ANALYTICAL
BIOCHEMISTRY
75, 646-648
(1976)
Use of Capillary Columns for the Analysis of Monosaccharides by Gas-Liquid Chromatography Gas-liquid chromatography has become popular for the determination of monosaccharides because it is convenient, specific, and relatively easy to quantitate. Several volatile derivatives such as trimethylsilyl ethers (l), alditol acetates (2), butane-boronates (3), etc., have been used for the separation of sugars (for review see 4). Conventional columns (0.125 or 0.15-m i.d.) packed with a suitable solid support were employed. Tesarik (5) used glass capillary columns (50-100 ft in length, 34,00070,000 theoretical plates) and resolved several pentoses and aldoses. During the course of our studies on the carbohydrate composition and sequence of human serum lipoproteins, we found that use of long capillary columns for the carbohydrate analysis increases the resolution and sensitivity compared to use of conventional columns. The method which we employed is described in this report. Monosaccharide standards were obtained from Sigma Chemical Co., St. Louis, MO. TRISIL-Z was purchased from Pierce Chemical Co., Rockville, Md. Pentasil was obtained from Applied Science Labs, State College, Pa. The column which we have used is a lOOO-ft stainless-steel capillary column (0.03-in. i.d., 200,000 theoretical plates), coated with Pentasil. The coating was applied as described by Apon and Nicholaides (6). Gas chromatography was performed on a Beckman Model 72-5 instrument equipped with a thermal conductivity cell, dual hydrogen flame ionization detector, and linear temperature programmer. Helium at a flow rate of 10 ml/min was the carrier gas. The temperatures of the column, inlet, and detectors, were 170, 250, and 250°C respectively. Samples were derivatized to trimethylsilyl ethers using TRISIL-Z. The amounts of sample introduced were 0.1 to 0.5 ~1. The attenuation was set at 2 x 100, and the column was operated isothermally at 170°C. The separation of an equilibrium mixture of 0.2 pg each of galactose, mannose, and fucose is shown in Fig. 1. The isomeric forms of the sugars were well resolved from each other and also from other sugars. The solvent peak appeared after 15 min, and the total analysis time was 45 min. The relative retention times for the various peaks are as follows: fucose (a, 0.49; p, 0.55); galactose (a, 0.68; j?, 0.78); mannose (a, 0.60; p, 0.80): glucose ((Y, 0.74; p, 1.00). The compositions of aqueous equilibrium solutions of the sugars were found to be as follows: fucose (a, 28%; p, 72%), mannose (a, 71%; p, 29%), galactose (a, 2%: p. 65%: y, 6%). 646 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
641
SHORT COMMUNICATIONS
45
-15 TIME
IN GNUTES
FIG. 1. Gas-liquid chromatography of trimethylsilyl ethers of sugars, a mixture of 0.2 pg each ofgalactose, mannose, and fucose. Peaks 1 to 6 are o-fucose, p-fucose, cy-mannose, a-galactose. P-galactose, and P-mannose, respectively.
y-Galactose, the furanose form of galactose (retention time, 0.61), was not well resolved from the a-mannose peak and appeared at the descending shoulder. Equilibrium compositions reported here are in agreement with published values (1,7). The carbohydrate composition of a glycopeptide obtained from the thermolysin digest of human serum low density lipoprotein apoprotein is shown in Fig. 2. The composition shows the presence of mannose and galactose, in addition to a trace amount of glucose. An aliquot of glyco-
15
30
TIME
IN MINUTES
45
FIG. 2. Gas-liquid chromatography of trimethylsilyl ethers of sugars obtained by hydrolysis of low density lipoprotein apoprotein glycopeptide. An aliquot of the glycopeptide containing I3 pg of total sugar was hydrolyzed with Dowex SO-H+ at 100°C for 16 hr (2). The acid was neutralized with Dowex I-HCO,-, deionized with MB-3 resin (Mallinckrodt). dried, and derivatized with TRISIL-Z. A 0. l-pi aliquot containing 0.1 kg of carbohydrate was injected. Peaks 1 to 6 are a-mannose, cu-galactose, o-glucose, pgalactose, pmannose and P-glucose, respectively.
648
SHORT COMMUNICATIONS
peptide containing 13 pg of total carbohydrate was enough for the study of its carbohydrate composition. This method has also been successfully employed for monitoring the release of mannose and galactose during enzymatic digestion of lipoprotein glycopeptides with glycosidases. This method is particularly useful when only small quantities of carbohydrate are available for analysis. We have detected as little as 0.01 pg of each of the sugars mentioned. REFERENCES 1. Sweeley, C. C.. Bentley. R., Makita. M.. and Wells, W. W. (1963) J. Amer. Chem. Sot. 85,2497-2507. 2. Lehnhardt, W. F., and Winzler, R. J. (1968)J. Chromatogr. 34, 471-479. 3. Eisenberg, F. (1971) Curbohyd. Res. 19, 13.5-138. 4. Dutton. G. G. S. (1973) in Advances in Carbohydrate Chemistry and Biochemistry (Tipson, R. S., and Horton, D.. eds.) Vol. 28, pp. 1 l-160, Academic Press, New York. 5. Tesarik, K. (1972) J. Chromatogr. 65, 295-302. 6. Apon, B., and Nicholaides, N. (1975) J. Chromatogr. Sci. 13, 467-473. 7. Dutton, G. G. S., Gibney, K. B., Jensen, G. D., and Reid, P. E. (1968) J. Chromafogr. 36, 152- 162.
