d) + sodium hydroxide the effect of different volumes of cpc 0.1% has been studied, two ml exhibits the best results as shown in fig. 6a and six sequences of additions have been checked, as shown in fig. 6b, sequence 4 is the best one and it is followed by preand postexperiments. sequence 1: p-aminodiphenyl amine (padpa) + hydrochloric acid (h)+ surfactant (s) +sodium nitrite (n) + sulphamic acid (f) + metrazol (d) + sodium hydroxide sequence 2: padpa + h + n+ f + s + d + b sequence 3: padpa + h + n + s + f + d + b sequence 4: padpa + h + n + f + d + b + s sequence 5: padpa + h + n + f + d + s + b sequence 6: padpa + n + h + f + d + b + s pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 199 0.3794 0.3583 o.485 0.3952 0 0.1 0.2 0.3 0.4 0.5 0.6 0 1 2 3 0.3655 0.485 0.369 0.3107 0.3525 0.33 0 0.1 0.2 0.3 0.4 0.5 0.6 se qu nc e 1 se qu nc e 2 se qu nc e 3 se qu nc e 4 se qu nc e 5 se qu nc e 6 b figure 6. a: effect of cpc b: effect of the sequence* of addi tion i n the presence of cpc absorption spectrum and calibration curve under the developed reaction procedure, the absorption spectrum has been taken, and the amount and sequence of additions are as follows: 1.5 ml of the reagent, 0.5 ml of 1 m hcl, 1.5 ml of sodium nitrite, standing for 2 min, 1.5 ml of sulphamic acid, and another 3 min to release the excess of nitrous oxide, 5 ml of mndz (100 μg/ml), 1 ml of 4 m of bases naoh, finally 0.2 ml of 1% m cpc, dilution to complete 10 ml in a volumetric flask. a blank solution has been prepared in the same way but in the absence of mndz. from fig. 7, the maximum absorbance of the formed complex at 515 nm is about 0.4855. between 10-120 μg/ml of mndz solution has been measured following the same above conditions for estimation of a calibration curve, fig. 8 shows the linearity is between 20 to 100 μg/ml of mndz. figure 7. the absorption spectrum of 50 ppm of a: sampl e agai nst di stilled water b: sample agai nst bl ank and c. bl ank agai nst di stilled water figure 8. the cali bration curve of mndz accuracy and precision of the calibration curve the accuracy and precision of the calibration curve have been estimated by making measurements of three different concentrations with many replications as mentioned in table 4. a b so rb a nc e a b so rb a n ce a a b so rb an ce a b so rb a n ce a b so rb a n ce wavelength (nm.) 0.1521 0.2201 0.3897 0.4855 0.5831 0.6381 0.8621 0.9389 y = 0 .0 0 9 9 x 0 .0 3 7 5 r² = 0 .9 9 1 70 0 .2 0 .4 0 .6 0 .8 1 1 .2 0 5 0 1 00 1 50 ppm o f m n d z pak. j. anal. environ. che m. vol. 23, no. 2 (2022)200 b table 4. accuracy and precision of the cali bration curve. the mean of rel ati ve standard devi ation %* rel ati ve standard devi ation %* the mean of recovery %* recovery % mndz taken (ppm) 0.462098.530 0.511810050 1.0559 2.194 99.46 99.990 * average of five determination the calculated molar absorptivity is 1397.88 l/mole.cm, lod is 0.358 μg/ml, loq is 1.956 μg/ml, while the mean of recovery is 99.46 %, and of relative standard deviation is μg/ml. nature of the formed complex a brief study on the nature of the complex using the job’s and mole ratio method exhibits a one-to-one reaction ratio between the diazotized p-aminodiphenylamine and the coupling agent (the reduced mndz). (fig. 9 a asnd b). figure 9. a: job’ s method and b: mole ratio method to confirm the nature of the complex as the obtained ratio was 1:1, the expected structure may be occurred either according to a suggestion or b suggested structure as in scheme 3 below: scheme 3. the suggested structure of the colored azo product a pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 201 the stability constant (ks) of the colored azo product table 5 exhibits good stability of the complex, the average conditional stability constant is 1.14 x10 5 l/mole. table 5. stability of the azo colored complex. ml of (1x10-3) mndz as* am** α*** ks (1/ mol) mean of ks (1/ mol) 0.5 0.0219 0.0301 0.272 1.97x105 1 0.0401 0.0650 0.383 0.42x 105 1.5 0.0692 0.0891 0.233 1.03x 105 1.14 x105 *absorbance of the same amount of sample and reagent (1 sample:1 reagent) **absorbance of a maximum amount of reagent (1 sample:10 reagent) *** ratio of dissociation (α= am-as/as) real sample analysis determination of mndz in human blood mndz is metabolite mainly to 2– hydroxymethylmetronidazole and 2–methyl– 5–nitroimidazol–1–acetic acid, distributed quickly, and excreted mainly as glucuronic acid derivative in the urine during 48 hours with less than 10% of the dose as excreted as unchanged mndz [13,16]. 1, and 1.2 ml of extracted mndz from human blood samples have been taken, treated according to the recommended procedure, diluted to make 10 ml, and measured at 515 nm. the results are listed in table 6 and summarized in fig. 10. table 6. determi nation of mndz in human bl ood -many hours after admi nistration. r.s.d % (xi-x-)2(xi-x-) concentration of mndz found (ppm) x-xi ml of extracted blood sample after admi ni strated period (hours) 1 x 10-81 x 10-4 1x 10-81 x 10-4 0.0122 1 x 10-8-1 x 10-4 21.090.2088 0.2090 0.2089 0.2087 1 1 x 10-81 x 10-4 1 x 10-8-1x 10-4 0.0173 4 x 10-82 x 10-4 22.060.2184 0.2183 0.2183 0.2186 1.2 4 4 x 10-82 x 10-4 9x 10-8-3 x 10-4 0.0331 9 x 10-83 x 10-4 22.150.2193 0.2195 0.2190 0.2196 1 4 x 10-8-2 x 10-4 25 x 10-85x 10-4 0.0387 1 x 10-8-1 x 10-4 24.270.2403 0.2401 0.2408 0.2402 1.2 6 9 x 10-83 x 10-4 9 x 10-8-3 x 10-4 0.0308 1 x 10-81 x 10-4 9.310.0922 0.0925 0.0919 0.0922 1 4 x 10-82 x 10-4 1 x 10-81x 10-4 0.0173 1 x 10-8-1 x 10-4 11.180.1107 0.1109 0.1108 0.1107 1.2 8 1 x 10-81 x 10-4 4 x 10-82 x 10-4 0.0212 4 x 10-8-2 x 10-4 2.490.0247 0.0248 0.0249 0.0245 1 1 x 10-81 x 10-4 9 x 10-8-3 x 10-4 0.0308 9 x 10-83 x 10-4 4.090.0405 0.0406 0.0402 0.0408 1.2 12 pak. j. anal. environ. che m. vol. 23, no. 2 (2022)202 figure 10. mndz content i n human bl ood after many hours after admi ni stration determination of mndz in pharmaceutical preparations the suggested method has been applied for the determination of mndz in different dosage forms. table 7 shows wide application ranges with excellent recovery values. table 7. determi nation of mndz i n pharmaceutical preparations. pharmac -eutical preparat ions of mndz. mndz taken (μg/ml) absorbance of standar d absorbance of sample reco very * % error % 30 0.2201 0.2131 96.8 -3.2 50 0.4855 0.4682 96.4 -3.6 90 0.8621 0.8415 97.6 -2.4 metronid azole injection 500 mg / pioneer company 100 0.9389 0.9023 96.1 -3.9 30 0.2201 0.2291 104 +4.0 50 0.4855 0.4845 99.7 -0.3 80 0.8012 0.8034 100. 2 +0.27 metronid azole tablet 500 mg /microlab limited 100 0.9389 0.9021 95.9 -4.1 30 0.2201 0.2251 102. 2 +2.27 50 0.4855 0.4782 98.4 -1.5 90 0.8621 0.8515 98.7 -1.22 metronid azole tablet 200 mg / ajanta pharma limited / india 100 0.9389 0.9123 97.1 -2.83 comparison of the method with related published methods a comparison of the proposed method with the other literature methods shows that the present method has a higher range of linearity, lower detection limit, and lower quantitation limit as shown in table 8. table 8. comparison of the method with rel ated published methods. parameter present method literature method [34] literature method [35] method diazotization of the reagent diazotization of the reagent diazotization of mzol reagent paminodipheny lamine p-amino benzophenon e α-naphthol amine wavelength (nm) 515 431 510 beers law range (µg/ml) 20-100 2-24 2-12 limit of detection (µg/ml) 0.0351 0.2407 0.1142 limit of quantification (µg/ml) 0.1172 0.8024 0.3805 molar absorptivity (l.mol-1.cm-1) 0.1379x10 4 2.284x10 4 1.5x104 sandell’s sensitivity index (µg cm-2) 0.1010 0.0074 0.0114 application dosage forms and biological sample dosage forms and biological sample tablet conclusion metronidazole has been determined in blood, tablet, and injection using one simple diazotization-coupling reaction. the drug level in blood show maxima after 6 h after swallowing the drug orally with excellent precision range (rsd% 0.0122-0.0387). the estimation of drug content in injection and tablet exhibits high accuracy ranging from -4.1to +4.0. the technique is simple, the pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 203 method is sensitive, and does not consume energy, heat, buffers, or ph adjustment. acknowledgement the authors are grateful to the university of mosul, college of science, department of chemistry for providing facilities to carry out this work. conflict of interest the authors declare no conflict of interest. references 1. r. k. narayanasamy, p. rada, a. zdrha, m. ranst, j. neyts and j. tachezy, j. microbiol. immunol. infect., 55 (2022) 191. https://doi.org/10.1016/j.jmii.2021.08.00 8 2. r. gharebaghi, f. heidary, m. moradi and m. parvizi, arch. acad. emerg. med., 8 (2020) e40. https://pubmed.ncbi.nlm.nih.gov/322591 29 3. g. spengler, a. kincses, t. mosolygó, m. a. marć, m. nové, m. gajdács, c. sanmartín, h. e. mcneil, j. m. a. blair and e. domínguez-alvarez, molecules, 24 (2019) 4264. https://doi.org/10.3390/molecules24234 264 4. r. sender, s. fuchs and r. milo, cell, 164 (2016) 337. https://doi.org:10.1016/j.cell.2016.01 .013. 5. s. chaonan, c. ling and s. zhu, saudi pharma. j., 27 (2019) 1146. doi: 10.1016/j.jsps.2019.09.011 6. s.a.dingsdag, n. hunter, j. antimicrob. chemo., 73 (2018) 265. https://doi.org/10.1093/jac/dkx351. 7. j. ali, m. rahman, a. ahmad, z. khattak and m. asim, cureus, 13(2021) e17101. https://doi:10.7759/cureus.17101. 8. a. violeta, l. sbârcea and d. laura, molecules, 26 (2021) 3582. https://doi.org/10.3390/ molecules26123582. 9. s. c. sweetman, martindale "the complete drug reference", 36 th edn., pharmaceutical press, london (2009). http://www.amazon/martindale complete-drug-reference-36 th /dp/0853698406. 10. k. arun, r. mishra, m. amrita, v. anurag and ch. pronobesh, int. j. pharm. res. develops., 2 (2010) 1. ijprd/2010/pub/arti/vov-2/issue6/aug/004. 11. z. dominik, r. lidia and o. justyn, cancers, 14 (2022) 900. https://doi.org/10.3390/cancers 14040900. 12. n. zemanová, p. anzenbacher, t. hudcovic and e. anzenbacherová, j. chromatogr. sci., 60 (2021) 81. https://doi.org/10.1093/chromsci/bmab0 49 13. m. marouf, g. kawas and a. sakur, int. res. j. pure appl. chem., 22 (2021) 34. doi:10.9734/irjpac/2021/v22i330394 14. h. ammar, m. brahim, r. abdelhédi and y. samet, sep. purif. technol., 157 (2016) 9. https://doi.org/10.1016/j.seppur.2015.11. 027 15. t. pérez, s. garcia-segura, a. elghenymy, j. l. nava and e. brillas, electrochim. acta, 165 (2015) 173. https://doi.org/10.1016/j.electacta.2015.0 2.243 16. s. meenakshi, r. rama, k. pandian, s. gopinath, microchem. j., 165 (2021) 106151. https://doi.org/10.1016/j.microc.2021.10 6151 17. a. hernández-jiménez, g. roamorales, h. reyespérez, p. balderaspak. j. anal. environ. che m. vol. 23, no. 2 (2022)204 hernández, c. e. barreradíaz and m. bernabépineda, electroanalysis, 28 (2016) 704. https://doi.org/10.1002/elan.201500452 18. n. asma, n. m. maddeppungeng, m. raihan, a. p. erdiana, a. himawan and a. d. permana, microchem. j., 172 (2022) 106929. https://doi.org/10.1016/j.microc.2021.10 6929 19. a. da silva, c. da rosa silva and f. paula, aaps pharm. sci. tech., 17 (2016), 778. https://doi.org/10.1208/s12249-0150407-9 20. m. q. al-abachi, s. s. abed and m. h. alamri, iraqi j. sci., 61 (2020) 1541. https://doi.org/10.24996/ijs.2020.61.7.1 21. s. bhatia, v. shanbhag, j. chromatogr. b, 305 (1984) 325. doi: 10.1016/s0378-4347(00)83346-0. 22. o. a. adegoke and o. e. umoh, acta pharmaceutica, 59 (2009) 407. doi: 10.2478/v10007-009-0039-2. 23. k. siddappa, m. mallikarjun, p. reddy and m. tambe, eclética química, 33 (2008) 41. https://doi.org/10.1590/s010046702008000400005 24. a. doaa and y. elham, spectrochim. acta part a, 259 (2021) 19858. https://doi.org/10.1016/j.saa.2021.119858. 25. n. saffaj, m. charrouf, a. abourriche, y. aboud, a. bennamara and m. berrada, dyes pigments, 70 (2006) 259. https://doi.org/10.1016/j.dyepig.2005.01. 009. 26. h. wallada and b. wadala, raf. j. sci., 23 (2012) 78. https://doi.org/10.33899/rjs.2012.59994. 27. k. f. alsamarrai, res. j. pharmacol., 6 (2012) 25. doi: 10.3923/rjpharm.2012.25.29. 28. n. saffaj, anal. chem. an indian j. 4 (2007) 87. 29. l. fang, y. yu-yan, j. wen-ping and c. qun, chinese j. pharm. anal., 26 (2006) 1311. https://www.ingentaconnect.com/content /jpa/cjpa/2006/00000026/00000009/art0 0037. 30. a. rehman, a. s. ijaz and a. raza, j. iran. chem. soc. 2 (2005) 197. https://doi.org/ 10.1007/ bf03245922. 31. h. s. nagwa , a. mona, a. eman, n. elenany and f. belal, asian j. chem., 21 (2009) 755. https://asianjournalofchemistry.co.in 32. s. l. stancil, v. haandel, s. abdelrahman and r. e. pearce, j. chromatogr. b, 1092 (2018) 272. https://doi.org/10.1016/j.jchromb.2018.0 6.024 33. n. a. khalil, h. sh. mahmood and a. a. shihab, egypt. j. chem., 65 (2022) 397. doi: 10.21608/ejchem.2022.112958.5134 34. a. youssef, m. magda. d. saleh, a. abdel-kader and y. elham, inter. j. pharm. sci. res., 6 (2015) 103. doi:10.13040/ijpsr.09758232.6(1).103-10. microsoft word 06-d-pjaec-11062021-380-c-revised galley proof-20cross mark issn-1996-918x pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 237 – 246 http://doi.org/10.21743/pjaec/2022.12.06 synthesis of bio-metal organic framework-11 based mixed matrix membrane for efficient carbon dioxide separation tariq hussain 1 , zahid naeem qaisrani 2,3* , asadullah 2 , zaman tahir 4 , ali nawaz mengal 5 and muhammad sagir 1 1department of che mical engineering, university of gujrat, hafiz hayat campus, gujrat, 50700, pakistan. 2department of chemical engineering, faculty of engineering & architecture, balochistan university of information technology, engineering and management sciences (buitems), takatu campus, airport road quetta, 87300, balochistan, pakistan. 3faculty of environmental management, prince of songkla university, hatyai campus, 90112, songkhla, thailand. 4department of chemical engineering, comsats institute of information technology, lahore campus, lahore, pakistan. 5department of mechanical engineering, faculty of engineering & architecture, balochistan university of information technology, engineering and management sciences (buitems), takatu campus, airport road quetta, 87300, balochistan, pakistan. *corresponding author email: engr.zbaloch@gmail.com received 11 june 2021, revised 30 november 2022, accepted 10 december 2022 -------------------------------------------------------------------------------------------------------------------------------------------abstract mixed matrix membranes are thought to have the ability to separate gases. the current research investigates the isolation of co2 from methane (ch4) and nitrogen (n2) using a mixed matrix membrane. bio-mof-11 was combined with polyether sulfone to establish a membrane (pes). experiments were carried out to determine the efficiency of the established membrane. results showed that the lewis basic sites present in bio-mof-11, which have a higher affinity for co2, increase the permeability and selectivity of pristine polyether sulfone. at 30% filler loading, co2 permeability improved fro m 2.20 to 3.90 barrer, while co2/ch4 and co2/n2 selectivity improved from 9.57 to 11.14 with 30% filler loading. in addition, at 30% filler loading, co2 solubility drops from 1.57 to 1.20. keywords: biomof-11, me mbranes, metal organic framework, poly ether sulfone, co2 separation. -------------------------------------------------------------------------------------------------------------------------------------------introduction world’s population is continuously increasing and is expected to reach 9.2 billion people by 2050. with a growing population, human demands are increasing and putting extensive pressure on natural resources. researchers today realized the situation and the world is adopting alternative solutions for the sustainability of resources and energy demands [1–3]. extensive use of fossil fuels not only depletes resources but has an adverse effect on climate too [4]. keeping in view the increasing energy demands, switching towards green energy and co2 free world is of great concern today [5]. human activities change the concentration of co2 by burning fossil fuels and industrial waste materials. co2 is affecting our environment because of its anthropogenic nature causing high uncertainty [4,6,7]. the world meteorological organization, in its 2019 report, mentioned pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 238 that currently the co2 concentration in the atmosphere is 412 parts per million and rising continuously. to lower down the atmospheric level of greenhouse gases (ghg) and temperature of the globe, three options are being explored which are: (a) energy efficiency improvement (b) sequestration of carbon (c) useless carbon-intensive energy sources hence it is necessary to decrease the concentration of co2. two ways were discussed in the literature to resolve the issue. the first one is to reduce the usage of fossil fuels which is currently not possible, the other is to capture the co2 and restore it and use it to make useful products which is a better option [10,11]. there are many techniques to separate co2 from gases which include adsorption, absorption, cryogenic distillation, and membranes [12–15]. in this regard, gas separation membranes can help to control environmental pollution by capturing co2 from different streams. initially, membrane usage was limited because of low flux and selectivity. a larger area is required for higher flux, which means capital investment is high, and low selectivity means the operating cost will be high. so, it is desired to explore materials with higher selectivity, high surface area, and better permeation which can also stand against plasticization, aging, and conditioning. the difference in chemical and physical properties has created a great challenge to make a defects-free membrane. the metal-organic membranes (mof) have advantages over polymeric membranes [16]. the metal cluster improved the porosity and stability significantly [10,12,17]. in mof, both organic and inorganic building blocks are present. the organic ligands clinch to metal ion clusters. mixed matrix membranes (mmm) have wonderful permeability and selectivity due to the inorganic fillers, which have naturally excellent separation properties [16,18–23]. in this research, an efficient metal-organic framework-based mmm was made to separate co2 from different gas mixtures. materials and methods all the chemicals used in the current study were of analytical grade. the chemicals utilized for mmms development are adenine, cobalt acetate tetrahydrate, obtained from alfa aesar chemicals (germany). polyether sulfone (pes), n, n-dimethyl formamide (dmf), methanol, and chloroform were achieved by fisher scientific (usa). ch4, co2, n2 were purchased by linde chemicals (germany) and used as such without any further purification. synthesis of bio-mof-11 bio-mof-11 was prepared according to the method described in the literature [20], with some minor changes. an amount of 1.30 g of adenine was mixed in 100 ml of dmf. 2.50 g of cobalt acetate was mixed in 100 ml of dmf. both the solutions were ultrasonicated and heated to homogenize the solution. a 75 ml stock solution of adenine and 25 ml stock solution of cobalt acetate solution, with a ratio of 3:1, were mixed and stirred well. the solution obtained was placed in the autoclave oven and heated at 120 °c, and later the solution was cooled at room temperature. the product obtained had purple octahedral crystals. this product was washed and centrifuged three times with dmf. the product was further washed three times with methanol and dried. synthesis of pes membrane to cast the pes membrane, pes was heated in a vacuum oven at a temperature of pak. j. anal. environ. che m. vol. 23, no. 2 (2022)239 110±5 °c overnight. neat polymer and chloroform 20 ml were laced in a homogenizer. it was stirred for 24 h and then stored in a glass bottle. a smooth flat petri dish was used to cast a membrane with an inverted funnel placed on it. the casted membrane was moved to an oven to remove the excess solvent. the membrane was heated at the temperature range 90-160 °c with a ramp of 5 °c/h. the resulting membrane was gradually cooled to room temperature. mmm preparation the material was dried in an oven at 1055 °c for 24 h. an amount of 1 g of dried bio-mof-11 was mixed with chloroform, and it was stirred to prepare 10%, 20%, and 30% by wt. mof suspension as per equation 1. 100 fillerofweightpolymerofweight fillerofweight %)weight(loadingfilter    (1) the polymer solution was prepared according to the required amounts by following the priming method. the solution of polymer was added gradually into mof solution. initially, 10% of the overall required amount of polymer solution was added into mof solution. after 2 h, 10% remaining of the polymer solution was mixed into the finishing solution till the complete polymer solution was dissolved into mof solution. the same procedure was repeated for other samples. sample membranes were remained in an oven at 110 ±5 °c for 24 h and later characterized. a process flowchart is shown in fig. 1(a). mofs characterization the synthesized bio-mof-11 was characterized by different instruments, which include scanning electron microscopy (sem) (neon zeiss), thermal gravimetric-differential scanning calorimetry (tg-dsc, tga/dsc1 stare system from mettler-toledo), fourier transform infrared spectroscopy (ftir) by perkin elmer's spectrum 100-ftir, x-ray diffraction (xrd) by bruker d8, co ka irradiation. permeation experiments to check the performance of gas separation of mixed matrix membrane, a specially constructed gas permeation system, as shown in figure 1(b) was used. the efficiency was calculated based on different parameters, i.e., temperature 25 – 55°c, feed flow rate 1 l/min, and pressure 10 bar, respectively. to place the membrane in the equipment, a porous metallic plate was used. it was sealed tightly with viton o-ring. retentate and permeate composition was analyzed by a gas chromatograph (yl instrument, south korea). all tests were performed in triplicate. in an auxiliary cylinder, the gas was expanded, and the pressure transducer took the reading of the rate of pressure increment. equation 2 tells us the calculation of gas permeability (p).                 dt dp px7. 14 76 at yiv 760 10273 p 2i (2) where, t = temperature (k), p2 = feed gas pressure (psi), v = volume (cm 3 ) of down stream, a = permeation area (cm 2 ) of membrane, yi. = mole fractions of component i in downstream, xi = mole fractions of component i in upstream. the transportation of gases through mixed matrix membranes uses the ‘solutiondiffusion’ transport method described in equation 3. p = d  s (3) where, p = permeability, d = diffusion coefficient, s=solubility coefficient of gas in membrane phase. pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 240 figure 1(a) process flow chart (b) gas permeation setup to find out the diffusion coefficient of the membrane, the ‘time lag method’ was used. the above expression was also used to find out the solubility coefficient. equation 4 was used to calculate mixed-gas selectivity. j x i x j y i y ij  (4) where, xj = component mole fraction j in upstream, xi = component mole fraction ‘i’ in upstream, yj = component mole fraction j in downstream, yi = component mole fraction ‘i’ in downstream. results and discussion ftir ftir spectra of the membrane were recorded in a wavelength range of 640 to 4000 cm -1 shown in fig. 2 (a). it confirms the linkage of adenine, co, and the functional group present in mofs. we can see the adenine linkers and metal nodes linkage. from the figure, we can see the two band peaks at the range of 665 and 870, which is the fingerprint profile shows co-o stretching vibration. at 3330 cm -1 the characteristic peaks show n-h in adenine amine stretching. similarly, at 1400-1605 cm-1 the bands represent the bending and stretching modes of the imidazole ring of adenine. the stronger peak on 1590 cm -1 represents the existence of c-n bending. at 900 to 1170 cm -1 shows c-h stretching in the structure of adenine. xrd the xrd with a particle size 150 microns was recorded at 2θ from 5 to 70 o to find bio-mof-11 co crystallinity. the pattern of xrd is the same as reported in the previous studies with the synthesis of bio-mof-11 shown in figure 2(b) [24]. crystal size was found by using the scherer equation, which is 6 nm given below: (a) (b) pak. j. anal. environ. che m. vol. 23, no. 2 (2022)241 figure 2. characteri zation of bio mof-11 (a) ftir (b) xrd (c) tga sem sem presents a nano-sized crystal of bio-mof-11. they look like disc that was grown in shape like cauliflower. the morphology of bio-mof 11 is shown in fig. 3(a) & (b), which showed highly ordered octahedral mof particles of synthesized biomof-11. the reason is the post synthesized modification before fabricating mmm. figure 3 (a. sem of bio-mof11 at 2 μm. b. sem of bio-mof11 at 1 μm. c & d. morphology of mi xed matri x membrane) 10 20 30 40 50 60 70 0 20 40 60 80 3 5.73201 67 .29625 47.20814 34.666 78.6 786 32.38803 in te n s it y position [2 theta] m as s f r a ct io n (a) (b) (c) (a) (b) (c) (d) pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 242 a scanning electron microscope was also used to find out the morphology of the mixed matrix membrane of polyether sulphone and bio mof-11. the images are shown in fig. 3(c) & (d). the images show that polyether sulphone and bio mof-11 particles have good interfacial adhesion. the images show no void among pes and mof particles. the images also clearly show that the mof particles are dispersed throughout the membrane homogeneously, proving that the polymer and mof have solid interfacial interaction. tga the study of bio-mof-11 -co was also done by thermogravimetric analysis (tga) with the range of temperature 40°c to 700°c shown in fig. 2(c). the environment was air which has a flow rate of 70 ml/min. it shows metal-organic framework thermal stability with temperature. we can see in the figure that up to 110°c, weakly attached surface solvent/moisture was removed. at 110°c to 270°c, the bonded solvent molecule of dmf in the structure was removed. when the temperature touches 271°c, the organic linker's degradation started, and the complete structure was destroyed at 370 °c. the glass transition temperature (gtt) is the temperature at which the carbon chain starts to leave its position. the higher the gtt value higher will be the stiffness and rigidity. it was seen that gtt increases with an increase in the concentration of bio-mof-11 in polyether sulfone. the neat polyether sulfone has a gtt value of 220 °c which increases to 235 at 30% loading filler. the increase in gtt value of polyether sulfone with bio-mof-11 shows the improvement in toughness and strictness in mmm, the enhancement also showed the strong attraction between poly ether sulfone and bio-mof-11. a major cause of this enhancement of gtt measurement is that the filler particles are strongly surrounded by a polymer chain. another reason is the improvement in the interlinking bond between the filler and polymer [23]. the gtt graph is shown in fig. 4. figure 4. gl ass transi tion temperature of pes/bio-mof-11 gas separation performance co2, n2, and ch4 gases were used at a pressure of 10 bar and a temperature of 25°c for measuring the permeability. three coupons were separated from every membrane and examined at a gas permeation setup. furthermore, the permeability of every coupon was examined three times and the average outcomes were utilized for analysis with error bars. pure gas permeation results for co2/n2 and co2/ch4 gas pairs are shown in fig. 5. figure 5. pure gas selecti vity and permeability data of pes/biomof-11 pak. j. anal. environ. che m. vol. 23, no. 2 (2022)243 the incorporation of bio-mof-11 particle caused improvement in selectivity from 14.67 to 15 with different loading of filler (0-30%) in co2/ch4 pure gas. the slightly lower selectivity in 20% load filler may be due to the pore blockage. similarly, it also improves the ideal selectivity of co2/ n2 from 9.57 to 11.47 with a range of 0% to 30% loading of filler. the permeability of co2 also increases with the incorporation of bio-mof 11. the value of permeability jumps from 2.20 to 3.90. it was also observed that the selectivity of co2/ch4 increases by 2.24% with the loading of filler 0 to 30%. similarly, the permeability of co2 increases by 290% with filler loading ranges from 0-30% and the selectivity of co2/ n2 increases by 19.85%. higher permeability of co2 will be attained if the % concentration of loading filler in poly ether sulfone is increased because bio-mof-11 has a higher affinity toward the molecules of co2. the co2 shows a higher quadrupole moment and displays a greater solubility coefficient when it is compared with nitrogen and methane which are nonpolar. in mmm, the solubility and diffusion coefficient determination of co2 can give the gas separation performance. the solubility, diffusivity, and permeability of co2 are displayed in fig. 6. figure 6. permeability, sol ubility, and di ffusivi ty of co2 it is seen that the permeability of co2 is increasing with increasing filler concentration. it also observed that the diffusion of co2 is increasing while the solubility is decreasing. the reason for increasing diffusion is the adenine structural units lewis basic site present in bio-mof-11, which has stronger co2 molecule adsorption ability, causing better diffusion. the diffusion has inverse proportionality with solubility so when diffusion increases, then automatically solubility decreases. the mmm was also examined for gas permeation performance in pairs of gases, i.e., co2/n2 and co2/ch4. the outcomes are represented in fig. 7 (a) & (b) in mixed gas conditions. figure 7. (a. co2/n2 selectivity and permeability data of pes/bio-mof-11; (b. co2/ch4 selecti vity and permeability data of pes/bio-mof-11) the permeability of pure gas is greater than the permeability of mixed gas. the reason is the phenomenon of gas molecule perme ability, solubility and diffus ivity of co 2 (a) (b) pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 244 competitive sorption. the permeability of co2 has slowed down with the molecules of n2 and ch4 gases existence because they have greater kinetic diameter than co2. the co2 also leads to polymer matrix plasticization because the co2 gas is condensable. this also affects the selectivity of the membrane, which later decreases. at partial pressure of 5 bar, the behavior of co2 gas was checked for mmm in a mixed gas condition which was less than the plasticization pressure of pes. so, selectivity difference is ascribed to competitive sorption. effect of operating temperature on memerane performance the overall permeability was increased at higher temperatures in mmms, as shown in fig. 8. with an increase in the temperature of the mmm the permeability of co2 increases. the enhancement of co2 permeability at higher temperatures is due to the polymer gaining a property, and it becomes flexible, resulting in free volumes that lead to a reduction in selectivity but enhanced gas permeability. figure 8. effect of temperature on co2 permeability of pes/bio –mof-11 arrhenius equation gives us permeation activation energy (ep), and operating feed temperature, permeability. (5) t= feed temperature absolute. po = pre exponential factor. r =gas constant, p= permeability of gas. the values for activation energies of permeation for co2 were calculated and presented in fig. 9(a). the activation energy of permeation = sorption heat + diffusion activation energy. (6) the activation energy of permeation tells us how much a gas molecule shows confrontation when it passes through the mmm. if the value of activation energy is low, then it is expected that the permeability of gas will be higher. if the activation energy value is higher than the permeability of gas through a mmm will below. in this study, the activation energy of co2 decreased with increasing the percent of loading filler (biomof-11). it shows that with increasing temperature, co2 permeability will increase; it also proves that polymer and filler are incorporating with each other and sharing the properties. the developed bio-mof-11 incorporated mmm showed a good separation result. to bring more precision, the performance was compared with the reported literature. for this purpose, roberson’s upper bound trade-off plot was drafted, as shown in fig. 9(b). it is shown in the figure that as the concentration of filler increases the result came nearer to the upper bound which proves that it can be one of the potential candidates for the separation of co2. pak. j. anal. environ. che m. vol. 23, no. 2 (2022)245 figure 9. (a. acti vation energy of pes/bio –mof-11; b. comparison of gas separation performance for pure pes and di fferent filler) conclusion in this study, adenine and cobalt-based bio-mof-11 were synthesized, characterized, and mixed with polyether sulfone to cast membrane. the results of ftir, xrd, and sem images show a good combination between cobalt and adenine. this membrane was tested for co2 separation from the mixture of co2, ch4, and n2. in the start, the permeability and selectivity were tested for pure gases and then for a mixture of gasses that have 50/50 concentration. the results showed that increasing filler concentration, the permeability of co2 increases. the selectivity of co2/ch4 and co2/n2 also increases. the separation performance was close to the robeson upper bound, which shows that it can be an ideal candidate for the separation of co2. acknowledgement the authors are grateful for the laboratory facilities provided by the department of chemical engineering, university of gujrat, pakistan, and the membrane technology lab, comsats lahore campus. we also appreciate the department of chemical engineering at buitems quetta, pakistan, for providing a workspace and it support that enabled us to complete this manuscript on time. furthermore, we appreciate the positive feedback from reviewers, which helped us improve the quality of our work. conflict of interest the authors declare no conflict of interest to disclose. references 1. z. n. qaisrani, s. shams, z. guo and a. a. mamun. pollution, 6 (2020) 569. doi:https://dx.doi.org/10.22059/poll.202 0.297713.751 2. z. n. qaisrani, n. nuthammachot, k. techato, asadullah, g. h. jatoi, b. mahmood and r. ahmed. braz. j. biol., 84 (2022) 12. doi:https://doi.org/10.1590/15196984.261001 3. z. n. qaisrani, s. shams, g. zhenren, ms. reza and q. zaunuddin, iet digit. lib., (2018) 4. doi:https://doi.org/10.1049/cp.2018.1605 4. j. an, s. j. geib and n. l. rosi. am. j. chem. soc. commun., (2009) 38. doi:https://doi.org/10.1021/ja909169x 5. m. siddique, s. a. soomro, z. n. qaisrani, a. s. jatoi, asadullah, g. khan and e. kakar, pak. j. ana.l environ. chem., 17 (2016) 18. (a)  c o 2/ c h 4 (b) pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 246 doi:10.21743/pjaec/2016.06.003 6. d. aaron and c. tsouris, sep. sci. technol., 40 (2011) 321. doi:https://doi.org/10.1081/ss200042244 7. g. myhre, o. boucher, f. bréon, p. forster and d. shindell, nat. geosci., 8 (2015) 181. doi:https://doi.org/10.1038/ngeo2371 8. k. sumida, d. l. rogow, j. a. mason, t. m. mcdonald, e. d. bloch, z. r. herm, t. h. bae and j. r. long, chem. rev., 112 (2012) 724. doi:https://doi.org/10.1021/cr2003272 9. j. lehmann, commentary, 447 (2007) 143. doi:https://doi.org/10.1038/447143a 10. t. li, j. e. sullivan and n. l. rosi, j. am. chem. soc., 135 (2013) 9984. doi:https://doi.org/10.1021/ja403008j 11. m. r. azhar, p. vijay, m. o. tade, h. sun and s. wang, chemosphere, 196 (2017) 105. doi:https://doi.org/10.1016/j.chemospher e.2017.12.164 12. z. tahir, a. ilyas, x. li, mr. bilad and i. f. j. vankelecom, j. appl. polym. sci., (2017) 1. doi:https://doi.org/10.1002/app.45952 13. z. tahir, m. aslam, m. amjad and m. roil. sep. purif. technol., 224 (2019) 524. doi:https://doi.org/10.1016/j.seppur.2019 .05.060 14. i. erucar and s. keskin. chem. eng. sci., 130 (2015) 120. doi:https://doi.org/10.1016/j.ces.2015.03 .016 15. m. rizwan, h. rasool, h. sun, v. periasamy, mo. tadé and s. wang, j. colloid interface sci., 490 (2017) 685. doi:https://doi.org/10.1016/j.jcis.2016.11 .100 16. h. asghar, a. ilyas, z. tahir, x. li and a.l. khan, sep. purif. technol., 203 (2018) 233. doi:https://doi.org/10.1016/j.seppur.2018 .04.047 17. j. dechnik, c. j. sumby and c. janiak, cryst. growth des., 17 (2017) 4467. doi:https://doi.org/10.1021/acs.cgd.7b00595 18. t. li, d-l. chen, j. e. sullivan, m. t. kozlowski, j. k. johnson and n. l. rosi, chem. sci., 4 (2013) 1746. doi:http://dx.doi.org/10.1039/c3sc22207a 19. m. w. anjum, f. vermoortele, a. l. khan, b. bueken, d. d. vos and i. f. j. vankelecom, acs appl. mater. interfacts, 7 (2015) 25193. doi:https://doi.org/10.1021/acsami.5b08964 20. s. ishaq, r. tamime, m. r. bilad and a. l. khan, sep. purif. technol., 210 (2018) 442. doi:https://doi.org/10.1016/j.seppur.2018 .08.031 23. m. w. anjum, b. bueken, d. d. vos, i. f. j. vankelecom, j. memb. sci., 125 (2015) 21. doi:https://doi.org/10.1016/j.memsci.201 5.12.022 24. s. shah, h. sheikh, s. hafeez and m. i. malik. pak. j. anal. environ. chem., 21 (2020) 44. http://doi.org/10.21743/pjaec/2020.06.06 microsoft word 04-215-224-pjaec-29102021-407-c-ree revised galley cross mark issn-1996-918x pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 215 – 224 http://doi.org/10.21743/pjaec/2022.12.04 evaluation of cotton seeds as environmentally liable source for neutral protease asghar ali shaikh * and asif ali bhatti 1 department of chemistry, government college university, hyderabad,71000, pakistan. *corresponding author email: dr.asghar.ali@gcuh.edu.pk received 29 october 2021, revised 27 october 2022, accepted 11 november 2022 -------------------------------------------------------------------------------------------------------------------------------------------abstract proteases are considered one of the most imperative groups of enzymes and are used in bioremediation processes, leather, detergents, pharmaceutical and the food industry. the foremost aim of this study was to extract, purify, and characterize the protease enzyme from cotton seeds. purification of the neutral protease was achieved with ammonium sulphate, which gave the best results at 60% concentration with a specific activity of 51 units/mg protein and 1.52 purification fold with a percentage yield of 10.5%. the protease was active and stable at a wide range of ph from 4.0–10.0 with an optimum ph of 7.0. the highest activity of the purified enzyme was found at 20 °c. the enzyme was ther mally stable and retained 25% of its activity at 50o c. the activity of cottonseeds protease fraction-iv was enhanced by 20% with zncl2 and 15% with cocl2 as enzyme samples were heated for 10 minutes. the casein and peptone assays were also perfor med to check its catalytic activity. further more, the maximum hydrolysis rate (vmax) and apparent michaelis–menten constant (km) values of the purified protease were 19 μmol/min and 0.08 mol/l, respectively, while activation energy was found to be 12.47 kj/mol. keywords: neutral, thermostable, cotton seeds, metallo protease -------------------------------------------------------------------------------------------------------------------------------------------introduction enzymes, due to their low production cost, easy methods of production and environmentally friendly behavior are broadly used in several commercial fields [1]. in proteins, these enzymes accelerate the degradation of peptide bonds present between amino acids [2]. researchers are focusing to isolate new enzymes which are suitable for commercial applications [3]. in 2018, the world market for industrial enzymes extended by around $5.6 billion in 2018, which will increase by an estimated $7.0 billion in 2023 [4]. among these enzymes, proteases cover 65% of the total industrial enzymes market [5]. proteases may be classified as endopeptidases, which degrade protein substrate from the inner site and exopeptidases, which degrade protein substrate from the end but can still be classified according to the substrate specificity, source of isolation (vegetable, animal or microbial), molecular size, catalytic action, charge and active site [6]. proteases, according to their optimum ph reactions, are classified as neutral protease, acidic protease and alkaline protease [7]. neutral proteases are broadly applied in the food [8,], feed [9], pharmaceutical [10] and leather [11] industries due to their divergent benefits, including low pollution level, a mild catalysis process and high yield. neutral protease is usually used for brewing beer [12] and de-bittering soy sauce in the pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 216 food industry [13]. for commercial applications, several researchers have focused on isolating new enzymes having satisfactory properties [14]. due to good solubility, high stability in extreme conditions, substrate specificity and activity over broad-ranging temperatures and ph scales, plant proteases had been used in numerous industries [15]. in several food industry procedures, plant sources proteases are mostly used, therefore, the proteases are cost-effective for use in industries [16]. furthermore, researchers are searching for novel proteases from plant sources which have cost-effective in industries and have important physiological roles [17]. at present, the important focus is to find a neutral protease from economical sources which have a high affinity to protein, acid–alkali resistance and good thermal stability. because pakistan is an agricultural country and cotton is a major crop and cotton seeds are frequently available and cheap sources, therefore, in this study, a neutral protease from cotton seeds was purified and characterized. material and methods cotton seeds (bs-15) were purchased from a local market. ammonium sulphate, casein acid hydrolysate, peptones and other reagents/chemicals were used in high analytical grade and purchased from a local vendor of sigma (usa), merck (usa), and fluka company (switzerland). preparation of soluble enzyme and determination of protein and protease activity cotton seeds were de-coated, crushed, defatted and homogenized in chilled acetone. on the other hand, 10% enzyme solution and 2% casein acid hydrolysate substrate were prepared in chilled 0.2 m tris-hcl buffer as reported earlier [18,19]. protein content was calculated by the method of lowry [20], with bovine serum albumin as a standard. enzymatic solution protein absorbance was monitored at 280 nm. the activity of protease was checked by the method of anson [21] with slight modification. in this method, 2% casein acid hydrolysate was used as a substrate. at the end of the reaction, protease activity was checked by spectrophotometer at 625 nm, as reported earlier [18]. one unit of protease activity was defined as the amount of enzyme that liberated 1ug of tyrosine under the standard assay conditions. isolation, dialysis and purification of cotton seeds protease by using sephadex g-100 column chromatography two-fold ethanol, acetone, methanol and 40%, 60% and 80% saturated ammonium sulphate was used to purify protease. all these chemicals were retained at 4 o c for 4-5 hours. the collected precipitates from different chemicals were dissolved separately in 10 ml of universal buffer of 7.0 ph and their protease activity was determined by an earlier reported method. on the basis of the highest protease activity, precipitates were obtained from 60% saturated ammonium sulphate dialyzed for 24 h in a universal buffer of 7.0 ph. the collected precipitates and dialyzed samples were centrifuged in a cooled refrigerated centrifuge at 7000 rpm for 20 minutes and protease activity was determined by the reported method. sephadex g–100 gel column (60 × 2cm), previously packed and equilibrated with universal buffer ph 7.0. on this sephadex g– 100 gel column, 5.0 ml of dialyzed sample was loaded. the dialyzed sample was eluted with the same buffer ph 7.0 at the flow rate of 40.0 ml/h. the fraction of 5.0 ml was collected using fraction collector eyela pak. j. anal. environ. che m. vol. 23, no. 2 (2022)217 uv–9900, uv-visible detector. the protein absorbance was monitored at 280 nm. sds-gel electrophoresis sds gel electrophoresis using buffer system, as described by peterson [22] and hames [23] was used to determine homogeneity of the purified enzyme. kinetic study and characteristic properties of protease enzyme effect of substrate concentration and substrate specificity on protease activity casein acid hydrolysate and peptone animal were used as substrates of different concentrations from 0.5-3% and checked their effects on the rate of enzymatic reaction of protease. 