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206 Multidimensional Chromatography

therefore overlapping the second-dimension runs. The improved injection scheme
resulted in enhancement of the apparent rate of the CZE separation (20).
The only other group to have performed comprehensive multidimensional
reverse-phase HPLC–CZE separations is at Hewlett-Packard. In 1996, a two-dimen-
sional LC–CE instrument was described at the Frederick Conference on Capillary
Electrophoresis by Vonda K. Smith (21). The possibility for a commercial multidi-
mensional instrument may have been explored at that time.

9.8 MICROCOLUMN SIZE EXCLUSION
CHROMATOGRAPHY–CAPILLARY ZONE ELECTROPHORESIS

In 1993, Lemmo and Jorgenson used microcolumn size exclusion chromatography
coupled to CZE for the two-dimensional analysis of protein mixtures. Under non-
denaturing conditions, SEC provides a description of the molecular weight distribu-
tion of a mixture, without destroying the biological activity of the analytes.
Microcolumn SEC was chosen due to its reasonable separation efficiency and its
ability to interface well with the CZE capillary. Here, a -SEC column of 250 m
ID was coupled to a CZE capillary of 50 m ID to generate a comprehensive two-
dimensional system. System efficiencies of over 100 000 theoretical plates per meter
have been obtained through the coupling of these two techniques.
A six-port valve was first used to interface the SEC microcolumn to the CZE cap-
illary in a valve-loop design. UV–VIS detection was employed in this experiment.
The overall run time was 2 h, with the CZE runs requiring 9 min. As in the reverse
phase HPLC–CZE technique, runs were overlapped in the second dimension to
reduce the apparent run time. The main disadvantage of this -SEC–CZE method
was the valve that was used for interfacing. The six-port valve contributed a substan-
tial extracolumn volume, and required a fixed volume of 900 nL of effluent from the
chromatographic column for each CZE run. The large fixed volume imposed restric-
tions on the operating conditions of both of the separation methods. Specifically, to
fill the 900 nL volume, the SEC flow rate had to be far above the optimum level and
therefore the SEC efficiency was decreased (22).
The second interface design that was developed for use with -SEC–CZE used
the internal rotor of a valve for the collection of effluent from the SEC microcolumn.
The volume collected was reduced to 500 nL, which increased the resolution when
compared to the valve-loop interface (20). However, a fixed volume again presented
the same restrictions on the SEC and CZE operating parameters. An entirely differ-
ent approach to the interface design was necessary to optimize the conditions in both
of the microcolumns.
Lemmo and Jorgenson developed a third interface for -SEC–CZE in 1993. This
design used a transverse flow of CZE buffer to prevent electromigration injections
from occurring into the CZE capillary until the appropriate time. Figure 9.7 shows a
block diagram of the “flow-gating interface” (18). The interface consisted of a Teflon