A systematic investigation of the effect of sample diluent on peak shape in hydrophilic interaction liquid chromatography

The aim of this study was to evaluate the importance of sample diluents to improve peak shapes in hydrophilic interaction liquid chromatography (HILIC), using low molecular weight (<1000 Da) analytes as well as peptides (with MW ranging between 1000 and 6000 Da) as model compounds. Various solvents were tested including water, acetonitrile, methanol, ethanol, propan-2-ol, dimethyl sulfoxide, and a number of combinations of them. For the analysis of small MW compounds, best peak shapes were obtained with sample dissolved in pure ACN but, IPA or a mixture of ACN/IPA (50:50, v/v) could represent a viable alternative in the case of solubility issues with pure ACN. For drug discovery applications, DMSO can be employed but in combination with at least 80% of ACN. For peptides analysis, acetonitrile, EtOH and IPA as sample diluents, provided similar chromatographic profiles, but pure EtOH or IPA were recommended to limit denaturation and samples solubility issues. Finally, whatever the nature of the compounds, it is recommended to add the lowest amount of water to the sample diluent, to maintain suitable peak shapes.