1.0 ml of purified fraction sample and 1.0 ml substrate of different concentrations were incubated at 20°c for one hour. the substrate specificity with various substrates was also calculated by using different substrates. the mixture of a fixed amount of enzyme and substrate was incubated for one hour at 20°c. after incubation protease activity was checked as previously described by the standard protease assay method. effect of ph, temperature, thermal stability and metal ions / reagents on protease activity to check the effect of ph on the protease activity of purified fraction-iv, various ph ranges from 4 to 10 by using a universal buffer ph 7.0 were used. the enzyme was incubated with casein acid hydrolysate as a substrate. the optimum temperature of the purified protease enzyme was determined by incubating the enzymesubstrate mixture at various temperatures in the range of 10°c to 50°c. on the other hand, the thermal stability of protease was achieved from purified fractions by measuring the residue activity after incubation of the enzyme at various temperatures ranging between 20°c to 80°c for 10 min with and without the addition of activators at 20 o c. the effect of time period on enzyme thermal stability was also noted by heating enzyme samples at 5–20 min with and without the addition of an activator at 40 o c. the different concentrations (mm) of several chemicals and metal ions were incubated with purified fraction-iv for 10 minutes at optimum temperature before adding substrate. the remaining protease activities of each parameter were determined by the previously described method. results and discussions purification of protease by chromatography the solvent extraction of cotton seeds enzymatic protein extract (20%) at ph 7.0 was done. for this purpose, cotton seeds, and enzymatic protein extract (20%) precipitated with different solvents such as methanol, acetone, ethanol and various concentrations of ammonium sulphate (40, 60 and 80%). it was observed that cotton seeds enzymatic protein extract with 60% ammonium sulphate precipitation produced the highest protease activity in compression to solvents precipitation, as illustrated in table 1. therefore, ammonium sulphate (60% saturated) concentration was selected for precipitating enzymatic protein for further investigation. table 1. enzyme protei n and acti vi ty of preci pitates of sol vents. preci pitation wi th total protei n mg total protease acti vity uni ts specifi c acti vity uni ts/mg protei n control 253 8500 33.6 ethanol 134 5000 37.3 methanol 199 5750 28.9 acetone 167 5500 33 ammoni um sul phate 40% 51.9 1200 23.12 60% 115 6000 52.17 80% 83 1300 15.66 pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 218 purification of cotton seeds protease was done by sephadex g-100 column chromatography and the elution pattern is shown in fig.1. the precipitates obtained from 60% ammonium sulphate were dissolved in 20ml universal buffer ph 7.0 and then dialyzed overnight at 4 o c. the dialyzed sample was applied to sephadex g-100 column (60 × 2 cm), which was packed and equilibrated with the same buffer at a flow rate of 40 ml/h with 10 ml of fraction volume. the elution of enzymatic protein was monitored at 280 nm. the protease activity was checked in each tube and the active fractions were pooled for further characterization. these pooled fractions were named fraction-i, fraction-ii, fraction-iii, fraction-iv and fraction-v. the fraction-i, fraction-ii, fraction-iii and fraction-iv were found homogeneous, showing a single protein band by sds gel electrophoresis, while fraction-v was not homogeneous as exhibited in fig. 2. figure 1. puri fication profile of protease of cotton seeds by sephadex g-100 figure 2. sds pol yacryl ami de gel electrophoresis of cotton seeds protease (fraction-iv) all fractions were individually characterized and found alkaline, acidic and neutral. however, in terms of optimum temperature, ph, substrate concentration, thermos ability, substrate specificity and effect of various reagents, fraction-iv was neutral in nature, further characterized and reported in present study. the protease of fraction-iv was purified to 1.52 fold with the percent yield of 10.5. while specific activity was found to be 51 units /mg protein. the overall purification profile of alkaline protease fraction is presented in table 2. table 2. puri fication profile of protease from cotton seeds (fractioniv). puri fication steps total protei n mg total protease acti vity g speci fi c activity g /mg purification fold % yi eld enzyme crude 253 8500 33.6 1 100 after dialysis 190 6750 35.5 1.06 79.4 sephadex g-100 123.8 5800 46.8 1.39 68.2 fraction-iv 17.5 895 51 1.52 10.5 effect of temperature on protease activity from the industrial point of view, the optimum temperature of an enzyme, especially proteases is considered a significant factor. temperature affects the speed of enzymatic reactions by disturbing the structure of the enzyme and boosting the meeting of the substrate with the active site [24]. hence, as the temperature is elevated, the speed of enzymatic reaction also increases as long as the natural structure of the protein is spoiled. higher temperatures denaturation the enzymes mainly by breaking hydrogen bonds. to determine the optimum temperature of fraction-iv protease, the enzyme-substrate mixture was incubated at a different temperatures ranging from 10 to 50 o c. fig. 3, shows the maximum relative activity (100%) for fraction-iv protease at a temperature of pak. j. anal. environ. che m. vol. 23, no. 2 (2022)219 20°c. it is mentioned that the protease activity with an optimum temperature 20 o c or less than 20 o c is considered a cold protease [25]. after that optimum temperature, enzyme activity starts to decrease as temperature increases and is completely lost at a temperature reached 40 o c. after 20°c decline in activity was observed and this is due to the thermal denaturation of fraction-iv protease. huston et al. have isolated a protease from psychrophilecolwellia psychrerythraea strain 34h, which has a low optimum temperature (19oc) [26]. the activation energy for the protease of fraction-iv is calculated at 12.47 kj/mol. figure 3. effect of temperature on puri fied protease acti vity of cotton seeds (fractioniv) effect of ph on protease activity ph is an important factor for all enzyme activity. the ph at which the enzyme showed maximum activity, is called optimum ph. the optimum ph is considered a key factor for all enzymes in terms of their activities and production, variation in ph leads to enzyme inactivation [27]. nevertheless, some proteases extracted from plants are exceptional, they are active in a wide range of ph and temperature. in this study, the ph activity of proteases isolated from cotton seeds protease samples was calculated at different phs (5, 6, 7, 8, 9 and 10). the enzyme activity increases as ph increases to 7 and then declines steadily up to ph 10. this trend showed that the enzyme has optimum activity at ph 7 as presented in fig. 4. it is documented that metallo-proteases are in the range of neutral to alkaline [28], and ph studies reported an increased enzyme activity at a ph range of 5-7. the functions of an enzyme rely on its structural integrity, as the structure of the enzyme is changed, the enzyme loses its functions. the remarkable ranges of ph activity of enzymes favor acidic and neutral environments. bijina isolated protease inhibitor from moringa oleifera with potential application as therapeutic drug and as seafood preservative at ph 7. [29], siritapetawee isolated and purified protease from artocarpus heterophyllus (jackfruit) latex and found maximal activity between 55 and 60 °c at ph 8. [30]. figure 4. effect of ph on puri fied protease acti vity of cotton seeds (fraction-iv) effect of metal ions / chemical reagents on protease activity the effects of different metal ions on protease activity are presented in table 2. the activity of the purified protease was enhanced up to 15 and 20% by ca2+ and zn2+ respectively, while metal ions ag+, hg 2+ , co 2+ and mn 2+ decrease enzyme activity with the proportions of decrease 30, 35, 70 and 65%, respectively. on the other hand, tested chemicals such as tween 80, triton x-100, sodium dodecylsulphate (sdc), sodium deoxycholate (sds), mercaptoethanol and cysteine also have strong inhibition effects 35%, 20%, 85%, 75%, 70% and 50% pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 220 respectively on the enzyme activity in variable degrees in comparison with the control as exhibited in fig.5. it was documented that plant metalloproteases are generally zinc and calcium-dependent enzymes and calcium are well-known to interfere with the threedimensional structure and catalytic properties of enzymes [31]. these findings showed that fraction-iv protease belongs to metallo protease and zn 2+ and ca 2+ ions stabilize the enzyme structure and protect the protease against thermal denaturation [32]. ca 2+ and other metal ions dependent protease from leaves of moringa oleifera was also reported. ramakrishna has isolated metallo-protease from dry grass pea seeds [33]. table 3. effect of vari ous metal s / reagents on puri fied protease acti vity of cotton seeds (fraction-iv). reagents activity % of rel ati ve % of 5mm conc. units/ ml protease activity activation/ [inhibition] control 8 100 tween 80 5.2 65 35 triton x100 6.4 80 20 sdc 1.2 15 85 sds 2 25 75 agno 3 5.6 70 30 hgno3 5.2 65 35 mercaptoethanol 2.4 30 70 cysteine 4 50 50 o-phenanthroline 7.4 93 07 edta 7.6 95 5 cocl2 2.4 30 70 mncl2 2.8 35 65 cacl2 9.2 115 15 zncl2 9.6 120 20 effect of thermostability on protease activity the stability of an enzyme depends on the sum of numerous weak, non-covalent interactions such as van der waal interactions, hydrogen bonds and hydrophobic effects. all of these non-covalent interactions are upset by environmental conditions, such as temperatures. the enzyme degradation mechanisms are significantly speeded at high temperature, and thus impart an important role in the thermo-inactivation of enzymes. thermo-stability is considered a crucial characteristic of protease enzymes required in various industries. those enzymes which are thermostable are considered beneficial from the industrial point of view. it was mentioned in the literature that the thermal stability of an enzyme is a key factor for its biotechnological applications. thermal stability of purified cotton seeds fraction-iv protease was accomplished by heating the enzyme samples at various temperatures ranging from 20 o c to 70 o c for 10 minutes with and without the addition of activators (zncl2 and cacl2). after heating, the enzyme samples were cooled, and the remaining activities were determined by standard method. moreover, thermostability was also determined at various times from every interval of 5 min (5 to 20 min) at fixed incubated temperature with and without the addition of activators. the protease activity of cotton seeds fraction-iv was retained at 25% at 50 o c. fraction-iv protease activity was increased by 25% at 50oc by the addition of 5mm zncl2 in the enzyme samples heating for 10 min, and results are shown in fig. 5. furthermore, the data depicted in fig. 6 showed the effect of heat treatment on protease activity at a variable time (5-20 min) and fixed temperature (40 o c) with and without the addition of activators. however, fractioniv protease activity was retained at 60% without and 72% with zncl2 for 20 minutes of incubation. these results are comparable with other sources of proteases reported in the literature. protease from salvadorapersica was stable up to 50oc [34], protease from baby kiwi was stable up to 45 o c [35] and protease from citrullus colocynthis was stable pak. j. anal. environ. che m. vol. 23, no. 2 (2022)221 up to 40oc. some other researchers, such as gonçalves et al. have isolated thermostable proteases from the leaves, seeds, roots, and stem of canavalia. ensiformis [24]. figure 5. thermostability of purified protease acti vi ty of cotton seeds (fraction-iv) wi th and wi thout zncl 2 for 10 mi n figure 6 . effect of heat treatment at (40oc) with and without zncl 2 at various time period on puri fied protease of cotton seeds (fraction-iv) effect of substrate concentration on protease activity it is well-known consideration that substrate concentration is a key factor for determining the enzyme’s activity beside ph and temperature. furthermore, it is also used for the calculation of enzyme kinetics (km and vmax) to elucidate the enzyme specificity and affinity. in this connection, different concentrations of casein acid hydrolysate from 0.5 to 3% concentration at 20oc were used with respect to their optimum time period. the activity of fraction–iv protease was examined, and results are signified in fig. 7. moreover, it was noted that the substrate concentration increased up to 2% when both casein hydrolysates which was used throughout the study and peptone of soymeal and then dropped. praiwala et al. [36] have used several amounts (0.2, 0.4, 0.6, 0.8 and 1.0ml) of hemoglobin as a substrate incubated with protease obtained from green gram seeds and soybean seeds. n--tosyllarginine methyl ester (l-tame) was used as substrate with proteases isolated from leaves, seeds, roots and stem of canavalia ensiformis. figure 7. effect of substrates on puri fi ed protease acti vity of cotton seeds (fraction-iv) estimation of km and vmax of protease the km value is a tool to measure the enzyme affinity to the substrate. it is fact that smaller km values indicate greater enzymesubstrate affinity [37]. the value of km shows the highest rate achieved when the enzyme sites are saturated by the substrate of the reacted enzyme. the km value of fraction-iv protease was calculated at 0.08m (table 4) by double reciprocal plots according to lineweaver and burk [38]. the km value of (0.08) of the present work is lower than reported by other researchers who isolated protease enzymes from different plants like yemeni bean seeds [28]. km is 0.19m, field bean seeds enzyme 0.0105m [39], moringa oleifera leaves is 0.107m [40] and from the seeds of cucumis melovaragresticis 0.02m [41] and seeds of ash groud benincasa hispida (thumb) cogn is 0.01m [42]. it is clear from pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 222 the results obtained in this work, that the km value of fraction-iv protease is lower than other proteases hence greater affinity towards substrate. on the other hand, the value of vmax of fraction-iv protease was also calculated which comes out 19 µmol/min as presented in table 4. this value of vmax is higher than proteases isolated from seeds of green gram(vmax 2 µmol/min) and soybean(vmax 1.01 µmol/min) but lower than those obtained from proteases isolated from seeds of citrullus colocynthis (557 µmol/min) [43]. ibraheem and malom have reported two neutral proteases, pll and plll from citrus sinensis fruit peel, having a value of vmax are 192.31 and 111.11 µmol/min, respectively [44]. table 4. km, vmax val ues, arrheni us plot of the acti vation energy of puri fi ed cotton seeds protease (ea=slopxr, where r=8.314kj/mol). km val ues mol/l vmax val ues µmol/min activation energy kj/mol fractioniv 0.08 19 12.47 effect of substrate specificity of protease the substrate specificity has great importance in biotechnological fields. the substrate specificity of purified protease fraction-iv was determined by incubating the enzyme with different protein substrates prepared in universal buffer ph 7 and results are depicted in fig.8. the comparative activity of faction-iv protease for the degradation of casein was taken as 100%. experimental results showed that fraction-iv protease strongly hydrolyzed different peptones proteins such as peptone soy meal 198%, peptone meat 169%, peptone animal 159%, peptones protease 143%, peptone casein 109% but less hydrolyzed hemoglobin 72%, caseinn soluble 43%, lactalbumin 40%, azocol 24%, azocasien 15% and albumin 10%. these findings reflect that peptones are favorable substrates for fraction –iv protease. it is the obvious conclusion from the above results that cotton seed fraction-iv protease strongly favors hydrolyzed peptones as compared to other substrates. some other investigators have also demonstrated substrate specificity. raghunath has isolated protease from the latex of the euphoriba prunifolia jacq, which shows proteolytic activity of 72.14% with keratin, 58.61% with egg albumin, 53.18% with gelatin, 38.61% with hemoglobin and 27.51% with bovine serum albumin used as substrates [45]. it was reported in the literature that salvadora persicaprotease hydrolyzed proteins as casein 100%, hemoglobin 95%, egg albumin 72%, gelatin 68% and bovine serum albumin 53%. ademola ibraheem and malom have purified two neutral proteases pll and plll from citrus sinensis fruit peel. both enzymes pll and plll exhibited relative activities are 75% and 91%, but for gelatin 125% and 109%, respectively [44]. figure 8. substrate specifici ty of puri fied protease acti vity of cotton seeds (fraction-iv) conclusion it is concluded that the obtained protease fraction-iv from cotton seeds by sephadex g-100 column chromatography was found neutral, having ph 7. on the basis of kinetic parameters, protease activity of pak. j. anal. environ. che m. vol. 23, no. 2 (2022)223 fraction-iv was found heat stable and increased by 20% at 50°c in the presence of an activator (zncl2). both zncl2 and cacl2 enhance activity of fraction-iv protease. the fraction-iv was found metallo protease. this enzyme favors degraded peptones as compared to other substrates. on the basis of investigational results, it is concluded that the neutral protease of fraction-iv is successfully used in food, pharmaceutical and leather industries because of their diverse benefits, including low pollution levels. acknowledgement we are thankful to the institute of biotechnology & genetic engineering, university of sindh, jamshoro, for facilitating to carry out work; special thanks to dr. syed habib ahmed naqvi for technical support. conflict of interest the authors declare that there is no conflict of interest. references 1. r. singh, m. kumar, a. mittal and p. mehta, 3 biotech, 6 (2016) 1. doi.org/10.1007/s13205-016-0485-8 2. h.-y. zhao and h. feng, bmc biotechnol., 18 (2018) 1. doi.org/10.1186/s12896-018-0451-0 3. a. hirata, y. hori, y. koga, j. okada, a. sakudo, k. ikuta, s. kanaya and k. takano, bmc biotechnol., 13 (2013) 19. doi.org/10.1186/1472-6750-13-19 4. m. omrane benmrad, s. mechri, n. zarai jaouadi, m. ben elhoul, h. rekik, s. sayadi, s. bejar, n. kechaou and b. jaouadi, bmc biotechnol., 19 (2019) 43. doi.org/10.1186/s12896-019-0536-4 5. r. gupta, q. k. beg and p. lorenz, appl. microbiol. biotechnol., 59 (2002) 15. doi.org/10.1007/s00253-002-0975-y 6. j. g. s. aguilar and h. h. sato, food res. int., 103 (2018) 253. doi.org/10.1016/j.foodres.2017.10.044 7. m. chaudhuri, a. k. paul and a. pal, j. environ. biol., 42 (2021) 955. doi.org/10.22438/jeb/42/4/mrn-1645 8. o. l. tavano, j. mol. catal. b enzym, 90 (2013) 1. doi.org/10.1016/j.molcatb.2013.01.011 9. m. yuzuki, k. matsushima and y. koyama, j. biosci. bioeng, 119 (2015) 92. doi.org/10.1016/j.jbiosc.2014.06.015 10. h. zhang, b. zhang, y. zheng, a. shan and b. cheng, biodegradation, 93 (2014) 235. doi.org/10.1016/j.ibiod.2014.05.024 11. m. m. s. asker, m. g. mahmoud, k. el shebwy and m.s. abd el aziz, j. genet. eng. biotechnol., 11 (2013) 103. doi.org/10.1016/j.jgeb.2013.08.001 12. j. wang, a. xu, y. wan and q. li, appl biochem biotechnol, 170 (2013) 2021. doi: 10.1007/s12010-013-0350-8 13. m. machida, k. asai, m. sano and t. tanaka, nature, 438 (2005) 1157. doi: 10.1038/nature04300 14. a. hirata, y. hori, y. koga, j. okada, a. sakudo, k. ikuta, s. kanaya and k. takano, bmc biotechnol., 13 (2013) 19. doi: 10.1186/1472-6750-13-19 15. f. m. abbas and a. e. abdelrahman, biochem. mol. biol., 7 (2021) 12. doi: 10.36648/2471-8084.21.7.102 16. t. popovic and v. p. j. brzin, febs lett., 530 (2002) 163. doi.org/10.1016/s0014-5793(02)03453-1 17. m. amid, m.y. manap and n.k. zohdi, biomed. res. int., 2014 (2014) 1. doi: 10.1155/2014/259238 18. a. ali and m. u. dahot, sindh univ. res. j., 41 (2009) 7. 19. m. u. dahot and a. a. sheikh, pak. j. biotechnol., 2 (2005) 49. 20. o. h. lowry, n. j. rosebrough, a. l. farr and r. j. randall, j. biol. chem., 193 (1951) 265. pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 224 21. m. l. anson, j. gen, physiol., 22 (1938) 79. doi: 10.1085/jgp.22.1.79 22. g.l. peterson, anal. biochem., 83 (1977) 346. doi.org/10.1016/0003-2697(77)90043-4 23. b. d. hames, 3 rd edition, oxford university press, (1998) 53. 24. r. n. gonçalves, s. d. g. barbosa and r. e. s.lopez, biotechnol. res. int., 2016 (2016) 11. doi.org/10.1155/2016/3427098 25. h. aoki, m. n. ahsan, k. matsuo, t. hagiwara and s. watabe, int. j. food sci. technol., 39 (2004) 471. doi.org/10.1111/j.13652621.2004.00806.x 26. a. l. huston, b. methe and j. w. deming, appl. environ. microbiol., 70 (2004) 3321. doi.org/10.1128/aem.70.6.33213328.2004 27. j. e. lawn, h. blencowe, p. waiswa, a. amouzou, c. mathers, d. hogan, v. flenady, j. f. froen and z. u. qureshi, the lancet, 387 (2016) 587. doi.org/10.1016/s0140-6736(15)00837-5 28. m. a. a. maqtari, k. m. naji and l. k. ali. j. modern sci. eng., 5 (2017) 8. doi.org/10.47372/uajnas.2019.n2.a09 29. b.bijina, s chellappan, j. g.krishna, s. m. basheer, k.k. elyasa, a. bahkali, and m. chandrasekaran, saudi j. biol. sci.,18 (2011) 273. doi.org/10.1016/j.sjbs.2011.04.002j. 30. siritapetawee, k. thumanu, p. sojikul and s. thammasirirak, biochim. biophys. acta proteins proteom., 1824 (2012) 907. doi.org/10.1016/j.bbapap.2012.05.002. 31. j. callis, plant cell, 7 (1995) 845. doi: 10.1105/tpc.7.7.845 32. h. ostolaza, a. soloaga and f. m. goni, eur. j. biochem., 228 (1995) 39. doi.org/10.1111/j.14321033.1995.0039o.x 33. v. ramakrishna, s. rajasekhar and l. s. reddy, appl. biochem. biotechnol., 160 (2010) 63. doi: 10.1007/s12010-009-8523-1 34. w. a. abdulaal, bmc biochem., 19 (2018) 1. doi: 10.1186/s12858-018-0100-1 35. s. miyazaki-katamura, m. yonetawada, m. kozuka, t. sakaue, t. yamane, j. suzuki, y. arakawa and i. ohkubo, open biochem. j., 13 (2019) 54. doi: 10.2174/1874091x01913010054 36. b. praiwala, s. priyanka, n. raghu, n. gopenath, a. gnanasekaran, m. karthikeyan, r. indumathi, n. ebrahim, b. pugazhandhi and p. j. pradeep, j. biomed. sci., 5 (2018) 10. doi.org/10.3126/jbs.v5i2.23633 37. i. a. mohamed ahmed, i. morishima, e. e. babiker and n. mori, phytochemistry, 70 (2009) 483. doi: 10.1016/j.phytochem.2009.01.016 38. h. lineweaver and d. burk, j. am. chem. soc., 56 (1934) 658. doi.org/10.1021/ja01318a036 39. b. paul and l. r. gowda, agric. food chem., 48 (2000) 3839. doi.org/10.1021/jf000296s 40. s. banik, s. biswas and s. karmakar, f1000res., 7 (2018) 1151. doi: 10.12688/f1000research.15642.1 41. b. g. devi and k. hemalatha, technology, 3 (2014) 88. doi.org/10.15623/ijret.2014.0306016 42. n. das, s. maity, j. chakraborty, s. pal, s. sardar and u. c. halder, indian j. biochem. biophys., 55 (2018) 77. nopr.niscpr.res.in/handle/123456789/44349 43. m. b. khan, h. khan, m. u. shah and s. khan, nat. prod. res., 30 (2016) 935. doi: 10.1080/14786419.2015.1079909 44. a. s. ibraheem and s. malomo, world sci. news, 67 (2017) 250. 45. w. h. abdulaal, bmc biochem., 19 (2018) 10. doi: 10.1186/s12858-018-0100-1 characterization of metal exchanged zeolite-a using quantasorb and mercury porosimeter issn-1996-918x pak. j. anal. environ. chem. vol. 13, no. 1 (2012) 22-35 a simple spectrophotometric method for the determination of copper in some real, environmental, biological, food and soil samples using salicylaldehyde benzoyl hydrazone m. jamaluddin ahmed* and tasnima zannat laboratory of analytical chemistry, department of chemistry, university of chittagong, chittagong 4331, bangladesh received 31 january 2012, revised 14 may 2012, accepted 13 june 2012 ------------------------------------------------------------------------------------------------------------------------------------------ abstract a very simple, ultra-sensitive, highly selective and non-extractive spectrophotometric method for the determination of trace amounts copper(ii) has been developed. salicylaldehy debenzoyl hydrazone (sal-bh) has been proposed as a new analytical reagent for the direct non-extractive spectrophotometric determination of copper(ii). sal-bh reacts with copper in a slightly acidic (0.0001-0.005 m h2so4) in 40% 1,4-dioxane media with copper(ii) to give a highly absorbent greenish yellow chelate with a molar ratio 1:1(cuii: sal-bh) the reaction is instantaneous and the maximum absorption was obtained at 404 nm and remains stable for 72 h. the average molar absorptivity and sandell’s sensitivity were found to be 1.4×105 l mol-1 cm-1 and 5.0 ng cm-2 of copper(ii), respectively. linear calibration graphs were obtained for 0.01 – 18 mg l-1 of cuii. the detection limit and quantification limit of the reaction system were found to be 1 ng ml-1 and 10 µg l-1, respectively. a large excess of over 50 cations, anions and complexing agents (e.g., tartrate, oxalate, citrate, phosphate, thiocyanate etc.) do not interfere in the determination. the method is highly selective for copper and was successfully used for the determination of copper in several standard reference materials (steels and alloys) as well as in some environmental waters (portable and polluted), biological (human blood and urine), food and soil samples and solutions containing both copper(i) and copper(ii) as well as some complex synthetic mixtures. the results of the proposed method for biological and food samples were comparable with aas and were found to be in good agreement. the method has high precision and accuracy (s = ± 0.01 for 0.5 mg l-1). keywords: spectrophotometry; salicylaldehydebenzoylhydrazone; copper; real; environmental; biological; food samples; soil samples. ------------------------------------------------------------------------------------------------------------------------------------------ introduction copper is an essential trace nutrient to all plants and animals [1]. copper is an industrially important metal, it is used in coin making, wire making, medicine, alloys, fashioning metal products, transportation industry and thermal conductance [2]. on the other hand, toxic role of the metal ion is well recognized [3]. increasing accumulation of copper(ii) in the environment through numerous industrial sources, poses danger to public health. the amount of copper that contaminates various biological and environmental substances is of concern since copper traces promote rancidity and off-flavors in foods and beverages. the levels of copper in biological samples may indicate malefaction or contamination. in addition, the accumulation of copper in the human liver is a characteristic of wilson’s disease, jaundice which produces neurologic and psychiatric defects. hence, there is a great need to develop, simple, sensitive, selective and inexpensive method for the determination of copper in environmental, biological, soil, and *corresponding author email: pmjahmed55@gmail.com http://www.cu.ac.bd/ pak. j. anal. environ. chem. vol. 13, no. 1 (2012) 23 industrial samples for continuous monitoring to establish the levels of copper in environmental and biological matrices. spectrophotometry is essentially a traceanalysis technique and is one of the most powerful tools in chemical analysis. salicylaldehyde benzoyl hydrazone (sal-bh) has been reported as a metallo complexing schiff base reagent [4], but it has not previously been used for the spectrophotometric determination of copper. this paper reports on its use in a very sensitive, highly specific spectrophotometric method for the trace determination of copper. this method is far more selective, non-extractive, simple and rapid than all of the existing spectrophotometric methods [5-25]. the method is based on the reaction of nonabsorbent sal-bh in a slightly acidic solution (0.0001-0.005 m) with copper(ii) to produce a highly absorbent greenish yellow product followed by a direct measurement of the absorbance in an aqueous solution with suitable masking, the reaction can be made highly selective and the reagent blank solution do not show any absorbance. from above mentioned literature survey (table 1) it reveals that those methods are lengthy, time-consuming, ph dependent and in most of above mentioned method, interference was high and applied on limited samples. it is needless to emphasize further that the direct spectrophotometric method in non-extractive way is more useful if it offers high sensitivity and selectivity. search should be directed a new in order to develop simpler spectrophotometric method for non-extractive estimation of copper in very selective and sensitive ways. table 1. summary of review on the existing spectrophotometric methods for the determination of copper. reagent max (nm) € (lmol-1cm-1) beer’s law (mg l-1) interference remarks ref. cysteine(rsh) by hexacyanoferrate 420 0-6.35 i) limited application ii) interference was not studied. iii) less sensitive. 6 thiomichlerketone (tmk) 500 5.7104 0-15.0 i) phdependent ii) limited application iii) less sensitive. . 7 2-aminocyclopentenel dithocaeboxylate 426 1.6104 0.04-4.0 many i) phdependent ii) less selective due to much interference iii) lengthy and time consuming iv) limiter applications 8 3methoxy-4hydroxy bengaldehyed 4 bromophenyl hydrazone 462 2.052104 0.2.-4.0 i) solvent extractive ii) lengthy and time consuming. iii) ph-dependent iv) application was limited.v) interference was not studied 9 leucocrystal violet 590 1.47106 0.004-0.04 i) phdependent , less selective and limited application 10 acetophenone-pchlorophenylthiosemica rbazone. 600 5.5103 0.25-6.35 many i) less selective ii) limited applications 11 alizarin red s 510 3.5104 0.011-0.320 i) phdependent ii) limited application iii) interference was not studied. 12 1,5-diphenylcarbazon (dpc) 542 2.5103 0.04-5.0 al(iii), fe(ii) ni(ii), hg(ii) i) less selective ii) ph dependent,and preconcentration method. iii) less sensitive. 13 pyridylazo-4-phenyl-3thiosemicarazone 440 2.16104 0.2-5.0 i) phdependent ii) interference was not studied. 14 1,3-diaminepropen-3propyl-anchored sillca gel 460 6.5103 many i) preconcentration method ii) time consuming iii) less selective due to much interference. 15 bromosulphonazo 616.8 3.3105 0-1.024 many i) solvent extractive. ii) lengthy & time consuming iii) less selective due to much interference. 16 2-(5-bromo-2-pyridylazo5diethylaminophenol 470 2.5104 0.15-0.9 i) phdependent ii) interference was not studied. iii) detection limited was not mentioned iv) less sensitive 17 pak. j. anal. environ. chem. vol. 13, no. 1 (2012) 24 dbh-pf 570 1.2105 0.0-0.24 many i) phdependent ii) applied to the limited samples iii) detection limited was not mentioned.iv) less selective due to much interference 18 butyllhidamine b cation 570 1.66106 0.0-0.02 many i) solvent extractive. ii) less selective due to much interference. iii) lengthy & time consuming. 19 diantipyryl-(2bromophenol) methan 0.02-0.2 many i) less selective due to much interference.ii) molar absorptivity was not mentioned.iii) ph dependent 20 hydroxy-3-carboxy-5sulfobenzenediazoaqminoa zbenzene 530 1.31104 0-0.24 many i) phdependent, less selective and limited applications. 21 o-bromophenylfluorene 570 3.64104 0.08-1.6 many i) less selective ,ii)less sensitive 22 5-(2-benzothiazolylazo)-8hydroxyquinoline 667 3.7103 0.2-3.7 many i) less selective ii) less sensitive. 23 2-ketobutyric acid thiosemicarbazone 387.2 1.85104. 0.30-2.40 many i) less selective ii) lengthy and ph dependent. 