( from A to B was administered ...)Separation of Peptides by Hydrophilic Interaction Chromatography Using TSKgel Amide-80

Results:
In the study to find suitable method conditions for the separation of peptides by HILIC, the effect of acid and ACN concentration were examined. It is commonly understood that residual silanols, present with functionalized silica based materials, can interact with the ionic residues of peptides. This interaction affects recovery and/or causes peak tailing. For this reason, TFA, acetic acid, formic acid and no acid were investigated to determine the appropriate acid strength for eliminating IEX interactions. As shown in Figure 1, elution order and peak shape were affected differently depending the acid investigated. Without the addition of acid, three of the peptides failed to elute and tailing was evident on the remaining peptides. The addition of weak acids (acetic or formic) allowed the elution of all components but did not eliminate tailing. TFA at 0.1% was strong enough to eliminate tailing and sequester the apparent IEX interactions. Subsequent work (not shown) indicates little improvement with the addition of equal molar amounts of TFA and TEA at 0.1% or 0.2% over what is possible with TFA alone(4). The advantage of using TFA to eliminate IEX interactions is the need for desalting is eliminated when a volatile mobile phase is used. PolyHydroxyethyl A, {poly (2-hydroxyethyl aspartamide) silica} a commonly used HILIC column for peptides, has been reported to exhibits IEX interactions(5,6). Thus, salts or nonvolatile acids such as orthophosphoric acid are recommended in the mobile phase to eliminate such effects. Peptide isolation su
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