1290
ANALYTICAL CHEMISTRY
The end of the filter paper nearest the spotted section is immersed in the buffered butyl alcohol of corresponding pH. The system is allowed to develop, descending, for about 15 hours in a Chromatocab. The papers are removed and the solvent front 1s marked and air dried. T o locate the compounds, the papers are first examined with a source of short-wave ultraviolet light for areas of fluorescence or absorption, then sprayed with iodoplatinate reagent to develop characteristic dark spots of the iodoplatinate complex of the basic drugs. The front of the spot is used to measure the movement and the Rj and codeine ratios distance traversed by the compound distance traversed by codeine
(
) are computed for each dr
T o recover the compounds, the iodoplatinate complex is cut out of the pa er, placed in a, separatory funnel, and covered with 5 ml. of a 4 8 sodium sulfite solution in half-saturated sodium borate (pH 9.5) to destroy the complex and free the basic compound. When the paper is decolorized, a suitable solvent is added and the separatory funnel is shaken. The solvent containing the basic drug is evaporated to dryness. Chemical teste, characteristic absorption spectra, or other tests can be made to confirm the identity of the compound. RESULTS
Table I lists the Rj values and codeine ratios of 44 pure coinpounds commonly encountered by toxicologists and pharmacologists. These values represent an average of a t least three determinations for a particular compound. The R , values and codeine ratios vary approximately 15% and 10%) respectively, a t the lower pH values, and about 5% and 2%, respectively, a t the higher pH values. An attempt was made to decrease the deviations by more careful control of such conditions as buffer saturation of the mobile phase, temperature, humidity, and the like but this was cumbersome, time-consuming, and not always successful. The compounds are grouped into seven classes according to the pattern produced by the codeine ratios a t four pH's, so that the identification of any one of these compounds can be more easily made. Consideration must be given to members of the adjoining class in borderline instances. When an unknown is found to have the following Rj values and codeine ratios, it is located as follows. pH 3 . 0 R f Ratio
R/ Ratio
1.90
0.62 3.20
0.31
pH 5 . 0
pH 6 5
Rj
Ratio
0.72 2.10
pH 7 . 5
R,
I n this group, ppibenzamine most closely resembles the Rj values and codeine ratios of the unknown. Methapyrilene and Chlor-Trimeton are possibilities. Because Chlor-Trimeton shows a pattern that retrogresses a t pH 6.5, the unknown is less likely to be this compound. The conclusive identification can be made by running another chromatogram m-ith the unknown along with pyribenzamine and methapyrilene or other suspected compounds. DISCUSSION
The above chromatographic procedure offers a relatively simple method for the identification of microgram quantities of an unknown drug even in the presence of other basic compounds. Along with the characteristic pattern of Rj values and codeine ratios, the procedure provides the following additional aids in the identification of an unknox-n. Examination of the paper with ultraviolet light (nave length, 255 mp) shous the presence of fluorescent compounds as ne11 as those compounds that strongly absorb ultraviolet light of this wave length. For example, quinine or quinacrine TI-ill be seen as fluorescent areas n hen present on the paper in concentrations of less than 1 y , n hile compounds like strychnine or adenine appear as dark areas n-hen present in concentrations of about 10 y . Khen the filter paper is sprayed n ith iodoplatinic acid reagent, all the compounds listed in Table I, ewept adenine, coramine, and pilocarpine, appear as blue or black spots when the concentration is approximately 50 y. As little as 10 y of many compounds listed in Table I can be detected with this reagent. Adenine and coramine produce yellow spots a t all pH values, pilocarpine produces a black spot at pH 3.0, a brown spot at pH 5.0, and a yelloE spot a t pH 6.5 and 7 5 . The ability to recover the compounds from the paper in a relatively pure state is also a great advantage. Confirmatory tests can be made on the recovered extract to make a conclusive identification of the unknoL7.n drug. When sufficient amounts of the basic compounds listed in Table I are available, as in pharmaceuticals and in many toxicological samples, the use of the above procedure has proved eff ertive. LITERATURE CITED
Ratio
0.95 1.25
From the key in Table I, the codeine ratios are replaced by their letter equivalents: 1.90 = C, 3.20 = F, 210 = D, and 1.25 = B. The pattern CFDB is checked against the classes in Table I. Only Class I V compounds include this particular pattern (Class IV: pH 3.0, BCD; 5.0, EFG; 6.5, CD; 7 . 5 , B ) .
