Detection and Paper Chromatography of Sugars and Sugar Phosphates in Picric Acid System HUBERT S. LORING, LUIS W. LEVY1,
and
LLOYD
K. MOSS
Department o f Chemistry and School of Medicine, Stanford University, Stanford, Calif.
Paper chromatography with the tert-butyl alcoholpicric acid-water solvent system has been limited to sugar phosphates. Its application to sugars in general has now been made possible by the development of a simple spray technique for their detection on paper chromatograms. The reaction involves the reduction of alkaline picrate to picramic acid or other reddishbrown products. Various sugars and sugar phosphates (including glyceraldehyde, erythrose, and glyceraldehyde 2- and 3-phosphate) have been examined in this system, and their R j and Rribosevalues determined.
THE
4 tei-l-butyl alcohol-picric acid-water solvent system has been u s d for the separation and identification of various sugar phosphates by paper chromatography ( 3 ) . I n this procedure the chromatograms are developed, after spraying the paper with a perchloric acid-molybdate reagent, by appropriate treatment leading to the formation of reduced phosphomolybdate in the areas containing phosphate (2). Reducing sugars are not readily detected in the presence of picric acid by the standard spray techniques such as ammoniacal silver nitrate (5) or aniline oxalate or phthalate ( 6 ) . It has now been found that the areas containing sugar or sugar phosphate are easily made visible after the dried Chromatogram is sprayed with ethanolic sodium hydroxide and heated. Under these circumstances the picric acid remaining in the paper reacts with the sugar or its alkaline decomposition products and forms a reddish-brown epot, probably picramic acid (4,7 ) , against the yellow background of the sodium picrate. Several sugars including glyceraldehyde, erythrose, arabinose, ribose, xylose, rhamnose, fructose, galactose, glucose, mannose, lactose, maltose, sucrose, and raffinose, as well as some of their phosphoric acid esters, \\-ere examined by descending paper chromatography in the picric acid system using the new development technique. The R, values of most of the substances studied arc sufficiently different to allon their separation and identification by the procedures described. The Rj and R r l b o s e values found are presented in this paper.
tal) was converted to glyceraldehyde 2-phosphate by hydrolysis a t 40" C. for 96 hours after treatment of a 2% solution with excess Dowex 50, acid form (1). Glyceraldehyde was obtained in solution from calcium glyceraldehyde 3-phosphate by hydrolysis a t p H 4 with purified semen phosphatase. The erythrose was in the form of a n aqueous solution. Procedure. Samples of 10 pl. containing 0.02 t o 0.3 pmole of sugar or sugar phosphate were applied to the starting line of the paper, which was exposed t o the vapor phrtse of the solvent for 3 t o 5 hours. The solvent was then added to the trough through a hole in the glass plate, which could be closed by means of a rubber stopper. After 16 t o 26 hours at room temperature, the paper sheets were removed from the cylinder, allowed to dry in air for about 30 minutes, sprayed with a freshly prepared solution consisting of equal parts of 1N sodium hydroxide and 95% ethanol, and heated in a n oven a t 85" C. for 7 to 10 minutes. The areas containing sugar appeared as reddish-brown spots, which retained their characteristic appearance for several weeks.
Table I.
