174
.
ANALYTICAL CHEMISTRY
ular weight or polysaccharides have not been tested with the new reagent. The prediction is offered that the reagent will react satisfactorily with these compounds if ( a ) they are readily hydrolyzed to hexose units during the period of color development and ( b ) the high concentration of sulfuric acid used in the reaction will not cause too extensive decomposition of the hydrolysis products to compounds other than 5-hydroxymethylfurfural. By this mechanism, pentoses (which form furfural on acid degradation) cannot give formaldehyde, as indeed they do not. Although furfural does react with chromotropic acid in 10 M sulfuric acid a yellow color is formed ( 7 )and its absorption spectrum is markedly different and does not display a sharply inflected maximum a t 570 mp. Hence hexoses may be determined in the presence of pentose. Of some interest in this present application is the fact that the reagent is specific for hexoses and will not be affected by the ‘‘saccharoids” ordinarily present in blood. These saccharoids would interefere with the determination of glucose by the commonly employed reduction methods (13). Hence this reagent gives “true glucose” values. This is evident in the close similarity between the values obtained by this new reagent and the Somogyi method, which gives true glucose values. In some respects, the chromotropic acid reagent resembles the anthrone reagent recently described by Durham, Bloom, Lewis, and Mandel ( 1 ) for use in the determination of blood sugar. However, in the present instance, the action of the chromotropic acid reagent has the advantage of being limited to hexoses (assuming that disaccharides under most circumstances are not found in circulating blood) and will not be affected by the presence of pentose in blood. The conditions of the determination are similar t o those reported for the quantitative estimation of formaldehyde by MacFadyen (6). In effect, it is formaldehyde which is actually being determined. Emphasis is placed on the use of freshly distilled water, or a t least distilled water which had not been allowed to stand open to laboratory air, for use as a blank substance and in the preparation of the reagents. It has been found that the air in this laboratory (and probably most chemical laboratories on hospital premises) is a t times heavily contaminated with formalin
fumes from adjacent histopathology laboratories. tend to produce high reagent blanks.
This would
ACKNOWLEDGMENT
The authors are indebted t o Louis Sattler of Brooklyn College and S. RI. Cantor, American Sugar Refining Co., for generous quantities of 5-hydrouymethylfurfural. They are also grateful to Louis Sattler for his interest and timely suggestions during the course of this investigatipn. They wish to acknowledge gratefully the assistance provided by Gloria Culpepper and Walter Duglass of this laboratory during the development of the blood glucose procedure and by Molly Vogel and Carmen Harrison, whose clerical contributions cannot be minimized. LITERATURE CITED
(1) Durham, W.F., Bloom, W. L., Lewis, J. T., and Mandel, E. E., Pub.Health Repis. (U.S.), 65, 670 (1950). (2) Eegriwe, E., 2.anal. Chem., 110, 22 (1937). (3) Gardner, J. H., J . Am. Chem. SOC.,67, 2111 (1945). (4) Hayden, R. L., J . Biol. Chem., 56,469 (1923). (5) Hunter, M. J., Wright, G. F., and Hibbert, H., Ber., 71B, 734 (1938). (6) MacFadyen, D. A , , J . Bid. Chem., 158, 107 (1947). (7) Pigman, W.W., and Goepp, R. M.,“Chemistry of the Carbohydrates,” p. 134, New York, Academic Press, 1948. (8) Shriner, R. L., and Fuson, R. C., “Systematic Identification of Organic Compounds,” 2nd ed., p. 65, S e w York, John Wiley & Sons, 1940 (9) Somogyi, M.,J . Biol. Chem., 160, 61 (1945). (10) Sowden, J. C., J . Am. Chem. Soc., 71,3568 (1949). (11) Ibid., 74,4377 (1952). (12) Sowden, J. C., Personal communication. (13) Sunderman, W., Am. J . Clin. Pathol., 21, 901 (1952). (14) Van Slyke, D. D., and Hawkins, J. A., J . B i d . Chem., 79, 739 (1928). (15) Rolfrom, M., Schuetz, R. D., and Cavalieri, L. F., J . Am. Chem. Soc., 70,514 (1948). RECEIVED for review September 25, 1952. Accepted February 31, 1953. Presented before the Division of Biological Chemistry at the 122nd Meeting of the AMERICAN C H E M I C ASOCIETY, L .4tlantic City, N. J.
