V O L U M E 2 7 , NO. 1, J A N U A R Y 1 9 5 5
33 acid in the region of p H 5 will coiisume acid, since the glutamate with a net negative charge is converted to the net uncharged aminobutyrate and carbonic acid. In this case advantage is taken of the fact that the alpha-carboxyl group of the amino acid ie a much stronger acid than carbonic. Thus, if the pH of the experiment is carefully selected, the types of enzyme-catalyzed reactions which can be investigated by constant pH titration are almost unlimited. Titration a t constant p H may be applied for a variety of uncatalyzed reactions. Amino groups may be determined, inasmuch as a stoichiometric amount of acid is generated when these groups are benzylated with reagents such as dinitrofluorobenzene (6). The alkaline decomposition of nucleoproteins such as tobacco mosaic virus may also be followed quantitatively, since this reaction liberates acid. The data in Figure 6 show that the apparatus is useful for determining completeness of reaction and equilibria as well as for rate measurements.
VOLUME OF TITRATION FLUID
Figure 6. Reduction of Diphosphopyridine Nucleotide by Lactic Dehydrogenase of Heart at pH 9.50 and 25” C. Initial concentrations. Sodium DL-lactate, 0.2.M; diphosphopyridine nucleotide, 1 O - a M . crystalline heart lactic dehydroOther ionditions same as for experiment genase, 2 X IO-aM. described in Figure 5
of its activity, a figure proportional to the enzyme concentration can be obtained. ,4 spectrophotometric method, when applicable, is undoubtedly the most convenient, sensitive, and accurate way to a m y an enzyme. For example, all pyridine nucleotiderequiring enzymes, including the lactic dehydrogenme used in the above experiments, are so assayed (8). Generally speaking, however, it is not possible to devise a spectrophotometric assay for the vast number of enzymes which catalyze hydrolytic reactions. Acetyl esterase (described above) and such important enzymes as acetylcholine esterase fall into the latter category. The use of constant p H titration as a means of assaying lactic dehydrogenase has been demonstrated here only to emphasize that this technique is not restricted to hydrolytic enzymes. The amino acid decarboxylases have been hitherto studied almost exclusively with cumbersome manometric methods, but there is no reason why such enzymes cannot also be amayed by titration (3). For instance, the decarboxylation of glutamic
ACKNOWLEDGMENT
The authors are indebted to R.A. Alberty and George Lauterbach for helpful discussions on certain phases of this work. Financial assistance was provided through a grant from Eli Lilly and Co. The apparatus described in this paper may be obtained by special order from the International Instrument Co., Canyon, Calif. LITERATURE CITED (1) Harris, S. A., Webb, T. J., and Folkers, K., J .
An. Chem. Sw., 62, 3198 (1940). (2) Jacobsen, C. F., and LQonis,J., Compt. rend. trao. lab. CarZabero, S&. chim., 27, 333 (1951). (3) Jang, R.,and Axelrod, B., personal communication. (4) Jansen, E. F.,Nutting, hI. D. F., and Balls, A. K., J. Biol. C h a . , 175,975 (1948). (5) Lew.8.L.. Dersonal communication. (65 Linpane, J.’ J., “Electroanalytical Chemistry,” Interscience Publishers,New York, 1953. (7) Neilands, J. B., J . B i d . Chem., 199, 373 (1952). (8) Neilands, J. B., in “Methods in Enzymology,” ed. by S. P. Colowick and N. 0. Kaplan, -4cademic Press, New York, in press.
(9) Parke, T.V., and Davis, W. W., - 4 s ~CHEM., ~ . 26,642 (1954). (10) Straub, F. B., B i o c h a . J., 34, 483 (1940). (11) Williams, V. R., and Neilands, J. B., ATch. Biochem. Bwphys., in press. RECEIVED for review July 23, 1954. Accepted September 25, 1954.
