Phases chiral stationary

An alternative approach for the direct resolution of enantiomers by HPLC is to use a chiral stationary phase. This technique relies on the formation of transient/temporary diastereoisomers between the sample enantiomers and the CSP. Differences in stability between the diastereoisomers is reflected in differences in retention times, the enantiomer forming the less stable complex being eluted first. 

A wide variety of CSPs are now commercially available for HPLC. These materials are commonly classified xmder five subgroupings (Table 6.9). 

The use of CSPs is the most popular approach towards the resolution of enantiomers because of convenience, the wide range of different CSPs available, that there are few practical problems as separation depends almost exclusively upon the chiral interactions between analyte and stationary phase and is little influenced by mobile phase composition. 

Pirkle or brush type bonded phases Helical chiral polymers (polysaccharides) Cyclodextrins and crown ethers Immobilised enzymes Amino acid metal complexes Three-point interaction Attractive hydrophobic bonding Host guest interaction within chiral cavity Chiral affinity Diastereomeric complexation 

The chiral recognition processes upon which the resolution of the enantiomers depends requires at least three points of interaction between the solute and the CSP, of which one must be stereochemically dependent. These points of interaction are provided by rr-donor, ir-acceptor aromatic fragments, the facility to hydrogen bond and the dipole stacking inducing structure in addition to the chiral centre within the stationary phase. 

A few years later, Blaschke designed purely synthetic chiral stationary phases obtained by emulsion polymerization of acrylamides prepared from amino acids [17]. These phases had been developed for preparative purposes and proved to be very efficient for the preparative resolution of various chiral drugs for which the enantiomers have been isolated for the first time. These earher applications include the separation of the enantiomers of the sadly well-known drug thalidomide (Fig. 6.2) [18], 

From the early 1980s, a growing number of analytical chiral columns became available and are now routinely used for the determination of the enantiomeric composition of mixtures of optical isomers from enantioselective syntheses, from biological investigations or from pharmacokinetic or toxicology studies. Some of these phases have also become extremely useful for enantioselective preparative separations [1-4, 16]. 

Basically, one can distinguish three kinds of CSPs, chiral polymers (Type 1), achiral matrices (mainly silica gel) modified with chiral moieties (Type 11), and imprinted materials (Fig. 6.3). 

In the first group (Type I), which comprises most organic polymeric phases, the 

Carrier material modified with chiral moieties 

CMP s advantages stem from the fact that it is cheaper, since it uses achiral stationary phases, and the chiral additive can be purchased at a low cost the approach is flexible, because after using a chiral additive, the chromatographic column can be washed out from the chiral additive and a new additive can be employed. On the other hand, the mechanism is difficult to predict due to the constant presence of a secondary chemical equilibrium in the column. Since the enantiomeric analytes are eluted out of the column as diastereomeric complexes, the detector response may be different for each complex. Also, the sample capacity is relatively small. 

The CSP approach also has advantages and disadvantages. The advantages stem from the fact that the mechanism of chiral separation is easier to predict. The enantiomers are eluted out of the column as enantiomeric entities thus, they have the same detector response. The disadvantages consist of the high price of chiral columns and the fact that, as in the case of CMPs, the sample capacity is relatively small. 

11 Guidelines for the analysis of acidic basic and neutral compounds. 104 

The separation scientist with experience gained from a LC background may tend to limit the modes of electrochromatography to reversed phase (RP), normal phase, ion-exchange and, maybe, size-exclusion. Analysts from an electrophoretic background typically use the term CE in a much broader sense to include the main modes of capillary zone electrophoresis, micellar electrokinetic chromatography, capillary gel electrophoresis, isoelectric focusing and isotachophoresis. 

As capillary electrochromatography (CEC) is a hybrid technique between CE and LC, there are actually many modes of operation ranging from those commonly used in CE to those described in LC. This is shown diagrammatically in Table 3.1. 

The section of the table topped by the heavy bar indicates the packed CEC region and it is these areas which are to be covered in this chapter. There is considerable scope for overlap between the described cells, and many further divisions of these artificial boundaries are possible, for example pressure-assisted electrochromatography (PEC) describes a continuum from pure CEC to pure pLC. 