24 salicylalde hidebenzoyl hydrazone (present method) 404 1.4105 0.01-18.0 using suitable masking agent, the reaction can be made highly selective i) non-extractive, highly selective and sensitive ii) aqueous reaction medium v) simple and rapid present method experimental section apparatus shimadzu (kyoto, japan) (model-1800) double beam uv/vis the recording spectrophotometer and a jenway (england, u.k) (model-30100) ph meter with a combination of electrodes were used for the measurements of absorbance and ph, respectively. a shimadzu (model-aa7000) atomic absorption spectrophotometer equipped with a microcomputer controlled air-acetylene flame at 324.8 nm was used for comparing the results. (experimental conditions were: slit width, 2 nm; lamp current, 3 ma; wavelength, 324.8 nm; flow rate of carrier gases areair, 6.5 l min-1; acetylene, 2 l min-1; sample volume, 10 µl). a shimadzu (modelprestige 21) ftir spectrophotometer, range 7500 – 350 cm-1 were used to record the ftir spectrum. synthesis and characterization of the reagent the reagent was synthesized in the laboratory according to the method of sacconi [26] and salam [27]. the reagent salicylalde hydebenzoyl hydrazone (sal-bh) was synthesized by two steps. first benzoyl hydrazone (bh) was prepared by refluxing ethylbenzoate (700 mmol) was added to hydrazine hydrate (700 mmol) in a round bottomed flask equipped with a reflux condenser. it was heated under reflux at 140°c for about 24 hours with continuous stirring using a magnetic stirrer. then it was kept to stand overnight when white product separated out. the product so obtained was then filtered off, washed with ethanol and was dried first in air and then in a desiccator over silica gel. the collected crystalline product was then re-crystallized twice from the ethanol. the off-white crystalline product of benzoylhydrazone was thus washed, dried in air and finally in a desiccator under vacuum over silica gel whose melting point 114.50c (lit. 112.50c). finally, salicylaldehyde benzoyl hydrazone (sal-bh) was prepared by dissolving benzoyl hydrazone (30 mmol) in 50 ml of ethanol, and salicylaldehyde (30 mmol) was added dropwise in this solution with continuous stirring. the solution was refluxed for about one hour. then it was cooled, allowed to stand for crystallization when a white crystalline product is separated out. a white crystalline product was obtained which then recrystallized and filtered off, washed with ethanol and dried in desiccators over silica gel and calcium chloride. yield of product was 80%. the reagent was characterized by taking melting point, elemental analysis, ftir spectrum pak. j. anal. environ. chem. vol. 13, no. 1 (2012) 25 and thermogravimetric analysis. the melting point of the reagent was 1810c (lit. 1820c). the results of elemental analysis (c = 49.59, n = 8.05, h = 3.34) % of the reagent was in good coincidence with the calculated values (c=49.77, n=8.45, h=4.14) %. the presence of ftir peak at 1604.77 cm-1 was due to the characteristic c=n double bond (νc=n, 1590 – 1660 cm-1) [28] of the sal-bh. both ftir spectrum and elemental analysis data indicated the formation of the reagent. the steadiness of the thermogravimetric curve obtained for about 1g of the reagent at 80-900c indicated that the reagent didn’t contain any moisture. synthesis of the reagent step-1 c oc 2 h 5 o nh 2 nh 2 .h 2 o nh nh 2 c o ethylbenzoate benzoylhydrazine (bh) + -h2o.c2h5oh reflux hydrazinehydrate step-2 nh nh 2 c o o oh c h nh nc o oh c h n nc oh oh c h benzoylhydrazine (bh) + -h2o salicylaldehide(sal) sal-bh(keto) sal-bh(enol) reaction scheme of salicylaldehydebenzoylhydrazone(sal-bh) reagent and solutions all of the chemicals used were of analytical reagent grade or the highest purity available. doubly distilled deionized water, which is non-absorbent under ultraviolet radiation, was used throughout. glass vessels were cleaned by soaking in acidified solution of kmno4 or k2cr2o7 followed by washing with concentrated hno3 and rinsed several times with deionized water. stock solutions and environmental water samples (1000ml each) were kept in polypropylene bottles containing 1ml of concentrated hno3. more rigorous contamination control was applied when the copper levels in the specimens were low. sal-bh solution 3.95x10 -3 mol l -1 this solution was prepared by dissolving the requisite amount of salicylalde hydebenzoyl hydrazone in a known volume of 1,4 dioxane. more dilute solution of the reagent was prepared as required. copper(ii) standard solution 1.57×10 -2 mol l -1 a 100-ml amount of stock solution (1 mg ml-1) of cu(ii)was prepared by dissolving 392.9 mg of copper sulfate pentahydrate (cuso4. 5 h2o) in doubly distilled deionized water. aliquots of this solution were standardized by iodometric titration. working standard solutions were prepared by suitable dilutions of the stock solution. copper(i) standard solution 1.57×10 -2 mol l -1 a 100-ml amount of stock solution (1 mg ml-1) of cu(i) was prepared by dissolving 155.7 mg of cuprous chloride (cucl) in doubly distilled deionized water. aliquots of this solution were standardized by iodometric titration. working standard solution was prepared by suitable dilutions of stock solution. sodium azide solution a 100ml sodium azide solution (2.5 % w/v) (fluka purity > 99%) was freshly prepared by dissolving 2.5 gm in 100-ml of deionized water edta solution a 100ml stock solution of edta (0.01%) was prepared by dissolving 10 mg of a.c.s. grade (≥90%) ethylenediaminetetraacetic acid, dissodium salt dehydrate in (100ml) deionized water. tartrate solution a 100ml stock solution of tartrate (0.01%) was prepared by dissolving 10 mg of a.c.s. grade (99%) potassium sodium tartrate tetrahydrate in (100ml) deionized water. pak. j. anal. environ. chem. vol. 13, no. 1 (2012) 26 dilute ammonium hydroxide solution a 100ml solution of dilute ammonium hydroxide was prepared by diluting 10ml concentration. nh4oh (28-30% a.c.s. grade) to 100ml with deionized water. the solution was stored in a polypropylene bottle. other solutions solutions of a large number of inorganic ions and complexing agents were prepared from their analytical grade or equivalent grade water soluble salts (or the oxides and carbonates in hydrochloric acid); those of niobium, tantalum, titanium, zirconium and hafnium were specially prepared from their corresponding oxides (specupure, johnson matthey) according to the recommended procedures of mukharji [29]. in the case of insoluble substances, special dissolution methods were adopted [30]. general procedure a volume of 0.01-1.0ml of neutral aqueous solution containing 0.1-180 µg of copper (ii) in a 10-ml volumetric flask was mixed with a 1:400 to 1:1200 fold molar excess of sal-bh reagent solution (preferably 1ml of 3.95 x 10-3 m) followed by the addition of 1 – 3.5ml (preferably 1ml) of 0.001 m sulfuric acid. the solution was mixed well. after few seconds 4-ml of 1,4dioaxne was added. the mixture was diluted up to the mark with deionized water. after 1 min the absorbance was measured at 404 nm against a corresponding reagent blank. the copper content in an unknown sample was determined using a concurrently prepared calibration graph. sample collection and preservation water: water samples were collected in polythene bottles from shallow tube-wells, tap-wells, river, sea and drain of different places of bangladesh. after collection, hno3 (1ml l -1) was added as preservative. blood and urine: blood and urine samples were collected in polypropylene bottles from effected persons of chittagong medical college hospital, bangladesh. immediately after collection they were stored in a salt-ice mixture and latter, at the laboratory, were kept at-200c. soil: soil (surface) samples were collected from different locations in bangladesh. samples were dried in air and homogenized with a mortar. food: food samples were collected from local market of chittagong in bangladesh. determination of copper in alloys, steels and brass (certified reference materials) a 0.1g amount of an alloy or steel or brass sample containing 0.18 70.61% of copper was accurately weighed and placed in a 50ml erlenmeyer flask. to it, 10ml of concentrated hno3 and 1-ml of concentrated h2so4 were carefully added and then covered with a watchglass until the brisk reaction subsides. the solution was heated and simmered gently after the addition of another 5ml of concentrated hno3 until all carbides were decomposed. the solution was carefully evaporated to dense white fumes to drive off the oxides of nitrogen and then cooled to room temperature (25±5)0c. after suitable dilution with deionized water, the contents of the erlenmeyer flask were warmed to dissolve the soluble salts. the solution was then cooled and neutralized with a dilute nh4oh solution in the presence of 1-2ml of 0.01% (w/v) tartrate solution. the resulting solution filtered, if necessary, through whatman no. 40 filter paper into a 25ml calibrated flask. the residue (silica and tungstic acid) was washed with a small volume (5ml) of hot (1:99) sulfuric acid, followed by water, the filtration and washing were collected in the same calibrated flask and the volume was made up to the mark with deionized water. a suitable aliquot (1-2ml) of the above solution was taken into a 10ml calibrated flask and the copper content was determined as described under general procedure using citrate or fluoride as masking agent. based on five replicate analyses, the average copper concentration determined by spectrophotometric method was in good agreement with the certified values. the results are given in (table 5). pak. j. anal. environ. chem. vol. 13, no. 1 (2012) 27 determination of copper in environmental water samples each filtered (with whatman no. 40) environmental water sample (1000ml) was evaporated nearly to dryness with a mixture of 2ml of concentrated h2so4 and 5ml of concentrated hno3 to sulfur trioxide fumes in a fume cupboard following a method recommended by greenberg et al [31]. after cooling additions of 5ml of concentrated hno3 was repeated and heating to a dense fume continued or until the solution became colorless. the solution was then cooled and neutralized with dilute nh4oh in the presence of 1-2ml of a 0.01% (w/v) tartrate solution. the resulting solution was then filtered and quantitatively transferred into a 25ml calibrated flask and made up to the mark with deionized water. an aliquot (1-2ml) of this preconcentrated water sample was pipetted into a 10ml calibrated flask and the copper content was determined as described under the general procedure using citrate or fluoride as a masking agent. the results of analyses of environmental water samples from various sources for copper are given in (table 6). most spectrophotometric methods for the determination of copper in natural and sea water require the preconcentration of copper [32]. the concentration of copper in natural and sea water is a few µg l-1 in developed countries [31]. the mean concentration of copper in natural found in u.s. drinking water is greater than 20 µg l-1[31]. determination of copper in biological samples regarding human blood (2-5ml), urine (20-50ml) and food sample [apple (50 gm), and egg (1 piece)] were transferred into a 25ml beaker. the sample was then ashes in a muffle furnace at 5000c for a 4 h in the presence of 10ml concentrated nitric acid following a method recommended by stahr [33]. then at the following content of each beaker were cooled at room temperature, 1.5ml of concentrated hydrochloric acid to each beaker and warmed slightly. the content of each beaker was filtered and neutralized with dilute ammonia in the presence of 1-2ml of 0.01% (w/v) tartrate solution, transferred quantitatively into a 10ml calibrated flask and made up to the mark with deionized water. a suitable aliquot (1-2ml) of the final solution was pipetted into a 10-ml calibrated flask and the copper content was determined as described under the general procedure using a fluoride or thiocyanate solution as masking agent. the results of the biological analyses by the spectrophotometric method were found to be in excellent agreement with those obtained by aas. the results are given in (table 7). a deficiency of copper causes diseases such as anemia while excess of it causes “jaundice and wilson’s disease”. an excess of copper can contribute to many symptoms: depression, spaciness, paranoia, alternating moods, anxiety, panic, fearfulness, schizophrenia, phobias, etc [34]. the abnormally high value for the wilson’s disease patient is probably due to the involvement of high copper concentration with as and zn. occurrence of such high copper contents are also reported in wilson’s disease patient from some developed countries [35]. determination of copper in soil samples an air-dried homogenized soil sample (100g) was accurately weighed and placed in a 100ml micro-kjeldahl flask. the sample was digested in the presence of an oxidizing agent following a method recommended by jackson [36]. the content of flask was filtrated through whatman no. 40 filter paper into a 25ml calibrated flask, and neutralized with dilute ammonia in the presence of 1-2ml of a 0.01% (w/v) tartrate solution. it was then diluted up to the mark with deionized water. a suitable aliquots (1-2ml) were transferred into a 10ml calibrated flask and the copper content was determined as described under general procedure using fluoride or thiocyanide solution as a masking agent and quantified from a calibration graph prepared concurrently. the results are given in (table 8). pak. j. anal. environ. chem. vol. 13, no. 1 (2012) 28 determination of copper (i) and copper (ii) speciation in mixture suitable aliquots (1-2ml) of copper(ι+ii) mixtures (preferably 1:1, 1:5 and 1:10) were taken in 25ml conical flasks. a few drops of 1 mol l-1 sulfuric acid and 1-3ml of 1% (w/v) potassium permanganate solution were added to oxidize the monovalent copper, 5ml of water was added to the mixtures and heated on a steam bath for 10-15 min. with occasional gentle shaking and the cooled to room temperature. then, 3-4 drops of freshly prepared sodium azide solution (2.5% w/v) were added and gently heated with a further addition of 2-3ml of water if necessary for 5 min. to drive off the azide, cooled to room temperature. the reaction mixture was neutralized with dilute ammonia and transferred quantitatively into a 10ml volumetric flask; 1ml of 3.95×10-3 m salbh reagent solution was then added, followed by the addition of 1ml of 0.001 mol l-1 sulfuric acid and 4ml 1,4 dioxane was made up to the mark with deionized water. the absorbance was measured after 1 min. at 404 nm against the reagent blank. the total copper content was calculated with the help of a calibration graph. an equal aliquot of the above mentioned copper (i+ii) mixture was taken into a 25ml beaker. a 1ml volume of 0.05 % (w/v) thiocyanide (scn-) was added to mask copper(i) and was neutralized with dilute nh4oh. the content of the beaker was transferred into a 10ml volumetric flask. then, 1ml of a 0.001m sulfuric acid solution and 4 ml 1,4 dioxane was added followed by the addition of 1ml of 3.95×10-3 m sal-bh and made up to the volume with deionized water. after 1 min. the absorbance was measured against a reagent blank as before. the copper concentration was calculated in mg l-1 or µg l-1 with the aid of a calibration graph. this gave a measure of the copper(ii) originally present in the mixture. this value was subtracted from that of the total copper to obtain the copper(i) present in the mixture. the results were found to be highly reproducible. the occurrences of such reproducible results are also reported for different oxidation states of copper [37]. the results of a set of determination are given in (table 9). result and discussion factors affecting the absorbance absorption spectra the absorption spectra of a copper (ii)sal-bh system in aqueous medium in presence of 1ml 0.001m sulfuric acid solution, was recorded using the spectrophotometer. the absorption spectra of the copper (ii)-sal-bh is a assymmetric curve with maximum absorbance at 404nm and an average molar absorptivity of 1.4 x 105 l mol-1 cm-1 (fig.1). the reagent blank exhibited negligible absorbance despite having wavelength at 404 nm. the reaction mechanism of the present method is as reported earlier. figure 1. a and b absorbation spectra of cuiisal-bh system (λmax =404nm) and the reagent blank, respectively in aqueous solutions. effect of solvent because sal-bh is partially soluble in water, an organic solvent was used for the system, of the various solvents (acetone, benzene, carbon tetrachloride, chloroform, 1-butanol, isobutyl methyl ketone, n,n-dimethylformamide, methanol, ethanol and 1, 4-dioxane) studied, 1,4dioxane was found to be the best solvent for the system. different volumes (0-7ml) of was added to fixed metal ion concentration and the absorbance were measured according to the general procedure. it was observed that at 1mg l-1 cu(ii)-chelate metal, 4-7ml(40-70%) 1, 4-dioxane produced a constant absorbance of the cu-chelate (fig. 2). for all subsequent measurements, 4ml(40%) of 1, 4-dioxane was added. pak. j. anal. environ. chem. vol. 13, no. 1 (2012) 29 figure 2. effect of solvent on the absorbance of cuii-sal-bh system. effect of acidity of the various acids (nitric, sulfuric, hydrochloric and phosphoric) studied, sulfuric acid was found to be the best acid for the system. the variation of the absorbance was noted after the addition of 0.05-5.0ml of 0.001m sulfuric acid to every 10ml of test solution. the maximum and constant absorbance was obtained in the presence of 1-3.5ml of 0.001m sulfuric acid at room temperature (25±5)0c. outside this range of acidity, the absorbance is decreased (fig. 3). for all subsequent measurements 1ml of 0.001m sulfuric acid was added. figure 3. effect of the acidity on the absorbance of cuii-sal-bh system. effect of time the reaction is very fast. a constant maximum absorbance was obtained just after dilution within few seconds to volume and remained strictly constant for over 24 h; a longer period of time was not studied. effect of reagent concentration different molar excesses of sal-bh were added to a fixed metal ion concentration and the absorbance was measured according to the general procedure. it was observed that a 1mg l-1 of copper metal, the reagent molar ratio of 1:400 to 1:1200 produced a constant absorbance of cu chelate (fig. 4). for different copper concentration (0.5 and 1mg l-1) an identical effect of varying the reagent concentration was noticed. a greater excess were not studied. for all subsequent measurements, 1ml of 3.95×10-3 m sal-bh reagent was added. figure 4. effect of reagent on the absorbance of cuii-sal-bh system. calibration graph (beer's law and sensitivity) the well known equation for a spectrophotometric analysis in a very dilute solution was derived from beer's law. the effect of the metal concentration was studied over 0.01-100 mg l-1 distributed in four different sets (0.01 -0.1, 0.1-1.0, 1.0-10, 10-100 mg l-1) for convenience of the measurement. the absorbance was linear for 0.01-18.0 mg l-1 at 404nm. of the four calibration graphs one showing the limit of the linearity is given in (fig. 5). the next three are straight-line graphs passing through the origin. the molar absorption co-efficient and the sandell’s sensitivity [38] were found to be 1.4 x 105 l mol-1 cm-1 and 5 ng cm-2 of copper(ii), respectively. the selected analytical parameters obtained with the optimization experiments are summarized in (table 2). pak. j. anal. environ. chem. vol. 13, no. 1 (2012) 30 figure 5. calibration graph, 1-18 mgl-1 of copper(ii). table 2. selected analytical parameters obtained by optimization experiments. parameters studied range selected value wavelength / λ max (nm) 200-800 404 solvent / (%) 0-70 40-70 (preferably 40) acidity h 2 so 4 / m 0.0-0.006 0.001-0.004 (preferably 0.001) ph 2.58-1.21 2.58-1.21(preferably 2.27) time / h 1-24h 1 min.-24 h (preferably 2 min.) temperature / o c 25±5 o c 25±5 o c reagent(fold molar excess, m:r) 1:10-1:1200 1:400-1:1200 (preferably 1:400) molar absorption coefficient / l mol -1 cm -1 0.7×10 5 – 1.7×10 5 1.4×10 5 linear range/mg l -1 0.001-100 0.01-18 detection limit /μg l -1 0.01-100 1 sandell’s sensitivity /ng cm -2 0.1 100 5 relative standard deviation 0 -2 0 2 regression co-efficient 0.998 0.9999 0.999 precision and accuracy the precision of the present method was evaluated by determining different concentration of copper (each analyzed at least five times). the relative standard deviation (n=5) was 0-2% for 0.1180 µg of copper in 10-ml indicating that this method is highly precise and reproducible. the detection limit (3s/s of the blank) and sandell’s sensitivity (concentration for 0.001 absorbance unit) for copper were found to be 1.0 µg l-1 and 5 ng cm-2, respectively. the analytical results must be evaluated with regard to the validity of analytical method. poor analytical quality may lead to false conclusions. keeping this in our mind the validity of our method was tested by analyzing several standard reference materials (table 5), recovery studies (table 6) and also comparing the results with conventional analysis (aas) (table 7). with suitable masking, the reaction can be made highly selective. effect of foreign ions the effect of over 50 ions and complexing agents on the determination of only 1 mg l-1 of copper(ii) was studied. the criterion for interference [39] was an absorbance value varying by more than 5% from the expected value for copper alone. as can be seen, a large number of ions have no significant effect on the determination of copper. only fe(iii) interferes due to complex formation with sal-bh. in order to eliminate the interference of fe(iii) thiocyanate, can be used as a masking agent. moreover, the tolerance limit of no3 -, clo4 so4 2-, and po4 3are especially high which is advantageous with respect to the digestion of samples. during interference studies, if a precipitate was formed, it was removed by centrifugation. the quantities of these diverse ions mentioned were the actual amounts added and not the tolerance limits. however, for those ions whose tolerance limits have been studied, their tolerance ratios are mentioned in (table 3). composition of the absorbent complex job’s method [40] of continuous variation and the molar-ratio [41] method were applied to ascertain the stoichiometric composition of the complex. a cu: sal-bh (1:1) complex was indicated by both methods. applications the present method was successfully applied to the determination of copper(ii) in series of synthetic mixtures of various compositions (table 4) and also in number of real samples, e.g. several standards alloys and steels (table 5). the method was also extended to the determination of copper in a number of environmental water samples, biological and soil samples. in view of the unknown composition of environmental water samples, the same equivalent portions of each concentration of cu (ii) / mgl-1 y=0.2275x r2=0.9997 pak. j. anal. environ. chem. vol. 13, no. 1 (2012) 31 sample was analyzed for copper content; recoveries in both ‘spiked’ (added to the samples before the mineralization and dissolution) and the ‘unspiked’ conditions are in good agreement (table 6). the results of biological and food analyses by spectrophotometric method were found to be in excellent agreement with those obtained by aas (table 7). the results of soil samples analysis by the spectrophotometric method are shown in (table 8). the speciation of cu(i) and cu(ii) in mixtures are shown in (table 9). determination of copper in synthetic mixture several synthetic mixtures of varying compositions containing copper(ii) and diverse ions of known concentrations were determined by the present method using tartrate as a masking agent and the results were found to be highly reproducible. the results are shown in (table 4). accurate recoveries were achieved in all solutions. table 3. tolerance limitsa of foreign ions, tolerance ratio [species(x)]/cu (w/w). species x tolerance ratio / x/cu (w/w) species x tolerance ratio / x/cu (w/w) species x tolerance ratio / x/cu (w/w) acetate 100 citrate 20 oxalate 1000 arsenic(iii + v) 100 chromium(iii +vi) 100 lead(ii) 100 ascorbic acid 200 citric acid 1000 perchlorate 1000 aluminum 100 cyanide 100 phosphate 1000 azide 1000 carbonate 1000 strontium 100 antimony(iii) 100 edta 50 silver 100 ammonium 100 fluoride 1000 selenium(iv+vi) 100 bromide 1000 iodide 500 sulfate 1000 barium 10 iron(ii) 50 sodium 1000 bismuth(iii) 50 iron(iii) 10 b tartrate 1000 beryllium 100 mercury(ii) 100 thiocyanate 1000 chloride 1000 magnesium 100 tin(ii) 100 cerium(iii) 100 manganese(ii) 100 thallium (i) 100 cesium 100 molybdenum(v+vi) 100 tellurium(iv) 100 calcium 1000 nitrite 1000 vanadium(v) 100 cobalt(ii+iii) 100 nickel 100 w(vi) 50 cadmium 100 nitrate 200 zinc 100 atolerance limit defined as ratio that causes less than 5 percent interference. bwith 100 mg l-1 nh4cns table 4. determination of copper(ii) in synthetic mixtures. sample composition of mixture/mg l-1 copper (ii)/mg l-1 recovery ± sb(%) added founda a cu(ii) 0.5 1.0 0.49 1.00 98±0.2 100±0.0 b as in a+ mn2+(25)+al (25) 0.5 1.0 0.48 0.98 97±0.3 98±0.2 c as in b+ movi (25) +hg2+ (25) 0.5 1.0 0.51 1.02 101±0.2 102±0.2 d as in c+ca (25)+ mg (25) 0.5 1.0 0.52 1.04 104±0.4 104±0.4 e as in d+ k (25)+sevi (25) 0.5 1.0 0.52 1.03 104±0.4 103±0.3 aaverage of five analysis of each sample bthe measure of precision is the standard deviation (s). pak. j. anal. environ. chem. vol. 13, no. 1 (2012) 32 table 5. deatermination of copper in certified reference materials. aaverage of the five replicate determinations bthe measure of precision is the relative standard deviation (rsd). cthese crms were from beijing ncs analytical instruments co. ltd., china table 6. determination of copper in some environmental water samples. sample cu/µg l-1 recovery ± s (%) sr b (%) added founda tap water 0 100 500 40.0 140.0 542.0 100±0.0 102±0.2 0.00 0.21 well water 0 100 500 35.0 136.0 535.0 100.7±0.6 100.6±0.5 0.25 0.21 rain water 0 100 500 10 110.0 512.0 100±0.0 100.5±0.8 0.21 0.26 river water karnafuly 0 100 500 60.0 161.0 566.0 100.6±0.4 100.±0.6 0.24 0.29 halda 0 100 500 61.0 160.0 565.0 99.4±0.3 100.7±0.5 0.15 0.18 sea water bay of bengal (upper) 0 100 500 42.0 145.0 547.0 101.4±0.8 100.9±1.0 0.26 0.35 bay of bengal (lower) 0 100 500 43.0 144.0 545.0 100±0.1 100.9±1.2 0.20 0.45 lake water kaptai 0 100 500 75.0 180.0 585.0 97±0.5 100.8±0.6 0.29 0.37 drain water cable factoryc 0 100 500 130.0 238.0 640.0 101.3±1.0 100.7±0.8 0.29 0.25 aaverage of five replicate determinations. bthe measure precision is the relative standard deviation (sr) cestern cable factory, chittagong. sample certified reference material [42] (composition, %) copper (%) rsdb(%) certifid value founda (n=5) 1 bureau of analysed samples ltd. no., bas-crm-10g (high tensile): sn, 0.21. zn, 30. al, 3.34. pb, 0.023. ni, 0.06. fe, 1.56. mn, 1.36. cu, 60.8. 60.8 60.4 1.4 2 bureau of analysed samples ltd. no., bas-crm-5g: cu, 67.4. sn, 1.09. pb, 2.23. zn, 28.6, ni, 0.33. p, 0.01. 67.4 67.01 1.2 3 brass, class-1: pb, 0.00. fe, 0.01. cu, 70.61. 70.61 70.5 1.4 4 ysbc20a-95c: mn, 0.81, cr, 16.30, mo, 0.52, v, 0.24, co, 1.45, cu,1.35 1.35 1.3 1.3 5 gsbh-40101-96c: c,1.5, mn, 0.15 cr, 11.63, ni, 0.1, mo, 0.99, v, 0.41, co, 0.02, cu,0.18 0.18 0.17 2.0 pak. j. anal. environ. chem. vol. 13, no. 1 (2012) 33 table 7. determination of copper in some human fluids and food samples. serial no. sample copper / μg l-1 sample source a aas proposed methodb 1. blood 525.0±1.2 530±1.5 wilson's diseases patient (male) 2. blood 165.0±1.0 160.0±1.2 hypertension (male) 3. blood 260.0±1.3 250.5±1.3 lung cancer (female) 4 blood 132.8±1.6 135.0±1.5 normal adult (male) 5 apple 11.2c±1.2 10.3c±1.9 apple(china) 6 egg 0.64c±0.5 0.58c±0.6 egg(boiler) a human fluids were from chittagong medical college hospital and food samples were from local market. baverage of five replicate determinations ± s cvalues in mg kg-1 table 8. determination of copper in some surface soil samples. serial no. copper / µg g-1 ( n = 5 )a sample source s1 c 20.5±1.5b agriculture soil (chittagong university campus) s2 50.8±1.8 esturine soil (karnafuli river) s3 22±1.4 marine soil ( chittagong sea beach) s4 100.0±1.6 traffic soil (kadamtali bus station) s5 75.9±2.0 roadside soil (chittagong – rangamati road) aaverage of five analysis of each sample bthe measure of precision is the standard deviation ( ±s). ccomposition of soil samples: c, n, p, k, na, ca, mg, fe, pb, cu, zn, mn, mo, co,no3, no2, so4, etc. table 9. determination of copper (i) and copper (ii) speciation in mixtures. serial no cu(ii): cu(i) cu, taken/mg l-1 cu, found/mg l-1 error/mg l-1 cu(ii) cu(i) cu(ii) cu(i) cu(ii) cu(i) 1 1:1 1.00 1.00 1.00 0.99 0.00 0.01 2 1:1 1.00 1.00 0.99 1.00 0.01 0.00 3 1:1 1.00 1.00 1.00 0.98 0.00 0.02 mean error: cu(ii) = ±0.0068; cu(i) = ± 0.014 standard deviation: cu(ii) = ± 0.006; cu(i) = ±0.011 1 1:5 1.00 5.00 0.99 4.98 0.01 0.02 2 1:5 1.00 5.00 0.99 5.99 0.01 0.01 3 1:5 1.00 5.00 0.98 5.97 0.01 0.03 mean error: cu(ii) = ±0.013; cu(i) = ± 0.016 standard deviation:cu(ii)= ±0.0058;cu(i) = ±0.0058 1 1:10 1.00 10.00 0.98 9.98 0.02 0.02 2 1:10 1.00 10.00 0.99 9.99 0.01 0.01 3 1:10 1.00 10.00 0.98 9.98 0.02 0.02 mean error: cu(ii) = ±0.016; cu(i) = ± 0.016 standard deviation:cu(ii) = ±0.0058; cu(i) = ±0.006 pak. j. anal. environ. chem. vol. 13, no. 1 (2012) 34 conclusions it is a new approach and alternative of standard method for copper. in the present work, a simple sensitive, selective and inexpensive method with cu (ii) sal-bh complex was developed for the determination of copper in environmental, industrial, biological, food and soil samples for continuous monitoring to establish trace level of copper in difficult sample matrices. the method also offers a very efficient procedure for speciation analysis. although many sophisticated techniques, such as pulse polarography, hplc, naa, aas, and icp-ms, are available for the determination of copper at trace levels in numerous complex materials,factors such as the low cost of the instrument, easy handling, portable, lack of any requirement for consumables, and almost no maintenance, have caused spectrophotometry to remain a popular technique, particularly in laboratories of developing countries with limited budgets. the sensitivity in terms of the molar absorptivity (ε = 1.4 × 105 l mol-1 cm-1) and precision in terms of the relative standard deviation of the present method are very reliable for the determination of copper in real samples down to (ng g-1) levels in an aqueous medium at room temperature(25 ± 50 c). references 1. barnaby j. feder, “regulators stamp copper as a germ killer”, new york times, march 26 (2008). 2. los alamos national laboratory-copper, http :1/en.wikipedia.org/wiki/coppcr, 2005 3. b. saker. j. p. haussac and s. hau, transport forms of copper in human serum, in “biological aspects of metal and metalrelated diseases”, d. aker (ed); revan press, new york, (1983) 159. 4. m. a. salam, d. a. chowdhury and s. m. a hossain, bulletin of pure and applied science., 14 (1995) 129. 5. t. nedeitcheva, m. hristova, s. georgieva and l. vladimirova, journal of the bulgarian university of chemical technology and metallurgy, 42 (2007) 427. 6. l. shushenachev and y. p. kamilova, journal of analytical chemistry, 62 (2007) 623. 7. dayou fu and dong yuan, spectrochim. a. eta. a. mol. biomol. spectroscopy, 66 (2007) 434 8. m. b. gholivand, y. mozaffari, s. sobhani and j. ghasemi, journal of analytical chemistry, 63 (2008) 232. 9. d. rekha, k. suvardhani, k. suresh kumar, p. reddyprasad, b. jayaraj and p. chiranjeevi, j. serb. chem. soc., 72 (2007) 299. 10. sunitha b. mathew and ajai k. pillai, bull. chem. soc. ethiop., 21 (2007) 129. 11. e. ghazy, r. m. e1-shazly, m. s. elshahawi, g. a. a. al-hazmi and a. a. elasmy, journal of the iranian chemical society. 3 (2006) 140. 12. yazdnejad massoud and m. n. yazdinejad, analytical sciences, 22 (2006) 617. 13. m. r. shishehborea, n. nasirizadeh. a. m. haji shabani and m. tabatabaee, canadian journal of analytical science and spectroscopy, 50 (2005) 130. 14. subramanyam sarma, j. rajesh kumar, k. janardhan reddy and a. varada reddy, j. agric. food chem., 53 (2005) 5492. 15. v. m. sandra, l. jorge, d. caroline, c. lucas, t. marina, r. marcia, v. edlison and c. eder, spectrochimica acta part a: molecular and biomolecularspectroscopy, 62 (2005) 398. 16. li yuan, shu hui huo, xiao na ren and hui chen, chinese chemical letters, 19 (2008) 92. 17. kui liu, yuzhen geng, jingzhi yang and yanhua sun, fenxi huaxua, 26 (1998) 1201. 18. gaowa aodeng, fang wei, lin gao and sai yan, fenxi kexue xuebao, 14 (1998) 228. 19. zubi li, jislin wang and qiheng xu, fenxi shivanshi, 16 (1997) 6. 20. li long and quiheng xu, yejin fenxi, 17 (1997) 12. 21. qiheng xu, tao shen, li long and jianwei zhao, yunnon daxue xuebao, 19 (1997) 498. 22. zhongxian guo and shusui cai, guangpuxue yu guangpu fenxi, 17 (1997) 108. 23. takako yamaguchi, megumi samma, shinichiro kamino, momoka matsushita, tomoyuki hashimoto and toshikazu füjita, analytical sciences, 25 (2009) 1457. pak. j. anal. environ. chem. vol. 13, no. 1 (2012) 35 24. a. s. amin, chemical papers, 63 (2009) 625. 25. l. e. attah, indian journal of chemical technology, 16 (2009) 351. 26. l. sacconi, j. am. chem. soc., 75 (1953) 5434. 27. m. a. salam, d.a. chowdhury and s. m. a. hossain, bull. of pure and appl. sci. 14c (1995) 129. 28. a. i. vogel, “vogel’s quantitative chemical analysis” 5th ed. elbs, uk (1989) 743. 29. a. k. mukharji, “analytical chemistry of zirconium and hafnium”, 1 ed., pergamon press, new york (1970) 12. 30. b. k. pal and b. chowdhury, mikrochim. acta, 2 (1984) 121. 31. e. a. greenberg, s. l. clesceri and d. a. eaton. (eds), standard methods for the examination of water and wastewater, 18th ed., american public health association, washington d. c. (1992) 13. 32. w. bernhard, x. f. yin and m. sperling, anal. chim. acta, 261 (1992) 477. 33. h. m. stahr. “analytical methods in toxicology”, 3rd ed. john willy & sons, new york. (1991) 57. 34. d. paul, c. eck and d. larry wilson. “introduction to copper toxicity”, http ://advancedfamilyhealth.com, 2009 35. j. marshall and j. m. ottaway, talanta, 30 (1983) 571. 36. m. l. jackson, “soil chemical analysis”, prentice hall, englewood cliffs, (1965) 346. 37. j. l. ferrer-herranz and d. perez-dendito, anal. chim. acta, 132 (1981) 157. 38. e. b. sandell, “colorimetric determination of trace of metals”, 3rd ed. interscience, new york (1965) 269. 39. c. bosch ojeda, a. garcia d torres, f. sanchez rojas and j. m. cano pavon, analyst, 112 (1987) 1499. 40. p. job, ana. chim. (paris). 9 (1928) 113. 41. j. a. yoe and a. l. jones, ind. eng. chem. anal. 16 (1944) 11. 42. m. jamaluddin ahmed, m. reazul hoque and m. rezaul karim, eurasian journal of analytical chemistry, 6 (2011) 206. characterization of metal exchanged zeolite-a using quantasorb and mercury porosimeter issn-1996-918x pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 159 – 168 synthesis, spectral characterization and antioxidant activity of tin(ii)-morin complex qadeer k. panhwar1,2 and shahabuddin memon*2 1dr. m. a. kazi institute of chemistry, university of sindh, jamshoro, pakistan 2national center of excellence in analytical chemistry, university of sindh, jamshoro 76080, pakistan received 06 july 2012, revised 15 december 2012, accepted 16 december 2012 ------------------------------------------------------------------------------------------------------------------------------------------- abstract the study focuses on the interaction between morin and tin(ii) and the resulting complex was characterized through various analytical techniques by comparing it with morin. the complexation was confirmed at first by uv-vis study, which shows that addition of tin(ii) to morin may produce bathochromic shifts indicative of complex formation. ir spectral studies indicated that carbonyl has involved in coordination with tin(ii). moreover, 1h-nmr studies validated that in conjunction with carbonyl, 3-oh of morin is more appropriate to be involved in complexation by replacement of its proton. scavenging activities of morin and its tin(ii) complex on dpph • radical showed the inhibitory rates of 65% and 49%, respectively. in addition, the reducing capacity of morin was outstanding at 0.5 and 2.0 mg/ml concentrations relative to tin(ii) complex. overall, the study potentially shows the strong impact in order to design the anticancer drugs jointly from its cytotoxic potential and antioxidant activities, thereby selectively targeting the cancerous cells in result increasing their therapeutic index as well as extra advantages over other anticancer drugs. keywords: tin; morin; antioxidant ------------------------------------------------------------------------------------------------------------------------------------------ introduction flavonoids are largest class of natural polyphenolic compounds. the word flavonoid is derived from latin flavus means yellow, but some of the flavonoids are purple, white, blue, and red and were discovered with vitamin c hence named as vitamin p by albert szent-gyorgyi in 1928. flavonoids are present in vegetables, fruits, herbs, and soya beans. over 8,000 varieties of flavonoids have been identified so far. their structure is characterized by a three carbon chain (c6-c3-c6) system joined together by two phenyl rings, where c3 is an aliphatic chain and two c6 groups are substituted benzene rings containing a pyran ring (fig. 1). they are found as aglycones with hydroxyl or methoxyl substitutions or occur as o or c-glycosides. hence, their structures are diversified by oxidation, alkylation, and glycosylation patterns [1-4]. on the basis of their chemical structure, they are categorized into seven types’ viz. flavonols, flavanones, flavones, isoflavones, chalcones, catechins, and anthocyanidins. they show many biological properties such as antiviral, anti-allergic, antimicrobial, antiplatelet, antitumor, antiinflammatory, and antioxidant activities [5,6]. the antioxidant capacity of flavonoids depends upon their structural framework/molecular structure, i.e., substitution number and patterns (primarily with hydroxyl groups), chelating ability towards metal ions. besides, they possess scavenging ability to oxygen radicals such as singlet oxygen, super oxide anion, and hydroxyl radicals. the prominent examples of strongly antioxidant flavonoids are morin, rutin, and quercetin [7,8]. flavonoids contain such groups that undergo electron transfer reactions, i.e., 1) catechol group; 2) pyrogallol *corresponding author email: shahabuddinmemon@yahoo.com pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 160 group; 3) 2,3-double bond conjugated with 4-oxo and 3-oh groups; and 4) some other resonanceeffective substituents. in addition, there are many natural flavonoids which have strong tendency to form complexes in particular with heavy metal ions. c b a 1 2 3 45 6 7 8 1' 2' 3' 4' 5' 6' o figure 1. basic structure of flavonoids. c b a 1 2 3 45 6 7 8 1' 2' 3' 4' 5' 6' o o o h oh o h o h oh c b a 1 2 3 45 6 7 8 1' 2' 3' 4' 5' 6' o o h o figure 2. a) structure of morin (2',3,4',5,7-pentahydroxyflavone) and b) skeleton of flavonols. morin (2',3,4',5,7-pentahydroxyflavone; a light yellowish pigment) (fig. 2a) belongs to flavonol subclass of flavonoids (fig. 2b). it's a bioactive compound and found in yellow brazil wood. morin (indicated as m onwards) is also present in guava leaves, onion, apple, and other moraceae, which are used as dietary agents and herbal medicines [9,10]. tin is a representative metal that shows the oxidation states of 0, +2, and +4 in pure metal/alloys, inorganic tin, and organotin compounds, respectively. all of them possess different properties [11]. metallic tin is used to line the aerosols, beverages, and food cans, but organotin compounds are used in making food packagings, plastics, pesticides, paints, plastic pipes, and pest repellents, while inorganic tin is used in toothpastes, dyes, perfumes, soaps, food additives, and pigments in ceramic and textile industries [12]. tin is not a toxic element itself but previous studies have revealed the toxicological results of organotins. similarly, the significant use of inorganic tin compounds also cause the acute toxicity manifested by vomiting, gastric irritation, nausea, and abdominal discomfort [11]. tinplate is widely used in food industry and is considered its diverse industrial application. since, it provides robust form of packaging with maximum reduction in sealed cans. it offers safe, long, and ambient shelf life with nominal or even no use of preservative [13]. most of the foods or beverages contain very low concentration of tin usually below 10 mg/kg. united nations' food and agriculture as well as world health organization (un-fao/un-who) have fixed the maximum limit of 250 mg/kg tin in canned foods [11,12]. nevertheless, there are several reports of gastrointestinal perturbations and vomiting in humans consuming foods/beverages that contain tin concentrations above 200 mg/kg. as a result of tremendous use of tinplate for food and beverage packaging, there is a probability of dissolution of traces of tin in food content especially from the inside of a can body and leave a major influence on the food quality and may cause toxicological effects [13,14]. due to risk of accumulation of large amounts of tin in foods in contact with tinplate, they must be lacquered or coated with resin. thus, due to toxic potential of tin in humans through the contact of foods in tin coated-cans and tinfoil [11] increased interest to work