Study on Retention Equation for Protein in Reversed Phase Liquid Chromatography

Study on the retention equation for protein in RPLC can contribute to optimizing gradient conditions for protein separation. In this study, five standard proteins, insulin, cytochrome C, lysozyme, transferrin and myoglobin were selected, and their retention time on three reversed-phase columns (Waters Symmetry 300 C-4, Waters Symmetry 300 C-18, C8HC) under six different linear gradients was obtained. According to nonlinear curve fit (CSASS software), the retention equation for five standard proteins on three columns was established. The regression coefficients of the 15 equations were all above 0: 999, indicating that the equation could accurately describe protein retention behavior on RPLC mode. Based on the equation, retention time for 5 standard proteins under other gradient conditions was predicted, and the relative errors for each protein were below 5%. This retention equation could be used in optimizing gradient conditions for protein separation on RPLC, leading to rapid baseline separation of five mixed standard proteins within 20 min.