RESULTS AND DISCUSSION
Xntimony(V) is reduced to antimony (111) by oxalic acid a t 150’ C., and if hydrofluoric acid is added, any arsenic (111) is volatilized as arsenic trifluoride by heating ( 3 ) . To determine if two treatments are sufficient for quantitative reduction of antimony(V) and removal of arsenic(III), some 1.00-ml. aliquots of the antimony(V) solution were mixed with 1.00-ml. aliquots of the arsenic(II1) solution; and the samples were carried through the procedure. The results of five replicate determinations showed that the antimony(V) is quantitatively reduced and the removal of arsenic(II1) is complete. The procedure was tried on some glass samples that had been analyzed
for total antimony by other accepted methods. A comparison of these results is given in Table I. On the basis of the satisfactory agreement it was concluded that this method is valid for determining total antimony in a variety of glasses. The antimony(II1) values were established in some glass samples containing arsenic(II1) by doing three replicate determinations using the second and third procedures given in the experimental section. Other portions of the same glasses were spiked with known amounts of antimony(II1) after dissolving, and the antimony(II1) was determined as before. The results are given in Table 11, and the data show that satisfactory recovery is obtained.
ACKNOWLEDGMENT
The authors are grateful for the help of P. R. Segatto who kindly offered valuable suggestions. LITERATURE CITED
(1) Van Aman, R. E., Hollibaugh, F. D.,
Kanzelmeyer, J. H., ANAL.CHEM.31, 1783 (1959). ( 2 ) Willard, H. H., Diehl, H., “Advanced Quantitative Analysis,” ppl 348-9, Van Nostrand, New York, 1943. (3) Williams, J. P., Schwenkler, T. A., J . Am. Ceram. SOC.38, 367 (1955). (4) Wise, W. M., Williams, J. P., ANAL. CHEM.36, 19 (1964). W. M. WISE J. P. WILLIAMS Glass Research and Development Corning Glass Works Corning, N. Y .
Thin Layer Chromatography of Vitamin A and Related Compounds SIR: Although several methods (4, 6) employing column and paper chromatography are available for the separation of vitamin Ai and related compounds, none is entirely satisfactory for the rapid separation and identification of trace amounts of these substances. Thin layer chromatography has been applied to the separation and detection of fat-soluble vitamins in general (Z> 3 ) and also to the separation of a few geometric isomers of the vitamin .Iland .Izseries ( 5 ) . I n these studies, only mixtures of pure components were employed and no quantitative data were given for the recovery of the samples put on the chromatogram. We have, therefore, attempted to separate a group of vitamin A compounds often encountered in fish liver oils and in liquid multivitamin preparations. These compounds include @-carotene, anhydrovitamin A,, retrovitamin A,, vitamin dl ester, vitamin A, alcohol, vitamin Az alcohol, vitamin -1,aldehyde, vitamin X p aldehyde, and vitamin A, epoxide Methods of separation, identification, and quantitative recovery of these materials employing the thin layer chromatographic technique are described. This technique was also applied to eluates from column chromatography to study the purity of fractions. EXPERIMENTAL
Crystalline all-trans vitamin AI alcohol, vitamin AI acetate, vitamin AI aldehyde, vitamin .Al acid, and @-carotene were obtained from Distillation Products Industries, Ltd., Rochester, S . Y. Anhydrovitamin .Al [E2 1814, 2700, and 2330 a t 350, 367, and 390 mp, respectively (all absorbancy values were measured in petroleum ether)] was prepared from crystalline all-trans
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ANALYTICAL CHEMISTRY
