The Behavior of Proteins on Reversed-Phase Supports During High-Performance Liquid Chromatography and Prediction of Protein Retention Time on Ymc Columns

Abstract

Protein molecules present more complex analytical challenges than conventional low molecular weight pharmaceutical compounds, and thus need powerful analytical approaches for the entire pharmaceutical development and quality control process. We used high-performance liquid chromatography to investigate the reversed-phase chromatographic behavior of eleven proteins (albumin, carbonic anhydrase, cytohrome c, L-glutamic dehydrogenase, enolase, α-lactoglobulin, Lysozyme, myoglobin and ribonuclease. By using a water/organic solvent/trifluoroacetic acid system the influence of experimental parameters was examined, and chromatographic results from two C4-chain-length supports were found to be comparable. The model enables prediction of initial conditions from two experimental data points for different types of reversed-phase columns with water-acetonitrile-TFA, water-methanol-TFA, and water-2-propanol-TFA mobile phases.