Evaluation of 1.0 mm i.d. column performances on ultra high pressure liquid chromatography instrumentation

The present study focuses on the evaluation of 1.0 mm i.d. (internal diameter) columns on a commercial Ultra-High Pressure system. These systems have been developed specifically to operate columns with small volumes, typically 2.1 mm i.d., by reducing extra-column volume dispersion. The use of columns with smaller i.d. results in a reduced solvent consumption and required sample volume. The evaluation of the columns was carried out with samples containing neutral and pharmaceutical compounds. In isocratic mode, the extra-column volume produced additional band broadening leading to poor performances compared to equivalent 2.1 mm i.d. columns. By increasing the length of the column, the influence of the extra-column bandspreading could be reduced and 75,000 plates were obtained when four columns were coupled. In gradient mode, the effect of the extra-column contribution on efficiency was limited and about 80% of the performance of the 2.1 mm i.d. columns was obtained. Optimum conditions in gradient mode were further investigated by changing flow rate, gradient time and column length. A different approach of the calculation of peak capacity was also considered for the comparison of the influence of these different parameters.