Making Sharper Peaks for Reverse-Phase Liquid Chromatography of Proteins

Protein separations have gained increasing interest over the past two decades owing to the dramatic growth of proteins as therapeutics and the completion of the Human Genome Project. About every decade, the field of protein high-performance liquid chromatography (HPLC) seems to mature, having reached what appears to be a theoretical limit. But then scientists well versed in the basic principles of HPLC invented a way around the limit, generating another decade of exciting progress. There is still the need for higher resolution and better compatibility with mass spectrometry because it is an essential tool for identification of proteins and their modifications. To make advances, the fundamental principles need to be understood. This review covers recent advances and current needs in the context of the principles that underlie the many contributions to peak broadening.