HPLC using CSPs

Irrespective of the mechanism of resolution, HPLC CSPs work by providing a chiral environment for analyte stereoisomers to interact with. Resolution relies upon the formation of reversible, transient diastereomers on the CSP that have different free energies of interaction and therefore stability. The stereoisomer forming the most stable diastereomer with the CSP will be the most retained and vice versa. Free energy differences are typically small in such systems but may be large enough to produce useable resolutions provided the column efficiency is sufficient [41]. If column efficiency is insufficient to allow complete separation of the stereoisomers, inaccurate integrations can result in erroneous 

The use of macrocyclic antibiotics as chiral selectors for HPLC was first proposed by Armstrong et al. [50] in 1994. The most successful of the CSPs are based on the glycopeptide antibiotics vancomycin, teicoplanin and ristocetin A and are commercially available through Advanced Separation Technologies Inc. (Astec Inc.) as Chirobiotic V , Chirobiotic 1 and Chirobiotic R , respectively. More recently, a number of other derivatives of these antibiotics have also been developed offering different stereoselectivities. A comprehensive handbook is now available from Astec Inc. [51 ] alongside a number of recent review articles 

Proteins are polymers of L-amino acids containing numerous chiral centres, each possessing a characteristic three-dimensional shape, or conformation. Most globular proteins such as albumins undergo extensive folding of the chains into 

---

BA 3,4-dihydrodiol metabolites were isolated by a reversed-phase HPLC using a Vydac C18 column (Chiu et al., unpublished results). DMBA dihydrodiol metabolites were isolated as described (42). The enantiomeric composition was determined either by CD spectral data or by CSP-HPLC (7.19.20). 

The chiral recognition ability of the insoluble (+)-l was estimated by HPLC using a column packed with small particles of l.25 However, this column showed a poor efficiency because of a low theoretical plate number. This defect was overcome by coating soluble poly(TrMA) with a DP of 50 on macroporous silica gel.26 The 1-coated silica gel had higher resistance against compression and longer lifetime than the CSP of insoluble 1. Moreover, the two 1-based CSPs show quite different chiral recognition for several race-mates, which may be attributed to the different orientation of 1 in bulk and on the surface of the silica gel.27... 

The cycloalkylcarbamates do not absorb UV light above 220 nm and therefore can be used as the CSPs for thin-layer chromatography (TLC).191 The TLC chromatogram was readily detected by UV radiation at 254 nm and showed the resolution of racemates into each enantiomer. The TLC results can be compared with those obtained by HPLC with the same CSP. The a values in HPLC are slightly larger than those in TLC, although a good correlation is observed between these a values. The cycloalkylcarbamates can be very useful CSPs for TLC as well as for HPLC resolution. 

For the majority of the direct chiral separations in HPLC, different kinds of CSPs are used. The CSP can consist of small chiral molecules or polymers [22] and is often immobilized on agarose [23], silica gel [23,24], or polymer particles [23]. During the two last decades, a number of new phases have been introduced. Most commonly used CSPs are listed in Table 17.1. 

Chiral separation can also be performed with packed capillaries. /3-CyD-bonded CSPs that are most frequently used in HPLC and CE were successfully applied in CEC. The separation of a variety of chiral compounds, such as some amino acid derivatives benzoin and hexobarbital was achieved by using CSPs bonded with different CyD derivatives [14,15]. Proteins are not ideal for use as buffer additives in CE because of their large detector response however, CEC may be a good way to use this type of chi-... 

The separation of the enantiomers of the chiral p-blocker propranolol in capillary HPLC using a polysaccharide type CSP is shown in Fig. 4 [120]. The capillary format requires aproximately 106 times less stationary and mobile phases, while offering separation characteristics at least adequate or better than that achieved with common-size columns. 

Figure 1 shows the distribution of the CSPs for HPLC used for the determination of enantiomeric excess (ee) that was reported in the Journal of the American Chemical Society in 2005 (a) and 2007 (b) [2, 9]. These statistics show that more than 90% of the ee determinations by chiral HPLC are carried out by the polysaccharide-based CSPs. 

Fig. 1 Distribution of CSPs for HPLC used for the determination of enantiomeric excess reported in Journal of the American Chemical Society in (a) 2005 and (b) 2007. The values in parentheses represent the number of the counted papers. Reprinted by permission from the Royal Society of Chemistry [2] and International Union of Pure and Applied Chemistry [9]...

Aromatic carbamate derivatives of polysaccharides are today the most used CSP in chiral HPLC, but unfortunately, they are not ideal selectors for TLC because of the detection difficulty by UV light. However, this problem may be overcome in the future by the use of cycloalkylcarbamates [54]. 