Determination of Vitamins A and E by Paper Chromatography JAMES A. BROWN, Eli Lilly and Co., Indianapolis, Znd. The problem of adequate separation and determination of vitamins A and E has never been satisfactorily solved. While the use of synthetic vitamins eliminates some problems, analytical results still are of doubtful accuracy and specificity. The vitamins are separated from each other, from impurities in the vehicle, and from oxidized vitamin A on impregnated paper strips in an acetonitrile-water chromatographic system. The substances are located on the strip by means of an automatic spec-
R
ECOGNITION of vitamin E interference with the spectrophotometric determination of vitamin A, particularly when the current United States Pharmacopoeia method (8) is used. has existed for some time ( 5 ) . Other interferences with the spectrophotometric methods for both vitamins exist because of ( a ) the different absorption patterns of the various biologically active forms of the vitamins, ( b ) the absorption pattern of closely related yet biologically inactive substances, and ( c ) the absorption of the vehicle and the countless miscellaneous substances that may be in the vehicle or in a given formulation. Various nonspectrophotometric methods exist for both vitamins ( 1 ), but all have objectionable features which cause their accuracy and specificity to be questioned.
trophotometric arrangement, which provides a graph of band position us. strip length. Quantitative results are obtained by measuring the zonal areas on the graph and comparing to standards. The technique can be made applicable to a variety of research problems o n both natural and synthetic vitamins. The quantitative method shows promise of being applicable to raw materials, to finished products such as pharmaceutical preparations, and to fortified foods.
As a potential means of resolving the situation, the application of paper chromatographic techniques t o the separation and estimation of vitamins A and E has been studied as a continuation of the previously reported work (3) which involved the use of a modified recording spectrophotometer to locate and measure the bands on the chromatograms. Datta and Overell ( 4 )have used alumina impregnated paper for the chromatography of vitamin -4by developing with petroleum ether, which is in reality a modification of the more frequently reported column chromatography of vitamin A using alumina. Brown ( 2 ) has reported on the separation of isomers of vitamin E on Vaseline-coated strips using 75% ethyl alcohol as a solvent. Kritchevsky and Tiselius (6) have described a technique for
V O L U M E 25, NO. 5, M A Y 1 9 5 3
175,
mixture of acetonitrile (redis’ commercial grade) tilled 99 % and of distilled water, the relative amounts of each varying as noted in the captions to the figures. .Samples of a solution of the v i t a m i n s (17.5-microliter) in chloroform, ‘n-hexane, or isopropyl alcohol were used per strip (3). The strips lvere dried, after application of the sample, in aspare develo ing jar which contained a smaE amount of dry ice to furnish a carbon dioxide atmosphere. After development, the strips were dried in a similar manner, carbon dioxide being famed in from a cylinder to urnish circulation. The strips were kept in the jay under carbon dioxide until they were scanned, which was done as described previously (3,7 ) . The quantitative area measurements were also made in the manner described (3).
