- Email: [email protected]
Chromatographic separation of glucose and mannose on cation-exchange resin
ANALYTICAL
BIOCHEMISTRY
73, 222-226
(1976)
Chromatographic Separation of Glucose and Mannose on Cation-Exchange Resin An analytical technique has been developed for separating mixtures of monosaccharides by partition chromatography in aqueous ethanol on columns of anion- (1) and cation- (2) exchange resins in the inorganic counterion forms. One disadvantage of the cation resin was that glucose could not be separated from mannose. This present report describes the optimum conditions for separating these sugars from each other and from other commonly occurring monosaccharides on cation-exchange resin in the potassium and sodium forms. MATERIALS
AND METHODS
All chemicals were of Analar grade. Monosaccharides and 3,3’dianisole-4,4’-bis-(3,5-diphenyl)chloride (tetrazolium blue) were purchased from Sigma (London) Chemical Co., Kingston, Surrey, U. K.; ethanol from James Burroughs Ltd., London S.E.11, U. K.; ethylene glycol (ethanediol) from BDH Chemicals Ltd., Poole, Dorset, U. K.; and sulphonated polystyrene resin (8% cross-linked divinyl benzene, 8-10 pm in diameter) from Locarte Instrument Co., Wendell Road, London W.12, U. K. The apparatus used for the automated quantitative analysis of monosaccharides was a modified Locarte amino acid autoanalyser. The column was glass (62 x 0.9 cm) with Teflon fittings and tubing at top and bottom, and it was placed in a glass heating jacket in which water was circulated at a constant temperature, the temperature being varied over the range .55-82°C. The resin was prepared by washing successively with 2 M HCl and water and converted into the lithium, potassium, or sodium form by washing with a 2 M solution of the appropriate base. To prepare the columns, a slurry of the resin was made in 0.2 M solutions of the base and packed into the column in sections at a 60-psi pressure of nitrogen and at the column temperature to be used during the chromatographic run. The resin was then equilibrated by pumping aqueous ethanol at the same composition and flow rate as that to be used during the chromatographic run for 3 hr. After equilibration the resin bed had shrunk by about 10 cm and more resin had to be added in 0.2--M solutians of the base and reequilibrated with the aqueous ethanol eluant was before. The height of the resin bed then remained constant. The aqueous eluants were stored in glass vessels sealed with Parafilm. Between chromatographic runs, the top 1 cm of the resin bed was 222 Copyright All rights
0 1976 by Academic Press. Inc. of reproduction in any form rexrved
223
SHORTCOMMUNICATIONS
L 0
I 25
1 50
I 75
I 100
I 125
Ttme Imtn)
FIG. 1. Separations of monosaccharides on the K+ form of resin. Resin bed, 52 x 0.9 cm; eluant composition, 62% (v/v) ethanol in water: temperature, 81°C; flow rate, 0.5 ml/min; and pressure drop across column, 190 psi. Each peak corresponds to 7 pg 3, of monosaccharide. 1, Glucuronic acid: 2, deoxyribose and N-acetylglucosamine: rhamnose and N-acetylgalactosamine; 4. fucose and xylose; 5. ribose. 6, glucose; 7, arabinose; 8, mannose; 9, galactose.
stirred in 0.2 M solutions of the base, and the resin was regenerated by forcing 3 ml of the base into the resin bed with a 60-psi pressure of nitrogen. Eluant was then pumped into the resin bed at a flow rate of 0.25 ml/min for 2 hr and for 1 hr at the flow rate to be used during the chromatographic run. Aliquots were taken from stock solutions (l%, w/v) of N-acetylD-galactosamine, N-acetyl-D-glucosamine, L-arabinose, 2-deoxyribose, L-fucose, D-galactose, D-glucose, D-glucuronic acid, D-mannose, Lrhamnose, D-ribose, and D-xylose, and standard mixtures (20 puglO. ml) were prepared in the eluant to be used in the chromatographic run. Standard solutions (~50 ~1) were placed on top of the resin column with a micropipet and forced into the resin with a 40-psi pressure of nitrogen, and the space above the resin was filled with eluant. The columns were then eluted with aqueous ethanol. The column eluate was analyzed for carbohydrate by the method of Mopper and Degens (3) which involved mixing with a solution of tetrazolium blue (0.2%, w/v) in 0.18 M NaOH pumped at a flow rate which was 40% that of the eluate. The colour of the reaction mixture was developed in a coil of Teflon tubing (7 m x 0.9 mm) immersed in a water bath at SO”C, and the absorbance measured at 570 nm in a 6-mm flow cell on a recorder with five-times scale expansion. All connecting
SHORT COMMUNICATIONS
224
I 0
I 20
I 60
I 100 Time
I IL0
J 160
Imd
FIG. 2. Separations of various monosaccharides on columns of cation-exchange resins (X 8.5%, DVB) in the Li+ form.* Resin bed, 50 x .09 cm; eluant composition, 83% (V/V) ethanol in water; temperature, 81”C, flow rate, 0.5 mlimin; and pressure drop across column, 150 psi. Each peak corresponds to 7 pg of monosaccharide. 1, Glucuronic acid; 2, deoxyribose; 3, rhamnose; 4. fucose: 5, xylose; 6, N-acetylglucosamine; 7, ribose; 8, arabinose; 9. N-acetylgalactosamine; 10. glucose and mannose; 11, galactose.
