Page 211 - Multidimensional Chromatography

204 Multidimensional Chromatography

Figure 9.5 The generic setup for two-dimensional liquid chromatography–capillary zone
electrophoresis as used by Jorgenson’s group. The LC separation was performed in hours,
while the CZE runs were on a time scale of seconds.

couplings of different chromatographic methods with capillary zone electrophoresis.
Figure 9.5 illustrates the major components of the two-dimensional LC–CZE sepa-
ration system used by Jorgenson and co-workers (18). The type of LC column (and
mode of the separation) and the interface design were both modified many times in
order to optimize multidimensional electrodriven separations for various different
samples.

9.7 MICROCOLUMN REVERSE PHASE HIGH PERFORMANCE
LIQUID CHROMATOGRAPHY–CAPILLARY
ZONE ELECTROPHORESIS

In 1990, Bushey and Jorgenson developed the first automated system that coupled
HPLC with CZE (19). This orthogonal separation technique used differences in
hydrophobicity in the first dimension and molecular charge in the second dimension
for the analysis of peptide mixtures. The LC separation employed a gradient at 20
L min volumetric flow rate, with a column of 1.0 mm ID. The effluent from the
chromatographic column filled a 10 L loop on a computer-controlled, six-port
micro valve. At fixed intervals, the loop material was flushed over the anode end of
the CZE capillary, allowing electrokinetic injections to be made into the second
dimension from the first.
The HPLC elution time was typically under 260 min, and the CZE analysis took
place in 60 s, which led to an overall run time of about 4 h. The 1 min CZE sampling
interval was problematic, as the LC column was probably slightly undersampled. A
shorter CZE analysis time, which would provide a more frequent sampling rate,
would improve this system a great deal. The second-dimension analysis time must
be short relative to the first dimension, lest resolution in the first dimension be sacri-
ficed.