Chromatography Oligosaccharides

Oligosaccharides.—Chromatography on paper of di-, tri-, and higher saccharides differs in one important respect from the resolution of monosaccharides. The larger molecules have very low Re values in the solvent... 

To allow all culture productiou to be coutrolled, a method for rapid analysis is required. Prior to development of an LC-MS method, the analysis was both complex and time-consuming, involving the purification of a relatively large amount of the antibody using affinity chromatography, enzymatic release, and subsequent derivatizafion of the oligosaccharides and their analysis by using capillary electrophoresis. 

The structures of many oligosaccharide chains can be elucidated by gas-liquid chromatography, mass spectrometry, and high-resolution NMR spectrometry. Glycosidases hydrolyze specific linkages in oligosaccharides and are used to explore both the structures and functions of glycoproteins. 

The compositions of high-mannose oligosaccharides isolated from urine of animals suffering from genetic or chemically induced mannosidoses have been assigned from their f.a.b.-mass spectra. After chromatography, some samples were sufficiently pure to be examined underivatized. Others required conversion into their peracetylated derivatives. 

Analyses were performed by gel permeation chromatography (GPC) and by high performance liquid chromatography (HPLC). Gel filtration of oligosaccharides was effected in a thermostated (65 <>C) column (210 X 1.5 i.d.) filled with polycrylamide gel (Bio-gel P2, 200-400 mesh Bio Rad-USA), using stilled water as eluent (flow rate 30 ml.h-1). 

Oligosaccharides were isolated preparatively by high-pH anion exchange chromatography carried out on a LC-system (Dionex Corporation, Sunnyvale CA) equipped with a CarboPac PA-1 column (9 x 250 mm), coupled to a Spectra System AS 3500 auto sampler. The detection was carried out using Pulsed Amperometric Detection (PAD-II). 

Hydrophilic interaction chromatography on Asahipak NH2P or Excel-pak CHA-P44 with pulsed amperometric detection has been used to fractionate malto-oligosaccharides.266 The Asahipak NH2P is a polyvinyl alcohol support with a polyamine bonded phase, and the Excelpak is a sulfonated polystyrene in the Zn+2 form. Amine adsorption of sialic acid-containing oligosaccharides was performed on a Micropak AX-5 column (Varian) using acetonitrile-water-acetic acid-triethylamine.267... 

Peelen, G. O. H., de Jong, J. G. N., and Wevers, R. A., High-performance liquid chromatography of monosaccharides and oligosaccharides in a complex biological matrix, Anal. Biochem., 198, 334, 1991. 

Blanken, W. M., Bergh, M. L. E., Koppen, P. L., and van den Eijnden, D.H., High-pressure liquid chromatography of neutral oligosaccharides effects of structural parameters, Anal. Biochem., 145, 322, 1985. 

Hardy, M. R. and Townsend, R. R., Separation of positional isomers of oligosaccharides and glycopeptides by high-performance anion-exchange chromatography with pulsed amperometric detection, Proc. Natl. Acad. Sci. U.S.A., 85, 3289, 1988. 

Watson, E., Bhide, A., Kenney, W. C., and Lin, F.-K., High-performance anion-exchange chromatography of asparagine-linked oligosaccharides, Anal. Bio-chem., 205, 90, 1992. 

Hernandez, L. M., Ballou, L., and Ballou, C. E., Separation of yeast asparagine-linked oligosaccharides by high-performance anion-exchange chromatography, Carb. Res., 203, 1, 1990. 

Thurl, S., Offermanns, J., Muller-Werner, B., and Sawatzki, G., Determination of neutral oligosaccharide fractions from human milk by gel permeation chromatography, /. Chromatogr., 568, 291, 1991. 

Suzuki-Sawada, J., Umeda, Y., Kondo, A., and Kato, I., Analysis of oligosaccharides by on-line high-performance liquid chromatography and ion-spray spectrometry, Anal. Biochem., 207, 203, 1992. 

Green, E. D. and Baenziger, J. U., Separation of anionic oligosaccharides by high-performance liquid chromatography, Anal. Biochem., 158, 42, 1986. 

Kotani, N. and Takasaki, S., Analysis of 2-aminobenzamide-labeled oligosaccharides by high-pH anion-exchange chromatography with fluorometric detection, Anal. Biochem., 264, 66, 1998. 

After the release of the oligosaccharides, they must be purified by a variety of methods before structural analyses can be undertaken. Ion-ex-change chromatography, gel-filtration chromatography, or some type of electrophoresis is usually used for the purification. At this point, structural analyses may begin. 

Jeffrey, A.M., Zopf, D.A., and Ginsburg, V. (1975) Affinity chromatography of carbohydrate-specific immunoglobulins coupling of oligosaccharides to sepharose. Biocbem. Biopbys. Res. Comm. 62, 608-613. 

Kakehi, K., Kinoshita, M., and Nakano, M. 2002. Analysis of glycoproteins and the oligosaccharides thereof by high performance capillary electrophoresis - significance in regulatory studies on biopharmaceutical products. Biomedical Chromatography 16(2), 103-115. 

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