Dependence of selectivity on eluent composition and temperature in the HPLC separation of taxanes using fluorinated and hydrocarbon phases

Optimized conditions of aqueous acetonitrile (ACN) eluent composition and temperature are established for the rapid separation of a standard mixture of 15 taxanes, on each of five different fluorinated phases and one C-8 hydrocarbonaceous phase. On both types of stationary phase, the retention factors (k) of most of the taxanes decrease at the same rate with increasing ACN concentration. However, the taxanes containing a xylosyl group show a higher rate of decrease, which necessitates careful control of eluent composition to achieve separation of all the taxanes. Temperature can have a remarkable and counter-intuitive effect on retention and selectivity. For the C8 phase with eluent compositions in the 40%-60% ACN range, the k' values of the xylosyl taxanes show an increase with increasing temperature over the range from 25 to 55 degreesC; the k' values for 10-deacetyl baccatin III and 10-deacetyl taxol go through a maximum over the same ranges. The other taxanes behave normally. The same pattern is observed on the propyl(perfluorophenyl) phase, although this and the other fluorinated phases are less retentive. This accounts for the common belief that fluorinated stationary phases offer resolution of taxanes superior to that on hydrocarbon phases. The higher retention on the latter requires eluent compositions near 50% ACN, where careful temperature optimization is required, which in practice is rarely performed. The lesser retention on the fluorinated phases allows use of lower ACN concentrations where the aberrant temperature effect is not found, so good separations without temperature optimization can be achieved. Further evidence of the lack of any fundamental difference in the selectivity of the two types of stationary phase is the similarity of the surface excess isotherms measured for ACN/H2O on both the fluorinated and hydrocarbon phases.