Evaluation and comparison of n‐alkyl chain and polar ligand bonded stationary phases for protein separation in reversed‐phase liquid chromatography

Abstract

Protein retention is very sensitive to the change of solvent composition in reversed-phase liquid chromatography for so called "on-off" mechanism, leading to difficulty in mobile phase optimization. In this study, a novel 3-chloropropyl trichlorosilane ligand bonded column was prepared for protein separation. The differences in retention characteristics between the 3-chloropropyl trichlorosilane ligand bonded column and n-alkyl chain modified (C2, C4, C8) stationary phases were elucidated by the retention equation l nk=a+cC(B). Retention parameters (a and c) of nine standard proteins with different molecular weights were calculated by using homemade software. Results showed that retention times of nine proteins were similar on four columns, but the 3-chloropropyl trichlorosilane ligand bonded column obtained the lowest retention parameter values of larger proteins. It meant that their retention behavior affected by acetonitrile concentration would be different due to lower |c| values. More specifically, protein elution windows were broader, and retentions were less sensitive to the change of acetonitrile concentration on the 3-chloropropyl trichlorosilane ligand bonded column than that on other columns. Meanwhile, the 3-chloropropyl trichlorosilane ligand bonded column displayed distinctive selectivity for some proteins. Our results indicated that stationary phase with polar ligand provided potential solutions to the "on-off" problem and optimization in protein separation.