vitamin AI alcohol by the reaction with p-toluenesulfonic acid ( 7 ) and purified by repeated chromatography on alumina. Crystalline all-trans vitamin -4, acetate was reacted with aq. HBr ( 1 ) and the reaction mixture was subjected to repeated chromatography on alumina to obtain retro-vitamin i l l acetate (E:,”, 1830 and 348 mp). Retro-vitamin AI alcohol was prepared by saponification of retro-vitamin X i acetate. From the nonsaponifiable fraction of a freshwater fiih liver oil, vitamin -A2 alcohol (E::, 790 a t 350 mp) was purified by chromatography on alumina. Vitamin .Az aldehyde ( E : z 64 a t 385 mp) was also separated from the same fish liver oil. Vitamin A, epoxide (A max. 270 mp; E::; 440 a t 270 mp) was purified from a deteriorated sample of crystalline all-trans vitamin hl alcohol by chromatography on alumina. A slurry was prepared by mixing aluminum oxide, G (neutral, for thin layer chromatography; 13rinkmann Instruments, Inc.), and water in the ratio 1 : 2 (w./v.) and applied to clean glass plates 2 X 71/2 inches or 7 ’ / 2 X 7 l / 2 inches with a glass rod. The plates were allowed to stand for 10 minutes a t room temperature and then placed in an oven at 100” C. for 30 minutes. Five micrograms of each of the compounds and 20 pg. of each of the mixtures, dissolved in petroleum ether or diethyl ether, were applied to the cool plates with a micropipet and the chromatograms developed with the appropriate solvent system. The solvent front was allowed to move 3 to 4 inches from the origin (requiring about 20 to 30 minutes) before the plates were removed from the chromatography chamber. The compounds were detected and characterized in the following manner : (1) characteristic fluorescence under UV light or color of the spot on the chromatogram; ( 2 ) mixed chromatography with standards or by compari-
son with standards on the same plate; (3) development of color when sprayed with Carr-Price reagent, and (4) elution of the compound, as described below, and measurement of the absorbance spectrum. The elution of the compounds from the plate was achieved by two methods. I n one, the alumina containing the spot was carefully scraped from the plate, transferred to a 10-ml. volumetric flask and shaken a few times with 20% (v./v.) diethyl ether in petroleum ether. The extract was decanted and the extraction repeated twice. The combined extract was evaporated to dryness under reduced pressure, the residue dissolved in petroleum ether, and the spectrum examined in a Beckman DL spectrophotometer. In the second method, the elution was carried out by the technique shown in Figure 1. Alumina on either side of the spot was uniformly scraped from the chromatogram with the flat end of a metal spatula at right angles to the direction of the solvent flow leaving a width of about 0.25 inch of the glass plate clear on either side of the spot. The plate was clamped on both ends and a solvent mixture consisting of 15% (v./v.) ethanol in cyclohexane was allowed to flow a t a slow rate from a separatory funnel along a v-shaped strip of filter paper attached to the tail of the funnel. The chromatogram was brought just behind the paper strip soaked with the solvent and the paper allowed to come into contact with the surface of the alumina. The paper was held firmly on the plate by surface tension. The elution was carried out with about 2 ml. of the solvent for every microgram of the compound spotted. Whenever the elution technique was employed for the spectroscopic characterization of the compounds, the quantities applied to the plates were sufficiently large so that the solution of the recovered material gave an absorbance of a t least
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z W 0
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FRONT V I E W
Figure 1. Apparatus for elution of compounds from thin layer chromatogram A. Tail of separatory funnel B. Glass strip attached to the tail of the funnel. C. Paper strip inserted between the glass strip, B, and the curved end of the separatory funnel. D. Thin layer chromatographic plate (arrow indicates the direction in which the solvent was run). E. Alumina-free area on the glass plate on either side of the spot
0.200 in a volume of 5 ml. With either method of elution, the recoveries were about 60 to 80%. Rechromatography of the solution of individual spots was carried out to ascerttiin that the substance was not altered in any way during chromatography. RESULTS AND DiSCUSSlON
I n Table I, the Rj values of vitamin A and 10 related compounds in different solvent systems are given. These values were obtained on the same chromatogram for individual solvent systems. They varied to the extent of +5y0 when different plates were employed. In Figure 2, the separation of components in the liver oil of a fresh-
Table I.
