High-performance liquid chromatography of amino acids, peptides and proteins. XCVIII. The influence of different displacer salts on the bandwidth properties of proteins separated by gradient elution anion-exchange chromatography.
The influence of eight different displacer salts on the bandwidth properties of four globular proteins separated by a high-performance anion-exchange chromatography has been investigated. Proteins were eluted under gradient conditions with a range of alkali metal halide salts, in which the anion and cation were varied in the series F-, Cl- and Br- and Li+, Na+ and K+, respectively. The experimentally observed bandwidths (sigma v,exp) were found to deviate significantly from peak widths (sigma v,calc) predicted on the basis of plate theory for small molecules. For data accumulated under conditions of varied gradient time and constant flow-rate the solute bandwidth ratios (sigma v,exp/sigma v,calc) increased in the order Br- less than Cl- less than F- at low values of the gradient steepness parameter, b, or increasing column residence times. In addition, systematic changes in the cation influenced the bandwidth ratios (sigma v,exp/sigma v,calc) in the order K+ less than Na+ less than Li+. Significant deviations between predicted and observed bandwidth values were also observed under elution conditions of constant gradient time and varied flow-rate. The results of the present study further demonstrate the complex nature of the interaction between protein solutes and coulombic chromatographic surfaces.
Hodder
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Department of Biochemistry, Monash University, Clayton, Victoria, Australia.
Aguilar
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Hearn
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D000838:Anions; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000302:isolation & purification; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D009113:Muramidase / Q000737:chemistry / Q000302:isolation & purification; D009211:Myoglobin / Q000737:chemistry / Q000302:isolation & purification; D010047:Ovalbumin / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D000644:Quaternary Ammonium Compounds; D012492:Salts
D000838:Anions; D009211:Myoglobin; D011506:Proteins; D000644:Quaternary Ammonium Compounds; D012492:Salts; D010047:Ovalbumin; D009113:Muramidase; D002256:Carbonic Anhydrases