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298
Analyst, April, 1976, Vol. 101, pp. 298-305
The Gas-chromatographic Determination of Selenium(V1) and Total Selenium in Milk, Milk Products and Albumin with 1,2- Diamino-4-nitrobenzene Yasuaki Shimoishi” De9artment of Chemistry, Faculty of Scielzce, Okayama University, Tsushima, Okayama-shi 700, Japan
Selenium(1V) reacts with lJ2-diarnino-4-nitrobenzene to form 6-nitropia-zselenol, which is detected by means of a gas chromatograph equipped with an electron-capture detector. Trace amounts of selenium(V1) and total selenium in milk, milk products and albumin were determined by this method with a practical detection limit of 0.005 pg. The amount of selenium(V1) found in milk was about 60% of the total selenium content.
Selenium is a substance that is toxic to animals, and its toxicity is several times greater than that of arsenic compounds. It is well known that selenium has been identified as the toxic substance in grains and forages that causes “alkali disease” and “blind staggers” in livestock,l and this finding has focused attention on the biological properties of selenium compounds. In recent years, it has also been shown that selenium is an essential micronutrient for animals,2 and that several important livestock diseases are in fact selenium deficiency syndromes. Schwarz and Foltz3 described the synergistic role of selenium with vitamin E. In order to study the biological properties of selenium, rapid, sensitive and accurate methods for its determination are needed at both toxicological and nutritional levels. Trace amounts of selenium are determined spectrophotometrically with 3,3’-diaminobenzidine,4-* fluorimetrically with 3,3’-diamin~benzidine~ and 2 ,3-naphthalenediamine8,9and by neutronactivation anafysis.1° The two reagents used in the fluorimetric method are unstable and give high blank readings, which should be avoided. The disadvantage with the neutron-activation method is that it frequently requires long periods of time for the decay of radioactive byproducts to occur. Young and Christianll determined the selenium in human serum and plasma by gas chromatography using 2,3-naphthalenediamine. No investigations for the presence of selenium(V1)have been reported. The author has recently proposed a much more sensitive method for determining selenium in sulphuric acid,12 telluriurn,l3 sea water,14 plant materials,15 copper metal and copper salts16 by means of a gas chromatograph equipped with an electron-capture detector, using lJ2-diamino-4-nitrobenzene(4-nitr0-o-phenylenediamine)~~J~ in order to concentrate the selenium. While selenium(V1) is known to occur in biological materials,lS no methods for its determination have been published. Only the total selenium content has been determined. This paper describes a gas-chromatographic method for the determination of selenium - toluene extraction procedure that is specific for selenusing a 1,2-diamin0-4-nitrobenzene ium(1V). Organoselenium and selenite are determined in a nitric acid digest of the sample, and total selenium is determined after further treatment of the digest with hydrochloric acid in order to convert selenium(V1) into selenium(1V). The difference between the two values obtained gives the content of selenium(V1). The percentage of selenium(V1)in milk is about 60% of the selenium content.
Experimental Reagents All of the reagents used were of analytical-reagent grade. 1,2-Diamino-4-nitrobenzene dihydrochloride solution, 1%. The pure dihydrochloride was was dissolved in about prepared as follows. The commercial lJ2-diamino-4-nitrobenzene
* Present address : School for X-Ray Technicians, Faculty of Medicine, Okayama University, Shikatacho, Okayama-shi, 700.
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299
1 M hydrochloric acid, activated charcoal was added and the mixture was filtered. The salt was precipitated by addition of concentrated hydrochloric acid in a volume equal to that of the filtrate. One gram of the salt was dissolved in 100 ml of 1 M hydrochloric acid. This solution can be kept for at least 1 week. Selenium(1V) stock solution, 0.97 mg ml-l of%. Selenium(1V)oxide (704 mg) was dissolved in 500 ml of distilled water ;the concentration of the solution was determined gravimetrically.l* Working solutions were prepared by diluting the stock solution, which was stable for at least 6 months. Selenium(V1) stock solution, 1.0 mg ml-l of Se. About 230 mg of selenic acid monohydrate were dissolved in 100 ml of distilled water, the concentration of the solution being determined by gas chromatography.20 Working solutions were prepared by diluting the stock solution. Extraction solvent. Toluene was used without further purification.
Apparatus A Shimadzu, Model GC-SAE, gas chromatograph equipped with an electron-capture detector was used. A glass column (1m long and 4 mm i.d.) was packed with 15% SE-30 on 60-80-mesh Chromosorb W. The column and the detector temperature were maintained at 200 "C. The nitrogen flow-rate was 27 ml min-l. A Shimadzu, Model 250A, recorder was used at a chart speed of 5mmmin-l. Recommended Procedure (I)For total selenium content One to two millilitres of liquid sample (40-500 mg of solid sample, accurately weighed) are transferred by pipette into a 100-ml conical flask; 10 ml of concentrated nitric acid are added, and the mixture is heated slowly on a sand-bath (140-150 "C) with the base of the conical flask inserted about 5 m m below the surface of the sand. (This amount of concentrated nitric acid is sufficient for up to 1 g of solid sample.) A vigorous oxidation reaction quickly occurs, and a copious brown vapour is evolved. After a few minutes, the main reaction comes to an end and the resulting clear solution is allowed to stand for 1 h. The digest is then strongly heated, by inserting the conical flask into the bottom of the sand-bath, until the volume is reduced to about 0.2-0.3 ml. If any charring has occurred, erroneous results may be obtained. Its occurrence can, however, be prevented by shaking the flask frequently. The digestion is usually completed by heating the mixture for about 2 h ; 3 ml of 1 M urea solution are then added and the mixture is heated on a sand-bath for 10min in order to decompose the oxides of nitrogen. By using this procedure, organic matter is decomposed and selenium is oxidised to the quadrivalent state, but selenium(V1) remains unchanged. Selenium(VI), however, must be reduced to the quadrivalent state, by adding 5 ml of concentrated hydrochloric acid and heating the mixture on a sand-bath for 10 min at about 100 "C. After cooling it, the solution is diluted with 10 ml of distilled water. The contents of the flask are transferred into a 100-ml separating funnel and the flask is rinsed with 10 ml of distilled water, the rinsings being transferred into the funnel; 5 ml of toluene are added, and the toluene-soluble materials extracted by vigorously shaking the mixture for 5 min, thus minimising interferences in the gas-chromatographic determination. After allowing the mixture to stand for 2 h, the aqueous phase is transferred into another solution are added, and 100-ml separating funnel; 2 mi of a 1% 1,2-diamino-4-nitrobenzene the solution is allowed to stand for 2 h. The 5-nitropiazselenol formed is then extracted by shaking the solution for 5 min with 1 ml of toluene on a mechanical shaker; the toluene extract is washed, first with 4 ml of 1 M sodium hydroxide solution and subsequently with 2 ml of 7.5 M hydrochloric acid, by shaking each mixture for 1 min. Two microlitres of the toluene extract are then injected into the gas chromatograph and the peak height is measured.