N. SWAMINATHAN B. APON F. ALADJEM Department of Microbiology University of Southern California, School of Medicine Los Angeles, California 90033 Received December 9, 1975; accepted April 27, 1976
BIOCHEMISTRY
75, 646-648
(1976)
Use of Capillary Columns for the Analysis of Monosaccharides by Gas-Liquid Chromatography Gas-liquid chromatography has become popular for the determination of monosaccharides because it is convenient, specific, and relatively easy to quantitate. Several volatile derivatives such as trimethylsilyl ethers (l), alditol acetates (2), butane-boronates (3), etc., have been used for the separation of sugars (for review see 4). Conventional columns (0.125 or 0.15-m i.d.) packed with a suitable solid support were employed. Tesarik (5) used glass capillary columns (50-100 ft in length, 34,00070,000 theoretical plates) and resolved several pentoses and aldoses. During the course of our studies on the carbohydrate composition and sequence of human serum lipoproteins, we found that use of long capillary columns for the carbohydrate analysis increases the resolution and sensitivity compared to use of conventional columns. The method which we employed is described in this report. Monosaccharide standards were obtained from Sigma Chemical Co., St. Louis, MO. TRISIL-Z was purchased from Pierce Chemical Co., Rockville, Md. Pentasil was obtained from Applied Science Labs, State College, Pa. The column which we have used is a lOOO-ft stainless-steel capillary column (0.03-in. i.d., 200,000 theoretical plates), coated with Pentasil. The coating was applied as described by Apon and Nicholaides (6). Gas chromatography was performed on a Beckman Model 72-5 instrument equipped with a thermal conductivity cell, dual hydrogen flame ionization detector, and linear temperature programmer. Helium at a flow rate of 10 ml/min was the carrier gas. The temperatures of the column, inlet, and detectors, were 170, 250, and 250°C respectively. Samples were derivatized to trimethylsilyl ethers using TRISIL-Z. The amounts of sample introduced were 0.1 to 0.5 ~1. The attenuation was set at 2 x 100, and the column was operated isothermally at 170°C. The separation of an equilibrium mixture of 0.2 pg each of galactose, mannose, and fucose is shown in Fig. 1. The isomeric forms of the sugars were well resolved from each other and also from other sugars. The solvent peak appeared after 15 min, and the total analysis time was 45 min. The relative retention times for the various peaks are as follows: fucose (a, 0.49; p, 0.55); galactose (a, 0.68; j?, 0.78); mannose (a, 0.60; p, 0.80): glucose ((Y, 0.74; p, 1.00). The compositions of aqueous equilibrium solutions of the sugars were found to be as follows: fucose (a, 28%; p, 72%), mannose (a, 71%; p, 29%), galactose (a, 2%: p. 65%: y, 6%). 646 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
641
SHORT COMMUNICATIONS
45
-15 TIME
IN GNUTES
FIG. 1. Gas-liquid chromatography of trimethylsilyl ethers of sugars, a mixture of 0.2 pg each ofgalactose, mannose, and fucose. Peaks 1 to 6 are o-fucose, p-fucose, cy-mannose, a-galactose. P-galactose, and P-mannose, respectively.
y-Galactose, the furanose form of galactose (retention time, 0.61), was not well resolved from the a-mannose peak and appeared at the descending shoulder. Equilibrium compositions reported here are in agreement with published values (1,7). The carbohydrate composition of a glycopeptide obtained from the thermolysin digest of human serum low density lipoprotein apoprotein is shown in Fig. 2. The composition shows the presence of mannose and galactose, in addition to a trace amount of glucose. An aliquot of glyco-
15
30
TIME
IN MINUTES
45
FIG. 2. Gas-liquid chromatography of trimethylsilyl ethers of sugars obtained by hydrolysis of low density lipoprotein apoprotein glycopeptide. An aliquot of the glycopeptide containing I3 pg of total sugar was hydrolyzed with Dowex SO-H+ at 100°C for 16 hr (2). The acid was neutralized with Dowex I-HCO,-, deionized with MB-3 resin (Mallinckrodt). dried, and derivatized with TRISIL-Z. A 0. l-pi aliquot containing 0.1 kg of carbohydrate was injected. Peaks 1 to 6 are a-mannose, cu-galactose, o-glucose, pgalactose, pmannose and P-glucose, respectively.
648
SHORT COMMUNICATIONS
peptide containing 13 pg of total carbohydrate was enough for the study of its carbohydrate composition. This method has also been successfully employed for monitoring the release of mannose and galactose during enzymatic digestion of lipoprotein glycopeptides with glycosidases. This method is particularly useful when only small quantities of carbohydrate are available for analysis. We have detected as little as 0.01 pg of each of the sugars mentioned. REFERENCES 1. Sweeley, C. C.. Bentley. R., Makita. M.. and Wells, W. W. (1963) J. Amer. Chem. Sot. 85,2497-2507. 2. Lehnhardt, W. F., and Winzler, R. J. (1968)J. Chromatogr. 34, 471-479. 3. Eisenberg, F. (1971) Curbohyd. Res. 19, 13.5-138. 4. Dutton. G. G. S. (1973) in Advances in Carbohydrate Chemistry and Biochemistry (Tipson, R. S., and Horton, D.. eds.) Vol. 28, pp. 1 l-160, Academic Press, New York. 5. Tesarik, K. (1972) J. Chromatogr. 65, 295-302. 6. Apon, B., and Nicholaides, N. (1975) J. Chromatogr. Sci. 13, 467-473. 7. Dutton, G. G. S., Gibney, K. B., Jensen, G. D., and Reid, P. E. (1968) J. Chromafogr. 36, 152- 162.
N. SWAMINATHAN B. APON F. ALADJEM Department of Microbiology University of Southern California, School of Medicine Los Angeles, California 90033 Received December 9, 1975; accepted April 27, 1976
