233 Journal homepage: www.fia.usv.ro/fiajournal Journal of Faculty of Food Engineering, Ştefan cel Mare University of Suceava, Romania Volume XII, Issue 3 – 2013, pag. 233 - 239 ATOM-ABSORPTION METHOD OF DETERMINATION OF SELENIUM CONTENT IN SOME RAW MATERIALS AND FOOD Igor KOBASA1 1Yu. Fedkovych National University of Chernivtsi, Ukraine, imk-11@hotmail.com * Corresponding author Received August 31st 2013, accepted September 14th 2013 Abstract: A novel atom-absorption method of determination of selenium in some raw materials, bio- logical objects and food is proposed. This method involves preliminary extraction and concentrating of the components to be analyzed with butylacetate/sodium diethyldithiocarbamate mixture. The high- est extraction degree for Se(IV) and Se(VI) ions is reached for pH ranged from 1 to 6. Investigation of influence of high concentrations of iron and zinc on accuracy of the Se determination proved only in- significant (under 5%) blurring of the Se atom-absorption peaks for Zn admixture contents 0÷6 g/kg and 0÷10 g/kg of Fe. Keywords: Selenium; atom-absorption analysis; environmental objects; food 1. Introduction Selenium and its compounds are known as biologically active and important compo- nents with clear limits of toxicity, suffi- cient and insufficient supply. Toxic con- centration of Se disturbs normal function- ing of the cell structures through formation of stable and reactionless compounds of Se and S. Selenites are easily reducible and can form free Se followed by active reac- tions between Se and organic compounds and its incorporation in the structure of proteins. Some authors report higher con- tent of Se found in malignant tumours. Other results prove radioprotection activity of the low-concentrated Se and some coun- teraction to negative effects of the heavy metals and irradiation after treatment with the Se microdoses. Therefore, it is essential to find the clear limits between useful mi- crodoses and harmful overdosage of this element. This issue is related to various methods available for continuous and pre- cise analytical determination of Se content in environmental objects and other materi- als. A series of experimental methods are available for experimental determination of Se content in water [1, 2], biomaterials [3], blood plasma and serum [4, 5] and food [5, 6]. However, all these methods are very complex and require deficient and expen- sive reagents and equipment. On the other hand, sensitivity and accuracy of some of these methods seem insufficient. Our investigation deals with development of the quantitative determination of Se content in the above objects using a meth- od of the extraction concentrating of Se followed by the atom-absorption spectros- copy determination of this element at the acetylene-air atomizer. 2. Experimental and Methods Dithiocarbonates and dithizone are used traditionally as extraction agents [7] and Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XII, Issue 3 – 2013 Igor KOBASA, Atom-absorption method of determination of selenium content in some raw materials and food, Food and Environment Safety, Volume XII, Issue 3 – 2013, pag. 233 - 239 234 chloroform, tetrachlorated carbon, various mixtures of the polar oxygen-containing and non-polar oxygen free liquids [8] are used as organic solvents for preliminary extraction of Se from aqueous solution re- quired for spectral determination methods. However, most of the above mentioned organic agents can complicate fine analyti- cal determination of Se since they are hard- ly combustible and form yellowish and smoking flame. Besides, emission of harm- ful products is an unavoidable result of combustion of the chlorine-containing compounds. Methylisobutylcetone (MIBC) is proposed in [9] as an alternative solvent. However, this agent cannot ensure sufficient concen- trating degree. In our experiments, we used the butyl ether of acetic acid as a solvent. Physico- chemical properties of this compound are similar to MIBC while concentrating per- formance is significantly higher. Technically, all extractions have been per- formed in two systems: bu- tylacetate/sodium diethyldithiocarbamate and butylacetate/dithizone. Extraction mixtures have been prepared using the 10 % aqueous solution of sodium diethylldithiocarbamate and 0.01 M solu- tion of dithizone in butylacetate. A series of the model solutions with prede- termined contents of Se has been prepared. All concentrations of Se were ranged with- in the values expected in real analytical samples. These