(11) For organoselenium and selenite, excluding selenate The procedure for the digestion of the sample is the same as that used in procedure I. After decomposing the oxides of nitrogen with urea, the contents of the flask must be cooled to room temperature, The solution is then diluted with 15 ml of distilled water and 5 ml
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Analyst, VoZ. 101 of concentrated hydrochloric acid are added to give the required pH. If the temperature of the solution is too high, a large proportion of the selenate may be reduced to selenite. Therefore, special care has to be exercised in order to prevent this reduction from occurring, The contents of the flask are then transferred into a 100-ml separating funnel, the toluene-soluble material is removed as before and the procedure completed as described for procedure I.
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SHIMOISHI : GAS-CHROMATOGRAPHIC DETERMINATION OF
Results and Discussion Digestion of the Biological Materials Biological materials are usually decomposed by an oxygen-flask combustion method21 or b y wet digestion. With the former method the amount of sample that can be taken for analysis is limited and it is difficult to apply this method to routine analysis. A mixture of nitric and sulphuric acids or nitric and perchloric acids is widely used in the wet-digestion method. A considerable loss of selenium has been reported by Gorsuch22when using the former mixture, and it has been our experience that selenium cannot be recovered completely by digesting with nitric acid in the presence of a large amount of SO,2- ions. Gorsuch, however, obtained a 100% recovery of selenium when using nitric and perchloric acids. I n order to hasten the digestion, a catalyst such as d i ~ h r o m a t evanadate24 ,~~ or molybdatellJ5 was used, but vigorous bumping was observed during the reaction. When the sample was digested with a mixture of nitric and perchloric acids, measurement of the peak height was made difficult by overlapping peaks of unknown origin in the cliromatogram. Although hydrogen peroxide is a familiar oxidising agent for biological materials, no advantage could be expected in its use. As fuming nitric acid reduced a small amount of selenate to selenite, this acid was not suitable for the digestion. When concentrated nitric acid alone was used for the digestion, the peak height of selenium in the sample was proportional to the sample mass and the graph obtained passed through the origin. Also, the amounts of selenium(1V) added were recovered completely. Concentrated nitric acid was therefore used for the digestion of the biological material. The effect of varying the amount of concentrated nitric acid used was studied on dried skim milk with and without the addition of 0.019 4 pg of selenium(1V). Fig. 1 shows that a minimum of 8 ml of concentrated nitric acid was required to digest 0.5 g of dried skim milk and a good recovery was obtained. Consequently, 10ml of concentrated nitric acid were used for the following experiments. The digestion temperature was examined for the same sample a t 140-150, 210-220 and 270-280 "C (sand-bath temperature). At temperatures higher than 210-220 "C, the digestion was not quantitative as the nitric acid had evaporated before the digestion was completed. The sample was therefore digested at 140-150 "C. At this temperature, the sample had to be digested for more than 2 h for complete decomposition to occur. Although the residual nitric acid did not affect the determination, the
5
10
15
Volume of concentrated HNO,/ml
Fig. 1. Effect of the volume of concentrated nitric acid: A, 0.5 g of dried skim milk 0.019 4 pg of selenium(V1); and B, 0.5 g of dried skim milk.
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April, 19% S E L E N I U M I N M I L K WITH 1 ,2-DIAMINO-4-NITROBENZENE 301 heating was continued a t this temperature until the volume of the digestion liquid was reduced to 0.2-0.3 ml; the smaller the volume of liquid that remains, the more satisfactory is the result. In the digestion procedure, small amounts of toluene-soluble materials and fats remained, which should be removed before gas chromatography. Although most of the interfering substances were eliminated by the extraction with 5 ml of toluene, the very small amounts that were not removed affected the determination. Thus, the 1ml of toluene that is used to extract the 5-nitropiazselenol formed should be washed again with 4 ml of 1 M sodium hydroxide solution and then with 2 ml of 7.5 M hydrochloric acid. Fig. 2 shows the gas chromatogram obtained by the use of the above procedure. B
A
I
I LO
A 4
-
8
2 12
L 16
21
0
U 2
4
6
%
T im e/m in Fig. 2. Determination of selenium in 0.5 g of dried skim milk: A, total selenium (recommended procedure I) ; and B, organoselenium and selenite (recommended procedure IT). (a), 5-Nitropiazselenol.
