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Multidimensional Electrodriven Separations 201

put into practice. Other planar two-dimensional electrodriven separations, however,
are still used today.
In 1975, O’Farrell determined that high-resolution separations of protein mix-
tures could be achieved with polyacrylamide gel isoelectric focusing (IEF) in one
dimension and polyacrylamide slab gel electrophoresis (PAGE) in the other (10).
This technique was used to separate 1100 different components from Escherichia
coli, some of which differed by as little as a single charge, while being similar in
molecular weight. Although this technique can be problematic due to the labor-
intensive complications of gel preparation and staining, IEF–sodium dodecyl sulfate
(SDS)–PAGE continues to be a very popular method for the separation of complex
biological samples, especially mixtures of proteins.
In 1988, Burton et al. developed a new analytical technique which they dubbed
the “chromatophoresis” process. This method coupled reverse-phase (RP) high-
performance liquid chromatography (HPLC) with SDS–PAGE in an automated sys-
tem used to separate proteins. Chromatophoresis involved a separation based on
differences in hydrophobicity in the first dimension and molecular charge in the sec-
ond. The chromatophoresis process is illustrated in Figure 9.3. After eluting from an
HPLC column, proteins were passed into a heated reaction chamber, where they
were denatured and complexed with SDS. The protein complexes in the eluate
stream were then deposited onto the surface of a polyacrylamide gradient gel, and an
electrophoretic separation subsequently occurred. The five-hour run-time, inconve-
nience of gel electrophoresis, and difficult detection were the main disadvantages of
this technique (11).

9.5 CHROMATOGRAPHY AND ELECTROPHORESIS
COMBINED IN NON-COMPREHENSIVE MANNERS

Many groups have used electrophoresis to enhance a primary chromatographic sepa-
ration. These techniques can be considered to be two-dimensional, but they are not
comprehensive, usually due to the loss of resolution in the interface between the two
methods. For instance, capillary electrophoresis was used in 1989 by Grossman and
co-workers to analyze fractions from an HPLC separation of peptide fragments. In
this study, CE was employed for the separation of protein fragments that were not
resolved by HPLC. These two techniques proved to be truly orthogonal, since there
was no correlation between the retention time in HPLC and the elution order in CE.
The analysis time for CE was found to be four times faster than for HPLC (12),
which demonstrated that CE is a good candidate for the second dimension in a two-
dimensional separation system, as will be discussed in more detail later.
In 1989, Yamamoto et al. developed the first technique that directly coupled
chromatography to capillary electrophoresis, although again in a non-comprehensive
fashion. Low-pressure gel permeation chromatography, which separates analytes
based on differences in molecular size, was combined with capillary isotachophore-
sis, which separates according to electrophoretic mobility. Capillary isotachophoresis