Determination of Sugars on Paper Chromatograms with p-Anisidine Hy drochIoride JOHN B. PRIDHAM1 The institute of Paper Chemistry, Appleton, Wis.
A new method for the determination of aldopentoses, aldohexoses, hexuronic acids, and 6-deoxyaldohexoses involves the paper chromatographic resolution of the sugar mixture, spraying of the chromatogram with p anisidine hydrochloride reagent, and heating the paper in an oven. The resulting colored spots are cut out, the colors eluted from the paper with aqueous methanol containing stannous chloride, and then the absorbance is measured with a spectrophotometer. The absorbances of the sugar-p-anisidine complexes are directly proportional to the weights of the sugars over a range of 5 to 50 y. The method has been used as a rapid analytical tool for the determination of the monosaccharide components of wood pulps and hemicelluloses.
P
APER partition chromatography has proved to be an in-
valuable aid to the quantitative determination of the eomponents of complex mixtures of sugars (1). However, the chromatographic procedure 1% hich is normally used has the following disadvantages: Relatively large amounts of the sugars must be applied t o the paper; the location of the bands by the use of marker strips may be inaccurate; and the complete elution of the sugars is time-consuming. In the procedure described here, the solution to be analyzed is applied to the paper as small discrete spots, thus ensuring good chromatographic separation when only a relatively short solvent development time is used. After development the chromatogram is dried, then sprayed with p-anisidine hydrochloride ( 2 ) reagent and heated. The resulting colored spots, the boundaries of which are clearly discernible, are then cut from the chromatogram. The colors can be rapidly eluted and measured in a spectrophotometer. The intensity of color bears a linear relationship to the amount of sugar over the range from 5 to 50 y. The method has been successfully used for the determination of aldohexoses, aldopentoses, hexuronic acids and 6-deoxyaldohexoses, and, therefore, has been particularly useful for the analysis of wood pulps and hemicelluloses.
then heated in an oven a t 130' C. for 10 minutes. The colored sugar spots, together v i t h suitable blanks, were cut out with a. scissors, the areas of the pieces of paper being kept constant for each sugar series and as small as possible. The color was eluted from the paper by shaking mechanically for 5 minutes with 3 ml. of methanol-stannous chloride solution in rubber-stoppered test tubes, 6 X 3/4 inch. The blanks were eluted sirnilail). The absorbances tvere then measured in 1-cm. cells in a Beckman 3Iodel DU spectrophotometer a t the following wave lengths: .ildopentoses and hexuronic acids Aldohexoses 6-Deoxyaldohexoses
510 mp 400 mp 385 mg
Absorbance measurements on aldopentoses or hexuronic acids were carried out within a period of 30 minutes after development of the color. Absorbance values were plotted against the weights of sugars for the standard determinations and the resulting graphs then used to determine the unknowns. RESULTS A h D DISCUSSIOY
-inexamination of the absorption spectra of thc colored compounds formed by the interaction of sugars with the p-arnsidinc hydrochloiide reagent showed that both aldopcntoscs and hexuronic acids gave compounds which had absorption maxima a t 510 mp. This suggests that the same reaction mechanism occurs v i t h both types of sugar, possibly an initial degradation to furfural followed by a condensation of the furfural n ith the p-anisidine to form a compound with a characteristic red color. Small
REAGENTS
p-Anisidine Hydrochloride Solution. Dissolve 1.0 gram of the crystalline reagent in 10 ml. of absolute methanol and make up to 100 ml. with 1-butanol. Add 0.1 gram of sodium hydrosulfite. Store the solution in the refrigerator. Eluent. Dissolve 1.0 gram of stannous chloride in 5 ml. of water. Add 90 ml. of absolute methanol and filter the solution. PROCEDURE
Quantitative analvsis of sugar solutions was carried out by spotting suitable volumes onto Whatman No. 1 filter paper with an ultramicroburet. Three or four spots of standard mixed sugar solutions, the components of which corresponded qualitatively t o those in the unknown samples, were also applied to the chromatograms. The standard solutions were spotted so that each sugar component was present in varying - - amounts over a range O F to ~ 50--y. The chromatograms were developed with l-butanol-wridinewater solvent (6-to 4 to 3 v./v.) br ethyl acetate-ace% acidwater (9 to 2 to 2 v./v.) solvent for a minimum of 20 hours. The chromatograms were air dried, then sprayed as uniformly as possible with the p-anisidine hydrochloride reagent. They were
Y OF SUGAR
Present address, Department of Chemistry, University of Edinburgh, Edinburgh 9, Scotland.
Figure 1.