I
+
I
I
:I
0 u)
m 4 w
2
c
4
-1
w ~
U . u
250
300 WAVELENGTH
350
- MU
i
400
I
RESULTS
-0
R E L A T I V E P O S I T I O N O N P A P E R STRIP Vitamin A Palmitate and a-Tocopherol Developed with 93Vo CH3CK-
The graphs resulting from the Figure 1. scanning of chromatograms con7% HzO V / V taining various combinations of A . Point of sample application G. Absorption spectrum of band B the vitamins along with the abB . Vitamin A palmitate band Vitamin A palmitate. 1,040,OOO U.S.P. units per C. a-Tocopherol band gram, 0.3291 gram per 180 ml. of isopropyl aleohol sorption spectra of the bands D. Scan of strip before development Pure eommercial grade a-tocopherol, 0.5139 g r a m E. Solvent front per 100 m l . of isopropyl alcohol from which the peak wave F. Absorption spectrum of band C lengths were obtained are given. Figures 1 to 3 show that vitamin A palmitate, acetate, and alcohol have been separated from a-tousing silicone-impregnated strips for reverse phase chromatogcopherol. Figure 4 shows that the three forms of vitamin A have raphy. The work reported here shows that the common forms of been separated from each other. Figures 3 and 5 show that oxivitamin A can be separated from each other and from vitamin E dized vitamin A has been separated from true vitamin 9. I n through use of silicone-impregnated strips and of acetonitrilethese figures, the curves have been shifted on the vertical axis. water solvent systems. -4basis for quantitative estimation as The relative absorbancies shown by each have no relationships well as qualitative determination is thus provided. to the others on a graph. I n all cases, the absorption spectra of APPARATUS
The apparatus and its use was essentially that described by Parke and Davis ( 7 ) and Brown and Marsh ( S ) , but the scanner was modified to eliminate some of the difficulties mentioned in (3). The aperture in the track for the paper strip was altered to by 15/32 inch so that a strip of paper 0.5 inch wide (commercially available in rolls) could be used rather than the odd 17/82-in~h width. The rubbercovered roller ahead of the aperture was replaced with a roughened brass roller. Its companion roller in the track was spring loaded and mounted so that its axle could move perpendicularly to the track to provide a positive pressure on the roughened brass roller. The set of rollers below the aperture was found to be unnrcessary and was eliminated. The one set of solid rollers drives the paper at constant speed without slippage. EXPERIMENTAL
Strips of 0.5-inch Whatman Xo. 1 filter paper were immersed in a suspension consisting of 25 grams of Dow Corning or Cenco silicone stopcock grease in 250 ml. of methylene chloride and dried in an oven a t 50 a C. iiscending chromatographic technique was used in 12 X 24 inch red borosilicate glass jars with flat lids or, where only one or two strips were developed, in a l-liter glass-stoppered cylinder. The jars and cylinders were lined with sheets of Whatman No. 1 filter paper and the equivalent of two 18 X 22.5 inch sheets were suspended in the large jar in addition, all being placed so that they dipped into the solvent. The developing solvent was a
328 t w 296 MU
Figure 2.
c-
O N PAPERSTRIP Vitamin -4Acetate and a-Tocopherol Developed with 60% CH3CN-40% Hz0 V / V RELATIVE P O S I T I O N
A . Point of sample application B . a-Tocopherol band C , Ci. Vitamin A acetate band D . Solvent front E . Abaorption spectrum of band Cat C El. Alisorption spectrum of band C at CI
F. Absorption spectrum of band B Merck crystalline commercial grade vitamin A acetate 99+%,0.1050 gram per 100 m l . of isopropyl alcohol Pure commercial grade a-tocopherol, 0.5139 gram per 100 ml. of isopropyl alcohol
ANALYTICAL CHEMISTRY
376 Table I. Solution A. Solution E.
Concentration Area Data
0.3291 gram of 1,040,000 U.S.P. units'gram of vitamin A palmitate per 100 ml. of isopropyl alcohol. 0.5139 gram of pure commercial grade a-tocopherol per 100 ml.
of isopropyl alcohol. Solution A, Solution E , 0.0175 MI. per Strip 0.0175 1\11, per Strip Strip Zonal area, Strip Zonal area, square inches square inches number number 7.11 6.46 6.45 7.20 7.34 6.91 +6.2% -6.7%
6 7 8 9 10
Average Dev. from av. Solutions A and E, 0.0175 MI. of Each per Strip Strip Area of 4 number band. souare inches
7: 57
11 12 13 14 15
Average Dev. from av.
2 55 2 78 2 69 2 80 2 46 2 66 + 5 3% -7.553
Solutions A and E , 0.0175 811. of Each per Strip Strip Area of E number band. souare inches 11
12 13 14 15
7.37 6.99 6.53 7.23 7.14 +6.0% -8.5%
i2
84
52 2 66
2 64 2 82 2.70
328 MI) RELATIVE POSITION
+5.2%
-6.770
the isolated bands appear to agree with the accepted spectra for the pure substances. Table I gives an example of the quantitative aspects of the procedure. The areas shown are those which resulted from running five replicate strips with each of two solutions, one containing vitamin A palmitate and the other or-tocopherol, and five replicate strips to each of which was applied the same amounts of both solutions.
O N P A P E R STRIP
c I
Figure 4. Vitamin A Alcohol, Vitamin A Acetate and Vitamin A Palmitate Developed with 60Yo CH3CY-4070H 2 0 V lV A.
