Development of a new highly sensitive and selective spectrophotometric method for the determination of selenium at nano-trace levels in various complex matrices using salicylaldehyde-orthoaminophenol European Journal of Chemistry 12 (4) (2021) 469-481 European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2021 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. https://dx.doi.org/10.5155/eurjchem.12.4.469-481.2137 European Journal of Chemistry View Journal Online View Article Online Development of a new highly sensitive and selective spectrophotometric method for the determination of selenium at nano-trace levels in various complex matrices using salicylaldehyde-orthoaminophenol Muhammad Jamaluddin Ahmed *, Muhammad Jihan Uddin and Muhammad Emdadul Hoque Laboratory of Analytical Chemistry, Department of Chemistry, University of Chittagong, Chittagong-4331, Bangladesh pmjahmed55@gmail.com (M.J.A.), jihan2141@gmail.com (M.J.U.), emdadulh994@gmail.com (M.E.H.) * Corresponding author at: Laboratory of Analytical Chemistry, Department of Chemistry, University of Chittagong, Chittagong-4331, Bangladesh. e-mail: mjahmed83@cu.ac.bd (M.J. Ahmed). 10.5155/eurjchem.12.4.469-481.2137 Received: 06 July 2021 Received in revised form: 14 September 2021 Accepted: 17 October 2021 Published online: 31 December 2021 Printed: 31 December 2021 A new spectrophotometric reagent, salicylaldehyde-orthoaminophenol (Sal-OAP) has been synthesized and characterized for the determination of selenium through novel reaction techniques. Also, a new highly selective, and sensitive spectrophotometric method for the nano-trace determination of selenium using salicylaldehyde-orthoaminophenol (Sal-OAP) has been developed. Sal-OAP undergoes reaction in a slightly acidic solution (0.0001-0.0002 M H2S04) with selenium (IV) to give an orange-red chelate, which has an absorption maximum at 379 nm. The reaction is instantaneous and absorbance remains stable for over 24 h. The average molar absorption co-efficient and Sandell’s sensitivity were found to be 6.4×105 L/mol.cm and 1.0 ng/cm2 of, respectively. Linear calibration graphs were obtained for 0.001-40.000 mg/L of Se having detection limit of 0.1 µg/L and RSD 0-2 %. The stoichiometric composition of the chelate is 1:2 (Se:Sal-OAP). A large excess of over 60 cations, anions and some common complexing agents, such as chloride, azide, tartrate, EDTA, SCN¯ etc., do not interfere in the determination. The developed method was successfully used in the determination of selenium in several Certified Reference Materials (Alloys, steels, human urine, bovine liver, drinking water, tea, milk, soil, and sediments) as well as in some environmental waters (Potable and polluted), biological fluids (Human blood, urine, hair, and milk), soil samples, food samples (Vegetables, rice, and wheat) and pharmaceutical samples (Tablet and syrup) and solutions containing both selenium (IV) and selenium (VI) as well as complex synthetic mixtures. The results of the proposed method for assessing biological, food and vegetables and soil samples were comparable with ICP-OES and AAS were found to be in excellent agreement. The method has high precision and accuracy (s = ±0.01 for 0.5 mg/L). Soil samples Biological samples Environmental samples Pharmaceutical samples Selenium determination Salicylaldehyde-orthoaminophenol Cite this: Eur. J. Chem. 2021, 12(4), 469-481 Journal website: www.eurjchem.com 1. Introduction Selenium is an essential element for humans at trace level, but it is toxic at higher concentration. A maximum acceptable concentration of selenium in drinking water is 10 ng/mL [1]. Food is the main source for intake of selenium for individuals who are not occupationally exposed; thus, toxic effects have most often been associated with food intake. A safe and adequate range of selenium intake of 50-200 g per person per day has been recommended for adults, with correspondingly lower ranges for infants and children. Selenium occurs in natural waters in trace amounts as a result of geochemical processes, such as weathering of rocks and erosion of soils, and is usually present in water as selenate or selenite. Excess of selenium causes toxic effects in living organism, and toxicity depends on many factors such as chemical form, pH, presence of other ions, etc. [2]. It rarely occurs in its elemental state or as pure ore compounds in the Earth’s crust. selenium is found in metal sulfide ores, where it partially replaces the sulfur. Commer- cially, selenium is produced as a byproduct in the refining of sulfide ores, most often during production. Minerals that are pure selenide or selenate compounds are known but rare. The chief commercial uses for selenium today are glass making and pigments. Selenium is a semiconductor and is used in photocells. Applications in electronics, once important, have been mostly replaced with silicon semiconductor devices. Selenium is still used in a few types of DC power surge protectors and one type of fluorescent quantum dot. Although trace amounts of selenium are necessary for cellular function in many animals, including humans, both elemental selenium and especially selenium salts are toxic in even small doses, causing selenosis. Selenium is listed as an ingredient in many multivitamins and other dietary supplements, as well as in infant formula, and is a component of the antioxidant enzymes glutathione peroxidase and thioredoxin reeducates (which indirectly reduce certain oxidized molecules in animals and some plants) as well as in three deiodinase enzymes. ABSTRACT RESEARCH ARTICLE KEYWORDS https://dx.doi.org/10.5155/eurjchem.12.4.469-481.2137 https://www.eurjchem.com/ https://dx.doi.org/10.5155/eurjchem.12.4.469-481.2137 mailto:pmjahmed55@gmail.com mailto:jihan2141@gmail.com mailto:emdadulh994@gmail.com mailto:mjahmed83@cu.ac.bd http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.12.4.469-481.2137&domain=pdf&date_stamp=2021-12-31 470 Ahmed et al. / European Journal of Chemistry 12 (4) (2021) 469-481 2021 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.12.4.469-481.2137 C O H OH + OH NH2 OH C N H OH Scheme 1. Synthesis of salicylaldehyde-ortho-aminophenol (Sal-OAP). Selenium requirements in plants differ by species, with some plants requiring relatively large amounts and others apparently requiring none [3]. Spectrophotometry is essentially a trace analysis technique and is one of the most powerful tools in chemical analysis. The aim of this study was to develop a simpler direct spectro- photometric method for nano-trace determination of selenium. In the search for a more sensitive reagent, in this work a new Schiff’s base reagent salicylaldehyde-ortho-aminophenol (Sal- OAP) was synthesized according to the method of Sacconi [4] and Uddin et al. [5] and a color reaction of Sal-OAP with Se(IV). Sal-OAP has not previously been used for the spectro- photometric determination of any metal. This paper reports its use in a very sensitive, highly specific spectrophotometric method of ultra-trace determination of selenium. The method possesses distinct advantages over existing methods [6-53] with respect to sensitivity, selectivity, range of determination, simplicity, speed, pH/acidity range, thermal stability, accuracy, precision and ease of operation. From above mentioned literature survey as shown in Table 1, it reveals that methods [16-52] are lengthy, time-consuming, pH dependent and in most of above-mentioned methods, inter- ference was high. 