V O L U M E 2 6 , NO. 8, A U G U S T 1 9 5 4 is 505.3 mp, checking with that giving the smaller coefficient of variation, 2.568 a t wave length 505 mp. The two prediction equations, a t 495 mp and 505 mp, may not be significantly different from each other. Errors in rounding out could have produced the observed differences between Equations 3 and 4. In terms of relative error, Equation 4 is better than Equation 3. It may be concluded that wave length 505 mp is the one a t which the mean attenuation index gives the minimum error when it is used to calculate from Equation 4 the total area bel017 the attenuation index curve within the visible region of the spectrum. Various attempts to reduce the observed deviations to less than 13% in one case did not lead to improvements. Those morkers who are inclined to place reliance on the average attenuation index over the visible spectrum (400 to 700 mp) may find it advantageous to use wave length 505 mp as the one giving a n approximate average measure of the concentration of coloring matter in raw sugars. ACKNOWLEDGMENT
Thr writers aish to express thcir thanks to Irving Lorge and
1365 Leo S. Goldstein, Teachers College, Columbia University, for the statistical analyses, and for reviewing the manuscript. LITERATURE CITED
Deitz, V. R., Pennington, S . L., and Hoffman, H. L., Jr., J . Research .Vat2. Bur. Standards, 49, 365 (1952). Hardy, A. C., "Handbook of Colorimetry," Cambridge, Mass., Massachusetts Institute of Technology, 1936. SOCIETY, Judd, D. B., 119th Meeting, .kMEHICAN CHEMICAL Boston, Mass., April 1951. Lorge, Irving, Sattler, Louis, and Zerban, F. R., IND.ESG. C H E M . , -4N.4L. ED.,4, 435 (1932). Peters, H. H., and Phelps, F. P., Bur. Standards, Technol. P a p e r , 338 (1927).
Zerban, F. W., and Sattler, Louis, ISD.ESG.CHEM.,ASAI.. ED., 9, 229 (1937). Zerban, F. W.,Martin, James, Erb, Carl, and Sattler, Louis, I b i d . , 24, 168 (1952). Sattler, Louis, and Martin, James, AN.4L. CHEM., Zerban, F. W., 23, 308 (1951). RECEIVED for review September 26, 1953. Accepted M a y 5 , 1954. Presented before the Division of Carbohydrate Chemistry, Symposium on .4nalytical Methods and Instrumentation Applied to Sugars and Other Carbohydrates, a t the 124th Meeting of the .4srERICnN CHEMICAL SOCIETY, Chicago, Ill.
Hydrolysis of Polysaccharides by a Cation Exchange Resin and Identification of Monosaccharide Components by Paper Chromatography R. E. GLEGG and DAVID ElDlNGER Department
of Anatomy, M c G i / / University, Montreal, Canada
C
-4TIOS exchange resins have been used for hydrolysis of di- and polysaccharides ( 1 , 7 , 9) and proteins (8). More recently, carbohydrate-protein complexes have been hydrolyzed in a similar manner prior to identifying monosaccharide components by paper chromatography ( 3 ) . The present paper describes the detailed analytical aspects of this technique, including control experiments designed to test the effect of the hydrolysis medium on monosaccharides representative of various chemical classes (pentoses, methylpentoses, aldohexoses, ketohexoses, hexuronic acids, hexosamines, and acetylated heyosamines), as well as di-, tri-, and polysaccharides. The treated and untreated solutions were analyzed qualitatively by chromatography. A comparison of the results provided information concerning the degree of decomposition of the monosaccharides and the hydrolysis of di-, tri-, and polysaccharides under various conditions. In this way, suitable conditions were established for the identification of monosaccharides by chromatography after the hydrolysis of carbohydrates or carbohydrate-protein complexes. EXPERIMENTAL
Resin. Permutit Q, a polystyrene sulfonic acid type cation exchange resin, was acid-regenerated by shaking with 4.4-V hydrochloric acid (900 ml. per liter of resin) in a separatory funnel. It was repeatedly washed with large volumes of distilled water until free of chloride ions. The final pH of the washings was 6. The resin was then air-dried. Effect of Hydrolysis Medium on Mono-, Di-, Tri-, and Polysaccharides. Two milliliters of 1% solutions of each of the saccharides listed in Tables I and I1 were heated a t 100" C. in sealed tubes for 48 and 96 hours with 1-gram portions of resin. As controls, 2 ml. of 1% solutions of the monosaccharides (Table I ) were heated (in the absence of resin) for 96 hours a t 100" C. to determine the degree of decomposition due to heating alone, and also incubated with 1 gram of resin a t 22" C. for 48 hours (likewise in sealed tubes) to determine the degree of adsorption of the monosaccharides by the resin. Three-microliter aliquots of the treated solutions were taken directly from the tubes for analysis by paper chromatography. (However, in the case of su-
Table I. Relative Intensitiesn of Sugar Spots after Treatment with and without Resin Substances
48
Resin a t 220
c.
