Size-exclusion chromatography considerations

Special Considerations for the Size Exclusion Chromatography of Proteins... 

Batas, B. and Chauduri, J.B., Considerations of sample application and elution during size-exclusion chromatography-based protein refolding, /. Chromatogr. A, 864, 229, 1999. 

HE FIELD OF SIZE EXCLUSION CHROMATOGRAPHY (SEC) remains a viable and lively area of polymer characterization. Over the past several years, there has been considerable research activity in the area of SEC detection and data analysis in order to obtain more comprehensive information concerning the composition and molecular architecture of complex polymer systems. 

Morr and Seo (22) reported that the MW of CMP as determined by size exclusion chromatography was 33 kDa at neutral pH. This MW was considerably higher than the theoretical vdue of 9 kDa based on the amino acid sequence of CMP. These findings suggested that CMP formed oligomers in neutral solutions owing to its self association, and that the association was partially disrupted under acidic conations. 

The presence of the different suPAR variants in EDTA plasma from a prostate cancer patient was analyzed by size exclusion chromatography and measurement of the fractions obtained with the three different TR-FIAs. This EDTA plasma contained detectable amounts of uPAR(I) in contrast to an EDTA plasma pool from healthy donors. The levels of suPAR measured with TR-FIA 2 were considerably higher and eluted at a later position in the patient sample than in the donor plasma, thus indicating the presence of suPAR(II nf) in the patient sample [108], In a recent study, the concentrations of uPAR(I) as well as the calculated suPAR(II-III) were found to be significantly elevated in serum samples from patients with prostate cancer compared to the concentrations in serum from men with benign prostatic conditions. Specific measurements of uPAR(I) were found to improve specificity of prostate cancer detection [142],... 

Size exclusion chromatography is the premier polymer characterization method for determining molar mass distributions. In SEC, the separation mechanism is based on molecular hydrodynamic volume. For homopolymers, condensation polymers and strictly alternating copolymers, there is a correspondence between elution volume and molar mass. Thus, chemically similar polymer standards of known molar mass can be used for calibration. However, for SEC of random and block copolymers and branched polymers, no simple correspondence exists between elution volume and molar mass because of the possible compositional heterogeneity of these materials. As a result, molar mass calibration with polymer standards can introduce a considerable amount of error. To address this problem, selective detection techniques have to be combined with SEC separation. 

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