Determination of Easily Hydrolyzable Fructose Units in Dextran Preparations C. S. WISE, R. J. DIMLER, H. A. DAVIS, and C. E. RlST Northern Utilization Research Branch,
U. S, Department o f Agriculture,
Characterization of dextran preparations required analytical methods for small amounts of free and combined fructose, present as impurities or minor constituent-units in the glucose polymers. A qualitative procedure was developed to detect as little as 0.01% free or easily liberated fructose in dextran samples. The two quantitative colorimetric methods which were developed measure, in addition, at least part of the fructose in such compounds as melezitose and leucrose, from which it is difficultly liberated. The method using a modified anthrone reagent also is particularly suitable for quantitative paper chromatography of fructose. In both this and a modified resorcinol method the color-forming power of glucose is limited to about ‘/uath that of fructose. These qualitative
Peoria, 111.
and quantitative methods should prove useful for obtaining information on the amount and relative ease of hydrolysis of fructose units in many natural and enzymically synthesized products.
T
H E glucose polymers known as dextrans are formed from sucrose by the action of microorganisms such as Leuconostoc mesenteroides or of enzyme solutions derived from cultures of the organisms ( 4 ) . The dextran preparations may contain fructose in any of several forms. Thus, levan, an easily acid-hydrolyzed polymer of fructose, often is formed along with dextran (8) and may remain as a contaminant during isolation of the dextran. Free fructose or sucrose in the medium may be carried down with the dextran. Because the disaccharide leucrose (5-D-glUCOpy-
34
ranosyl-n-fructose) has been obtained in the enzymic synthesis of dextran (14), this and other difficultly hydrolyzed compounds of fructose also are potential contaminants of dextran preparations, Finally, fructose units may be present in small amounts as structural parts of dextran molecules-for example, as easily hydrolyzed terminal fructosyl units (6) or as difficultly liberated units carrying glucosidic linkages on one or more of the hydroxyl groups. For fundamental studies of dextrans and their structures it is essential, therefore, that analytical evidence be obtained on the presence of fructose in dextran samples. To fill the need for methods of fructose determination adapted t o the research on dextran, one qualitative and two quantitative methods were developed. These methods were devised with particular emphasis on the ability to measure amounts of fructose below 1% of the dextran sample. They are based, in general, on known procedures and principles, with modifications as necessary to meet the specific requirements of their proposed use. I t was particularly important to avoid interference from glucose and its polymers. QUALITATIVE CHROMATOGRAPHY PROCEDURE FOR FREE OR EASILY LIBERATED FRUCTOSE
The qualitative procedure was developed specifically to detect free fructose and its easily hydrolyzed combinations, such as levan or sucrose, in contrast to fructose in more difficultly hydrolyzed combinations such as leucrose ( 5-D-glucopyranosylD-fructose, 14), melezitose, and possibly some dextran molecules. The procedure provides for chromatographic identification of the liberated fructose and gives evidence of the ease of hydrolysis. I n addition, quantitative determinations have been performed by an extension of the method. The detection of easily liberated and free fructose in dextran preparations is based on paper chromatography of the ethyl alcohol-soluble sugars formed by limited acid hydrolysis of the sample. When free fructose only is to be detected, the hydrolysis step is omitted. Under the conditions selected for hydrolysis (0.2N sulfuric acid a t 70' C. for 1 hour) the recoveries of fructose from sucrose and levan samples were over 90%, as measured by quantitative paper chromatography. The hydrolysis of dextran, however, amounted to only about 0.2 to 0.9%, as measured by reducing power expressed as glucose equivalent. Even so, suitable developing solvents and a selective spray reagent must be used to avoid interference from glucose in the chromatography step, especially when dextran samples containing only a small amount of fructose-e.g., 0.5% or less-are being studied. An improved spray reagent for the selective detection of fructose in the presence of aldoses on the paper chromatograms was obtained by replacing the hydrochloric acid in the urea-hydrochloric acid reagent of Hough et al. (7) with phosphoric acid, as suggested for several other reagents by Bryson and hIitchell(2). The urea-phosphoric acid reagent provides a better color differentiation