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 selenium in very selective and sensitive ways. The method is based on the reaction of non-absorbent Sal-OAP in a slightly acidic (0.0001-0.0002 M H2SO4) solution with selenium to produce a highly absorbent bright orange-red chelate 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 solutions do not show any absorbance. 2. Experimental 2.1. Apparatus A Shimadzu (Kyoto, Japan) (Model-1800) double beam UV/VIS spectrophotometer and Jenway (England, U.K) (Model- 3010) pH meter with a combination of electrodes were used for the measurements of absorbance and pH, respectively. A Shimadzu (Model: AA7000) atomic absorption spectrophoto- meter equipped with microcomputer-controlled air-acetylene flame and A Shimadzu (Japan) (Model: 9800) Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES), (λ = 418 nm, plasma gas flow rate (L/min) = 15, LOD: below 1 μg/L of Se, RF Power (W) = 1400, Nebulizer gas flow rate (L/min) = 1-10) were used for comparison of the results. The elemental Analyzer (Exeter Analytical Inc., Model CE 440) equipped with supersensitive thermal conductivity detector for simultaneous determination of CHN was used (The National Center of Excellence in Analytical Chemistry, University of Sindh, Pakistan). Infrared spectrum was recorded with FTIR Spectrophotometer, Shimadzu (Model-IR Prestige 21, Detector- DTGS KBr) in the range 7500-350 cm-1 (Department of Chemistry, University of Chittagong) and model: JEOL 500SS, magnetic field strength: 500 MHz solvent used: DMSO-d6, standard: TMS, four channel NMR spectrometer with signal-to- noise ratio of ~500:1 for proton were used for characterization of the ligand (Jahangirnagar University, Savar, Dhaka). 2.2. Live subject statement We were not aiming to carry out detailed human studies, but some samples from individuals were used in our study and as such we abided by all the necessary procedures and regulations and our University gave consent. University of Chittagong, Bangladesh, is committed to the protection and safety of human subjects involved in research. 2.3. Synthesis and characterization of the reagent The reagent was synthesized in the laboratory according to the method recommended by Sacconi [4] and Uddin et al. [5]. The reagent salicylaldehyde-ortho-aminophenol (Sal-OAP) was synthesized by following steps (Scheme 1). A mixture of salicylaldehyde and ortho-aminophenol (25 mmol each) in ethanol (70 mL) was stirred at 30 °C and refluxed for one hour when an orange red crystalline solid appeared. This was filtered off, washed with and recrystallized from ethanol. Salicylaldehyde-orthoaminophenol (2-((2-hydroxybenzyli dene)amino)phenol) (Sal-OAP): Color: Orange red. Yield: 84%. M.P.: 182-184 °C. FT-IR (KBr, ν, cm-1): 3600-3300 (OH), 1631 (C=N), 1535 (C=C), 1362 (C-N), 1313 (C-O). 1H NMR (500 MHz, DMSO-d6, δ, ppm): 8.89 (s, 1H, CH=N), 4.52-6.91 (m, 8H, Ar-H). Anal. calcd. for C13H11NO2: C, 73.23; H, 5.20; N, 6.57. Found: C, 73.15; H, 5.15; N, 6.57%. UV/Vis (C2H5OH, λmax, nm, (0.45)): 326. HPLC (Isocratic: CH3CH2OH: H2O = 80:20, 120 min; 0.1%): r.t. = 7.5 min., purity = 99.5%. Λm(S.m2.mol-1): 25. nD25 = 1.281. [α]D25: -68.5 (c 0.5, CH3CH2 OH). 2.4. Reagents and solutions All the chemicals used were of analytical reagent grade of the highest purity available. High-purity absolute ethanol and high-purity de-ionized water were used throughout. High- purity water was obtained by passing tap water through cellulose absorbent and to mixed-bed ion exchange columns, followed by distillation in a corning AG-11 unit. Glass vessel were cleaned by soaking in acidified solutions of KMnO4 or K2Cr2O7 followed by washing with concentrated HNO3 and rinsed several times with high purity de-ionized water. Stock solutions and environmental water sample (1000 mL each) were kept in polypropylene bottles containing 1 mL concent- rated HNO3. More rigorous contamination control was used when the selenium levels in the specimens were low. 2.4.1. Sal-OAP solution This solution (1.17×10-3 M) was prepared by dissolving the requisite amount of salicylaldehyde-orthoaminophenol in a known volume solution of distilled absolute ethanol. More dilute solution of the reagent was prepared as required. Ahmed et al. / European Journal of Chemistry 12 (4) (2021) 469-481 471 2021 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.12.4.469-481.2137 2.4.2. Selenium (IV) standard solution A 100 mL amount of stock solution (1.72×10-2 M) (1 mg/mL) of tetravalent selenium was prepared by dissolving 297.45 mg of sodium selenite (Na2SeO3), (Merck pro-analysis grade, purity 99.6%) in doubly distilled de-ionized water and it was subsequently standardized iodometrically with standard sodium thiosulphate [53]. More dilute standard solutions were prepared by appropriate dilution of aliquots from the stock solution with de-ionized water as and when required. A freshly standardized solution was always used. 2.4.3. Selenium (VI) standard solution A 100 mL amount of stock solution (1.72×10-2 M) (1 mg/mL) of hexavalent selenium was prepared by dissolving 325.97 mg of sodium selenate (Na2SeO4) (Aldrich A.C.S. grade) in doubly distilled de-ionized water. Aliquots of this solution were standardized with iodometrically [53]. More dilute standard solutions were prepared by appropriate dilution of aliquots from the stock solution with de-ionized water as and when required. A freshly standardized solution was always used. 2.4.4. Potassium dichromate solution A 100 mL amount of stock solution (0.1 N) was prepared by dissolving 490.3 mg of finely powdered K2Cr2O7 (E. Merck) in 100 mL deionized water. 2.4.5. Sodium azide solution Sodium azide solution (2.5%, w/v) (A.C.S grade 99.5% pure) was freshly prepared by dissolving 2.5 g in 100 mL of deionized water 2.4.6. Tartrate solution A 100 mL stock solution of tartrate (0.01%, w/v) was prepared by dissolving 10 mg of A.C.S. grade (99% pure) potassium sodium tartrate tetrahydrate in 100 mL de-ionized water. 2.4.7. Aqueous ammonia solution A 100 mL solution of an aqueous ammonia solution was prepared by diluting 10 mL concentrated NH4OH (28-30%, A.C.S. grade) to 100 mL with de-ionized water. The solution was stored in a polypropylene bottle. 2.4.8. EDTA solution A 100 mL stock solution of EDTA (0.01%, w/v) was prepared by dissolving 10 mg A.C.S.-grade (≥99% pure) ethylenediaminetetraacetic acid as disodium salt dehydrate in 100 mL deionized water. 2.4.9. Other solutions Solutions of a large number of inorganic ions and complexing agents were prepared from their AnalaR 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 (Specpure, Johnson Matthey) according to the recommended procedures of Mukherji [54]. In the case of insoluble substances, special dissolution methods were adopted [55]. 2.5. General procedure A volume of 0.1-1.0 mL of neutral aqueous solution containing 0.01-400 μg of selenium in a 10 mL volumetric flask was mixed with a 1:80 to 1:600-fold molar excess (preferably 2 mL of 1.17×10-3 M) of salicylaldehyde-ortho-aminophenol (Sal- OAP) reagent solution followed by the addition of 0.8-1.6 mL (preferably 1 mL) of 0.0001 M sulfuric acid. The solution was mixed well. After 1 minute 2.0 mL of ethanol was added. The mixture was diluted up to the mark with deionized water. The absorbance was measured at 379 nm against a corresponding reagent blank. The selenium content in an unknown sample was determined using a concurrently prepared calibration graph. 