(1) Carless, J. E., Woodhead, H. B., A-ature 168, 203 (1951). (2) Moerloose, P. de, Mededel. Vlaam. Chem. Ver. 15, 13-18 (1953). (3) Munier, R., hlachehoeuf, AI., Cherrier, N.. Bull. soc. chim.biol. 34, 204 (1952). RECEIVED for review February 8, 1956. Accepted M a y 1, 1966. Preliminary report presented a t American Academy of Forensic Sciences, 5th Annual Meeting. Chicago, Ill., February 1953.
Quantitative Paper Chromatography of Methylated Aldose Sugars Improved Colorimetric Method Using Aniline Hydrogen Phthalate W. C. SCHAEFER and J. W. VAN C L E V E Northern Utilization Research Branch, Agricultural Research Service,
A n improved procedure is presented for the colorimetric determination of methyl ethers of D-glucose which have been separated by paper chromatography. Aniline hydrogen phthalate is the chromogenic agent in this procedure, and the optimum range of sugar is 90 to 200 y. Similar color development was observed with methyl ethers of D-mannose, D-galactose, and D-xylose, indicating the general value of this method for the determination of methyl ethers of aldose sugars.
0
U. S.
Department o f Agriculture, Peoria,
111.
F CONSIDER-\BLE importance in structural studies of
polysaccharides by the methylation technique is a reliable method for the determination of methylated reducing sugars after their separation from mixtures by means of paper chromatography. Such a method has been developed by modification of a known colorimetric procedure using aniline hydrogen phthalate as chromogenic agent. The color-forming reaction of the simple aldose sugars with this reagent was first demonstrated by Partridge (20), v-ho used
1291
V O L U M E 28, NO. 8, A U G U S T 1 9 5 6 a solution of aniline and phthalic acid in butyl alcohol as a spray reagent for the qualitative detection of sugars on paper chromatograms. The same spray reagent has recently been used for determining methylated reducing sugars by a densitometric procedure ( 1 7 ) . The first colorimetric method using aniline phthalate for the quantitative determination of the simple aldose sugars was proposed by Blass, IIacheboeuf, and Nunez (4). Shortly thereafter, Gardell ( 1 1 ) reported a quantitative procedure using aniline in trichloroacetic acid. Application of the method of Blass and others to the determination of the methyl ethers of glucose then was reported by Bartlett, Hough, and Jones in a preliminary note ( 2 ) .
Table 1. Analyses of Iiiiown Mixtures Recovery from Chromatogram %,of
A
Spotted, Mixture t y 2,3,4,6-Tetra-O-methyl-n-glucose 145 140 2,3,4-Tri-O-methyl-~-glucose 150 143 2,3-Di-O-methyl-~-glucose 152 149 142 2,3,4,6-Tetra-O-methyl-~-giucose 145 148 2,1-Di-O-methyl-~-gluooae 153 97 2,3,4,6-Tetra-O-methyl-~-glucose 98 2046 2,3,4-Tri-O-methyl-~-glucose 2191: 2,4-Di-O-meth~l-~-glucose 100 110
Av. Dev. orig- from M e a n , inal %
97
with a slight excess (only a few crystals) of potassium permanganate in the presence of magnesium sulfate (0.26 gram per liter) prior to azeotropic distillation. The spray reagent ( 5 )was 0.4% .V,N-dimethyl-p-aminoaniline hydrochloride in aqueous 2YGtrichloroacetic acid. This reagent is usable for 1 week when kept a t '2 C. The colorimetric reagent consisted of a 2.410% f 0.006% (w./v.) solution of crystalline aniline hydrogen o-phthalate in methanol. When kept a t 2" C. the reagent was stable for 3 months. The solvent used for extracting sugars from chromatograms consisted of methyl alcohol containing 0.900% =k 0.006% (w./v.) of tetraethylene glycol dimethyl ether (2,5,8,11114-pentoxapentadecane). Approximately 1.5y0aqueous sugar solutions were found convenient in applying known sugars to paper chromatograms. The 2,3,4,6-tetra-, 2,3,4-tri-, and 2,3-di-0-methyl-~-glucoses were repared by known rocedures (6); 2,3,4-tri-O-methyl-~-xyPose, 2,3,4,6-tetra-O-metfyl-~-galactose, and 2,3,4,6-tetra-O-methyl-~mannose were prepared through methylation of their methyl glycosides by a modified Muskat procedure (19); and 2,4di-0methyl-D-glucose ( 2 5 ) and 1,3,4,6-tetra-O-methyl-~-fructose (26) were prepared by new and improved procedures. PROCEDURE
3.1 2.2 1.2 1.5 2.6 1.3
Separation. The mixture of sugars is applied to a paper 95 Chromatogram in I-pl. aliquots with the micropipet-buret in an 98 arrangement that will provide space for guide spots and in an 98 B amount t h a t will give a t least 90 y of the smallest constituent in 97 the mixture. I n the present studies the following arrangement 99 C of spots was adopted. 93 0,7 110 2.1 Chromatograms were prepared on 15 by 40 cm. sheets of Khatman No. 1 filter paper, having the long dimension parallel with the machine direction and the starting line 7.5 cm. from one end of the sheet. Three spots were applied on the starting line, tn o of rn hich were guide spots placed 2 cm. from the edges of the paper. The third spot, containing the sugars to be eluted, was This present procedure is also based on the colorimetric method in the form of a band 2 cm. long composed of five adjacent spots placed a t the center of the starting line This arrangement allows of Blass, Macheboeuf, and Sunez ( 4 ) >with the unique modifia space of 4.5 cm. between the guide spots and the center band cation that the color-producing reaction is carried out in a small of spots. volume of tetraethylene glycol dimethyl ether, a high-boiling After the paper has been spotted, the chromatogram is develsolvent. This modification provides a homogeneous reaction oped with a suitable partitioning solvent. Ammoniacal methyl ethyl ketone-water azeotrope provided good separation of mixture and enables better reproducibility and higher sensitivity methylated glucoses in less than 3 hours, duiing which time the than the original method, in which the reactants were heated in solvent front advanced approximately 35 cm This solvent the solid state. Thus, an 84Ycincrease in color intensity was obevaporated rapidly from the developed chromatogram and left tained in the determination of 2,3,4,6-tetra-0-rnethyl-~-glucose.no residues which interfered with the sugar determinations if the aqueous ammonia was added immediately before use. Best A further modification involves the use of crystalline aniline resolution was obtained by avoiding equilibration of the hydrogen phthalate (IS, 15), instead of an equimolecular mixture chromatogram in a solvent-saturated atmosphere prior to deof aniline and phthalic acid, for preparation of the reagent. The velopment. salt is stable indefinitely, and is easily purified by recrystallizaStrips containing the guide spots are cut from the developed chromatogram, sprayed with a suitable reagent, and heated under tion to eliminate dark-colored oxidative impurities commonly the groper conditions to indicate the osition of sugar spots formed in aniline. Use of the salt has the added advantage of S-dimethyl-p-amino(125 C. for 1 to 2 minutes in the case of ease of accurate weighing in making the reagent solution. aniline). With the aid of the treated guide strips, appropriate sections are then cut from the iinsprayed portion of the chromatogram, and the area of each sugar-containing section is measured APPARATUS A\-D 3IATERIALS so that a correction can be appl5ed to compensate for the interfering substances extracted from the paper. The usual apparatus for descending chromatography niay be Elution. Each section of paper containing a sugar component used ( 2 1 ) . is rolled, secured with a coil of nickel wire, and extracted under The Gilmont combination micropipet-buret (Emil Greiner Co., reflux ( 4 , IO) for 20 minutes (4)a i t h 5 ml. of 0.9% methanolic S e w Tork), a modification of the earlier ultramicroburet ( 1 2 ) ) tetraethylene glycol dimethyl ether, added from a pipet. proved convenient and accurate for use in applying sugar solutions In some cases-e.g , 2,3,4-tri-0-methy1-~-glucose in Table I, to chromatograms. mixture C, and in Table 11-the eluate must be diluted in order to obtain an absorbance within the optimum range of the spectroThe extraction apparatus, which enabled rapid and complete removal of sugars from cutout sections of paper chromatograms, photometer. In the present work, greatest accuracy was obtained x a s similar to that of previous workers (4,IO) except that a small in the range of 90 to 200 y of methylated glucose. Dilutions, of glass ring was fused to the drip tip of the reflux condenser. Paper course, must be made with the eluting solvent, 0.9% tetraethylene to be eluted was attached t,o this ring by means of a small wire glycol dimethyl ether in methanol, in order to provide the standhook. ard amount of glycol ether in the 5-ml. aliquots upon which