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Coupled-Column Liquid Chromatography 129

isolate tangeretin and heptamethoxyflavon, a large quantity of tangeretin was
collected from the peak front and tail on the first pass. As the heavy sample load was
reduced on each successive pass by shaving, the column’s effective separation effi-
ciency increased, thus reducing the number of passes necessary to separate and
recover the two compounds, which were completely separated and collected by
employing only four recycles. Under the same conditions, other oxygen heterocyclic
compounds of citrus essential oils were isolated (63) and used to prepare a library of
mass spectra to identify these compounds in samples of genuine cold-pressed citrus
oils by HPLC hyphenated techniques (64). Recycling techniques included the simu-
lated moving bed method (65, 66), mainly proposed for the large-scale chromato-
graphic separation of enantiomers (67–73). Such methods are rather complex and
require dedicated equipment. However, they usually require less solvent to separate a
given quantity of enantiomers and the operating costs are therefore significantly
lower than with batch chromatographic methods (74).

5.4 CONCLUSIONS

Today, the various chromatographic techniques represent the major parts of modern
analytical chemistry. However, it is well known that the analysis of complex mix-
tures often requires more than one separation process in order to resolve all of the
components present in a sample. This realization has generated a considerable inter-
est in the area of two-dimensional separation techniques. The basics of LC–LC and
its practical aspects have been covered in this chapter.
LC–LC systems can be divided into two different approaches; namely (i) where a
small-sized column (or SPE cartridge) is mainly used as the first column for fast
sample enrichment and/or clean-up, and (ii) an LC–LC coupling employing two
full-sized separation columns operating in orthogonal mode, which provides, rela-
tive to one-dimensional or linear techniques, a greatly enhanced peak capacity. In
many cases, the LC–LC coupling of conventional columns, as well as microbore
columns, provides the optimum efficiency and selectivity for the separation of a wide
range of compounds of interest in the biological field, as well as in environmental
analysis.
Using LC–LC systems, a high degree of automation with a lower amount of sam-
ple and low detection limits is usually obtained. However, even with manual valve
switching, these techniques are less time-consuming than most alternative HPLC
methods. Furthermore, the combination of on-line coupling of LC–LC methods and
a spectroscopic detection device which provides structural sample information, is a
promising option for use in systems combining automated sample pretreatment and
efficient and selective separations with high sensitivity detection. Reviews of com-
bined LC–MS systems have been extensively published over the last few years
(75–77) and the use in conjunction with hyphenated LC–LC methods has been
proposed (18, 48, 78, 79), and its potential recently demonstrated (80).