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
1368
ANALYTICAL CHEMISTRY
sodium bicarbonate which is dissolved, and acidimetric titration of an aliquot of the extract to measure the dissolved bicarbonate for use as correction in the Karl Fischer titration. Experiments were conducted to define optimum conditions for extracting moisture from the sample quantitatively. Consideration was given to the duration of the extraction period and the duration of the digestion or settling period which followed extraction. These experiments indicated optimum recovery of moisture from the sample by extracting the mixture vigorously and continuously for 1 minute and digesting for 5 minutes prior to filtration. The accuracy of the method was measured by analyzing three series of synthetic samples containing known amounts of moisture. These samples were prepared by weighing 100-gram portions of freshly prepared, free flowing sodium bicarbonate into a series of widemouthed sample bottles equipped with tightfitting screw caps, and adding the requisite weight of distilled water to each specimen with a dropping pipet having a finely drawn tip. Immediately after the addition, the bottles were closed tightly and mixed thoroughly by vigorous shaking. The level of moisture added to the three series was within the calculated range of 0.01 to 0.10%; as the moisture was increased from 0.01 to 0.10% the observed flowability of the samples decreased and the samples became lumpy. Periods of 24, 48, and 72 hours of conditioning in the respective moist environments were allowed to transpire before the moisture tests by the Karl Fischer method were conducted. These conditioning periods provided time for reaching equilibrium between the free moisture and the sodium bicarbonate components. The precision of the method was measured by conducting duplicate analyses of a series of typical production lots of sodium bicarbonate which had been stored for varying periods under normal conditions. MOISTURE EXTRACTION AND TITRATION APPARATUS
The extraction flask is a conventional 500-ml. separatory funnel equipped with a ground-glass stopper and a medium-porosity sintered-glass filter disk sealed permanently to the inner walls of the funnel near the vertex. Provision is made for attaching a drying tube charged with Drierite to the top opening of the funnel to protect the system from atmospheric contamination of moisture during the transfer of the extract to the reservoir. The reservoir is a dispensing funnel of 125-ml. capacity graduated a t 50- and 100-ml. volumes and is connected to the extraction flask by a tightcfitting rubber stopper. The stopper is fitted with a drying tube charged with Drierite to which an aspirator pump is attached. A two-way stopcock a t the bottom of the reservoir facilitates removal of extract from the reservoir directly into the Karl Fischer titration cell or into a beaker for acidimetric titration. The apparatus for the Karl Fischer titration is of conventional design involving the dead-stop principle RE4GENTS
The reagents required for the test include Karl Fischer reagent, reagent grade anhydrous methanol, Fleisher’s methyl purple indicator solution, and standardized 0. layhydrochloric acid.
Withdraiv the remaining 50-ml. aliquot of the extract into a 250-ml. beaker. Bdd 125 ml. of distilled water and titrate to a steel gray end point with standardized 0.1N hydrochloric acid solution, employing Fleisher’s methyl purple indicator. Conduct a blank determination involving all of the operations detailed above with a portion of the absolute methanol used for the extraction. AXALYSIS OF SYNTHETIC MIXTURES AYD TYPICAL PRODUCTION LOTS OF SODIUM BICARBONATE
Analrsis of the synthetic mixtures of sodium bicarbonate and determination of moisture by the Karl Fischer method are shown in Table I. The calculated recovery of moisture from the samples conditioned 24, 48, and 72 hours is shown in this table and ranges between 72 and 103%. The absolute accuracy of the method is unknown because of the lack of a standard basic material or a suitable alternative method for determining moisture in the control material. This limitation waq resolved by selecting a freshlv prepared. free-flowing product and adding weighed amounts of water, then subjecting the entire misture to the test procedure. Residual moisture in the control samples, subjected to the same conditioning periods as the synthetic miutures. was determined by the Karl Fischer method described. Random specimens of refined sodium bicarbonate stored under plant conditions were analyzed in duplicate by the Karl Fischer method. The moisture content of these commercial specimens ranged between 0.01 and 0.16% with a calculated precision within =to 005%.
T a b l e 1. €1.0 Control
H20
ddded, %
Total HqO Present, %
Total H20 Found, %
Recover>, %
24-Hour Conditioning Time 0 0?5
0 0 0 0
0,025
0.019 0.036 0.051 0.071
0.005
0.010 0.018 0.038 0.066 0.086 0.094
018 039 055 OB1
0 0 0 0
043 OR4 080 086
0 039 0 058 0 079 0 081
91 91 99 98
48-Hour Conditioning Time 0 044 0 081 0.076 0.096
0.043 0.058 0.074 0.099
98 95 97 103
72-Hour Conditioning Time 0.015 0.015 0.023 0.019 0,043 0.031 0.061 0.046 0.091 0.075 0 104 0.083
100 83 72 75 82 80
ACKNOWLEDGMENT
The authors wish to express their appreciation to Donald Varner and Hiram Bell for helpful suggestions in the design of the apparat,us, and to B. J. DeWitt for advice in preparing the manuscript. LITERATURE CITED
PROCEDURE
Rinse the extraction flask and reservoir with four 15-ml. portions of absolute methanol, using suction and the drying tube devices after each rinse. Place 100 grams of the sodium bicarbonate sample in the extraction flask and add 130 ml. of absolute methanol. Stopper the flask immediately and shake vigorouely for 1 minute after the sample has been completely wetted with the methanol. Rapidly replace the stopper with the drying tube device and allow the solids to settle for 5 minutes. Attach the reservoir to the extraction flask, apply gentle suction to the system with a water aspirator and withdraw 100 ml. of the extract. Disconnect the water aspirator, close the cock a t the bottom of the extraction flask, and measure a 50-ml. aliquot of the extract into the Karl Fischer titration cell Stir the extract and titrate directly to a dead-stop end point with standardized Karl Fischer reagent. Record this titration which measures the moisture plus diqwlved sodium bicarbonate
Recovery of M o i s t u r e from Sodium Bicarbonate
(1) ANAL.CHEM.,23, 1058 (1951), Symposium Papers, Division of Analytical Chemistry, 118th Meeting of the AMERICAN CHEMICAL SOCIETY, Chicago, Ill. (2) Bryant, W. M. O., hZitchell. J., Jr., Smith, D. 31.,and Ashby, E. C., ,J. Am. Chem. SOC.,63, 2924 (1941). (3) Fischer, Karl, Angew. Chem., 48, 394 (1935). (4) Foulk, C. W., and Bowden, A. T., J . A m . Chem. SOC.,48, 2045 (1926). (5) Hou, Te-Pang, “Manufacture of Soda,” A. C.
(6) (7)
(8) (9)
S. Monograph Series, p. 148, Kew York, Chemical Catalog CO., 1933. Mannheimer, M., IND. ENG.CHEM.,ANAL.ED.,1 , 154 (1929). Mitchell, J., and Smith, D. &I., “Aquametry,” p. 206, New York, Interscience Publishers, 1948. U.S.Pharmacopoeia, XIV, 541 (1950). Wernimont, G., and Hopkinson, F. J.. IND. EXG.C A E Y . . ANAL. ED.,15, 272 (1943).
RECEIVED for review January 13, 1954. .4ccepted M a y
6, 1954.