Cyclodextrin stationary phases utilize cyclodextrins bound to a soHd support in such a way that the cyclodextrin is free to interact with solutes in solution. These bonded phases consist of cyclodextrin molecules linked to siUca gel by specific nonhydrolytic silane linkages (5,6). This stable cyclodextrin bonded phase is sold commercially under the trade name Cyclobond (Advanced Separation Technologies, Whippany, New Jersey). The vast majority of all reported hplc separations on CD-bonded phases utilize this media which was also the first chiral stationary phase (csp) developed for use in the reversed-phase mode. 

In this context, the enantiomeric pair containing the eutomer of cyclothiazide can be resolved by HPLC on cellulose-derived coated CSPs. Nevertheless, the poor solubility of this compound in solvents compatible with this type of support makes this separation difficult at preparative scale. This operation was achieved with a cellulose carbamate fixed on allylsilica gel using a mixture of toluene/acetone as a mobile phase [59]. 

The type of CSPs used have to fulfil the same requirements (resistance, loadabil-ity) as do classical chiral HPLC separations at preparative level [99], although different particle size silica supports are sometimes needed [10]. Again, to date the polysaccharide-derived CSPs have been the most studied in SMB systems, and a large number of racemic compounds have been successfully resolved in this way [95-98, 100-108]. Nevertheless, some applications can also be found with CSPs derived from polyacrylamides [11], Pirkle-type chiral selectors [10] and cyclodextrin derivatives [109]. A system to evaporate the collected fractions and to recover and recycle solvent is sometimes coupled to the SMB. In this context the application of the technique to gas can be advantageous in some cases because this part of the process can be omitted [109]. 

Gas chromatography (GC) has also been used for preparative purposes, but is restricted to relatively volatile racemates such as anesthetics, pheromones or monoterpenes and, therefore, very few applications are reported. Nevertheless, in the cases to which GC may be applied, it could be considered as an economical alternative to HPLC. Most of the resolutions of enantiomers were performed on cyclodex-trin-derived CSPs [109, 144-153], and only on very few occasions were other chiral selectors used [153]. 

Another important issue that must be considered in the development of CSPs for preparative separations is the solubility of enantiomers in the mobile phase. For example, the mixtures of hexane and polar solvents such as tetrahydrofuran, ethyl acetate, and 2-propanol typically used for normal-phase HPLC may not dissolve enough compound to overload the column. Since the selectivity of chiral recognition is strongly mobile phase-dependent, the development and optimization of the selector must be carried out in such a solvent that is well suited for the analytes. In contrast to analytical separations, separations on process scale do not require selectivity for a broad variety of racemates, since the unit often separates only a unique mixture of enantiomers. Therefore, a very high key-and-lock type selectivity, well known in the recognition of biosystems, would be most advantageous for the separation of a specific pair of enantiomers in large-scale production. 

Fig. 3-9. Preparative HPLC of 100 mg of the test racemate 8 in a single 2 mL injection using a 250 x 4.6 mm i.d. column containing (5)-Glu-(5)-Leu-DNB CSP. Conditions mobile phase ethyl acetate, flowrate 2.0 mL min , UV detection at 380 nm. Injection 2 mL of 50 mg mL racemate solution. Fractions collected before and after the indicated cut point were 98.4 % ee and 97 % ee pure, respectively. (Reprinted with permission from ref. [86]. Copyright 1999, American Chemical Society.)...

Our group also demonstrated another combinatorial approach in which a CSP carrying a library of enantiomerically pure potential selectors was used directly to screen for enantioselectivity in the HPLC separation of target analytes [93, 94]. The best selector of the bound mixture for the desired separation was then identified in a few deconvolution steps. As a result of the parallelism advantage , the number of columns that had to be screened in this deconvolution process to identify the single most selective selector CSP was much smaller than the number of actual selectors in the library. 

As expected from the design of the experiment, the HPLC column packed with CSP 14 containing all 36 members of the library with tt-basic substituents separated 7t-acid substituted amino acid amides. Although encouraging since it suggested the presence of at least one useful selector, this result did not reveal which of the numerous selectors on CSP 14 was the most powerful one. Therefore, a deconvolution process involving the preparation of series of beads with smaller numbers of attached selectors was used. The approach is schematically outlined in Fig. 3-17. 

Despite the difficulties caused by the rapidly expanding literature, the use of chiral stationary phases (CSPs) as the method of choice for analysis or preparation of enantiomers is today well established and has become almost routine. It results from the development of chiral chromatographic methods that more than 1000 chiral stationary phases exemplified by several thousands of enantiomer separations have been described for high-performance liquid chromatography (HPLC). 

The solvent used was hexane-isopropanol(4 1). Later, Allenmark and colleagues obtained enantioselective HPLC retention of a series of alkyl carboxymethyl sulphoxides (and other sulphoxides and classes) on a column of (R)-lV-(3,5-dinitrobenzoyl)phenylglycine covalently bound via an amide bond (CSP 1), or ionically bound (CSP 2), to 3-aminopropylsilica the mobile phase was again hexane-isopropanol(4 l, or, also 19 1) and determination carried out at 254 or 280nm. The sulphoxides were better resolved on CSP 2. 

---