Molecular weight determination by Chromatography and Viscometry: A case study using novel polysaccharide of Eupatorium adenophorum L.

Molecular weight analysis of natural polysaccharides is necessary for diverse nutraceutical, pharmaceutical, biological, chemical and other industrial applications. The present study focuses on the determination of the molecular weight of polysaccharide from Eupatorium adenophorum using HP-GPC and Viscometry independently, apart from the chemical composition of constituent monosaccharides. The novel polysaccharide was isolated from leaves of the species, followed by purification using repetitive precipitation, centrifugation, dialysis and chromatographic purification to obtain the polysaccharide in 0.62 ± 0.08 % yield. To analyze monosaccharides constituting polysaccharide, it was hydrolyzed, followed by reduction of hydrolysates and derivatization to corresponding alditol acetates by acetylation. Weight average and number average molecular weights were determined by chromatography (HPGPC) with the help of known MW dextran standards. The viscosity average molecular weight was determined by intrinsic viscosity measurement using the Ubbelohde dilution viscometer and applying the Mark-Houwink equation. GC-MS/FID analysis indicated the presence of l-arabinose, d-xylose, d-mannose, d-glucose and d-galactose as the constituent monosaccharides in 1.61:1.00:0.25:5.89:3.24 M ratios. The different molecular weights were determined to be 4.05 × 10 Da (M), 2.28 × 10 Da (M) and 3.59 × 10 Da (M). The polysaccharide has a polydisperse nature with PDI value of 1.793. Such prudent studies are useful for third-world countries and undergraduate laboratories requiring the guided molecular weight-based screening for diverse applications, but do not have HPGPC in the laboratory. Results indicate that a simple glass Ubbelohde Dilution Viscometer fulfils the goal of molecular weight determination. The molecular weight and monosaccharide composition of E. adenophorum leaf polysaccharide have been reported for the first time.