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.

An understanding of why hydrophilic interaction liquid chromatography gives a higher resolution for glycans than for glycoproteins would facilitate column improvements. Separations of the glycoforms of ribonuclease B compared to its released glycans were studied using a commercial hydrophilic interaction liquid chromatography column. The findings were used to devise a new hydrophilic interaction liquid chromatography column. For the commercial column, chromatograms and van Deemter plots showed that selectivity and efficiency are comparable factors in the higher resolution of the released glycans. The higher selectivity for the released glycans was associated with more water molecules displaced per added mannose. To investigate why, three-dimensional structures of the glycoprotein and the glycan were computed under chromatographic conditions. These showed that hydrogen bonding within the free glycan makes its topology more planar, which would increase contact with the bonded phase. The protein sterically blocks the hydrogen bonding. The more globular-shaped glycan of the glycoprotein suggests that a thicker bonded phase might improve selectivity. This was tested by making a column with a copolymer bonded phase. The results confirmed that selectivity is increased. The findings are possibly broadly relevant to glycoprotein analysis since the structural motif involved in internal hydrogen bonding is common to N-linked glycans of human glycoproteins.

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