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Authors: T.J. Higley, Tosoh Bioscience LLC, Tatsunari Yoshida, Tosoh Corporation
Abstract:
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.
Introduction:
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.
Experimental Conditions:
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
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Source:
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