Many of the modes of CEC illustrated in Table 3.1 are applicable to both gradient and isocratic elution, aqueous and non-aqueous conditions, as well as to chiral and achiral separations and these will be discussed within the appropriate sections. The complex mechanisms responsible for selectivity will not be discussed, rather this chapter will be limited to describing the scope for application of the different CEC modes. 

---

Twenty-eight chiral compounds were separated from their enantiomers by HPLC on a teicoplanin chiral stationary phase. Figure 8-12 shows some of the structures contained in the data set. This is a very complex stationary phase and modeling of the possible interactions with the analytes is impracticable. In such a situation, learning from known examples seemed more appropriate, and the chirality code looked quite appealing for representing such data. 

An alternative model has been proposed in which the chiral mobile-phase additive is thought to modify the conventional, achiral stationary phase in situ thus, dynamically generating a chiral stationary phase. In this case, the enantioseparation is governed by the differences in the association between the enantiomers and the chiral selector in the stationary phase. 

Thin-Layer Chromatography. Chiral stationary phases have been used less extensively in tic as in high performance Hquid chromatography (hplc). This may, in large part, be due to lack of avakabiHty. The cost of many chiral selectors, as well as the accessibiHty and success of chiral additives, may have inhibited widespread commerciali2ation. Usually, nondestmctive visuali2ation of the sample spots in tic is accompHshed using iodine vapor, uv or fluorescence. However, the presence of the chiral selector in the stationary phase can mask the analyte and interfere with detection (43). 

Chiral stationary phases in tic have been primarily limited to phases based on normal or microcrystalline cellulose (44,45), triacetylceUulose sorbents or siHca-based sorbents that have been chemically modified (46) or physically coated to incorporate chiral selectors such as amino acids (47,48) or macrocyclic antibiotics (49) into the stationary phase. 

Table 2. Classes of Hplc Chiral Stationary Phases...

Fig. 5. The stmcture of the chiral selector in the Whelk-O-1 chiral stationary phase.

Limitations with the chiral selectivity of the native cyclodextrins fostered the development of various functionalized cyclodextrin-based chiral stationary phases, including acetylated (82,83), sulfated (84), 2-hydroxypropyl (85), 3,5-dimethylphenylcarbamoylated (86) and... 

Proteias, amino acids bonded through peptide linkages to form macromolecular biopolymers, used as chiral stationary phases for hplc iaclude bovine and human semm albumin, a -acid glycoproteia, ovomucoid, avidin, and ceUobiohydrolase. The bovine semm albumin column is marketed under the name Resolvosil and can be obtained from Phenomenex. The human semm albumin column can be obtained from Alltech Associates, Advanced Separation Technologies, Inc., and J. T. Baker. The a -acid glycoproteia and ceUobiohydrolase can be obtained from Advanced Separation Technologies, Inc. or J. T. Baker, Inc. 

The chirobiotic chiral stationary phases (103,104) are based on macrocycHc antibiotics such as vancomycin (4) and teicoplanin (5). 

Mobile phases used with this stationary phase are typically 0.01 N perchloric acid with small amounts of methanol or acetonitrile. One significant advantage of these phases is that both configurations of the chiral stationary phase are commercially available and can be obtained from J. T. Baker Inc. and Chiral Technologies, Inc. (Crownpak CR). 

Another type of synthetic polymer-based chiral stationary phase is formed when chiral catalyst are used to initiate the polymerisation. In the case of poly(methyl methacrylate) polymers, introduced by Okamoto, the chiraUty of the polymer arises from the heUcity of the polymer and not from any inherent chirahty of the individual monomeric subunits (109). Columns of this type (eg, Chiralpak OT) are available from Chiral Technologies, Inc., or J. T. Baker Inc. 

