Almost all the peptides I have synthesized were subsequently purified using a reversed-phase C18 column. Sometimes this worked, but sometimes it didn’t work so well. When my C18 purifications failed, I questioned whether or not I could have predicted this outcome prior to extensive HPLC efforts. Since then, I have learned that the amino acid composition of the peptide may give some clues to the peptide’s chromatographic behavior.
While there are numerous stationary phase functionalization types for reversed-phase chromatography, in today’s post I will describe some differences I have observed when purifying peptides using C18- or C4- functionalized stationary phases for peptide purification.