2.6. Sample collection and preservation Environmental samples: Water and soil samples were collected in polythene bottles from different places of Bangladesh. After collection, HNO3 (1 mL/L) was added as preservative. Blood, urine and milk: Blood and urine samples were collected in polypropylene bottles from effected persons of Chittagong Medical College Hospital, Bangladesh. Milk sample was collected from a Bangladeshi lactating mother. Imme- diately after collection they were stored in a salt-ice mixture and later, at the laboratory, were at 20 °C. Soil samples: Soil samples were collected from different locations of Bangladesh. Samples were dried in air and homogenized with a mortar. Food samples: Food samples (Rice, wheat, fruits, and vegetables) were collected from local market of Chittagong. After collection the samples (fruits and vegetables) were stored in refrigerator for preservation. Samples (Rice and wheat) were used as dry condition and homogenized with a mortar. Pharmaceutical samples: Pharmaceutical samples tablets of different companies were collected from local Pharmacy of Chittagong. Samples (Tablet) were homogenized with a mortar. 3. Results and discussion 3.1. Characterization of the reagent The reagent was characterized by taking melting point, elemental analysis, FTIR, 1H NMR spectrums and thermo- gravimetric analysis. The melting point of the reagent was 182- 184 °C (Lit. 182 °C) [4]. The results of elemental analysis of the reagent was in good coincidence with the calculated values [5]. The presence of FTIR peak at 1631 cm-1 was due to the characteristic C=N double bond (νC=N, 1590-1631 cm-1) of the Sal-OAP. The presence of 1H NMR peak at δ 8.89 ppm assigned for CH=N and aromatic protons of Sal-OAP are observed between δ 4.52 and 6.91 ppm. Both FTIR and 1H NMR spectrums and also elemental analysis data indicated the formation of the reagent. The steadiness of the thermo- gravimetric curve obtained for about 1 g of the reagent at 80-90 °C indicated that the reagent didn’t contain any moisture. 3.2. Factors affecting the absorbance 3.2.1. Absorption spectra The absorption spectra of a Se(IV)-Sal-OAP system in aqueous medium in presence of 1 mL 0.0001 M sulfuric acid solution, was recorded using the spectrophotometer. The absorption spectrum of Se(IV)-Sal-OAP is a symmetric curve with maximum absorbance at 379 nm and an average molar absorptivity of 6.4×105 L/mol.cm (Figure 1). The reagent blank exhibited negligible absorbance despite having wavelength at 379 nm. The reaction mechanism of the present method is as reported earlier [56]. 472 Ahmed et al. / European Journal of Chemistry 12 (4) (2021) 469-481 2021 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.12.4.469-481.2137 Figure 1. A and B absorbance spectra of Se(IV)-Sal-OAP (λmax = 379 nm) and the reagent blank in aqueous solutions, respectively. Figure 2. Calibration graph of 10-40 mg/L of selenium (IV). 3.2.2. Optimization of some parameters on the absorbance 3.2.2.1. Effect of solvent As Sal-OAP is partially soluble in water, an organic solvent was used for the system, consideration of cost, availability, toxicity and volatility of the solvent etc. Of the various solvents (Acetone, benzene, carbon tetrachloride, chloroform, ethanol, 1-butanol, isobutyl methyl ketone, N,N-dimethylformamide, methanol, and 1,4-dioxane) studied, ethanol was found to be the best solvent for the system. Different volumes (0-5 mL) of ethanol were added to fixed metal ion concentration and the absorbance were measured according to the general procedure. Maximum absorbance was observed in 20±2% (v:v) ethanol/ water medium, hence, a 20% ethanol solution was used in the determination procedure. It was observed that 10-70% (1-7 mL) ethanol produced a constant absorbance of the Se-chelate. For all subsequent measurements, 20.0 % (2 mL) of ethanol was added. 3.2.2.2. Effect of acidity Among the various acids (Nitric, sulfuric, hydrochloric, and phosphoric acids) studied, sulfuric acid was found to be the best acid for the system. The variation of the absorbance was noted after the addition of 0.1-2.5 mL of 0.0001 M sulfuric acid to every 10 mL of test solution. The maximum and constant absorbance was obtained in the presence of 0.8-1.6 mL of 0.0001 M sulfuric acid at room temperature 25±5 °C. Outside this range of acidity, the absorbance decreased. For all subsequent measurements 1.0 mL of 0.0001 M sulfuric acid was added. 3.2.2.3. 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. 3.2.2.4. Effect of temperature The Se-Sal-OAP system attained maximum and constant absorbance at room temperature 25±5 °C. Outside this range of temperature, the absorbance decreased. 3.2.2.5. Effect of reagent concentration Different molar excesses of Sal-OAP were added to a fixed metal ion concentration and the absorbance was measured according to the general procedure. It was observed that selenium metal, the reagent molar ratio of 1:80 to 1:600 produced a constant and maximum absorbance of Se-chelate. For different (0.5 and 1 mg/L) selenium concentrations an identical effect of varying the reagent concentration was noticed. Further concentration of the reagent was not studied. For all subsequent measurements, 2 mL of 1.17×10-3 M Sal-OAP reagent was added. 3.3. 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 0.001-100 mg/L distributed in five different sets (0.001-0.01, 0.01-0.1, 0.1-1.0, 1.0-10 and 10-100 mg/L) for convenience of the measurement. Of the five calibration graphs, one showing the limit of the linearity is given in Figure 2. The other four were straight-line graphs passing through the origin (R2 = 0.9996). The molar absorption co-efficient and the Sandell’s sensitivity [57] were found to be 6.4×105 L/mol.cm and 1.0 ng/cm2 of selenium (IV), respectively. The selected analytical parameters obtained with the optimization experiments are summarized in Table 1 and 2. y = 0.034x 0.0 0.5 1.0 1.5 2.0 2.5 0 10 20 30 40 50 60 70 80 90 Ab so rb an ce Concentration of Se(IV) (mg/L) Ahmed et al. / European Journal of Chemistry 12 (4) (2021) 469-481 473 2021 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.12.4.469-481.2137 Table 1. Summary of reviews on the existing spectrophotometric methods for the determination of selenium. Reagent Solvent Medium aqueous/ surfactant/ organic Acidity/ pH λmax, Molar absorb. coef. Beer’s law Detection limit RSD (%) Interference Remarks Hydrazine Dihydrochloride [16] Chloroform Acidic 3.0 507 532×104 0.05-0.12 14 6 Many i) Less sensitive ii) pH dependent iii) Less selective due to much interference iv) Solvent extractive Dithizone [34] CCl4 Solvent extractive 3.0 620 5.8×103 0.2-1.0 70 6 Many i) Less sensitive ii) pH dependent iii) Time consuming iv) Solvent extractive Thionine [44] Chloroform Acidic 5.8 598 3.59×104 0.5-1.5 50 11 Many i) Solvent extractive ii) Less sensitive iii) Less selective due to much interference Gallocyanine by sodium sulfide [46] Chloroform Acidic 1.5 620 5.8×103 0.06-5.00 20 25 Many i) Solvent extractive ii) Less sensitive iii) Less selective due to much interference 4,5-Diamino-6- thiopyrimidine [50] Chloroform Solvent extractive 3.0 600 7.33×104 0.1-5.0 100 4 Many i) Lengthy and time consuming ii) Less sensitive iii) Less selective due to much interference. iv) Solvent Extractive. 