I 0
I 20
I 60
I 100 Tfme Imin)
I 1LO
J 160
FIG. 3. Separation of monosaccharides on the Na+ form of the resin. Resin bed, 54 x 0.9 cm; eluant composition, 8% (v/v) ethylene glycol, 66% ethanol in water; temperature, SlS’C; flow rate, 0.38 ml/mitt; and pressure drop across column, 290 psi. Each peak corresponds to 7 pg of monosaccharide. I, Glucuronic acid; 2, deoxyribose; 3, 4. N-acetylgalactosamine; 5. xylose; 6. fucose; 7, ribose: 8, N-acetylglucosamine; arabinose; 9. mannose; 10, glucose: 11. galactose.
SHORT COMMUNICATIONS
225
tubing was Teflon (diameter, 0.8 mm). To prevent the formation of air bubbles at high column temperatures a back pressure was created by attaching a length of narrow-bore Teflon tubing (10 m x 0.8 mm) between the exit tube of the flow cell and the line to waste. RESULTS AND DISCUSSION
To optimize the conditions for the separation of glucose from mannose and from other commonly occurring monosaccharides on columns of cation-exchange resin in potassium, lithium, and sodium forms, the effects of the composition of the water-ethanol eluant, the column length, the temperature, and the flow rate were investigated. The general order of elution was deoxy-sugars, pentoses, and hexoses. Figure 1 shows a typical chromatogram for separations carried out on resin in the potassium form. The peaks are stylized because of ease of presentation. It can be seen that glucose (peak 6) was completely separated from mannose (peak 8). Although the peak of arabinose (peak 7) overlapped those of glucose and mannose, the separations were sufficient to allow the integration of all three peaks. Considering the separation of other monosaccharides, one disadvantage of this resin form was the failure to resolve fucose and xylose. When the resin was in the lithium form, glucose could not be separated from mannose or N-acetylgalactosamine but fucose and xylose were resolved (Fig. 2). Although no separation between glucose and mannose was observed when the resin was in the sodium form, some separation was achieved by including ethylene glycol in the aqueous ethanol eluant and developing the chromatogram at a column temperature of 57.5”C (Fig. 3). The separation although incomplete was sufficient to allow integration of both peaks. Also, the sodium form of the resin gave sufficient resolution of fucose and xylose for the peaks to be integrated. The equipment used for the automated analysis of the sugars was a Locarte amino acid autoanalyzer which had been modified. The eluted sugars were quantitated from the original chromatograms by a method which involved measuring the colour produced after reaction with alkaline tetrazolium blue solution. There was a linear relationship between the amount of each sugar in the range 3-15 pg and the peak area, and the analysis for any one sugar was reproducible within the limits ?5%. This supported the results of Mopper and Degens (3). ACKNOWLEDGMENT This research was supported in part by the Science Research Council.
226
SHORT
COMMUNICATIONS
REFERENCES I. Larsson, L. I., and Samuelson, 0. (1965) Acre Chem. Scund. 19, 1357-1364. 2. Jonsson, P.. and Samuelson, 0. (1967) Anal. Chem. 39, 1156-1158. 3. Mopper, K., and Degens, E. T. (1972) AnuI. Biochem. 147-153.
J.
THOMAS
Department of Biochemistry University College, P. 0. Box 78 Cardiff CFI IXL, U. K.
