278-291 LCE May04

2 Introduction Ever since the introduction of liquid-phase chromatography,1 efforts have been made to increase the speed and resolution of the technology. Undoubtedly, the major breakthrough came with the introduction of high-pressure instruments and the availability of uniform packing materials, leading to the advent of high performance liquid chromatography (HPLC) in the 1970s.2–3 To further increase speed, open-tubular columns4–6 and ultra-high pressures7 have been explored. Most recently, the use of more open-porous packings (monolithic columns) with smaller flow resistances have demonstrated increased separation speeds.8–9 Generally, maximum performance limits for HPLC lie around H 5 μm, and Nmax 100 000 plates, and plate generation speeds of approximately 500 plates/s can be obtained.10 With the highest possible pressures (5000 bar), and using 1 μm particles, theoretical plate heights as small as H 2 μm and plate generation velocities of N 200 000 plates in 6 min (solute with k » 1) have been reported.7 Another large research area focusing on the development of higher separation efficiencies and shorter separation times is the field of electrically driven separations. The consideration of electrically driven separation methods also comes hand-in-hand with the advent of lab-on-a-chip techniques,11–13 which use radically new column manufacturing approaches, directly derived from the micro-electronics industry. These chip-based techniques enable a maximal degree of miniaturization and integration as injection, separation, detection and flow propagation are all integrated onto the same surface. Researchers have demonstrated very high separation efficiencies and plate generation velocities (respectively, up to N 106 plates and 20 000 plates/s)14 and ultra-short separation times (down to the millisecond level).15 The highest separation efficiencies are reported for capillary electrophoresis (CE) separations, but this method, based on differences in molecular charge rather than on differences in chemical affinity for a stationary phase, is less general than liquid chomatography (LC). Research efforts have also been devoted to the HPLC/CE hybrid, commonly referred to as capillary electrochromatography (CEC), wherein the pressure-force used to propagate the mobile phase in HPLC and OT-LC is replaced by an electrical force, while the selective retention-based separation principle of LC is to a large extent retained. With this method, separation efficiencies of 700 000 plates/m (packed-bed CEC)16 and 220 000 plates/m (monolithic column CEC)17 have been demonstrated. The method, however, suffers from poor column-to-column and even run-to-run flow reproducibility.18–19 Joule heating effects also impose an upper limit on the applicable voltage gradient, and additional peak broadening stemming from the inlet and outlet frits remains a point of concern for packed-bed CEC applications.19 In many instances, the generated electroosmotic flow is also too small to operate the system at its optimum mobile-phase velocity.20