Analysis of oligosaccharides from heparin by reversed-phase ion-pairing high-performance liquid chromatography.
An ion-pairing high-pressure liquid chromatography procedure was developed for analysis of mixtures of oligosaccharides generated by nitrous acid cleavage of heparin. Oligosaccharides were eluted from a Hi-Chrom 5S ODS (C18) column using mixtures of acetonitrile and buffers containing 40 mM ammonium phosphate and 1 mM tetrabutylammonium phosphate. Isocratic conditions were developed for optimal separation of a number of individual disaccharides and tetrasaccharides that were characterized previously (M.J. Bienkowski and H.E. Conrad (1985) J. Biol. Chem. 260, 356-365). These isocratic conditions were then coupled to obtain gradient elution conditions for the ion-pairing separations of mixtures of disaccharides and mixtures of tetrasaccharides. A comparison of the elution profiles obtained in the ion-pairing chromatography procedure with profiles obtained by anion-exchange high-pressure liquid chromatography profiles showed markedly better overall resolution by the ion-pairing procedure. As a result of this improved resolution, the new procedure showed the presence of previously unidentified products in the heparin oligosaccharide mixtures.
Guo
Y C
YC
Department of Biochemistry, University of Illinois, Urbana 61801.
Conrad
H E
HE
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006493:Heparin; D009844:Oligosaccharides / Q000032:analysis*; D012997:Solvents
D009844:Oligosaccharides; D012997:Solvents; D006493:Heparin