Effect of column temperature and eluent flow rate on the high performance liquid chromatographic analysis of cyclosporin a and d.

On a reversed-phase C18 analytical column using an eluent of 70:30 acetonitrile and water, the following effects were observed with increasing column temperature (from 25 to 75°C) for Cyclosporin A (CSA) and Cyclosporin D (CSD). The peak heights and number of theoretical plates (N) increased. The height equivalent to a theoretical plate (HETP) decreased. The areas under the peaks, retention times and capacity factors (k') for both compounds did not vary with temperature. With increasing eluent flow rate (from 0.5 to 2.5ml/ min), the peak heights, peak areas, retention times and N all decreased for both compounds. A slight decrease in k' for CSA and CSD was also observed. HETP increased with increasing flow. The separation factor, α, remained relatively constant for the ranges of temperatures and flow rates investigated.