Molecular Weight Determination of Polysaccharides Using Size Exclusion Chromatography Coupled with Multi-Angle Laser Light Scattering and Refractive Index Detectors.

: Size exclusion chromatography (SEC)/refractive index (RI) were used to determine molecular weight (MW) and molecular weight distributions (MWD) of polysaccharides. In aloe product research and quality control, commercially available pullulan and dextran are most commonly employed as calibration standards. Significant difference in the MW and MWD were found in literature when different methods were used. This study was to investigate the traditional methods and more recent technologies used to determine the MW and MWD of polysaccharides. In this study, multi-angle laser light scattering (MALS) detection was studied on three polysaccharides, 1, 2, and 3, that were isolated and purified from leaf. The chemical structures of 1-3 were characterized as 1, 4-β-linked glucomannans by monosaccharide composition and glycosidic linkage analysis. Absolute MW and root-mean-square radius were determined by MALS on the isolated aloe polysaccharides. The conditions to obtain reliable results from MALS measurement were examined. MALS analysis demonstrates that the 1, 4-β-linked glucomannan adopt the conformation of random coils or hard spheres in the analytical environment of a 0.1 M NaCl solution. Non-size exclusion effects and interactions between polysaccharide molecules were also observed in some aloe polysaccharides in the current analysis. The weight-average MW obtained by MALS measurement for 1, 2, and 3 are 55, 129, and 962 kDa, respectively. Comparing the results with SEC/RI calibrated by pullulan and dextran standards, marked differences in the MWD are found. Both overestimated the MW of 1 and 2 by factors of 4.4 and 4.2, and 2.4 and 1.6, when using dextran and pullulan calibration, respectively. Using pullulan calibration underestimated the MW of 3 by a factor of 3.1, but a similar result was obtained from dextran calibration compared to MALS measurement. The two isolated aloe polysaccharides were employed to be broad calibration standards or to be combined with narrow polydispersity pullulan calibration standards. Several aloe samples were tested using the different calibration curves, and the determined MWs were compared with the results obtained by MALS measurement. The results clearly indicated that until true polysaccharide standards become available MW and MWD's will be simply relative to the standards employed and the technologies used.