Page 183 - Multidimensional Chromatography

176 Multidimensional Chromatography

These workers used binary solvent systems over a range of mole fractions to
determine, for each solute, the constants a and b of equation (8.2). For methyl and
phenacyl esters, TLC was used, while overpressured layer chromatography (OPLC)
was used for dansyl amino acids. Nurok and co-workers (11) also evaluated how the
quality of a simulated separation varies with changing solvent strength by using the
inverse distance function (IDF) or planar response function (PRF), as follows:
k 1 k 1

IDF (8.4)
i 1 j i 1 SD ii
k 1 k SD ii

PRF ln SPEC (8.5)
i 1 j i 1 SD
where SD is the center-to-center spot separation and SD SPEC is the spot separation
desired for a mixture of k solutes. Larger values of SD SPEC reflect the easier separa-
tion of a mixture containing fewer solutes, when many solute pairs will make a zero
contribution to the PRF if too small a value of SD SPEC is selected (11).
Härmälä et al. (12) reported a very easy method of searching for appropriate
mobile phases for 2-D separations. These authors suggested starting the solvent
selection according to the first part of the ‘PRISMA’ system for planar chromatogra-
phy (13), with a selection of ten solvents chosen from the eight Snyder (14) selec
tivity groups, which can be evaluated in parallel in unsaturated chromatographic
chambers. The compounds to be separated should be applied to the TLC plates in
groups of three or four, for ease of identification. The solvent strength (s i ) of each
neat solvent is adjusted individually, by the use of n-hexane, so that the zones of the
compounds to be separated are distributed in the R f range 0.2–0.8. The exact posi-
tions of the spots must be located densitometrically. If solvents afford good separa-
tion, their homologues or other solvents from the same group can also be tested. The
R f values of the compounds to be separated are obtained from the TLC runs by using
the most promising binary solvents and must be compared with each other in a corre-
lation matrix. The next step is validation of the solvent systems by regression analy-
sis, where the solvents with the poorest correlation values are selected. After choice
of an appropriate pair of solvents, the development technique (e.g. OPLC or rotation
planar chromatography (RPC)) and other separation conditions must be selected,
according to the third part of the ‘PRISMA’ system. In this way, an excellent separa-
tion can be achieved for closely related compounds in the minimum time, generally
within a few hours.

8.5 TWO-DIMENSIONAL DEVELOPMENT ON BILAYERS

Use of a bilayer plate (15) affords the special chromatographic possibility of being
able to perform two different multidimensional separations on the same chromato-
graphic plate (Figure 8.5), either with the same mobile phase or with mobile phases
of different composition.