Comparison of peak shapes obtained with volatile (mass spectrometry-compatible) buffers and conventional buffers in reversed-phase high-performance liquid chromatography of bases on particulate and monolithic columns.
Retention factor, column efficiency and asymmetry factor were recorded for nine basic compounds on a number of RP-HPLC columns using phosphate and a variety of (MS-compatible) volatile mobile phase buffers of acid and neutral pH, in order to assess any effects of the buffer on performance. With formic or acetic acid, some phases gave partial or complete solute exclusion effects (reduced or negative k) compared with results using phosphate buffers at low pH. Despite its possible suppression of mass spectrometer sensitivity, trifluoroacetic acid was useful in enhancing retention times of relatively hydrophilic protonated bases, due to ion-pair effects. Peak shape was relatively poor on some pure silica-based ODS phases at pH 7 compared with results at acid pH. At low pH and at pH 7, ammonium and potassium phosphate gave very similar k, but the former may be preferable due to its volatile cation. Improved peak shapes, attributed to superior silanol masking effects, were obtained with ammonium phosphate at pH 7, but not at acid pH. Ammonium acetate gave acceptable peak shape at pH 7, but due to very limited buffer capacity, poor results were obtained for solutes having a pKa close to the mobile phase pH. Due to its instability, ammonium hydrogen carbonate is not a viable alternative buffer at pH 7.
McCalley
David V
DV
Centre for Research in Biomedicine, University of the West of England, Frenchay, Bristol BS16 1QY, UK. david.mccalley@uwe.ac.uk
D002021:Buffers; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation* / Q000379:methods; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*
D002021:Buffers