Chondroitin sulfate is used to treat arthritis and other conditions involving cartilage. Different preparations of the product have different molecular properties, which are related in turn to therapeutic effectiveness. Such properties can be studied—and quantified—by light scattering.
Two chondroitin sulfate samples were compared by means of a DAWN multi-angle light scattering (MALS) instrument (Wyatt Technology) coupled to size-exclusion chromatography (SEC). A differential refractive index (dRI) detector served to measure concentration. MALS and dRI measurements are combined in ASTRA software to calculate molar mass distributions, moments such as the weight-average molar mass Mw, and the polydispersity index Mw/Mn.
Analysis by MALS relies on first principles and therefore overcomes the uncertainties associated with column calibration, which arise from differences in elution properties between the sample and reference molecules related to conformation, density, and column interactions. SEC–MALS also eliminates the need to perform frequent recalibration.
Figure 1 shows chromatograms and molar masses obtained from MALS measurements for two samples, CS and CSLM. The dRI trace alone is shown for CSLM in red, while both dRI and MALS signals are shown for CS, in blue. Not all of the differences between the two samples appear in standard SEC analysis, but are readily revealed by SEC–MALS.
MALS indicates that CS has a Mw of 14.8 ± 0.2 kDa and the polydispersity index (Mw /Mn) is 1.21 ± 0.03. The low molar mass chondroitin sulfate LMCS sample has a Mw of 6.9 ± 0.2 kDa and a polydispersity of 1.4 ± 0.1. MALS analysis in fact determines a full set of moments including Mn, Mw, and Mz, as well as cumulative and differential molar mass distributions (not shown).
The SEC–MALS method provides complete information about the molar mass of chondroitin sulfate, determining distributions and a full set of moments. The analysis indicated similarities and discrepancies between the two samples. The presence of a high-molecular-weight peak in CS, not observed by dRI, was detected unequivocally by MALS, thanks to its enhanced sensitivity to high-molecular-weight species.
The fact that similar molar masses and sizes of each sample were eluted at the same time is a good indication of similar conformation and chemical properties. Absolute macromolecular characterization by SEC–MALS provides rich, detailed information without the biases inherent in standard analytical SEC, for confidence in essential physiochemical properties.
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