the A constant temperature water bath, suitable for operation a t determinations are performed. I t is not feasible to make dilutions 90" to 98" C., was used. The water may be covered with a layer after color development, for such dilutions result in low values. of paraffin to minimize evaporation. Determination. The eluate or a suitable aliquot of it is mixed Selected borosilicate glass culture tuhes \yere used as cuvettes with 1 ml. of the aniline phthalate reagent, and the methanol is in conjunction with a Coleman Junior Model 6A spectrophotomremoved by evaporation in vacuo a t 30' C. (bath temperature). eter. The residual liquid is then heated for 35 minutes (Figure 1) a t The water azeotrope of methyl ethyl ketone ( 5 ) ,containing 1% either 98" C. in the case of aldohexose methyl ethers or 90' C. for by volume of aqueous concentrated ammonium hydroxide, was aldopentose methyl ethers. Shorter heating periods result in lower reproducibility used for developing chromatograms. In the absence of ammonia, this solvent mixture partially resolves individual sugars into their The flasks are then removed from the hot water bath and anomeric forms, causing elongation of sugar spots. The ketone was quickly cooled to room temperature. The small, red-brown pool saturated wit,h water and treated a t room t,emperature overnight of reaction products is dissolved in 10 ml. (from a pipet) of 95Y0
x',
ANALYTICAL CHEMISTRY
1292 ethyl alcohol, and the absorbance is measured with the epectrophotometer against 95% ethyl alcohol a t 415 mp for the methylated aldohexoses and a t 460 mp for the methylated aldopentoses. The curves in Figure 2, which were obtained with a Cary recording spectrophotometer, are representative of those of other methylated aldose sugars. The curve of the reagent blank (heated 35 mmutes a t 98' C.) indicates little interference a t the wave lengths of absorbance measurement. Because the colors fade after development (1 to 27, during the first hour, 5 to 10% overnight), the time interval between the end of the heating step and the measurement of absorbance should be held reasonably constant. The authors found a 30minute interval to be convenient. The reagent and paper blanks are subtracted from the absorbance reading, giving the net absorbance due to sugar in the eluate. As will be explained later, the use of mean values of the blanks, established by multiplicate (at least 10) determinations, is preferable to evaluation of the blanks with each sugar determination. The mean values apparently are applicable over extended periods of time. In the determination of methylated hexoses, the absorbance of the reagent blank was equal to that of 40 y of 2,3,4,6tetra-0-methyl-n-glucose, and the paper blank gave an equivalent of 0.4 y per sq. cm. of paper. For the methylated pentoses, which were heated a t a lower temperature, the reagent blank was one third of the value given for hexoses. For the determination of the paper blank, a known amount of sugar should be added to the extracting solvent to raise the absorbance to within the optimum range of the spectrophotometer. The determination is made on paper that has been chromatographically washed with the developing solvent. The reagent blank determination is made on extracting solvent plus reagent in the absence of sugar.
n
0
2,3,4-Tri-O-Mcfhyl-~-Xylore 0
o
u
Table 11.
Analysis of Methylated Dextran Hydrolyzate
(Dextran from L. mesenteroides N R R L B-512) Weight, Mole Sugar Presenta Y % 2,3,4,6-Tetra-O-methy~-n-giuoose 158 6.8 2,3,4-Tri-O-methyl-~-glucose 1929 88.7 2,4-Di-O-h1ethyl-~-glucose 92 4.5
Molar Ratio 1.5 19.8 1.0
Identified by derivative formation after separation on a cellulose column (23). (1
W
z 204Ln
4
\\
\.
'>.
\\
'$.
'?\
0
Figure 2. Spectral absorbance curves for reaction produ c t s of methylated sugars heated with aniline p h t h a l a t e
io
20
I
30
I
40
50
TIME, MINUTES
Figure 1. Intensity of color related to time of heating for two representative sugars
After the reagent and the paper blanks have been deducted, the weight of eluted sugar is determined by referring to a standard absorption curve which has been prepared previouely (Figure 3). The curves for all sugar8 de arted from Beer's law (in a reproducible manner) a t quantities elow 90 y . This was possibly due to instrument error.
