High performance liquid chromatography analysis of wine anthocyanins revisited: effect of particle size and temperature.

The complex anthocyanin fraction of red wines poses a demanding analytical challenge. We have found that anthocyanins are characterised by extremely low optimal chromatographic velocities, and as a consequence generic HPLC methods suffer from limited resolving power. Slow on-column inter-conversion reactions, particularly between carbinol and flavylium species, are shown to occur on the same time scale as chromatographic separation, leading to increased plate heights at normal chromatographic velocities. In order to improve current routine HPLC separations, the use of small (1.7 microm) particles and high temperature liquid chromatography (HTLC) were investigated. 1.7 microm particles provide better efficiency and higher optimal linear velocities, although column lengths of approximately 20 cm should be used to avoid the detrimental effects of conversion reactions. More importantly, operation at temperatures up to 50 degrees C increases the kinetics of inter-conversion reactions, and implies significantly improved efficiency under relatively mild analysis conditions. It is further demonstrated using relevant kinetic data that no on-column thermal degradation of these thermally labile compounds is observed at 50 degrees C and analysis times of <2h.