between fructose and glucose and excellent freedom from background color on the paper, as compared with the urea-hydrochloric acid reagent. Fructose gives a characteristic bluegray color, in sharp contrast to the light brown color given by much higher concentrations of glucose. On standing overnight and longer, the fructose spot becomes gray and then gray-brown, but otherwise is stable. About 25 to 50 times as much glucose is required to give as intense a spot as fructose if the heating period is not too long. I t is possible, after development of the chromatogram, to detect as little as 4 y of fructose in the presence of a t least 100 times as much glucose. After the limited hydrolysis of the dextran sample, careful control of the p H of the solutions is advisable to avoid alkaline rearrangement of the glucose present to fructose. The use of bromothymol blue as indicator together with neutralization of the sulfuric acid with barium hydroxide permitted the pH to be held easily a t about 6.1 to 6.6. Earlier trials involving neutralization with an excess of barium carbonate gave much less satisfactory control of p H (see also 11). The indicator does not interfere
ANALYTICAL CHEMISTRY with paper chromatography of the sugars, as its R/ value is nearly unity in the developing solvent used. Apparatus and Reagents. Thermoregulated water bath a t 70' c. Apparatus for paper chromatography ( 3 , Q ) . Solution of 0.2,V sulfuric acid. Approximately 0 . W barium hydroxide. Bromothymol blue indicator. Urea spray reagent made up as follows: To 100 ml. of 1 X phosphoric acid in water-saturated butanol (about 80% butanol by m i g h t ) add 3 grams of urea, followed by about 5 ml. of ethyl alcohol to eliminate the water phase which forms when the urea dissolves. The reagent is stable for several months. Procedure. To 5 grams, or less, of dextran sample add enough 0.2,V sulfuric acid to give a 5% carbohydrate concentration and heat the mixture a t 70' C. for 1 hour. Keutralize the cooled solution with approximately 0.4hr barium hydroxide, using bromothymol blue as an indicator. Add, with stirring, enough absolute ethyl alcohol to give a solution Thich contains 85% alcohol by volume. Remove the barium salts together with the precipitated dextran by decanting and centrifuging the solution. Evaporate the supernatant solution in vacuo to dryness. When low concentrations of combined fructose-e.g., below about 1% of the sample-are involved, a second alcohol precipitation from a smaller aqueous volume may be necessary to reduce further the amount of par tially degraded dextran, the presence of which can cause elongation and streaking of the sugar spots on the chromotogram. I n extreme cases, where spot elongation still has not been avoided because of remaining dextran or salts, the fructose-containing area of a duplicate chromatogram can be eluted with water (S),the eluate evaporated to dryness, and the residue rechromatographed. Dissolve the dried solubles from the precipitation step in a drop or two of water and transfer the solution with an ultramicroburet to a paper chromatogram, using patterns of contiguous spots ( 3 ) . Develop the chromatogram once or twice with the butanol-pyridine-water (6 to 4 to 3) solvent mixture ( 9 ) and then dry and spray with the urea-phosphoric acid reagent. After drying the sprayed paper a t room temperature, heat it in an oven for several minutes a t 100' to 110" C. The presence of fructose is indicated by a blue-gray spot whose position corresponds to that of a known sample of fructose on the same chromatogram. For quantitative paper chromatograph.;, perform the spotting and eluting of strips containing the unknown as previously described ( 3 ) and meagure the fructose by the alcoholic anthrone method described below. If the amount of fructose is smalle.g., 50 to 100 y per strip-collect the eluate directly in weighed test tubes, dilute xith water to a weight of 2.00 grams and use the entire quantity for one anthrone determination. QUANTITATIVE FRUCTOSE METHOD USING ALCOHOLIC ANTHRONE
