Chiral Stationary Phases in SFC

Polysaccharide-based CSPs incorporate derivatives of cellulose and amylose adsorbed on silica gel. The selectivity of these CSPs depends upon the nature of the substituents introduced during the derivatization process. The secondary structure of the modified polysaccharide is believed to play a role in selectivity, but the chiral recognition mechanisms have not been fully elucidated [55]. 

Benzodiazepine enantiomers have also been resolved on the Chiralcel OD CSP. Wang et al. utilized this CSP to determine the enantiomeric composition of camazepam and its metabolites [59]. SFC provided improved resolution of the compounds of interest in a shorter period of time than LC. Phinney et al. demonstrated the separation of a series of achiral and chiral benzodiazepines. An amino column was coupled in series with the Chiralcel OD CSP to achieve the desired separation [41]. 

The selectivity of another cellulose-based CSP, Chiralcel OJ, has also been examined in SFC [60]. Separations of racemic drugs such as benoxaprofen, temazepam, and mephobarbital were obtained. Acetonitrile proved to be a better modifier than methanol for some of the compounds investigated. The four optical isomers of a calcium channel blocker were resolved by Siret et al. on the Chiralcel OJ CSP [30]. In LC, two CSPs were required to perform the same separation. 

Derivatized amylose is the basis for the Chiralpak AD CSP. This CSP has been utilized for the resolution of ibuprofen and flurbiprofen, as well as other members of the family of nonsteroidal inflammatory drugs (NSAIDs) [39, 61]. Ibuprofen was not resolved on the Chiralpak AD CSP in LC. Pressure-related effects on stereoselectivity were observed by Bargmann-Leyder et al. on a Chiralpak AD CSP [58]. No corresponding effect of pressure on selectivity was observed with a Chiralcel OD CSP. The authors speculated that the helical conformation of the amylose-based CSP is more flexible than that of the cellulose-based CSP. 

This relatively new class of CSPs incorporates glycopeptides attached covalently to silica gel. These CSPs can be used in the normal phase, reversed phase, and polar organic modes in LC [62]. Various functional groups on the macrocyclic antibiotic molecule provide opportunities for tt-tt complexation, hydrogen bonding, and steric interactions between the analyte and the chiral selector. Association of the analyte 

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The high diffusivity and low viscosity of sub- and supercritical fluids make them particularly attractive eluents for enantiomeric separations. Mourier et al. first exploited sub- and supercritical eluents for the separation of phosphine oxides on a brush-type chiral stationary phase [28]. They compared analysis time and resolution per unit time for separations performed by LC and SFC. Although selectivity (a) was comparable in LC and SFC for the compounds studied, resolution was consistently... 

Since all the physical properties of two given enantiomers are the same in the absence of a chiral, or optically active, medium, their chromatographic resolution needs a different approach from the relatively simple separation of geometrical isomers, stereoisomers or positional isomers. Two methods are used. The older technique of indirect resolution, requires conversion of the enantiomers to diastereoisomers using a suitable chiral reagent, followed by separation of the diastereoisomers on a non-chiral GC or LC stationary phase. This technique has now been largely superseded by direct resolution, using either a chiral mobile phase (in LC) or a chiral stationary phase. A variety of types of chiral stationary phase have been developed for use in GC, LC and SFC(21 23). 

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. 

In this chapter, approaches to fast chiral separations using SFC, including fundamental considerations, influences of chiral stationary phases, modifiers, and additives are discussed. The thermodynamic aspects of SFC are also presented. 

Pirkle and coworkers [59] compared retention and selectivity factors between HPLC and SFC using Poly Whelk-O chiral stationary phases and a-naphthyl-1-ethylamine carbamates. The results indicate that both retention and selectivity factors in SFC were higher than those in HPLC. This can be mainly attributed to the weaker solvating power of the carbon dioxide supercritical fluid as compared to a liquid such as methanol or hexane. 

Chiral separations result from the formation of transient diastereomeric complexes between stationary phases, analytes, and mobile phases. Therefore, a column is the heart of chiral chromatography as in other forms of chromatography. Most chiral stationary phases designed for normal phase HPLC are also suitable for packed column SFC with the exception of protein-based chiral stationary phases. It was estimated that over 200 chiral stationary phases are commercially available [72]. Typical chiral stationary phases used in SFC include Pirkle-type, polysaccharide-based, inclusion-type, and cross-linked polymer-based phases. 

The enantiomeric purity is determined by chiral stationary phase, supercritical fluid chromatographic (CSP-SFC) analysis (Berger Instruments, Daicel Co. CHIRALCEL OD column 4% methanol, 180 psi, 3.0 mUmin flow rate detection at 220 nm). Racemic 1-phenylpropanol exhibited base-line separation of peaks of equal intensity arising from the R-isomer (tp, 2.74 min) and the S-isomer (tp, 3.10 min) whereas the synthetic alcohol showed these peaks in the ratio 97.7 / 2.3. This chromatographic method allowed for identification of the trace contaminants propiophenone (tp, 1.63 min) and benzyl alcohol (tp 3.40 min). 

To a larger degree than separations of drugs and related substances, packed-column SFC has generated considerable interest for use in enantiomeric separations. SFC has shown compatibility with a large variety of chiral stationary phases, including cyclodextrins (native and derivatized), Pirkle type, and derivatives of cellulose and amylose. The chief advantage potentially is the improved separation efficiency, which increases of the enantiomers but also reduces the likelihood that impurities will interfere with the analysis. 

A number of groups have shown how enantiomeric resolution of amino acids derivatized with non-chiral reagents is possible in SFC with chiral stationary phases. N-Acetylamino acid t-butyl ester racemates were rapidly resolved [17] on (N-formyl-L-valylamino)propyl silica with CO2 modified with methanol, acetonitrile and diethyl ether. A similar stationary phase allowed (18 rapid (< 5 min) separation of racemic N-4-nitrobenzoyl-amino acid isopropyl esto-s with methanol-modified CO2 the enantioselectivity in SFC was comparable with that in HPLC with isopropanol/n-hexane as mobile phase. Capillary column SFC on polysiloxane stationary phases containing chiral side chains has been employed... 

Owing to its intermediate position between GC and LC, SFC can be performed equally well in open capillaries and packed columns. The separation can be influenced by the type of stationary phase and of modifier, by pressure, pressure drop, and temperature. In contrast to GC, SFC can also be used for the separation of nonvolatile or thermally labile compounds (although some temperature compatibility is necessary). The separation of enantiomers on chiral stationary phases can be very attractive because the temperature is lower than in GC, which increases the separation factors. SFC is an alternative to normal-phase LC because it is fast and carbon dioxide is ecologically sound. An example of an SFC separation can be found in the previous article. Principles, where Figure 2 shows the separation of orange oil components. 

There are a wide range of polar stationary phases available for SFC. They include bare silica, cyano, amino, diol, ethylpyridine, and many others. Virtually all chiral stationary phase types have been used in SFC. 

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