1
1967
Increase in absorbance with increase in weight of various sugars
ANALYTICAL CHEMISTRY
1968 Table I. Quantitative Analysis of Spruce Pulp Hydrolyzed with 7270 Sulfuric Acid Volume of Hydrolyzate Determined Calcd. Sugar Applied t o Weight Concn. in Chromatogram, of Sugar, Hydrolyzate, Sugar PI. 7 Mg. /Ml. Glucosea 5 12.2 24.4 10 24.9 24 9 Mannose b 20 28.3 1.30 30 38.2 1.40 Xylose b 40 39.0 0.97 60 55.0 0 92 Sum of av. of sugar in hydrolyzate, 27.0 mg. /nil. Hydrolyzate diluted 10 times; calculated sugar values corrected or dilution. b Hydrolyzate undiluted.
variations in the shade of this color for different sugars, however, indicate that the reaction mechanism may be somewhat more complex than this. The absorption maxima of the brown-colored compounds formed with aldohexoses and 6-deoxyaldohexoses were a t 400 nip and 385 mp, respectively. The colors obtained with these sugars were relatively stable, but for best results, the absorbance should be measured shortly afler color development. The compound produced by reaction of aldopentoses or hexuronic acids with the p-anisidine hydrochloride reagent was rather unstable, but the addition of stannous chloride to the eluting solution enhanced the color stability. Nevertheless, for a pentose determination, it is advisable to measure the absorbance of the solution within a period of 30 minutes after the development of the color on the chromatogram.
The determination of standard sugars together with the unknowns on the same paper chromatogram has alleviated the use of exactly standardized conditions, so that small variations in the procedure given will not affect the final results. Some typical examples of curves obtained with standard sugars are shown in Figure 1. The errors are within &4%. Table I shows data obtained for the quantitative analysis of spruce pulp hydrolyzed with 7’2% sulfuric acid, using the procedure developed by Saeman and coworkers ( 3 ) . In order to determine glucose, which was present in a high concentration in the pulp, the hydrolyzate was diluted 10 times before it was applied to the chromatogram. Mannose and xylose determinations were made using undiluted hydrolyzate. A determination of the total sugar present in the hydhlyzate was carried out by cautious evaporation of a known volume a t 50” C. under reduced pressure to a constant weight sirup. This gave a value of 27.3 nig. per nil. On this basis the figure given for the total sugar concentration in Table I represents a 99% recovery. ACKNOWLEDGMENT
The author wishes to thank Maija Lakstigala for her interest and criticism. LITERATURE CITED
(1) Hough, L., in “Methods of Biochemical Analysis.” T’ol. I, D. Glick, ed., p. 205-42, Interscience, New York, 1954. (2) Hough, L., Jones, J. K. K.,Wadman, W. H., J. Chem. SOC.1950, 1702. (3) Saeman, J. F., Moore, W.E., hlitchell, R. L., Alillett, AI. 1.. Tappi 37, 3 3 6 4 3 (1954). RECEIVED for review .4pril7, 1956. Accepted August 16,1956.
Colorimetric Estimation of Dialkyl Phosphites in Presence of Trialkyl Phosphites, Phosphates, and Phosphonates SAMUEL SASS
and
JAMES CASSIDY
Chemical Research Division, Chemical W a r f a r e Laboratories, A r m y Chemical Center,
A method for the detection and quantitative estimation of dialkyl hydrogen phosphites in the presence of other phosphatics is based on the reaction of these phosphites with trinitrobenzene to form a red color measurable on a colorimeter or spectrophotometer at 465 m p . The method is sensitive to 0.2 to 0.5 y of dialkyl hydrogen phosphite per ml. of solution.
ANY phosphorus compounds used for insecticides and
related purposes are made in several synthetic steps which require alkyl phosphites or yield them as intermediates. No micromethod was available for distinguishing between dialkyl hydrogen phosphites and the monoalkyl and trialkyl phosphites, among others. I n this particular case a method was required for the estimation of dialkyl hydrogen phosphites in the presence of larger quantities of compounds with varying similarity in chemical properties, Evidence that the dialkyl hydrogen phosphite exists as an equilibrium between the hydrogen phosphite and phosphonate came from unpublished infrared and nuclear magnetic resonance studies made in these laboratories, and from its hydrolytic characteristics (Reactions 1, 2, 3), studied here and also reported by Fox (S), among others.
/
/
CHiO
CHIO
90 %
CHIO
\P
CHIO/
10%
0
P-H+HOH
M
Md.
CHIO
\7
HCL
-+
P
HO’
0
+ CHsOH
(2)
H ‘
However, its oxidative characteristics in the presence of iodine or bromine show an apparent 100% trivalent phosphorus character or 100% as the apparent hydrogen phosphite form. The tautomeric character of the dialkyl hydrogen phosphite appeared to be reminiscent of the enol keto equilibrium of carbon ketones, thus suggesting the use of aldehyde and ketone techniques. The use of dinitro- and polynitrobenzenes to form colored compounds with ketones ( I , 9,4 ) was of interest in these latora-
