Effect of anionic ion-pairing reagent hydrophobicity on selectivity of peptide separations by reversed-phase liquid chromatography.
Despite the continuing dominance of trifluoroacetic acid (TFA) as the anionic ion-pairing reagent of choice for peptide separations by reversed-phase high-performance liquid chromatography (RP-HPLC), we believe that a step-by-step approach to re-examining the relative efficacy of TFA compared to other ion-pairing reagents is worthwhile, particularly for the design of separation protocols for complex peptide mixtures, e.g., in proteomics applications. Thus, we applied RP-HPLC in the presence of different concentrations of anionic ion-pairing reagents - phosphoric acid, TFA, pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA)--to a mixture of three groups of four 10-residue peptides, these groups containing peptides of +1, +3 or +5 net charge. Overall separation of the 12-peptide mixture improved with increasing reagent hydrophobicity (phosphate- < TFA- < PFPA- < HFBA-) and/or concentration of the anion, with reagent hydrophobicity having a considerably more pronounced effect than reagent concentration. HFBA, in particular, achieved an excellent separation at a concentration of just 10 mM, whereby the peptides were separated by charged groups (+1 < +3 < +5) and hydrophobicity within these groups. There was an essentially equal effect of reagent hydrophobicity and concentration on each positive charge of the peptides, a useful observation for prediction of the effect of varying counterion concentration hydrophobicity and/or concentration during optimization of peptide purification protocols. Peak widths were greater for the more highly charged peptides, although these could be decreased significantly by raising the acid concentration; concomitantly, peptide resolution increased with increasing concentration of ion-pairing reagent.
Shibue
M
M
Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center at Fitzsimons, Aurora, CO 80045, USA.
Mant
C T
CT
Hodges
R S
RS
D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D007202:Indicators and Reagents / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D014269:Trifluoroacetic Acid / Q000737:chemistry
D007202:Indicators and Reagents; D010455:Peptides; D014269:Trifluoroacetic Acid