Separation on CSPs

The mobile phases used for chiral separations on CSPs differ depending on the type of column and range from normal-phase systems containing high amounts of nonpolar solvent (e.g., hexane) to... 

A majority of the chiral purity assays made with HPLC published during the last decade are based on separation on CSPs and subsequent UV detection (Table 17.3). The polysaccharide phases seem to be the dominating CSPs, but there is an even distribution in the methods that uses normal-and reversed-phase modes. A few of the methods utilize CMPA [39,59,60] or indirect separation by chemical derivatization [16,18]. However, it seems that the majority of the published papers... 

TABLE 21-4. Summary of SMB Separations Reported in the Literature (Only Enantiomeric Separations on CSP and Purification of Intermediates and Drug Substance in Pharmaceutical Development)... 

Analytical-scale enantiomer separations on CSPs are usually carried out under linear (non-overloaded) chromatographic conditions, under which the retention of the individual enantiomers can be expressed as... 

Figure 9, Chromatogram showing the separation on CSP 1 of the eight possible P ydroxy sulfoxides derived from racemic disparlure. Bands bearing the same letter designation arise from enantiomers, A and B differ in relative stereochemistry from C and D, A and B (and C and D) are identical stereochemically but are...

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. 

Fig. 2-5. Examples showing the eomplementary separations on glyeopeptide CSPs. (A) Separation of N-CBZ-norvaline on vaneomyein (left) and teieoplanin (right). The mobile phase was methanol 1 % triethylammonium aeetate (20/80 v/v) pH 4.1. (B) Separation of warfarin on teieoplanin (left) and vaneomyein (right) CSPs. The mobile phase was aeetonitrile 1 % triethylammonium aeetate (10/90 v/v) pH 4.1. (C) Separation of naproxen on teieoplanin (left) and ristoeetin A (right). The mobile phase was methanol 0.1 % triethylammonium aeetate (30/70 v/v) pH 4.1. All eolumns were 250 x 4.6 mm i.d. The flow rate for all the separations was 1 mL min at ambient temperature (23 °C).

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. 

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. 

Select a set of compounds resolved on a given CSP, calculate the similarity indices between all possible molecule pairs, and then use these indices to build a similarity matrix containing relevant information about the structural diversity within the set of samples separated on this CSP. 

Free amino acids can be derivatized with isothiocyanates to phenyl- or methyl-thiohydantoin derivatives. The thiohydantoins can be separated on a CSP with poly-[Af-acryloyl-L-phenylalanine ethylester] (Chiraspher ) as a chiral selector [25]. This CSP offers a known selectivity for many five-membered heterocyclic rings. 

A derivatization with acid chlorides is also possible. Amino acids can be derivatized with 9-fluorenylmethyl chloroformate (FMOC) and separated on a CSP with X-cyclodextrin (ChiraDex gamma ), a cyclic oligosaccharide which consists of eight glucose units. 

An example for this approach is the immobilization of (5 )-(-)-a-A-(2-naph-thyl)leucine, a 7t-donating group on silica. This chiral selector exhibits excellent recognition for 3,5-dinitrobenzoyl (DNB)- and 3,5-dintroanilido (DNAn)-deriva-tives. Amines and alcohols can be derivatized with DNB- or DNAn-chloride to the esters or carbamates and separated on the CSP, as shown by Pirkle for a wide variety of compounds [27]. 

This method represents the most common and traditional application of computational tools to rational drug design. From a list of molecules of known activity, one can establish a 3D-pharmacophore hypothesis that is then transformed into a 3D-search query. This query is then used to search a 3D database for structures that fit the hypothesis within a certain tolerance. If the yield of active molecules is significant, then the query can be used to predict activities on novel compounds. In our situation, the enantiophore is built from the superposition of a list of sample molecules, which are all well separated on a given CSP. Hence, the common features of this series of molecules can become a good enantiophore hypothesis for the enantiores-olution on this CSP. 

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