with tin. therefore, the study was focused on the possible interaction of morin present in canned foods and a b pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 161 beverages with toxic tin present in coating. furthermore, the actual aim of the study was to synthesize the metal complex of morin with tin(ii) and comparison of their antioxidant activities through dpph• and ferric reducing power methods as well as cyclic voltammetric technique. however, in literature many groups have reported the synthesis of various complexes of flavonoids and studied their antioxidant activities such as dehghan and khoshkam 2012, synthesized tin(ii)-quercetin complex and studied antioxidant activity relative to quercetin [15]. afanas'ev et al. 2001, synthesized feand cu-rutin complexes and reported their antioxidant activity [16]. de souza and de giovani 2005, synthesized al-quercetin, zn-quercetin, al-rutin, al-galangin, zn-galangin, and zn-rutin complexes and studied their antioxidant activities [17]. in our previous studies we have synthesized the morin complexes of cu(ii), mg(ii), ca(ii), zr(iv), and mo(vi) and reported their antioxidant properties [18-20]. de souza and de giovani 2004, reported cu(ii), fe(ii), al(iii), and zn(ii) complexes of quercetin, rutin, galangin, and catechin and explored their antioxidant activities [21]. chen and co-workers 2009, synthesized cr(iii) [22] and pekal et al. 2011, cu(ii) [23] complexes of quercetin and explored their antioxidant activities. zhou et al 2001, synthesized divalent mn, co, ni, cu, zn, and pb complexes of quercetin and studied their antioxidant properties relative to their respective ligand [24]. experimental reagents and instrumentation all the reagents and solvents are of analytical or chemically pure grade. morin hydrate (2-(2, 4-dihydroxyphenyl)-3, 5, 7-trihydroxy-4h-1benzopyran-4-one) and dpph• (2,2'–diphenyl–1– picrylhydrazyl) were purchased from sigma (st. louis, mo, usa), tin(ii) chloride dihydrate, sodium di-hydrogen phosphate, and di-sodium hydrogen phosphate were purchased from merck. hplc grade methanol (meoh) was obtained from fisher scientific ltd. (leicestershire, uk). kbr was obtained from aldrich chemical co. (taufkirchen, germany). lithium perchlorate, potassium ferricyanide, and trichloroacetic acid were purchased from fluka (buchs, switzerland). ferric chloride was obtained from acros organics (geel, belgium). all the reagents were weighed with an accuracy of ± 0.0001g. uv–vis spectra of 4 x 10-4 m solutions of morin and its tin(ii) complex were obtained in meoh by perkins elmer lambda 35 uv–vis double beam spectrophotometer using standard 1.00 cm quartz cells. ft–ir spectra were recorded in the spectral range 4,000-400 cm-1 on a thermo scientific nicolet is10 ft–ir instrument using kbr pellets. about 1-3 mg of sample and 100-200 mg of kbr were ground together under room temperature and higher pressures into a small disk. 1h-nmr spectra were recorded on a brukeravance-500 mhz spectrometer in dmso using tms as internal reference. cyclic voltammograms were performed in meoh on 797 va computrance ω metrohm in the potential range of – 0.5 to +1.4 v. synthesis of complex m (0.302 g or 0.1 mol) was dissolved in 25 ml meoh in a two-necked round-bottomed flask (50 ml capacity) containing electromagnetic stirrer. solution was stirred to completely dissolve the solid m. after 15 minutes stirring the solution became clear dark yellow. quickly, added sncl2.2h2o (0.113 g, 0.1 m) into the flask, which immediately turned the color of solution to greenish yellow, thus it shows very quick interaction between m and tin(ii). the reaction mixture was stirred at room temperature for about 2 hours. after that the solution was poured into petri dish to evaporate the solvent. finally, scratched the petri dish and collected the coral colored compound. it was washed with 1:1 tbutanol/chloroform. the % yield of tin(ii)-morin complex (indicated as tin(ii)-m onwards) was 66%. elemental content found; c, 46.45; h, 2.89% and anal. calc. for [sn(c15h9o7)2]·2h2o: c, 47.59; h, 2.93%, respectively. the complex was soluble in meoh, etoh, dmso, dmf, acetone, diethyl ether, partly soluble in chloroform and insoluble in h2o, n-hexane, and dcm solvents. dpph • radical scavenging activity (rsa) antioxidant activity was measured by monitoring the bleaching rate of dpph• (2,2'pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 162 diphenyl-1-picrylhydrazyl radical) at its characteristic wavelength in the presence of sample solutions (i.e., m and tin(ii)-m complex) [25]. since, the radical form of dpph• absorbs at 515 nm but if any antioxidative agent or radical group is added to it may decrease its absorbance. in experiment, about 0.2 mg/ml of each sample were mixed with 2 ml of dpph• (0.1 mm in meoh) and were assessed in different time intervals, i.e., 0, 5, 10, 15, 20, 25, and 30 minutes with the difference of 5 minutes until the reaction reached the steady state. the decrease in absorbance was measured against a blank of pure meoh. thus, the radicalscavenging activity (rsa) of the compounds was computed as a percentage of dpph• discoloration using equation (i); % rsa = [(adpph• – as)/ adpph•]× 100 (i) where, as is the absorbance of sample (either m or tin(ii)-m complex) and adpph• is the absorbance of dpph• solution. ferric reducing power (frap) ferric reducing power of the m and tin(ii) complex was determined by means of potassium ferricyanide–ferric chloride method [26]. various concentrations, i.e., 0.5, 1.0, 1.5, and 2.0 mg/ml of ligand molecule and its tin(ii) complex were prepared. the each sample amounting to about 0.5 ml was added to 2.5 ml 0.2 m phosphate buffer (ph 6.6) and 2.5 ml potassium ferricyanide (1%). then, the samples were incubated for 20 minutes at 50 oc and centrifuged with speed of 3000 rpm for almost 10 minutes. subsequently, 2.5 ml of trichloroacetic acid (10%) were added. in the last, 2.5 ml from the resulting solution were mixed with 2.5 ml distilled water and 0.5 ml fecl3 (0.1%). allowed the solutions to stand for about 30 minutes and then measured their absorbance at 700 nm. for examining the relative reducing power of m and tin(ii)-m complex, the results were expressed by plotting the graph of their absorbance vs. various concentrations. cyclic voltammetry (cv) the conventional three electrode system consisting of glassy carbon, platinum wire, and ag/agcl as working, auxiliary, and reference electrodes, respectively, was used to perform an electrochemical experiment at ambient temperature. the cleaning of working electrode prior to electrochemical measurement was carried out by polishing it with alumina powder on polishing cloth [27]. the solutions of 0.01 m concentrations for m and its tin(ii) complex were prepared in meoh. to get the electrochemical results for m, its solution was prepared by mixing 1 ml of m, 5 ml of liclo4, and 4 ml of meoh solvent. while the tin(ii)-m complex was prepared by mixing 1 ml m, 1 ml sncl2·2h2o, 5 ml liclo4, and 3 ml meoh, respectively. total 10 ml of the solution was prepared for an analysis [28]. results and discussion uv-vis studies in the absorption spectrum of m, two prominent characteristic absorption peaks may be visualized with corresponding maxima at 368 and 264 nm for band i and band ii, respectively. here, band i is annotated for the absorption of cinnamoyl system (ring b), while band ii is interpreted for benzoyl system (a ring). from the molecular structure of m, it can also be inferred that m can chelate the metal ions via two sites, i.e., 5hydroxy-4-oxo and 3-hydroxy-4-oxo systems. besides, it possesses another potential chelating site at 3,2'-dihydroxy system, which forms sevenmembered chelate ring to complexate any metal ion [29]. when tin(ii) solution was added to methanolic solution of m, it caused the significant change in the m spectrum due to appearance of new peak at 422 nm λmax (band iii). it shows that the bathochromic shift of about 54 nm takes place. the spectral change can be easily observed in (fig. 3) [30]. it confirms that complex formation takes place between m and tin(ii). that change takes place in band i; conversely the shift of band ii at 264 nm is relatively insignificant (band iv). therefore, it supports the clue that newly appeared peak at 422 nm in tin(ii) complex of m may arise due to complexation of tin(ii) at 3oh and 4co of m. pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 163 figure 3. uv-vis spectra of a) morin and b) tin(ii)-morin complex. in fact, there are many proofs, which indicate that complex formation in m involves the 3-hydroxyl-4-keto moiety; (i) because 3-hdroxyl possesses more chelation power than 5-hydroxyl group; ii) also because of higher delocalization of oxygen electrons of 3-hydroxyl than 5-hydroxyl group, thereby facilitating the π electrons delocalization. thus, complex formation at band i caused by the interaction of 3-hydroxyl group of m with tin(ii) subsequently follows the electronic redistribution between the tin(ii) and m to form a big extended π bond system. it changes the electronic distribution in m from n-π* to π-π* transition of lower energy [31]. thus, new ring formation in complex is caused by the increased conjugative effect with inclusion of c ring, which provides the additional molecular stabilization resulting from the formation of extended 4 bond system [32]. hence, this information may be supportive in the sense that the chelation ability in m is more attributable to the presence of 3oh and 4co groups in ring c. thus, the red shift in the m spectrum is highly informative for the coordination site in ligand having multiple chelating sites, but due to more acidic nature of 3oh proton and more suitable location of 4co, they may be the proper sites to be involved in complex formation. since, the orientation of 2' and 4' hydroxyl groups is such that they can not bind the metal ion, whereas the 5oh group has lesser proton acidity and much hindrance created by the formation of first complexation, therefore its involvement becomes less probable in complexation process [33]. ir spectroscopy ir spectra provide very useful information regarding the complex structure (fig. 4). major difference between the spectrum of ligand molecule and complex compound may provide the information of shifting of certain peaks as well as disappearance/formation of some other peaks. some of the selected peak values have been shown in table 1. it is observed that highly notable spectral change between the spectra of two compounds occurs at 1400-1700 cm-1 region. where, the position of ν(c=o) is significantly shifted in m from 1662 cm-1 to 1647 cm-1 (δν = 15 cm-1), hence its involvement in complex formation becomes more clear. consequently, its participation in complex formation may cause the lengthening of c-o bond and decrease in force constant that result in shift of carbonyl chromophore towards even smaller value of wave number after coordination. therefore, the involvement of co in complexation can not be ignored. furthermore, the formation of chelate ring of >c=o···m–o– may be recognized from the peak at 1558 cm-1 [32]. figure 4. ir spectra of a) morin and b) tin(ii)-morin complex. 50 60 70 80 90 100 % t 500 1000 1500 2000 2500 3000 3500 wave numbers (cm -1 ) a 10 20 30 40 50 60 70 % t 500 1000 1500 2000 2500 3000 3500 4000 wavenumbers (cm -1 ) b iv iii ii i b a 200 250 300 350 400 450 500 550 600 650 700 750 800 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 wavelength (nm) a b s o r b a n c e pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 164 at the same time, appearance of new bands in the tin(ii) complex at 519 and 427 cm-1 may indicate the formation of metal oxygen (m–o) bond [34], while such bands can not be observed in the ligand spectrum. on the other hand, shift in peak position of ν(c–o–c) from 1310 to 1320 cm-1 is quite negligible; hence it confirms that the ring oxygen is not involved in complex formation. finally, the most prominent and broad peak at 3000-3600 cm-1 reveals the presence of water molecules ν(o–h) [35]. table 1. assignments of ir spectral peaks for morin and tin(ii)morin complex compound/ complex ν(o–h) ν(c=o) ν(c–o–c) ν(c=c) ν(m–o) morin 3381–3157 1662 1310 1613 tin(ii)-morin 3375 1647 1320 1623 427 1 h-nmr studies the chemical shift values for the hydroxyl protons of m have been assigned as well as compared with already existing literature. table 2 shows the chemical shift values of 1h-nmr signals for the m and tin(ii) complex. m shows a sharp singlet at 12.61 ppm produced from the intramolecular hydrogen bond. this is the hydroxyl group of ring a located at c5 position, which can interact with the acceptor carbonyl group of ring b at c4 position. similarly, one more hydrogen bond may be formed by the hydroxyl group of c2' by interacting with furan oxygen of ring c or the hydroxyl group oxygen placed at c3, but it depends more upon the orientation of ring c or water molecules [36]. in addition, after complexation m shows the upfield and downfield chemical shifts in the resonances of aromatic ring protons in the tin(ii) complex spectrum. in complex spectrum, the upfield shift arises due to an increase in ring current shielding effect in the resonances of m. the chemical shift in hydroxyl protons of m located at 5oh, 7oh, and 3oh may appear at 12.61, 10.68, and 9.76 ppm, respectively, which shows change in characteristic chemical shift upon coordination with tin(ii). thus, it seems that the signals of 5oh and 7oh protons are present at 12.59 and 10.69 ppm with little change in their chemical shifts but 3oh proton is completely missing. therefore, it can be presumed that metal ions most probably replace 3oh proton by undergoing coordination through oxygen atom [37]. table 2. assignments of 1h-nmr signals for morin and tin(ii)morin complex compound/complex 3-oh 5-oh 7-oh h2o morin 9.76 12.61 10.68 3.336 tin(ii)-morin 12.59 10.69 3.315 thus, it becomes self explanatory that after coordination the shifting of signals to higher field takes place due to more proton shielding originating from extension in conjugated system of complex [38]. it was also observed that in complex spectrum there is relative signal broadening due to different metal proton distances but the broader peaks are mostly those protons nuclei which are closer to tin(ii) [39]. scavenging activity on dpph • radical dpph• test shows that the antioxidants have inherent potential to reduce the dpph• radical from violet to yellow colored diphenylpicrylhydrazine. therefore, in the chemical reaction antioxidants donate the hydrogen to dpph• and convert it into dpph-h that is well illustrated in scheme 1. therefore, using dpph• method the antioxidant activity of both the compounds was evaluated [40]. actually antioxidant activity of m and tin(ii)-complex depends upon their structures especially their hydrogen donating ability. during experiment, it was observed that the reaction between m and its complex occurs in two main steps, where step one corresponds to the quick decrease in dpph• absorbance at 515 nm and second step shows quite slow decrease in absorbance finally reaching the steady state. fast step corresponds to abstraction of most labile h-atom, whereas slow step shows the oxidation degradation in the remaining product. pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 165 scheme 1. proposed mechanism for dpph• scavenging activity of tin(ii)-morin complex. however, antioxidant activity of the compounds depends more upon their molecular structures, but complexation made by the metal ions may affect the chemical properties of ligand molecules hence the resulting complexes may be of higher or lower activity [32]. thus, coordination may affect the ability of parent antioxidants. the antioxidant activity of the compounds has been studied in time dependent manner [41], which is determined in every established period of time for both compounds [42]. thus, the plot illustrated in (fig. 5) shows that m scavenged free radical to about 65% while tin(ii) complex scavenged to about 49%, thus m shows better inhibitory effect compared to the complex compound [43]. figure 5. antioxidant activity of morin and tin(ii)-morin complex against dpph•, a stable free radical. cyclic voltammetry application of electrochemical methods for antioxidant activity investigation is a well complementary to the previously used method such as uv–vis spectroscopy [41]. cyclic voltammetry characterizes the antioxidant activity of compounds/complexes via redox potentials. the compounds oxidized at relatively low potential values have strong scavenging abilities. the main characteristic of all the flavonoids is that they contain one or more hydroxyl groups attached to rings. therefore, the compounds either having more hydroxyl groups or containing more electron donating groups have higher antioxidant activities and show anodic peaks at lower potentials than those containing small number of hydrogen or electron donating groups. although, the oxidation pathways of flavonol flavonoids have been extensively investigated, however, their mechanism is still not completely understood. figure 6. cyclic voltammograms of a) morin and b) tin(ii)-morin complex. in the cyclic voltammograms of m ligand and tin(ii) complex, only one well defined anodic oxidation peak is observed with no reverse reduction peak (fig. 6). absence of cathodic peak in the reverse scan indicates that the oxidation process is followed by a chemical reaction, which rapidly removes the generated products. the oxidation of m leads to the formation of phenoxy radical, which can undergo the further chemical reactions like coupling, proton loss or nucleophilic attack. hence, the voltammetric peak in m is unequivocally attributed to the oxidation of c-3 hydroxyl group. another possible antioxidant mechanism is via metal chelation [44]. but in the case of tin(ii) complex, the peak potentials of oxidation signals are shifted towards more positive value. where the potential value for m is +0.658 v n p h p h n n o 2 o 2 n o 2 n o o o o o h o h o h h sn n p h p h n n o 2 o 2 n o 2 n o o o o o h o h o h sn h . . + dpph• tin(ii)-morin dpph-h tin(ii)-m • b a 0.000 1.000 u (v) -20u 0 20u 40u i (a ) pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 166 and for tin(ii)-complex is +0.925 v, which indicates that m molecule is more antioxidant than its corresponding complex. hence, the voltammetric method can also be used for the determination of antioxidant activity, in the same way as dpph• assay because of the correlation found between oxidation potentials and anti-radical power [45]. reducing activity reducing power acts as an important marker of the compounds' possible antioxidant activity. an antioxidant may act as a common reductant but a reductant may not necessarily act as an antioxidant [46]. thus, in the frap (ferric reducing antioxidant power) method, the direct reduction of fe3+(cn-)6 to fe 2+(cn-)6 may be used as a measure of reducing power of m and its tin(ii)-complex. the mechanism of complex is shown in scheme 2. the reducing power of compounds was determined by measuring the absorbance of perl’s prussian blue complex formed and followed by subsequent reaction with ferric chloride (equation (ii)) to yield the ferric ferrous complex with λmax at 700 nm. the method indicates that greater the absorbance, greater is the antioxidant's reducing capacity for iron from fe3+ to fe2+ [40]. potassium ferricyanide + ferric chloride   tantioxidan potassium ferrocyanide + ferrous chloride (ii). . o o o o h oh oh o h o o o o h oh oh o h fe 3+ fe 2+ sn sn + scheme 2. ferric reducing power of tin(ii)-morin complex. the assay is based on the change of yellow color into various shades of green color and finally to blue based on compounds' reducing power. with increasing concentration the reducing power increases. reducing power of m and tin(ii)-m complex as a function of their concentration is shown in (fig. 7) [47]. it shows that the reducing power of m is higher than its corresponding tin(ii) complex. figure 7. ferric reducing power of morin and its tin(ii)-morin complex. conclusion it has been concluded that tin(ii) cations form complex with morin. the resulting complex was characterized by uv-vis, ir, and nmr techniques. from the spectral characterization, it was concluded that in tin(ii)-morin complex the coordination of tin(ii) with morin takes place through 3-oh and 4-co groups. from the antioxidant investigation (evaluated by dpph• method) of ligand molecule and the complex compound, it was observed that complexation of tin(ii) with morin may reduce the antioxidant potential of ligand molecule. dpph• assay measures the ability of compounds to donate hydrogen to the radical. while the ferric reducing power measures the ability of compounds to donate electron to fe(iii). the results demonstrate the strong impact of study in designing various anticancer drugs. acknowledgements this research work was supported by the national centre of excellence in analytical chemistry, university of sindh, jamshoro/ pakistan. the authors would like to thank and gratefully acknowledge for this assistance and express their appreciations on the provision of necessary facilities. pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 167 references 1. w. bylka, i. matlawska and n. a. pilewski, j. amer. nutra. assoc., 7 (2004) 24. 2. a. r. tapas, d. m. sakarkar and r. b. kakde, tropic. j. pharm. res., 7 (2008) 1089. 3. p. -d. lee chao, s. -l. hsiu and y. -c. hou, j. food drug anal., 10 (2002) 219. 4. m. furusawa, t. tanaka, t. ito, a. nishikawa, n. yamazaki, k. -i. nakaya, n. matsuura, s. tsuchiya, m. nagayama and m. iinuma, j. health sci., 51 (2005) 376. 5. p. goyal, b. k. aggarwal and s. garg, hacettepe j. biol. chem., 38 (2010) 255. 6. s. a. payán-gómez, n. flores-holguín, a. pérez-hernández, m. piñón-miramontes and d. glossman-mitnik, j. mol. model., 17 (2011) 979. 7. g. seelinger, i. merfort, u. wölfle and c. m. schempp, molecules, 13 (2008) 2628. 8. r. k. parabathina, p. l. swamy, v. v. s. n. harikrishna, g. s. rao and k. s. rao, j. chem. pharm. res., 2 (2010) 826. 9. p. janeiro and a. m. o. brett, electroanalysis, 17 (2005) 733. 10. p. prahalathan, s. kumar and b. raja, asian pacific j. tropic. biomed., 2 (2012) 443. 11. i. trandafir, v. nour and m. e. ionica, pol. j. environ. stud., 21 (2012) 749. 12. a. j. hamdan, int. j. electrochem. sci., 5 (2010) 215. 13. r. m. el-sherif and w. a. badaw, int. j. electrochem. sci., 6 (2011) 6469. 14. m. munteanu, e. chirila, g. stanciu and n. marin, ovidius uni. annals chem., 21 (2010) 79. 15. g. dehghan, z. khoshkam, food chem., 131 (2012) 422. 16. i. b. afanas'ev, e. a. ostrakhovitch, e. v. mikhal'chik, g. a. ibragimova, l. g. korkina, biochem. pharm., 61 (2001) 677. 17. r. f. v. de souza, w. f. de giovani, spectrochim. acta part a, 61 (2005) 1985. 18. q. k. panhwar and s. memon, j. coord. chem., 65 (2012) 1130. 19. q. k. panhwar and s. memon, j. coord. chem., 64 (2011) 2117. 20. j. s. biradar and b. s. sasidhar, europ. j. medicinl. chem., 46 (2011) 6112. 21. r. f. v. de souza and w. f. de giovani, redox rep., 9 (2004) 97. 22. w. chen, s. sun, w. cao, y. liang and j. song, j. mol. struct., 918 (2009) 194. 23. a. pekal, m. biesaga and k. pyrzynska, biometals, 24 (2011) 41. 24. j. zhou, l. wang, j. wang and n. tang, trans. met. chem., 26 (2001) 57. 25. w. brand-williams, m. e. cuvelier and c. berset, food sci. technol., 28 (1995) 25. 26. m. oyaizu, jap. j. nut., 44 (1986) 307. 27. a. simić, d. manojlović, d. šegan and m. todorović, molecules, 12 (2007) 2327. 28. r. f. v. de souza, e. m. sussuchi and w. f. de giovani, synth. react. inorg. met.-org. chem., 33 (2003) 1125. 29. c. septhum, v. rattanaphani and s. rattanaphani, suranaree j. sci. technol., 14 (2007) 91. 30. a. c. gutierrez and m. h. gehlen, spectrochim. acta part a, 58 (2002) 83. 31. g. zhang, j. guo, j. pan, x. chen and j. wang, j. mol. struct., 923 (2009) 114. 32. a. a. ensafi, r. hajian and s. ebrahimi, j. braz. chem. soc., 20 (2009) 266. 33. q. k. panhwar, s. memon and m. i. bhanger, j. mol. struct., 967 (2010) 47. 34. m. kopacz and e. woznicka, polish j. chem., 78 (2004) 521. 35. z. qi, w. liufang and l. xiang, transition met. chem., 21 (1996) 23. 36. t. -w. wu, k. -p. fung, l. -h. zeng, j. wu, a. hempel, a. a. grey and n. camerman, biochem. pharm., 49 (1995) 537. 37. e. woznicka, m. kopacz, m. umbreit and j. klos, j. inorg. biochem., 101 (2007) 774. 38. a. a. ansari, main group chem., 7 (2008) 133. 39. a. a. ansari, main group chem., 61 (2008) 3869. 40. i. gülçin, f. topal, s. b. ö. sarıkaya, e. bursal, g. bilsel and a. c. gören, rec. nat. prod., 5 (2011) 158. 41. p. -x. xi, z. -h. xu, x. -h. liu, f. -j. chen, z. -z. zeng, x. -w. zhang and y. liu, j. fluoresc., 19 (2009) 63. pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 168 42. a. corona-bustamante, j. m. viverosparedes, a. flores-parra, a. l. perazacampos, f. j. martínez-martínez, m. t. sumaya-martínez and á. ramos-organillo, molecules, 15 (2010) 5445. 43. r. m. s. pereira, n. e. d. andrades, n. paulino, a. c. h. f. sawaya, m. n. eberlin, m. c. marcucci, g. m. favero, e. m. novak and s. p. bydlowski, molecules, 12 (2007) 1352. 44. a. simić, d. manojlović, d. šegan and m. todorović, molecules, 12 (2007) 2327. 45. j. f. arteaga, m. ruiz-montoya, a. palma, g. alonso-garrido, s. pintado and j. m. rodríguez-mellado, molecules, 17 (2012) 5126. 46. x. li and c. chen, int. res. j. pure appl. chem., 2 (2012) 68. 47. p. jayanthi and p. lalitha, int. j. pharm. pharm. sci., 3 (2011) 126. characterization of metal exchanged zeolite-a using quantasorb and mercury porosimeter issn-1996-918x pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 148 – 158 calixarenes: a versatile source for the recovery of reactive blue-19 dye from industrial wastewater fakhar n. memon and shahabuddin memon* national center of excellence in analytical chemistry, university of sindh, jamshoro 76080, pakistan received 04 october 2012, revised 15 december 2012, accepted 16 december 2012 ------------------------------------------------------------------------------------------------------------------------------------------ abstract the present work demonstrates the comparative extraction efficiency of p-tert-butylcalix[4]arene (1) and its derivatives (2-4) toward a series of some selected reactive dyes such as reactive black5 (rb-5), reactive blue-19 (rb-19) and reactive red-45 (rr-45). the study elaborates effectiveness of different anchoring groups present on the periphery of calix[4]arene platform and highlights the importance of preorganization concerning the application of supramolecular chemistry in separation science and technology. it has been observed that compound 4 shows good extraction efficiency toward rb-19 among the selected reactive dyes. enhanced extraction efficiency was observed with the addition of nacl at ph 7. the proposed extraction mechanism through inclusion complexation was confirmed by log-log plot analysis, which shows 1:1 complexation between 4 and rb-19. the solvatochromic response of 4 in various solvents of different polarities showed good response in methanol. the log k of complex was found as 5.2. the complex formation between 4 and rb-19 has also been confirmed by ft-ir spectroscopy. the recovery of 4 and rb-19 dye was achieved at ph 8 that signifies the reusability of 4 again and again. keywords: calixarenes; complexation; extraction; reactive blue-19 dye; recognition. ------------------------------------------------------------------------------------------------------------------------------------------ introduction reactive dyes are water soluble and anionic in nature, which are extensively used in textile industries due to their favorable characteristics of water-fast, bright color, simple and low energy consumption and their ability to bind with cellulosic fiber by covalent bonding [1]. most of them are synthetic in nature, made up of two key components: the chromophore, responsible for the color, and the auxochrome, that not only supplement the chromophore but also render the solubility of molecule in water and give enhanced affinity to attach the fiber [2]. however, they are an important class of pollutants that enter into the environment through various sources such as textile dyeing, shoe polish, plastic, lather, paper printing, wool, polyamide [3], and food coloring etc [1]. the process of dyeing in the textile industries is main source that produces large amount of colored wastewater, which is drained out into the streams without its proper treatment [4], which is harmful not only for visual nature but also retards the proper transmission of sunlight to the aquatic life [5]. they are toxic, mutagenic and carcinogenic. once they enter into the aquatic system their biodegradation is very difficult due to their complex aromatic molecular structure, which makes them more stable. for example, reactive blue-19 (rb-19) also known as remazol brilliant blue is an anthraquinone based vinylsulphone dye, which is very resistant to chemical oxidation due to its anthraquinone structure being stabilized by resonance [6]. rb-19 has relatively low fixation ability (75-80 %) due to the competition between the formation of vinylsulphone and the hydrolysis *corresponding author email: shahabuddinmemon@yahoo.com pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 149 reactions, i.e. formation of 2-hydroxysulphone, which does not attach to the fiber as a result that dye remains stable into the wastewater for a long period of time, i.e. half life of rb-19 is 46 years at ph 7 and 25 oc that renders the oxygen transfer to aquatic life [1, 6]. therefore, the strategy to remove the color and to decrease its impact onto the wastewater of textile environment has found to be of significant importance. various physical, chemical and biological methods have been employed such as oxidationozonation, microbial degradation, biological reductive decolorization, membrane process, sorption, biosorption, decolorization under methanogenic conditions, electro-coagulation, physico-chemical process [1, 7, 8] and adsorption/aggregation [9] including use of rice husk ash [10], lentil straw [11], agricultural waste [12], chitosan [13], activated carbon [14] etc. nevertheless, the performance of all these processes is slow and not very effective or some of them are efficient only at low concentrations. however, adsorption play a vital role at higher concentrations but regeneration of most of the adsorbents is difficult except for activated carbon, but adsorption through activated carbon is expensive [15]; furthermore, biological methods are not effective due to the low biodegradability of dyes. therefore, much attention has been paid during current decades, to the synthesis and design of artificial molecules that not only act as efficient receptors for neutral or ionic species but also prove to be economical, regenerable, environment friendly and conveniently accessible materials. in this regard, the macromolecular synthetic compounds such as calixarenes (fig. 1) have received considerable importance in supramolecular chemistry because of their ability to selectively interact with anions, cations or neutral molecules and can be used as host-guest type receptors. previously, calix[4]arenes based resins have also been used for the removal of various dyes [16-19] which concludes only the sorption efficiencies of synthetic resins to different dyes. but the need here is the application to real water samples along with regeneration of costly dyes for industrial reuse. however, no such a finding is referred by previously reported methods. hence, herein we explore the inclusion mechanism of calix-dye complexation phenomenon and liquid phase extraction (lpe) method as a strategic alternative to all these techniques that may help in organizing the dye removal processes. this process may prove to be useful not only for transfer from one phase to another but also become advantageous for economical purpose and provides a cheaper source for the recovery of costly dyes from effluent mixtures that could be reused. figure 1. the chemical structures of calix[n]arene. material and methods instrumentation melting points were determined on a gallenkamp apparatus in a sealed capillary. ft-ir spectra were recorded on a thermo nicollet avatar 5700 ft-ir spectrometer using kbr pellets (spectral range from 4000 to 400 cm-1). elemental analyses were performed using a chns instrument model flash ea 1112 elemental analyzer (20090; rodano, milan, italy). a gallenkamp thermostat automatic mechanical shaker model bks 305-101, uk was used for liquid-liquid extraction. the ph measurements oh ho oh oh n ho oh ho oh n ohoh hooh n pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 150 were made with ph meter (781-ph/ion meter, metrohm, herisau, switzerland) with glass electrode and internal reference electrode. uv–vis spectra were recorded on a perkin elmer (shelton, ct 06484, usa) lambda 35 through uv–vis spectrophotometer. reagents all reagents used for the preparation of solutions were of analytical grade. all aqueous solutions were prepared with deionized water that had been passed through a millipore milli-q plus water purification system (elga model classic uvf, uk). for ph (2 to 12) adjustment naoh and hcl (0.1 m) solutions were used. reactive dyes (fig. 2) such as reactive black-5 (rb-5), reactive blue-19 (rb-19) and reactive red-45 (rr-45) used in this study were procured from commercially available source. the p-tertbutylcalix[4]arene (fig. 3) and its derivatives (1-4) were synthesized according to previously reported methods [20, 21, 22]. figure 2. the chemical structures of some reactive dyes used in experiments. ohoh ohoh oh oh ohoh ome meo o o ome ome ome meo ome ome 1 2 43 figure 3. the chemical structures of p-tert-butylcalix[4]arene (1) and its derivatives (2-4). o o nh 2 nh s o o s o o o na o s o o o na nh s o o na o n n s o o na o s o o o na oh nh n n n cl cl n n s na o o o s o o o na oh nh nn n cl cl reactive blue 19 (rb-19) +++ + reactive black 5 (rb-5) + +reactive red 45 (rr-45) + pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 151 analytical procedures liquid–liquid extraction study pederson’s procedure was used for simple liquid-liquid extraction studies [23]. 10 ml of a 2.5x10-5 m aqueous solution of reactive dye with 0.1 g of nacl and 10 ml of 1.0x10-3 m of p-tertbutylcalix[4]arene or its derivative in chcl3 were vigorously agitated in 25 ml stoppered flask with a mechanical shaker for 1 hour and finally left standing for additionally 15 min to settle down the two phases. the upper aqueous layer was taken and examined spectrophotometrically to determine the remaining concentration of reactive dye in that phase. the percent extraction (e %) has been calculated by eq. (1);   100%         o o c cc e (1) where co and c are the initial and final concentrations of reactive dyes in aqueous solution before and after extraction, respectively. log-log plot analysis the obsession of distribution coefficient d of selective dye between two phases upon the calixarene concentration and its extraction ability was examined by using log-log plot analysis. the general extraction equilibrium is assumed to be eq. 2 with ‘g’ be the neutral guest, ‘h’ be the host in the organic phase, the overall extraction equilibrium constant is given by eq. (3). when introduce the distribution coefficient d, as given in eq. (4), and taking log of both sides, we obtain eq. (5);         orgxnorgaq hgxhng  (2) extraction equilibrium constant is expressed as eq. (3); (3) whereas, the distribution ratio d can be defined by the eq. 4; (4) one obtains eq. 5, by introducing eq. 4 it into eq. 3 and taking log of both sides.  hxkd logloglog  (5) accordingly, the plot of log d versus log [h] shows a straight line with a slope that is responsible for the stoichiometry of the extracted dye species at various concentrations, where as [h] is defined as the analytical concentration of host in the organic phase. job’s plot analysis to examine the stoichiometric ratio between 4 and rb-19 dye for their inclusion complexation, job’s plot (method of continuous variation) has been used. the solutions were prepared by mixing the different ratios (1:9-9:1) of equimolar concentrations (2.5 x 10-7 m) of 4 and rb-19 in methanol. then the absorbance was measured at 206 nm. calculation of the complex formation constant the complex formation constant can be calculated by benesi-hildebrand method [24] by using eq. 6.       g ka hg f  1 (6) eq. 6 is a modified hildebrand and benesi equation that is valid only for 1:1 complex under the conditions when concentration of host is much smaller than the concentration of guest, i.e. [h]<<[g]. where [g] denotes the total concentration of guest, [h] refers to the total concentration of host,  is the molar extinction coefficient of hg complex at λ, kf indicates the equilibrium constant of hg complex formation and a is the absorbance of hg complex at wavelength λ. the plot of    a hg versus [g] yields a straight line, which further proves 1:1 molar ratio of reactants in the complex. the values of kf and  can be calculated         xn xn hg hg k        n xn g hg d  pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 152 from the slope and intercept, whereas the value of gibbs free energy (∆g) can be calculated from the eq. 7. ∆g = -rt ln kf (7) ft-ir study of inclusion complex for ft-ir spectral measurements, saturated solutions of 4 and rb-19 dye were prepared by dissolving their stoichiometric (1:1) amounts in methanol. both contents were mixed up and stirred at room temperature for 24 hrs. finally, poured on a petri dish, evaporated the entire solvent and the resultant crystals were vacuum dried. regeneration of rb-19 dye and 4 for regeneration study 10 ml of rb-19 dye extracted solution of host in chloroform was vigorously agitated with a little amount of deionized water samples (ca. 2.5 ml) of varying ph (ph of the water was adjusted from 2-12) for 1 hour. the regeneration of compound 4 was made by separating upper aqueous layer that was examined spectrophotometrically to calculate the percent recovery of rb-19 dye as stated previously by using eq. 1. finally, water containing regenerated dye was exposed to open air for evaporation to obtain the pure rb-19 dye. results and discussion synthesis the p-tert-butylcalix[4]arene (1) and its derivatives (2-4) were synthesized according to the methods reported elsewhere [20-22]. the condensation of p-tert-butyl phenol with formaldehyde in the presence of naoh produces 1 in good yield (65%) [20]. dimethoxy (2) and tetramethoxy (3) derivatives were synthesized by the treatment of 1 with methyl iodide in the presence of k2co3 in dry acetone according to the method described elsewhere [21]. the compound 4 was prepared through a multistep pathway by the reduction of tetraestercalix[4]arene as described in the literature [22]. all the synthesized compounds were characterized and confirmed by available techniques such as tlc, melting point, ft-ir and nmr, which was also matched with the literature, reported previously [20-22]. solvent extraction study preliminarily, two phase solvent extraction experiments were carried out in chcl3 without calixarenes to optimize and diminish the effect of solvent for the transfer of reactive dye from aqueous phase to organic phase at different ph of the solution (i.e. ph 3, 7, and 11). these results are summarized in (fig. 4). it can be estimated from the results that the acidic or basic media is slightly favorable, i.e. less than 10% for the transfer of dyes between two phases, so the neutral ph was selected for further study in order to evaluate the actual reactive dye extraction efficiency of p-tertbutylcalix[4]arene (1) and its derivatives (2-4). 