1-Naphthylo amine- 7-sulfonic acid (Cleve’s acid) [52] H2SO4 Acidic 2.0 350 8.9×103 0.04-3.00 20 3.0 Many i) Less sensitive ii) Time consuming iii) Less selective due to much interference iv) Solvent extractive Salicylaldehyde- orthoaminophenol (Sal-OAP) (Proposed method) Ethanol Aqueous Slightly Acidic, 0.0001 - 0.0002 M H2SO4 379 6.4×105 0.001-40.000 0.1 0-2 i) Ultra-sensitive ii) Highly selective iii) Aqueous reaction medium. iv) Simple and rapid. v) Color stable more than 24 h at room temperature. vi) Non-extractive. vi) Application in various real, environmental, biological, soil, food and pharmaceutical samples * Units: λmax (nm), Beer’s Law (mg/L), Molar absorption co-efficient, ε (L/mol.cm), and Detection limit (ng/mL). Table 2. Summary of selected analytical parameters obtained with optimization experiments. Parameters Studied value Selected value Wavelength, λmax (nm) 200 - 800 379 Solvent, mL (Ethanol) 0 - 8 1 - 8 (Preferably 2.0) Acidity, M H2SO4 0.00001 - 0.01 0.0001 - 0.0002 (Preferably 0.0001) pH 2.1 - 6.5 3.5 - 5.8 (Preferably 3.5) Time 0 - 24 h 1 min-24 h (Preferably 2 min) Temperature, ᵒC 10 - 80 25±5 Reagent (fold molar excess, M:R) 1:1 - 1:700 1:80 - 1:600 (Preferably 1:100) Linear range, mg/L 0.0001 - 100 0.001 - 40 Molar absorptivity, L/mol.cm 5.6×105 - 7.2×105 6.4×105 Limit of quantification, µg/L 0.1 - 10 1.0 Detection limit, µg/L 0 - 100 0.1 Sandell’s sensitivity, ng/cm2 0 - 100 1.0 Reproducibility (% RSD) 0 - 10 0 - 2 Regression coefficient, R2 0.9993 - 0.9999 0.9996 3.4. Effect of foreign ions The effect of over 60 anions, cations and complexing agents on the determination of only 1 mg/L of selenium (IV) was studied. The criterion for an interference [58] was an absor- bance value varying by more than 5% from the expected value for selenium alone. The results are summarized in Table 3. As can be seen, a large number of ions have no significant effect on the determination of selenium. The interferences were from Fe(III) and Cr(VI) ions. Interference from these ions is probably due to complex formation with Sal-OAP. The greater tolerance limits for these ions can be achieved by using several masking methods. In order to eliminate interference of Fe(III) and Cr(VI), EDTA and SCN- are used as masking agents, respectively. During the interference studies, if a precipitate was formed, it was removed by centrifugation. The amount mentioned is not the tolerance limit but the actual amount studied. However, for those ions whose tolerance limit has been studied, their tolerance ratios are mentioned in Table 3. 3.5. Composition of the absorbent complex Job’s method [59] of continuous variation method was applied to ascertain the stoichiometric composition of the complex under the optimum conditions (Table 2). A Se(IV)-Sal- OAP (1:2) complex was indicated by this method. The stoichiometry was found to be 1:2 (Metal: Ligand). The molar- ratio method [60] was also applied to ascertain the stoichiometric composition of the complex. A Se(IV)-Sal-OAP complex was indicated by both methods and the stoichiometry was also found to be 1:2 (Metal: Ligand). 474 Ahmed et al. / European Journal of Chemistry 12 (4) (2021) 469-481 2021 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.12.4.469-481.2137 Table 3. Tolerance limits of foreign ions, tolerance ratio [Species (x)/V (w/w)] a. Species x Tolerance ratio (x/Se) (w/w) Species x Tolerance ratio (x/Se) (w/w) Aluminum 20 Lead(II) 50 Arsenic(III) 20 Magnesium 100 Arsenic(V) 100 Mercury(II) 100 Antimony 100 Molybdenum(VI) 100 Azide 100 Manganese(II) 50 Ammonium 50 Manganese(VII) 20 Bismuth(III) 100 Nickel(II) 50 Bromide 100 Oxalate 100 Barium 100 Phosphate 100 Cadmium 50 Potassium 100 Calcium 50 Cobalt (II & III) 50 Carbonate 100 Selenium(VI) 50 b Citrate 50 Silver 50 c Chromium(III) 100 b Strontium 50 b Chromium(VI) 20 b Sulphate 100 Cesium 100 Sodium 50 Copper(II) 50 Tartrate 1000 Cerium(III) 100 Tin(II & IV) 20 c Chloride 50 Tellurium(IV) 100 Dimethylglyoxime 100 Titanium(IV) 100 EDTA 1000 Thallium(I &III) 50 Fluoride 100 Thiocyanate 100 Iron(II) 50 b Tungsten(VI) 20 Iron(III) 20 b Thiosulphate 100 Iodide 100 Uranium(VI) 20 b Lithium 50 Zinc 50 a Tolerance limit was defined as ratio that causes less than ±5 percent interference. b with 10 mg/L EDTA. c with 10 mg/L Tartrate. Table 4. Determination of selenium levels in a variety of synthetic mixtures. Sample Composition of mixtures (mg/L) Se(IV) (mg/L) Added Found a (n=5) Recovery± SD b (%) A Se(IV) 0.50 1.00 0.50 0.99 100±0.0 99±0.8 B As in A + TeIV (25) + Pb2+ (25) + Zn (25) + Ba (25) + Cu2+ (25) + EDTA (50) 0.50 1.00 0.49 1.00 98±0.8 100±0.0 C As in B + K( 25) + V V (25) + Na (25) + Ni2+ (25) + Mn2+ (25) 0.50 1.00 0.51 0.99 102±0.9 99±1.0 D As in C + AsIII (25) + AsV (25) + Li (25) + WVI (25) + SeIV (25) + TlIII (25) 0.50 1.00 0.52 1.03 104±1.5 103±1.6 E As in D + Hg2+ (25) + FeIII (25) + Sr (25) + Ag (25) + CrVI (25) + Tartrate (50) 0.50 1.00 0.53 1.05 106±1.6 105±1.8 F As in E + CeIII (25) + MoVI (25) + Sn2+ (25) + MnVII (25) + BiIII (25) + UVI (25) 0.50 1.00 0.54 1.08 108±1.8 108±2.0 a Average of five analyses of each sample. b The measure of precision is the standard deviation (SD). 3.6. Precision and accuracy The precision of the present method was evaluated by determining different concentrations of selenium (each analyzed at least five times). The relative standard deviation (n = 5) was 0-2.0 % for 0.01-400 μg of selenium in 10 mL, indicating that this method is highly precise and reproducible. The detection limit 3s/S (s = Standard deviation of the blank, S = Slope of calibration graph) and Sandell’s sensitivity (concentration for 0.001 absorbance unit) for selenium were found to be 0.1 μg /L and 1.0 ng/cm2, respectively. The method was also tested by analyzing several synthetic mixtures containing selenium(IV) and diverse ions (Table 4). The results for total selenium were in good agreement with certified values (Table 5). The reliability of our Se-chelate procedure was tested by recovery studies. The average percentage recovery obtained for addition of selenium (IV) spike to some environmental water samples was quantitative as shown in Table 6. The results of biological analyses by the spectrophotometric method were in excellent agreement with those obtained by AAS and ICP-OES (Table 7). The results of soil analyses by the spectrophoto- metric method were excellent agreement with those obtained by AAS (Table 8). The results of food and vegetables analyses by spectrophotometric method were also found to be in excellent agreement with those obtained by AAS and ICP-OES (Table 9). The results of pharmaceutical samples by the spectrophoto- metric method were in excellent agreement with those obtained by claimed values and by ICP-OES (Table 10). The results of speciation of selenium (IV) and selenium(VI) in mixtures were highly reproducible (Table 11). Hence, the precision and accuracy of the method were excellent. With suitable masking, the reaction can be made highly selective. 4. Applications The proposed method was successfully applied to the determination of selenium (IV) in a series of synthetic mixtures of various compositions (Table 4) and also in a number of real samples e.g. several Certified Reference Materials (CRMs) (Table 5). The method was also extended to the determination of selenium in a number of environmental, biological, food, soil and pharmaceutical samples. In view of the unknown composition of environmental water samples, the same equivalent portions of each such sample were analyzed for selenium content; the recoveries in both the “spiked” (added to the samples before the mineralization or dissolution) and the “unspiked” samples are in good agreement (Table 6). The results of biological analyses by spectrophotometric method were found to be in excellent agreement with those obtained by AAS and ICP-OES (Table 7). The results of soil samples analyses by the spectrophotometric method were found to be excellent agreement with those of obtained by AAS (Table 8). Ahmed et al. / European Journal of Chemistry 12 (4) (2021) 469-481 475 2021 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.12.4.469-481.2137 Table 5. Determination of selenium in some certified reference materials. Sample Certified reference materials (Composition, mg/kg) Selenium, mg/kg Certified value Found a (n=5) RSD b (%) 1 GBW 01620 * (Ag = 4.6, As = 11, Bi = 0.4, Ca = 21, Cd = 4.6, Ga = 32, In = 2.6, Mg = 16, Pb = 4.1, Sb = 9.5, Se = 16, Sn = 53, Te = 11, Ti = 22, Zn = 32) 16.0 15.88 1.5 2 GBW 01622 * (Ag = 0.3, As = 72, Bi = 0.5, Ca = 32, Cd = 1.9, Ga = 28, In = 0.4, Mg = 53, Pb = 2.2, Sb = 7.4, Se = 43, Sn = 10.4, Te = 0.5, Ti = 8.3, Zn = 20) 43.0 42.98 2.0 3 GBW01637 * (As = 14, Bi = 0.19, Ag = 1.0, Ga = 34, In = 7.2, Pb = 3.7, Sb = 3.3, Se = 12, Sn = 8.3, Te = 3.1, Ti = 0.16, Zn =13) 12.0 11.88 1.8 4 CRM-TMDW-A-100: Water Standards **: (Sb = 55, As = 55, Be = 15, Cd = 10, Se = 11, Ag = 2, Tl = 10, Mn = 40, Ni = 60, Zn = 75) 11.00 c 10.95±0.8 2.0 5 CEC®-CRM®-277, Estuarine Sediment *** 2.04 1.95 1.5 6 NIST®-SRM®-2711: Soil *** 4.95 4.93 1.8 7 NIST®SRM®1577b: Bovine liver *** 1.68 1.67±0.14 1.5 8 NRC®-CRM®-C85-05, Tea *** 0.041 0.043 0.5 9 NIST®SRM®-2670a-Toxic Elements in Freeze-Dried Urine *** 17.8 c 17.5 c 2.0 10 BCR®-CRM®-150: Skim Milk Powder *** 0.127 0.128 1.0 * The CRMs were obtained from the NCS Analytical Instruments Co. Ltd. Beijing, China. ** This CRM was obtained from High Purity Standards (HPS), Amazon, North Charleston, USA. *** These CRMs were obtained from the National Research Council, Govt. of Canada. a Average of five analyses of each samples b The measure of precision is the relative standard deviation (RSD) c Values in µg/L. The results of some vegetable and food samples by the spectrophotometric method were found to be excellent agreement with those obtained by AAS and ICP-OES (Table 9). The results of some pharmaceutical samples by the spectro- photometric method were found to be excellent agreement with those obtained by claimed values and by ICP-OES (Table 10). The results of speciation of selenium(IV) and selenium(VI) in mixtures were highly reproducible (Table 11). The precision and accuracy of the method were excellent. 4.1. Determination of selenium in some synthetic mixtures Several synthetic mixtures of varying compositions containing selenium (IV) and diverse ions of known concent- rations were determined by the present method using tartrate or EDTA as 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 in the range 98±0.5 to 100±0.01%. The reliability of our Selenium-Sal-OAP procedure was approved by quantitative recovery of selenium (IV) spiked in several synthetic mixture containing selenium(IV) and diverse ions. The method has high precision and accuracy (s = ±0.01 for 0.5 μg/L). 4.2. Determination of selenium in some certified reference materials A 0.1-g amount of an alloy or steel sample containing 0.411- 50.16 % of selenium was weighed accurately and placed in a 50- mL Erlenmeyer flask in presence of excess reducing agent to reduce selenium(VI) to selenium(IV) following a method recommended by Mitra [61]. To it, 10 mL of 20% (w/v) sulfuric acid was added and while carefully covering with a watch glass until the brisk reaction subsided. The solution was heated and simmered gently after the addition of 10-mL of concentrated HNO3 until all residual carbides were decomposed. Then a further 2 mL of 1+1 H2SO4 and 2 mL 2.5% (w/v) freshly prepared sodium azide solution were added and the solution was evaporated carefully to dense white fumes of excess azide, then cooled to room temperature, 25±5 °C. After suitable dilution with de-ionized water, the contents of the Erlenmeyer flask were warmed so as to dissolve the soluble salts. The solution was then cooled and neutralized with dilute NH4OH solution in presence of 1-2 mL of 0.01% (w/v) EDTA solution. The resulting solution was filtered if necessary, through a Whatman No. 40 filter paper into a 100-mL calibrated flask. The residue (silica and tungstenic acid) was washed with a small volume of hot 1+99 H2SO4, followed by water; the volume was made up to mark with de-ionized water. A suitable aliquot (1-2 mL) of the above-mentioned solution was taken into a 10 mL calibrated flask and the selenium (IV) content was determined; as described under general procedure using EDTA or tartrate as masking agent. The proposed procedure for the spectrophotometric determination of selenium was applied to the analysis of single element CRM of Se, estuarine sediment (CRM®-MESS®-3), Soil (CRM®029), human serum (CRM®-ASTMRCVD®-74231), Bovine liver (NIST® SRM®-1577c), These CRMs obtained from the National Research Council, Govt. of Canada, using tartrate or EDTA as masking agents, following a method recommended by Sun et al. [62]. Based on five replicate analyses, average selenium concentration determined by the spectrophotometric method was in an excellent agreement with the certified values. The results are given in Table 5. 4.3. Determination of selenium in environmental water samples Each filtered (with Whatman No. 40) environmental sample (25 mL) contained in a 50 mL Pyrex beaker were added 1 mL of concentrated H2SO4 and 2 mL of concentrated HNO3 in the presence of excess freshly prepared (2.5% (w/v) sodium azide solution in a fume cupboard to reduce selenium(VI) to selenium(IV) and the mixture was heated on a hot plate until white fumes of azide to remove completely, following a method recommended by AWWA [63]. The solution was cooled and neutralized with dilute NH4OH solution in presence of 1-2 mL of 0.01% (w/v) EDTA solution. Resulting solution was then filtered through a Whatman No. 40 filter paper and quantita- tively transferred into a 25 mL calibrated flask and made up to the mark with de-ionized water. An aliquot (1-2 mL) of this water sample was pipetted into a 10 mL calibrated flask and the selenium content was deter- mined as described under the general procedure using tartrate or EDTA as masking agent. To test the validity of our method, we have analyzed different types of portable and polluted waters in spike and un-spike conditions. The reliability of our spectrophotometric method was tested by recovery studies. The average percentage recovery obtained for the addition of a selenium (IV) spike to some environmental water samples was quantitative. The results of analyses of environmental water samples from various sources for selenium are shown in Table 6. Most spectrophotometric methods for determination of selenium in natural and sea-water require preconcentration or standard addition of selenium [63]. 476 Ahmed et al. / European Journal of Chemistry 12 (4) (2021) 469-481 2021 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.12.4.469-481.2137 Table 6. Determination of selenium in some environmental water samples. Samples Selenium (µg/L) Recovery±s (%) sr b (%) Added Found a Tap Water 0 100 500 13.0 112.0 515.0 - 100±0.0 100.5±0.6 - 0.00 0.22 Mineral Water 0 100 500 25.0 125.0 530.0 - 100±0.0 100.9±0.6 - 0.00 0.21 Well Water 0 100 500 15.0 112.0 525.0 - 97.4±0.5 101.9±0.6 - 0.31 0.40 Rain Water 0 100 500 10.0 112.0 520.0 - 101.8±0.8 102.0±0.5 - 0.29 0.23 River Water Karnaphuli River Kanaphuli (upper) 0 100 500 45.0 150.0 540.0 - 103.4±0.6 99.1±0.4 - 0.25 0.21 Karnaphuli (lower) 0 100 500 50.0 150.0 560.0 - 100±0.0 101.8±1.0 - 0.00 0.30 Halda (upper) 0 100 500 31.0 126.0 536.0 - 96.2±0.5 100.9±0.8 - 0.22 0.28 Halda (lower) 0 100 500 36.0 136.0 540.0 - 100±0.0 100.9±0.6 - 0.00 0.24 Lake Water Kaptai Lake Rangamati 0 100 500 55.0 156.0 560.0 - 100.6±0.8 100.3±0.5 - 0.21 0.35 Fayez Lake Bhatiary, Chittagong 0 100 500 26.0 126.0 530.0 - 100±0.0 100.9±0.8 - 0.00 0.34 Sea Water Potenga Sea Beach Bay of Bengal (upper) 0 100 500 45.0 145.0 548.0 - 100±0.0 100.5±0.6 - 0.00 0.45 Bay of Bengal (lower) 0 100 500 50.0 152.0 550.0 - 101.3±0.8 100.0±0.0 - 0.39 0.00 Laboni Beach, Cox’s Bazaar 0 100 500 45.0 145.0 550.0 - 100±0.0 100.9±0.7 - 0.00 0.29 Kolatoli Beach, Cox’s Bazaar 0 100 500 48.0 148.0 552.0 - 100±0.0 100.7±0.6 - 0.00 0.21 Drain Water Eastern Cablesc 0 100 500 140.0 245.0 650.0 - 102±1.0 101.6±0.8 - 0.58 0.35 Eastern Refineryd 0 100 500 225.0 330.0 735.0 - 100.6±1.0 101.4±0.8 - 0.49 0.37 Elite Painte 0 100 500 175.0 280.0 670.0 - 101.8±0.6 99.3±0.5 - 0.28 0.21 PHP Glassf 0 100 500 125.0 230.0 635.0 - 102±0.8 01.6±0.7 - 0.29 0.27 a Average of five replicate determinations of each sample. b The measure precision is the relative standard deviation(sr). c Eastern Cables Ltd., North Patenga, Chittagong. d Eastern Refinery Ltd., North Potenga, Chittagong e Elite Paint and Chemical Industries Ltd., Baized Bostami Road, Chittagong. f PHP Glass, Kumara, Chittagong. The concentration of selenium in natural and sea water is a few µg/L in developed countries. The mean concentration of selenium found in US drinking water is greater than 10 µg/L [64]. 