Packed column SFC

The analysis of mefloquine in blood, using packed-column sfc, a mobile phase consisting of / -pentane modified with 1% methanol and 0.15% -butylamine, and electron capture detection has been reported (92). The method compares favorably to a previously pubflshed hplc-based procedure having a detection limit of 7.5 ng/mLin 0.1 mL blood sample. 

An on-line supercritical fluid chromatography-capillary gas chromatography (SFC-GC) technique has been demonstrated for the direct transfer of SFC fractions from a packed column SFC system to a GC system. This technique has been applied in the analysis of industrial samples such as aviation fuel (24). This type of coupled technique is sometimes more advantageous than the traditional LC-GC coupled technique since SFC is compatible with GC, because most supercritical fluids decompress into gases at GC conditions and are not detected by flame-ionization detection. The use of solvent evaporation techniques are not necessary. SFC, in the same way as LC, can be used to preseparate a sample into classes of compounds where the individual components can then be analyzed and quantified by GC. The supercritical fluid sample effluent is decompressed through a restrictor directly into a capillary GC injection port. In addition, this technique allows selective or multi-step heart-cutting of various sample peaks as they elute from the supercritical fluid... 

SFC has been performed with either open capillary columns similar to those used in GC or packed columns transferred from LC, and the instrumentation requirements differ for these two approaches [12]. This chapter will focus on the use of packed column technology because of its dominance in the area of pharmaceutical compound separations. Current commercial instrumentation for packed column SFC utilizes many of the same components as traditional LC instruments, including pumps, injection valves, and detectors. In fact, most modem packed column SFC instm-ments can also be used to perform LC separations, and many of the same stationary phases can be used in both LC and SFC [9]. 

Mourier s report was quickly followed by successful enantiomeric resolutions on stationary phases bearing other types of chiral selectors, including native and deriva-tized cyclodextrins and derivatized polysaccharides. Many chiral compounds of pharmaceutical interest have now been resolved by packed column SFC, including antimalarials, (3-blockers, and antivirals. A summary is provided in Table 12-2. Most of the applications have utilized modified CO, as the eluent. 

The difference in resolution between LC and SFC can be significant enough to turn a marginal LC separation into a viable chromatographic method in SFC. String-ham et al. reported that packed column SFC yielded satisfactory chiral analysis... 

Packed column SFC has also been applied to preparative-scale separations [42], In comparison to preparative LC, SFC offers reduced solvent consumption and easier product recovery [43]. Whatley [44] described the preparative-scale resolution of potassium channel blockers. Increased resolution in SFC improved peak symmetry and allowed higher sample throughput when compared to LC. The enhanced resolution obtained in SFC also increases the enantiomeric purity of the fractions collected. Currently, the major obstacle to widespread use of preparative SFC has been the cost and complexity of the instrumentation. 

The nature of the modifier and the modifier concentration impact both retention and selectivity in packed column SFC. SFC offers considerable flexibility in modifier selection because nearly all commonly used organic modifiers, including methanol and acetonitrile, are miscible with CO,. In contrast, methanol and acetonitrile are rarely used as modifiers in normal phase LC because they are immiscible with hexane [68]. 

Optimum flowrates are higher in packed column SFC than in LC. Flowrates as high as 5.0 mL min generally do not dramatically reduce efficiency in SFC [12]. Bier-manns and co-workers reported the separation of (3-blockers at a flowrate of 4.0 mL miiT a rate eight times higher than the flowrate recommended for LC [56]. No deterioration of column performance was observed. 

Figure 6.12 Schematic diagram of a solventless injector used for packed column SFC. (Reproduced with permission from ref. 132. Copyright Dr. Alfred Huethig Publishers).

The selection of the column type is mainly determined by the composition of the sample. In general open-tubular (capillary) columns are preferred for low-density (gas-like) SFC, whereas packed columns are most useful for high-density (liquid-like) SFC. Open-tubular columns can provide a much larger number of theoretical plates than packed columns for the same pressure drop. Volumetric flow-rates are much higher in packed column SFC (pSFC) than in open-tubular column SFC (cSFC), which makes injection and flow control less problematic. 

