Page 62 - Subyek Encyclopedia - Encyclopedia of Separation Science

Sepsci*21*TSK*Venkatachala=BG
I / CHROMATOGRAPHY 57

incremental multiple development can be used with ment and sample clean-up on short pre-columns is
a decreasing solvent-strength gradient. In this case, Rnding increasing use in the automated determina-
the Rrst development distance is the shortest and tions of drugs in biological Suids and crop protection
employs the strongest solvent composition, while agents in water by LC-LC. Figure 21 illustrates the
subsequent developments are longer and employ separation of a mixture of deoxyribonucleosides and
mobile-phase compositions of decreasing solvent their 5 -monophosphate esters using LC-LC with an
strength. The Rnal development step is the longest anion exchange column and a reversed-phase column
and usually corresponds to the maximum useful de- connected in series by a microvolume valve interface.
velopment length for the layer and employs the The neutral deoxyribonucleosides are switched as
weakest mobile phase. In this way sample compo- a single peak for separation on the reversed-phase
nents migrate in each development until the strength column while the phosphate esters are resolved by the
of the mobile phase declines to a level at which some anion exchange column. The separation time remains
of the sample zones are immobile, while less retained acceptable since both separations are performed al-
zones continue to be separated in subsequent devel- most simultaneously. TLC-TLC is commonly called
opment steps, affording the separation of the two-dimensional TLC and is a widely used qualitative
mixture as a single chromatogram (Figure 20). In- method of analysis. It is very easily performed by
cremental multiple development with a decreasing placing a sample at the corner of the layer and devel-
solvent-strength gradient is easily automated. oping the plate in the normal way, evaporating the
solvent, turning the plate through a right angle and
developing the plate a second time at 903 to the Rrst
Multidimensional and Multimodal development. If adequately optimized this is a very
Chromatography

The analysis of complex mixtures requires a very
large peak capacity since the probability of peak
overlap increases with the number of compounds
requiring separation. Multidimensional and multi-
modal chromatographic systems provide a better
route to achieving high peak capacities than is
possible with single-column systems. The necessary
characteristic of these systems is that the dominant
retention mechanism should be different for each
dimension. Other uses of multidimensional and
multimodal chromatography include trace enrich-
ment, matrix simpliRcation, increased sample
throughput, and as an alternative to gradient elution
in LC.
Multidimensional column chromatography in-
volves the separation of a sample by using two or
more columns in series where the individual columns
differ in their capacity and/or selectivity. Multi-
modal separations involve two or more chromato-
graphic methods in series, for example, the online
coupling of LC and GC (LC-GC) or SFC and GC
(SFC-GC). Both methods involve the transfer of the
whole or part of the eluent from the Rrst column to
another via some suitable interface. The function of
the interface is to ensure compatibility in terms of Figure 21 Separation of the major deoxyribonucleosides and
their 5 -monophosphate esters by multidimensional LC-LC. The
Sow, solvent strength and column capacity. The de- first column is a strong anion exchange column and the second
sign requirements and ease of coupling differ a reversed-phase column. The unseparated nucleosides, A, are
signiRcantly for the different chromatographic switched to the second column after which the 5 -monophosphate
modes. Coupling GC-GC, SFC-GC, SFC-SFC, LC- esters, B to D are separated on the IEC column and the parent
LC, LC-GC and LC-TLC are routine and other com- deoxyribonucleosides,E to H, are separated on the RPC column.
(Reproduced with permission from Sagliano N, Hsu SH, Floyd
binations such as SFC-TLC, SFC-LC and GC-TLC TR, Raglione TV and Hartwick RA (1985) Journal of Chromato-
have been described in the literature. Trace enrich- graphic Science 23: 238, copyright Preston Publication, Inc.)