Rapid Chromatographic Method for Sugars Using Glass Paper

orange background. Choline chlo- ride gave a brown-purple spot on an orange background. Phosphomolybdic acid-stannous chloride. Quaternary ammonium...
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light. However, ethanolamine and choline chloride could not be detected by the charring technique, and sodium glycerophosphate gave only a weak spot. Dragendorff reagent [potassium iodobismuthate (III)]. Quaternary ammonium compounds were detected by spraying the chromatogram with the modified Dragendorff reagent. Lecithin, lysolecithin, and sphingomyelin appeared as orange-red spots on an orange background. Choline chloride gave a brown-purple spot on an orange background. Phosphomolybdic acid-stannous chloride. Quaternary ammonium compounds were also detected by use of the phosphomolybdic acid reagent of Levine and Chargaff (9). The prompt appearance of a blue spot on a white background is a positive test. Ninhydrin. Compounds containing a primary amino group !{-ere detected by spraying the chromatogram with ninhydrin reagent and heating in a n oven a t SOo C. for 5 minutes. A pink to purple spot on a white background is a positive test. RESULTS AND DISCUSSION

The R, values of the compounds examined ale given in Table I. The substances produced well defined, slightly elliptical spots. Separation of saturated and unsaturated lecithins (dimyristoyllecithin and egg lecithin) or of saturated and unsaturated fatty acids (stearic acid and linoleic acid) was not achieved by this method. KO significant difference was observed in the Rfvalues of a given sutistance when it \vas iun singly or in a mixture.

To determine the reproducibility of the procedure, several 12-inch chromatograms were run on a mixture of lecithin, lysolecithin, sphingomyelin, and phosphatidylethanolamine. The strips were spotted with 5 to 10 pl. of a methanolic solution containing 2.5 y per ~ 1of. each component of the mixture. The sulfuric acid reagent was used for detection of the spots. The results obtained are given in Table 11. The mode of preparation of the paper appears to have an effect on the reproducibility of the R/ values. Less variation in the R, value of a given compound was observed for a given preparation than for different preparations. R i values obtained with a given batch of paper mere apparently not affected by the batch of solvent used. A tank of developing solvent could be used for several days without affecting the reproducibility. At the present stage of development of the method, the R f values are not reproducible enough to be used alone for identification of the phospholipides and their hydrolytic products. As a control, reference compounds should be run concurrently with the unknown on the same strip. The phenolic solvent system offers certain advantages over the methanolether solvent for examination of the conipounds included in this investigation. For example, traces of impurities in some egg lecithin preparations which had been chromatographed on aluniina m r e detected with the phenolic solvent but were not observed when the methanolether solvent was used. More compact spots are obtained, with less t i d i n g .

with the phenolic solvent. The phenol system also permits the application of larger samples of phospholipides to the paper. ACKNOWLEDGMENT

The dimyristoyllecithin used in this work was kindly supplied by Ericli Baer, University of Toronto, and the sphingomyelin by H. E. Carter, University of Illinois. This investigation was supported in part by funds from the Office of the Surgeon General. LITERATURE CITED

Bregoff, H. M., Roberts, E., Delwiche, C. C., J . Biol. Chem. 205, 565 (1953). Dieckert, J. W., Morris, N.J., A 4 ~ CHEM.29. 31 11957). Dieckert, J,’ W.,’ Reiser, R., Federation Pwc. 14, 202 (1955). Hanahan, D. J., J . Biol. Chem. 211, 321 (1954). Hanahan, D. J., Rodbell, hI ,Turner, L. D., Ibid., 206, 431 (1954). Huennekens. F. M.. Hanahan. D. .J.. Uziel, M.,’ Ibid., 206, 443 (i954). ’ Lea, C. H , Rhodes, D. N., Biochem. J . (London) 54, 467 (1953). Lea, C. H.. Rhodes, D PIT.. Stoll. R. D., Ibzd., 60, 353 (1955): Levine, C., Chargaff, E., J . Bid. Chem. 192. 465 (19.513. Rlarinetti, G. V . , ’StoG, E., J . Am. Chem. SOC.77, 6668 (1955). Smith, I., Nature 171, 43 (1953). RECEIVED

for review RIav 7, 1956. Ac-

cepted September 10, 1956. Division of

Analytical Chemistry, 129th Meeting, ACS, Dallas, Tex., April 1956. Mention of specific trade-names does not imply endorsement of the product or its maniifnctiirer.