Ranre Sugars Glyceraldehyde Erythrose ~-Brabinose Ribose Xylose L-Rhamnose Fructose Galactose Glucose Mannose Lactose Maltose Sucrose Raffinose
Ri
RR b
0.25-0.32 0.49-0.60 0.38-0.39 0.37-0.49 0.43-0.45 0.46-0.48 0.31-0.34
0.60-0.64 1.2-1.4 0.74-0.78 1 .o
0.24-0.28 0.33-0.35 0.32-0.37 0.09-0.11 0.12-0.15 0.22 0.06-0.07
0.84-0.90 1.1-1.2 0.72-0.76 0.56-0.57 0.65-0.67 0.74-0.79 0.21 0.29-0.30 0.51-0.52 0.13-0.15
Sugar Phosphates 0.33-0.40 Glyceraldehyde 2-phosphatec 0.77-0.79 Glyceraldehyde 3-phosphate 0.19-0.26 0.44-0.52 Ribose 5-phosphate 0.46-0.49 0.90-0.92 Fructose 6-phosphated 0.34-0.39 0 82-0.90 Fructose 1,6-diphosphated 0.32-0.34 0.76-0.82 Glucose 6-phosphated 0.25-0.27 0.60-0.62 a All sugars and sugar phosphates, with the exceptions noted, were of the D- configuration. distance traveled by compound RR = distance traveled by n-ribose on same chromatogram Values shown are the lowest and highest found on a t least two chromatograms. C The dimethyl acetal from which glyceraldehyde 2-phosphate was obtained gave Ej and RR values of 0.79 t o 0.83, and 1.9, respectively. d The free acid prepared by treatment of the salt with Dowex 50, acid form, gave identical Rj values. 1
EXPERIMENTAL
Apparatus. -4borosilicate glass cylinder, 24 cni. in diameter and 4G cm. in height, covered with a tight-fitting glass plate was used. The paper and the solvent trough for descending chromatography were supported on a stainless steel rack. A container with solvent was placed in the bottom of the jar to provide a saturated atmosphere of the vapor phase. The data presented were obtained with Whatman No. 1 filter paper, but in several instances where Schleicher and Schuell No. 597 filter paper was used, similar results were found. Solvent. A solution was used consisting of 4 grams of picric acid dissolved in 80 ml. of fert-butyl alcohol and 20 ml. of water (3). Materials. JJ'ith the exception of glyceraldehyde and erythrose, the sugars were the usual commercial preparations. Ribose 5-phosphate, fructose G-phosphate, fructose 1,6-diphosphate, and glucose G-phosphate were purchased as barium salts from Nutritional Biochemicals Corp., Cleveland, Ohio. Cyclohesylammonium 2-phosphoryl n-glyceraldehyde dimethyl acetal (hex~lamrnon~um-2-o-~,hosplionyl-~-glyceraldeliyde dimethyl aceI hlonsanto Chemical Co. Fellow, Stanford University, 1964-55. of absence from Escuela Polit6cnica Nacional, Quito, Ecuador.
Paper Chromatography of Sugars and Sugar Phosphatesa in Picric Acid System
RESULTS AND DISCUSSION
The tert-butyl alcohol-picric acid solvent moves with two fronts, a faster colorless one and a slightly slower picric acid front, mhich remains visible on drying. The picric acid front vms used for the calculation of the Rj values. The ranges of these values for the respective compounds are shown in Table I. In each instance a t least two chromatograms were prepared and run on different days. The relatively wide ranges of Rj values shown for some compounds-e.g., ribose and erythrose-were found over a period of 2 to 3 months, during which there was considerable fluctuation between day and night temperatures. This probably accounts for the wide variation found in these cases. Ribose was present on many of the chromatograms showing variations in R , values; therefore, it was possible to calculate RriboBevalues, or the ratio
On leave
539
ANALYTICAL CHEMISTRY
540 of the distance traveled by the particular compound t o t h u traveled by ribose. These RR values were much more uniforri, and chromatograms were prepared for all the substances studied, in comparison with ribose. The resulting RR values are alto given in Table I. I n some instances compounds which appeared to have similar R, values-e.g., erythrose and rhamnose-could be distinguished when compared with ribose. A single, characteristic spot was found for each of the compounds studied, with the exception of the nonreducing sugars sucrose and raffinose, and the hexose phosphates. The first two compounds showed evidence of slight hydrolysis during :I run, and the hexose phosphates gave faint “shadows” as described by Hanes and Isherwood ( 3 ) . In these instances, and with thi: other sugar phosphates studied, the respective spots were also located on separate chromatograms by means of the perchloric acid-molybdate spray (2). No significant differences in R; or Rx values were found. Al’PLlCATIOK TO OTHER CIIRORIATOGRAPIIIC SYSTEJIS
Reducing sugars can also be detected in systems where picric acid is not used, such as acetone-boric acid (S), by spraying the chromatogram with a fresh solution containing picric acid (1
gram), IN sodium hydroxide (25 ml.), and ethanol (25 m!.), then heating as described above. ACKNOWLEDGMENT
The authors would like to express their thanks to C. E. Ballou, University of California, Berkeley, for samples of glyceraldehyde phosphates, and to A. S. Perlin, National Research Laboratories, Saskatoon, Sask., Can., for the sample of erythrose. The semen phosphatase was prepared in this laboratory by Forrest H. Riordan, 111. This investigation was supported by research grants from the American Cancer Society, the Rockefeller Foundation, and the United States Public Health Service. LITERATURE CITED
(1) Ballou, C. E., Fischer, H. 0. L., (1955).
