Page 125 - Multidimensional Chromatography

Coupled-Column Liquid Chromatography 117

analytes have been separated, the first eluted peaks on the first column are then
separated on the longer or more selective second column. When comparing the chro-
matogram obtained on eluting by the single-dimension system with the chromato-
graphic profile achieved by LC–LC column switching, it is evident that the elution
order of analytes will be reversed. Under the proposed conditions, the chromato-
graphic profile shows first the most retained analytes and than the least retained com-
ponents. Peaks are usually eluted in a smaller volume of the mobile phase with less
dead space between each of them, and as sharper peaks they are easier to detect.
Moreover, separation may be carried out isocratically, thus allowing detection with
electrochemical (EC) and refractive index (RI) detectors, which are very sensitive to
mobile phase changes and are best used under isocratic conditions.
An LC–LC coupling experiment system can be performed by employing a com-
mercially available HPLC apparatus and involving various combinations of HPLC
columns, eluents, additives, switching devices and detectors.
LC–LC coupling can be subdivided into both homomodal and heteromodal sys-
tems (11).
Homomodal LC–LC. In this type of development, the chromatographic improve-
ment occurs by switching columns of analogous selectivity. Mainly the goal is to
optimize an already satisfactory separation, that is, to concentrate a dilute sample
(sample enrichment) or to shorten an analysis time.
Heteromodal LC–LC. Essentially, this type of development is achieved by varying
the separation mechanism during the separation process; selectivity changes may be
made by varying the nature of the stationary phase, which can posses complemen-
tary separation characteristics. The high power of heteromodal LC–LC is repre-
sented by equation (5.16) and mainly by equatiuon (5.18) for a multidimensional LC
system. The term LC–LC, and more generally Multidimensional, is usually
restricted to these LC systems, which involve separation modes which are as differ-
ent as possible (orthogonal), and in which there is a distinct difference in retention
mechanisms (2).
Trace enrichment and sample clean-up are probably the most important applica-
tions of LC–LC separation methods. The interest in these LC–LC techniques has
increased rapidly in recent years, particularly in environmental analysis and clean-up
and/or trace analysis in biological matrices which demands accurate determinations
of compounds at very low concentration levels present in complex matrices
(12–24). Both sample clean-up and trace enrichment are frequently employed in the
same LC–LC scheme; of course, if the concentration of the analytes of interest are
sufficient for detection then only the removal of interfering substances by sample
clean-up is necessary for analysis.
Trace enrichment or preconcentration by LC–LC methods are based on the
possibility that the analytes will be retained as a narrow zone on the top of the first
column when a large volume of sample is pumped trough the column. Good repro-
ducibility can be achieved when the column capacity is not exceeded and the column
is not overloaded. Trace enrichment is usually performed when relatively non-polar