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Biomedical and Pharmaceutical Applications 253

analyte enrichment prior to LC analysis. In LC, a popular way towards enhanced
selectivity and sensitivity is the use of small solid-phase extraction (SPE) cartridges
filled with a more or less selective sorbent for the preconcentration of samples (3).
The application of the on-line combination of SPE and LC for bioanalytical purposes
will be discussed to some extent in the next section. Another viable approach
towards more chromatographic selectivity is coupled-column LC (LC–LC) in which
two or more analytical columns are combined in an on-line fashion to accomplish
the isolation of the compound(s) of interest. LC–LC can be used either for the pro-
filing of a complete sample or for the analysis of target compounds (4).

11.2.1 COMPREHENSIVE LC–LC

The objective of the profiling mode of LC–LC is to fractionate all components of
the analysed mixture. This may be accomplished by so-called comprehensive two-
dimensional LC in which the entire chromatogram eluting from the primary column
is submitted to the secondary column. The secondary instrument must operate fast
enough to preserve the information contained in the primary signal. That is, it should
be able to generate at least one chromatogram during the time required for a peak to
elute from the primary column. Until now, a limited number of studies involving
comprehensive LC–LC for the analysis of compounds of biological interest have
been reported. Most of these studies were carried out within the group of Jorgenson
and mainly deal with the design, construction and implementation of comprehensive
LC–LC systems for the separation of either a complex protein mixture or an enzy-
matic digest of a protein (i.e. a mixture of peptides) (5–10). For these purposes,
orthogonal on-line combinations of ion-exchange chromatography, size-exclusion
chromatography (SEC) or reversed-phase (RP) LC are used. In a recent study, pep-
tide fragments generated in the tryptic digests of ovalbumin and serum albumin are
separated by SEC in the first dimension (run time, 160 min) and fast RPLC in the
second (run time, 240 s) (7). Following RPLC, the peptides flow to an electrospray
mass spectrometer for on-line identification. The complete LC system yields a peak
capacity of almost 500, thereby maximizing the chance of completely resolving each
peptide of the digest and, thus, permitting highly reliable peptide mapping. In addi-
tion, a comprehensive ion-exchange LC–RPLC–mass spectrometry (MS) system
for the analysis of proteins was demonstrated in which a 120-min ion-exchange LC
run is sampled by 48 RPLC runs of 150 s, leading to a peak capacity of over 2500
(8). The system was succesfully applied to the screening of an Escherichia coli
lysate without any prior knowledge of the characteristics (e.g. molecular weight,
isoelectric point, hydrophobicity, etc.) of its individual components.
Other bioanalytical applications of systems in which the eluate of a first LC
column is sampled in continuous and repetitive intervals and subjected to a second
LC dimension are, for example, described by Wheatly et al. (11) and Matsuoka et al.
(12). Wheatly coupled gradient affinity LC with RPLC for the determination of the
isoenzymatic- and subunit composition of glutathione S-transferses in cytosol