Vitamin A pulmitate band (at point of sample application)
B. Vitamin A acetate band C. Vitamin A alcohol band D. Solvent front
.
E. Absorption spectrum of band 4 F. Absorption spectrum of band B G. Absorptiou spectrum of band C
Eastman crystalline reference standard vitamin A alcohol, 0.1053gram per 100 ml. isopropyl alcohol Merck crystalline commercial grade vitamin A acetate 99+7& 0.1218 gram per 100 ml. of isopropyl alcohol 1,040,000 U.S.P. units of Vitamin A palmitate per gram, 0.1764 grarq per 100 ml. of isopropyl alcohol DISCUSSION
W
1 I-
< d W
D !
80
MU
'96MU
I28 MU R E L A T I V E POSITION O N PAPER STRIP
Figure 3. A.
Vitamin A Alcohol and a-Tocopherol Developed with 50% CH3CN50% HZO V / V
a-Tocopherol band (at point of samplf: applicntion)
G. Absorption spectrum of band B H . Absorption spectrum of band C
Solvent front Absorption spectrum of material at point of application before development Absorption spectrum of band A
Eastman crysthlline reference standard vitamin A alcohol, 0.1053 gram per 100 ml. of isopropyl alcohol: after 19 days storage i n refrigerator Pure commercial grads a-tocopherol, 0.5139 gram per 100 ml. of isopropyl alcohol
B. Vitamin A alcohol band 12. Deteriorated vitamin A alcohol band D.
E.
F.
The coating of the strips is a simple operation and seems to provide uniform strips, judging by the uniformity of the back-. ground absorption (Figure 1). The strips are not sticky 01: greasy after the treatment and can be handled in the same way as any other strips. The lining of the jars and the addition of extra paper is necessary in order to get reasonably constant R f values and relatively uniform band dimensions. Because of the high volatility of the solvent, there seems to be a tendency for a dynamic equilibrium to be set up in the jar which results in the evaporation of the solvent from the exposed strip and a starving of the strip for solvent. The addition of the extra paper minimizes this tendency. The use of a reasonably high-boiling solvent for the sample solutions is desirable in order to get good preciion during the pipetting of $he s a m p l e . C h l o r o f o r m , n-hexane, high-boiling petroleum ether, isopropyl alcohol, qtc., can be used. Chloro--
777
V O L U M E 25, NO. 5, M A Y 1 9 5 3
I R E L A T I V E POSITION O N PAPER STRIP
-’
Figure 5 . Titamin A Acetate and Deteriorated Vitamin A Acetate Developed with 60% CH&N-40% H20 V / V A . Point of sample application B . Vitamin A acetate band C. Deteriorated vitamin A acetate band (spread out from B to C) D . Solvent front E . Absorption spectrum of undeveloped spot of t h e deteriorated vitamin A acetate solution only on paper strip F . Absorption spectrum of band B G. Absorption spectrum of band C Vitamin A acetate. Merck crptalline commercial grade, 99+ 7 ’0 0.1050 gram per 100 m l . of isopropyl alcohol Deteriorated vitamin A acetate. Merck 99+ 9 ’‘ aged in an uncovered Petri dish i n an oven at 50’ C. for 1 year and having less than 1% of original potency by bioassay: 0.1429 gram per 100 m l . of isopropyl alcohol
form, becausr of its high density, is somewhat difficult to pipet ~ i t ah capillary-type pipet but, with care, it can be done successfully. Chloroform is often otherwise desirable, particularly if extraction of a pharmaceutical preparation or other material is necessary to get a suitable solution. For example, vitamin preparations in gelatin capsules have been prepared for chromatography by dissolving the capsules in hot water, digesting with ficin to hj-drolyze the gelatin, and extracting with chloroform. The protection of the strips with carbon dioxide is necessary to prevent oxidation of vitamin A. So far, however, it has not been necessary to try to protect the strips during the actual scanning since the time required is very short. The obtaining of quantitative results is hampered-as it is with all vitamin A and E methods-by the difficulty in obtaining and keeping a suitable standard. Since the esters and alcohol of vitamin A are separated by this method (Figure 4), one should have available a pure standard of each ester and the alcohol in order to evaluate properly the amount of each present-unless a saponification is performed to convert all of the vitamin to the alcohol form. The saponification procedure is time consuming, subject to errors due to mechanical loss, and, in addition, the alcohol form is more subject to oxidation than the esters. Because many pharmaceutical preparations are