Page 207 - Multidimensional Chromatography

200 Multidimensional Chromatography

Chromatographic and electrophoretic separations are truly orthogonal, which
makes them excellent techniques to couple in a multidimensional system. Capillary
electrophoresis separates analytes based on differences in the electrophoretic mobili-
ties of analytes, while chromatographic separations discriminate based on differ-
ences in partition function, adsorption, or other properties unrelated to charge (with
some clear exceptions). Typically in multidimensional techniques, the more orthogo-
nal two methods are, then the more difficult it is to interface them. Microscale liquid
chromatography ( LC) has been comparatively easy to couple to capillary elec-
trophoresis due to the fact that both techniques involve narrow-bore columns and
liquid-phase eluents.
The sampling of the first dimension by the second is an extremely important
aspect in multidimensional separations. In heart-cutting techniques, fractions of elu-
ent from the first column are separated by a second column of different selectivity.
Comprehensive sampling is similar to heart-cutting in practice, except with consid-
erably elevated sampling frequency. In a comprehensive sampling arrangement, each
peak that elutes from the first column is sampled by the second column numerous
times. This results in the maximum retention of resolution and information obtained
in the first separation. Another characteristic unique to a comprehensive sampling
scheme is that all components are analyzed by the second dimension. Non-compre-
hensive heart-cutting techniques may not allow for complete gathering of informa-
tion on all components of the sample, since some analytes may elute between
fractions and fail to be sampled by the second dimension. The tremendous resolving
power and orthogonality of comprehensive multidimensional techniques are the
characteristics that make multidimensionality so attractive in separations.

9.4 PLANAR TWO-DIMENSIONAL SEPARATIONS

Planar two-dimensional separation methods can offer the distinct advantage of truly
comprehensive sampling of the first dimension by the second, since transfer of ana-
lytes from one medium (or separation conduit) to another is not a requirement. The
first comprehensive two-dimensional separations were achieved with paper chro-
matography in 1944 (6). This discovery led to a number of other multidimensional
techniques, with many of these involving electrodriven separations. In 1948,
Haugaard and Kroner separated amino acids by coupling paper chromatography
with paper electrophoresis, thus performing the original multidimensional electro-
driven separation. This study involved the use of a 100 V electric field applied across
one dimension of the paper, while a phosphate buffer was used as a chromatographic
eluent to move analytes in the orthogonal direction (7).
Two-dimensional planar electrophoresis was first used in 1951 (8), while elec-
trophoresis was coupled with thin-layer chromatography (TLC) in 1964 to separate
mixtures of nucleosides and nucleotides (9). These techniques were novel and led to
other great discoveries, but did not survive the test of time, and they are no longer
commonly used. TLC–electrophoresis in particular was an awkward technique to