Table 4.29 Main features of packed column SFC Advantages...

Packed column SFC and CE are both able to make inroads into the application area served by HPLC, but from opposite extremes of polarity and with little overlap. CE is likely to be more efficient and faster, but mostly applicable to very polar molecules and ions. SFC qualifies as a more reproducible, trace technique, with greater selectivity and multiple detection options. HPLC and CE have been compared [365], Owing to their orthogonality, CZE and SFC are worth developing, not in competition or as an alternative to HPLC, but as an additional method in order to augment the information obtained from the analysis. With the broad scope of possible eluents and stationary phases, HPLC has fewer constraints than SFC and CZE. The parameters influencing selectivity may be used as a guide to optimisation (Table 4.44). 

T.A. Berger, Packed Column SFC, The Royal Society of Chemistry, Cambridge (1995). 

Applications APCI-MS is often more widely applicable than ESI-MS to the analysis of classes of compounds with a low molecular weight, such as basic drugs and their metabolites, antibiotics, steroids, oestrogens, benzodiazepines, pesticides, surfactants, and most other organic compounds amenable to El. LC-APCI-MS has been used to analyse PET extracts obtained by a disso-lution/precipitation procedure [147]. Other applications of hyphenated APCI mass spectrometric techniques are described elsewhere LC-APCI-MS (Section 7.33.2) and packed column SFC-APCI-MS (Section 73.2.2) for polar nonvolatile organics. 

SFE can be combined with several forms of SFC, i.e. with conventional packed columns (l-4.6mm i.d. packed-column SFC or pSFC), with capillary columns (10-250 xm i.d. capillary SFC or cSFC), and recently with packed capillary columns (200-530 p,m i.d., 3-10 xm particles packed capillary SFC or pc-SFC). 

A limitation in the use of API sources results from the frequent application of mobile-phase composition programming in pSFC. Pinkston el al. [411] have compared electrospray and electron impact for open-tubular and packed-column SFC-MS. Direct on-line coupling of SFC to FAB/MS (as well as SFC-ELSD) is also very promising to detect components which give no response in a UV detector [412]. 

Jedrzejewski and Taylor [408] have evaluated microbore pSFC-PB-MS. Microbore separations can take full advantage of the simple DFI design, and are still being pursued. Packed-column SFC-MS was recently reviewed [13]. The two techniques, cSFC and pSFC, are complementary compounds in complex mixtures may be more easily identified with cSFC-MS, while pSFC-MS may be more suitable for target component analysis. 

Alternatively, the LC dimension of LC-GC may be replaced by packed-column SFC, in order to improve the compatibility between two mobile phases and to allow the FID to be used for both separations. Because of the relatively nonpolar nature of scCCL. SFC-GC is particularly recommended as a substitute for many normal-phase LC-GC analyses. The techniques developed for solvent evaporation at the LC-GC interface are often not required in SFC-GC, because the solutes are deposited at the front of the GC column when CCL decompresses into a gas at the end of the SFC column. 

Enantiomeric separations have proven to be one of the most successful applications of packed column SFC. Despite initial reluctance, many analysts now use SFC routinely for both analytical and preparative chiral separations. Additional studies of chiral recognition in SFC and continued improvements in instrumentation will ensure a prominent role for SFC in chiral separations methodology in the future. 

Enantioselective separation by supercritical fluid chromatography (SFC) has been a field of great progress since the first demonstration of a chiral separation by SFC in the 1980s. The unique properties of supercritical fluids make packed column SFC the most favorable choice for fast enantiomeric separation among all of the separation techniques. In this chapter, the effect of chiral stationary phases, modifiers, and additives on enantioseparation are discussed in terms of speed and resolution in SFC. Fundamental considerations and thermodynamic aspects are also presented. 

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