now made with synthetic vitamins, it seems desirable in these and similar cases not to saponify and to refer assays for vitamin A acetate to pure crystalline A acetate and assays for A palmitate to samples of pure A palmitate, if available, or to samples for which the United States Pharmacopoeia assay is accurately known. For the vitamin E, quantitative data can be referred to pure a-tocopherol or to some other form of tocopherol for which the purity is known by other methods, as the occasion demands. For quantitative results, then, it is necessary to run a series of strips on the sample and a series on the pertinent standard a t about the sanie concentration as the sample or a t several concen-
trations to establish a graph if the approximate concentration of the sample is unknown. It is believed that the slightly lower precision shown in Table I when compared to the data previously reported ( 3 ) is due to the high volatility of the acetonitrile in the solvent system. When the jars were unlined and contained no paper other than the test strips, extremely erratic results were obtained, but when one strip was developed in an individual I-liter glass-stoppered cylinder or a series was developed in the large jars with extra paper added, the results vere much improved. From this evidence, it appears that better precision might be obtained either with a less volatile solvent system or through some means-such as the use of smaller chambers-to reduce the effects of the high volatility. The acetonitrile system is very advantageous with respect to the development time and appears to have only the objection of high volatility. Figures 3 and 5 show that oxidized vitamin A, even though its nature may not be fully known, is apparently separated from the true vitamin. The spreading of band C in the latter figure may be an indication that the nearly completely oxidized A acetate is not a single substance. Severtheless, the A acetate band is well defined and from the absorption spectrum appears to contain only pure A acetate. The individual identification of the bands in Figure 4 is not immediately apparent from the absorption spectra. The ideal curves vary only slightly for the different forms of A, the most well-known variation being a shift of the peak of the alcohol form to 325 mp. The strip scanning for vitamin A shown in this and the other figures was arbitrarily done a t 328 mp since the plot a t either wave length would be indistinguishable from that a t the other. The identification was made here by the approximate RJ values which decrease R-ith the decrease in concentration of acetonitrile in the developing solvent. It was deduced from Figures l and 3 that the palmitate has the lowest Rt, and the alcohol the highest. The acetate falls intermediate between the two, its R, value being shown in Figure 2 since this chromatogram was developed xvith the same solvent as that of Figure 4. From all of the data, it appears that vitamin .Z is separated from ultraviolet absorbing impurities, vehicle, vitamin E, etc., by this paper chromatographic technique which, therefore, has an immediate important advantage as an analytical tool. In addition, two means of identification are immediately provided : R, value and an absorption spectrum of the band. The material in a band can also be extracted and identification tests can be applied to the substantially pure material thus obtained. ACKNOWLEDGMENT
Ackno~ledgmentis made to K. IT. Hilty and Max M. lIarsh of these laboratories for constructive criticism and assistance and to Frieda Lillis of the Lilly Advertising Department for the layout of the figures. LITERATURE CITED
( 1 ) Association of Vitamin Chemists, Inc., “Methods of Vitamin Assay,” 2nd ed., New York, Interscience Publishers, 1952. (2) Brown, F., Biochem. J., 51,237-9 (1952). . 24, 1952 (1952). (3) Brown, J. A,, and Marsh, AI, M., A N ~ LCHEM., (4) Datta, S. P., and Overell, B. G., Biochem. J . (Proc.), 44, xliii (1949). (5) Fox, S. H., and Mueller, A , , J . Am. Pharm. Assoc., 39, 621-3 (1950). (6) Kritchevsky, T. H., and Tiselius, A , Scicnce, 114, 299-300 (1951) . (7) Parke, T. V., and Davis, W.W., ANAL. CHEM.,24, 2019 (1952). (8) “United States Pharmacopoeia,” 14th revision, Easton, Pa., Mack Publishing Co , 1950. RECEIVED for review Sovember 14, 1952. Accepted February 13, 1953. Presented before the Division of Analytical Chemistry a t the 123rd Meeting of the AMERICANCHEMICAL SOCIETT. Los Angeles, Calif.
