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Improved isolation of L-arabinose from mesquite gum
NOTES FROM THE BIOCHEMICAL RESEARCH FOUNDATION ELLICE McDONALD, DIRECTOR IMPROVED ISOLATION OF L-ARABINOSE FROM MESQUITE GUM BY FRANCIS B. CRAMER
L-Arabinose is most easily obtained by the acid-catalyzed hydrolysis of mesquite gum. It has been shown by White (1) that controlled hydrolysis frees most of the arabinose leaving the rest of the polysaccharide molecule in the form of oligosaccharide fragments. The arabinose has ordinarily been separated from the by-products by one of two time-consuming methods. The first, the most recent refinement of which was contributed by the late C. S. Hudson (2), involves a tedious and hazardous fractional precipitation and extraction procedure. The second (3) necessitates prolonged mild hydrolysis, repeated dialyses and a final solvent fractionation. A less cumbersome method has now been discovered for the removal of impurities with improved yields of L-arabinose. It is based on the fact that the major portion of the by-products contains uronic acid residues which are ionizable and may be easily removed by the use of ion-exchange resins. After hydrolysis in sulfuric acid, neutralization and filtration by established procedures (4), the solution is passed through columns of Amberlite IR-120.(S) and Duolite A-4 (6). The water is evaporated and the residual sirup dissolved in methanol. Arabinose separates in good yield as white, odorless crystals of purity sufficient for most uses without recrystallization. EXPERIMENTAL
A 300-gm. quantity of crude mesquite gum (7) was tied in a square of cheese cloth and suspended in 1100 ml. of water in a 2-liter beaker. The gum dissolved on standing overnight. The cheese cloth bag containing bark, etc. was discarded. The solution was heated to 85 ° C. and acidified with 200 ml. of 27 per cent sulfuric acid (37 ml. of concentrated sulfuric acid diluted to 200 ml. with water). Hydrolysis was continued for 6 hours at 84o-86 ° C. after which the liquid was cooled and the acid neutralized with 70 gm. of calcium carbonate, capryl alcohol being used as foam breaker. The solution was left overnight at room temperature and filtered after the addition of 150 gin. of decolorizing charcoal. (The use of less charcoal may result in bothersome foaming during subsequent evaporation.) The filter cake was washed with 500 ml. of water which was kept separate from the main filtrate. 93
94
T H E FRANKLIN INSTITUTE
[J. F. I.
The solution and then the washings were passed slowly through a 110 X 5 era. column of Amberlite IR-120 (5, s) and then through a similar column of Duolite A-4 (6, s). The columns of resin were washed with distilled water until a test of the washings with Fehling's solution showed negligible reducing power. The colorless effluent was concentrated in v a c u u m to a thin sirup. (If foaming occurs, it may be controlled by adding about 10 per cent of isopropyl alcohol to the solution before evaporation. The liquid is then passed slowly and continuously into the distilling flask through a separatory funnel.) A 50 ml. portion of methanol was then added and evaporated in v a c u u m to assist in dehydration. This was repeated until crystallization began, whereupon the material was rinsed into a beaker with 200 ml. of methanol. After crystallizing overnight at room temperature, the arabinose was filtered out and washed with 100 ml. of methanol. The yield was 114 gin. of material with an equilibrium specific rotation of +99.8 ° and a melting point of 147°-155 ° C. The m o t h e r liquor was concentrated in v a c u u m and the residue dissolved in 100 ml. of methanol. After one m o n t h at room temperature, this solution yielded an additional 27.5 gm. of crude material with a final rotation of + 9 5 °. When the size of the batch was doubled, the capacity of the ionexchange columns used was apparently exceeded. As a result, a less pure product was obtained. For example, 600 gm. of mesquite gum yielded 194 gm. of arabinose with a final rotation of 4-98.5 ° and a melting point of 145°-152 ° C. Resin columns of the size described could probably hold the ionizable impurities from as much as 450 gin. of mesquite gum without difficulty. The method here described combines the older rapid hydrolysis procedure with a quick and easy method for removing by-product impurities. This results in a substantial saving in time and materials and removes the hazard involved in the use of large volumes of flammable organic solvents. REFERENCES
(I) (2) (3) (4) (5) (6) (7) (8)
E. V. WHITE,J. Am. Chem. Sot., 69, 622 (1947). C. S. HUDSON,J. Am. Chem. Soc., 73, 4038 (1951). E. V. WroTE, J. Xm. Chem. Soc., 69, 715 (1947). Circular of the Natl. Bur. Stand. C 440, "Polarimetry, Saccharimetry and the Sugars," F. J. Bates and Associates, Govt. Printing Office, Washington, D. C., 1942, p. 457. A product of Resinous Products and Chemical Co., Washington Square, Philadelphia 5, Pa. A product of Chemical Process Co., 901 Spring St., Redwood City, Calif. Obtained from Martin Drug Co., Tucson, Arizona. H. G. FLETCHER,JR., H. W. DIEItL ANDC. S. HUDSON,J. Am. Chem. Soc., 72, 4546 (1950).
