Page 118 - Multidimensional Chromatography

110 Multidimensional Chromatography

or by various combinations of columns possessing complementary separation char-
acteristics. This technique provides the optimum efficiency and selectivity for sepa-
rations of the component of interest, while simultaneously minimizing the analysis
time by decreasing the time spent in separating those components of the sample that
are of no analytical interest. Multidimensional chromatography (also known as cou-
pled-column chromatography or column switching) represents a powerful tool and
an alternative procedure to classical one-dimensional HPLC methods.
Multidimensional liquid chromatography can be performed either in an on-line or
off-line mode. With off-line operation, the fractions eluted from the primary column
are collected manually or by a fraction collector and then reinjected, either with or
without concentration, into a second column. This approach has the advantage of
being simple, does not need any switching valve, and the mobile phases used in each
column need not be mutually compatible. However, these procedures are labour-
intensive and time-consuming and the recovery of sample is often low. On-line tech-
niques have the advantage of automation by using pneumatic or electronically
controlled valving, which switches the column effluent directly from the primary
column into the secondary column. Automation improves reliability and sample
throughput, and shortens the analysis time, as well as minimizing sample loss or
change since the analysis is performed in a closed-loop system. Obviously, on-line
techniques are preferred, although they are not always feasible from an operational
point of view. The main limitation is that the mobile phase system used in the cou-
pled columns must be compatible in both miscibility and solvent strength. This
requirement arises since the eluent from the first column is the injection solvent for
the second column; consequently, not all column types are mutually compatible.
Furthermore, the use of two different separation principles may lead to an inversion
of the elution order on the two subsequent columns. Hence, the separation achieved
on the first column can be substantially reduced on the second column. A compari-
son of advantages and disadvantages of off- and on-line multidimensional liquid
chromatographic techniques is shown in Table 5.1.
Multidimensional LC separation has been defined as a technique which is mainly
characterized by two distinct criteria, as follows (1). The first criterion for a multidi-
mensional system is that sample components must be displaced by two or more sep-
aration techniques involving orthogonal separation mechanisms (2), while the
second criterion is that components that are separated by any single separation
dimension must not be recombined in any further separation dimension.
Coupled-column liquid chromatography (LC–LC coupling) refers to the conven-
tional two-dimensional mode of chromatography in which fractions from one col-
umn are selectively transferred to one secondary column for a further separation.
What characterizes LC–LC coupling when compared to conventional multistep
chromatography is the requirement that the whole chromatographic process be car-
ried out on-line. The transferred volume of the mobile phase from the first column to
the second column can correspond to a group of peaks, a single peak or a fraction of
a peak, so that different parts of the sample may follow different paths through the
LC–LC configuration. A large number of factors play a role in the development of
an LC–LC procedure; these include the separating power of the chromatographic