L. H. Locarte Instrument Cornpun) Wendell Road London Wl2, U.K. Received November 24. 1975; uccepted Murch 16. 1976
LOBEL
BIOCHEMISTRY
73, 222-226
(1976)
Chromatographic Separation of Glucose and Mannose on Cation-Exchange Resin An analytical technique has been developed for separating mixtures of monosaccharides by partition chromatography in aqueous ethanol on columns of anion- (1) and cation- (2) exchange resins in the inorganic counterion forms. One disadvantage of the cation resin was that glucose could not be separated from mannose. This present report describes the optimum conditions for separating these sugars from each other and from other commonly occurring monosaccharides on cation-exchange resin in the potassium and sodium forms. MATERIALS
AND METHODS
All chemicals were of Analar grade. Monosaccharides and 3,3’dianisole-4,4’-bis-(3,5-diphenyl)chloride (tetrazolium blue) were purchased from Sigma (London) Chemical Co., Kingston, Surrey, U. K.; ethanol from James Burroughs Ltd., London S.E.11, U. K.; ethylene glycol (ethanediol) from BDH Chemicals Ltd., Poole, Dorset, U. K.; and sulphonated polystyrene resin (8% cross-linked divinyl benzene, 8-10 pm in diameter) from Locarte Instrument Co., Wendell Road, London W.12, U. K. The apparatus used for the automated quantitative analysis of monosaccharides was a modified Locarte amino acid autoanalyser. The column was glass (62 x 0.9 cm) with Teflon fittings and tubing at top and bottom, and it was placed in a glass heating jacket in which water was circulated at a constant temperature, the temperature being varied over the range .55-82°C. The resin was prepared by washing successively with 2 M HCl and water and converted into the lithium, potassium, or sodium form by washing with a 2 M solution of the appropriate base. To prepare the columns, a slurry of the resin was made in 0.2 M solutions of the base and packed into the column in sections at a 60-psi pressure of nitrogen and at the column temperature to be used during the chromatographic run. The resin was then equilibrated by pumping aqueous ethanol at the same composition and flow rate as that to be used during the chromatographic run for 3 hr. After equilibration the resin bed had shrunk by about 10 cm and more resin had to be added in 0.2--M solutians of the base and reequilibrated with the aqueous ethanol eluant was before. The height of the resin bed then remained constant. The aqueous eluants were stored in glass vessels sealed with Parafilm. Between chromatographic runs, the top 1 cm of the resin bed was 222 Copyright All rights
0 1976 by Academic Press. Inc. of reproduction in any form rexrved
223
SHORTCOMMUNICATIONS
L 0
I 25
1 50
I 75
I 100
I 125
Ttme Imtn)
FIG. 1. Separations of monosaccharides on the K+ form of resin. Resin bed, 52 x 0.9 cm; eluant composition, 62% (v/v) ethanol in water: temperature, 81°C; flow rate, 0.5 ml/min; and pressure drop across column, 190 psi. Each peak corresponds to 7 pg 3, of monosaccharide. 1, Glucuronic acid: 2, deoxyribose and N-acetylglucosamine: rhamnose and N-acetylgalactosamine; 4. fucose and xylose; 5. ribose. 6, glucose; 7, arabinose; 8, mannose; 9, galactose.
stirred in 0.2 M solutions of the base, and the resin was regenerated by forcing 3 ml of the base into the resin bed with a 60-psi pressure of nitrogen. Eluant was then pumped into the resin bed at a flow rate of 0.25 ml/min for 2 hr and for 1 hr at the flow rate to be used during the chromatographic run. Aliquots were taken from stock solutions (l%, w/v) of N-acetylD-galactosamine, N-acetyl-D-glucosamine, L-arabinose, 2-deoxyribose, L-fucose, D-galactose, D-glucose, D-glucuronic acid, D-mannose, Lrhamnose, D-ribose, and D-xylose, and standard mixtures (20 puglO. ml) were prepared in the eluant to be used in the chromatographic run. Standard solutions (~50 ~1) were placed on top of the resin column with a micropipet and forced into the resin with a 40-psi pressure of nitrogen, and the space above the resin was filled with eluant. The columns were then eluted with aqueous ethanol. The column eluate was analyzed for carbohydrate by the method of Mopper and Degens (3) which involved mixing with a solution of tetrazolium blue (0.2%, w/v) in 0.18 M NaOH pumped at a flow rate which was 40% that of the eluate. The colour of the reaction mixture was developed in a coil of Teflon tubing (7 m x 0.9 mm) immersed in a water bath at SO”C, and the absorbance measured at 570 nm in a 6-mm flow cell on a recorder with five-times scale expansion. All connecting
SHORT COMMUNICATIONS
224
I 0
I 20
I 60
I 100 Time
I IL0
J 160
Imd
FIG. 2. Separations of various monosaccharides on columns of cation-exchange resins (X 8.5%, DVB) in the Li+ form.* Resin bed, 50 x .09 cm; eluant composition, 83% (V/V) ethanol in water; temperature, 81”C, flow rate, 0.5 mlimin; and pressure drop across column, 150 psi. Each peak corresponds to 7 pg of monosaccharide. 1, Glucuronic acid; 2, deoxyribose; 3, rhamnose; 4. fucose: 5, xylose; 6, N-acetylglucosamine; 7, ribose; 8, arabinose; 9. N-acetylgalactosamine; 10. glucose and mannose; 11, galactose.
