Is hydrophilic interaction chromatography with silica columns a viable alternative to reversed-phase liquid chromatography for the analysis of ionisable compounds?
The separation of acidic, neutral and particularly basic solutes was investigated using a bare silica column, mostly under hydrophilic interaction chromatography (HILIC) conditions with water concentrations >2.5% and with >70% acetonitrile (ACN). Profound changes in selectivity could be obtained by judicious selection of the buffer and its pH. Acidic solutes had low retention or showed exclusion in ammonium formate buffers, but were strongly retained when using trifluoroacetic acid (TFA) buffers, possibly due to suppression of repulsion of the solute anions from ionised silanol groups at the low (s)(s)pH of TFA solutions of aqueous ACN. At high buffer pH, the ionisation of weak bases was suppressed, reducing ionic (and possibly hydrophilic retention) leading to further opportunities for manipulation of selectivity. Peak shapes of basic solutes were excellent in ammonium formate buffers, and overloading effects, which are a major problem for charged bases in RPLC, were relatively insignificant in analytical separations using this buffer. HILIC separations were ideal for fast analysis of ionised bases, due to the low viscosity of mobile phases with high ACN content, and the favourable Van Deemter curves which resulted from higher solute diffusivities.
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
D000097:Acetonitriles / Q000737:chemistry; D000327:Adsorption; D000641:Ammonia / Q000737:chemistry; D002021:Buffers; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D002853:Chromatography, Liquid / Q000295:instrumentation*; D005561:Formates / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007477:Ions / Q000737:chemistry* / Q000302:isolation & purification*; D009994:Osmolar Concentration; D012822:Silicon Dioxide / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D014269:Trifluoroacetic Acid / Q000737:chemistry; D014867:Water / Q000737:chemistry*
D000097:Acetonitriles; D002021:Buffers; D005561:Formates; D007477:Ions; D014867:Water; C030544:formic acid; D012822:Silicon Dioxide; D000641:Ammonia; D014269:Trifluoroacetic Acid; C032159:acetonitrile