4.4. Determination of selenium in some biological samples The biological samples were digested accordingly following a method reported by Khayatian et al. [65]. The samples were initially dried in an oven at 120 °C for 24 h. Blood serum samples were further dried in an oven at 20°C for an additional 24 h. Then, the biological samples were dry-ashed in a Muffle furnace at 300 °C for 24 h, then at 450 °C for 4 h. After dry- ashing, samples were wet-ashed with 5 mL concentrated nitric acid and 2mL of freshly prepared 2.5% sodium azide solution. The mixture was heated to just below boiling until complete reduction of selenium(VI) to selenium(IV). The samples were cooled and wet-ashed three more times in the same manner. At completion, the white residue was dissolved with 10 mL of 1 M HNO3 by heating of an excess reducing agent according to the method recommended by Stahr [66] and diluted to 20.0 mL for analysis. After neutralizing pH by addition of dilute NH4OH in the presence of 1-2 mL of a 0.01% (w/v) tartrate or EDTA solution. The resultant solution was then filtered and trans- ferred quantitatively into a 25 mL calibrated flask and made up to the mark with deionized water. Ahmed et al. / European Journal of Chemistry 12 (4) (2021) 469-481 477 2021 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.12.4.469-481.2137 Table 7. Determination of selenium in human fluids. Serial Sample Selenium (µg/L) Sample source a AAS (n=5) Proposed method (n=5) ICP-OES (n=5) Found b RSD (%) Found b RSD (%) Found b RSD (%) 1 Blood Urine 101.5 25.9 1.7 1.0 103.5 29.5 1.5 1.0 102.5 28.8 1.6 1.1 Normal adult (Male) 2 Blood Urine 414.8 121.5 3.0 2.0 415.8 120.5 2.0 1.5 416.5 122.0 3.0 1.8 Hair loss, nail discoloration patient (Female) 3 Blood Urine 315.5 81.5 2.5 2.0 319.8 87.5 2.0 1.8 320.0 88.0 2.5 1.8 Neurological disorder (Male) 4 Blood Urine 274.8 71.5 2.0 1.9 276.5 77.5 2.0 1.8 277.6 79.0 2.5 2.0 Dental caries patient (Female) 5 Blood Urine 287.5 72.8 3.0 2.5 288.8 75.5 2.5 2.0 290.2 76.8 3.0 2.5 Pulmonary edema (Female) 6 Blood Urine 298.5 77.8 3.5 2.8 300.8 78.9 3.0 2.0 302.8 80.5 3.5 2.5 Conjunctivitis patient (Female) 7 Blood Urine 226.8 65.8 3.0 2.5 228.5 66.6 2.8 2.5 229.5 68.0 3.0 2.5 Heart disease patient (Female) 8 Blood Urine 245.0 62.5 3.2 2.8 248.5 65.6 2.5 2.0 250.0 67.8 3.2 2.5 Kidney Dialysis patient (Male) 9 Blood Urine 200.8 53.5 2.0 1.8 202.5 56.8 1.8 1.5 203.0 58.5 1.8 1.5 Hypothyroidism (Female) 10 Hair c 1.58 c 2.0 1.65 1.5 1.63 2.0 Normal human hair (Female) 11 Nail c 0.85 c 1.5 0.88 1.5 0.89 1.8 Normal human nails (Female) a Samples were collected from Chittagong Medical College Hospital, Chittagong. b The measure of precision is the relative standard deviation (RSD). c Values in mg/kg. Table 8. Determination of selenium in some soil samples. Serial Selenium (µg/g) Sample sources c Proposed method (n=5) AAS (n=5) Found a (n=5) RSD b (%) Found a (n=5) RSD b (%) S1 2.8 1.0 3.0 1.3 Road-side soil (Dhaka-Chittagong) S2 0.68 1.0 0.71 1.0 Agricultural soil (Chittagong University Campus) S3 26.9 1.5 27.8 1.8 Industrial soil (Eastern Cables Ltd.) S4 58.5 2.5 59.6 2.8 Industrial soil (Eastern Refinery Ltd.) S5 65.6 2.8 68.0 3.0 Industrial soil (PHP Glass Ltd., Kumara, Chittagong) S6 25.5 2.0 26.5 2.5 Madina Tannery soil (Jalalabad, Chittagong) S7 75.8 2.5 78.5 3.0 Paint soil (Elite Paint & Chemical Industries Ltd., Baized, Chittagong) S8 7.5 1.5 8.6 1.5 Karnafuli River Bank soil (Chittagong) S9 25.8 2.5 26.5 2.8 T.S.P complex soil (Patenga, Chittagong) S10 18.9 2.0 19.5 2.2 Estuarine soil (Karnafuli River) S11 23.5 1.8 24.2 2.0 Industrial soil (Delhi Aluminum Factory Ltd.) S12 0.35 0.8 0.36 1.0 Normal Soil of Science Faculty (Chittagong University) a Average of five analyses of each sample. b The measure of precision is the relative standard deviation (RSD). c Composition of the soil samples: C, N, P, K, Na, Ca, Mg, Ce, Cu, Mo, Fe, Pb, Zn, Mn, Co, NO3-, SO4- etc. A suitable aliquot (1-2 mL) of the final solution was pipetted into a 10 mL calibrated flask and the selenium(IV) content was determined as described under the procedure using tartrate or EDTA as masking agent. The results of biological analyses by the spectrophotometric method were found to be in excellent agreement with those obtained by AAS and ICP-OES. The results are shown in Table 7. The abnormally high values for the hair loss and nail discoloration and neurological disorder patient are probably due to the involvement of high selenium concentration with As and Zn. The occurrence of such high selenium contents is also reported in hair loss and neurological disorder patients from some developed countries [67]. 4.5. Determination of selenium in some surface soil samples An air-dried homogenized soil sample (10 g) was accurately weighed and placed in a 100 mL micro-Kjeldahl flask. The sample was digested in the presence of an excess reducing agent (2 mL of 2.5% freshly prepared sodium azide solution) to reduce selenium(VI) to selenium(IV) following method recom- mended by Jackson [68]. As the heating process continued 1-mL of H2SO4 is added and heated for about 5 minutes to dense white fumes to remove excess azide. The solution was then cooled at room temperature and neutralized with dilute NH4OH solution in presence of 1-2 mL of 0.01% (w/v) EDTA solution. The content of the flask was then filtered through a Whatman No. 40 filter paper and quantitatively transferred into a 25 mL calibrated flask and made up to the mark with de-ionized water. 478 Ahmed et al. / European Journal of Chemistry 12 (4) (2021) 469-481 2021 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.12.4.469-481.2137 Table 9. Determination of selenium in some food, fruit and vegetable samples Serial Sample Selenium (µg/kg or µg/L) Sample Source Proposed method (n=5) AAS (n=5) ICP-OES (n=5) Found a RSD b (%) Found a RSD b (%) Found a RSD b (%) 1 Chicken meat (Gallus cibum) 22.0 2.0 21.8 2.0 21.98 2.2 Local Market, Chittagong 2 Chicken liver (Gallus jecur) 25.0 2.0 25.5 2.3 25.8 2.5 Local Market, Chittagong 3 Egg (Gallus domesticus) 14.5 1.8 14.8 2.0 15.0 1.8 Local Market, Chittagong 4 Radish (Raphanus sativus) 12.5 1.5 12.8 1.8 13.3 2.0 Local Market, Chittagong 5 Carrot (Daucus carota) 15.9 1.8 16.5 2.0 16.8 2.0 Local Market, Chittagong 6 Tomato (Lycopersicon esculentum) 31.5 2.5 31.8 2.5 32.1 2.8 Local Market, Chittagong 7 Rice (Oryza sativa) 20.5 2.0 21.2 2.2 21.8 2.5 Local Market, Chittagong 8 Wheat (Triticum aestivum) 25.8 2.5 26.5 2.5 27.0 2.8 Local Market, Chittagong 9 Spinach (Spinacia oleracea) 8.5 1.5 9.0 1.6 8.9 1.8 Local Market, Chittagong 10 Mushrooms (Agaricus bisporus) 21.8 2.1 22.0 2.5 22.5 2.8 Local Market, Chittagong 11 Dried Loitta Fish (Harpadon nehereus) 28.5 2.5 29.8 3.0 30.0 3.0 Local Market, Chittagong 12 Prawn (pandulus jordani) 35.8 2.8 35.5 3.0 35.8 3.5 Local Market, Chittagong 13 Tea (Camellia sinensis) 22.5 2.0 23.0 2.5 23.5 2.8 Local Market, Chittagong 14 Banana (Musa) 2.5 1.5 2.8 1.8 2.6 1.8 Local Market, Chittagong 15 Milk (Diary Milk) 4.8 1.8 5.1 2.0 5.2 2.5 Local Market, Chittagong a Average of five replicate analyses of each sample. b The measure of precision is the relative standard deviation (RSD). A suitable aliquot (1-2 mL) of the final solution was pipetted out into a 10 mL calibrated flask and the selenium content was determined as described under the general procedure using tartrate or EDTA as masking agent. The selenium content was then determined by the above procedure and quantified from a calibration graph prepared concurrently. The results of soil analyses by spectrophotometric method were also found to be in excellent agreement with those obtained by AAS. The average value of selenium in the Chittagong region surface soil was found to be 43.29 mg/kg [69]. The results are shown in Table 8. 