Page 215 - Multidimensional Chromatography

208 Multidimensional Chromatography

9.10 PACKED CAPILLARY REVERSE PHASE HIGH
PERFORMANCE LIQUID CHROMATOGRAPHY–
FAST CAPILLARY ZONE ELECTROPHORESIS

In order to improve the speed of CZE analysis, Monnig and Jorgenson developed on-
column sample gating in 1991. The gating procedure allowed for rapid and auto-
mated sample introduction into the CZE capillary. In traditional CZE techniques, a
plug of material is mechanically introduced at one end of the capillary, thus yielding
a relatively slow sampling rate. In the on-column optical gating method, analytes
were first tagged with fluorescein isothiocyanate, a fluorescent label, and then con-
tinuously introduced into one end of the capillary. A laser constantly photodegraded
the tag near the entrance of the capillary. A sample zone was created by momentarily
blocking the laser so that a narrow plug of fluorescent material was created in the
column. This method allowed for the rapid injection to be made while the capillary
was maintained at operating voltage.
The experimental setup for high-speed CZE can be seen in Figure 9.8. High-
speed CZE, or fast CZE (FCZE), yielded 70 000 to 90 000 theoretical plates for the
separation of amino acid mixtures. Complete separation was achieved in under 11 s,
using a capillary length of 4 cm (24).
In 1995, Moore and Jorgenson used the optically gated CZE system to obtain
extremely rapid separations with HPLC coupled to CZE. The rapid CZE analysis
made possible more frequent sampling of the HPLC column, thus increasing the
comprehensive resolving power. Complete two-dimensional analyses were per-
formed in less than 10 min, with the CZE analyses requiring only 2.5s. A peak

Figure 9.8 Schematic illustration of the two-dimensional HPLC/fast–CZE instrumental
setup. The argon laser beam was set at 488 nm, which was used to photodegrade and detect the
fluorescein isothiocyanate tag.