solutions were prepared from the deionized water and analytically pure selenium acid. Hydrochloric acid and analytically pure ammonium solution were used to adjust pH of the systems. A process of extraction was performed ac- cording to [10]. The flame atom-absorption spectropho- tometer C-115-M1 has been used for de- termination of Se content. The stage of at- omization was realized with the acetylene- air flame at the temperature up to 2600 °C. A high frequency gas discharge tube filled with microamount of Se was used as a source of the discrete spectrum. All atom absorption determinations were performed at the wave length 196.1 ± 0.4 nm. Rest of the samples preparation stages were made according to [11]. 3. Results and Discussion First, we had to select the most effective atomization mode and a value of electric current of the gas discharge tube. The mode of atomization had to provide the highest values of the wanted signal and signal/noise ratio. Results of the atomiza- tion mode selection are shown in Fig. 1. As seen in Fig. 1, the most optimal compo- sition of the combustible mixture can be reached at feeding the system with acety- lene at the pressure 0.4 kg/sm2 and air at 0.75 kg/sm2. This composition of the com- bustible mixture insures the highest and comparatively stable value of the wanted signal (light absorption value). A dependence of the wanted signal value on the tube current is shown in Fig. 2. It can be seen that the highest wanted signal was obtained at the lowest current value (90 mA). However, under this current we had to feed the photocell with almost the highest possible current, which caused generation of quite intense ‘noise’. Taking into account the above conditions, we set the photocell feeding current value at 100 mA. This value was still close to the most optimal wanted signal area and did not cause generation of excessive interfer- ing noise. This mode of the gas discharge tube feeding was realized using the super- high frequency electric generator 2PBL- 3MV. It was also found that the hollow cathode tube produced less relevant results. At the next stage we analyzed a character of influence of pH on the Se extraction de- gree. A series of extractions has been per- formed for solutions with predetermined Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XII, Issue 3 – 2013 Igor KOBASA, Atom-absorption method of determination of selenium content in some raw materials and food, Food and Environment Safety, Volume XII, Issue 3 – 2013, pag. 233 - 239 235 content of SeО3 2-. Then the content of the extracted Se in the organic phase has been determined using similar atom absorption method and the degree of absorption (R) was calculated using the formula below: R=(C0V0/CВVВ+C0V0)·100 %, where V0 and VB – volumes of the aqueous and organic phases; C0 and CB – concentrations of Se in the aqueous and organic phases respectively. 0 50 100 150 200 250 300 0,4 0,45 0,5 0,55 0,6 0,65 0,7 0,75 0,8 0,85 Oxygen pressure, kg/sm2 Li gh t a bs or pt io n, c .u . 1 2 3 4 5 Figure 1. A series of dependencies of the light absorption (conventional units, c.u) on the oxygen pressure (kg/sm2) for the following acetylene pressures: Pac = 0.40 kg/sm 2 (curve 1); Pac = 0.45 kg/sm 2 (curve 2); Pac = 0.50 kg/sm2 (curve 3); Pac = 0.55 kg/sm 2 (curve 4); Pac = 0.60 kg/sm 2 (curve 5). 0 50 100 150 200 250 300 350 400 90 95 100 105 110 115 120 125 130 I, mA L ig ht a bs o rp tio n , c .u . Figure 2. Dependence of the wanted signal value on the tube feeding current. It should be noted that accurate determina- tion of Se distribution ratio between the phases was quite difficult due to very low initial concentration in the aqueous phase. A dependence of the distribution ratio val- ues on pH is shown in Fig. 3. One can see that the mixture bu- tylacetate/sodium diethyldithiocarbamate (Fig. 3, line 1) shows the highest extraction performance in the range of pH values 1-6 where this ratio is between 90-95 %. Ex- traction performance of the mixture dithi- zone/butylacetate is much lower and its highest value (about 10 %) is reached within pH range 0-1.5 (see Fig. 1, line 2). Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XII, Issue 3 – 2013 Igor KOBASA, Atom-absorption method of determination of selenium content in some raw materials and food, Food and Environment Safety, Volume XII, Issue 3 – 2013, pag. 233 - 239 236 Figure 3. Dependence of the Se extraction ratio (R) on pH for the system butylacetate sodium diethyl- dithiocarbamate (line 1) and dithizone/butylacetate (line 2). Therefore, one can conclude that the ex- traction mixture butylacetate/sodium di- ethyldithiocarbamate ensures very high extraction ratio of Se and should be used for this purpose. The Se- diethyldithiocarbamate chelate is well sol- uble in butylacetate and this facilitates bet- ter atomization of the element. The narrower pH range of the best Se ex- traction is 4-6 and our next experiments were performed within this range. A series of the model solutions was prepared and Se contents were ranged within the limits that can be expected for the real analytical systems. A model solution with some predefined Se content was mixed with the extraction mix- ture butylacetate/sodium diethyldithiocar- bamate with pH 4-6. After the extraction process completed, the organic mixture with extracted Se was analyzed at the at- om-absorption spectrophotometer and the dependence of its optical density on initial Se concentration is shown in Fig. 4 (line 1). This dependence is almost exactly linear, which proves correct selection of the work- ing concentrations range. A slope of the line is determined by the spectrophotome- ter’s sensitivity and in our case it seems quite sufficient for quantitative determina- tion of the selected concentrations of Se. It should be taken into account that many other inorganic elements would also be co- extracted from real experimental systems after their mineralization with strong inor- ganic acids. These side elements may sig- nificantly raise the noise level of the ana- lytical signal complicating reliability of Se determination in the real systems. Our next experiments were aimed onto in- vestigation of relevance of the Se determi- nation in more ‘noisy’ systems containing Se on the background of some side ele- ment(s). A series of biological samples were miner- alized and then some known amounts of selenite were added to the probes. All natu- ral samples contain some background amounts of Se and this admixture method has to show relevance of determination of small extra amounts of Se on the back- ground of its natural content in presence of the other elements. Results of this investi- gation are represented in Fig. 4 (line 2). Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XII, Issue 3 – 2013 Igor KOBASA, Atom-absorption method of determination of selenium content in some raw materials and food, Food and Environment Safety, Volume XII, Issue 3 – 2013, pag. 233 - 239 237 0 200 400 600 800 1000 1200 0 5 10 15 20 C, mg/dm3 Li gh t a bs or pt io n, c .u . 1 2 Figure 4. Dependence of light absorption on Se concentration in the initial model solution for extraction mixture butylacetate/sodium diethyldithiocarbamate. Line 1 – a mixture prepared with the pure deionized water; line 2 – a mixture prepared from a real biological sample with some admixture of extra selenite. It is seen that the line 2 (‘noisy’ samples taken from the biological objects) is simi- lar to the pure one (line 1) but has slightly lesser slope. This effect can be caused by influence of the ‘matrix’ of other elements present in the natural objects and some slackening of the wanted signal by this ma- trix. It was found that the bottom limit of this method is 0.2 mg/kg Se in the organic ex- tract. According to peculiarities of the method of the samples preparation, this concentration corresponds to the content of Se in the source biological objects or food 0.04 mg per 1 kg dry weight. This sensitiv- ity is about one order of magnitude below background and maximum permissible contents of Se in these objects. Therefore, this analytical method can be recommend- ed for analytical determination of Se in food and other biological objects. As reported in [12], ions of iron, nickel, magnesium and zinc can also be intensive- ly co-extracted by the mixture bu- tylacetate/sodium diethyldithiocarbamate in the weakly acid medium. Many biologi- cal objects can release significant amounts of these ions so, it is essential to investi- gate possible overlapping and shadowing of the wanted signal because of presence of these ions. 950 955 960 965 970 975 980 985 990 995 0 2 4 6 8 10 C, mg/dm3 Li gh t a bs or pt io n, c .u . Ряд1 Ряд2 Figure 5. Influence of iron (line 1) and zinc (line 2) on light absorption of the test solution with the con- stant concentration of Se. