Rationalisation of unusual changes in efficiency and retention with temperature shown for bases in reversed-phase high-performance liquid chromatography at intermediate pH.
Despite the possibility of poorer peak shapes, analysis of pharmaceuticals and other bases using reversed-phase high-performance liquid chromatography (RP-HPLC) at intermediate pH gives useful increases in retention, selectivity and column loading capacity compared with low pH. Retention times of some bases showed anomalous increases with column temperature. Peak shapes for bases improved significantly at elevated temperature (up to 70 degrees C), with the weakest bases studied (pKa approximately 8) giving greater improvement than the strongest bases (pKa approximately 10). In contrast, quaternary ammonium compounds showed reduced retention with increasing column temperature (normal behaviour) and only modest improvements in peak shape. Considering these results, and pKa measurements of the bases made using capillary electrophoresis, it appears that increases in retention and improvement in efficiency may be influenced significantly by reduction in the pKa of bases with temperature, leading to reduction in protonation. It is less likely that efficiency improvements are due to the speeding up of the kinetics of silanol ion exchange, at least in the temperature range studied here.
Buckenmaier
Stephan M C
SM
Centre for Research in Biomedicine, University of the West of England, Frenchay, Bristol BS16 1QY UK.
McCalley
David V
DV
Euerby
Melvin R
MR
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature