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Optimization of conditions for size-exclusion chromatography of proteins

D. Hagarová, M. Horváthová, V. Žúbor, and A. Breier

Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, CS-833 34 Bratislava

 

Abstract: Chromatographic properties of bead celluloses (types Perlose MT 100 M, 200 M, 500 M and the low porous Perlose MT 5L) were investigated in conditions for size-exclusion chromatography. With mobile phase containing no detergent the retention time for elution of proteins was considerably long, the sample remained on the top of the column. Increasing content of sodium dodecyl sulfate (SDS) present in the mobile phase lowered the retention volume. At w(SDS) = 0.1% the retention volumes could be established safely for all proteins tested. From values of retention volumes the distribution coefficients K(av) could be calculated for all proteins applied. For Perloses MT 100 M, 200 M, and 500 M the relationship between K(av) and the relative molecular mass of proteins could be described by the equation Kav = a' + b' Mw1/3. An extrapolation of respective equations for Kav = 0 provided the following estimated values for upper exclusion limits: 155 000 +/- 28 000, 263 000 +/- 75 000, and 547 000 +/- 75 000 for MT 100 M, 200 M, and 500 M, respectively. The exclusion limit for low porous cellulose MT 5L was less than 12 400. Results indicate that Perloses may be utilized in size-exclusion chromatography of proteins.

Full paper in Portable Document Format: 453a341.pdf

 

Chemical Papers 45 (3) 341–348 (1991)

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