High-performance liquid chromatography of amino acids, peptides and proteins : LIII. Evaluation of the effect of several stationary phase parameters on the chromatographic separation of polypeptides on alkylsilicas

Abstract

The separation of several polypeptides and proteins of varying molecular size by reversed-phase high-performance liquid chromatography on octadecylsilicas of different pore diameters has been studied. In particular, the retention behaviour of phenylalanine oligomers has been further investigated with these stationary phases as the volume fraction, ψ s, of the organic solvent modifier was systematically increased up to ψ s, = 0.7. The results are compared with data obtained in related experiments with commercially available μBondapak C 18 columns. Quantitative relationships between relative retention, the stationary phase surface area, alkyl ligand density, and ψ s have been utilised to aid the interpretation of the role of pore size effects in the separtion of these and other polypeptides on alkylsilicas. The results confirm that the s value for a given polypeptide, as evaluated from the linear dependency of the logarithmic capacity factor, log k′, on ψ s, and the ω-value as evaluated from the plots of log k′ versus the surface tension of the eluent, γ, over the range of k′ values of interest in isocratic or gradient elution optimisation, are essentially constant for all the octadecylsilicas. Further, the results confirm that retention under regular reversed-phase conditions is directly related to the accessible non-polar surface area within the column. Comparison of the data for small- and large-pore octadecylsilicas suggests that not all of the bonded surface area of packings with small pores ( e.g., 7.3 nm) is available to polypeptide solutes. Predictions based on solvophobic theory have been further tested and the linear dependency of the logarithmic selectivity factor (log α = τ) on the bulk surface tension, γ, confirmed. Based on this analysis, the magnitudes of the difference in Van der Waals interaction energies and the free energies of association per phenylalanine residue have been estimated. Finally, the data suggest that the size exclusion component has little effect on reversed-phase retention other than to restrict entropically the access to the inside of the pore. Once inside the pore, retention appears to be predominantly a mobile phase driven phenomenon.