L-Arabinose is most easily obtained by the acid-catalyzed hydrolysis of mesquite gum. It has been shown by White (1) that controlled hydrolysis frees most of the arabinose leaving the rest of the polysaccharide molecule in the form of oligosaccharide fragments. The arabinose has ordinarily been separated from the by-products by one of two time-consuming methods. The first, the most recent refinement of which was contributed by the late C. S. Hudson (2), involves a tedious and hazardous fractional precipitation and extraction procedure. The second (3) necessitates prolonged mild hydrolysis, repeated dialyses and a final solvent fractionation. A less cumbersome method has now been discovered for the removal of impurities with improved yields of L-arabinose. It is based on the fact that the major portion of the by-products contains uronic acid residues which are ionizable and may be easily removed by the use of ion-exchange resins. After hydrolysis in sulfuric acid, neutralization and filtration by established procedures (4), the solution is passed through columns of Amberlite IR-120.(S) and Duolite A-4 (6). The water is evaporated and the residual sirup dissolved in methanol. Arabinose separates in good yield as white, odorless crystals of purity sufficient for most uses without recrystallization. EXPERIMENTAL
A 300-gm. quantity of crude mesquite gum (7) was tied in a square of cheese cloth and suspended in 1100 ml. of water in a 2-liter beaker. The gum dissolved on standing overnight. The cheese cloth bag containing bark, etc. was discarded. The solution was heated to 85 ° C. and acidified with 200 ml. of 27 per cent sulfuric acid (37 ml. of concentrated sulfuric acid diluted to 200 ml. with water). Hydrolysis was continued for 6 hours at 84o-86 ° C. after which the liquid was cooled and the acid neutralized with 70 gm. of calcium carbonate, capryl alcohol being used as foam breaker. The solution was left overnight at room temperature and filtered after the addition of 150 gin. of decolorizing charcoal. (The use of less charcoal may result in bothersome foaming during subsequent evaporation.) The filter cake was washed with 500 ml. of water which was kept separate from the main filtrate. 93
94
T H E FRANKLIN INSTITUTE
[J. F. I.
The solution and then the washings were passed slowly through a 110 X 5 era. column of Amberlite IR-120 (5, s) and then through a similar column of Duolite A-4 (6, s). The columns of resin were washed with distilled water until a test of the washings with Fehling's solution showed negligible reducing power. The colorless effluent was concentrated in v a c u u m to a thin sirup. (If foaming occurs, it may be controlled by adding about 10 per cent of isopropyl alcohol to the solution before evaporation. The liquid is then passed slowly and continuously into the distilling flask through a separatory funnel.) A 50 ml. portion of methanol was then added and evaporated in v a c u u m to assist in dehydration. This was repeated until crystallization began, whereupon the material was rinsed into a beaker with 200 ml. of methanol. After crystallizing overnight at room temperature, the arabinose was filtered out and washed with 100 ml. of methanol. The yield was 114 gin. of material with an equilibrium specific rotation of +99.8 ° and a melting point of 147°-155 ° C. The m o t h e r liquor was concentrated in v a c u u m and the residue dissolved in 100 ml. of methanol. After one m o n t h at room temperature, this solution yielded an additional 27.5 gm. of crude material with a final rotation of + 9 5 °. When the size of the batch was doubled, the capacity of the ionexchange columns used was apparently exceeded. As a result, a less pure product was obtained. For example, 600 gm. of mesquite gum yielded 194 gm. of arabinose with a final rotation of 4-98.5 ° and a melting point of 145°-152 ° C. Resin columns of the size described could probably hold the ionizable impurities from as much as 450 gin. of mesquite gum without difficulty. The method here described combines the older rapid hydrolysis procedure with a quick and easy method for removing by-product impurities. This results in a substantial saving in time and materials and removes the hazard involved in the use of large volumes of flammable organic solvents. REFERENCES
(I) (2) (3) (4) (5) (6) (7) (8)
E. V. WHITE,J. Am. Chem. Sot., 69, 622 (1947). C. S. HUDSON,J. Am. Chem. Soc., 73, 4038 (1951). E. V. WroTE, J. Xm. Chem. Soc., 69, 715 (1947). Circular of the Natl. Bur. Stand. C 440, "Polarimetry, Saccharimetry and the Sugars," F. J. Bates and Associates, Govt. Printing Office, Washington, D. C., 1942, p. 457. A product of Resinous Products and Chemical Co., Washington Square, Philadelphia 5, Pa. A product of Chemical Process Co., 901 Spring St., Redwood City, Calif. Obtained from Martin Drug Co., Tucson, Arizona. H. G. FLETCHER,JR., H. W. DIEItL ANDC. S. HUDSON,J. Am. Chem. Soc., 72, 4546 (1950).