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

Abstract

: 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.

Purification and Structural Characterization of Sulfated Polysaccharides Derived from Brown Algae, Sargassum binderi: Inhibitory Mechanism of iNOS and COX-2 Pathway Interaction

“…MALS is used to check molar mass, polydispersity, and rms radius [ 46 ]. We found that SBP-F4 had a number-average molecular weight (Mn) of 1.986 × 10 5 g/mol, a weight average molecular weight (Mw) of 2.351 × 10 5 g/mol, and a Z-average molecular weight (Mz) of 2.867 × 10 5 g/mol.…”

Membrane Stabilization of Poly(ethylene glycol)-b-polypeptide-g-trehalose Assists Cryopreservation of Red Blood Cells

“…The significant difference in the molecular weight and its distribution could be caused by using the PEG calibration standard. 47 Compared to the polypeptides, the The feeding composition is the stoichiometric molar ratio of Tre to the amino groups in the main chain of PEG-b-polypeptides where "Tre" refers to carboxylated trehalose. b The grafting ratio and the molecular weight M n,NMR of the synthesized glycopeptides were calculated from the 1 H NMR spectra by the integral ratio of Tre and the molecular weight of PEG-b-polypeptides.…”

“…Similarly, the molecular weights of PKDT , PKDF1T , and PKDF2T measured by GPC were 1.45 × 10 4 , 2.18 × 10 4 , and 3.10 × 10 4 with narrow distribution ( Đ ) in 2.53, 1.99, and 1.41, respectively. The significant difference in the molecular weight and its distribution could be caused by using the PEG calibration standard . Compared to the polypeptides, the higher polydispersity of glycopeptides could be found in Table and Table S1, which was presumably caused by the introduction of trehalose in GPC measurement .…”

Exploiting the Amazing Diversity of Natural Source-Derived Polysaccharides: Modern Procedures of Isolation, Engineering, and Optimization of Antiviral Activities

“…Weak anion-exchangers, such as diethylaminoethyl (DEAE), tertiary amine-functionalized media [93,94] or strong anion-exchangers including quaternary (Q) amine-functionalized media [95], have been successfully utilized to purify polysaccharide sulfates. SEC is extensively employed for the measurement of total molecular masses and molecular mass distributions [96][97][98][99][100]. Moreover, this analytical technique has been effectively employed in many examples for the elucidation of fine-structures of oligosaccharides and polysaccharides [94] and to produce size classes for the determination of structure-activity relationship [101][102][103].…”

“…Weak anion-exchangers, such as diethylaminoethyl (DEAE), tertiary amine-functionalized media [93,94] or strong anion-exchangers including quaternary (Q) amine-functionalized media [95], have been successfully utilized to purify polysaccharide sulfates. SEC is extensively employed for the measurement of total molecular masses and molecular mass distributions [96][97][98][99][100].…”