4.6. Determination of selenium in some vegetable, food and fruit samples The vegetable and fruit samples collected prior to the determination were pretreated in the following way: Edible portion of samples was first washed clean with tap water followed by rewashing with de-ionized water. After removing de-ionized water from the surface of vegetables and fruits, the samples were cut into small pieces and dried at 65 °C in oven. An air-dried vegetables and fruits samples (10 g) were ground in a mortar and taken in a 100 mL micro-Kjeldahl flask in presence of excess reducing agent and digested following a method recommended by Stahr [66] and 10 mL of concentrated nitric acid were added and the flask was placed on the digester under gentle heating. When the initial brisk reaction was over, the solution was removed and cooled at room temperature. 1 mL volume of concentrated sulfuric acid was added carefully, followed by the addition of 1 mL of concentrated HF, and heating was continued for at least 30 min and then cooled. In the resulting solution 2 mL of 2.5% (w/v) of freshly prepared sodium azide solution was added. The mixture of each foodstuff was heated below the boiling point for 5-10 min to reduce selenium(VI) to selenium(IV). Excess azide was removed by further heating. The solutions were then cooled and neutralized with dilute NH4OH in presence of 1-2 mL of 0.01% (w:v) EDTA solution. The resulting solution was filtered through a Whatman No. 40 filter paper and quantitatively transferred into a 25 mL calibrated flask and mixed well and made up to the mark with de-ionized water. The food samples used were rice and wheat and these were used under dry conditions. Each sample was first ground in a mortar. Fruit samples (2 g) or rice and wheat samples (1 g) were weighed accurately and placed in a porcelain crucible and charred in an electric furnace; the sample was ashen at 555 °C in a muffle furnace in presence of excess oxidizing agent following a method recommended by Mitra [61]. To it, 2.0 mL of HCl and 10 mL of water were added to the ash. The mixture of each foodstuff was heated with 2 mL of 2.5% (w/v) freshly prepared sodium azide solution was added below the boiling point for 5-10 min to complete reduction from Se(VI) to Se (IV). Then the solution was heated for another 5 min to remove excess azide. The solutions were cooled and neutralized with dilute NH4OH in presence of 1-2 mL of 0.01% (w/v) EDTA solution and filtered. The resulting solution was quantitatively transferred into a 25 mL calibrated flask and mixed well and made up to the mark with de-ionized water. A suitable aliquot (1-2 mL) of the final digested solution was pipetted into a 10 mL calibrated flask and the selenium content was determined as described under the general procedure using EDTA or tartrate as masking agent. The results of food and vegetables analyses by spectrophotometric method were also found to be in excellent agreement with those obtained by AAS and ICP-OES. The results are shown in Table 9. 4.7. Determination of selenium in pharmaceutical samples Finished pharmaceutical samples (each Se containing tablet or 10 mL insulin or required weight) were quantitatively taken in a beaker and digested following a method recommended by Ahmed et al. [70]. 10 mL of concentrated nitric acid was added and heated to dryness and then added 10 mL of 20% (v/v) of H2SO4. Ahmed et al. / European Journal of Chemistry 12 (4) (2021) 469-481 479 2021 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.12.4.469-481.2137 Table 10. Determination of selenium in some pharmaceutical samples. Sample Sample type a Brand name Trade name Selenium (mg/kg or µg/kg) Reported value / Claimed value Proposed method (n=5) ICP-OES (n=5) Found a (n =5) RSD b (%) Found a (n =5) RSD b (%) 1 Tablet Square Pharmaceuticals Ltd. (Selenoprotine) Mulvit plus (Multivitamin-mineral)/20 mg 20 mg 20.8 2.0 21.0 2.0 2 Tablet Beximco Pharmaceuticals Ltd. (Selenoprotine) Bextram Gold (A to Z) (Multivitamin-mineral)/20 mg 20 mg 19.8 1.8 20.0 2.0 3 Tablet Opsonin pharma (Selenoprotine) Zovia Gold (A to Z) (Multivitamin-mineral)/20 mg 20 mg 19.85 1.6 19.92 1.8 4 Tablet Drug Int. Ltd. (Selenoprotine) Supravit-G (Multivitamin-mineral)/20 mg 20 mg 20.5 1.8 21.0 2.0 5 Tablet Holland and Barrett (Selenoprotine) Selenium 50 µg 50 µg 49.6 2.0 49.8 2.0 6 Tablet Incepta Pharmaceuticals Ltd. Evagren 70 µg 70 µg 69.8 2.5 71.0 2.5 a Samples were collected from local market, Chittagong. b The measure of precision is the relative standard deviation. Table 11. Determination of selenium(IV) and selenium(VI) speciation in mixtures. Serial Se(IV): Se(VI) Se taken (mg/L) Se, found (mg/L) Error (mg/L) Se(IV) Se(VI) Se(IV) Se(VI) Se(IV) Se(VI) 1 1: 1 1.00 1.00 0.98 0.99 0.02 0.01 2 1: 1 1.00 1.00 1.00 1.02 0.00 0.02 3 1: 1 1.00 1.00 0.97 0.99 0.03 0.01 Mean error: Se(IV) = ±0.016 Se(VI) = ±0.013 Standard deviation: Se(IV)= ± 0.015 Se(VI) = ±0.011 1 1: 5 1.00 5.00 0.98 4.98 0.02 0.02 2 1: 5 1.00 5.00 0.99 4.99 0.01 0.01 3 1: 5 1.00 5.00 0.98 4.98 0.02 0.02 Mean error: Se(IV) = ±0.016 Se(VI) = ±0.016 Standard deviation: Se(IV) = ± 0.0058 Se(VI) = ±0.0058 1 1:10 1.00 10.00 0.99 9.99 0.01 0.01 2 1:10 1.00 10.00 0.98 9.98 0.02 0.02 3 1:10 1.00 10.00 0.98 9.98 0.02 0.02 Mean error: Se(IV) = ±0. 016 Se(VI) = ±0.016 Standard deviation: Se(IV) = ± 0.015 Se(VI) = ±0.015 The mixture was heated with 2 mL of 2.5 % (w/v) freshly prepared sodium azide solution was added below the boiling point for 5-10 min to complete reduction from Se(VI) to Se (IV). Excess azide was removed by heating and the volume was reduced to 2.5 mL and then cooled to room temperature. The solution was then neutralized with dilute NH4OH in the presence of a 1-2 mL of 0.01% (w/v) EDTA or tartrate solution. The resulting solution was then filtrated and quantitatively transferred to a 25 mL calibrated flask and made up to the mark with deionized water. An aliquot (1-2 mL) of this digested sample was pipetted into a 10 mL calibrated flask and then selenium content was determined as described under the general procedure using tartrate as a masking agent. The results of some pharmaceutical analyses by the spectrophotometric method were found to be in excellent agreement with those obtained by reported values and ICP-OES. The analyses of pharmaceutical samples from several Pharmaceutical Companies for selenium are given in Table 10. 