Evaluation of the Effects of Sample Solutions and Injector Wash Solutions on Separation Efficiency in Hydrophilic Interaction Liquid Chromatography

Abstract

Hydrophilic interaction liquid chromatography (HILIC) is advantageous for separation of polar compounds and sensitive detection in mass spectrometry. Sample dilution can negatively affect separation. We previously reported that separation efficiency in HILIC is affected by sample water content and perhaps the wash solution used in the injector sample loop. In this study, the effects of sample composition, sample volume, and wash solutions, on separation in HILIC was investigated using a mixture of catechol compounds including basic, acidic, and zwitterionic analogs. A ZIC-cHILIC column with a phosphorylcholine stationary phase was used for analysis. In the investigation of the effect of sample composition and volume, it was shown that separation efficiency was dependent on the amount of water injected into the column. Furthermore, we found that the most distorted peaks had similar elution times to water. We also investigated the effect of the sample loop wash solution, with separation efficiency reduced by increasing water content in the wash solutions. These results indicated that the water content of both sample and washing solutions should be kept to a minimum in HILIC separation.