%
RESULTS
Application of the above procedure of quantitative paper chromatography to synthetic mixtures of methylated glucoses yielded the results shown in Table I. The experimental values, which represent the mean of results from triplicate chromatograms, are given with the average deviation of the individual values from their mean. Recoveries from the chromatograms averaged within 4% of the amount spotted on the starting lines. The highest variation, lo%, was encountered in the analysis of mixture C, which contained the most unfavorable proportion of sugars. Within the sets of triplicate determinations the average deviation of individual values from the mean for the set was 2.17 0 , with a maximum of 4.7%.
The application of the method to a hydrolyzate of methylated dextran (Leuconostoc rnesenterozdes NRRL B-512) yielded the results in Table 11. The data reveal that 95.57, of the anhydroglucose units in the dextran studied were linked a t either the 1- or the 1- and 6- positions, in good agreement with 94.6% found by periodate oxidation studies ( 1 6 ) and 95.5 to 96% found by periodate oxidation followed by reduction and hydrolysis ( 1 ) . Analyses of mixtures of this type, in which one component sugar was present in a high proportion, were made possible by the excellent resolution of sugar spots obtained with the ammoniacal methyl ethyl ketone-water azeotrope. The dextran studied had been methylated to 45.37" methoxyl in a 60% yield and had been hydrolyzed to constant rotation with a hydrochloric acid-glacial acetic acid mixture ( 2 4 ) similar to that of Bell (3) In addition to analyses of hydrolyzates, this procedure was also applied to a study of the extent of demethylation which occurred when various partially methylated glucoses were subjected to conditions of acid hydrolysis (@), and to the analysis of cellulose column fractions in which overlapping of sugars occurred. Exploratory experiments with 2,3,4,6-tetra-o-methyl-n-galactose and 2,3,4,6-tetra-O-methyl-n-mannose, as well as the work with 2,3,4tri-0-rnethyl-n-xylose, indicated that this method is generally applicable to methylated aldose sugars. The above fully methylated sugars gave colors of somewhat greater intensity than that produced by the same weight of 2,3,4,6-tetra-O-methyln-glucose. When the color intensity of the latter was made equal to 100, the intensity of the galactose derivative was 124; that of the mannose derivative, 106; and that of the xylose derivative (heated a t 90" instead of 98' C.), 121. Determinations of 1,3,4,6-tetra-0-methyl-~-fructose gave negative results. This was to be expected because aniline phthalate does not produce colors with ketoses.
V O L U M E 2 8 , NO. 8, A U G U S T 1 9 5 6
1293
Table 111. Variations in Experimental Factors Which Produce Change of =!=1$7~ in Absorbancea Relative Standardized Tolerance, Variable Value f Aniline phthalate reagent 2 1 1 mg. 0 . 3 mg. Tetraethylene glycol dimethyl ether 45 0 mg. 1 4 mg. Temperature of heating 980 c. 30 c. Time of heating 35 min. 4 minutes Wave length 0-Methyl aldohexoses 415 m e 10 mu 0-Methyl aldopentoses 460 mp 10 m p a Determined for 150 y of 2,3,4,G-tetra-O-methyl-~-giucose.
DISCUSSION
The present colorimetric procedure is particularly well adapted
to the determination of aldoses resolved by paper chromatography.
I n contrast to procedures using strong mineral acids
determined graphically, represents the amount of change in the given variable that would produce a change of i1yo in a determination of 150 y of 2,3,4,6-tetra-C)-methyl-n-ghcose. The tolerances are presented in this manner in order to indicate the relative influence exerted by variations in the factors encountered in this procedure. Solvent Media for Color Reaction. An important consideration was the choice of solvent to serve as the medium for the color-producing reaction. I n the present studies, several highboiling solvents were examined, but none proved so satisfactory as tetraethylene glycol dimethyl ether When glycerol or tetraethylene glycol was used, the reaction mixtures became so viscous during the heating step that difficulty was encountered in dissolving the colored products in 9570 ethyl alcohol to give the solution needed for spectrophotomet1 ic measurement. This increase in viscosity was possibly due to the formation of acetals (14) or to polymerization (18). iinisole was found to be too volatile, and mineral oil was eliminated because it failed to dissolve aniline hydrogen phthalate.