Rapid Chromatographic Method for Sugars Using Glass Paper Impregnated with Silicic Acid JULIUS W. DIECKERT and NELLE

J. MORRIS

Southern Regional Research Laboratory, U. S. Department of Agriculture, New Orleans, La.

b Sugars have been successfully separated by chromatographyon glass fiber filter paper impregnated with silicic acid. The developing solvent used was a mixture of ethyl ether, phenol, acetone, and water. N o equilibration period was necessary prior to development of the chromatogram. Less than 2 hours was required to develop a chromatogram 10 to 12 inches long and less than 45 minutes for one 7.5 inches long. Concentrated sulfuric acid or panisidine phosphate spray reagents were used to locate the chromatographed sugars.

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on glass paper inipregnated with silicic acid has been used to separate mono-, di-, and triglycerides, to separate cholesterol and cholesteryl esters ( 3 , 4 ) ,and to separate some of the phosphatides (1, 2 ) . This medium permitted the use of indicating reagents of very low specificity, such as dichromate-sulfuric acid cleaning solution and sulfuric acid, for locating the chromatographed substances. The present report is concerned with the separation of some of the sugars. HROJIATOGRAPHY

EXPERIMENTAL

Preparation of Silicic Acid-Impregnated Glass Fiber Paper. Glass fiber paper (No. X-934-AH, H. R e e w Angel and Co., 52 Duane St., NenYork, N. Y., distributors for Hurlbut Paper Co.) was prepared substantially as described by Dieckert and Reiser ( S ) , except that the waterwashing process was repeated six times. Chromatographic Tank. The tank consisted of a battery jar of suitablr size and two glass cover plates separated from each other and from the jar n-ith neoprene gaskets. The cover VOL. 29, NO. 1, JANUARY 1957

e

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plates were held in place with weights. This tank is essentially the same as that described by Dieckert and Reiser (S), except that neoprene gaskets were used in place of plasticine for sealing the tank. This innovation permitted a simplification of the tank and increased the useful life of a given batch of developing solution to a t least 5 days. Developing Solution. One hundred grams of phenol (ACS grade), 200 ml. of ethyl ether (ACS grade), and 126 ml. of acetone were placed in the chromatographic tank and stirred until the phenol had dissolved. Finally, 25 ml. of water was added, giving a clear solution. Spot Test Reagents. After the developing solution was removed one of the following spot test reagents was applied t o the chromatogram. SULFURIC ACID. The chromatogram was sprayed with concentrated sulfuric acid and then heated over a hot plate until charring took place. Heat should be applied cautiously a t first for best results, particularly if rhamnose is present. However, it is usually necessary to heat the chromatogram until the sulfuric acid distills in order to get maximum charring. L-Rhamnose gave a characteristic yellowish tan spot while the other sugars gave brownish black spots. ~ A N I S I D I NPHOSPHATE. E This reagent was prepared according to the procedure of Mukherjee and Srivastava ( 5 ) . The sugars appear as yellow spots on a white background, when the chromatogram is heated for several minutes a t 95' to 100" C. Method. Each sugar was spotted on the impregnated glass paper in 10t o 50-7 amounts as an alcoholic solution. The paper, after drying, was suspended in the tank according to the method of Dieckert and Reiser (3) and, without prior equilibration, was developed by the ascending technique. Two hours or

less were required to develop a chromatogram 10 to 12 inches long and about 30 minutes for a 7.5-inch chromatogram. ilfter development, the ether and acetone were allowed to evaporate. Heat was then applied carefully to remove all of the phenol without igniting the vapors. After the chromatogram was treated n-ith the appropriate spot test reagent and heated, if required, the spots were viewed best by transmitted light. RESULTS AND DISCUSSION