Time, Hours 96 96 48 Treatment of Solution Without resin a t 100' C. Resin a t 100' C.
Pentoses Arabinose Xylose Ribose Nethylpentoses Fucose Rhamnose -4ldohexoses Galactose Glucose +++T ++t+ Mannose ++T+ A++ Ketohexoses Fructose Sorbose Hexuronic acids Galacturonic acid + Glucuronic acid a Control untreated solutions, containing amounts of sugar which would have been present if treatment had no effect, were also placed on chromatograms a s markers. Control spots were given a rating of regardless of intensity. Spots obtained after treatment were compared visually and given relative ratings.
++++ ++++
++ +
++
++ ++ + ++ +++
++++ ++++ ++++
+++ +++ +++
++ +
++++
++' +++ +++
+++ +++ ++++ ++++
++++ +++
+++
++ + ++ + +
++ + +++
n o s e and raffinose, 6 and 9 pl., respectively, were used, as sucrose contains two, and raffinose three different monosaccharide units). Effect of Hydrolysis Medium on Amino Sugars and Acetyl Derivatives. Two milliliters of 1% solutions of glucosamine and S-acetyl glucosamine R ere subjected to the three treatments described above for monosaccharides. The resin which was recovered from the last treatment was thoroughly washed with water, and then eluted three times with 2-ml. portions of 0.5N hydrochloric acid. The eluates were evaporated to dryness, and the residues were dissolved in 0.5 ml. of water.
1366
ANALYTICAL CHEMISTRY RESULTS AND DISCUSSION
T a b l e 11. Relative Intensities" of Sugar Spots Produced b y Resin Hydrolysis of Di-, Tri-, and Polysaccharides Substanaes
48
Time of Hydrolysie. Hours 96 48 96
48
Products of Hydrolysis Galactose Glucose Cellobiose Maltose Sucrose Raninose Glycogen Starch
+++
++
Fructose
++++ ++++ ++ ++ ++++ ++
+++ +++ +++
SO
-
++
-
++ ++
I n addition, to determine whether amino sugars could be recovered from mucopolysaccharides. 50 m& each of chondroitin ~
~
~~~
~~
evaporated to dryness, and th; residues were dissolved in 0.1 &I: of water. Six-microliter aliquots of each of the solutions were used for chromatographic aialysis. Paper Chromatography. The solutions were analyzed by unidimensional ascending paper chromatography. Rectangular sheets (27 X 38 cm.) of Schleicher and Schuell No. 580 ivhitc r i b b o n p a p e r were used. The sheets were cut so that the long sides were in the machine direction. Aliquot8 of the solutions u-ere plaoed a t individual points of origin along a line 4 em. above one of the narrower RHAh edges of the paper. The long sides of the paper were approximated, stapled securely, and Fl the resultant cylinder was developed in a butyl alcohol-pyridine-water solvent ( 8 ) . T h e solvent was allowed to rise to the tap of t,he MAP Dauer three times in the same direction ( b ) , eaoh development requiring about 22 hours. Reducing sugars GLI and uronic acids were identified by spraying the dried pitper uniformly GALA with aniline hydrogen oxalate ( 4 ) ; amino sugars by spraying Kith acetyl acetone dimethylaminobenzaldehyde
Monosaccharides (Table I, Figure 1). Pentoses, methylpentoses, and aldohexose8 were easily identified on paper chromatograms after their solutions were heated a t 100' C. with the resin for 48 or even 96 hours, although the intensities of the spots indicated varying degrees of decomposition (last two columns of Table I). None of these substances u-as significantly adsorbed by the resin a t 22" C. (column 2). The slight decomposition of the aldohexoses can thus be attributed to heating in the presence of mater a t 100' C. (column 3),but the resin appeared to catalyze the decomposition of pentoses and methylpentoses. Ketohexoses uwe not detectable after heating with the resin for 48 hours. Thus the decomposition observed by heating with mater alone is catalyzed further by the resin. However, mineral acid hydrolysis also completely destroys ketoheaoses. Hexuronic acids are destroyed by heating at 100' C. with or without resin for 96 hours, but enough WRS preserved for identification when the hydrolysis was limited to 48 hours. Under all these conditions decomposition was accompanied by develop ment of dark brown solutions. When the solutions were chromatographed, no interference was caused by the brown material, since the uronic acids traveled a8 umal and were the only substances observed above the origin. Glucosamine and N-acetyl glucosamine were destroyed by hesting without resin a t 100" C . However, glucosamine waa protected from destruction by the presence of resin even after heating for 96 hours. This effect may be explained by the adsorption of glucosamine on the resin. N-Bcetyl glucosamine was PENTOSES
SES
I X ARAB
(a).