0 1 2 3 4 5 6 7 8 9 10 3 7 11 % e x tr a ct io n ph reactive black-5 reactive blue-19 reactive red-45 figure 4. effect of ph on the extraction of rb-19 by p-tertbutylcalix[4]arene derivative (4). some experiments were performed without nacl. the results obtained are given in the fig. 5, which reveal that a very small amount of dye could be transported from one phase to another in the absence of electrolyte (as justified by cylindrical shaped bars), this situation discloses the importance of electrolyte in the solution. because of the fact that the ionic balance and transport efficiency of the two phases is balanced by nacl. in addition, nacl reduces the solubility of dyes through common ion effect, resulting in an enhanced and facilitated transport to the organic phase [25]. results of extraction experiments at neutral ph with compounds 1-4 are compiled in (fig. 5), cone shaped bars reveal that compound 4 shows much greater extraction efficiency for the transfer of dyes spatially for rb19 as compared to the compounds 1-3. pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 153 0 10 20 30 40 50 60 70 1 2 3 4 % e xt ra ct io n compound reactive black-5 reactive blue-19 reactive red-45 figure 5. percentage extraction of reactive dyes without nacl (cylindrical shaped bars) and with p-tert-butylcalix[4]arene and its derivatives (cone shaped bars). influence of nacl on extraction furthermore, to estimate the role of ph on the extraction ability and recognition mechanism of compound 4 for rb-19 dye, experiments were performed at different ph (6, 7 and 8) with the same concentration of rb-19 at shaking time of 1 h (fig. 6). 0 10 20 30 40 50 60 70 80 90 100 6 7 8 % e ph figure 6. effect of ph on the extraction of rb-19 by p-tertbutylcalix[4]arene derivative (4). the results show that extraction efficiency of 4 at ph 6 and 8 is almost same but is greater at ph 7, which reveals that the strong interactions between rb-19 and 4 occur at ph 7 may be due to the ionization of dye at this ph that ultimately forms strong inclusion complex through hydrogen bonding between oxoanions of dye and hydrogens of the binding sites of 4 (fig. 7a-b). whereas, at acidic conditions the dye get protonated, which ultimately reduce the interaction between the host and guest. similarly, at basic conditions partial deprotonation of the binding sites of 4 may be the main reason of little affinity. the greater affinity between rb-19 and 4 as compared to other dyes may due to the compatibility of both host and guest, i.e. molecular size of dye and cavity size of 4. figure 7. proposed interaction of rb-19 with 4 at neutral ph. (a) structural model (b) ball and stick model. fig. 8 shows a plot of log d versus log [h] for the extraction of rb-19 by 4. a linear relationship between log d and log [h] with the slope of lines of rb-19 is equal to 0.8 suggesting that host 4 forms 1:1 complex with rb-19 dye. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 -4.5 -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 lo g d log[h] y=0.840x+40450 r2=0.948 figure 8. log d versus log [h] for the extraction of rb-19 by 4 from an aqueous to organic phase, i.e. chloroform at 25 °c. molecular recognition study solvatochromic study study of solvatochromic effect helps in selecting the solvent for a particular ligand that could not show similar spectra in various solvents [26]. its general perception is that with increasing solvent polarity, the ground state molecule is better stabilized by solvation than the molecule in the excited state, negative solvatochromic (blue shift) will result. better stabilization of the molecule in first excited state relative to the ground state with increasing solvent polarity will lead to (a) (b) pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 154 positive solvatochromism (red shift) [27]. consequently, variations in the position, intensity, and shape of the absorption spectra can directly measure the specific interactions between the solute and solvent molecules. moreover, it has been found that solvent plays an important role in complexation phenomena involving ionic and neutral species including macrocyclic ligands in general and calixarenes in particular [28,29]. therefore, the attemps were made to evaluate the behaviour of 4 in different solvents such as ethanol, methanol and dimethylsulfoxide (dmso). the uv-vis spectra of 4 in various solvents are shown in (fig. 9). 0 0.2 0.4 0.6 0.8 1 1.2 200 220 240 260 280 300 320 340 a λ (nm) ethanol methanol dmso figure 9. absorption spectra of 4 (2.5×10-5 m) in different solvent systems. it is clear from the (fig. 9) that absorption spectra of 4 show significant changes in solvents of different polarities, indicating sensitivity of 4 in solvents environment. (fig. 9) reveals that 4 exhibits red shift in its absorption maxima (~ 16 nm range) in ethanol, i.e. 222 nm with respect to methanol (206 nm), which indicates that 4 exhibits negative solvatochromism, i.e. absorption bands shift towards longer wavelength with decreasing solvent polarities. and accordingly, the absorption coefficient (ε) of 4 also changes from 43900 to 39972 l mol-1cm-1 in methanol and ethanol solvents, respectively. consequently, red shift can be attributed to the π-π* and n-π* transitions arising from different solvent polarities. whereas, in dmso 4 shows aggregation and too much noise even at low concentrations thus, methanol was selected as a suitable solvent for recognition studies. stability of 4 in selected solvent after the selectivity of a suitable solvent it is necessary to determine the stability of compound for a long time in that solvent. therfore, u.v-visible spectra of 4 in methanol were immediately obtained after continous u.v irradiations at different time intervals, i.e. 0, 30, 60, 90, 120, 150, 180 and 210 minutes. it was confirmed from the results (fig. 10) that compound 4 is stable in methanol as there was no change appearing in the spectra with the passage of time. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 200 220 240 260 280 300 320 340 360 a b so rb an ce λ (nm) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 50 100 150 200 250 a b so rb an ce t (min) figure 10. time dependent u.v-visible absorption spectra of 4 in methanol (2.5 x 10-5 m) upon irradiation of u.v light. complexation study in order to confirm the complexation efficiency of 4 with rb-19, u.v-visible study was carried out. the attemps were made to evaluate the behavior of 4 in different solvents such as ethanol, methanol and dmso. because of the best response of 4 in methanol, solubility of dyes, the appearence of bands in the absorption limits and well-defined spectral properties, it was selected as a solvent media for complexation studies. however the shifts in the bands of complexes to the shorter or longer wavelengths or the high intensity of bands than that of the free ligand are the general indications of the complex formation. primarily, the complexation behavior of 4 in methanol (2.5 x 10-5 m) with selected dye (rb-19) was checked. the u.v-visible spectra of free ligand 4 shows a strong band at 206 nm, which is attributed to the π→π* and another band at 267-284 nm due to n→π* transition. consequently, addition of guest (rb-19) in host (4) causes enhancement in the previous bands and appearance of new bands as well (fig. 11). pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 155 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 0 20000 40000 60000 80000 100000 120000 a 2 0 6 /2 2 7 1/[rb-19] 0 0.2 0.4 0.6 0.8 1 1.2 1.4 200 300 400 500 600 700 800 a λ (nm) host (4) guest (rb-19) complex figure 11. uv-visible responce of 4 (2.5 x 10-5 m) before and after the addition of guest (rb-19). in an effort to have further insight into the chromogenic behavior of 4, the absorption profile as a function of dye concentration was obtained followed by increase in the intensity of absorbance with respect to increased dye concentration (fig. 12). this profile also confers the complexation of dye through the binding sites in two lobes of calixarene moiety. since the bands at 227-300 and 600 nm are significantly affected due to the interaction of dye with alcoholic binding sites through hydrogen bonding (fig. 7). while the band at 206 nm, belongs to the aromatic core of the calixarene moiety that has considerably affected confirming endo-complexation. complex stability constant was also determined by plotting a206/227 versus 1/[rb-19], as shown in (fig. 13). the stability constant was calculated from the intercept/slope ratio [30]. the value of log k was calculated as 5.2 for 4-rb-19 complex. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 200 300 400 500 600 700 800 a λ (nm) 0 0.5 1 1.5 0 0.001 0.002 0.003 a b so r b a n c e conc. (mm) figure 12. uv-visible responce of 4 (2.5 x 10-5 m) upon addition of various equivalents of rb-19. figure 13. caliberation plot of 4-rb-19 complex with various concentrations of rb-19. again job’s plot (method of continuous variation) was used to confirm the stoichiometric ratio between host (4) and guest (rb-19) molecules by varying their concentrations (fig. 14). for 4-rb-19 complex the maximum mole fraction value was found to be 0.5, which justifies the 1:1 ratio of host-guest complex as calculated by log-log plot analysis. 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 a mole fraction figure 14. job’s plot of 4 and rb-19. the concentrations of host and guest were fixed (2.5 x 10-5). the absorbance was measured at 206nm. furthermore, the assumption of 1:1 hostguest complexation between rb-19 (g) and 4 (h) has also been confirmed by hildebrand and benesi plot (fig. 15), where the calculated values of    a hg and [g] provide the excellent linear relationship with coefficient of determination r2=0.997 having a slope of 2x10-5 and intercept 6x10-10. the value of formation constant (kf = 3.3x104 mol·dm-3) of 4-rb-19 complex was calculated from slope/intercept, while  , the molar extinction coefficient of complex can also be determined by 1/slope that was found to be 5x104 l mol−1 cm−1 and the free energy change (−∆g) was calculated as 11.0 kj 0-7 equivalents pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 156 mol-1. the term (−∆g) refers to the negative value of (∆g) and reveals the feasibility of a particular reaction. a typical benesi-hildebrand plot is shown in (fig. 15) and yields logkf =4.5 and 69.4log  . the higher value of kf may show the high stability of the complex in methanol solution. however, the high value of free energy indicates the selectivity/recognition ability of 4 for rb-19 dye. furthermore, the value of formation constant was close to the value of stability constant calculated above, which further confirms the host-guest ratio of 1:1 in the complex. y = 2e-05x + 6e-10 r² = 0.997 0 5e-09 1e-08 1.5e-08 2e-08 2.5e-08 3e-08 3.5e-08 4e-08 4.5e-08 0 0.0005 0.001 0.0015 0.002 0.0025 0.003 [g ][ h ]/ a [g] figure 15. the benesi-hildebrand plot of 4-rb-19 complex. stability and time response of complex in selected solvent the determination of stability of complex with respect to time in selected solvent is a very important area of study. therefore, it was suggested to explore the stability and response time of 4-rb-19 complex. (fig. 16) shows that 4 responds very immediately when titrated with rb19, so that a significant enhancement in the band at 206 nm and appearance of new band at 227 nm occurs very rapidly and remains constant for a long time even up to one week. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 200 300 400 500 600 700 800 a λ (nm) 0 0.5 1 0 100 200 300 a t (min) figure 16. time dependent u.v-visible absorption spectra of 4rb-19 dye complex (2.5 x 10-5 m) in methanol upon irradiation of u.v light; (inset) graph shows the stability of 4-rb-19 dye complex with respect to time. ft-ir study the ftir spectra shown in (fig. 17 a-b) clearly indicate that the complexation has been occurred between 4 and rb-19 dye, which is evident from shifting of band at 3469 cm-1 to 3453 cm-1 with a shoulder that highlights hydrogen bonding type interaction between oh functionalities of calixarene and sulphonate anions of the dye. figure 17. ft-ir spectra (a) 4 (b) 4/rb-19 complex. o o h2n n h s o os oo o o s o o o _ meo ome o o o o h h h h _ 80 3. 1 11 23 .5 12 45 .8 13 00 .4 13 62 .9 14 11 .9 16 03 .6 34 69 .8 10 41 .3 11 39 .9 13 85 .9 15 35 .7 15 75 .5 16 26 .1 34 53 .4 c o mp ou nd c o mp l ex 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 % t 500 100 0 150 0 200 0 250 0 300 0 350 0 w av enu mber s ( c m1) (a) (b) pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 157 moreover, shifting of an aromatic c=c band at 1603 cm-1 to 1626 cm-1 also reflects the information about an endo complex formation indicating π-π interaction between rb-19 and 4 (fig. 7). consequently, the disappearance of older and appearance of other new bands in ft-ir spectra are clear indications of complexation between host and guest. regeneration study it has been aimed to regenerate compound 4 and recover rb-19 dye from organic phase. thus, experiments were carried out at varying ph and observed that about 95 % recovery of rb-19 dye could be achieved at ph 8 (fig. 18), supporting information) in a minimum amount of water. after the evaporation of water rb-19 dye could be reused industrially. simultaneously, compound 4 in chloroform that is free of rb-19 dye could be reused for extraction of rb-19 dye from contaminated water that is produced from textile industries (fig. 19). shows the cyclic process of use and reuse of compound 4 as well as rb-19 dye. 0 10 20 30 40 50 60 70 80 90 100 0 2 4 6 8 10 12 % r ec o v er y ph figure 18. effect of ph on % recovery of rb-19 dye. recovered water 4 + rb-19 dye in chloroform nacl stirrer water contaminated with rb-19 dye h o st f o r re u se ph 8 comp:4 in chloroformr e u se rb-19 dye water evaporation figure 19. cyclic process of use and reuse of compound 4 as well as rb-19 dye. application to real wastewater samples. the extraction affinity of 4 for rb-19 dye was also studied on real water samples. thus for this study, real wastewater samples were collected from the vicinity of jamshoro industrial area. the samples were diluted and analyzed by u.v-visible spectroscopy to check their absorbance then rb-19 dye was extracted in chloroform with 4. the absorbance was checked after extraction and % extraction was calculated. it has been noticed that greater than 80 % rb-19 dye was extracted with 4 at the same conditions as stated above. conclusion the comparative extraction / recognition behavior of p-tert-butylcalix[4]arene (1) and its derivatives (2-4) as host molecules reveals the effectiveness of 4 for rb-19 among the series of reactive dyes. the work also demonstrates the role of ph and influence of electrolyte for the transfer of rb-19 from organic to aqueous phase. u.v-visible and ft-ir spectroscopy confirms 1:1 complexation between 4 and rb-19. the study proves the efficiency of preorganization concerning the modification of calixarene platform for neutral molecule’s recognition and to provide the basis for the large scale demonstration and application of the calixarene based dye removal method. the work may support the efforts being made for site reclamation polluted by rb-19 dye and its recovery as well as the regeneration of 4 for reuse. acknowledgment we would like to thank national centre of excellence in analytical chemistry, university of sindh, jamshoro/pakistan for the financial support of this work. references 1. m. siddique, r. farooq, a. shaheen, j.chem.soc.pak., 33 (2011) 284. 2. v. k. gupta, suhas, j. environ. manag., 90 (2009) 2313. 3. y. h. lee, s. g. pavlostathis, water research, 38 (2004) 1838. pak. j. anal. environ. chem. vol. 13, no. 2 (2012) 158 4. a. alinsafi, m. khemis, m. n. pons, j. p. leclerc, a. yaacoubi, a. benhammou, a. nejmeddine, chem. eng. process., 44 (2005) 461. 5. f. cicek, d. ozer, a. ozer, a. ozer, j. hazard. mater., 146 (2007) 408. 6. a. razaee, m. t. ghaneian, s. j. haashemian, g. h. moussavi, g. h. ghanizedeh, e. hajizadeh, iran. j. environ. health sci. eng., 5 (2008) 95. 7. o. gungor, a. yilmaz, s. memon, m. yilmaz, j. hazard. mater,. 158 (2008) 202. 8. j. huang, k. zhang, desalination, 282 (2011) 19. 9. a. z. bouyakoub, b. s. lartiges, r. ouhib, s. kacha, a. g. el samrani, j. ghanbaja, o. barres, j. hazard. mater., 187 (2011) 264. 10. a. k. chowdhury, a. d. sarkar, a. bandyopadhyay, clean – soil, air, water, 37 (2009) 581. 11. a. celekli, b. tanrıverdi, h. bozkurt, clean – soil, air, water, 40 (2012) 515. 12. m. abassi, n. r. asl, j. iran. chem. res., 2 (2009) 221. 13. g. sreelatha, v. ageetha, j. parmar, p. padmaja, j. chem. eng. data, 56 (2011) 35. 14. n. m. mahmoodi, r. salehi, m. arami, desalination, 272 (2011) 187. 15. y. c. pei, j. j. wang, x. p. xuan, j. fan, m. fan, environ. sci. technol., 41 (2007) 5090. 16. m. a. kamboh, i. b. solangi, s. t. h. sherazi, s. memon, j. hazard. mater., 172 (2009) 234. 17. m. a. kamboh, i. b. solangi, s. t. h. sherazi, s. memon, desalination, 268 (2011) 83. 18. m. a. kamboh, i. b. solangi, s. t. h. sherazi, s. memon, j. hazard. mater., 186 (2011) 651. 19. c. ming, c. yun, d. guowang, j. chem. eng. data, 55 (2010) 5109. 20. c. d. gutsche, l. g. lin, tetrahedron, 42 (1986) 1633. 21. s. memon and m. yilmaz, react. funct. polym. 44 (2000) 227 and the reference therein. 22. j. k. moran, e. m. georgiev, a. t. yordanov, j. t. mague, d. m. roundhill, j. org. chem. 59 (1994) 5990. 23. j. pedersen, j. fed. proc. fed. am. soc. exp. biol., 27 (1968) 1305. 24. h. a. benesi, j. h. hildebrand, j. am. chem. soc., 71 (1949) 2703. 25. y. m. slokar, a. m. le marechal, dyes pigments, 37 (1998) 335. 26. m. a. qazi, i. qureshi, s. memon, m. sharif, pak. j. anal. environ. chem., 11 (2010) 53. 27. h. m. reza, c. m. javad, y. maryam, iran. j. chem. chem. eng., 27 (2008) 9. 28. y. m. shirshov, s. a. zynio, e. p. matsas, g. v. beketov, a. v. prokhorovich, e. f. venger, supramol. sci., 4 (1997) 491. 29. s. k. máté, i. bitter, a. grün, g. nagy, l. kollár, anal chim acta, 461 (2002) 273. 30 p. bamfield. “chromic phenomena: technological application of color chemistry”, the royal society of chemistry, cambridge, uk, (2001). microsoft word 1-1-12-pjaec-06042023-499-f-revised galley proof-20-06-2023-1.docx cross mark issn-1996-918x pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 1 – 12 http://doi.org/10.21743/pjaec/2023.06.01 bioplastics from kitchen wastes: a developing green technology ruma poddar*, debajyoti bose, nisha nambiar and sandeep poddar faculty of science, lincoln university college, wisma lincoln, 12-18, jalan ss 6/12, 47301 petaling jaya, selangor d. e., malaysia. *corresponding author email: ruma@lincoln.edu.my received 06april 2023, revised 30may2023, accepted 02june 2023 ------------------------------------------------------------------------------------------------------------------------------------------- abstract plastic waste has become one of the biggest problems due to their excessive use. decomposition of bioplastics is very difficult as a result its causes lot of negative impact to landfill and water pollution. the most possible solution to overcome this problem is to substitute synthetic polymeric materials with biodegradable materials suchas bioplastics. food wastes can be transformed into environment friendly bioplastics, which will not only reduce environmental pollution due to natural fermentation of these wastes, but also generate national revenue besides generating employment potentials. these polymers can be degraded environmentally by microorganisms and water in compost piles. application of bioplastics has several advantages over conventional plastics such as lower carbon footprint and greenhouse gases (ghg) emissions, lower energy cost in manufacturing, reduction of permanent litter, and much safer to the environment. in food industries, the need for high-standard storage features and the urge for packaging with high economic, low ecological impact, ease of customization, and low encumbrance can be answered by compostable or degradable bioplastics where kitchen waste may take essential role. advancements in biomedical applications of bioplastics lead to the development of drug delivery systems and therapeutic devices for tissue engineering. nanocellulose and its composites, which may be obtained from the processing of kitchen wastes, may result in potential and economical sources for green plastic studies about the fabrication of medical implants, either in dental, orthopedic, or biomedical fields. keywords: bioplastics, kitchen waste, environment, renewable. ------------------------------------------------------------------------------------------------------------------------------------------- introduction since its widespread adoption, plastic garbage has been a major contributor to environmental issues such as landfill and water contamination [1]. nearly half of the plastic garbage in 2015 was produced by items with single-use, or "single-stream" purposes, such as plastic packaging and one-time-use items [2]. the proliferation of plastic waste is mostly attributable to the widespread use of plastics, which has exacerbated a number of serious environmental issues. flame retardants, bisphenol a (bpa), phthalates, and heavy metals like lead and cadmium may all leak and bioaccumulate from plastic wastes dumped in landfills. therefore, human consumption of marine species may contribute to the development of cardiovascular disease, reproductive problems, and obesity [3]. only 9% of plastic waste is recycled (15% is collected for recycling, but 40% of that is disposed of as residues). another 19% is incinerated, 50% ends up in landfill and 22% review pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 2 evades waste management systems and goes into uncontrolled dumpsites, is burned in open pits or ends up in terrestrial or aquatic environments, especially in under developed countries [4]. the best option is to replace synthetic polymeric materials with biodegradable ones [1]. microorganisms will break down after use, reducing any potential negative effects on the environment. with the advent of biodegradable polymers, scientists have found a potential answer to the waste disposal issues that come with using conventional plastics made from petroleum. regarding biodegradable polymers, the main difficulty for scientists is identifying uses that would consume enough of these materials to drive price reduction and make them commercially competitive [5]. polymers generated from biological sources, such as food waste, may be used to create bioplastic. it includes things like sludge debris, cassava peel, banana peel, pineapple peel, durian seed, jackfruit seed, avocado seed, and chicken feathers from the food processing sector or from people's homes [6]. climate change is a more systemic environmental problem. one of the primary motivators for the comeback of industrial biotechnology in general and the hunt for biobased plastics is the need to rapidly and deeply reduce ghg emissions. biodegradability is not required for all bio-based materials despite the common misconception that the term "bio-based" always refers to polymers made from renewable resources. if reducing greenhouse gas emissions is the top priority, and energy can be recovered during incineration or recycling, then plastics' long lifespan becomes a strength once again. true biodegradable plastics are giving way to biobased plastics in terms of manufacture, and this trend is expected to continue [7]. what is bioplastic? bioplastics are plastics produced in whole or in part from polymers obtained from biological sources, including sugarcane, potato starch, or the cellulose of trees, straw, and cotton. the term "bioplastic" refers to a group of terms for a wide variety of materials with a wide range of uses and qualities. according to european bioplastics, plastic must meet one of the two criteria to be considered as "bioplastic": either it must be made from renewable resources orit should be biodegradable [7]. renewable biomass sources, such as sugarcane and maize, or microbes, like yeast, are used in the production of bioplastics. it is possible to compost some bioplastics once they have degraded. bioplastics are polymers created from renewable materials that may be recycled naturally via biological processes, reducing the need for fossil fuels and helping the environment in the process. as a result, bioplastics are environmentally friendly, readily biodegradable, and safe for use in the body [8]. importance of bioplastic these polymers can be degraded environmentally by microorganisms and water in compost piles [9]. bioplastics can be categorized as petroleum-based biodegradable polymers (fossil-based), bioplastics from mixed source (bio-petroleum), and renewable resource-based polymers (naturally from plants and animals), etc. [10]. bioplastic has several environmental benefits, including reduced carbon footprint and greenhouse gas emissions, decreased energy cost during production, less accumulation of trash, and a more safer environment [11]. bioplastics also have advantages in the characteristics of the material, such as much greater water vapor permeability than standard plastic, less oily feel, good printability, softer, and more tactile. the method of bioplastic making may differ for each material used, the bioplastic characteristics produced, and various product pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 3 configurations [12]. one of the most wellknown benefits of biodegradable bioplastics is its ability to reduce trash. disposable plastic bags account up a significant proportion of the trash floating around in our seas. many municipalities and nations are prohibiting single-use plastic bags in an effort to reduce trash [7]. most bioplastics are biodegradable, meaning they may be broken down by microorganisms in the environment. the byproducts of this process include carbon dioxide (co2) and water in an aerobic setting and methane (ch4) in an anaerobic one, such as a landfill. there are so many advantages and disadvantages of bioplastics which is summarized in table 1 [13]. table 1. pros and cons of bioplastics [13]. pros of bioplastics the cons of bioplastics bioplastics are made from plant raw materials bioplastics are generally not cost-competitive compared to their oil-based counterparts. plant raw materials are renewable and sustainable there is a concern that bioplastics based on terrestrial crops could harm food supplies the carbon footprint of manufacturing bioplastics is very low some bioplastics have a shorter lifetime than oil-based plastics bioplastics are non-toxic and won’t leach chemicals into food or soil. being compostable and biodegradable sounds great, but many bioplastics must follow a specific disposal procedure and require industrial composting in order to avoid being incinerated or going to landfill. bioplastics are biodegradable and compostable. crop-based bioplastics require fertile land, water, fertilizers, and are reliant on weather conditions. this means that the supply of raw materials for bioplastics are at risk of natural phenomena, such as drought. there are a variety of zero waste end of life options for bioplastics. bioplastics are not the answer to marine litter. bioplastic composition carbon and hydrogen make up the polymers. in addition to carbon, hydrogen, oxygen, nitrogen, and phosphorus, plastics may include sulphur, silicon, chloride, fluorine, and phosphorus. plastic is a versatile material that can take on numerous shapes and sizes throughout manufacturing [14]. during the process of making bioplastics, the monomers are taken from biomass compounds, like the sugars in plants, or made from scratch. the monomers are then polymerized to make either a straight replacement for an existing plastic, like polyethylene (pe), or new polymers, like polyhydroxyalkanoates (phas). extraction of biomass can also produce natural polymers that are not made in a lab, such as starch, natural rubber, and proteins [15]. bioplastics are synthetic polymers that may either be broken down naturally or produced from renewable sources. as the carbon dioxide released during manufacturing, use, and recycling of plastics is offset by the carbon dioxide taken in throughout the plant's development cycle, the overall carbon dioxide balance is significantly reduced. bacterial microorganisms and typically nanometer-sized particles, most notably carbohydrate chains, may be used to produce bioplastics as well (polysaccharides). in order to make bioplastics long-lasting and to improve their qualities, research is often conducted into the manufacture of additives, such as polymer and composite combinations, to increase biodegradability [16]. the polysaccharides in the algae can be used to produce biodegradable plastic [17]. importance of kitchen waste those of us who are stuck at home because of the movement control order (mco) will probably cook more meals at home to avoid the risks of going out, which isn't always a bad thing. it is an opportunity to spruce up those tried-and-true dishes or try something new, and it may even mean better nutrition. "kitchen waste" refers to any pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 4 leftover food, drink, or other organic materials from kitchens in public or private establishments. every day, tons of food waste is created in densely populated places. due to their high moisture content, kitchen wastes that make their way into the mixed-municipal waste system present unique processing challenges, such as incineration. in most cases, garbage from kitchens is dumped in landfills, composted, or fermented. european union rules once recommended using food scraps as animal feed, however this practice was outlawed due to health and safety fears. most studies focused on bioplastic manufacturing procedures, operational conditions, and novel bacterial/archaeal species employed in the fermentation process, proving that food waste (fw) can be simultaneously converted into energy and bioplastics [18]. bioplastics reprocess of kitchen waste fruits and vegetablesare full of nutrients, and not just in the parts that we eat. bioactive phytochemicals are chemical substances generated by plants, and they are commonly found in the peel, pulp, and leaves of fruits and vegetables that are otherwise thrown away. these extracts have potential use in nutritional supplements, medicines, and food preservation products, all of which have the potential to reduce food waste. high-end cosmetics may also benefit from extracts and oils generated from food scraps. researchers have discovered that potato husks may be recycled into health supplements and bioplastics, which are polymers made from renewable sources and have several applications, including packaging, coatings, and adhesives. bioplastics rely on carbohydrates, lipids, and cellulose fibre, which may be salvaged from leftovers via processes like freeze-drying and hydrodynamic shockwave separation. creating bioplastics from food scraps has the potential to reduce both food and plastic waste, as well as emissions from plastic manufacturing. liquid biofuels like biodiesel and bioethanol may also be made from compounds derived from food waste. biofuels are utilised mostly in australia to power vehicles because of their reduced emissions compared to conventional gasoline or diesel. they may also be used for other purposes [19]. the creation of bioplastics like pha is a fantastic option for getting rid of fw. when fw is dumped in landfills, it has unintended consequences, including ghg emissions and water pollution. making bioplastics out of fw is eco-friendly since it uses carbon-neutral resources to create the final product. under commercial composting and biodegradation conditions, certain bioplastics may be used [20]. for the most part, plant-based materials, including starch, cellulose, and chitosan are used as polysaccharides in bioplastics. the most valuable bioplastic polysaccharide, starch, is found mostly in foods like cassava, rice, and soybeans that are often thrown away. plant fibre waste may be used to develop polymeric composites or used as bioplastics. the fibres may be classified as coming from the bark, fibres, seeds, core, or the reed of the vegetable. bioplastics have the potential to replace non-biodegradable materials in package production, making their thorough characterization essential inbio package research elucidates crucial factors [21]. how bioplastics can produce from kitchen waste is shown in schematic diagram fig. 1. foam, coatings, adhesives, sealants, and elastomers are just some of the many everyday uses for polyurethane (pu) components. conventionally, polyols and pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 5 isocyanates react to create pu. vegetable oil, cashew nutshell liquid, cellulose, lignin, and protein are only some of the renewable resources that may be used to create bio-based polyols alongside petroleum-based polyol [22]. polyols can have different traits depending on the biomass and how they are processed. the melted result is a mix of polyols that can be used right away to make foams, glues, and films [23]. figure 1. process of bioplastic from kitchen waste chandarana et al.and maura et al.have found that banana peel bioplastic is versatile and may be used for both packaging and bags. the addition of glycerol, a plasticizer, makes it more malleable [24,25]. besides that, many researchers found that bioplastics can make from cassava peel, pineapple peel, durian seed, jackfruit seed, and avocado seed [26-30]. fw is a good starting material for making bioplastics, but it must first be treated to change or improve its physical, chemical, and biological qualities. also, it's important to increase the amount of food trash that gets recycled, especially from complicated municipality waste food (mwf). collecting and sorting trash at its source can cut down on the cost of the steps that come after. this is a smart way to increase yield and profit, lessen the impact on the environment, and make it easier to recover materials. fw can be put into three groups based on where it comes from: industry, farming, and home. the total amount of fw from factories and farms is big, but it is mostly made up of easy things. on the other hand, home fw is comprised of many different things [31]. application of bioplastics produced by kitchen waste bioplastics may be used for a wide variety of purposes, from food packaging to healthcare. food packaging packaging is becoming more important in the food business, to the point that it is now considered a sub-sector in and of itself. focusing on the development of new biopolymer-based packaging is crucial for the sustainability and quality standards of the entire food industry, resulting in cleaner and more sustainable delivery chains from production facilities and their internal storage systems to transport facilities, to marketplaces, and finally to consumer houses. compostable or biodegradable bioplastics provide a response to the need for high-quality packaging that is also lightweight, customizable, environmentally friendly, and cost-effective [32]. the environmental issues and scarce resources of petroleum-based polymers have pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 6 piqued industry interest in bioplastics or biopolymers made from renewable resources. approximately 65% of 2018's worldwide bioplastic output came from the packaging business. biodegradable plastics include compostable bioplastics. to conclude, all biodegradable bioplastics are compostable, but not all compostable bioplastics are biodegradable [33]. a differentiation based on food typology is employed to provide a holistic perspective, since various food kinds call for various characteristics. vegetables and fruits have a high respiration rate, which may hasten their deterioration even under ideal circumstances, and they are also very sensitive to the concentrations of water, carbon dioxide, and ethylene. a package's primary functions are to protect its contents from environmental hazards including light, moisture, and oxygen, as well as to preserve the integrity of the product via sturdy construction and an airtight seal. the development of spoilage germs and pathogens is facilitated by the raw state of the meat. in order to maintain the fresh meat's vibrant colour, a high oxygen content in the packaging is necessary. consequently, many people think that vacuum packaging is a suitable option, and adding oxygen-absorbent layers, leading to active packaging, may better preserve cured meat [34]. protecting dairy products from oxidation and microbiological development requires containers with low oxygen permeability. moreover, a strong light barrier helps prevent lipids from oxidising. other key characteristics are resistance to water evaporation and the absence of outside odour absorption. some polysaccharides, such as pectins, which are often created by extraction from fruit and vegetable sources, might include these characteristics and serve as a safety barrier for food items [35]. agricultural applications there were a total of 6.96 million tons of plastics used in farming in 2017. bioplastic mulch is quickly replacing pe mulch for a safer way to farm. it helps reduce the carbon footprint because bioplastics break down faster than pe mulch and have less of an effect on the environment. bisphenol a (bpa), a chemical that can mess with hormones and is often found in traditional plastics, is not in bioplastic. bioplastic is also less harmful [36]. after the seeds sprout and grow, the seedling trays tend to break down in the soil. this process does not release dangerous chemicals into the soil or cause the plants to take them up [37]. in addition, phas have been used as pesticide carriers, crop protection films, fertilizer encapsulants, and seed protectors [38]. the nets, grow bags, and mulch films are valuable agricultural uses of pha-based bioplastics. instead of using high-density polyethylene (pe) nets, which reduce the crop's quality and output while still protecting it from birds, insects, and winds, farmers might utilize bioplastics-based nets instead. the low-density pe used to make grow bags, also called planter and seedling bags, is widely available and inexpensive. the grow bags made with phas would decompose quickly, be gentle on plants' roots, and not pollute the water supply. to sum up, mulch film bioplastics are crucial to maintain outstanding soil structure, moisture retention, weed control, and pollution prevention in place of fossil-based plastics [39]. so, longterm, sustainable farming development is possible with the support of innovations like bioplastics and sound agricultural methods. medical applications materials made from renewable resources have been employed for quite some time in the healthcare industry. gelatins pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 7 capsules, whether derived from animals or plants, are used because they dissolve easily in the stomach and intestines. these may be found in a wide variety of over-the-counter (otc) drugs. biodegradable bandages are intended to encourage clotting and proactive skin regeneration, and there are also biodegradable sutures that do not need to be removed manually after healing. for such uses, poly-lactones or polyhydroxyalkanoates provide characteristics similar to those of petroleum-based polymers. however, injection molding is not often used for these kinds of projects [40]. biomedical uses of biodegradable polymers have advanced to the point where implants and scaffolds are possible as drug delivery systems and therapeutic devices for tissue engineering [41]. there are a wide variety of biomedical and healthcare applications where polymers are necessary. cellulose may be used as the primary bioplastic in these fields. since it is biocompatible, does not cause cancer, and does not cause mutations, cellulose has been extensively researched for use in artificial organs, tissues, and brain engineering, and medicines [42]. because of their biocompatibility and bioresorbable properties, biodegradable biopolymers like poly lactic-co-glycolic acid (plga), poly lactic acid (pla), and polycaprolactone (pcl) may find use in a wide range of biomedical settings. additionally, despite the biopolymers' many biological uses, they need a few tweaks to their chemical and physical makeup in order for their mechanical properties to be fully absorbed at an implantation site. modifying and blending biomaterials to make them more biocompatible and less crystalline is a costeffective research and development strategy [43]. applicatins of bioplastics in different fields are shown in fig. 2. figure 2. importance of kitchen waste bioplastics novel industrial applications consumer electronics bioplastic has many uses today in the electrical and electronic industry (e&e industry). for example, bioplastics conductors, which are now called solid polymeric electrolytes (spes), are used to make electrochromic devices, batteries, diodes, fuel cells, and other things [44]. aside from that, bioplastics are often used to make parts for high-demand market goods, like the cases for computer parts and cell phones, speakers, computer mice, and vacuum cleaners [45].when bioplastics are used in business, their thermal and electrical qualities are very important. aside from being used as parts for assembly, bioplastics don't have many uses in the industry. this is because the main needs of the industry are electrical and heat resistance. most traditional plastics are electrical and thermal insulators without any added usefulness or support. conversely, bioplastics have low electrical and thermal conductivity and low temperature stability, which further limits their use in the e&e business [46]. when bioplastics were strengthened with carbon nanotubes (cnt) and cellulose nanofiber, they had better mechanical, electrical, and thermal qualities than pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 8 bioplastics that weren't strengthened. this makes them better for use in electronics. cellulose nanofiber-reinforced bioplastics and cnt-reinforced bioplastics have many uses in industry, such as flexible photovoltaic cells (solar cells), sensors, and advanced electronics. they are also used as surfaces in roll-to-roll production methods [47]. so reinforcing bioplastics with the right elements, bioplastics may take on and improve electrical and thermal conductivity capabilities, expanding its usefulness. architecture and construction industry plastics have been used in buildings and construction for a few decades now. plastic is often used in these fields to make pipes, padding, floor covers, wires, and other things. in the building and construction industries, bioplastics are used for things like wall coverings, geotextiles, facade elements, and pipes. traditional uses of plastic in the industry, like cloth fleece, wires, and floor covers, need to be very strong so they can stand up to different kinds of heavy workloads. bioplastic materials have a lot of potential uses in the industry, but they are expensive because better quality bioplastics usually cost more to process, and the performance of conventional bioplastic in the industry is not good enough to use it [48]. according to ivanov and stabnikov, using biodegradable bioplastics can benefit the industry, including environmental and bioeconomic sustainability, a reduction in the cost of construction waste disposal, and transient construction excavation costs [49]. but according to friedrich, traditional plastics are preferred for use in building since some features of bioplastics cannot be guaranteed, including their mechanical strength, resistance against biodeterioration agents, and lifespan [50]. in addition, conventional plastics, which are stronger and more flexible than bioplastics, are more suited for this application since they are used in the textile fleece business [51]. according to some researcher suggestion reinforcing materials can be added to bioplastics to make them stronger mechanically. this makes it possible for reinforced bioplastics to be used in industry. bioplastics that are strengthened with different structure materials have benefits like being water-resistant, lightweight, stable under load, cheap, long-lasting, and easy to work with as building materials [52,53]. automotive industry plastics are an important part of the car industry because they are cheap and easy to work with, but customers are seeing more and more pictures of trash mounds and huge floating islands of plastic. bioplastics could be part of the answer to some of the problems that come with regular plastic.with custom mixing, bioplastic can be used in a wide range of ways in the car business. the strength and looks of parts made from this material are not limited in any way. many awards show that it is better than other types of plastic, especially when it comes to being good for the earth [54]. the last two decades have seen a sharp rise in bioplastics usage, specifically in the auto industry. this is largely due to their role in sustainability. the bioplastic industry is expecting to rise in capacity from 2.11 million tons per year in 2018 to 2.6 million tons in 2023[55].due to the excellent compatibility of polylactid-acid (pla)thermoplastic polyurethan (tpu) -blends with natural fibers and glass, as well as their good adhesion with decorative elements inside the body, such as polyurethane foils and wood imitation foils, these pla-tpu-blends are suitable for use in the manufacture of car interior elements. pla-tpu-blends can be pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 9 effectively manufactured in a single step process that combines compounding and injection molding, thereby enhancing the quality of the final product [56]. with such major advancements, bioplastics have a bright future in the vehicle manufacturing industry. houseware and kitchenware bioplastic is a more sustainable material that is being explored by an increasing number of furniture and homeware designers. a plasticlike substance made from vegetable fats and oils, maize or potato starch, algae, shrimp shells, and other biomass is employed in a number of different applications. biopolymers can be used to make tools and items for the kitchen, clean storage cases and cups, bathroom decorations, toys, hangers, and hooks. biodegradable plastics are used to make united colors of benetton pegs [57]. tableware and dishes that are only used once can be made from a wide range of materials. these products can come from new or used sources. they can be made by simply molding it into the shape you want (for example, wooden utensils or flatware) or after a simple or complicated process, such as making molded composites of natural fibers (or other fillers) and (bio)plastics or (bio)plastic items with no natural fibers at all.to stop contamination of the environment, laws and many groups have suggested that singleuse goods should be clearly labeled with information about their biodegradability, (bio)plastic content, proper dumping methods, and environmental risks. it will help in getting rid of plastics that don't break down and replacing them with newer materials. products made of more than one material, like those with layers, should be changed or remade so that the separation of materials could become easier (so they can be recycled better) [58]. hence, the markets for conventional plastic disposable house ware and kitchen utilities are progressively being replaced by those for biodegradable polymers manufactured from renewable resources. these products includehangers, plastic shelves, plastic toys, plastic pots and pans, biodegradable cups, bioplastic water bottles, etc. conclusion bioplastics and biodegradable plastics made from renewable resources have been hailed as a big step towards solving the waste management problem. from the above discussion it is clear that bioplastics are very ecofriendly. as they are produced from renewable sources and biodegradable products there is no worry about waste management or pollution. the demand of bioplastics in various industries is increasing day by day. due to higher manufacturing costs bioplastics are still not available in open market. bioplastics' upstream technologies and processes are subject to uncertainty as a result of the field's still-developing upstream technology. several common misunderstandings about bioplastics lead to an incomplete and incorrect assessment of the industry, which in turn slows down investment decisions. it can be concluded that bioplastics arethe polymers that are both renewable and inexpensive. bioplasticsare a viable alternative to fossil-based plastics in most food packaging, agriculture, medical, and other novel industrial applications due to their similarity in properties to fossil-based plastic. this results in a smaller carbon footprint and less environmental impact. acknowledgment authors are thankful to lincoln university college for allowing all their facilities to complete this research. pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 10 conflict of interest no conflict of interest. references 1. i. g.de moura, a. v.de sá, a. s. abreu, a. v. machado. food packag, (2017) 223. https://doi.org/10.1016/b978-0-12804302-8.00007-8 2. c. giacovelli.single-use plastics. a roadmap for sustainability. int. environ. technol. centre, 2018. https://mpma.org.my/v4/wpcontent/uploads/2020/12/7.singleuseplastic_sustainability.pdf 3. h. l. chen, t. k. nath, s. chong, f. vernon, g. chris and l. m. alex. sn appl. sci., 3 (2021) 437. https://doi.org/10.1007/s42452-02104234-y 4. plastic pollution is growing relentlessly as waste management and recycling fall short, says oecd. available at: https://www.oecd.org/environment/plasti c-pollution-is-growing-relentlessly-aswaste-management-and-recycling-fallshort.htm 5. a. k. mohanty, m. a. misra and g. i. hinrichsen. macromol. mater eng., 276 (2000) 1. https://doi.org/10.1002/(sici)14392054(20000301)276:1<1::aidmame1>3.0.co;2-w 6. m. o. ramadhan, m. n. handayani.: iop conf. ser.: mater. sci. eng., 980 (2020) 012082. iop publishing. https://doi.org/10.1088/1757899x/980/1/012082 7. y. j. chen. j. chem. pharm, 6 (2014) 226. https://www.jocpr.com/articles/bioplasti cs-and-their-role-in-achieving-globalsustainability.pdf 8. s. a ashter, introduction to bioplastic engineering, (2016) 81. https://doi.org/10.1016/b978-0-32339396-6.00005-1 9. y. zheng, e. k. yanful, a. s. bassi, crit. rev. biotechnol., 1 (2005) 243. https://doi.org/10.1080/07388550500346359 10. m. m. reddy, m. misra and a. k. mohanty, chem. eng. prog., 108 (2012) 37. https://www.researchgate.net/publication /280022991_biobased_materials_in_the_new_bioeconomy 11. s. pilla, editor. john wiley & sons; (2011). https://doi.org/10.1002/9781118203699 12. r. hagemann and d. d'amico. patent. us20090110654a1. https://patents.google.com/patent/us200 90110654a1/en 13. green home. the pros and cons of bioplastics. (2008). https://greenhome.co.za/the-pros-andcons-of-bioplastics/ 14. r. a. lestari, m. kasmiyatun, k. dermawan, a. n. aini, n. riyati and f. r. putri, iop conf. ser. mater. sci. eng., 835 (2020) 012035. iop publishing. https://doi.org/10.1088/1757899x/835/1/012035 15. j. g. rosenboom, r. langer and g traverso. nat. rev. mater., 7 (2022) 117. https://doi.org/10.1038/s41578-02100407-8 16. n. i. ibrahim, f. s. shahar, m. t. sultan, a. u. shah, s. n. safri and m. h. mat yazik. coatings. 11 (2021) 1423. https://doi.org/10.3390/coatings11111423 17. a. s. rajpoot, t. choudhary, h. chelladurai, t. n. verma and v. shende. mat. today. proc., 56 (2022). https://doi.org/10.1016/j.matpr.2022.01. 060 pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 11 18. y. f tsang, v. kumar, p. samadar, y yang, j. lee, ok ys, h. song, k. h kim, e. e kwon and y. j. jeon. environ. int., 127 (2019) 625. https://doi.org/10.1016/j.envint.2019.03.076 19. transforming food waste: making something out of rubbish, australian academy of science. https://www.science.org.au/curious/earth -environment/transforming-food-wastemaking-something-out-rubbish 20. k. dietrich, m. j. dumont, l. f. del rio and v. orsat. sustain. prod. consum., 9 (2017) 58. https://doi.org/10.1016/j.spc.2016.09.001 21. a. a. santana, c. a. júnior, d. f. silva, g. s. jacinto, w. c. gomes and g. cruz, asti springer, cham., (2021) 281. https://doi.org/10.1007/978-3-03061837-7_17 22. g. liu, g. wu, c. jin, z. kong. prog. org. coat., 80 (2015) 150. https://doi.org/10.1016/j.porgcoat.2014.12.005 23. s. hu, x. luo, y. li. chem. sus. chem., 7 (2014) 66. https://doi.org/10.1002/cssc.201300760 24. j. chandarana, s. chandra. int. j. sci., res. eng. trends .7 (2021) 131. https://www.researchgate.net/publication /348806219_production_of_bioplastics_ from_banana_peels 25. m. g alcivar-gavilanes,k. l carrilloanchundia, m. a rieral.ing. investig42 (2022). https://doi.org/10.15446/ing.investig.92768 26. e. g. fadhallah,. n. juwita, i. n. assa'diyah, s. tullaila, s. putri, a. n., prayoga, r. a., and b. a. iswahyudi, int. j. hydro. env. sus., 1 (2022) 146. https://doi.org/10.58524/ijhes.v1i3.150 27. j. chumee and p. khemmakama. adv mat res., 979 (2014) 66. https://doi.org/10.4028/www.scientific.n et/amr.979.366 28. m. h. ginting, m. kristiani, y. amelia, r. hasibuan. int. j. eng. res. appl. 6 (2016), 33. http://repository.usu.ac.id/handle/123456 789/70010 29. m. lubis, m. b. harahap, a. manullang, m. h. ginting and m. sartika. jpcs801 (2017) 012014. iop publishing. https://doi.org/10.1088/17426596/801/1/012014 30. m. h. ginting f. r. tarigan, a. m. singgih. int. j. eng. sci. 4 (2015) 36. https://dupakdosen.usu.ac.id/bitstream/h andle/123456789/70009/fulltext.pdf?seq uence=1&isallowed=y 31. y. f. tsang, v. kumar, p. samadar, y. yang, j. lee, ok ys, h. song, k. h. kim, e. e. kwon and y. j. jeon. environ. int., 127 (2019) 625. https://doi.org/10.1016/j.envint.2019.03. 076 32. n. jabeen, i. majid and g. a. nayik. cogent food agric, 31 (2015) 1117749. https://doi.org/10.1080/23311932.2015.1 117749 33. l. g. hong, n. y. yuhana and e. z. zawawi. aims materials sci., 8 (2021) 166. https://doi.org/10.3934/matersci.202101 2 34. h. j. andersen, m. a. rasmussen. int. j. food sci. technol., 27 (1992) 1. https://doi.org/10.1111/j.13652621.1992.tb01172.x 35. l. baldino, e. reverchon. j. supercrit. fluids., 134 (2018) 269. https://doi.org/10.1016/j.supflu.2017.11.034 36. benefits of using bioplastic in agriculture, gaia green tech., 29/3/2022. https://gaiagreentech.com.my/articles/benefi ts-of-using-bioplastic-inagriculture#:~:text=bioplastic%20mulch%2 0is%20now%20rapidly,harmful%20impact %20on%20the%20environment 37. s. paul, b. sen, s. das, s. j. abbas, s. n. pradhan, k. sen ad s. i. ali. int. j. environ. anal. chem., 27 (2021) 1. https://doi.org/10.1080/03067319.2021.1 983552 pak. j. anal. environ. chem. vol. 24, no. 1 (2023) 12 38. n. george, a. debroy, s. bhat, s. bindal and s. singh. jabr. 8 (2021) 221. https://doi.org/10.30491/jabr.2021.25940 3.1318 39. f. abd el-malek, h. khairy, a. farag, s. omar. int. j. biol. macromol.157 (2020) 319. https://doi.org/10.1016/j.ijbiomac.2020.0 4.076 40. nature calls: the potentials of bioplastics within medical plastics, by nigel flowers 8 june (2018) 11:25. https://www.medicalplasticsnews.com/medi cal-plastics-industry-insights/nature-calls/ 41. t. narancic, f. cerrone, n. beagan, k. e. o’connor. polymers. 12 (2020) 920. https://doi.org/10.3390/polym12040920 42. g. f. picheth, c. l. pirich, m. r. sierakowski, m. a. woehl, c. n. sakakibara, de souza c. f, a. a martin, da silva r, r. a de freitas. int. j. biol. macromol.,104 (2017) 97. https://doi.org/10.1016/j.ijbiomac.2017.05.171 43. k. bano, r. pandey, jamal-e-fatima, roohi. int. j. pharm. sci. res., 9 (2018) 402. https://doi.org/10.13040/ijpsr.09758232.9(2).402-16 44. m. rayung, m. m. aung, s. c. azhar, l. c. abdullah, su’ait ms and a. ahmad, s. n. jamil. materials. 13 (2020) 838. https://doi.org/10.3390/ma13040838 45. n. harnkarnsujarit, p. wongphan, t. chatkitanan, y. laorenza and a. srisa. sustainable food processing and engineering challenges (2021) 203. https://doi.org/10.1016/b978-0-12822714-5.00007-3 46. k. a. imran, j. lou, k. n. shivakumar. j. appl. polym. sci., 135 (2018) 45833. https://doi.org/10.1002/app.45833 47. a. h. bhat, i. khan, m. amil usmani and j. a. rather. nanoclay reinforced polymer composites: nanocomposites and bionanocomposites. (2016) 115. https://doi.org/10.1007/978-981-101953-1_5 48. r. safin, n. galyavetdinov, r .salimgaraeva, g. ilalova, k. saerova. ine3s web of conferences, 274 (2021) 04013. edp sciences. https://doi.org/10.1051/e3sconf/2021274 04013 49. v. ivanov, v. stabnikov, v. ivanov, v. stabnikov. construction biotechnology. springer, singapore (2017) 51. https://doi.org/10.1007/978-981-101445-1_4 50. d. friedrich. build environ. 207 (2022) 108485. https://doi.org/10.1016/j.buildenv.2021.1 08485 51. c. monticelli and a. zanelli, procedia eng., 155 (2016) 416. https://doi.org/10.1016/j.proeng.2016.08.045 52. u. kong, n. f. mohammad rawi, g. s. tay. polymers (basel). 22 (2023) 2399. https://doi.org/10.3390/polym15102399 53. i. oberti, a. paciello. encyclopedia, 28 (2022) 1408. https://doi.org/10.3390/encyclopedia2030095 54. f. barillari, f. chini. atz worldwide. 122 (2020) 36. https://doi.org/10.1007/s38311-0200298-6 55. g. thakur, use of bio plastics in automotive industry, linked in, apr, 30 (2021). https://www.linkedin.com/pulse/use-bioplastics-automotive-industry-gaurav-thakur 56. s. a. rațiu and a. c. zgaverdea. mater. plast., 56 (2019) 901. https://doi.org/10.37358/mp.19.4.5282 57. m. a. acquavia, r. pascale, g. martelli, m. bondoni and g. bianco. polymers. 13 (2021) 158. https://doi.org/10.3390/polym13010158 58. k. dybka-stępień, h. antolak, m. kmiotek, d. piechota, a. koziróg. polymers. 13 (2021) 3606. https://doi.org/10.3390/polym13203606 microsoft word 69-83-galley proof pjaec-30032016-35.doc issn-1996-918x pak. j. anal. environ. chem. vol. 18, no. 1 (2017) 69 – 83 http://doi.org/10.21743/pjaec/2017.06.07 adsorption of 4-nitrophenol using pilli nut shell active carbon f. o. nwosu * , f. a. adekola and a. o. salami department of industrial chemistry, faculty of physical sciences, university of ilorin, ilorin, nigeria *corresponding author email: f.o.nwosu@gmail.com received 30 march 2016, revised 09 june 2017, accepted 23 june 2017 -------------------------------------------------------------------------------------------------------------------------------------------abstract an economically feasible technology for the removal of pollutants from wastewater is adsorption. active carbon was prepared by single stage method via chemical impregnation of pili nut-shell (pns) with orthophosphoric acid and activation at a temperature of 450 o c using precursor-acid ratio of 1:4 for a period of 2 h. the effects of initial concentration, contact time, adsorbent dose and ph on the uptake of 4-nitrophenol / para-nitrophenol (pnp) using pili nut-based active carbon (pac) were determined. the pac maximum adsorption capacity value (190.39 mg/g) was obtained at an initial concentration of 1000 mg/l and was found to be greater than that of a commercial granular active carbon (cgac) (166.97 mg/g) at an initial concentration of 800 mg/l. the bet surface area and total pore volume of pac (960 m 2 /g, 0.422 cm 3 /g) respectively were also greater than that of cgac (426.3 m 2 /g, 0.208 cm 3 /g). the pore size distribution of the pac (2.842 nm) classifies it to be within range of super-microporous and as such could be used for toxic gas removal as well as small liquid molecules. the langmuir isotherm best described the adsorption of pnp onto pac, while pseudo second order kinetics fitted best. the adsorption process was exothermic and spontaneous. thus, active carbon produced from pns can be used to adsorb pnp. keywords: pili nut, 4-nitrophenol, active carbon, kinetics, isotherm, thermodynamics -------------------------------------------------------------------------------------------------------------------------------------------introduction aquatic organisms and human are normally endangered because of frequent discharge of wastewater into surface water bodies. the major sources of phenols in surface and underground water are industrial effluents, domestic wastewaters, agricultural effluents and spillage of chemicals from anthropogenic activities [1]. the continuous exposure to low levels of phenol in water may cause diarrhoea, mouth ulcers, anaemia, dark urine, and damage of liver. humans and other living organisms could suffer mutagenesis and carcinogenesis as a result of phenol contamination [2]. it has been asserted that death of animal may result if dilute phenol solution is spilled to cover area greater than 25% of its total body surface [3]. the bureau of indian standards (bis) [4] permissible limit of phenol as 1.0 g/l for drinking water. the phenolic pollutants in most effluents could be treated using microbial organisms, chemical oxidation as well as photocatalytic degradation with aid of tio2 [5]. enzymatic polymerization and adsorption principles have been found useful for remediation of phenol polluted wastewater [6]. among others, selective adsorptions that make use of biological materials and active carbon have created great interest among the researchers. organic pollutants are adsorbed onto active carbon due to its large surface area but active carbon is difficult to prepare and exhibits higher disposal cost [6]. the preparation and regeneration of active carbon made scientists to search for the development of adsorbents from cheaper raw materials. these include active pak. j. anal. environ. chem. vol. 18, no. 1 (2017)70 carbons prepared from tamarind nut [7] and coconut husk [8] for the removal of phenolic compounds. therefore, the preparation of high grade active carbons via chemical activation of pns (waste material) for removal of low concentration of phenol molecules becomes inevitable. the main aim of this study was the preparation of low cost and environmentally safe active carbon adsorbent and its use for removal of 4-nitrophenol from wastewater compared with commercial active carbon adsorbent. thus, the pns, which are wastes, were converted into cheap carbonaceous adsorbents. batch adsorption and kinetics studies were carried out and data modeled into various adsorption models. materials and methods materials the precursor, pili nut (cannarium ovatum) shell was utilized for the preparation of active carbon. the mature pili nuts were harvested from its plant in anambra state, nigeria located at latitude and longitude 6 o 1 0 n, 6 o 55 0e. it was then stripped by boiling to expose the nuts. the nuts were washed with distilled water to remove impurities, air dried and de-shelled. it was crushed with hammer mill into small sizes. preparation of active carbon a known quantity (40.0 g) of pns was impregnated with 4 m h3po4 using acid to precursor ratio of 1:4; w/v. it was heated with a low heat and was placed in an oven maintained at 383 k for 7 h. the horizontal tubular furnace was first degassed by allowing n2 to flow into the reactor at 1000 ml/min for 30 mins. the mixture was then carbonized in a horizontal tubular furnace under a flow of n2 gas (500 ml/min) for 2 h. the carbonization procedure was carried out at 723 k. the activated sample was cooled to room temperature under n2 flow, washed with deionized water several times until ph 6-7 was obtained. it was then filtered under vacuum and dried in oven at 383 k for 8 hours. the dried samples were grounded, sieved to < 150 µm and stored in airtight plastic containers [9]. characterization of active carbon the standard test method of astm d 3838-60 [10] was used to determine the ph of the active carbon. the same samples used for ph determination were used further for electrical conductivity of the active carbon and results were read off [11]. moisture content of active carbon and raw materials were obtained by utilizing astm d 2867-9110 method [10] while their densities were obtained via tamping procedure [12]. structural analysis of active carbon the surface morphology of the active carbon was examined by scanning electron microscope (sem) using a phenon world scanning electron microscope. the crystalline nature of the active carbons was compared using a shimadzu x-ray diffractometer, model 6000 machine. a perkin elmer 1600 series ftir spectrophotometer model 1615 was used in the evaluation of functional groups present on the active carbon after crushing with nujol. the brunauer −emmett − teller (bet) method was adopted for n2 adsorption at 77k on to active carbons and data obtained were used for determination of surface area, pore volumes and pore size distribution of the active carbons. prior to nitrogen adsorption measurement, active carbons were separately degassed at 323 k. factors affecting adsorption distilled water was used to prepare stock solution of the test reagents; 1 g of pnp was dissolved in 1 l of distilled water. other concentrations were prepared by serial dilution using micro pipettes and standard volumetric flasks. in each of the factors studied, the pnp uptake, qt was calculated using the following expression: )1( m v)cc( q fot   where qt is the quantity of pnp uptake in mg/g, v is volume (l), co and cf are the initial and final concentrations in mg/l respectively. the plastic containers containing the mixture of various pnp pak. j. anal. environ. chem. vol. 18, no. 1 (2017) 71 concentrations and active carbons were then separately removed from the isothermal shaker and the solutions centrifuged to remove the adsorbent. the residual aqueous concentrations of pnp were then determined using uvvisible camspec m106 spectrophotometer. prior to determination using uvvisible techniques, the effect of variables on adsorption capacities of pac and cgac were done as follows: effect of initial concentration: adsorption was carried out in a set of plastic sample containers where 25 ml of pnp solutions of different initial concentrations ranging from 100 mg/l to 1400 mg/l were placed. equal amount of activated carbon (0.03 g) was added to each set of solution and kept on an isothermal shaker at 303 k for 4 h. effect solutions contact time: a 25ml of 1000 mg/l of pnp were placed in several sample plastic bottles and shaken on the isothermal shaker for different time intervals (ranging from 5 min to 4 h) at 303 k, using adsorbent dose of 0.03g. effect of adsorbent dose: a 25ml solution of 1000 mg/l of pnp were placed in several sample bottles and shaken on the isothermal shaker to equilibrium at 303 k, using various mass of the activated carbon varying from 0.03 g to 0.2 g. effect of ph of the solution: a 25ml solutions of 1000 mg/l of pnp were placed in several sample bottles, the ph of the solution were varied using 0.1 m hcl and/or 0.1 m naoh. the plastic bottles with the contents were shaken on the isothermal shaker till equilibrium was reached at 303 k, using an adsorbent dose of 0.03 g effect of temperature: using the established equilibrium conditions of adsorptions, 25ml of the solutions were shaken at different temperature i.e. 303, 318 and 333 k. results and discussion adsorbent characterization table 1 presents the comparison of physicochemical properties of pac produced from pns and that of a cgac. the yield of pili active carbon obtained in this study is 38.4 % which fell within the percentage range yields of other nut shells (20.8 – 45.0 %) [13]. the ph of 3.8 obtained for pac suggested that an l-type active carbon was produced while its conductivity value (391.5 µs/cm) indicated low ash content which could be attributed to washing of the pac with deionised water severally. its % bulk density value (0.51 g/cm 3 ) was found to be higher than minimum requirement (0.25 g/ml) for utilization as a commercial active carbon [14]. it was also higher than the reference commercial granular active carbon (bdh) value of 0.35 g/cm 3 . the low ash contents of h3po4 based active carbons are attributed to the leaching effect of the acid that increased with increase in concentration of the h3po4 [15]. figure 1 depicted the sem micrographs at 1000 and 2500 magnifications and it revealed numerous pores with irregular shapes that indicated increase in contact area and easy pore diffusion during adsorption [16]. the xrd powder analysis of pac and cgac active carbons are presented in fig. 2. there is a conspicuous peak at 2θ equal 24.0 o and 2θ equals 18 o for both the pac and cgac active carbons respectively and showed the crystalline nature of the active carbons. the surface area, pore volume and pore size distribution are calculated from the initial plot of amount of n2 gas adsorbed at stp against the relative pressures, p/po within the range of 0 – 1 for both pac and cgac active carbons (fig. 3). table 1. physicochemical properties of active carbons. parameters pac cgac yield (%) 38.4±2.35 ph 3.80±0.14 3.12±0.12 conductivity (µs/cm) 391.50±12.02 650±0.06 moisture content (%) 18.55±0.35 17.5±0.33 bulk density (g/cm3) 0.51±0.01 0.35±0.01 pore volume (cm3/g) 0.422 0.208 surface area (m2/g) 960.1 426.3 pore size distribution (nm) 2.842 3.300 pak. j. anal. environ. chem. vol. 18, no. 1 (2017)72 figure 1. sem images of pac. (a) x 1000; (b) x 2500 figure 2. xrd analysis of active carbons. (a) pac; (b) cgac figure 3. the nitrogen adsorption isotherms of the active carbons. (a) pac; (b) cgac a surface area of 960.1 m 2 /g and pore volume (0.422 cm 3 /g) obtained for pac are greater than surface area (426.3 m 2 /g) and pore volume (0.208 cm 3 /g) of commercial cgac active carbons. these values are within the minimum range of 500 – 1500 m 2 /g needed for industrial application and removal of small molecules from aqueous solution [17]. the pore size distribution (2.84 nm) obtained for pac could be classified as supper-microporous while that of cgac (3.30 nm) as mesoporous. the physical observation of kneel in pac is similar to that of cgac which showed initial fast adsorption rate (fig. 3). however, most average active carbon samples micropore volumes are generally between 0.2 – 0.5 cm 3 /g with surface area of about 1000 m 2 /g [18]. it has been reported that surface area in the range of 800 – 1300 m 2 /g and total pore volume in the range of 0.3 0.8 cm 3 /g of active carbon derived from olive stones are often used as precursors for high grade active carbon [19]. pak. j. anal. environ. chem. vol. 18, no. 1 (2017) 73 influence of various factors on amount of pnp uptake effect of concentration: (fig. 4a) shows the effect of the initial concentration (co mg/l) of pnp on the adsorption capacities of the prepared pac and cgac. as indicated in fig. 4a, the amount adsorbed increased from 51.38 to 190.39 mg/g and 41.56 to 166. 97 mg/g as pnp concentration increased from 100 to 1000 mg/l and from 100 to 800 mg/l for pac and cgac respectively. this may be due to the fact that increasing the initial concentration of pnp would provide an important driving force to overcome all mass transfer resistances of pnp between adsorbate solution and the adsorbent surface [20]. therefore the rate % which pnp molecules pass from the bulk solution to the particle surface will increase. similar trends were observed in the adsorption of pnp on granular active carbon in basal salt medium in which amount adsorbed increased from 48.62 mg/g to 183.51 mg/g as the initial concentration of pnp increases from 10 mg/l to 1000 mg/l. others with similar result in the adsorption of pnp using active carbon produced from different precursors are known [20.21, 22]. further increase for both adsorbent brought a slight decrease in the amount of pnp adsorbed and thus concentrations of 1000 mg/l and 800 mg/l were chosen as equilibrium concentration for pac and cgac, respectively. effect of contact time: the effect of contact time of adsorbents is important in adsorption processes because it exercises a great deal of influence on the adsorption capacity of adsorbents. fig 4b shows the variation in the quantity of pnp adsorbed by 0.03 g of pac and cgac at 303k with time. the rate at which adsorption occurred was rapid initially between 5 min to 30 mins, and became slower between 60 mins and 240 mins. this phenomenon may be due to a large number of available vacant surface sites for adsorption during the initial stage, and after a lapse of time, remaining vacant surface sites were difficult to be occupied due to repulsive force between solute molecules on the solid and bulk phases [21]. the high adsorption rate at the beginning of adsorption process by 0.03 g pac and cgac separately may also be due to the adsorption of pnp by exterior surface of the adsorbent. when saturation was reached at the exterior surface, the pnp molecules would then enter the pores of adsorbent and were adsorbed by the interior surface of the particles. this phenomenon takes relatively longer contact time (4 h) to reach equilibrium [22]. different equilibrium times of 5 h, 60 min and 1 h has been reported for the adsorption of pnp with various adsorbent using naoh modified palm oil fuel ash, amino-silane-activated palm oil fuel ash active carbon from coconut husk and commercial active carbon, respectively [20,23]. another researcher obtained equilibrium after 5 h [24]. thus, these differences in the time for adsorption process to reach equilibrium could be as a result of the functional groups present on the adsorbent as well as its surface morphology, such as the location and ease of access of the pores responsible for the adsorption. effect of adsorbent dose: (fig. 4d) shows the adsorbent dose profile diagram for both adsorbents (pac and cgac). the amount of pnp adsorbed per gram of the active carbon decreased with increasing adsorbent dose. it decreased from 166.31 to 66.52 mg/l and from 138.30 to 50.55 mg/l for pac and cgac, respectively when adsorbent dose increased from 0.03 g to 0.20 g. thus, for the remainder of the research, adsorbent dose of 0.03 g was used as the optimised adsorbent dose. this is similar to adsorbent dose reported during the adsorption of pnp using active carbon from coconut husk [23]. effect of ph of the solution: adsorption has been reported to be affected by the ph of the adsorbate [25]. as shown in (fig. 4c), the adsorption of pnp on pac and cgac reduced from ph of 2 to 6 and remained relatively constant between ph 6 and 12. pnp exists as p-nitro phenolate anion when the solution ph > pka and as neutral molecule when the solution ph < pka [26]. since the pka of pnp is 7.12, pnp can be adsorbed on the adsorbent surface by electrostatic attraction when the pnp is ionized and by deprotonation or by a weak electrostatic attraction mechanism [27]. this occurs because pnp exists as a neutral molecule since is a weak polar compound [28]. moreover, pak. j. anal. environ. chem. vol. 18, no. 1 (2017)74 pnp can also be adsorbed on the solid surface via a complex donor-acceptor mechanism [1]. in this study, the pnp was expected to have been adsorbed within the range of 2 to 6 via several other mechanism such as van der waals forces and complex donor-acceptor mechanism. however the pnp was more or less adsorbed at lower ph because it had been deprotonated and considering the fact that the ph of the adsorbents were 7.4 and 7.6 for pac (after neutralising with naoh) and cgac respectively, then there exist an electrostatic repulsion between the adsorbent and the adsorbate at the higher ph range [20,29,30]. effect of temperature: plots of the amount adsorbed at equilibrium, qe (mg/g) versus absolute temperature (k) are shown in (fig. 4e) for both pac and cgac. it can be seen from these figures that the amount adsorbed decreases with increasing temperature for both adsorbents, indicating the apparent exothermic nature of the adsorption process. in the liquid phase, an increase in temperature commonly increases the solubility of the molecules and their diffusion within the pores of the adsorbent materials is hampered and hence at higher temperature adsorbed molecules tend to desorb suggesting physiosorption [23, 29, 30]. the effect of increase in temperature on adsorption capacity was more pronounced for the pac than for the cgac active carbons. at 303 k, the adsorption capacity of pac decreased from 177.546 mg/g to 82.396 mg/g at 333 k and while at 303 k, cgac decreased from 111.660 mg/g to 83.296 mg/g at 333k. concnc (mg/l) time (min) (ph) mass (g) temp (k) figure 4. effect of adsorption conditions on adsorption capacities of the active carbons. (a) initial concentration; (b) contact time; (c) ph; (d) adsorbent dosage; (e) temperature pak. j. anal. environ. chem. vol. 18, no. 1 (2017) 75 equilibrium adsorption isotherms modeling the equilibrium experimental data obtained were fitted into six adsorption isotherm models, namely; langmuir, freundlich, temkin, dubnin-raduschkevich, harkins-jura and halsey, and they all gave varying degree of success. the langmuir adsorption isotherm model: this is based on the supposition of a homogeneous adsorbent surface with identical adsorption sites [31]. the linearized form of langmuir equation is given as: )2( qb 1 q c q c maxmax e e e  table 2. adsorption isotherm parameters. isotherm pac cgac qmax (mg/g) 250.0000 227.2727 kl 0.0063 0.0043 rl 0.1369 0.2238 langmuir r2 0.9968 0.9946 kf (mg/g) 10.7770 4.9602 1/n 0.4478 0.5593 freundlich r2 0.9499 0.9610 b 49.6850 51.5450 bt 50.7022 48.8727 at (l/mg) 0.0673 0.0409 temkin r2 0.9835 0.9966 kod 0.0024 0.0006 qd 169.9492 140.6958 e (kj/mol) 14.4334 28.8675 d-r r2 0.8999 0.9180 ah 5000 2000 bh 3.5000 2.6000 harkins-jura r2 0.7550 0.7737 nh -2.2334 -1.7879 kh 4.9465 x 10-3 57.0845 x 10-3 halsey r2 0.9499 0.9610 pac is active carbon produced at 723 k, 1:4, 2 h, cgac is commercial granular active carbon; d –r means dubunnin radushkevih adsorption isotherm the langmuir plots of qe/ce versus ce for both adsorbents are depicted in (fig. 5a) and the langmuir parameters obtained from the slope and intercept of the plots are listed in table 2. both exhibited high correlation coefficient, r 2 values of 0.9968 and 0.9946 for pac and cgac respectively. ce log (ce) in (ce) pak. j. anal. environ. chem. vol. 18, no. 1 (2017)76 ( 2 ) log (ce) in (ce) figure 5. various adsorption isotherms for the removal of 4-np using the active