DIRECT AND INDIRECT METHODS FOR MOLAR-MASS ANALYSIS OF FRAGMENTS OF THE CAPSULAR POLYSACCHARIDE OF HAEMOPHILUS-INFLUENZAE TYPE-B

Two methods are described for direct molar-mass measurement of low-mol ar-mass fragments obtained by oxidative cleavage of the capsular polys accharide of Haemophilus influenzae type b. Absolute molar masses were determined by size-exclusion chromatography (SEC) with detection by m ultiangle laserlight-scattering photometry (MALLS) and differential re fractometry (RI). The end-group structure of the polysaccharide fragme nts allowed the direct measurement of average chain length by quantita tive H-1 NMR, fi-om which molar masses were derived. Variation between the molar masses obtained by the two methods ranged from 5 to 7%. Whe n molar masses estimated by indirect methods were compared to SEC-MALL S/RI data, significant deviations were observed. Analysis by SEC with secondary calibration with dextran standards gave molar masses that ex ceeded the SEC-MALLS/RI data by as much as 2.5-fold. Molar masses esti mated by a combination of colorimetric assays varied from the SEC-MALL S/RI data by as much as 50%. These results demonstrated the applicabil ity and superior accuracy of the direct methods of molar-mass determin ation of the polysaccharide fragments. (C)1997 Academic Press.