I 0
I 20
I 60
I 100 Tfme Imin)
I 1LO
J 160
FIG. 3. Separation of monosaccharides on the Na+ form of the resin. Resin bed, 54 x 0.9 cm; eluant composition, 8% (v/v) ethylene glycol, 66% ethanol in water; temperature, SlS’C; flow rate, 0.38 ml/mitt; and pressure drop across column, 290 psi. Each peak corresponds to 7 pg of monosaccharide. I, Glucuronic acid; 2, deoxyribose; 3, 4. N-acetylgalactosamine; 5. xylose; 6. fucose; 7, ribose: 8, N-acetylglucosamine; arabinose; 9. mannose; 10, glucose: 11. galactose.
SHORT COMMUNICATIONS
225
tubing was Teflon (diameter, 0.8 mm). To prevent the formation of air bubbles at high column temperatures a back pressure was created by attaching a length of narrow-bore Teflon tubing (10 m x 0.8 mm) between the exit tube of the flow cell and the line to waste. RESULTS AND DISCUSSION
To optimize the conditions for the separation of glucose from mannose and from other commonly occurring monosaccharides on columns of cation-exchange resin in potassium, lithium, and sodium forms, the effects of the composition of the water-ethanol eluant, the column length, the temperature, and the flow rate were investigated. The general order of elution was deoxy-sugars, pentoses, and hexoses. Figure 1 shows a typical chromatogram for separations carried out on resin in the potassium form. The peaks are stylized because of ease of presentation. It can be seen that glucose (peak 6) was completely separated from mannose (peak 8). Although the peak of arabinose (peak 7) overlapped those of glucose and mannose, the separations were sufficient to allow the integration of all three peaks. Considering the separation of other monosaccharides, one disadvantage of this resin form was the failure to resolve fucose and xylose. When the resin was in the lithium form, glucose could not be separated from mannose or N-acetylgalactosamine but fucose and xylose were resolved (Fig. 2). Although no separation between glucose and mannose was observed when the resin was in the sodium form, some separation was achieved by including ethylene glycol in the aqueous ethanol eluant and developing the chromatogram at a column temperature of 57.5”C (Fig. 3). The separation although incomplete was sufficient to allow integration of both peaks. Also, the sodium form of the resin gave sufficient resolution of fucose and xylose for the peaks to be integrated. The equipment used for the automated analysis of the sugars was a Locarte amino acid autoanalyzer which had been modified. The eluted sugars were quantitated from the original chromatograms by a method which involved measuring the colour produced after reaction with alkaline tetrazolium blue solution. There was a linear relationship between the amount of each sugar in the range 3-15 pg and the peak area, and the analysis for any one sugar was reproducible within the limits ?5%. This supported the results of Mopper and Degens (3). ACKNOWLEDGMENT This research was supported in part by the Science Research Council.
226
SHORT
COMMUNICATIONS
REFERENCES I. Larsson, L. I., and Samuelson, 0. (1965) Acre Chem. Scund. 19, 1357-1364. 2. Jonsson, P.. and Samuelson, 0. (1967) Anal. Chem. 39, 1156-1158. 3. Mopper, K., and Degens, E. T. (1972) AnuI. Biochem. 147-153.
J.
THOMAS
Department of Biochemistry University College, P. 0. Box 78 Cardiff CFI IXL, U. K.
L. H. Locarte Instrument Cornpun) Wendell Road London Wl2, U.K. Received November 24. 1975; uccepted Murch 16. 1976
LOBEL