Authors: T.J. Higley, Tosoh Bioscience LLC, Tatsunari Yoshida, Tosoh Corporation
The TSKgel Amide-80 column provides excellent selectivity for separating peptides in hydrophilic interaction chromatography due to polar carbamoyl functionalities bonded to a silica base material.
The separation of peptides with many acidic and basic residues has always proved to be problematic with RPLC gradient elution runs common in LC/MS and/or high throughput methodologies. Several options are available to increase retention of polar peptides including mobile phase adjustment(1) or switching to a polar adsorptive mechanism used in both normal phase and hydrophilic interaction chromatography (HILIC)(2,3). HILIC methods are often advantageous because aqueous and polar organic mobile phase systems, common to RPLC, are acceptable. The data presented within will highlight the rational, recovery and reproducibility associated with a HILIC method developed within Tosoh laboratories for the separation of peptides.
A 5m TSKgel Amide-80 column (4.6mm ID x 25cm) was used in conjunction with a Tosoh HPLC system. The peptides were purchased from the Peptide Institute (Osaka, Japan) and Sigma (St. Louis, MO). Final mobile phase conditions: Eluent A (initial eluent) contained 0.1% trifluoroacetic acid (TFA) in acetonitrile (ACN): water (97:3) and Eluent B contained 0.1% TFA in ACN-water (55:45). The peptides were dissolved in 10L of ACN-water-formic acid (5:45:50) and subsequently diluted to a final volume of 50L with Eluent A. A linear gradient