No abstract

A comparison is made between the efficiency of microparticulate capillary columns and silica and polymer-based monolithic capillary columns in the pressure-driven (high-performance liquid chromatography) and electro-driven (capillary electrochromatography) modes. With packed capillary columns similar plate heights are possible as with conventional packed columns. However, a large variation is observed in the plate heights for individual columns. This can only be explained by differences in the quality of the packed bed. The minimum plate height obtained with silica monolithic capillary columns in the HPLC mode is approximately 10 microm, which is comparable to that of columns packed with 5-microm particles. The permeability of wide-pore silica monoliths was found to be much higher than that of comparable microparticulate columns, which leads to much lower pressure drops for the same eluent at the same linear mobile phase velocity. For polymer-based monolithic columns (acrylamide, styrene/divinyl benzene, methacrylate, acrylate) high efficiencies have been found in the CEC mode with minimum plate heights between 2 and 10 microm. However, in the HPLC mode minimum plate heights in the range of 10 to 25 microm have been reported.

The kinetic-plot approach, in which experimental t(0) and N-values are extrapolated to the performance at maximum system pressure by increasing the column length, was validated by coupling 250×3 mm columns packed with 3 μm particles. The extra-column volume introduced by coupling columns could be neglected with respect to the peak volumes. Plate numbers of up to 132,000 were experimentally achieved by coupling four columns. The maximum deviation between the experimental and predicted plate numbers was 7% for two coupled columns, and decreasing to 0.1% for four coupled columns. Kinetic plots were used to find the conditions to separate a critical pair, with a preset value for the effective plate number, in the shortest possible time. For high-efficiency separations yielding 100,000 effective plates, the optimum critical-pair retention factor was around 4.5. Kinetic plots are presented to find the optimal column length to obtain the fastest possible 100,000 effective-plate separation, taking into account the effect of mobile-phase viscosity on column pressure, and consequently the optimum column length.

A new hardware solution is proposed that allows one to automatically change the length of a chromatographic bed. The setup is based on the serial coupling of chromatographic columns using two rotor-stator valves (with N positions, N + 1 ports). Despite the use of a prototype setup requiring rather long connection tubing, only 9% loss in efficiency is observed for compounds with a retention factor above 4 compared to the efficiency expected on the basis of the individual column results. It has been demonstrated for a number of isocratic and gradient separations that the system allows one to realize considerable analysis time savings by adapting the total column length to the specific sample requirements and/or to the stage of method development wherein one is working. During method development, a separation on a short column length can first be used to rapidly gain insight into the composition of the sample, leaving fewer runs to be done on a column of maximal length (offering efficiencies that are inaccessible with individual column systems). The ease with which information can be obtained on columns of different lengths can furthermore be exploited for screening purposes to detect coeluting components in a stage wherein they still appear completely unresolved (i.e., have a resolution well below R(s) = 0.5).