Study of retention and peak shape in hydrophilic interaction chromatography over a wide pH range.
Retention factor and column efficiency measurements were made for 14 test compounds comprising acids, bases and neutrals on two pairs of amide and bare silica HILIC columns, each pair obtained from a different manufacturer. The columns were tested with up to 6 different mobile phases with acetonitrile-water containing formic (FA), trifluoroacetic (TFA), heptafluorobutyric acids (HFBA) and ammonium salt buffers at w(w)pH 3, 6 and 9. Measurements of mobile phase pH in water (w(w)pH) and in the aqueous-organic mixture (w(s)pH) were performed, and calculations of ionic strength made, in order to aid interpretation of the chromatographic results. Stronger acids like TFA produced very different selectivity compared with ammonium formate buffers at similar aqueous pH. On a given column using TFA as additive, the retention of strongly acidic solutes was considerably increased relative to that of bases. Some bases even showed exclusion on both amide, and on a hybrid silica column. Conversely, in ammonium formate buffers of similar aqueous pH, bases had increased retention compared with acids, particularly on the bare silica columns. This result can be attributed to the higher pH of the salt buffers when measured in the aqueous-organic phase and interaction with negatively charged silanols. It is possible that the silica surface becomes positively charged at the low pH of TFA, leading to anion exchange properties that become competitive with the cation exchange properties normally attributed to silanol dissociation, although other explanations of these results are possible. Very marked selectivity differences were obtained by use of TFA in the mobile phase. Useful selectivity differences may also be obtained with salt buffers at different pH if the use of TFA is not desired due to its relatively unfavourable properties in mass spectrometry.