The fact that fructose reacts more rapidly than glucose in the color-forming reaction of the anthrone determination of total carbohydrate had been noted in this laboratory ( I S ) as well as by Koehler (10). This observation, together with experience prrviously gained ( 3 ) in the use of the anthrone reaction. prompted studies leading to the present modification of the anthrone reaction for the determination of fructose in free and most combined forms. The fructoPe procedure differs from the total carbohydrate method in the use of a lower temperature (50') and a lower concentration of acid in the reaction mixture, so that incomplete reaction of fructose and very limited reaction of glucose occur. Dilution of the anthrone-sulfuric acid reagent before use avoids heat of mixing which xould result in uncontrolled color formation. The use of ethyl alcohol, instead of water, as diluent provides a twofold advantage of a more intense color and avoidance of precipitation of anthrone a t the lower concentration of sulfuric acid. The alcoholic anthrone-sulfuric acid reagent is advantageous in several respects. The small sample size required makes the reagent suitable for the measurement of fructose by quantitative paper chromatography, since 25 to 100 y of fructose suffices for a determination. I n the analysis of dextran samples, the small sample size permits analysis of dextrans which give hazy solutions without need for the corrective steps described for the resorcinol procedure below. For known mixtures the results were
V O L U M E 2 7 , N O . 1, J A N U A R Y 1 9 5 5 accurate within &2% for concentrations of fructose in dextran a t least as l o a as 0.04% (see Table I). In addition, the precision of the method was very good, as shown by the fact that each of the values under column 3 of Table I is the average of duplicates having an average deviation within 0.0003 or 0.0004 mg. of fructose. Apparatus and Reagents. Spectrophotometer-e.g., Coleman Model 11-adapted, if necessary, for use of the reaction tubes in the cuvette carrier. Borosilicate glass tubes, 18 X 150 mm., selected for uniformity in spectrophotometric measurements. Thermoregulated water bath a t 50" f 0.5" C. and ice bath, with a suitable basket or rack for the test tubes. Alcoholic anthrone reagent. To 60 ml. of absolute ethyl alcohol add slowly, with cooling, 100 ml of concentrated sulfuric arid. When the mixture is a t room temperature, add 200 mg. of anthrone. The reagent is ready for use immediately and can be kept in a refrigerator (about 6' C.) for a t least 3 weeks. Procedure. Weigh samples of dextran up to 60 mg. (or transfer up to 2.0 ml. of solution) containing the e uivalent of 25 to 100 y of fructose or levan into the tubes. AId 2 ml. of water or sufficient water to give a total of 2 ml. if a solution was used. Include a fructose standard (60 y) and a reagent blank in each run. In addition, if the fructose content is below lo%, have a control for the dextrans consisting of a high purity sample of the same type of dextran a t approximately the same concentration. To each of the solutions, cooled in ice water, layer in 8 ml. of the cold alcoholic anthrone reagent. Transfer the well-stirred cold mixtures to the 50' C. water bath. After 20 Z!Z 0.1 minutes, return the tubes to the ice bath for 1 minute to cool the solutions t o a little below room temperature. Measure the absorbance promptly a t 620 mp against the reagent blank. As the color formation still is continuing slowly, the tubes should be read in order from the first to the last, then from the last to the first, and the average of the two readings used. In making these readings a uniform rate schedule should be maintained. Calculation of Results. From the absorbance of the fructose standard, calculate the factor for converting absorbance to weight of fructose: mg. of fructose K = absorbance This factor \vi11 vary somewhat from run to run, mainly because of variations in the time required to read the tubes and also because of other variations in conditions Therefore, a fructose standard is included in each set of tubes t o be heated. Calculate the weight of the fructose in the dextran sample using the absorbances, A, of the "unknown" dextran and the control dextran sample: Weight of fructose = K (A unknown
-A
control)
SAMPLE CALCULATION. Absorbances. 0 OGO mg of fructose 10 mg of unhnown dextran 10 mg of control dextran
0.524 0 599 0 182
Calculations. 0.060 -= 0.1145 0.524 Fructose in unknown sample = 0.1145 (0.599 - 0.182) = 0.048 mg. or 0.48% Expressed a s levan = 0.048 X 0.9 = 0.043 mg. or 0.43%
K =
The K value under a given set of conditions is constant over a wide range of absorbances in conformance to Beer's law, as shown in Figure 1 for the alcoholic anthrone reagent. A similar linear relationship was observed with the resorcinol-hydrochloric acid method described below. QUANTITATIVE FRUCTOSE METHOD USING RESORCINOL
Initial studies on the determination of fructose in dextran samples were directed toward adaptation of the colorimetric Seliwanoff reaction using resorcinol in the presence of hydrochloric acid, for which several detailed reports had appeared (1, 6, 12). The modified method which was developed is described briefly here because of its apparent usefulness, in combination with the alcoholic anthrone method, for getting a partial differentiation beitween easily and difficultly liberated forms of fructose in a sample.
35 Table I.
Typical Results on Known Mixturesa and Fructose-Containing Sugars
Combinations
++ + +
Dextran fructose Dextran fructose Dextran levan Dextran levan Raffinose.5HzO Sucrose Melezitose. 2Hz0 Leucrose
Total Weight Sample, Rfg.
Fructose Found
Alcoholic Anthrone Method 16.0 0.0480 16.0
0.5 10.0 0.165 0.095 0.150 0.095
0.0060
0.0508 0.0507 0.0498 0.0502 0.0520
0.0097
Recovery
%
70 of Theo;y
0.300 0.038 10.2 0.507 30.2 52.8 34.7 10.2
100 100 99 98 100 100 104 19
Mg.
Resorcinol Method 203.0 0.377 Dextran fructose 0.186 94 Dextran fructose 204.0 0.045 0.022 90 Dextran levan 5.0 0.486 9.72 98 Dextran levan 100.0 0.465 0.465 94 1.65 0.492 Raffinose. 5H20 29.8 98 0.95 Sucrose 0.492 51.8 98 Melezitose, 2H20 1.50 0.178 11.9 36 Leucrose 0.95 0.010 1.0 2 a I n all cases the dextran was a highly purified sample from Leuconostoc nesenteroides NRRL B-512 prepared as described (8).
++ ++
0.6 u)
D-FRUCTOSE. MICROGRAMS
Figure 1. Absorbance Curve, Alcoholic Anthrone hlethod
The procedure described by Gray ( 5 )was used as a basis for the present method. Ferric chloride was added to the hydrochloric acid, as done by Bacon and Bell ( 1 ) in order to intensify the color produced and minimize the effect of possible traces of iron in the hydrochloric acid. The temperature a t which the color-forming reaction was conducted was lowered from 80' C., used by Gray, to 50" C. This lowering of the reacting temperature, together with a reaction time of 20 minutes, allowed a greater differentiation between fructose and glucose in mixtures of the two. The selectivity thus was increased threefold-Le., 240 parts instead of 80 parts (6) of glucose were required to give the same absorbence as 1 part of fructose. Ice-bath cooling of the dextran solution and reagents before mixing was adopted to eliminate erratic results attributed to variable amounts of reaction before the timed period of heating. Application of the procedure to known mixtures containing as little as 0.02% of fructose, sucrose, or levan in dextran gave recoveries of fructose ranging from 90 to 100 f 2%. The decrease in accuracy with the lower percentages of fructose, shown in Table I, tentatively is attributed to a lowering of the effective acidity of the reaction mixture by the larger weights of dextran sample required for fructose contents below about 1%. Apparatus and Reagents. The spectrophotometer, borosilicate glass test tubes, and the water and ice baths are the same as for the alcoholic anthrone method. Hydrochloric acid, concentrated, specific gravity 1.18 to 1.19, to which has been added 0.0124 gram of ferric chloride hexahydrate per liter. Resorcinol reagent, consisting of a 0.1% solution of resorcinol in absolute ethyl alcohol.
