Guo Y C, Conrad H E
Department of Biochemistry, University of Illinois, Urbana 61801.
Anal Biochem. 1988 Jan;168(1):54-62. doi: 10.1016/0003-2697(88)90009-7.
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.
开发了一种离子对高压液相色谱法，用于分析肝素经亚硝酸裂解产生的寡糖混合物。使用乙腈与含有40 mM磷酸铵和1 mM磷酸四丁铵的缓冲液的混合物，从Hi-Chrom 5S ODS（C18）柱上洗脱寡糖。针对先前已表征的多种单糖和四糖（M.J. Bienkowski和H.E. Conrad（1985）J. Biol. Chem. 260, 356 - 365），开发了等度洗脱条件以实现最佳分离。然后将这些等度洗脱条件相结合，以获得用于二糖混合物和四糖混合物离子对分离的梯度洗脱条件。离子对色谱法获得的洗脱曲线与阴离子交换高压液相色谱法获得的曲线相比，离子对法的整体分辨率明显更高。由于分辨率的提高，新方法显示肝素寡糖混合物中存在先前未鉴定的产物。