I n order to estimate the effect of the various treatments on the saccharides of known structure, the intensities of the spots resulting from 3-pl. aliquots of the treated 801"tions were compared visually with those from 3 pl. of 1% untreated solutions of the corresponding sugars (or known m o n o s a c c h a r i d e c o m ponents in the cme of di-, tri; and polysaccharides) placed on the same piece of paper and developed a t the same time. Because the various control untreated sugars gave spots of different colors and intensities, they were all given an arbitrary rating regardless of the intensity, and the resin-treated material compared with these as standards. The treated materials (Tables I and 11) were rated or -, depending on whether the spots showed no, a very slight, a moderate, or a drastic reduction of intensities, or were completely invisible, respectively. (The spots rated as were still easily visible.)
++++,
++++, +++, ++, +, +
C
C
Figure I. L-,,=X U ~ L V ~ U ~ L V E T ~ Ianuwxng I I Positions ana intensities of >pots tor Methylpentoses (Greenish Brown), Aldohexoses (Reddish Brown), and Pentoses (Pink) Columns demonstrate e5e0ts oi various trektrnent.8 on these monoeaooharides A . Untreated B . 1noubatedr.itIloutresinat 1OO'C. for96 hours Cand D . Incubated vxhresin a t 100' C. for48andS6 hours, respeetivel Original ~olutionswere 1%, and 3 - 4diqnote were used for ohrom&togrspXy
Chromatograms were smayed ivith aniline hydrogen oxalate A l l monosaccharides were run o n eame piece of paper from common line of odd"; photograph was in twofor eonvenioneein labeling
V O L U M E 2 6 , NO. 8, A U G U S T 1 9 5 4 deacetylated under the influence of the resin and the free glucosamine was likewise absorbed and protected. It was found possible to elute the hexosamine from the resin with 0.5N hydrochloric acid and then t o identify it on chromatograms as the hydrochloride. These results indicated that, with the exception of ketoses, the monosaccharides were detectable after heating with the resin for 48 hours. Di-, Tri-, and Polysaccharides. The di-, tri-, and polysaccharides n ere hydrolyzed with the resin for periods of time ranging from 12 to 96 hours. In general, maximum intensities of sugar spots were obtained after 48-hour hydrolysis. Sucrose, cellobiose, maltose, starch, and glycogen gave rise to glucose only, and raffinose gave rise to glucose and galactose (Table 11). In agreement nith the fact that ketohexoses were easily destroyed. no fructose was detected in the hydrolyzate from sucrose or raffinose. Since uronic acids were only just detectable after their solutions were treated with the resin for 48 hours (Table I), polysaccharides known t o contain these substances as structural units (pectin, chondroitin sulfuric acid, and heparin) n-ere hydrolyzed for periods of time ranging from 12 to 96 hours and the hydrolyzates were chromatographed. The uronic acids in these three substances were easily detectable up to 72 hours, with maximum intensities a t 48 hours. The data suggest that the hydrolysis of the polysaccharides is initially a slow process, so that the free uronic acid liberated during the latter part of the hydrolysis is not completely destroyed. Hexosamines eluted from the resin after the hydrolysis of heparin and chondroitin sulfuric acid were identified as glucosamine and galactosamine, respectively. (As N-acetyl galactosamine is a component of chondroitin sulfuric acid, this provided further evidence that the resin deacetylates N-acetylhexosamines). The resin hydrolyzates of carbohydrate-protein complexes were almost colorless (in contrast to the dark brown solutions obtained