ANALYTICAL CHEMISTRY
36 Procedure. The procedure is essentially the same as for the alcoholic anthrone method, except that the sample consists of up to 500 mg. of dextran (or up to 2.0 ml. of solution) containing the equivalent of 200 to 800 y of fructose or levan, while the fructose standard is 500 y. To each sample in 2.0 ml. of cold aqueous solution is added 5 ml. of the cold hydrochloric acid followed by 2 ml. of cold resorcinol reagent. The well-mixed solutions are heated and cooled, and their absorbances measured as for the alcoholic anthrone method, except that a wave length of 505 mp is used. The calculation of results is similar to that for the alcoholic anthrone method. If any of the solutions are hazy, haze blanks must be prepared of those dextrans and the control dextran. For a haze blank, the 2 ml. of resorcinol reagent is replaced with an equal volume of cold absolute ethyl alcohol. The absorbances of these solutions then are used as a correction as follows: Weight of fructose = K ( A unknown 9 unknown haze blank A control A control haze blank).
-
-
+
DISCUSSION
Glucose gives the same color reaction as fructose with the anthrone or resorcinol methods but requires longer heating to attain a similar absorbance. Under the conditions of limited reaction described here, for either method, 1 mg. of fructose is equivalent in absorbance to about 240 mg. of glucose. This very favorable ratio of color formation from fructose and glucose is achieved by restricting the extent of reaction. Thus, heating for more than 20 minutes or a t higher temperatures than 50’ C. gives a higher absorbance per unit weight of either sugar. However, the increase for glucose is relatively greater than for fructose, so that more interference is obtained from glucose in mixtures under such conditions. The incompleteness of the colorforming reaction, together with the low reaction temperature of 50” C., results in a slow continuation of reaction a t room temperature during the spectrophotometric measurements. Since the absorbances of the reaction mixtures in either method are increasing a t a rate of about 0.4% per minute a t room temperature, a set of tubes must be read twice and in a uniform manner from first to last and then from last to first if highest accuracy is desired. Any attempt to wait until the change in absorbance decreases or ceases will result in greater interference from glucose and its polymers. Both colorimetric methods measure a t least part of the fructose in combinations less easily hydrolyzed than sucrose or levan, as shown by the results with melezitose and leucrose (5-D-glUCOpyranosyl-D-fructose) ( 1 4 ) ,Table I, neither of which yields fructose under the hydrolytic conditions of the qualitative chromatographic procedure. The two methods differ significantly in the extent to which they measure such forms of fructose, the alcoholic anthrone reagent being the more effective. This difference probably results, at least in part, from the greater hydrolyzing power of the anthronesulfuric acid reagent, as has been indicated by preliminary estimates of the extents of hydrolysis of dextran by the two reagents under the conditions of the fructose determination. Advantage can be taken of this difference by using the resorcinol method where the measurement is to be limited more nearly to the easily hydrolyzed fructose units and employing the alcoholic anthrone method for obtaining more nearly a “total fructose” value. Even with the latter method, however, only a minor part of the fructose units may be measured in structures reacting like leucrose, whether present in impurities or as part of the dextran molecule. The use of a control dextran in the colorimetric analysis of samples containing low concentrations of fructose is required to compensate for color formation from glucose liberated from the dextran sample by the hydrolytic action of the reagent. The measurement of fructose content, therefore, is relative rather than absolute, in so far as the control sample may be more easily hydrolyzed (liberation of glucose) or may contain free or combined fructose. The effect of differences in rate of liberation of glucose, which probably would result from differences in the proportion and kind of non-l,6’-glucosidic linkages, must remain well below the equivalent of about 0,4y0fructose in the sample, since