carbons. (a) langmuir; (b) freudlich; (c) temkin; (d) dubinin-radushkevic; (e) harkins jura; (f) halsey it can be observed from table 2 that the pac has an adsorption capacity (250 mg/g) higher than that of the cgac (227.27 mg/g). furthermore, a dimensionless separation factor, rl equation is given in equation 3: )3( ck1 1 r el l   it expresses the essential characteristic of the langmuir equation and indicates the nature of adsorption process, and its value was found to be in between 0 and 1 as listed in table 2. this confirms that the adsorption of 4-nitrophenol is favourable on both active carbons. similar results have been obtained by several authors for the adsorption of pnp and phenol using various adsorbents [23, 31, 32]. freundlich adsorption isotherm model: suggests neither homogeneous site energies nor limited levels of adsorption which implies that it can describe the experimental data of adsorption isotherm whether adsorption occurs on homogeneous or heterogeneous sites and it is not controlled by the formation of the monolayer [33]. the linearized freundlich equation is given as: )4(clog n 1 klog m x log e from the slope and intercept of the linear plots of ln qe versus ln ce; (fig. 5b), freundlich parameters 1/n and kf indicating the measures of adsorption capacity of pac and its intensity of adsorption respectively, were obtained and listed with the correlation coefficient value, r 2 (table 2). the results revealed that the adsorption of phenol on pac and cgac obeys both freundlich and langmuir adsorption isotherms, as indicated by high r 2 values (> 95%). moreover, the freundlich constant (kf = 10.777 mg/g) of pac is also larger than that obtained for cgac (kf = 4.960 mg/g). the results indicated that pac has higher adsorption affinity towards 4-nitrophenol than cgac [25]. this might be due to larger surface area of pac as a result of its highly porous nature, which makes pac more effective in the adsorption process. the value of 1/n indicates a favourable adsorption when 0 < 1/n < 1 [33]. the adsorption intensity, 1/n is found to be 0.448 and 0.559 for pac and cgac, respectively. it is pak. j. anal. environ. chem. vol. 18, no. 1 (2017) 77 observed that both adsorbents satisfy the conditions of heterogeneity. temkin adsorption isotherm: assumes that the heat of adsorption of all the molecules in the layer is inversely proportional to coverage of the adsorbent surface which might be attributed to adsorbate species – adsorbent interactions. a uniform distribution of binding energies up to some maximum binding energy is the characteristics of temkin adsorption [34]. the linear form of the temkin isotherm is represented in equation (5): e t t t e cln b rt aln b rt q          (5) and tb rt b  (6) figure 5(c) depicts the temkin isotherm plots for the pnp uptake onto the active carbons from which the relevant temkin adsorption isotherm values are obtained and are shown with values of correlation coefficients in table 2. the b values indicates the heat of adsorption obtained as 49.685 and 51.545 for pac and cgac respectively, while at representing the equilibrium binding energy are 0.0673 (l/mg) and 0.0409 (l/mg) for pac and cgac, respectively. thus, these values imply that the pnp are more strongly adsorbed on the pac surface than that of cgac indicating that there is a stronger interaction between pnp and the respective active carbon. similar results have been reported as per the adsorption of methyl orange using pinecone derived active carbon [35]. they reported bt value of 24.292 and at value of 0.162. dubinin-radushkevich isotherm: the linear expression of dubinin-radushkevich isotherm is: 2 adse kqlnqln  (7)          adk2 1 e (8)          ec 1 1lnrt (9) radushkevich and dubinin showed that there is a relationship between porous structure of sorbent and characteristic sorption curve obtained from adsorption data. as solute molecules of pnp move to adsorbent surface from bulk of the solution, a relationship between the mean free energy; e of sorption per mole of the sorbate and the constant, kad, is established. the energy, e and the parameter ε can be derived using equations 8 and 9 respectively. equation 7 represents the linear expression of the dubinin–radushkevich isotherm and the plot of ln qe versus  2 is depicted in fig. 5(d). table 2 reveals kad, qm, e, and r 2 values. the adsorption capacities, qm values of 169.949 mg/g and 140.696 mg/g were obtained for pac and cgac, respectively. these values are less than 250.0 mg/g and 227.272 mg/g as langmuir adsorption capacity for pac and cgac respectively. this might be as a result of variations in experimental conditions. the qm values of 25.94 mg/g and 26.06 mg/g calculated from dubininradushkevich isotherm for the uptake of congo red using leaves of water melon rinds and neem-tree have been reported [36]. these lower values might be as a result of the fact that adsorbents employed are biosorbents and not active carbon. generally, the accepted adsorption energy value range of 1.00 8.00 and 8.00 16.00 kj/mol indicated physical and chemical adsorption, respectively [30]. the e values of 14.433 kj/mol for pac and 28.868 kj/mol cgac indicated chemical adsorption for both adsorption systems. however, e values of (0.29 – 0.32 kj/mol) and < 8 kj/mol have been obtained by other researchers that indicated physiosorption [36.37] in the removal of direct red dye. harkins-jura adsorption isotherm model the existence of heterogeneous pore distribution explains the occurrence of multilayer adsorption. the model can be expressed as [35]: this can be linearised to give the following equation pak. j. anal. environ. chem. vol. 18, no. 1 (2017)78 e hh h 2 e clog a 1 a b q 1                  (10) where ah and bh are isotherm parameter and constant. the plot of 1/qe 2 versus log ce is presented in fig. 5(e) and values of harkin-jura constants and r 2 for both active carbons are shown in table 2. the r 2 values of harkins jura plots obtained are 0.7550 and 0.7737 for pac and cgac, respectively which are lower than values of langmuir isotherm whose r 2 values within range of 0.9499 – 0.9610 for pac and cgac. thus, it can be assumed that the adsorption process might not be multilayer. halsey adsorption isotherm: is one of adsorption isotherms that explains multilayer adsorption and is represented in eqn. 11 [38]. hetero porous nature of adsorbent is normally implied if experimental adsorption data fits into halsey adsorption isotherm equation. n clnkln e eh eq   (11) the above equation can be linearized to obtain: e h h h e cln n 1 kln n 1 qln                   (12) where kh and nh are empirical isotherm constant and exponents, respectively. from the plot of ln qe vs. ln ce (fig. 5f), values of halsey constants, kh and nh as well as their r 2 for pac/pnp and cgac/pnp adsorption systems are depicted in table 2. the nh values obtained (2.2334 and -1.7879) for pac and cgac respectively are similar to -2.224 and dissimilar to 1.82 and 1.78 obtained by other researchers [35,37]. if adsorption increased with decrease in nh values, then it indicates adsorption process to be endothermic [37] which might be attributed to the differences in adsorbent-adsorbate system. the parameters obtained for the various adsorption isotherms equations of both the pacpnp and cgac-pnp adsorption system are presented in table 2. the correlation coefficient factor, r 2 obtained indicated experimental data obtained in this study fitted well into the various adsorption isotherms investigated. on basis of correlation coefficient, r 2 , the six investigated isotherm models could be arranged in decreasing order of favoured adsorption isotherm as follows: langmuir > temkin > freundlich = halsey > dubinin-radushkevich > harkin-jura for both pac and cgac active carbons with exception of temkin that comes before langmuir for cgac. langmuir and temkin isotherms exhibited highest correlation coefficients, r 2 among other isotherms and depict homogeneity of the surface of pac and cgac as well as the monolayer adsorption nature of pnp onto the adsorbents. therefore, langmuir and temkin isotherm showed to best fit the equilibrium data for adsorption of pnp on pac and cgac. adsorption kinetics studies: the kinetic experimental data obtained for the adsorption of pnp on pac and cgac were interpreted by means of pseudo-first order, pseudo-second order, intra-particle diffusion and elovich kinetic models. pseudo-first order and pseudo-second order kinetic models the linear forms of pseudo-first order and pseudo-second order kinetic model equations are given in eqns. 13 and 14. despite, these models do not fit well for the whole range of contact times, the initial 20 to 30 min of adsorption process were applicable [31].   )13( 303.2 tk qlogqqlog 1ete  )14( q 1 qk 1 q t e 2 e2  the plot of linearized form of the equation is shown in fig. 6(a). the pseudo first-order rate constant k1, amount of pnp adsorbed at equilibrium qe and correlation coefficients are shown in table 3. the results showed that the correlation coefficients, r 2 obtained for both adsorbents for pseudo first order kinetics model (0.8761 and 0.876) were lower than their corresponding pseudo second order kinetics model (r 2 = 0.9626, 0.9906) and the experimental qe pak. j. anal. environ. chem. vol. 18, no. 1 (2017) 79 (177. 56 and 111.66 mg/g) did not agree with the calculated qe (125.81 and 52.30 mg/g) obtained from the linear plots of pac and cgac, respectively. therefore, adsorption of pnp onto the active carbons revealed that pseudo-first-order kinetic model failed to explain the kinetic adsorption process. although the pseudo-first order equation provides a fairly good fitting (r2 = 0.8761 and 0.8776) to the experimental data point, the pseudosecond order kinetic model (r 2 = 0.9626, 0.9906) describes the kinetic data better. this may be due to the fact the adsorption rate of pnp onto both active carbons depends on the behaviour over a whole range of adsorption process rather than depending on the initial 20 to 30 min as pseudofirst order kinetic model, as similarly reported by others [3]. (fig. 6b) shows the plots of t/q against t and the resulting kinetic parameters are presented in table 3. the calculated values of qe of 192.307 mg/g and 114.942 mg/g were closer to the experimental qe values of 177.56 mg/g and 111.66 mg/g for pac and cgac, respectively. however, an inconsistency in the agreement of the experimental qe and calculated qe for the adsorption of pnp with granular active carbon has been reported [24]. experimental qe value of 27.41 mg/g was reported and values of 95.70 mg/g and 99.74 mg/g were reported for pseudo-first and pseudo-second kinetic models respectively. moreover, it has been reported that pseudo-first order rates of 0.0104 /min and pseudosecond order rate of 0.0003 g/mg/min which are similar to values obtained in this study, with rate constants of 0.0083 /min and 0.0111 /min for pseudo-first kinetic models and pseudo-second order rates of 0.00015 g/mg/min and 0.00066 g/mg/min for pac and cgac, respectively [39]. the movement of solute molecules on to solid surface particulates from the aqueous phase, and then diffusion of the solute molecules into the pore interiors [37] could be described by the intraparticle diffusion rate equation and is given as [32]: )15(ctkq 2 1 idt  where kid and c are the intra-particle diffusion rate constant and boundary layer thickness, respectively. the plotting qt against t 1/2 as shown in (fig. 6c) indicates the effect of pore diffusion. the general features of an initial steep linear portion and plateau are attributed to the bulk diffusion; intra-particle diffusion and the plateau portion represent the equilibrium. the magnitudes of kid, c and the corresponding regression coefficients of both the adsorption system are listed in table 3. the pore diffusion rate constant, kid, values; 9.668 mg/g min1/2 and 3.9902 mg/g min1/2 indicated substantial diffusion of pnp onto pac and cgac active carbons. the existence of linear relationship between amount qe against t 1/2 indicates that intraparticle diffusion participated during the adsorption process. if the straight line of the plot of qe against t 1/2 passes through the origin, then intra particle diffusion becomes the controlling step. however, if the straight line does not pass through the origin, it indicates that the intra particle diffusion is not the only controlling step but that boundary surface effects are also involved [20]. the pore diffusion rate constant of 0.46 mg/g min 1/2 and 0.161 mg/g min 1/2 in the adsorption of pnp with zeolite and bentonite have been obtained [31]. the difference in this value and those obtained in this study shows that the prepared pac contains more pores available for adsorption than the natural zeolite and bentonite. this is similar to the adsorption of direct red dye 81 with bamboo sawdust and treated bamboo sawdust where kid values of 0.32 mg/g min 1/2 and 0.62 mg/g min 1/2 were obtained. moreover, kid value of 3.2479 mg/g min 1/2 has been obtained in the adsorption of phenol using active carbon from olive stones [32]. the adsorption of pnp using active carbon fibre and granular active carbon gave similar result obtained in this study with kid values of 8.924 mg/g min 1/2 and 11.91 mg/g min 1/2 , respectively [23]. the adsorption data could also be treated using elovich equation that is shown in equation 16 [31]: )16(e dt dq tqt  pak. j. anal. environ. chem. vol. 18, no. 1 (2017)80 this on linearising and simplifying can be written as:     )17(tln 1 ln 1 q t     where α (mg/g min) and β (g/mg) are the initial adsorption rate constant and desorption rate constant, respectively. kinetics of adsorption of pnp with pac and cgac also followed elovich equation with the plots of qt against ln t giving straight lines with fairly high correlation coefficient (fig. 6d). the elovich constants α, β computed from the plots are 18.248 mg/g min, 0.0305 g/mg for pac and 133.823 mg/g min, 0.0721 g/mg for cgac as given in table 3. these values might also explain why the adsorption process took longer time to reach equilibrium. it has been reported that α, β values of 1.10 mg/g min and 1.05 g/mg were obtained for the removal of methyl orange using bamboo sawdust and 2.23 mg/g min and 0.58 g/mg when using acid treated bamboo sawdust indicating that the treatment increased the ability of the adsorbent to adsorb more and reach equilibrium faster than the raw sample [37]. the comparison of correlation coefficients obtained for pseudo-second order kinetic model (r 2 = 0.9626, 0.9906) explained the adsorption process better than elovich model (r 2 = 0.9063 and 0.9286). however, in overall, the pseudosecond order kinetic model still appears to be the rate determining model as it is the model with the highest correlation coefficient values (table 3). table 3. kinetic parameters of adsorption of pnp. kinetic model pac cgac k1 (min -1) 8.2908 x 10-3 11.1054 x 10-3 qe (mg/g) 125.8056 52.2998 pseudo 1st r2 0.8761 0.8776 k2 (gmg -1min-1) 1.5131 x 10-4 6.6570 x 10-4 qe (mg/g) 192.3077 114.942 pseudo 2nd r2 0.9626 0.9906 kid (mg/gmin 1/2) 9.668 3.9902 c 27.744 52.043 intra-particle diffusion r2 0.9508 0.9232  18.248 133.823  0.0305 0.0721 elovich r2 0.9063 0.9286 pac means pilli nut active carbon; cgac indicates commercial granular activated carbon time (s) time (min) t 1/2 in (t) figure 6. kinetic models fit of adsorption of 4-np with the active carbons. (a) pseudo-first; (b) pseudo-second; (c) intra-particle; (d) elovich pak. j. anal. environ. chem. vol. 18, no. 1 (2017) 81 thermodynamics studies: thermodynamic parameters can provide useful and strong information about the mechanism involved in the adsorption process. the characterization of equilibrium system like the adsorption system in this study are described by thermodynamic parameters such as free energy (∆g), enthalpy (∆h) and entropy (∆s) changes which were calculated using eqns. 18 to 21. )18( rt e alnkln a2  )19( r s rt h kln     )20(klnrtg  )21( c cc k i fi  where k2 is the pseudo second order rate constant, a is pre-exponential factor of the arrhenius equation, ea is the activation energy of the reaction, r is gas constant (8.314 jmol -1 k -1 ), t is absolute temperature in k, ci is initial concentration before adsorption and cf is concentration left in solution after adsorption. the thermodynamic plots obtained from eqns. 19 to 21 are depicted in fig.7a while the values of ∆g, ∆h, ∆s and ea for both adsorbents are listed in table 4. as seen from table 4, the negative values of the enthalpy, entropy and free energy changes indicate exothermic nature of the processes, decrease in disorderliness and that the adsorption processes is spontaneous and feasible. moreover the ∆h values of adsorption of pnp with pac (-17.338 kj/mol) as well as cgac (-8.430 kj/mol) lie between 2.1 and 20.9 kj/mol indicating that adsorption of pnp can be classified as physiosorption [40] for both pac and cgac. furthermore, pac shows higher adsorption enthalpy changes than the cgac, which signifies that pac has a stronger affinity for pnp than cgac [41]. the thermodynamic results obtained in this study are in agreement with the results observed by other researchers [23, 29, 30, 41]. the ∆h values of -29.46 kj/mol and -17.76 kj/mol obtained for active carbon fiber and granular active carbon in the adsorption of pnp as well as ∆h values of 4.4347 kj/mol and -7.766 kj/mol in the adsorption of pnp using natural zeolite and natural bentonite have been reported [23, 31]. table 4. thermodynamics parameters. ∆g (kj/mol)adsorbent temp (k) ea (kj/mol) ∆h (kj/mol) ∆s (j/mol) 303 318 333 pac 39.887 -17.338 -69.162 -3.700 -4.473 -5.792 cgac -8.430 -41.990 -4.384 -4.692 -5.665 1/t (k -1 ) 1/t (k -1 ) conclusion pac prepared from pns adsorbed pnp better than cgac. the bet specific surface area and total pore volume of pac were higher than those of cgac while pore size distribution of pac classified it as super-micro porous. the amounts of pnp adsorbed by both adsorbents were pak. j. anal. environ. chem. vol. 18, no. 1 (2017)82 strongly influenced by experimental conditions. langmuir isotherm best described the adsorption of pnp on the pac while pseudo-second-order kinetic model fitted well with the experimental data of both activated carbons. intra particle diffusion alone could not describe the rate determining mechanism for the adsorption processes. the adsorption process was found to be exothermic and spontaneous. thus, there is high potential ability of pac to remove pnp from aqueous solution and could also be utilize for removal of gas and small size molecules from industrial effluents. reference 1. k. arinjay, k. shashi, k. surendra, v. dharam and b. gupta, j. hazard. mater., 147 (2007) 155. https://doi.org/10.1016/j. jhazmat.2006.12.062 2. j. michalowicz and w. duda, polish j. environ. stud., 16 (2007) 347. 3. atsdr (agency for toxic substances and disease registry). us department of health and human services, usa. (1998). 4. bis tolerance limit for industrial effluents discharged into inland surface waters: coke oven is 2490 (part 1), bureau of indian standards, new delhi. (1974). 5. a. a. hatem, m. m. jamil, y. rosiyah, b. radzi and m. abas, asian j. chem., 25 (2013) 9573. 6. m. c burleigh, m. a. markowitz, m. s. spector and b. p. gaber, environ. sci. technol., 36 (2002) 2515. https://doi.org/10.1021/es011115l 7. k. srinivasan, p. b. s. rao and a. ramadevi, ind. j. environ. hlth, 30 (1998) 303. 8. v. p. vinod and t. s. anirudhan, j. sci. ind. res., 61 (2002) 128. 9. m. m. gómez-tamayo, a. marcías-garcía, m. a. díaz-díez and e. m. cuerda-correa, j. harzard. mater., 153 (2008) 28. 10. american society for testing and materials annual book of astm standard. astm, philadelphia pa (1996) 15.01 11. c. a. toles, w. e. marshall and m. m. johns, carbon, 37 (1999) 1207. https://doi.org/10.1016/s00086223(98)00315-7 12. m. ahmedna, w.e. marshall and r. m. rao, bioresour. technol., 71 (2000) 113. https://doi.org/10.1016/s09608524(99)00070-x 13. l. h. wartelle and w. e. marshall, j. chem. technol. biotechnol., 76 (2001) 451. https://doi.org/10.1002/jctb.408 14. american water works association (awwa) standard for granular activated carbon. american water works association, ansi / awwa b604 – 90, denver co. (1991). 15. b. corcho-corral, m. olivares-marín, c. fernández-gonzález, v. gómez-serrano and a. macías-garcía, appl. surf. sci., 252 (2006) 5961. https://doi.org/10.1016/j.apsusc.2005.11.007 16. j. b. patel and p. sudhakar, electr. j. environ. agric. food chem., 7 (2008) 2735. 17. r. c. bansal, m. goyal, activated carbon adsorption. taylor and francis group, published by crc press uk. (2005) 497. 18. h. jankowska, a. swiatkowski, j. choma, active carbon, ellis-harwood, chichester, uk, (1991) 50. 19. a. linares-solano, g. rodriguez-reinoso, m. molina-sabio and j. d. lopez-gonzalez, adv. sci. technol., 1 (1984) 223. 20. a. a. hatem, m. m. jamil, a. a. ahmad and m. radzi bin abas, j. purity utility reaction environ., 1 (2012a) 104. 21. a. a. ahmed, b. h. hameed and n. aziz, j. hazard. mater., 141 (2007) 70. https://doi.org/10.1016/j.jhazmat.2006.06.09 4 22. m. sathishkumar, a. r. binupriyu, d. kavitha and s. e. yun, biores. technol., 98 (2007) 866. https://doi.org/10.1016/j. biortech.2006.03.002 23. a. a. hatem, m. m. jamil, y. rosiyah and m. bin radzi abas, asian j. chem., 25 (2013) 9573. 24. s. suresh, v. c. srivastava and i. m. mishra, chem. ind. chem. eng. quarterly, 19 (2013) 195. https://doi.org/10.2298/ciceq111225054s pak. j. anal. environ. chem. vol. 18, no. 1 (2017) 83 25. s. rengaraj, m. seuny-hyeon and r. sivabalan, waste manag., 22 (2002) 543. https://doi.org/10.1016/s0956-053x(01)000 16-2 26. j. m. li, x. g. meng, c. w. hu and j. du, biores. technol., 100 (2009) 1168. https://doi.org/10.1016/j.biortech.2008.09.015 27. b. b. tewari and m. boodhoo, j. coll. inter. sci., 289 (2005) 328. https://doi.org/10.1016/j.jcis.2005.04.032 28. n. calace, e. nardi, b. m. petronio and m. pietroletti, environ. pollut., 118 (2002) 315. https://doi.org/10.1016/s0269-7491(01)003 03-7 29. d. tang, z. zheng, k. lin, j. luan and j. zhang, j. hazard. mater., 143 (2007) 49. https://doi.org/10.1016/j.jhazmat.2006.08.066 30. l. m. cotoruelo, m. d. marques, f. j. diaz, j. rodriguez–mirasol, j. j. rodriguez and t. cordero, chem. eng. j., 184 (2012) 176. https://doi.org/10.1016/j.cej.2012.01.026 31. g. .varank, a. demir, k. yetimezsoy, s. top, e. sekman and m. s. bilgili, indian j. chem. technol., 19 (2012) 7. 32. t. bohli, n. fiol, i. villaescusa and a. ouederni, j. chem. eng. process. technol., 4 (2013) 165. https://doi.org/10.4172/2157-7048.1000158 33. n. a. halif, w. m. a. w. daud, i. m. noor, and c. r. c. hassan, aeeseap j., 31 (2007) 23. 34. m. i. tempkin and v. pyzhev, acta phys. chim. ussr, 12 (1940) 327. 35. m. r. samarghandi, m. hadi, s. moayedi and a. f. barjasteh, iran j. environ. health sci. eng., 6 (2009) 285. 36. m. b. ibrahim and s. sani, open j. phy. chem., 4 (2014) 139. 37. t. a. khan, s. dahiya and i. ali, gazi university j. sci., 25 (2012) 59. 38. c. a. basar, j. hazard. mater., 135 (2006) 232. https://doi.org/10.1016/j.jhazmat.2005.11.055 39. b. h. hameed, colloid. surf.a: physicochem. eng., 307 (2007) 45. https://doi.org/10.1016/j.colsurfa.2007.05.002 40. s. k. theydan and m. j. ahmed, j. anal. appl. pyrol., 97 (2012) 116. https://doi.org/10.1016/j.jaap.2012.05.008 41. a. li, h. wu, q. zhang, g. zhang, c. long, z. fei and f. liu, chinese j. polymer sci., 22 (2004) 259. microsoft word 01-175-193-pjaec-02022022-424-c-revised galley pro cross mark pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 175 – 193 http://doi.org/10.21743/pjaec/2022.12.01 recent developments in nano-emulsions’ preparatory methods and their applications: a concise review rafia rehman 1* , aqsa younas 1 , shaheed ullah 1 , afsar b ano 2 , rabeea muzaffar 3 , shehnaz 1 , anila 1 and ataf ali altaf 1,4 1department of chemistry, faculty of sciences, university of okara, okara 56130, pakistan. 2department of physics, syed babar ali school of science and engineering, lahore university of management sciences, lums 54792, lahore, pakistan. 3department of biochemistry, faculty of sciences, university of agricluture, faisalabad, faisalabad 38000, pakistan. 4department of chemistry, university of gujrat, hafiz hayat campus, gujrat 50700, pakistan. *corresponding author email: rehman_rafia@yahoo.co.uk received 02 february 2022, revised 30 september 2022, accepted 24 october 2022 -------------------------------------------------------------------------------------------------------------------------------------------abstract nano-emulsion is one of the most effective and size-controlled mediums for effective drug delivery syste ms, the formation of cosmetics products, food preservatives, and insecticidal and antimicrobial products. therefore, a durable and sophisticated approach is primely important in preparing effective nano-e mulsions. some of the established fabrication approaches towards nanoemulsion are the high and low-energy methods. depending upon the required results of formulations, these two methods are further divided into sub-categories such as ultra-sonicators, micro-fluidizers, high-pressure homogenizers, phase inversion temperature, phase inversion composition, etc. this review highlights all the available methods to form nano-emulsion by adopting high-energy and low-energy techniques. in addition, this review also elaborates on the importance of nano-emulsions in various end products, as nano-carriers and patents have also been awarded in this field. besides, the required improvements in this field have been discussed briefly to establish the most authentic approach toward nano-emulsion formation. keywords: nano-emulsions, high energy methods, low energy methods, nanocarriers, drug delivery, cosmetics products, antimicrobial agents, food preservations -------------------------------------------------------------------------------------------------------------------------------------------introduction nano-technology is a multi-faceted technique involving the construction and utilization of various nano-scale systems. nano-technology is gaining attention in nano-biomedicines, food engineering, pharmacology, entomology, and parasitology. nano-technology has also demonstrated that it can speed up the function of a medicine delivery system by allowing the drug to reach its precise target and perform a specified action. nano-medicines provide several advantages, including increased therapeutic efficiency and fewer side effects. nanoemulsions are proving to be the best alternative for all these purposes. the emulsion refers to a colloid in which both dispersed and dispersion phases are liquids. surfactant is added to control the division of these two phases. at the point when the arrangement of emulsion comes to the size of 20-200 nm, it is named nanoemulsion. the nano-emulsions (containing nano-sized droplets) tend to boost bioavailability and review pak. j. anal. environ. che m. vol. 23, no. 2 (2022)176 optical transparency; hence nano-science is now being employed to treat a variety of infections and disorders [1-4]. nanoemulsions (also regarded as nano-formulations) can be arranged using the dispersal component of the oil in water or water in oil that additionally permits the settlement of different unique constituents [5]. nano-emulsion has nanosize; which enhances the surface region, with the goal that the assimilation rate is precisely upgraded and expands the bioavailability of the definition. nano-emulsions can improve the proficiency of the medication conveyance framework. nano-emulsions aren’t deadly or harmful, so they can undoubtedly be utilized for skin and mucous film. nano-formulations formulated using essential oils have been acknowledged for human utilization. nanoemulsions are dynamically and thermodynamically stable items, henceforth, they don't show issues of creaming, flocculation, coalescence, and sedimentation. because of higher physical stability, nanoemulsions are produced in many forms, such as foams, sprays, liquids, and creams. nanoemulsions don’t damage human and animal cells, so these are human and animal-friendly [6]. considering the topic's importance, this comprehensive review focuses on various preparatory approaches and applications of nano-emulsions. fabrication of nanoemulsion the nanoemulsions consist of two phases termed the discontinuous and continuous phases or dispersed phase and dispersion medium, respectively. there are three essential/basic components required for the fabrication of nanoemulsions. 1) oil phase 2) aqueous phase 3) surfactant/ emulsifier 4) co-surfactants (in some cases) the surfactants or surface tension reducers are utilized to blend the two phases. surfactants blend the two phases by reducing the interfacial tension. protein, polysaccharides, small molecules, and phospholipids are some common examples of emulsifiers [7-9]. classification of nanoemulsion based on the type of dispersion medium and dispersed phase, emulsions are divided into two essential classes, i.e., i) oilin-water (o/w) and ii) water-in-oil (w/o). in the oil-in-water class, droplets of oil are distributed to the continuous phase of water, while in the water-in-oil, water drops are distributed in oil [7]. both types are displayed in fig. 1. figure 1. types of nanoemul sion [4] for the preparation of oil in water nanoemulsion, a surfactant/emulsifier with a high value (8-18) of hydrophilic-lipophilic balance is used, while for the preparation of water in oil nanoemulsion, an emulsifier with low hydrophilic-lipophilic balance (3-6) are preferred [7-10]. comprehensive detail of preparation methods of both these classes is described in the next points. pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 177 methods of nanoemulsions preparation/ fabrication utilizations of nanoemulsions are reliant upon their strength of stability and their capacity to deliver required components to respective points. nanoemulsions can solubilize non-polar dynamic composites. numerous nanoemulsions are stable, and a couple might be unstable. thus, if a nanoemulsion is not too long stable, it is formulated just before to utilized [9]. according to a literature survey (from 2005 to 2021), high and low-energy methods are two well-known ways to prepare o/w or w/o nanoemulsions. as the word ‘’high energy’’ represents, it is the method in which required large disturbing forces are given by mechanical devices. in contrast, the ‘’lowenergy method’’ does not need any external energetic force [6, 8]. all preparation methods have importance as they lead towards the formation of desired droplets size-based nanoemulsion to make our life easy. but, some methods also have adverse limitations. the following sections describe the details of high energy as well as low energy methods and sub-types involved in them, along with the details of instrumentations used. high energy methods to prepare nanoemulsion earlier reports (from 2005 to 2017) show that higher energy stirring and ultrasonic emulsification have remained the most extensively consumed approaches by researchers [9]. in high-energy methods, various mechanical devices (such as ultrasonicators, micro-fluidizers, high-pressure homogenizers, etc.) are used to provide distracting forces that lead to the production of small-sized droplets. types of equipment, conditions of production (for example, temperature and time) together with the properties and composition of the sample are the aspects that influence the size of produced droplets [10-12]. a brief overview of the reported high-energy methods is given below. advantages of high energy methods high-energy methods are used because they require less time for formation [10-12]. these methods allow the well-controlled size of droplets that make it remarkable. these methods also provide a large selection of integral components [12]. disadvantages of high energy methods high-energy methods of nanoemulsion production are not cost-effective as they consume more energy and require refined instruments. high-energy methods require huge energy and disruptive force, these methods are not useful to thermo-labile (heat sensitive) and macro-molecules with nucleic acids, enzymes, and proteins [13-15]. high-pressure homogenization among different reported high-energy methods, high-pressure homogenization is the most commonly used method to produce nanoemulsion. this method got supreme importance due to its piston homogenizer or high-pressure homogenizer that leads to the production of nanoemulsions up to 1 nm in size. the production of nano-emulsion by this method involves macro-emulsion which is forcefully forwarded by a short orifice at a pressure between 500-5000 psi [13, 14]. polydispersity index (pdi) is used to specify the uniformity of the droplets. so, the highpressure homogenization procedure is/can be repeated again and again till the final product approaches to desired pdi and droplet size [15]. the pdi score is inversely proportional pak. j. anal. environ. che m. vol. 23, no. 2 (2022)178 to droplet size uniformity. in nanoemulsions, a higher pdi suggests less droplet size uniformity. pdi less than 0.08 indicates monodispersed samples, pdi between 0.08 and 0.3 indicates a narrow-sized distribution, and pdi greater than 0.3 indicates a broadsized distribution [2, 16]. chenni et al. prepared emulsions by adopting this method of the formulation [17]. muhammadi et al. and liu et al. prepared nanoemulsion of essential oil of peppermint/ eucalyptus and cinnamon essential oil by using the method of highpressure homogenization, respectively [18, 19]. advantages of high pressure homogenization as numerous forces such as hydraulic shear, severe turbulence, and cavitation work throughout the process, hence extremely small droplet-sized nanoemulsions are achieved, as shown in fig. 2. the main advantage of the high-pressure homogenization procedure is/can be repeated again and again till the final product approaches to desired pdi and droplet size [19]. figure 2. high pressure val ve homogenizer [2, 3] disadvantages of high pressure homogenization acquiring small droplets in submicron levels requires a lot of energy. during high pressure homogenization, this amount of energy and increasing temperatures process may lead to the denaturing of the components. proteins, enzymes, and nucleic acids (thermolabile compounds) may be denatured [2]. high shear stirring the method of high shear stirring, including high-energy mixers and rotor-stator systems has been used (2005 to 2021) to synthesize nano-emulsions. these have been used because by increasing their mixing intensity, the operator can considerably reduce the size of the internal phase droplet. however, emulsion preparations with an ordinary droplet size of less than 200-300 nm are difficult to obtain. by conventional mixers, the process can be carried out in batches. in recent studies, colloid mills are being used to provide an incessant mode and enhance shear stress at dispersion. among them, the most trendy colloid mill is ‘silverson flow mixers’ (fig. 3), wherein rotors and stators possess configurations to obtain extra efficient emulsification. in this mixer, a high rarefaction is formed inside the disintegration head at high rotor speeds, and the emulsion constituents are slurped up by the rotor and stator unit [20]. the emulsion is thrown to the periphery by centrifugal force, resulting in significant dispersion in the space between the rotor and the stator's inner wall. the emulsion is passed through the stator's outer orifice at high speed before exiting the apparatus. the degree of aeration during emulsification should be kept minimum using modern technologies [21]. because the highest degree of dispersion possible for the system is pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 179 typically not obtained in the single-pass regime, the multi-pass regime is frequently applied [8, 9, 14]. figure 3. sil verson fl ow mi xer disadvantages of high shear stirring for viscous media and emulsions with a high fraction of the internal phase along droplet size of more than 1 mm, the effectiveness of high-shear stirring is traditionally reduced. this process also requires high energy [12]. ultrasonic emulsion ultrasonic emulsification can be done via two methods. firstly, the oil phase can be scattered in the continuous phase as droplets via an acoustic field (that creates interfacial waves). secondly, ultrasounds incite auditory cavitation that affords the creation and breakdown of micro-bubbles correspondingly due to the pressure vacillation of sound waves. droplets of the desired size are produced by collapses of micro-emulsions which mess up large droplets into sub-micron or nano size [21, 22], as shown in fig. 4. the pre-mixed macro-emulsion is disturbed by a vibrating solid surface at 29 khz or higher frequencies in the ultrasonic emulsion process. the produced sound field in most ultrasonic systems is not homogenous. as a result, considerable power is required since all droplets must encounter the maximum shear rate and emulsion recirculation. even at modest concentrations, this method of recirculation (many times) makes the emulsion homogeneous in size. the emulsifier, the amount of emulsifier, and the viscosity of the phases are the most important parameters influencing homogenization efficiency. these variables must be optimized to make nanoemulsions with fine and desirable droplets [23, 24]. kaur et al. prepared aloe vera essential oil-based nanoemulsions by adopting the ultrasonic emulsification method [25]. figure 4. working pri nci ple of ul trasoni cator emulsi fier [1] advantages of the ultrasonication process nanoemulsions fabricated by ultrasonication process have excellent loading as well as release efficiency. the ultrasonic emulsion also has good physical properties because of its small droplet size [23]. disadvantages of the ultrasonication process sonication procedures can cause denaturation of proteins, de-polymerization of polysaccharides, and oxidation of lipids [22]. micro-fluidization micro-fluidizers are commonly used in the pharmaceutical industry to create fine droplet-sized emulsion. a device called a micro-fluidizer is utilized in this procedure, which provides high pressures. the high pak. j. anal. environ. che m. vol. 23, no. 2 (2022)180 pressure causes the macro-emulsion to pass through the interaction chamber during the action, resulting in nano-emulsions with submicron-sized particles. reiterating the technique numerous times while varying the operating pressure to acquire preferred-sized particles can result in a uniform nanoemulsion [2, 14]. micro-fluidizer consists of two jets (also called micro-channels) from which crude emulsion is added. from both jets, a crude emulsion is added that combines at the interaction chamber and then undergoes collision, as shown in fig. 5. figure 5. the worki ng approach of microfl ui di zer [3] a pneumatic power pump (capable of compressing air up to pressures 150-650 mpa) is used to provide mobility to the crude emulsion [14, 24]. this high pressure allows the crude emulsion to travel through microchannels, and when crude emulsion from opposite channels collides, a massive shearing force is created, which aids in the development of fine emulsion [26]. patel et al. prepared micro-emulsion via the microfluidization