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XII, Issue 3 – 2013 Igor KOBASA, Atom-absorption method of determination of selenium content in some raw materials and food, Food and Environment Safety, Volume XII, Issue 3 – 2013, pag. 233 - 239 238 Influence of ions of iron and zinc on the wanted signal value at the given (un- changed) concentration of Se is represent- ed in Fig. 5. We ranged contents of the both interfering elements from 0 to 6 g/kg (Zn) and 10 g/kg (Fe). It should be under- stood that such concentrations are about 100 times higher than the concentrations found usually in any real extractions taken from the biological objects. It can be seen that despite some decrease of the analytical signal it remains quite strong and easily detectable. Maximum slackening of the wanted signal does not exceed 1-2 % of its value. Such high selectivity is caused by absence of any characteristic lines close to 196.1 nm (Se characteristic line of light absorp- tion) in the spectra of Fe and Zn. The spec- trophotometer is equipped with precision monochromator that allow to distinguish the lines with accuracy up to 0.4 nm. Then the calibration line (Fig. 4, line 2) was employed to recalculate results of de- termination of Se content in a series of the soil samples taken from the grass cover in a park located in Chernivtsi, Ukraine. In order to assess relevance and accuracy of the method each sample has been analyzed in a row of 5 consequent tests. Results of the Se content determination are shown in Table 1. As seen, statistical relevance of the atom absorption determination of Se content is quite high. Relative error of the method is 1.26-4.47 %, statistical dispersion of the results is ranged from 1.7∙10-4 to 3.68∙10-3 within the confidence interval 0.0334 - 0.0697. Table 1 Results of the atom absorption determination of Se content in the soil samples Added selenium. mg/kg Determined selenium. mg/kg Relative error. % Statistical dis- persion Average* experi- mental content of Se (mg/kg) 0.40 0.395 0.418 0.408 0.384 0.398 1.4 1.678∙10-4 0.4006 ± 0.0334 0.80 0.792 0.792 0.808 0.812 0.806 4.47 9.79∙10-4 0.802 ± 0.0359 1.20 1.26 1.14 1.10 1.14 1.18 2.33 3.68∙10-3 1.164 ± 0.0697 1.60 1.64 1.58 1.48 1.48 1.52 1.39 2.325∙10-3 1.54 ± 0.054 2.0 2.0 1.92 1.86 1.92 1.88 1.26 2.88∙10-3 1.916 ± 0.062 * _ x ± n St  Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XII, Issue 3 – 2013 Igor KOBASA, Atom-absorption method of determination of selenium content in some raw materials and food, Food and Environment Safety, Volume XII, Issue 3 – 2013, pag. 233 - 239 239 4. Conclusion It can be concluded that the method of the atom absorption determination of Se with preliminary extraction by the bu- tylacetate/sodium diethyldithiocarbamate mixture proves high relevance and accura- cy and can be recommended for monitor- ing of content of this metal in food and other biological objects. This method is characterized by high selectivity and rela- tively low errors even for the ‘noisy’ sam- ples containing some side elements. 5. References [1]. NAZARENKO I.I. and KISLOVA I.V. Determination of various forms of Se in water. Lab and technological investigations and con- centrating of mineral raw materials. 6. 1-8. (1977). [2]. NAZARENKO I.I. and KISLOVA I.V. A highly sensitive method of determination of the migrating forms of selenium in natural water. J. Anal. Chem.. 32. 1857-1859. (1978). [3]. WATKINSON J.H.. Fluorometric deter- mination of selenium in biological material with 2. 3 - diaminonaphthalene. Anal. Chem.. 38. 92- 97. (1966). [4]. BAYFIELD R.F.. ROMALIS L.F.. pH control in the fluorometric assay for selenium with 2.3-diaminonaphthalene. Anal. Biochem.. 144. 569-576. (1985). [5]. LEBEDEV P.A and LEBEDEV A.A.. A modification of the spectral fluorimetric method of determination of selenium content in blood. J. Chem. and Pharm.. 30. 54-55. (1996). [6]. GOLUBKINA N.A.. et al.. Determination of selenium in wheat flour from various regions of USSR. Nutrition problems.. 59. 64-66. (1990). [7]. PILIPENKO A. and SAMCHUK A.. Ex- traction-atom-absorption analysis of natural ob- jects. J. Anal. Chem.. 61. 957-961. (1989). [8]. GINDIN L. Extraction process and their usage.. Nauka. Moscow. 481 p. (1984). [9]. GRANDZHAN A.. KUCHUK A. and CHARYKOV A.. Extraction-atom-absorption determination of heavy metals. J. Anal. Chem.. 63. 711-717. (1991). [10]. ZOLOTOV Yu. and KUZMIN N.. Extrac- tion concentrating.. Khimiya. Moscow. 320 p. (1971). [11]. BILOGOLOVKA V. T. and KOBASA I. M.. Atom-absorption analysis of heavy metals content in raw materials. food and environmen- tal objects.. Chernivtsi University. Chernivtsi. 108 p. (2010). [12]. ZOLOTOV Yu. Extraction in inorganic analysis.. Moscow University. Moscow. 18-21. (1988).