this is the difference in color formation between glucose itself (equivalent to immediate complete hydrolysis) and the blank (equivalent to no hydrolysis of dextran). The possible presence of fructose units in the control is a potential source of greater difficulty. Free or easily hydrolyzed fructose can be detected and measured by the chromatographic procedure. Some indication of the presence of difficultly hydrolyzed fructose units can be obtained from the total color formation in the reaction of the control. The B-512 dextran used as a control in the present studies gave absorbances equivalent to about 0.06 and 0.25% fructose in the sample by the resorcinol and anthrone methods, respectively. Preliminary estimates of the estent of hydrolysis of this dextran sample to glucose by these t n o reagenb have suggested that approximately half of this apparent fructose may arise from sources other than glucose liberation. This dextran, however, contained not over 0.02% fructose in free or easily hydrolyzed form, as shown by application of the chromatographic procedure described and comparison with known amounts of fructose on the chromatograms. The possibility thus suggested that difficultly hydrolyzed fructose units, or sugar units giving comparable color reactions, may be present in dextrans is given further support by observations on other dextran samples. Some highly purified, partially degraded dextrans, for example, have given apparent fructose contents, measured against controls, as high as 0.3’%,although no easily hydrolyzed fructose units could be detected by the qualitative procedure. The interpretation of such data must await the results of further studies of the chemical structure of dextrans. These observations emphasize, hen-ever, that samples of dextran to be used as controls should be selected on the basis not only of tests by the qualitative procedure but also of determinations of the actual amount of color, compared with reagent blanks, produced in the quantitative procedures. The extent to which other sugars may interfere in the determination of fructose was investigated. Only the ketohexose sorbose gave an appreciable amount of color. The absorbance developed in the resorcinol and anthrone methods was 52 and 74%, respectively, of that given by an equal weight of fructose. Xylose gave only 0.4 and 1.8%,respectively. For either method, densities below 0.5% were obtained with mannose, arabinose, and glucuronic acid. 4CKNOWLEDGMENT
The authors are grateful to F. H. Stodola, C. L. Mehltretter, and Allene Jeanes for furnishing the samples of leucrose, glucuronic acid, and dextrans and levan, respectively. (1) Bacon, J.
LITERATURE CITED Bell, D. J., Biochem J . (London), 42, 397
S.D . , and
(1948). (2) Bryson, J. L., and Mitchell, T. J., .Vatwe, 167, 864 (1951). (3) Dimler, R.J., Schaefer, W. C., Wise, C. S., and Rist, C. E., ANAL. CHEM.,24, 1411 (1952). (4) Evans, T. H., and Hibbert, H. “bdvances in Carbohydrate Chemistrv.” Vol. 2. D. 203. Academic Press. New York, 1946. (5) Gray, D . J. S., Analyst,*75,314 (1950). (6) Hehre, E. J., private communication. (7) Hough, L., Jones, J. K. K.,and Wadman, W. H., J. Chem. Soc., 1950,1702. (8) Jeanes, A., Wilham, C. A,, and Miers, J. C . , J . Bid. Chem., 176, 603 (1948). (9) Jeanes, S . , Wise, C. S., and Dimler, R.J., ANAL.CREM.,23, 415 (1951). (10) Koehler, L. H., Ibid.,24,1576 (1952). (11) Laidlaw, R.A., and Reid, S.G . , J . Sei. Food Agr., 3, 19 (1952). (12) Roe, J. H., J . Bid. Chem., 107, 15 (1934). . 25, 1656 (1953). (13) S c o t t , T. A,, and Melvin, E. H., A x . 4 ~CHEM., (14) Stodola, F. H., Koepsell, H. J., and Sharpe, E. S., J . Am. Chem. Soc., 74,3202 (1952). RECEIVED for review May 19, 1954. Accepted August 25, 1954. Presented before the Division of Carbohydrate Chemistry at the 124th Meeting of the AMERICAN CHEMICAL SOCIETY, Chicago, Ill., 1953. The mention of firm names or trade products does not imply that they are endorsed or recommended by the Department of Agriculture over other firms orsimilar products not mentioned.