Chiral separations present special problems for vaUdation. Typically, in the absence of spectroscopic confirmation (eg, mass spectral or infrared data), conventional separations are vaUdated by analysing "pure" samples under identical chromatographic conditions. Often, two or more chromatographic stationary phases, which are known to interact with the analyte through different retention mechanisms, are used. If the pure sample and the unknown have identical retention times under each set of conditions, the identity of the unknown is assumed to be the same as the pure sample. However, often the chiral separation that is obtained with one type of column may not be achievable with any other type of chiral stationary phase. In addition, "pure" enantiomers are generally not available. 

Gc chiral stationary phases can be broadly classified into three categories diamide, cyclodextrin, and metal complex. 

Diamide Chiral Separations. The first chiral stationary phase for gas chromatography was reported by GH-Av and co-workers in 1966 (113) and was based on A/-trifluoroacetyl (A/-TFA) L-isoleucine lauryl ester coated on an inert packing material. It was used to resolve the tritiuoroacetylated derivatives of amino acids. Related chiral selectors used by other workers included -dodecanoyl-L-valine-/-butylamide and... 

Gyclodextrins. As indicated previously, the native cyclodextrins, which are thermally stable, have been used extensively in Hquid chromatographic chiral separations, but their utihty in gc appHcations was hampered because their highly crystallinity and insolubiUty in most organic solvents made them difficult to formulate into a gc stationary phase. However, some functionali2ed cyclodextrins form viscous oils suitable for gc stationary-phase coatings and have been used either neat or diluted in a polysiloxane polymer as chiral stationary phases for gc (119). Some of the derivati2ed cyclodextrins which have been adapted to gc phases are 3-0-acetyl-2,6-di-0-pentyl, 3-0-butyryl-2,6-di-0-pentyl,... 

Although the chiral recognition mechanism of these cyclodexttin-based phases is not entirely understood, thermodynamic and column capacity studies indicate that the analytes may interact with the functionalized cyclodextrins by either associating with the outside or mouth of the cyclodextrin, or by forming a more traditional inclusion complex with the cyclodextrin (122). As in the case of the metal-complex chiral stationary phase, configuration assignment is generally not possible in the absence of pure chiral standards. 

Chiral Hplc Columns. There are about 40 commercially available chiral columns which are suitable for analytical and preparative purposes (57). In spite of the large number of commercially available chiral stationary phases, it is difficult and time-consuming to obtain good chiral separation. In order to try a specific resolution meaninghilly, a battery of chiral hplc columns is necessary and this is quite expensive. 

Among various types of chiral stationary phases, the host-guest type of chiral crown ether is able to separate most amino acids completely (58). 

Gas chromatography (gc) is inferior to hplc in separating abiUty. With gc, it is better to use capillary columns and the appHcation is then limited to analysis (67). Resolution by thin layer chromatography or dc is similar to Ic, and chiral stationary phases developed for Ic can be used. However, tic has not been studied as extensively as Ic and gc. Chiral plates for analysis and preparation of micro quantities have been developed (68). 

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. 

Table 6. Chiral Stationary Phases, Manufacturers, and Prices...

Chiral Chromatography. Chiral chromatography is used for the analysis of enantiomers, most useful for separations of pharmaceuticals and biochemical compounds (see Biopolymers, analytical techniques). There are several types of chiral stationary phases those that use attractive interactions, metal ligands, inclusion complexes, and protein complexes. The separation of optical isomers has important ramifications, especially in biochemistry and pharmaceutical chemistry, where one form of a compound may be bioactive and the other inactive, inhibitory, or toxic. 

Separation of enantiomers by physical or chemical methods requires the use of a chiral material, reagent, or catalyst. Both natural materials, such as polysaccharides and proteins, and solids that have been synthetically modified to incorporate chiral structures have been developed for use in separation of enantiomers by HPLC. The use of a chiral stationary phase makes the interactions between the two enantiomers with the adsorbent nonidentical and thus establishes a different rate of elution through the column. The interactions typically include hydrogen bonding, dipolar interactions, and n-n interactions. These attractive interactions may be disturbed by steric repulsions, and frequently the basis of enantioselectivity is a better steric fit for one of the two enantiomers. ... 

---