Page 124 - Multidimensional Chromatography

116 Multidimensional Chromatography

which by a combination of equations (5.19) and (5.3), can be approximated by the
following:

1/2
S n (5.20)
where it is evident that the benefits implied by equation (5.20) for columns of corre-
lated selectivity are small when compared to the exponential effect described by
equation (5.18), regarding two columns of different selectivities.
Thus, if two identical column with a peak capacity of 35 are coupled in series, the
resultant peak capacity calculated from equation (5.20) will be 49.5, compared to
a value of 1225 if the same columns are used in multidimensional mode. However,
the exponential effect of equation (5.18) will be observed only if each column hav-
ing a distinct selectivity will give elution sequences that are unrelated to those
obtained by any of the other chromatographic mechanisms being used. If the
columns are related (redundant selectivity), the system selectivity can be
expressed by equation (5.20). For instance, in size exclusion chromatography,
columns packed with porous particles having different exclusion limits are
connected in series, thus allowing separation of sample components over a wide
molecular-weight range.

5.3 LC–LC TECHNIQUES

As reported above, the coupling of individual separation techniques increases the
total peak capacity of the chromatographic system, which is given by the product of
the peak capacities of the individual dimensions (1). The full separation power of an
LC–LC system can be better understood by considering a multicomponent mixture
comprised of analytes having a wide range of distribution coefficients. In a single-
dimension system, when employing a column of high selectivity, less retained ana-
lytes will be eluted as well-resolved peaks. On the other hand, the more retained
peaks may become excessively broad and detection may be difficult. In contrast, by
selecting a column with lower selectivity, the retention time and width of the later
peaks will become acceptable, but early peaks will be poorly resolved.
In LC–LC mode, two columns are linked together via a switching valve in such a
manner that any component flowing through the first column can be directed to the
detector, to waste or into the second column in which further resolution can occur
before the sample passes into the detector cell. A typical LC–LC arrangement con-
sists of two columns having the same packing material, but of different length, or
two columns of similar length, but of different selectivity. In both systems there is a
distinct difference in retention in the columns used. The first column is short or of
lower selectivity and is employed to separate the most retained analytes of the multi-
component mixture. The less retained analytes which are eluted from the first
column are switched to the second column and remain there, while components
which are most retained are selectively eluted on the first column. When these