| Investigations were performed to characterize the broadening of analyte bands in ultra-high pressure liquid chromatography (UHPLC). UHPLC has demonstrated over 700,000 plates/m in fused silica capillary columns packed with 1.0-μm C18 non-porous silica (NPS) particles using isocratic elution. Despite such high efficiencies, reduced C-term values are consistently higher than what theory predicts for well-packed columns. Experimental values fall in the range of 0.2–0.6, which is several-fold higher than the theoretical value of ∼0.05 for non-porous materials. The origins of this additional band-broadening were explored.; At ultra-high pressures, the liquid mobile phase can no longer be assumed to be incompressible. In fact, the mobile phase was found to compress from 8–16% of the length of the column. Upon application of the run pressure, a burst of velocity is experienced by the analytes as compression of the mobile phase occurs. This velocity burst is a major source of peak broadening—C-terms were found to increase 32–51% due to the compression phenomenon alone.; Retention factors of analytes change not only as a function of the column backpressure, but across the pressure drop of the column during the course of a single run. Large retention factors at the high pressure end cause a disproportionate amount of broadening to occur toward the inlet of the column. This effect was calculated to increase the C-term by between 14% and 22%.; Other sources of band broadening were investigated, although quantitative conclusions could not be reached. From the analysis of SEM images of packed beds, and from data indicating column diameter affects the structure of the packing, it is likely that the geometry of the packing plays a significant role in band broadening. Chromatography at elevated temperatures was also investigated. Heating the column was found to allow much faster separation times, although no overall improvement in efficiency was observed.; Finally, the use of sub-micron particles was explored. Sub-micron particles were bonded in-house with stationary phase and analyzed. Separation performance of columns packed with these particles was poor. Results were comparable to those of 1.0-μm particles, yet at the price of much higher backpressures. |
