Inclusion complex stationary phases

These stationary phases separate enantiomers on the basis that one isomer fits in the pocket and the other does not. In this fashion, the relative speed of the isomers is different, and separation results. There are three main types of inclusion chiral stationary phases a-cyclodextrin, /Tcyclodextrin, and y-cyclodextrin. From these three native cyclodextrins, several derivations can be made to alter the selectivity of the inclusion complex, including formation of acetates, esters, and carbamates. Astec produces all three native cyclodextrin stationary phases as well as several derivatized phases (called the Cyclobond series), and as with their macrolide polypeptide phases, they are covalently bonded. 

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. 

Analytically, the inclusion phenomenon has been used in chromatography both for the separation of ions and molecules, in Hquid and gas phase (1,79,170,171). Peralkylated cyclodextrins enjoy high popularity as the active component of hplc and gc stationary phases efficient in the optical separation of chiral compounds (57,172). Chromatographic isotope separations have also been shown to occur with the help of Werner clathrates and crown complexes (79,173). 

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. 

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. 

When a CSP is applied, the separation mechanism is based on the differences in the interaction between the chiral selector in the stationary phase and the enantiomers of the solute. Depending on the nature of the selector and the type of the solute, the stereoselective interaction can be based on interactions of one or more different types such as inclusion complexation, Tr-jr-interaction, dipole stacking, hydrogen bonding, electrostatic interaction, hydrophobic interaction, and steric interaction [35]. In order to obtain chiral discrimination between the enantiomers, a three-point interaction is required between at least one of the enantiomers and the CSP [36]. The interactions can be of attractive as well as repulsive nature (e.g., steric and electrostatic interactions). 

Many types of chiral stationary phase are available. Pirkle columns contain a silica support with bonded aminopropyl groups used to bind a derivative of D-phenyl-glycine. These phases are relatively unstable and the selectivity coefficient is close to one. More recently, chiral separations have been performed on optically active resins or cyclodextrins (oligosaccharides) bonded to silica gel through a small hydrocarbon chain linker (Fig. 3.11). These cyclodextrins possess an internal cavity that is hydro-phobic while the external part is hydrophilic. These molecules allow the selective inclusion of a great variety of compounds that can form diastereoisomers at the surface of the chiral phase leading to reversible complexes. 

Chiral crown ethers based on IB-crown-6 I Fig. 4> can form inclusion complexes with ammonium ions and proionated primary amines. Immobilization of these chiral crown ethers on a chromatographic support provides a chiral stationary phase which can resolve most primary amino acids, amines and amino alcohols. However, the stereogenic center must be in fairly close proximity in the primary aininc lor successful chiral separalion. Significantly, ihe chiral crown ether phase is unique in that ii is one of the few liquid chromatographic chiral stationary phases that does not require the presence of an aromatic ring to achieve chiral separations. 

Generally, CD-based chiral stationary phases have been used in the reversed-phase mode. Earlier, it was assumed that in the normal phase mode, the more nonpolar component of the mobile phase would occupy the CD cavity, thereby blocking inclusion complexation between the chiral analyte and CD [4,11], But with the development of CD derivatives, it has become possible to use the normal phase mode too [45,74], Among the various CSPs based on CD derivatives, one based on a naphthylethyl carbamoylated derivative has shown excellent enantioselectivity in the normal phase mode [46,59]. Armstrong et al. [45] synthesized several /CCD derivatives and had them tested in the normal phase mode to resolve the enantiomers of a variety of drugs hexane-2-propanol (90 10, v/v) served as the mobile phase. The authors discussed the similarities and differences of the enantioselectivities on the native and derivatized CD phases. 

The use of chiral stationary phases (CSP) is a technique that relies on the formation of transient, temporary diastereoisomers between the sample enantiomers and the chiral stationary phase.1 Differences in stability between the diastereoisomers are reflected in differences in retention times the enantiomer that forms the least stable complex is eluted first. Chiral stationary phases may be classified into five different groups on the basis of mechanism of retention 77 (1) chiral phases with cavities78 80 (inclusion mechanism), shown in Figure 2.19, (2) chiral affinity phases,81,82 (3) chiral phases based on multiple hydrogen bond formation,83 (4) chiral jr-donor and... 

The inclusion of basic additives in the run buffer leads to a reduction in the EOF. This is due to the reduction in the number of free silanol sites on the silica surface. However, above 50 mM the continued reduction in the EOF is less pronounced [63]. In practice, sufficient EOF is generated, even in the presence of mobile phase additives, to elute neutral species in acceptable times. The upper limit on the additive concentration is most frequently due to excessive baseline noise arising from high background absorbance. The inclusion of mobile phase additives leads to a further level of complexity in method development and prohibits coupling to mass spectrometry. However, this approach is a practical solution until better stationary phases are developed. 

The use of cyclodextrins as the mobile phase components which impart stereoselectivity to reversed phase high performance liquid chromatography (RP-HPLC) systems are surveyed. The exemplary separations of structural and geometrical isomers are presented as well as the resolution of some enantiomeric compounds. A simplified scheme of the separation process occurring in RP-HPLC system modified by cyclodextrin is discussed and equations which relate the capacity factors of solutes to cyclodextrin concentration are given. The results are considered in the light of two phenomena influencing separation processes adsorption of inclusion complexes on stationary phase and complexation of solutes in the bulk mobile phase solution. 

To improve the effectiveness of the chromatographic separation, a comparison study has been carried out on cyclodextrin and liquid crystal stationary phases Both materials function as "ordered" media with cyclodextrins the inclusion complex formation predominates, whereas the liquid crystals enable interaction of compounds with the ordered structure of the mesophase ... 

In Section 22.3 the main types of interactions occurring between the enantiomeric analytes and the stationary phase (hydrogen bonding, charge transfer, and inclusion complexes) was described. In the following section,... 

The use of chiral stationary phases (CSP) in liquid chromatography continues to grow at an impressive rate. These CSPs contain natural materials such as cellulose and starch as well as totally synthetic materials, utilizing enantioselective and retentive mechanisms ranging from inclusion complexation to Ti-electron interactions. The major structural features found in chiral stationary phases include cellulose, starch, cyclodextrins, synthetic polymers, proteins, crown ethers, metal complexes, and aromatic w-electron systems. 

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