Selenium(V1)in Biological Samples It is known that a considerable amount of selenate is contained in biological materials. However, the object of all previous methods of determination was to determine the total selenium content in these materials; with these methods little attention was paid to selenium(V1). 1,2-Diamin0-4-nitrobenzenereacts only with selenium(1V). If, therefore, selenium(V1) co-exists in the sample, it cannot be determined without being reduced to the quadrivalent state. Selenium(V1) can readily be converted by hydrochloric acid into the lower state, but prolonged reduction may be responsible for the loss of selenium by formation of volatile compounds such as SeO.Cl, and Se02.2HC1. Consequently, the effect of the amount of the concentrated hydrochloric acid and the reduction time were studied; the results obtained are shown in Figs. 3 and 4. As shown in Fig. 3, use of more than 4 mi of concentrated hydrochloric acid could reduce the selenium(V1) quantitatively to the quadrivalent state a t about 100 "C. Taking this and the pH required for the extraction into account, 5 m l of hydrochloric acid were considered to be a suitable volume for the reduction. Also, curves A and B in Fig. 3 crossed
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SHIMOISHI : GAS-CHROMATOGRAPHIC DETERMINATION OF
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Analyst, Val. 101
a t the same point on the ordinate, and the intersection of curve C on the ordinate coincided with the blank reading. These findings showed that no reduction of selenium(V1) occurred during the digestion procedure in the absence of hydrochloric acid. As shown in Fig. 4, by heating for 10 min with the addition of 5 ml of hydrochloric acid the reduction was quantitative, and no loss of selenium was found to occur during this procedure. loo
100
%
1
-4
/--
0
1.0
u 2.0
% ,
3.0
4.0
5.0
I
U
6.0
I
I
10
5
I
I 25
I
20
15
Reduction time/min
Volume of concentrated HCl/mi
Fig. 3. Effect of the volume of concentrated hydrochloric acid: A, 0.5 g of dried skim milk 0.020 p g of selenium(V1); B, 0.5 g of dried skim milk; and C, 0.020 pg of selenium(V1).
Fig. 4. Effect of reduction time [0.5 g of dried skim milk 0.020 pg of selenium(VI)].
+
+
Accuracy and Precision The accuracy of the procedure was evaluated by recovery experiments , the recovery being established by digesting dried skim milk to which a known amount of both selenium(1V) and selenium(V1) were added and treating the samples as described above. In all instances, theoretical recovery was obtained within experimental error (Table I). The precision of the method was evaluated by using dried skim milk and the results obtained are shown in Table 11. TABLE
RECOVERY OF Dried skim milk/g 0.502 0.502 0.502 0.501 0.502 0.503 0.500 0.502 0.502 0.503 0.502
Added Added selenium(IV)/ p g selenium(V1)/ p g None None 0.019 4 0.019 4 0.019 4 0.019 4 0.019 4 0.020 0.020 0.020 0.020 0.020
-
1
ADDED SELENIUM
(PROCEDURE
Selenium found/pg 0.050 7 0.070 6 0.069 6 0.068 8 0.071 3 0.070 7 0.071 3 0.070 4 0.071 3 0.071 7 0.071 3
I)
Selenium recovered /,US
0.019 9 0.018 9 0.018 2 0.020 6 0.019 9 0.020 8 0.019 7 0.020 6 0.020 9 0.020 6
Recovery,
%
103 97 94 106 103 104 99 103 105 103
Preparation of Calibration Graph The calibration graph was constructed with known amounts of selenous acid in solution in hydrochloric acid. The graph obtained was coincident with that obtained with the same solutions by procedure I. Therefore, the calibration graph can be prepared from aqueous standard selenium(1V) solutions. A known amount of each selenium standard solution is added to about 30 ml of 1 M hydrochloric acid that had previously been washed with pure toluene. After the addition of 2 ml of 1 yo 1,2-diamino-4-nitrobenzeneJ the reaction mixture is allowed to stand for 2 h. The complex formed is extracted into toluene and 2 p1 of the toluene extract are then injected into the gas chromatograph after washing with 2 m l of 7.5 M hydrochloric acid.
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April, 1976
SELENIUM I N MILK WITH
1 ,%DIAMIN0-4-NITROBENZENE
303
TABLEI1 REPRODUCIBILITY OF SELENIUM DETERMINATION DRIED SKIM MILK (PROCEDURE
Dried skim milk/g 0.408 0.496 0.503 0.502 0.503 0.501 0.502 0.502 0.503 0.502 Mean .. .. Standard deviation Coefficient of variation,
.. .. % ..
Peak height/ arbitrary units 57.1 57.7 55.7 57.5 58.5 58.8 56.7 55.6 57.5 57.0
.. ..
..
..
..
IN
I)
Selenium determined/pg g-' 0.101 0.103 0.098 0.101 0.102 0.103 0.100 0.098 0.100 0.101 .. 0.101
.. -J=o.ooa .. 1.7
..
Calibration graphs for two sensitivity settings of the gas chromatograph are shown in Fig. 5. The detector response was linear over the range 0-0.04 pg of selenium. The smallest amount of selenium actually injected into the gas chromatograph was only 0.000 01 pg.
0
0.Q2
0.04
0.06
0.08
Se Ien iu m/p g
Fig. 5. Calibration graphs for selenium. Sensitivity setting: A, 16 x 0.01 V ; and I3, 32 x 0.01 V. 8, Selenium(1V) in about 1 M hydrochloric acid (no digestion); and 0,recommended procedure I.
Determination of Selenium in Milk, Milk Products and Albumin The determination of both total selenium and selenium(V1) was carried out on milk, milk products and albumin by the recommended procedure. To these samples, a known amount (0.809 7-0.019 4 pg) of quadrivalent selenium was added and the recovery was examined. In all instances the results were satisfactory within experimental error. The selenium(V1) in these samples was determined as follows. Firstly, the total selenium contained in these samples was determined by procedure I. Secondly, the organoselenium and selenite were determined by procedure 11. The amount of selenium(V1) was obtained by subtracting the value for the latter from that for the former. The amount of selenium(V1) in milk was about 60% of the total selenium content. The results obtained are shown in Tables I11 and IV. Included in the latter table are results for L-cystine and L-methionine as it has been reported that the combined use of selenium and cystine could decrease the toxicity of mercury. The state of oxidation and content of selenium in albumin, cystine and methionine are therefore of fundamental importance in a biological study of the role of selenium.