J. Am. C h e m SOC.77, 3320
( 2 ) Bandursky, R. S., Axelrod, B., J . BioE. C h e m 193, 405 (1951). (3) Hanes, C. S., Isherwood, F. A., Nature 164, 1107 (1949). (4) Lewis, R. C., Benedict, S. R., J . Biol. C h e n . 20, 61 (1915). (5) Partridge, S. hI., Biochem. J . 42, 238 (1948). (6) Partridge, S. M., h‘ature 164, 443 (1949). (7) I’igman, W. W., Goepp, R. A i . , Jr., “Chemistry of the Carbohydrates,” p. 135, Academic Press, Yew York, 1948. (8) Seepmiller, J. E., Horecker, B. L., J. Bid. Chem. 194, 2G1 (1052). KECEZVED for review November 1 % 1955.
Accepted December 12. 1856.
Peroxytrifluoroacetic Acid as a Reagent for Determination of the Carbonyl Function in Aldehydes and Ketones M. FREDERICK HAWTHORNE Redstone Arsenal Research Division, Rohm
& Haas Co., Huntsville, Ala.
The Bacyer-Villigcr reaction of simple aliphatic aldehydes and ketones is quantitative with peroxytrifluoroacetic acid in cthyleno chloride solution and affords a new method for the determination of these compounds. Samples of aldehydes or ketones are treated with excess standard peroxytrifluoroacetic acid, the reacton is allowed to proceed to conipletion, and the residnal perony acid is deterniined iodonletrically. The method is accurate to &lo%in the most unreliable cases invcstigatcd and recoveries high by 3Yo are most o€ten obtained. A procedure for the preparation of standard anhydrous solutions of peroxytriflnoroacetic acid in ethylenc chloride is described.
B
ECAUSE the most commonly employed method for the quantitative estimation of the carbonyl function in aldehydes and ketones is based on the slow and reversible oximation reaction (S), a new. procedure was sought, which might be executed volumetrically with high precision. Such a method has been made available through the discovery of the extremely reactive oxidizing agent, peroxytrifluoroacetic acid, and the pieparative oxidation of ketones to mixtures of esters (Baeyer-V-illiger reaction) by this reagent has been recently described (2). The basis of this method lies in ease with which solutions of peroxytrifluoroacetic acid containing trifluoroacetic acid enter into the Baeyer-Villiger reaction ( 1 ) Tyith aldehydes and ketones. As these reactions RCOR’
+ CF,C03H
-*.
RCOOR and R’COOR
+ CF3COlH
are relatively rapid and peroxytrifluoroacetic acid solutions in ethylene chloride lose their active oxygen only very slowly,
these determinations may be carried out by the simple iodometric titration of excess peroxy acid after a reasonable reaction time (up to 1 hour a t 50” C.) without the use of a reagent dccomposition blank determination, Solutions of peroxytrifluoroacetic acid may be conveniently prepared by the reactions of a weighed sample of 90% hydrogen peroxide with the calculated volume of a standard solution of trifluoroacetic anhydride in ethylene chloride. Under these eonditions the reactions
HZO
+ (CF3CO),O
+ 2CF3COzH
rapidly take place and the resulting standard solution of perosytrifluoroacetie acid mill retain its active oxygen titer throughout a normal working day. SOLUTIONS AND REAGENTS
Ethylene Chloride. Three liters of U.S.P. ethylene chloride was washed three times n3,h concentrated sulfuric acid and three times with water, and dried over sodium sulfate. This material mas then distilled through a helices-packed 2-foot column until water ceased t o be removed from the system by azeotropic distillation. Sufficient phosphorus pentoxide was added t o the boiler to remove any extraneous moisture and thc distillation continued in a system protected from atmospheric moisture. Two liters of material boiling a t 84’ C. was collected in a flask and protected from atmospheric moisture. Standard Trifluoroacetic Anhydride Solution. Two hundred grams of trifluoroacetic anhydride (obtained from the Minnesota *Mining and Manufacturing Co.) was slowly distilled into the ethylene chloride described above, using a distillation systcni