R, values, obtained by chromatographing six sugars singly and by chromatographing a mixture of the same six sugars, are listed in Table I. The agreement between the two sets of values indicates that the sugars migrate similarly, whether chromatographed singly or in admixture.

was permitted to remain in the paper and the sulfuric acid test was applied, a pink spot appeared for each sugar upon the application of heat. This phenomenon resembles the Molisch test. The p-anisidine phosphate test performed satisfactorily on the silicic acidimpregnated glass paper but mas found to be less sensitive than the sulfuric acid test. I n addition, all the sugars investigated gave the same color reactions, contrary to their behavior in a conventional paper system. The technique of glass paper chromatography has proved useful as a rapid means for following the progress of fractionation of constituents of extracts from peanuts, tung meal, and other natural products. ACKNOWLEDGMENT

The authors wish to thank John Table I.

Sugar >Rhamnose D-Xylose >Fructose n-Glucose Sucrose Raffinose

R,

L. White and Katherine M. Formusa for suggesting the use of the neoprene

Values

Single

Mixture

0.75 0 63 0.52 0.43 0 19 0 02

0.74 0.65 0.52 0.44 0.22 0.02

gaskets. LITERATURE CITED

Brown, M., Yeadon, D. A,, Goldblatt, L. A., Dieckert, J. W., AXAL. CHEM.29, 30 (1957). Dieckert, J. W., Reiser, R., Federation Proc. 14, 202 (1955).

Dieckert, J. W., Reiser, R., J. Am. Oil Chemists' Soc. 33, 123 (1956).

At the present stage of development of the method, the R, values are not reproducible enough to be used alone for the identification of the sugars. As a control, reference sugars should be run concurrently with the unknown. Sulfuric acid proved t o be a satisfactory reagent for locating the spots containing these sugars. If a little phenol

Dieckert, J. W., Reiser, R., Science 120, 678 (1954).

Mukherjee, S., Srivastava, H. C., Nature 169, 330 (1952). RECEIVED for review April 27, 1956. Accepted September 10, 1956. Division of Analytical Chemistry, 129th Meeting, ACS, Dallas, Tex., April 1956. Mention of specific tradenames does not imply endorsement of the product or of its manufacturer.

Quantitative Chromatog rap hic Procedure for Determining Dextrose in Sugar Mixtures EMMA

J. MCDONALD

National Bureau of Sfandards, Washington 25,

b Dextrose can be transferred from a paper chromatogram to glass fiber paper and the sugar subsequently determined in the presence of the glass fiber. Results are given for the procedure as applied to the determination of dextrose in honey.

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sugar methods are not completely selective; therefore, the determination of individual sugars in a mixture offers problems not present in the analysis of individual sugar samples. It is desirable to separate

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ANALYTICAL CHEMISTRY

D. C.

mixtures into their components prior t o analysis, and paper chromatography offers a means of accomplishing such a separation. The advantage gained by thus eliminating interfering sugars prior to a particular analysis is somewhat reduced by the size of sample that can be chromatographed on paper. In spite of this limitation, paper chromatography has been used in the analysis of many sugar products. Following paper chromatographic separation, sugars are generally removed from the chromatogram before quantitative determination. Such a proce-

dure is imperative if analysis is to be made with reagents which have an appreciable effect on cellulose. The colorimetric methods employing anthrone or phenol in concentrated sulfuric acid are representative of this group. Although the use of filter papers of heavy weight, such as Whatman Nos. 3 and 17, has increased the size of sample that can be separated chromatographically, the fibers of these papers interfere with titration when the sugars are determined by copper reduction methods. Also, an appreciable correction must be made for the effect of the'