Page 257 - Multidimensional Chromatography

252 Multidimensional Chromatography

pharmaceutical field by means of typical examples. In this respect it should be noted
that multidimensional separation systems are much more important in biomedical
analysis than in pharmaceutical analysis, because the usefulness of such systems
strongly depends on the complexity of the samples to be analysed. No attention will
be paid here to thin-layer chromatography (TLC). Over the past decade, new devel-
opments in two-dimensional TLC for biomedical purposes have been scarce and in
addition the amount of research in coupled chromatographic systems with TLC as
one dimension has been very small (1, 2).
The discussions in essence will be confined to the application of on-line (i.e.
directly coupled) chromatographic techniques, since with respect to off-line coupling,
these systems offer improved precision and accuracy as a result of the elimination of
intermediate steps such as fraction collection, evaporation and manual transfer.
Moreover, on-line coupling offers shorter analysis times and good possibilities for
automation, with both of these being important aspects in modern bioanalysis. In addi-
tion, with off-line systems commonly only an aliquot of the fraction collected from the
first chromatographic dimension is introduced into the second dimension. It should be
added that efficient on-line coupling requires thorough attention to the instrumental
aspects, and that the interfacing is not always easy and/or can give some limitations to
the total system. The main advantages of an off-line combination of two separation
systems are the higher flexibility in the choice of the operating conditions and the pos-
sibility of independent optimization. At present, the availability of efficient robotic
analysers allows full automation of off-line methods. However, generally the other
disadvantages of the off-line mode are still valid for these so-called ‘at-line’systems.

11.2 LIQUID CHROMATOGRAPHY–LIQUID CHROMATOGRAPHY

LC is an important and widely used analytical separation technique in the pharma-
ceutical, biomedical and clinical field. The technique is well established with a
variety of sophisticated instrumentation and high performance columns readily
available. The range of biologically interesting substances that can be analysed
directly by LC is very large as it includes charged, polar, thermolabile, non-volatile
and high-molecular-weight compounds. These type of substances are not amenable
to GC without using time-consuming (off-line) chemical derivatization procedures.
Furthermore, various modes of LC are carried out using (partly) aqueous eluents
and, therefore, are highly suitable for the processing of aqueous samples, a fre-
quently encountered sample type in biomedical analysis. However, biological sam-
ples are generally highly complex mixtures in which the compounds of interest may
appear as minor constituents. As a consequence, a large excess of interferences fre-
quently hinders the direct determination of target compounds. Moreover, the sensi-
tivity and specificity of UV absorbance detection–this being the standard detection
principle in LC–is often too low to allow the analysis of trace-level components of
biological samples. Hence, in biomedical analysis usually some kind of off-line or
on-line sample preparation procedure is required to achieve sample clean-up and