Rj
PLATE 2
PLATE 3
PLATE 4
Plates 1 and 2. Thin layer chromatograms of a fresh-water Figure 2. fish (pike) liver oil (solvent systems; S3 and S g (Table I), respectively) Plate 1: a, vitamins AI and A2 esters; b, vitamin A1 aldehyde; c, vitamin A2 aldehyde; d, unidentified mixture. Plate 2: a, mixture of vitamins AI and A2 esters and aldehydes; b, vitamin A1 alcohol; c, vitamin A2 alcohol; d, unidentified mixture
Plate 3. Thin: layer chromatogram of the nonsaponifiable fraction of the liver of a rat fed refro-vitamin A1 acetate (solvent system: S d ) a, retro-vitamin AI alcohol; b, vitamin A1 alcohol; c, unidentified mixture
Plate 4. Thin layer chromatogram of the nonsaponifiable fraction of a liquid multivitamin sample (solvent system: Sn) a, anhydrovitamin AI; b, retra-vitamin A1 alcohol; c, vitamin A, alcohol; d, unidentified campound; e, unidentified mixture
water fish, the nonsaponifiable fraction of the liver oil of a r a t fed retro-vitamin AI acetate, and the nonsaponifiable fraction of a liquid multivitamin preparation is shown. Thin layer chromatography takes less time than the column and paper chromatographic methods; this permits its use in routine analysis. The amount of material needed is small and the degree of separation remarkable considering the short time required for
Values of Vitamin A and Related Compounds in Different Solvent Systems
Solvent system and R, value (24~5%) Compound s1 S2 SI SI Sb S8 S? 0.63 0.90 0.97 Anhydrovitamin AI 0.06 0.80 p-Carotene 0.36 0.90 rstro-Vitamin A1 acetate 0.00 0.19 0.88 Vitamin AI acetate 0.00 0.66 Vitamin A1 aldehyde 0.59 1.00 Vitamin A2 aldehyde 0.12 0.36 0.42 retro-Vitamin AI alcohol 0.06 0.16 0.28 0.45 Vitamin A1 alcohol 0.03 0.12 0.32 Vitamin AI e oxide 0.00 0.08 0.26 0.28 0.58 Vitamin A2 aycohol 0.00 0.00 0.05 Vitamin A1 acid Solvent systems: SI = cyclohexane, SZ = 57, (v./v.) benzene in cyclohexane, S3 = 0.25YG (v./v.) methanol in cyclohexane, S4 = 17, (v./v.) methanol in cyclohexane, Sa = 37, (v./v.) methanol in cyclohexane, Ss = 3y0 (v./v.) ethanol in cyclohexane, S, = 87, (v./v.) ethanol in cyclohexane.
development of the chromatogram. Reference standards and several mixtures can be run side by side as in paper chromatography. The method may prove useful in detecting radioactive impurities in labeled preparations and in characterizing metabolites when isotopic methods are employed. LITERATURE CITED
(1) Beutel, R. H., Hinkley, 1). F., Pollak, P. I., J . Am. Cheni. SOC.77,5166 (1955).
(2) . . Blattna, J., Davidek,. J.,. Ezperientia . 17, 474 ( M i ) . (3) Davidek, J., Blattna, J., J . Chromatog. 7. 204 (1962). (4) ’Jungalwala, F. B., Cama, H. R., J . Chromatog. 8, 535 (1962). (5) Planta, C. V.,Schweiter, U., Choparddit-Jean, L., Ruegg, R., Kofler, M., Isler. 0.. Helv. Chim. Acta 45, 548 (1962). ’ (6) Porter, J. W., Anderson, D. G., in E. Heftman, “Chromatography,” p. 465, Reinhold, Kew York, 1961. ( 7 ) Shantz, E. M., J . Riol. Chem. 182, 515 (1950). T. N. R. VARMA’ THAVIL PANALAKS T. K. h l U R R A Y Vitamins and Nutrition Section Food and Drug Laboratories Department of Xational Health and Welfare Ottawa, Canada Xational Research Council of Canada postdoctoral fellow. VOL. 3 6 , N O . 9, AUGUST 1 9 6 4
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