Gerald L. Bachman

Development and optimization of a SE-HPLC method for proteins using organic mobile phases

Publisher Summary This chapter describes the development and optimization of an organic mobile phase size exclusion high-performance liquid chromatography (SE-HPLC) method using organic mobile phases for several proteins. With an acetonitrile/trifluoroacetic acid (TFA) mobile phase, non-ideal SE-HPLC (ionic, hydrogen bonding and hydrophobic interactions) is minimized. SE-HPLC is the method to monitor removal of the oligomers during purification of the proteins. Most SE-HPLC methods for proteins use aqueous mobile phases containing salts or chaotropic agents, such as sodium dodecyl sulfate (SDS) or urea. Hydrophobic and ion-exchange interactions occurring between the protein and the chromatography resin with these mobile phases may result in nonideal size exclusion chromatography. Limited sample solubility and non-covalent oligomer formation may also occur with these mobile phases resulting in inconsistent performance of the method. The chapter demonstrates that acetonitrile/TFA mobile phases can be effectively used for analyzing several different proteins.