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Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS
Published on: June 20, 2019
Precise Determination of the Band-Broadening Function for the Size-Exclusion Chromatography using Multi-Angle Light
1Department of Polymer Chemistry, Kyoto University, Kyoto, Japan.
Abstract:
Size-exclusion chromatography (SEC) equipped with refractive index (RI) and multi-angle light scattering (MALS) detectors was carried out on a polystyrene (PS) sample with the weight-average molecular weight 2.07 ×€104 along with the matrix-assisted laser desorption flight time mass spectrometry (MALDI-TOF-MS). The MALDI-TO-MS result was corrected by the relative sensitivity, which was found to decay exponentially with the molecular weight. Exchanging the elution volume for molecular weight by the calibration relation, the molecular-weight distribution (MWD) curves were obtained from the chromatograms. The MWD curve determined by the SEC chromatogram was much broader than that obtained by MALS and MALDI-TOF-MS because of the peak-broadening effect. Based on the MWD determined by MALDI-TOF-MS, the RI chromatogram was closely described by the calculated values using the Gaussian function. Based on the results of the PS sample, RI and MALS data of polyethylene oxide samples in water were analyzed using the exponentially modified Gaussian equation.
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