(7-9), this method does not require the removal of extraneous
cellulose particles, which are inevitably present in eluates from filter papers. While the present method is less sensitive than those using mineral acids, the amount of aldose required (90 to 200 y ) is readily obtained in most chromatographic separations. Accuracy and Reproducibility. I n 14 single determinations, not involving chromatographic separations, on samples of 2,3,4,6tetra-0-methyl-n-glucose which contained 145 y of sugar, the mean value obtained colorimetrically m-as 145 y , with a standard deviation of 5.6 y (3.8Y0of the mean). These determinations m-ere performed over a period of 3 months and were based on the standard curve of Figure 3. The reagent used was of varying age a i d was obtained from a number of successive preparations. Thus, it is seen that a known solution need not be included viith each set of determinations. During the course of the work, i t was observed that variations of the reagent blank were not consistent with variations of the absorbance of the corresponding sugar sample-Le., it appeared that the absorbances of the sugar samples and of the blanks varied independently. Therefore, sugar determinations have been calculated on the basis of an established mean value for the blank. If, instead, the above 14 determinations are calculated on the basis of the individual blanks which were run simultaneously nith each determination, the mean value becomes 143 y r i t h a standard deviation of 6.2 y (4.47, of the mean). .4 study of the effects of some selected variable factors yielded the data given in Table 111. I n each case the relative tolerance,
ACKNOWLEDGMENT
The authors are grateful to I. A. Wolff for his many valuable suggestions, both during the experimental work and in the preparation of the manuscript. They are aleo indebted to E. H. Melvin and T. A. Scott for spectral absorbance curves, and to J. E. Hodge for the sample of methylated NRRL B-512 dextran. L I T E R A T U R E CITED
;Ibdel-Akher, AI., Hamilton, J. K., Montgomery, R., Smith, F. J . Am. Chein SOC.74, 4970 (1952).
Bartlett, J . K., Hough, L., Jones, J. K. S . , Chemistry & Indu8t r y 1951, 76.
Bell, D. J . , Biochem. J . 29, 2031 (1935). Blass, J., hlacheboeuf, lI.,liunez, G., BuZl.
SGC.
chim. biol. 32,
130 (1950).
Boggs, L., Cuendet, L. S.,Ehrenthal, I., Koch, R., Smith, F., .Vature 166, 520 (1950).
Bourne, E. J., Peat, S., “Advances in Carbohydrate Chemistry,” vol. V, pp. 160, 172, 186, Academic Press, New York, 1950. Devor, A. W., J . Am. Chem. Soc. 72, 2008 (1950). Dimler, R. J., Schaefer, W.C., Wise, C. S., Rist, C. E., ANAL. CHEM.24, 1411 (1952). Dubois, hl., Gilles, K., Hamilton, J. K., Rebers, P. A., Smith, F., A’ature 168, 167 (1951).
Flood, -4. E., Hirst, E. L., Jones, J. K. N., J . Chem. SOC.1948, 1679.
Gardell, S.,Acta Chem. Scand. 5, 1011 (1951). Gilmont, R., AKAL.CHEM.20, 1109 (1948). Graebe, C., Ber. 29, 2803 (1896). Hoover, K. H., U. S. Patent 1,934,309 (Xov. 7, 1934). Hough, L., Jones, J. K. N., ’Tadman, TT’. H., J . Chem. Soc. 1950, 1702.
Jeanes, A., Wilham, C. -4., J . Am. Chem. SOC.72, 2655 (1950). Kent, P. W.,Chemistry &. I n d u s t r y 1952, 1176. hleyer, K. H., “High Polymers,” vol. IV, 2nd ed., p. 243, Interscience, New York, 1942. Muskat, I. E., J . Am. Chem. S O C .56, 693 (1934). Partlidge, S. AI., A’ature 164, 443 (1949). Stenard, F. C., Stepka, W.,Thompson, J. F., Science 107, 461 (1948).
Van Cleve, J. W., unpublished work. Van Cleve, J. W., Schaefer, W. C., J . Am. Chem. SOC.77, 5341 (1955).
’,‘ /:,
++’
I 40
I
I
80 I20 M I C R O G R A M S OF SUGAR
I 160
J
Van Cleve, J. W., Schaefer, W. C., Rist, C. E., Abstracts of Papers of 125th Meeting, AMERICAN CHEMICAL SOCIETY, p. 8D, 1954.
200
Figure 3. Typical absorbance curves for colored products obtained from methylated sugars
RECEIVED for review September 21, 1955. ilccepted March 29, 1956. Division of Carbohydrate Chemistry, 124th Meeting, ACS, Chicago, Ill., September 1953. T h e use of trade names in this paper does not necessarily constitute endorsement of these products nor of t h e manufacturers thereof.