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SHIMOISHI : GAS-CHROMATOGRAPHIC DETERMINATION O F
Analyst, Vol. 101
TABLE I11 SELENIUM CONTENT Material Milk (human) . . Milk (human) . . Milk (human) . . Raw milk .. Raw milk .. Raw milk .. Bottled milk . . Processed milk Condensed milk
.. ..
.. .. ..
.. .. .. ..
Sample takenlml 2.0 1.o 1.o 1.0 1.0 1.0 1.0 1.0 0.50
OF MILK
Selenium(VI)*/ pug ml-l 0.003
-
0.011 0.012 0.017 0.014 0.009 0.012 0.058
Total selenium?/ p g ml-l 0.010 f 0.000 4 0.083 & 0.004 0.039 f 0.004 0.021 f 0.002 0.027 f 0.002 0.022 f 0.002 0.019 f 0.002 0.033 f 0.004 0.089 f 0.004
Selenium(VI), 30
y'
-
28 57 63 64 47 36 65
* Results are the means of two determinations. t Results are the means of five determinations. Morris and Lavender26 determined the selenium content in various biological materials by a fluorimetric method using 2,3-naphthalenediamine. The concentrations of selenium found were 0.01, 0.10 and 0.09 p.p.m. in raw milk, dried skim milk and cheese, respectively. These values compared well with the mean values of 0.023, 0.10 and 0.07 p.p.m., respectively, obtained for similar samples by our method.
TABLE IV SELENIUM CONTENT Material Dried skim milk .. Milk powder (baby food) . . .. Instant cream powder Cheese . . .. .. Cheese . . .. .. Albumin, human (fraction V) . . Albumin, egg . . .. Albumin, egg, crystalline . . .. Albumin, bovine (fraction V) . . .. L-Cystine . , .. L-Methionine . . ..
OF MILK PRODUCTS AND ALBUMIN
Mass of sample taken/g 0.5
Selenium(V1)*/ Pg g-l 0.058
0.2-0.4 0.1-0.2 0.1 0.1
0.064 N.D. 0.008 0.034
0.227 0.235 0.050 0.087
0.05-0.1 0.05
3.48 4.37
4.77 f 0.21 5.85 0.24
73 75
0.04
6.84
5.64 f 0.37
79
0.2 0.05 0.5
0.106 N.D. 0.010
0.226 f 0.007 5.87 f- 0.26 0.010 f 0.001
..
Total seleniumt/ Pg g-l 0.101 f 0.002
f 0.017 f 0.010 f 0.002 & 0.008
Selenium(VI), % 67
28 0 16 39
47 0 100
* Results are the means of two determinations. t Results are the means of five determinations. N.D. denotes not detected.
Conclusions By using the proposed methods, trace amounts of selenium in milk, milk products and albumin have been determined. The procedures recommended are simple, rapid, selective and very sensitive for selenium. The detection limit is 0.005 ,ug of selenium. The author thanks Professor Kyoji T8ei for his encouragement and helpful discussion throughout this work.
References 1. Franke, K. W., and Painter, E. P., J . Nutr., 1935, 10, 599. 2. Thompson, J. N., and Scott, M. L., J . Nutr., 1969, 97,335. 3. Schwarz, K.. and Foltz, C. M., J . A m . Chem. Soc., 1957, 79, 3292. 4. Hoste, J., Analytica Chim. Acta, 1948, 2, 402. 5. Cheng, K. L., Analyt. Chem., 1956, 28, 1738.
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April, 1976
SELENIUM IN MILK WITH 1,2-DIAMINO-4-NITROBENZENE
305
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Parker, C. A., and Harvey, L. G., Analyst, 1961, 86, 54. Watkinson, J. H., Analyt. Chem., 1960, 32, 981. Lott, P. F., Cukor, P., Morber, G., and Solga, J., Analyt. Chem., 1963, 35, 1159. Watkinson, J. H., Analyt. Chem., 1966, 38, 92. Rook, H. L., Analyt. Chem., 1972, 44, 1276. Young, J . W., and Christian, C. D., Analytica Chim. Acta, 1973, 65, 127. Shimoishi, Y., and Taei, K., Talanta, 1970, 17, 165. Shimoishi, Y., Bull. Chem. SOC.Japan, 1971, 44, 3370. Shimoishi, Y., Analytica Chim. Acta, 1973, 64, 465. Shimoishi, Y., Bull. Chem. SOC.Japan, 1974, 47, 997. Shimoishi, Y., Bull. Chem. SOC.Japan, 1975, 48, 2797. Tanaka, M., and Kawashima, T., Talanta, 1965, 12, 211. Zingaro, R. A., and Cooper, W. C., “Selenium,” Van Nostrand Reinhold, New York, Cincinnati, Toronto, London and Melbourne, 1974, pp. 568 and 680. 19. Erdey, L., “Gravimetric Analysis,” Part 2, Pergamon Press, Oxford, New York, Toronto, Sydney and Braunschweig, 1965. 20. Shimoishi, Y., Talanta, 1970, 17, 784. 21. Allaway, W. H., and Cary, E. E., Analyt. Chem., 1964, 36, 1359. 22. Gorsuch, T. T., Analyst, 1959, 84, 135. 23. Lindberg, P., Acta Vet. Scand., 1968, Supplement 23. 24. McNulty, J. S., Analyt. Chem., 1947, 19, 809. 25. Cummins, L. M., Martin, J . I., Maag, G. W., and Maag, D. D., Analyt. Chem., 1964, 36, 382. 26. Morris, V. C., and Lavender, 0. A., J . Nutr., 1970, 100, 1383. Received August 8th, 1975 Accepted October 16th, 1976
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298
Analyst, April, 1976, Vol. 101, pp. 298-305
The Gas-chromatographic Determination of Selenium(V1) and Total Selenium in Milk, Milk Products and Albumin with 1,2- Diamino-4-nitrobenzene Yasuaki Shimoishi” De9artment of Chemistry, Faculty of Scielzce, Okayama University, Tsushima, Okayama-shi 700, Japan
Selenium(1V) reacts with lJ2-diarnino-4-nitrobenzene to form 6-nitropia-zselenol, which is detected by means of a gas chromatograph equipped with an electron-capture detector. Trace amounts of selenium(V1) and total selenium in milk, milk products and albumin were determined by this method with a practical detection limit of 0.005 pg. The amount of selenium(V1) found in milk was about 60% of the total selenium content.
Selenium is a substance that is toxic to animals, and its toxicity is several times greater than that of arsenic compounds. It is well known that selenium has been identified as the toxic substance in grains and forages that causes “alkali disease” and “blind staggers” in livestock,l and this finding has focused attention on the biological properties of selenium compounds. In recent years, it has also been shown that selenium is an essential micronutrient for animals,2 and that several important livestock diseases are in fact selenium deficiency syndromes. Schwarz and Foltz3 described the synergistic role of selenium with vitamin E. In order to study the biological properties of selenium, rapid, sensitive and accurate methods for its determination are needed at both toxicological and nutritional levels. Trace amounts of selenium are determined spectrophotometrically with 3,3’-diaminobenzidine,4-* fluorimetrically with 3,3’-diamin~benzidine~ and 2 ,3-naphthalenediamine8,9and by neutronactivation anafysis.1° The two reagents used in the fluorimetric method are unstable and give high blank readings, which should be avoided. The disadvantage with the neutron-activation method is that it frequently requires long periods of time for the decay of radioactive byproducts to occur. Young and Christianll determined the selenium in human serum and plasma by gas chromatography using 2,3-naphthalenediamine. No investigations for the presence of selenium(V1)have been reported. The author has recently proposed a much more sensitive method for determining selenium in sulphuric acid,12 telluriurn,l3 sea water,14 plant materials,15 copper metal and copper salts16 by means of a gas chromatograph equipped with an electron-capture detector, using lJ2-diamino-4-nitrobenzene(4-nitr0-o-phenylenediamine)~~J~ in order to concentrate the selenium. While selenium(V1) is known to occur in biological materials,lS no methods for its determination have been published. Only the total selenium content has been determined. This paper describes a gas-chromatographic method for the determination of selenium - toluene extraction procedure that is specific for selenusing a 1,2-diamin0-4-nitrobenzene ium(1V). Organoselenium and selenite are determined in a nitric acid digest of the sample, and total selenium is determined after further treatment of the digest with hydrochloric acid in order to convert selenium(V1) into selenium(1V). The difference between the two values obtained gives the content of selenium(V1). The percentage of selenium(V1)in milk is about 60% of the selenium content.
Experimental Reagents All of the reagents used were of analytical-reagent grade. 1,2-Diamino-4-nitrobenzene dihydrochloride solution, 1%. The pure dihydrochloride was was dissolved in about prepared as follows. The commercial lJ2-diamino-4-nitrobenzene
* Present address : School for X-Ray Technicians, Faculty of Medicine, Okayama University, Shikatacho, Okayama-shi, 700.
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1 M hydrochloric acid, activated charcoal was added and the mixture was filtered. The salt was precipitated by addition of concentrated hydrochloric acid in a volume equal to that of the filtrate. One gram of the salt was dissolved in 100 ml of 1 M hydrochloric acid. This solution can be kept for at least 1 week. Selenium(1V) stock solution, 0.97 mg ml-l of%. Selenium(1V)oxide (704 mg) was dissolved in 500 ml of distilled water ;the concentration of the solution was determined gravimetrically.l* Working solutions were prepared by diluting the stock solution, which was stable for at least 6 months. Selenium(V1) stock solution, 1.0 mg ml-l of Se. About 230 mg of selenic acid monohydrate were dissolved in 100 ml of distilled water, the concentration of the solution being determined by gas chromatography.20 Working solutions were prepared by diluting the stock solution. Extraction solvent. Toluene was used without further purification.
Apparatus A Shimadzu, Model GC-SAE, gas chromatograph equipped with an electron-capture detector was used. A glass column (1m long and 4 mm i.d.) was packed with 15% SE-30 on 60-80-mesh Chromosorb W. The column and the detector temperature were maintained at 200 "C. The nitrogen flow-rate was 27 ml min-l. A Shimadzu, Model 250A, recorder was used at a chart speed of 5mmmin-l. Recommended Procedure (I)For total selenium content One to two millilitres of liquid sample (40-500 mg of solid sample, accurately weighed) are transferred by pipette into a 100-ml conical flask; 10 ml of concentrated nitric acid are added, and the mixture is heated slowly on a sand-bath (140-150 "C) with the base of the conical flask inserted about 5 m m below the surface of the sand. (This amount of concentrated nitric acid is sufficient for up to 1 g of solid sample.) A vigorous oxidation reaction quickly occurs, and a copious brown vapour is evolved. After a few minutes, the main reaction comes to an end and the resulting clear solution is allowed to stand for 1 h. The digest is then strongly heated, by inserting the conical flask into the bottom of the sand-bath, until the volume is reduced to about 0.2-0.3 ml. If any charring has occurred, erroneous results may be obtained. Its occurrence can, however, be prevented by shaking the flask frequently. The digestion is usually completed by heating the mixture for about 2 h ; 3 ml of 1 M urea solution are then added and the mixture is heated on a sand-bath for 10min in order to decompose the oxides of nitrogen. By using this procedure, organic matter is decomposed and selenium is oxidised to the quadrivalent state, but selenium(V1) remains unchanged. Selenium(VI), however, must be reduced to the quadrivalent state, by adding 5 ml of concentrated hydrochloric acid and heating the mixture on a sand-bath for 10 min at about 100 "C. After cooling it, the solution is diluted with 10 ml of distilled water. The contents of the flask are transferred into a 100-ml separating funnel and the flask is rinsed with 10 ml of distilled water, the rinsings being transferred into the funnel; 5 ml of toluene are added, and the toluene-soluble materials extracted by vigorously shaking the mixture for 5 min, thus minimising interferences in the gas-chromatographic determination. After allowing the mixture to stand for 2 h, the aqueous phase is transferred into another solution are added, and 100-ml separating funnel; 2 mi of a 1% 1,2-diamino-4-nitrobenzene the solution is allowed to stand for 2 h. The 5-nitropiazselenol formed is then extracted by shaking the solution for 5 min with 1 ml of toluene on a mechanical shaker; the toluene extract is washed, first with 4 ml of 1 M sodium hydroxide solution and subsequently with 2 ml of 7.5 M hydrochloric acid, by shaking each mixture for 1 min. Two microlitres of the toluene extract are then injected into the gas chromatograph and the peak height is measured.
(11) For organoselenium and selenite, excluding selenate The procedure for the digestion of the sample is the same as that used in procedure I. After decomposing the oxides of nitrogen with urea, the contents of the flask must be cooled to room temperature, The solution is then diluted with 15 ml of distilled water and 5 ml
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Analyst, VoZ. 101 of concentrated hydrochloric acid are added to give the required pH. If the temperature of the solution is too high, a large proportion of the selenate may be reduced to selenite. Therefore, special care has to be exercised in order to prevent this reduction from occurring, The contents of the flask are then transferred into a 100-ml separating funnel, the toluene-soluble material is removed as before and the procedure completed as described for procedure I.
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Results and Discussion Digestion of the Biological Materials Biological materials are usually decomposed by an oxygen-flask combustion method21 or b y wet digestion. With the former method the amount of sample that can be taken for analysis is limited and it is difficult to apply this method to routine analysis. A mixture of nitric and sulphuric acids or nitric and perchloric acids is widely used in the wet-digestion method. A considerable loss of selenium has been reported by Gorsuch22when using the former mixture, and it has been our experience that selenium cannot be recovered completely by digesting with nitric acid in the presence of a large amount of SO,2- ions. Gorsuch, however, obtained a 100% recovery of selenium when using nitric and perchloric acids. I n order to hasten the digestion, a catalyst such as d i ~ h r o m a t evanadate24 ,~~ or molybdatellJ5 was used, but vigorous bumping was observed during the reaction. When the sample was digested with a mixture of nitric and perchloric acids, measurement of the peak height was made difficult by overlapping peaks of unknown origin in the cliromatogram. Although hydrogen peroxide is a familiar oxidising agent for biological materials, no advantage could be expected in its use. As fuming nitric acid reduced a small amount of selenate to selenite, this acid was not suitable for the digestion. When concentrated nitric acid alone was used for the digestion, the peak height of selenium in the sample was proportional to the sample mass and the graph obtained passed through the origin. Also, the amounts of selenium(1V) added were recovered completely. Concentrated nitric acid was therefore used for the digestion of the biological material. The effect of varying the amount of concentrated nitric acid used was studied on dried skim milk with and without the addition of 0.019 4 pg of selenium(1V). Fig. 1 shows that a minimum of 8 ml of concentrated nitric acid was required to digest 0.5 g of dried skim milk and a good recovery was obtained. Consequently, 10ml of concentrated nitric acid were used for the following experiments. The digestion temperature was examined for the same sample a t 140-150, 210-220 and 270-280 "C (sand-bath temperature). At temperatures higher than 210-220 "C, the digestion was not quantitative as the nitric acid had evaporated before the digestion was completed. The sample was therefore digested at 140-150 "C. At this temperature, the sample had to be digested for more than 2 h for complete decomposition to occur. Although the residual nitric acid did not affect the determination, the
5
10
15
Volume of concentrated HNO,/ml
Fig. 1. Effect of the volume of concentrated nitric acid: A, 0.5 g of dried skim milk 0.019 4 pg of selenium(V1); and B, 0.5 g of dried skim milk.
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April, 19% S E L E N I U M I N M I L K WITH 1 ,2-DIAMINO-4-NITROBENZENE 301 heating was continued a t this temperature until the volume of the digestion liquid was reduced to 0.2-0.3 ml; the smaller the volume of liquid that remains, the more satisfactory is the result. In the digestion procedure, small amounts of toluene-soluble materials and fats remained, which should be removed before gas chromatography. Although most of the interfering substances were eliminated by the extraction with 5 ml of toluene, the very small amounts that were not removed affected the determination. Thus, the 1ml of toluene that is used to extract the 5-nitropiazselenol formed should be washed again with 4 ml of 1 M sodium hydroxide solution and then with 2 ml of 7.5 M hydrochloric acid. Fig. 2 shows the gas chromatogram obtained by the use of the above procedure. B
A
I
I LO
A 4
-
8
2 12
L 16
21
0
U 2
4
6
%
T im e/m in Fig. 2. Determination of selenium in 0.5 g of dried skim milk: A, total selenium (recommended procedure I) ; and B, organoselenium and selenite (recommended procedure IT). (a), 5-Nitropiazselenol.
Selenium(V1)in Biological Samples It is known that a considerable amount of selenate is contained in biological materials. However, the object of all previous methods of determination was to determine the total selenium content in these materials; with these methods little attention was paid to selenium(V1). 1,2-Diamin0-4-nitrobenzenereacts only with selenium(1V). If, therefore, selenium(V1) co-exists in the sample, it cannot be determined without being reduced to the quadrivalent state. Selenium(V1) can readily be converted by hydrochloric acid into the lower state, but prolonged reduction may be responsible for the loss of selenium by formation of volatile compounds such as SeO.Cl, and Se02.2HC1. Consequently, the effect of the amount of the concentrated hydrochloric acid and the reduction time were studied; the results obtained are shown in Figs. 3 and 4. As shown in Fig. 3, use of more than 4 mi of concentrated hydrochloric acid could reduce the selenium(V1) quantitatively to the quadrivalent state a t about 100 "C. Taking this and the pH required for the extraction into account, 5 m l of hydrochloric acid were considered to be a suitable volume for the reduction. Also, curves A and B in Fig. 3 crossed
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SHIMOISHI : GAS-CHROMATOGRAPHIC DETERMINATION OF
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Analyst, Val. 101
a t the same point on the ordinate, and the intersection of curve C on the ordinate coincided with the blank reading. These findings showed that no reduction of selenium(V1) occurred during the digestion procedure in the absence of hydrochloric acid. As shown in Fig. 4, by heating for 10 min with the addition of 5 ml of hydrochloric acid the reduction was quantitative, and no loss of selenium was found to occur during this procedure. loo
100
%
1
-4
/--
0
1.0
u 2.0
% ,
3.0
4.0
5.0
I
U
6.0
I
I
10
5
I
I 25
I
20
15
Reduction time/min
Volume of concentrated HCl/mi
Fig. 3. Effect of the volume of concentrated hydrochloric acid: A, 0.5 g of dried skim milk 0.020 p g of selenium(V1); B, 0.5 g of dried skim milk; and C, 0.020 pg of selenium(V1).
Fig. 4. Effect of reduction time [0.5 g of dried skim milk 0.020 pg of selenium(VI)].
+
+
Accuracy and Precision The accuracy of the procedure was evaluated by recovery experiments , the recovery being established by digesting dried skim milk to which a known amount of both selenium(1V) and selenium(V1) were added and treating the samples as described above. In all instances, theoretical recovery was obtained within experimental error (Table I). The precision of the method was evaluated by using dried skim milk and the results obtained are shown in Table 11. TABLE
RECOVERY OF Dried skim milk/g 0.502 0.502 0.502 0.501 0.502 0.503 0.500 0.502 0.502 0.503 0.502
Added Added selenium(IV)/ p g selenium(V1)/ p g None None 0.019 4 0.019 4 0.019 4 0.019 4 0.019 4 0.020 0.020 0.020 0.020 0.020
-
1
ADDED SELENIUM
(PROCEDURE
Selenium found/pg 0.050 7 0.070 6 0.069 6 0.068 8 0.071 3 0.070 7 0.071 3 0.070 4 0.071 3 0.071 7 0.071 3
I)
Selenium recovered /,US
0.019 9 0.018 9 0.018 2 0.020 6 0.019 9 0.020 8 0.019 7 0.020 6 0.020 9 0.020 6
Recovery,
%
103 97 94 106 103 104 99 103 105 103
Preparation of Calibration Graph The calibration graph was constructed with known amounts of selenous acid in solution in hydrochloric acid. The graph obtained was coincident with that obtained with the same solutions by procedure I. Therefore, the calibration graph can be prepared from aqueous standard selenium(1V) solutions. A known amount of each selenium standard solution is added to about 30 ml of 1 M hydrochloric acid that had previously been washed with pure toluene. After the addition of 2 ml of 1 yo 1,2-diamino-4-nitrobenzeneJ the reaction mixture is allowed to stand for 2 h. The complex formed is extracted into toluene and 2 p1 of the toluene extract are then injected into the gas chromatograph after washing with 2 m l of 7.5 M hydrochloric acid.
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SELENIUM I N MILK WITH
1 ,%DIAMIN0-4-NITROBENZENE
303
TABLEI1 REPRODUCIBILITY OF SELENIUM DETERMINATION DRIED SKIM MILK (PROCEDURE
Dried skim milk/g 0.408 0.496 0.503 0.502 0.503 0.501 0.502 0.502 0.503 0.502 Mean .. .. Standard deviation Coefficient of variation,
.. .. % ..
Peak height/ arbitrary units 57.1 57.7 55.7 57.5 58.5 58.8 56.7 55.6 57.5 57.0
.. ..
..
..
..
IN
I)
Selenium determined/pg g-' 0.101 0.103 0.098 0.101 0.102 0.103 0.100 0.098 0.100 0.101 .. 0.101
.. -J=o.ooa .. 1.7
..
Calibration graphs for two sensitivity settings of the gas chromatograph are shown in Fig. 5. The detector response was linear over the range 0-0.04 pg of selenium. The smallest amount of selenium actually injected into the gas chromatograph was only 0.000 01 pg.
0
0.Q2
0.04
0.06
0.08
Se Ien iu m/p g
Fig. 5. Calibration graphs for selenium. Sensitivity setting: A, 16 x 0.01 V ; and I3, 32 x 0.01 V. 8, Selenium(1V) in about 1 M hydrochloric acid (no digestion); and 0,recommended procedure I.
Determination of Selenium in Milk, Milk Products and Albumin The determination of both total selenium and selenium(V1) was carried out on milk, milk products and albumin by the recommended procedure. To these samples, a known amount (0.809 7-0.019 4 pg) of quadrivalent selenium was added and the recovery was examined. In all instances the results were satisfactory within experimental error. The selenium(V1) in these samples was determined as follows. Firstly, the total selenium contained in these samples was determined by procedure I. Secondly, the organoselenium and selenite were determined by procedure 11. The amount of selenium(V1) was obtained by subtracting the value for the latter from that for the former. The amount of selenium(V1) in milk was about 60% of the total selenium content. The results obtained are shown in Tables I11 and IV. Included in the latter table are results for L-cystine and L-methionine as it has been reported that the combined use of selenium and cystine could decrease the toxicity of mercury. The state of oxidation and content of selenium in albumin, cystine and methionine are therefore of fundamental importance in a biological study of the role of selenium.
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SHIMOISHI : GAS-CHROMATOGRAPHIC DETERMINATION O F
Analyst, Vol. 101
TABLE I11 SELENIUM CONTENT Material Milk (human) . . Milk (human) . . Milk (human) . . Raw milk .. Raw milk .. Raw milk .. Bottled milk . . Processed milk Condensed milk
.. ..
.. .. ..
.. .. .. ..
Sample takenlml 2.0 1.o 1.o 1.0 1.0 1.0 1.0 1.0 0.50
OF MILK
Selenium(VI)*/ pug ml-l 0.003
-
0.011 0.012 0.017 0.014 0.009 0.012 0.058
Total selenium?/ p g ml-l 0.010 f 0.000 4 0.083 & 0.004 0.039 f 0.004 0.021 f 0.002 0.027 f 0.002 0.022 f 0.002 0.019 f 0.002 0.033 f 0.004 0.089 f 0.004
Selenium(VI), 30
y'
-
28 57 63 64 47 36 65
* Results are the means of two determinations. t Results are the means of five determinations. Morris and Lavender26 determined the selenium content in various biological materials by a fluorimetric method using 2,3-naphthalenediamine. The concentrations of selenium found were 0.01, 0.10 and 0.09 p.p.m. in raw milk, dried skim milk and cheese, respectively. These values compared well with the mean values of 0.023, 0.10 and 0.07 p.p.m., respectively, obtained for similar samples by our method.
TABLE IV SELENIUM CONTENT Material Dried skim milk .. Milk powder (baby food) . . .. Instant cream powder Cheese . . .. .. Cheese . . .. .. Albumin, human (fraction V) . . Albumin, egg . . .. Albumin, egg, crystalline . . .. Albumin, bovine (fraction V) . . .. L-Cystine . , .. L-Methionine . . ..
OF MILK PRODUCTS AND ALBUMIN
Mass of sample taken/g 0.5
Selenium(V1)*/ Pg g-l 0.058
0.2-0.4 0.1-0.2 0.1 0.1
0.064 N.D. 0.008 0.034
0.227 0.235 0.050 0.087
0.05-0.1 0.05
3.48 4.37
4.77 f 0.21 5.85 0.24
73 75
0.04
6.84
5.64 f 0.37
79
0.2 0.05 0.5
0.106 N.D. 0.010
0.226 f 0.007 5.87 f- 0.26 0.010 f 0.001
..
Total seleniumt/ Pg g-l 0.101 f 0.002
f 0.017 f 0.010 f 0.002 & 0.008
Selenium(VI), % 67
28 0 16 39
47 0 100
* Results are the means of two determinations. t Results are the means of five determinations. N.D. denotes not detected.
Conclusions By using the proposed methods, trace amounts of selenium in milk, milk products and albumin have been determined. The procedures recommended are simple, rapid, selective and very sensitive for selenium. The detection limit is 0.005 ,ug of selenium. The author thanks Professor Kyoji T8ei for his encouragement and helpful discussion throughout this work.
References 1. Franke, K. W., and Painter, E. P., J . Nutr., 1935, 10, 599. 2. Thompson, J. N., and Scott, M. L., J . Nutr., 1969, 97,335. 3. Schwarz, K.. and Foltz, C. M., J . A m . Chem. Soc., 1957, 79, 3292. 4. Hoste, J., Analytica Chim. Acta, 1948, 2, 402. 5. Cheng, K. L., Analyt. Chem., 1956, 28, 1738.
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SELENIUM IN MILK WITH 1,2-DIAMINO-4-NITROBENZENE
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6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Parker, C. A., and Harvey, L. G., Analyst, 1961, 86, 54. Watkinson, J. H., Analyt. Chem., 1960, 32, 981. Lott, P. F., Cukor, P., Morber, G., and Solga, J., Analyt. Chem., 1963, 35, 1159. Watkinson, J. H., Analyt. Chem., 1966, 38, 92. Rook, H. L., Analyt. Chem., 1972, 44, 1276. Young, J . W., and Christian, C. D., Analytica Chim. Acta, 1973, 65, 127. Shimoishi, Y., and Taei, K., Talanta, 1970, 17, 165. Shimoishi, Y., Bull. Chem. SOC.Japan, 1971, 44, 3370. Shimoishi, Y., Analytica Chim. Acta, 1973, 64, 465. Shimoishi, Y., Bull. Chem. SOC.Japan, 1974, 47, 997. Shimoishi, Y., Bull. Chem. SOC.Japan, 1975, 48, 2797. Tanaka, M., and Kawashima, T., Talanta, 1965, 12, 211. Zingaro, R. A., and Cooper, W. C., “Selenium,” Van Nostrand Reinhold, New York, Cincinnati, Toronto, London and Melbourne, 1974, pp. 568 and 680. 19. Erdey, L., “Gravimetric Analysis,” Part 2, Pergamon Press, Oxford, New York, Toronto, Sydney and Braunschweig, 1965. 20. Shimoishi, Y., Talanta, 1970, 17, 784. 21. Allaway, W. H., and Cary, E. E., Analyt. Chem., 1964, 36, 1359. 22. Gorsuch, T. T., Analyst, 1959, 84, 135. 23. Lindberg, P., Acta Vet. Scand., 1968, Supplement 23. 24. McNulty, J. S., Analyt. Chem., 1947, 19, 809. 25. Cummins, L. M., Martin, J . I., Maag, G. W., and Maag, D. D., Analyt. Chem., 1964, 36, 382. 26. Morris, V. C., and Lavender, 0. A., J . Nutr., 1970, 100, 1383. Received August 8th, 1975 Accepted October 16th, 1976
