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284 Multidimensional Chromatography

Figure 11.15 also shows that cytochrome C is displaced by lysozyme during extrac-
tion, i.e. at longer extraction times (cf. Figure 11.15(b) and (c)) the amount of
lysozyme is increased as the amount of cytochrome C is decreased.
Generally speaking, SPME is still relatively slow and/or yields are relatively low,
but significant improvements are curently being made. Research effort is directed at
the present time towards the development of new SPME fibre coatings in the search
for new selectivities. Incorporation of other principles as, for instance, membrane
technologies, antibodies, receptors and molecularly imprinted polymers could
greatly enhance the development of special fibres and further promote future
applications. The combination of SPME with micro-separation techniques also
seems very interesting.

11.7 SUPERCRITICAL FLUID EXTRACTION COUPLED
WITH SUPERCRITICAL FLUID CHROMATOGRAPHY

The gas-like mass transfer and liquid-like solvating properties of supercritical fluids,
together with considerations of automation, speed and cost, make supercritical fluids
very attractive for application in coupled systems. Supercritical fluid extraction
(SFE) lends itself to either off-line or on-line coupling with various separation tech-
niques (144). For on-line coupling, the general advantages are also true in this case,
i.e. high sensitivity, no contamination and easy automation. For the off-line
approach, the following advantages can be mentioned: larger sample sizes, different
applicable separation methods and operational simplicity. Many applications of the
off-line combination of SFE and LC or GC in the bio-pharmaceutical area have been
described (145–148).
Prostaglandins have been extracted from drugs (149), as well as from aqueous
solutions, by loading the sample on an SPE cartridge and, subsequently, carrying out
on-line SFC (150). A glycoside, ouabain, was used as a model compound to study
the coupling of SFE and SFC, combined with fraction collection, thus allowing
determination of the biological activity of the collected fractions (151). Important
information pertaining to solute elution density, efficiency of extraction, solute trap-
ping, and supercritical fluid chromatography (SFC) was obtained. A cytostatic, mito-
mycin C, has been determined in plasma by SPE–SFC after application on an
XAD-2 sorbent. After washing and drying the sorbent, the drug was supercritically
desorbed and chromatographed with 12% methanol in CO 2 (152). This phase-
system-switching approach prevents the direct injection of a polar matrix or solvent
into an SFC system. Up to 1 ml of plasma containing 20 ng of mitomycin C has been
analysed (Figure 11.16). The UV chromatogram obtained at 215 nm shows that the
isolation step was surprisingly selective. The choice of 360 nm as the detection
wavelength has further improved the selectivity.
In principle, the sample transfer from the supercritical state is relatively easily
adaptable to other systems, due to the high volatility of the fluid at atmospheric pres-
sure, particularly for carbon dioxide which is the most frequently used fluid.