1367 after hydrolysis with I S hydrochloric acid at 100" C. for 8 hours), except when glucuronic acid was a component,. Monosaccharide components were clearly ident'ified without further treatment of the solutions ( 3 ) . The paper chromatographic technique provided clear separations of galactose, glucose, mannose, fucose, and rhamnose in a single mixture (Figure 1). Ribose when added to such a mixture could also be identified despite its proximity to fucose, owing to the differences in color produced by aniline hydrogen oxalate. The clarity of the separations is a result of the combination of several factors-the solvent used, the technique of t'riple development,, and the fact that t,he solvent &-asallowed to move in the machine direction of the paper. S o n e of the monosaccharides yielded decomposition products which could be identified on the chromatograms; and di-, tri-, and polysaccharides yielded only those monosaccharides which were expected on the basis of their known structural units. The overall analytical technique, involving hydrolysis in the presence of a cation exchange and paper chromatography of the hydrolyzates is therefore dependable and free from artifacts. LITERATURE CITED
(1) Bodamer, G., and Kunin, R., I n d . Eng. Chem., 43, 1082 (1951). (2) Chargaff, E., Levine, C., and Green, C. J., B i d . Chem., 175, 67
(1948).
(3) Glegg, R. E., Eidinger, D., and Leblond, C. P., Science, 118,
614 (1953). (4) Horrocks, R. H., and Manning. G. B., Lancet. 256, 1042 (1949). (5) Jeanes, A, Wise, C. S., and Dimler, R . J., Ax.4~.CHEM.,23, 415 (1961). (6) Partridge, S. AT., and Westall, R. G.. Bioehem. J., 42, 233 (1945). (7) Sussman, S., Ind. Eng. C h e n . , 28, 1228 (1946). (8) Underwood, G. E., and Deatherage, F. E., Science, 115,95(1952). (9) Wadman, W. H., J . Chem. Soc., 3061 (1952). RECEIVED for review September 23, 1953. Accepted .4pril 20, 1954. Work supported by a grant from the National Cancer Institute of Canada t o C. P. Lehlond.
Determination of Moisture in Sodium Bicarbonate Karl Fischer Method L E A V I T T N. G A R D and ROBERT C. BUTLER' Columbia-Southern Chemical Corp., Barbetton, O h i o
T
HE presence of small amounts of moisture in sodium bicar-
bonate inhibits the flowability of the product and promotes lump formation during storage ( 6 ) . The occurrence of these undesirable effects has created a need for an accurate and sensitive method for measuring the moisture content of commercial sodium bicarbonate. The U. S. Pharmacopoeia (8) directs the drying of 3-gram portions of sodium bicarbonate contained in a low-form weighing bottle over concentrated sulfuric acid in a desiccator for 4 hours prior to assay, No recommendation is made to utilize the weight loss from this operation as an index of the moisture content of the sample. In fact, tests of this type have indicated the inadequacy of the desiccation period under such conditions. Actually, dessication periods of 24 hours rarely showed weight loss values (moisture) greater than 0.02%, with values of 0.01% predominating in virtually all cases. Occasionally samples showed a slight gain in weight resulting from the initial desiccation treatment and, in such cases, an additional 24hour dessication treatment was conducted to obtain a measurable weight 1
Present address, Faultless Rubber Co.. riahland, Ohio.
loss of the sample. Analyses obtained by a procedure of this type are unsatisfactory and are often open to considerable question and doubt. A review of general methods for determining moisture in solids ( 1 ) suggested potential application of the Karl Fischer method ( 3 ) described by Wernimont (9) involving the use of the deadstop end point (4). Application of this method to sodium bicarbonate involves an extraction technique with anhydrous methanol (6, 7 ) and takes into account the interference of bicarbonate ( 2 ) which is also extracted slightly from the sample along with the moisture. It was the purpose of the work to adapt the Karl Fischer technique of moisture determination to sodium bicarbonate and to give a typical analysis of the commercial product. EXPERIMENTAL
The general procedure for determining moisture in sodiuni bicarbonate by the Karl Fischer method involved extraction of the moisture in the sample with dr,v methanol, filtration to separate the extract, titration of an aliquot of the extract with Karl Fischer reagent to measure the moisture and a small amount of
