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Prediction of a protein band profile in preparative reversed‐phase gradient elution chromatography
Author(s) -
Zoubaïr M.,
Fallah El,
Guiochon Georges
Publication year - 1992
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260390810
Subject(s) - chromatography , acetonitrile , chemistry , lysozyme , elution , adsorption , solvent , high performance liquid chromatography , phase (matter) , analytical chemistry (journal) , reversed phase chromatography , langmuir adsorption model , saturation (graph theory) , organic chemistry , biochemistry , mathematics , combinatorics
The overloaded band profiles of lysozyme in reversedphase preparative chromatography were recorded on a C18 chemically bonded silica column, with acetonitrile/water as the mobile phase. These experiments were carried out under isocratic conditions at 31.6, 31.9, and 32.2% acetonitrile (ACN) for loading factors up to 43% of the column saturation capacity and under linear‐solvent‐strength gradientelution with gradient slopes of 0.5 and 1% ACN/min, for loading factors up to 11.3%. The adsorption isotherms of lysozyme were measured for the same solvent compositions and found to be accurately accounted for by a bi‐Langmuir isotherm model. With the use of a Craig model implementation of the equilibrium‐dispersive model of chromatography, the band profiles of lysozyme were calculated. An excellent agreement was observed between these calculated profiles and the experimental profiles recorded at loading factors below 5%. By contrast, band profiles calculated using a Langmuir isotherm failed to describe the experimental bands. At column loadings exceeding 8%, a slight but systematic deviation takes place between calculated and experimental profiles. It is most probably explained by the considerable concentration effect of the gradient, making the band experience phase equilibrium in a concentration range that exceeds largely the one where the isotherm data have been measured.