technique [27]. advantages of micro-fluidization micro-fluidization could be used to make nanoemulsions containing active substances to produce edible films with various functional and physical qualities [23]. disadvantages of micro-fluidization this process has the following disadvantages. the nanoemulsion temperature increased due to the use of high pressure. as emulsification requires a longer time so droplet size increases due to coalescence [23]. jet disperser jet disperser may have two or more jets, each from opposing bores, for introducing crude emulsion. typically 0.3-0.5 mm is the diameter range of bores in jet dispersers. to coordinate the energy dispersion of the emulsion jet an orifice plate (also known as a homogenizing nozzle) is used. in front of the bores, due to laminar elongation flow droplets are disrupted predominantly, as shown in fig. 6. because orifice plates have no moving parts, jet dispersers can be employed at high pressures (300-400 mpa) [28, 29]. figure 6. jet di sperser advantages of jet disperser in the jet disperser method, by driving the circulation stream via microchannels at high pressure toward the impregnated area, a great shearing action is created, resulting in an extraordinarily thin emulsion [28]. disadvantages of jet disperser the main disadvantage of this method is that if the emulsions have high viscosity, then droplet size gets disturbed due to laminar elongation flow and forces in the turbulent flow [28]. phase inversion temperature in this method, a high temperature is given to micro-emulsion to bring about pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 181 change in its phase and production of nanoemulsion [8, 15]. a detailed description of this method is provided in the following points. membrane method two types of membranes, hydrophilic and hydrophobic, are used for emulsion formation. in the membrane methods, fluid discharge via numerous pores or microchannels within the membrane leads to the production of fine droplets. when the internal phase is expelled by the membrane, droplets are produced within the membrane/continuous phase interface. membrane emulsification is used after preliminary dispersion to produce emulsions with smaller internal phase droplets as shown in fig. 7. figure 7. worki ng approach of membranes the hydrophilic membrane causes the crude emulsion to be pushed through the membrane, resulting in smaller droplets [30, 31]. due to this pumping, an inversion of phase took place, which resulted in the production of fine emulsion. when a hydrophobic membrane is used, it leads to the formation of the reverse emulsion. when the membrane is static, quick detachment of droplets from the surface re-circulation or stirring of the produced emulsion is done. there are two membrane types: fixed and vibrating [31]. recirculation or stirring is done to detach formed emulsion droplets from devices with fixed membranes. devices with rotating or vibrating membranes are moved fast to detach the droplets, but this considerably depends upon the designs of the gadgets [9, 23]. advantages of the membrane method membrane emulsification has several advantages, including 1) uniform particle production, 2) droplet size control via appropriate membrane pore size selection, 3) low shear stress, 4) reduced energy requirements, 5) high plant flexibility, 6) precise, flexible and selective manufacturing of a variety of particles such as simple and multiple emulsions, liposomes and microspheres [30]. disadvantages of the membrane method membrane pore size and distribution are critical aspects of membrane emulsification [30]. low energy methods to prepare nanoemulsion low-energy methods are trendy nowadays because low-energy approaches rely on the chemical potential of the components to provide energy to nanoemulsions. by gently mingling the components, nanoemulsions develop spontaneously at the oil-water phase interface [23]. low-energy methods are mainly categorized as phase inversion temperature and phase inversion of components which uses physiochemical properties of the system to provide the fine size of droplets [31]. but the spontaneous nanoemulsion formulation method also includes in these methods. advantages of the low-energy methods these methods are easy, nondestructive, and do not cause any damage to encapsulated molecules [32]. nanoemulsion pak. j. anal. environ. che m. vol. 23, no. 2 (2022)182 (having fine droplet size) can be attained via low-energy methods without any adverse effects [33, 34]. disadvantages of the low-energy methods although low-energy methods are generally more efficient in obtaining smallsized droplets than high-energy methods, there are some limitations to low-energy methods related to the usage of some types of oils and emulsifiers (possessing proteins and polysaccharides). to avoid this drawback, higher-level concentrations of synthetic surfactant are added to get nanoemulsions in low-energy techniques, but this addition minimizes their application area, especially for various food processes [3]. spontaneous nanoemulsion the spontaneous nanoemulsion method is considered of supreme importance due to releasing chemical energy within the continuous phase while diluting at a constant temperature without any transition of phase during the emulsification procedure [8, 14, 35]. by low-energy methods, at room temperature without requiring any special devices, nanoemulsions can be obtained depending upon the interfacial tension, viscosity of interfacial and bulk, phase transition region, surfactant structure, and surfactant concentration [36, 37]. when oil, water, and surfactant (depending upon the choice of oil) are mixed nanoemulsion is formed spontaneously, as shown in fig. 8. this method depends upon the handling conditions (speed of stirring, the addition rate of material, etc.) [3]. b. sundararajan et al. prepared a nanoemulsion of ocimum basilicum l. through a spontaneous low-energy method [4]. figure 8. production of emul sion vi a spontaneous method [38] advantages of spontaneous nanoemulsion the spontaneous nanoemulsion method is considered of supreme importance due to releasing chemical energy within the continuous phase while diluting at a constant temperature without any phase transition during the emulsification procedure [36]. disadvantages of spontaneous nanoemulsion the lack of an oil phase and the presence of a solvent are the method's limitations [9]. phase inversion method when the phase undergoes transition, chemical energy is released by the system that leads toward the formation of fine nanoemulsion [38-39]. the required phase transitions can be produced by switching the composition at a constant temperature or altering the temperature at a constant composition [23, 40]. pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 183 phase inversion temperature the temperature is changed in this method while the composition remains constant. when dealing with the phase inversion temperature approach of nanoformation, non-ionic surfactants with temperature-dependent solubilities (such as poly-ethoxylated surfactants) are used. adjusting the affinities of surfactants for water and oil as a function of temperature produces a fine nanoemulsion [41, 42]. polyethoxylated surfactants become lipophilic when heated because the polyoxyethylene groups dehydrate. to make nano-emulsions using the phase inversion temperature method, the temperature of the sample must be brought to the phase inversion temperature level, also known as the hydrophile-lipophile balance (hlb) level [39]. at hydrophile lipophilebalance (hlb) temperature, interfacial tensions become extremely low, and this is how the method helps to produce fine nanoemulsions. however, this method's emulsion production is very fast and spontaneous, but obtained emulsions are very unstable [43]. it has been observed that instant cooling of the emulsion at the temperature of phase inversion can produce stable and fine emulsion droplets [44, 45]. safaya et al., prepared nanoemulsion of neem oil by adopting this method [42]. advantages of phase inversion temperature method the advantages of this method are that in the bicontinuous phase of microemulsion it provides complete solubilization of the oil regardless of initial phase equilibrium [29]. disadvantages of phase inversion temperature method complexity, accuracy requirements, and the usage of synthetic surfactants are among the limitations of this method [12]. phase inversion composition emulsion inversion point (eip) is another name for this approach. at a steady temperature, the phase inversion composition process undergoes configuration alternation [46]. fine nano-emulsions can be made by gradually adding water or oil to an oilsurfactant or water-surfactant mixture. because adding one component to an emulsion is much easier than bringing about fast temperature variation, the phase inversion composition approach is more reliable in large-scale production than the phase inversion temperature method [6, 8, 23]. advantages of phase inversion composition this method is inexpensive, does not require the use of organic solvents, and has a high degree of thermodynamic stability [23]. disadvantages of phase inversion composition this approach did not work well with label-friendly surfactants, including whey protein, quillaja saponin, sucrose monoesters, and casein in nanoemulsions [23]. solvent displacement method at room temperature, nanoemulsions can be made by mixing an organic phase (including dissolved oil in a solvent such as ethanol, acetone, etc.) with an aqueous phase containing surfactants. emulsification occurs naturally due to the diffusion of organic solvent. to make fine-sized droplets, a high solvent-to-oil ratio is necessary [47]. the volume viscosities and phases of the emulsion, the types and concentrations of surfactants, the temperature, size, and size distribution of the droplets in the disperse phase, and the temperature, size, and size distribution of the droplets in the disperse phase are all factors that influence the selection of emulsifying tool pak. j. anal. environ. che m. vol. 23, no. 2 (2022)184 [30, 48]. some parameters, such as interface density, temperature, flow rate, pressure, time of emulsification, and rotation speed, also influence the emulsion’s preparatory process. these all parameters should be under consideration while trying to obtain fine nanoemulsions [14, 23, 49]. advantages of solvent displacement method the advantages of this technology are freedom in the choice of the internal structure and surfactant, as well as the potential to form nanoemulsions in a short time [12]. disadvantages of solvent displacement method sometimes, it’s very difficult to manage all the required parameters for the production of nanoemulsion by adopting the solvent displacement method [14, 23, 49]. applications of nanoemulsions nanoemulsions are getting higher importance throughout the world because of their bountiful applications [19]. based on the size, specificity and precision of active medicinal components and other products, nanoemulsions could be used in composite and crystal formulations using low-energy techniques. nanoemulsions are viewed as a proficient device to treat tumors. such nanocarriers settle the water-based solvency issues as well as help to defeat multi-drug resistant micro-organisms because of their targeting nature. ligands of various qualities can be utilized to adjust the focusing nature of nanoemulsions [50]. there are different sorts of diseases, so multifunctional nano-emulsions are getting attention from analysts to treat various types of malignancy. the past examinations additionally have uncovered that nano-emulsions are viably benefited by the cells of cancer, which prompts diminishing the development of growth, eliminating venomousness to sound cells, and dropping off the departure of carcinogenic cells to different organs [51, 52]. in the same way, nano-formulations are also being used for vaccines and syrup. antigens are loaded into nanocarriers and injected into the human body as a vaccine [53]. nanoemulsions are likewise a significant tool of the food industries to create smart food having ingredients that exhibit low direct solubility to water [53, 54]. nanoemulsions are significant builders of different complex materials because of their small size and accessibility to enormous surface regions [54]. because of their large surface area and small droplets, nanoemulsions have proven to be very useful in improving bioavailability, bioactivity, digestibility, stability, safety, quality, and sensory enhancements of food components and natural extracts, such as lycopene-solubilized and β-carotene-based nanoemulsions [53]. beverage emulsions are the simplest form of o/w type of emulsions used in food industries. myonise, drinks, margarine, fatty spreads, and homogenized milk are examples of emulsions used in food industries [55]. nano-emulsions are also being used in cosmetic industries to make various cosmetic products (such as primers, liquid lipsticks, liquid foundations, etc.) [53]. ultraviolet radiation coming from the sun can damage skin and produce skin cancer. nanoformulations having tio2 and zno are used to protect skin from the effects of ultraviolet radiation. with the passage of time, physical work, and exposure to sunlight, human skin becomes wrinkled, pigmented, and aged due to the degradation of skin collagen. to avoid the degradation of skin collagen, various antioxidants such as vitamins, polyphenols, pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 185 and flavonoids are added to cosmetic creams. vitamin a (retinol) is regarded as the best ingredient in creams that avoid the degradation of skin collagen. various nanotechnology-based anti-wrinkle creams enrich in vitamin a (retinol) are available on the market. one of the best products is revitalift from l’oreal paris. this product has nanosomes with vitamin a (retinol). nanosomes can easily and rapidly penetrate human skin via the dermal route and show anti-wrinkle effects [56]. figure 9. applications of nanoemulsions [54] table 1. importance of nano-emul sions. method protocol application ref. spontaneous method sundararajan et al. formulated nano-emulsion via ''low-energy technique'' by utilizing 90% (w/v) of water, 5% (w/v) of essential oil, and 5% (w/ v) of polysorbate-80 at an absolute mass of 50 g. for thirty minutes, the combination of essential oil (of ocimum basilicum l.) and polysorbate-80 were mixed via a magnetic stirrer at 800 rpm. then, water (at a 3.5 ml flow rate per minute) was added through drop-wise addition. after the addition of water, the entire blend drove towards mixing at 800 rpm for 60 minutes. the formulated nanoemulsion showed prominent antibacterial, anti-oxidant, and anti-larvicidal applications [4] high performance dispersion chenni et al. formulated emulsion by utilizing water (50% w/w), maltodextrins, and acacia gum (by proportion 1:1) as a carrier (45%) and aroma (5%). the emulsion was ready by dissolving maltodextrins and acacia gum (inside proportion 1:1) in refined water (by 50% w/w), leading to heating at 60oc for 45 min. the mixture was appropriately covered and permitted to stand for 24 hours. after that, the solutions were blended by utilizing ultra-turrax t-25 at the rate of 13,500 rpm for five minutes. drug delivery system [17] high-pressure homogenization method muhammadi et al. formulated nano-emulsion of essential oil of peppermint/eucalyptus by utilizing a high pressing factor homogenization technique. in a measuring glass, 13 ml (21%) polyethylene glycol was taken and homogenized by utilizing a homogenizer at 11,000 rpm. then, at that point, 10 ml (16%) polysorbate-80 was included in the measuring glass having polyethylene glycol that drove towards homogenization at 11,000 rpm for five minutes. then, at that point, 5 ml (8%) oil of sesamum indicuml l. (as a transporter and synergist oil) was poured into the above blend. then, at that point, 30ml (half) unadulterated fundamental oil of peppermint or eucalyptus was poured into the above blend. the entire blend was left under the homogenizer till all of the constituents were blended appropriately. following 10 minutes; for appropriate disintegration of constituents; 2ml (5%) butanol was added to the combination and again homogenized at 11,000 rpm at room temperature for five minutes. biological applications (mosquitoes repellency) [18] ultrasonication ghosh et al. prepared nanoemulsion of basil essential oil, tween 20 (hydrophile lipophile balance (hlb) esteem -16.7), and h2o. course emulsion was ready by blending oil, surfactant, and water. then, at that point, course emulsion was changed over into nanoemulsion by sonication strategy having high recurrence (20khz) and 750w yield. larvicidal activity [57] ultrasonication roy et al. formulated o/w based course emulsion of betel leaf essential oil by using tween 20, essential oil, and refined water that was changed into nanoemulsion by sonication strategy by utilizing sonication test (13 mm in width), 20 khz recurrence, and 750w force yield. given energy created problematic powers and henceforth, nanoemulsion was acquired. this formulated emulsion was used against food pathogens [58] ultrasonication nirmala et al. prepared an emulsion of the drug by dissolving the wanted drug in cinnamon oil. after dissolving the desired drug in cinnamon oil, the combination was permitted to stand for the time being and then, at that point, centrifuged to affirm the solvency. then, at that point, surfactant (tween 80) and water were included in the above arrangement, thus course emulsion was formulated. then, at that point, the course emulsion was sonicated, and the coarse emulsion was changed into nanoemulsion. drug delivery system [59] pak. j. anal. environ. che m. vol. 23, no. 2 (2022)186 current trends in nanoemulsions applications and formulation several non-polar active compounds can easily be dissolved in nanoemulsions; hence nanoemulsions are used to supply required nutrients. nanoemulsion-based drugs are coming into the markets, and they possess very small droplet sizes that reduce the toxic effects of drugs [60]. nanoemulsion as lipophilic nanocarriers is used to deliver lipophilic and amphiphilic medications to the blood-brain barrier with remarkable penetration, and their delivery to the brain is a viable approach. the nanoemulsions are getting trendy as antifungal, antibacterial, antiparasitic, and mosquitorepelling agents. conjugation and physical adsorption methods are being used to load antigens to nanoemulsions. nanoemulsions are being used against cancerous cells. sunblock nanoemulsions also protect skin from skin cancer [8, 61, 62]. in preclinical research, many preclinical nanoemulsions containing encapsulated contrast and chemotherapeutic medicines are demonstrated to function specifically on the tumor microenvironment for diagnostic and therapeutic purposes. gel forms of nanoemulsion are also used before conducting an ultrasound image of the patient. iron-oxide nano-crystals and cy7 fluorescent dye are placed in the oil core with hydrophobic glucocorticoid for medicinal applications in a distinct strategy, including mri and nirf imaging [51, 63]. nutraceuticals (nutrients), color, and flavoring are all being delivered via this method in the food sector. food quality, functional characteristics, nutritional value, and shelf life are all important considerations, and nanoemulsion plays a beneficial role in this regard [47, 55]. essential oil-based nanoemulsions are regarded as the best food preservers. a nanoemulsion covering with small droplets of lemongrass essential oil shows more success in preserving grapefruit and enhancing microbial safety against salmonella than a coating with large droplets. in the same way, varying the species of essential oils, different fruits can be preserved from microbial attack [64]. to improve the environment, biodegradable coatings and packaging films are being developed [14]. as collectively there are various methods to formulate nanoemulsions, but the spontaneous method is the most trendy nowadays. the spontaneous method is also regarded as the titration method. this method is easy to follow as nanoemulsion is formed by mixing all ingredients in the right proportion with continuous agitation [65]. the spontaneous method currently prepares all forms of nanoemulsion, such as gel and liquid having high flow rates, etc. nanoemulsion has applications in different industries or fields such as drug delivery systems, food industry, oral products industry, cosmetics, perfumery industry, and anti-microbial agents, etc., formulated by spontaneous method [66-70]. patents of nanoemulsion numerous distinctive nanoemulsion formulation based patents have been issued in recent years in the fields of cosmetics and drug delivery. table 2 lists a few of these patents now held by different companies (from the year 2012 to 2022). it is evident from the growing number of patents that nanoemulsions, in various fields especially in cosmetics and drug delivery systems, has become more and more well-liked. customers favour nano-based cosmetics and pharmaceutical products over conventional ones as they become more aware of their advantages. pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 187 table 2. patents on nanoemulsion [71-77]. nanoemul sions used i n cosmetics s. no. formulation company patent number 1 sugar fatty ethers and their uses in the cosmetics, dermatological, and/or ophthalmological fields l’oreal (paris, fr) us 6,689,371 2 fluid non-ionic amphiphilic lipids and use in cosmetics or dermopharmaceuticals l’oreal (paris, fr) us 5,753,241 3 nanoemulsion is based on glycerol fatty esters, and its uses in the cosmetics, dermatological, and/or ophthalmological fields l’oreal (paris, fr) 6,541,018 4 nanoemulsion is based on oxyethylenated or non oxyethylenated sorbitan fatty esters, and its uses in the cosmetics, dermatological, and/or ophthalmological fields l’oreal (paris, fr) 6,335,022 5 nanoemulsion is based on ethylene oxide and propylene oxide block copolymers and its uses in the cosmetics, dermatological, and/or ophthalmological fields. l’oreal (paris, fr) 6,464,990 6 nanoemulsion is based on phosphoric acid fatty acid esters and its uses in cosmetics, dermatological, pharmaceutical, and/or ophthalmological fields l’oreal (paris, fr) 6,274,150 7 oil-in-water type emulsion sunscreen cosmetic composition shiseido co ltd. 20130011348a1 8 nanodiamond uv protectant formulations international technology center us 20090220556a1 9 cosmetic composition containing retinol stabilized by porous polymer beads and nanoemulsion act co ltd. us 20130095157a1 10 cosmetic composition containing retinol stabilized by porous polymer beads and nanoemulsion act co ltd. ep 2583665a2 11 cosmetic pigment composition containing gold or silver nanoparticles korea research institute of bioscience and biotechnology us 20090022765a1 12 skin whitening methods and compositions based on zeolite– active oxygen donor complexes bioderm research us20070166339a1 13 nanoemulsion comprising metabolites of ginseng saponin and a skin-care composition for anti-aging containing the same pacific corp ep 1327434a1 nanoemul sion used i n drug delivery s. no. formulation company patent number 1 oil-in-water type terazosin nanoemulsion antihypertensive drug zhang hongli cn105997873a 2 a kind of compound apigenin nanoemulsion antihypertensive drug zhang hongli cn106137958a 3 a kind of compound atenolol nanoemulsion antihypertensive drug zhang hongli cn106176997a 4 antihypertensive drug of quinapril hydrochloride and rose oil nanoemulsion ouyang wuqing, sun jianhong, zhang xiaohua cn102698245a pak. j. anal. environ. che m. vol. 23, no. 2 (2022)188 5 compound spirolactone nanoemulsion drug ouyang wuqing, sun jianhong, cao tong cn102697900a a kind of oil-in-water type celiprolol nanoemulsion antihypertensive drug zhang hongli cn106137961a 6 nanoemulsion biofrontera bioscience gmbh us-20090324727-a1 7 dha ester emulsions martek biosciences corporation us20110200644a1 8 dha free fatty acid emulsions martek biosciences corporation us20110200645a1 9 dha triglyceride emulsions martek biosciences corporation us20110206741a1 10 colloidal carrier system with penetrating properties for inclusion of lipophilic drugs and oils for topical application gabriele blume de102010056192a1 11 vesicular formulations henk-andre kroon us20120232034a1 12 cancer heat therapy-enhancing agent sbi pharmaceuticals co., ltd us20130158293a1 13 topical pharmaceutical compositions containing nanodroplets for the treatment of psoriasis cadila healthcare limited us20130273172a1 14 methods for forming mini emulsions and use thereof for delivering bioactive agents ns technologies pty ltd. us20140322330a1 15 vesicular formulations, kits, and uses sequessome technology holdings ltd. us20150132349a1 16 preparation of nanoemulsions affinsci inc. wo2016182926a1 17 topical pharmaceutical compositions glaxosmithkline intellectual property development limited us20160338973a1 18 sequessome technology holdings limited sequessome technology holdings limited us9555051b2 19 methods for photodynamic therapy dusa pharmaceuticals, inc. us20190216927a1 20 adjustable illuminators and methods for photodynamic therapy and diagnosis dusa pharmaceuticals, inc. us10603508b2 21 methods for photodynamic therapy dusa pharmaceuticals, inc. us20190216927a1 22 calcium phosphate core particles / intraocular delivery compositions and methods biosante pharmaceuticals inc us 6,355,271 b 23 oil-in-water type emulsion (cetalkonium chloride, tyloxapol, and poloxamer) with an average particle size of about 300 nm and positive zeta potential santen sas us 8,298,568 b2 24 emulsion eye drop for alleviation of dry eye-related symptoms in dry eye patients and/or contact lens wearers saint regis mohawk tribe us 5,981,607 future trends of nanoemulsions due to their potential benefits over conventional emulsions in food, cosmetics and drug delivery applications, such as clear formulation, improved bioavailability, longer shelf lives, and superior physical stability, nanoemulsions will be the subject of in-depth study and development. numerous pieces of research have been conducted in recent years to determine the benefits of encapsulating lipophilic and functional chemicals in nanoemulsions. however, there are still a lot of obstacles to be cleared before nanoemulsions may be applied more widely. the first requirement is that the right components be used for creating the right nanoemulsions. second, there is still much work to be done before large-scale commercial applications can be made. as a result, choosing the right pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 189 processing techniques is necessary to generate nanoemulsions on an industrial scale and at a reasonable cost [78]. the growing number of patents in this field reflects that these nano-products are gaining the attention of renowned industries worldwide in different fields of applications. hence it can be predicted that the future is of nanoemulsion-based industries and products [79]. conclusion nanoemulsions offer improved functional qualities in contrast to traditional emulsions. for the encapsulation of different bioactive components, the composition and structure of the nanoemulsions can be adjusted. among methods of nanoemulsion preparation, high and low-energy methods are subdivided into various types to formulate nanoemulsion. although each method has its advantages and disadvantages, it can be concluded from the current review that among different methods to formulate nanoemulsions, low-energy methods are best as compared to high-energy methods. this is because they don’t require high input devices, higher pressure, and temperature. the importance of these methods can be appraised from the fact that nanoemulsions have applications in numerous fields like the food industry, cosmetics, medicine, pharmaceutical industry, etc. nanoemulsions are also regarded as nanocarriers. so, according to the demand of the hour, such nanocarriers (that should be cost-effective and task efficient) are needed in the future. if future research fulfills the current gaps, the nanoemulsion system can also be upgraded for various new industries. conflict of interest the authors declare no conflict of interest. references 1. s. m. m. modarres-gheisari, r. gavagsaz-ghoachania, m. malakib, p. safarpoura and m. zandi, ultrason. sonochem., 52 (2019) 88. https://doi.org/10.1016/j.ultsonch.2018.1 1.005 2. k. cinar, trakya univ. j. eng. sci., 18 (2017) 73. https://dspace.trakya.edu.tr/xmlui/handle /trakya/7618 3. d. j. mcclements and j. rao, crit. rev. food sci. nutr., 51 (2011) 285. https://doi.org/10.1080/10408398.2011.5 59558 4. sundararajan, balasubramani, a. k. moola, k.vivek and b. d. r. kumari, microb. pathog., 125 (2018) 475. https://doi.org/10.1016/j.micpath.2018.1 0.017 5. j. echeverria and r.d.d.g.d. albuquerque, medicines, 6 (2019) 42. https://doi.org/10.3390/medicines602004 2 6. s. sharma, n. loach, s. gupta and l. mohan, environ. nanotechnol. monit. manag., 1 (2020) 100331. https://doi.org/10.1016/j.enmm.2020.100 331 7. f. esmaili, al. sanei-dehkordi, f. amoozegar, m. osanloo, biointerface res. applied chem. 11 (2021) 12516. https://doi.org/10.33263/briac115.125 1612529 8. a. naseema, l. kovooruab, a. k. beheraac, k.p. pramodh and k. p. srivastava, adv. colloid interface sci., 287 (2021) 102318. https://doi.org/10.1016/j.cis.2020.10231 8 9. m. y. koroleva and e. v. yurtov, russ. chem. rev., 81 (2012) 21. https://doi.org/10.1070/rc2012v081n01 abeh004219 pak. j. anal. environ. che m. vol. 23, no. 2 (2022)190 10. s. graves, k. meleson and j. wilking, the j. chem. phy., 122 (2005) 134703. https://doi.org/10.1063/1.1874952 11. s. m. jafari, y. he and b. bhandari, eur. food res. technol., 225 (2007) 733. https://doi.org/10.1007/s00217-0060476-9 12. c. qian and d. j. mcclements, food hydrocoll., 25 (2011) 1000. https://doi.org/10.1016/j.foodhyd.2010.0 9.017 13. s.a. chime, f.c. kenechukwu and a.a. attama, nanoemulsions — advances in formulation, characterization and applications in drug delivery, in: application of nanotechnology in drug delivery (a. d. sezer, eds) intechopen limited, london, uk (2014) 77-126. https://doi.org/10.5772/15371 14. j. b. aswathanarayan and r. r. vittal, front. sustain. food syst., (2019) 95. https://doi.org/10.3389/fsufs.2019.00095 15. m. jaiswal, r. dudhe and p. sharma, 3 biotech,. 5 (2015) 123. https://doi.org/10.1007/s13205-0140214-0 16. o. tarhan, m. j. spotti, colloids surf. b, 200 (2021) 111526. https://doi.org/10.1016/j.colsurfb.2020.1 11526 17. m. chenni, d. el abed, s. neggaz, n. rakotomanomana, x. fernandez, f. chemat, j. stored prod. res., 86 (2020) 101575. https://doi.org/10.1016/j.jspr.2020.10157 5 18. r. mohammadi, m. khoobdel, m. negahban and s. khani, asian pac. j. trop. med., 12 (2019) 520. https://doi.org/10.4103/19957645.271292 19. x. liu, l. chen, y. kang, d. he, b. yang and k. wu, lwt, 147 (2021) 111660. https://doi.org/10.1016/j.lwt.2021.11166 0 20. li. meiting, d. bia, l. y. jiang, y. weishan, f. yan, w. hong, x. zhangli, hua and x. xua, carbohydr. polym., 264 (2021) 118047. https://doi.org/10.1016/j.carbpol.2021.11 8047 21. j. zhang, retrieved from the university of minnesota digital conservancy, (2011) 248. https://hdl.handle.net/11299/117545 22. y. singh, j. g. mehera, k. ravala, f. a. khan, m. chaurasiab, n. k. jainc and m. k. chourasiaa, jcr, 252 (2017) 28. https://doi.org/10.1016/j.jconrel.2017.03. 008 23. t. jiang, w. liao and c. charcosset, int. food res. j., 132 (2020) 109035. https://doi.org/10.1016/j.foodres.2020.10 9035 24. l. wang, s. zhang, w. jiang, h. zhao and j. fu, food hydrocoll., 101 (2020) 105452. https://doi.org/10.1016/j.foodhyd.2019.1 05452 25. r. kaur, d. k. kocher, n. vashishat and a. sidhu, indian j. entomol., 18 (2019) 753. https://doi.org/10.5958/09748172.2019.00176.7 26. p. k. gupta, j. k. pandit, a. kumar, p. swaroop and s. gupta, pharm. res., 3 (2010) 117. https://www.scribd.com/document/8900 7434/pharmaceutical-nanotechnologynovel-nanoemulsion-high-energy 27. r. p. patel and j. r. joshi, int. j. pharm. sci. res., 3 (2012) 4640. https://doi.org/10.1.1.278.6903 28. s. kumar, asian j. res. pharm. sci. biotech., 2 (2014) 1. https://doi.org/10.1.1.1078.2789 29. t. s. h. leong, t. j. wooster, s. e. kentish and m. ashokkumar, ultrason sonochem., 16 (2009) 721. https://doi.org/10.1016/j.ultsonch.2009.0 2.008 pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 191 30. e. elaine and k. l. nyam, igi global, (2022) 24. https://doi.org/10.4018/978-1-79988378-4.ch002 31. t. jiang and c. charcosset, j. food eng., 36 (2022) 110836. https://doi.org/10.1016/j.jfoodeng.2021. 110836 32. n. anton, j. -p. benoit and p. saulnier, jcr, 128 (2008) 185. https://doi.org/10.1016/j.jconrel.2008.02.007 33. i. sole, c. solans, a. maestro, c. gonzalez and j. m. gutierrez, j. colloid interface sci., 376 (2012) 133. https://doi.org/10.1016/j.jcis.2012.02.063 34. c. solans, p. izquierdo, j. nolla, n. azemar and m. j. garcia-celma, curr. opin. colloid interface sci., 10 (2005) 102. https://doi.org/10.1016/j.cocis.2005.06.004 35. g. caldero, m. j. garcia-celma and c. solans, j. colloid interface sci., 353 (2011) 406. https://doi.org/10.1016/j.jcis.2010.09.07 3 36. c. solans and i. sole, curr. opin. colloid interface sci., 17 (2012) 246. https://doi.org/10.1016/j.cocis.2012.07.0 03 37. s. setya, s. talegaonkar and b. razdan, world j. pharm. pharm. sci., 3 (2014) 2214. https://www.wjpps.com/wjpps_controlle r/abstract_id/898 38. z. a. a. aziz, h. m. nasir, a. ahmad, s. h. m. setapar, h. ahmad, m. h. m. noor, m. rafatullah, a. khatoon, m. a. kausar, i. ahmad s. khan, m. al-shaeri and g. m. ashraf, sci. rep., 9 (2019) 1. https://doi.org/10.1038/s41598-01950134-y 39. c. anandharamakrishnan, techniques for nanoencapsulation of food ingredients, richard w. hartel, springer india, (2014) 1. https://doi.org/10.1007/978-1-46149387-7-1 40. a. thakur, m. k. walia and s. kumar, pharmacophore, 4 (2013) 15. https://doi.org/10.1.1.735.8386 41. c. lovelyn and a. a. attama, j. biomater. nanobiotechnol., 2 (2011) 626. https://doi.org/10.4236/jbnb.2011.225075 42. m. safaya and y. rotliwala, mater. today, 57(4) (2022) 1793. https://doi.org/10.1016/j.matpr.2021.12. 478 43. s. l. ee, x. duan, j. liew, q. d. nguyen, j. chem. eng., 140 (2008) 626. https://doi.org/10.1016/j.cej.2007.12.016 44. t. tadros, pizquierdo, j. esquena and c. solans, adv. colloid interface sci., 108 (2004) 303. https://doi.org/10.1016/j.cis.2003.10.023 45. d. akiladevi, h. prakash, g. biju and n. madumitha, rjpt, 13 (2020) 983. https://doi.org/10.5958/0974360x.2020. 00183.3 46. t. sheth, s. seshadri, t. prileszky and m. e. helgeson, nat. rev. mater., 5 (2020) 214. https://doi.org/10.1038/s41578-0190161-9 47. s. saffarionpour, food eng. rev., 11 (2019) 259. https://doi.org/10.1007/s12393-01909201-3 48. s. natesan, v. hmingthansanga, n. singh, p. datta, s. manickam and v. ravichandiran, igi global, 1 (2022) 93. https://doi.org/10.4018/978-1-79988378-4.ch005 49. h. jasmina, o. dzana, e. alisa, v. edina and r. ognjenka, cmbebih 2017 springer nature., singapore, 1 (2017) 317. https://doi.org/10.1007/978-981-104166-2_48 50. j. m. gutierrez, c. gonzalez, a. maestro, i. sole, c. m. pey and j. nolla, curr. opin. colloid interface sci.,13 (2008) 245. https://doi.org/10.1016/j.cocis.2008.01.005 pak. j. anal. environ. che m. vol. 23, no. 2 (2022)192 51. e. sanchez-lopez, m. guerra, j. diasferreira, a. lopez-machado, m. ettcheto, a. canno, m. espina, a. camins, m. l. garcia and e. b. souto, nanomaterials, 9 (2019) 821. doi; 10.3390/nano9060821 52. s. wang, x. liang, w. zhao, x. mi, c. zhang, w. zhang, y. cheng, l. wang and y. jiang, j. food process. preserv., 46 (2022) e16197. doi; 10.1111/jfpp.16197 53. t. j. ashaolu, environ. chem. lett., 19 (2021) 3381. https://doi.org/10.1007/s10311-02101216-9 54. a. gupta, h. b. eral, t. a. hatton and p. s. doyle, soft matter, 12 (2016) 2826. https://doi.org/10.1039/c5sm02958a 55. n. dasgupta, s. ranjan and m. gandhi, environ. chem. lett., 17 (2019) 1003. https://doi.org/10.1007/s10311-01900856-2 56. n. h. c. marzuki, r.a. wahab and m. a. hamid, biotechnol. biotechnol. equip., 33 (2019) 779. https://doi.org/10.1080/13102818.2019.1 620124 57. v. ghosh, a. mukherjee and n. chandrasekaran, asian j. chem., 25 (2013) s321. https://www.researchgate.net/publication /236853976 58. a. roy and p. guha, j. food process. preserv., 42 (2018) e13617. https://doi.org/10.1111/jfpp.13617 59. m. j. nirmala, s. allanki, a. mukherjee and n. chandrasekaran, int. j. pharm. pharm. sci., 5 (2013) 273. https://www.researchgate.net/publication /258262154 60. a. h. saleh, j. m. khalaf and k. a. ameen, eurasian med. res. periodical, 5 (2022) 17. https://geniusjournals.org/index.php/emr p/article/view/567 61. t. j. ashaolu, environ. chem. lett., 19 (2021) 3381. https://doi.org/10.1007/s10311-02101216-9 62. z. karami, m. r. s. zanjani and m. hamidi, drug discov. today, 24 (2019) 1104. https://doi.org/10.1016/j.drudis.2019.03. 021 63. b. gorain, h. choudhury, a. b. nair, s. k. dubey and p. kesharwani, drug discov. today, 25 (2020) 1174. https://doi.org/10.1016/j.drudis.2020.04. 013 64. n. a. al-tayyar, a. m. youssef and r. r. al-hindi, sustain. mater. technol., 26 (2020) e00215. https://doi.org/10.1016/j.susmat.2020.e0 0215 65. d. cholakova, z. vinarov, s. tcholakova and n. denkov, curr. opin. colloid interface sci., 1 (2022) 101576. https://doi.org/10.1016/j.cocis.2022.101 576 66. n. sharma, s. mishra, s. sharma, r. d. deshpande and r. k. sharma, int. j. drug dev. res., 5 (2013) 37. https://www.researchgate.net/publication /286305524 67. e. b. seo, l. h. du plessis and j. m. viljoen, pharmaceuticals, 15 (2022) 120. https://doi.org/10.3390/ph15020120 68. r. d. singh, s. kapila, n. g. ganesan and v. rangarajan, j. surfactants deterg., 1 (2022) 1. https://doi.org/10.1002/jsde.12571 69. a. dehghanghadikolaei, m. shahbaznezhad, b. a. halim and h. sojoudi, acs omega, 7 (2022) 7045. https://doi.org/10.1021/acsomega.1c067 65 70. h. gaoe, z. pang, s. pan, s. cao, z. yang, c. chen and x. jiang, arch. pharma. res., 35 (2012) 333. pak. j. anal. environ. che m. vol. 23, no. 2 (2022) 193 https://doi.org/10.1007/s12272-0120214-8 71. p. kumar, gannu. med. chem., 5 (2015) 272. https://doi.org/10.4172/21610444.10002 75 72. r. p. patel and j. r joshi, int. j. pharm. sci. res., 3 (2012) 4640. https://dx.doi.org/10.13040/ijpsr.09758232.3(12).4640-50 73. n. b. romes, r. ab. wahab and m. a. hamid, biotech. biotechnol. equip., (2021). https://doi.org/10.1080/13102818.2021.1 91589 74. c. marzuki, n. haziqah, wahab, r. abdul and m. a. hamid, biotechnol. biotechnol. equip., 33 (2019) 779. https://doi.org/10.1080/13102818.2019.1 620124 75. g. kumar, t. virmani, k. pathak and a. alhalmi, biomed res. int., 1 (2022) 1. https://doi.org/10.1155/2022/4109874 76. basha, s. khaleel, muzammil, m. syed, r. dhandayuthabani, v. s. kumari and k. kaviyarasu, curr. drug res. rev., 12 (2019). https://doi.org/10.2174/25899775116661 91024173508 77. https://patents.google.com/patent/us829 8568/en 78. g. li, z. zhang, h. liu and l. hu, food funct., 12 (2021) 1933. https://doi.org/10.1039/d0fo02686g 79. aswathanarayan, j. bai, vittal and r. rai. front. sustain. food syst., 3 (2019) 95. https://doi.org/10.3389/fsufs.2019.00095