Page 131 - Multidimensional Chromatography

Coupled-Column Liquid Chromatography 123

calculated from the following:
V 0
(1 k ) (5.23)
v S
√N
By combining equations (5.22) and (5.23), equation (5.21) can be rewritten as follows:
S
V (1 k ) 1 √ N 0 (5.24)
2.3
V
B
However, for very highly retained solutes direct measurement of the capacity factor
k S is not possible, and this parameter must be predicted on the basis of retention data
determined with a stronger mobile phase. The determination of V B is an essential
step in the optimization of trace enrichment and clean-up procedures.
Loading of the analytes on the analytical column can be carried out either in the
back-flush or forward-flush modes. The back-flush mode allows a better resharpen-
ing of the solute band than the forward-flush mode. However, such a flow reversal
may lead to precolumn packing disturbances. In addition, back-flushing does not
protect the analytical column as well as forward-flushing (10). In fact, other than
analytes, a large number of contaminants can be simultaneously sorbed on, and then
eluted from the precolumn. In the development of a new LC–LC enrichment
method, we have to deal with the following: (i) the elaboration of a carefully
designed gradient profile to achieve a more or less stepwise elution of the retained
components from the precolumn; (ii) the choice of a more selective stationary phase
for the trace-enrichment step; (iii) the use of a selective detection principle.
An LC–LC separation system may be used in either the profiling or targed mode
(11). The purpose in the profiling mode is to separate all single components from a
complex mixture. Every component from the first column (primary column) is frac-
tionated and transferred in the second column (secondary column). In contrast, the
purpose of LC–LC separation in the targeted mode is to isolate either a single or a
few components of similar retention in a complex mixture containing components
having a wide range of capacity factor values. Targeted component analysis is car-
ried out by transferring a wide or narrow cut of the chromatographic effluent from
the primary column to the secondary column by flow switching and the mobile phase
is thereby diverted or reversed. The fraction of interest to be transferred on to the sec-
ondary column may be early-eluting analytes (first eluted zone, usually the named
the ‘front-cut’), or components eluted in the middle of the chromatographic effluent
(‘heart-cut’) or at the end of the chromatogram (‘end-cut’) (38).
A schematic diagram of a ‘heart-cut’ LC–LC system is depicted in Figure 5.4.
The column switching technique was developed by employing two high-pressure
four-way pneumatic valves inserted before and after the precolumn (39). The ‘front-
cut’ and the ‘end-cut’ of the sample eluted from the first column were vented to
waste. The valves were manipulated to transfer only the ‘heart-cut’ of the analyte of
interest to the analytical column. The detailed operational conditions for the four-
step sequence of this system can be described as follows:
(Step 1) Divert initial portion of chromatogram to waste. The sample is injected
with the valve A (left) closed; mobile phase flows through the precolumn to valve B
(right), which is opened to waste.