Towards a high peak capacity of 130 using nanoflow hydrophilic interaction liquid chromatography

Abstract Hydrophilic interaction liquid chromatography, HILIC, is a relatively new HPLC mode. Compared with other HPLC modes, HILIC is a high resolution chromatographic mode with high peak capacity for separations of complex mixtures. Although the separation mechanism is still not completely clear, HILIC has been widely used for analysis of hydrophilic compounds which are difficult for reversed phase chromatography to retain and separate. In this study, we fabricated and investigated nanoHILIC columns in terms of separation efficiency, van Deemter curves and more importantly, we focused on long packed capillary columns, and studied their extreme resolution for protein digests. Using meter long nanoHILIC columns packed with 5 μm particles, we realized a high peak capacity of 130. Based on nanoLC-MS, we compared the resolution and protein identification capabilities of nanoHILIC and nanoRPLC. The results indicate both nanoHILIC and nanoRPLC can provide high resolution for protein sequencing but neither mode is significantly better than the other. Among the 99 digest peptides identified, 17 were uniquely identified by nanoHILIC-MS and 20 were uniquely identified by nanoRPLC-MS and 62 were identified by both methods. Although at this moment in time, nanoRPLC is the most popular microseparation tool in proteomics, the excellent complementarity of nanoHILIC and nanoRPLC suggests their combined use in achieving deep-coverage in MS-based proteomics.