4.8. Determination of selenium (iv) and selenium (vi) speciation in mixtures Suitable aliquots (1-2 mL) of selenium (IV+VI) mixtures (preferably 1:1, 1:5, 1:10) were taken in a 250 mL Pyrex conical flask. A few drops (3–5 drops) of 4M H2SO4, and 5–10-mL of 2.5 % (w:v) freshly prepared sodium azide were added to reduce hexavalent selenium to tetravalent selenium and the mixture was heated gently with further addition of 10-mL water, if necessary, for 5 minutes to drive off the excess azide, then the mixture was cooled to room temperature 25±5 °C following the method recommended by Abrarin et al. [71]. The reaction mixture was then cooled and neutralized with dilute NH4OH in presence of 1-2 mL of 0.01% (w:v) EDTA solution. The solution was transferred quantitatively into a 25-mL volumetric flask and 2.0 mL of 1.17×10–3 M Sal-OAP reagent solution was added followed by the addition of 1.0 mL of 0.0001 M H2SO4. It was made up to the mark with de-ionized water. The absorbance was measured then being cooled at room temperature, 25±5 °C, at 379 nm against a reagent blank. The total selenium content was calculated with the help of a calibration graph prepared concurrently. An equal aliquot (1-2 mL) of the above selenium (IV + VI) mixture was taken into a 250 mL Pyrex conical flask. The solution was neutralized with dilute NH4OH in presence of 1-2 mL of 0.01% (w:v) EDTA solution. After, the content of the beaker was transferred quantitatively into a 25 mL volumetric flask, 2.0 mL of 1.17×10–3 M Sal-OAP reagent solution was added, followed by the addition of 1.0 mL of 0.0001 M H2SO4. It was made up to the mark with de-ionized water. After 5 min the absorbance was measured following the general procedure at 379 nm against a reagent blank, as before. The selenium concentration was calculated in mg/L or μg/L with the aid of a calibration graph. This gives a measure of selenium (IV) originally present in the mixture. This value was subtracted from that of the total selenium to determine the selenium (VI) present in the mixture. The results of the assessment of speciation of Se(IV) and Se(VI) were found to be highly reproducible. The occurrence of such reproducible results is also reported for different oxidation states of selenium [72]. The results of a set of determination are given in Table 11. 5. Conclusions A new simple, sensitive, selective and inexpensive method with the Selenium-Sal-OAP complex was developed for the 480 Ahmed et al. / European Journal of Chemistry 12 (4) (2021) 469-481 2021 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.12.4.469-481.2137 ultra-trace determination of selenium in some real, environ- mental, biological, soil, food and pharmaceutical samples, for continuous monitoring to establish the trace levels of selenium in different sample matrices. Compared with other methods in the literature Table 1, the proposed method has several remarkable analytical characteristics: i) The proposed method is highly sensitive with molar absorptivity of the complex of 6.4×105 L/mol.cm. Thus, amount of ng/g of selenium can be determined without preconcentration. ii) The proposed method is very simple, rapid and stable. The reaction of selenium (IV) with Sal-OAP is completed rapidly in 1 min at room temperature so it does not involve any stringent reaction conditions and offer the advantage of high complex stability (24 h). iii) The method has added the advantage of determining individual amounts of Se(IV) and Se(VI). With suitable masking agents, the reaction can be made highly selective. The proposed method using Sal-OAP in aqueous solutions not only is one of the most sensitive methods for the ultra –trace determination of selenium but also is excellent in terms of selectivity and simplicity. Therefore, this method will be successfully applied to the routine monitoring of trace and ultra-trace amounts of selenium in real, environmental, biological, soil, food and pharmaceutical samples. It is a new method needs neither heating nor extraction to organic phase, works satisfactorily and could be an alternative method for the rapid determination of selenium in a wide variety of sample solutions and found superior to spectrophotometric methods described in different literature [6-52,73-82]. Acknowledgements We are indebted to the Chittagong Medical College and Hospital for supplying biological samples. In addition, express our gratitude to “Training Institute for Chemical Industries” (TICI), Polash, Norsindi, for analyzing water, biological, food samples by Inductively Coupled Plasma-Optical Emission Spectrophotometer (ICP-OES). We are thankful to Center of Excellence in Analytical Chemistry, University of Sindh for Elemental analysis and Mass spectrum of the ligand Sal-BH. Disclosure statement Conflict of interests: The authors declare that they have no conflict of interest. Ethical approval: All ethical guidelines have been adhered. Biochemistry of the Essential Ultra Trace Elements Sample availability: Samples of the compounds are available from the authors. CRediT authorship contribution statement Conceptualization: Muhammad Jamaluddin Ahmed, Muhammad Jihan Uddin; Methodology: Muhammad Jamaluddin Ahmed; Software: Muhammad Emdadul Hoque; Validation: Muhammad Jihan Uddin; Formal Analysis: Muhammad Jihan Uddin, Muhammad Emdadul Hoque; Investigation: Muhammad Emdadul Hoque, Muhammad Jihan Uddin; Resources: Muhammad Jamaluddin Ahmed, Muhammad Jihan Uddin; Data Curation: Muhammad Jihan Uddin, Muhammad Emdadul Hoque; Writing - Original Draft: Muhammad Jamaluddin Ahmed; Writing - Review and Editing: Muhammad Emdadul Hoque, Muhammad Jihan Uddin; Visualization: Muhammad Emdadul Hoque; Validation: Muhammad Jihan Uddin; Funding acquisition: Muhammad Jamaluddin Ahmed; Supervision: Muhammad Jamaluddin Ahmed; Project Administration: Muhammad Jamaluddin Ahmed, Muhammad Jihan Uddin. 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This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). https://www.who.int/publications/i/item/9789241549950 https://en.wikipedia.org/w/index.php?title=Selenium&oldid=1048104284 https://en.wikipedia.org/w/index.php?title=Selenium&oldid=1048104284 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Apparatus 2.2. Live subject statement 2.3. Synthesis and characterization of the reagent 2.4. Reagents and solutions 2.4.1. Sal-OAP solution 2.4.2. Selenium (IV) standard solution 2.4.3. Selenium (VI) standard solution 2.4.4. Potassium dichromate solution 2.4.5. Sodium azide solution 2.4.6. Tartrate solution 2.4.7. Aqueous ammonia solution 2.4.8. EDTA solution 2.4.9. Other solutions 2.5. General procedure 2.6. Sample collection and preservation 3. Results and discussion 3.1. Characterization of the reagent 3.2. Factors affecting the absorbance 3.2.1. Absorption spectra 3.2.2. Optimization of some parameters on the absorbance 3.2.2.1. Effect of solvent 3.2.2.2. Effect of acidity 3.2.2.3. Effect of time 3.2.2.4. Effect of temperature 3.2.2.5. Effect of reagent concentration 3.3. Calibration graph (Beer’s law and sensitivity) 3.4. Effect of foreign ions 3.5. Composition of the absorbent complex 3.6. Precision and accuracy 4. Applications 4.1. Determination of selenium in some synthetic mixtures 4.2. Determination of selenium in some certified reference materials 4.3. Determination of selenium in environmental water samples 4.4. Determination of selenium in some biological samples 4.5. Determination of selenium in some surface soil samples 4.6. Determination of selenium in some vegetable, food and fruit samples 4.7. Determination of selenium in pharmaceutical samples 4.8. Determination of selenium (iv) and selenium (vi) speciation in mixtures 5. Conclusions Acknowledgements Disclosure statement CRediT authorship contribution statement ORCID References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField16: PrintField17: PrintField18: PrintField19: PrintField110: PrintField111: PrintField112: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: PrintField26: PrintField27: PrintField28: PrintField29: PrintField210: PrintField211: PrintField212: