Page 278 - Multidimensional Chromatography

Biomedical and Pharmaceutical Applications 273

selectively recognize and bind a specific compound in a similar manner as
immunoaffinity sorbents, but can be tailor-made (117). A disadvantage of most cur-
rent MIPs is that the analyte needs to be dissolved in a non-protic organic solvent
and, as a consequence, the use of MIPs in on-line SPE of aqueous biofluids has been
very limited so far. In order to circumvent this problem, Boos et al. (118) have pro-
posed an on-line SPE–SPE–LC scheme which comprises a RAM and a MIP pre-
column. After sample loading, the RAM sorbent is desorbed with a pure organic
solvent which is led to the MIP pre-column for molecular recognition of the target
analyte. A practical evaluation of this approach, as well as other multidimensional
set-ups involving MIPs, can be expected in the near future.

11.4 LIQUID CHROMATOGRAPHY –GAS CHROMATOGRAPHY

LC is not only a powerful analytical method as such, but it also allows effective sam-
ple preparation for GC. The fractions of interest (heart-cuts) are collected and intro-
duced into the GC. The GC column can then be used to separate the fractions of
different polarity on the basis of volatility differences. The separation efficiency and
selectivity of LC is needed to isolate the compounds of interest from a complex
matrix.
Traditionally, LC and GC are used as separate steps in the sample analysis
sequence, with collection in between, and then followed by transfer. A major limita-
tion of off-line LC–GC is that only a small aliquot of the LC fraction is injected into
the GC (e.g. 1–2 l from 1 ml). Therefore, increasing attention is now given to the
on-line combination of LC and GC. This involves the transfer of large volumes of
eluent into capillary GC. In order to achieve this, the so-called on-column interface
(retention gap) or a programmed temperature vaporizor (PTV) in front of the GC
column are used. Nearly all on-line LC–GC applications involve normal-phase (NP)
LC, because the introduction of relatively large volumes of apolar, relatively volatile
mobile phases into the GC unit is easier than for aqueous solvents. On-line LC–GC
does not only increase the sensitivity but also saves time and improves
precision.

11.4.1 NPLC–GC

The first bioanalytical application of LC–GC was presented by Grob et al. (119).
These authors proposed this coupled system for the determination of diethylstilbe-
strol in urine as a replacement for GC–MS. After hydrolysis, clean-up by solid-
phase extraction and derivatization by pentafluorobenzyl bromide, the extract was
separated with normal-phase LC by using cyclohexane 1% tetrahydrofuran (THF) at
a flow-rate of 260 l min as the mobile phase. The result of LC–UV analysis of a
urine sample and GC with electron-capture detection (ECD) of the LC fraction are
shown in Figures 11.8(a) and (b), respectively. The practical detection limits varied
between about 0.1 and 0.3 ppb, depending on the urine being analysed. By use of