Protein surface area and retention in hydrophobic interaction chromatography |
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Authors: | A. Katti Y. -F. Maa Cs. Horváth |
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Affiliation: | (1) Department of Chemical Engineering, Yale University, 06520 New Haven, CT, USA;(2) Present address: Oak Ridge National Laboratory, Analytical Chemistry Division, 37831 Oak Ridge, TN, USA |
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Abstract: | Summary Molecular surface areas accessible to a 4 ? diameter spherical probe were calculated from crystallographic data for five proteins: α-chymotrypsinogen A, lysozyme, trypsinogen, ribonuclease A and ribonuclease S. The retention factors of various proteins were measured on stationary phases having polyether- and phenylligates and with aqueous eluents containing (NH4)2SO4, Na2SO4 or NaCl at pH 7.0. The logarithmic retention factors were plotted against the salt molality and the hydrophobic interaction parameters evaluated from the limiting slopes of the plots at high salt concentrations for the proteins in the chromatographic systems investigated. The hydrophobic interaction parameters thus obtained were linear in both the molecular surface areas of the proteins and the molal surface tension increments of the salts. The experimental results obtained with these relatively simple proteins of known molecular structure, which were available in high purity, support earlier theoretical predictions for the dependence of the hydrophobic interaction parameter on the surface area of the protein and the surface tension raising effect of the salt. |
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Keywords: | Column liquid chromatography Hydrophobic interactions Proteins Surface tension Salt effect |
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