Phases using cyclodextrins

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. 

Early attempts to use cyclodextrins and their simple derivatives as chira stationary phases in gas chromatography met... 

The mobile phases used with cyclodextrin CSPs are similar to those used in reverse phase chromatography, ie water or buffer solutions modified with methanol or acetonitrile. 

Using a chiral column, coated with a definite modified cyclodextrin as the chiral stationary phase, the elution orders of furanoid and pyranoid linalool oxides are not comparable [11, 12]. Consistently, the chromatographic behaviour of diastereomers and/or enantiomers on modified cyclodextrins is not predictable (Fig. 17.1, Table 17.1). Even by changing the non-chiral polysiloxane part of the chiral stationary phase used, the order of elution may significantly be changed [13]. The reliable assignment of the elution order in enantio-cGC implies the coinjection of structurally well defined references [11-13]. 

Kreis P, Dietrich A, Mosandl A (1996) Elution order of the furanoid linalool oxides on common gas chromatographic phases and modified cyclodextrin phases. J Essent Oil Res 8 339 Weinert B, Wiist M, Mosandl A Hanssum H (1998) Stereoisomeric flavour compounds LXX-Vlff. Separation and structure elucidation of the pyranoid linalool oxide stereoisomers using common gas chromatographic phases, modified cyclodextrin phases and nuclear magnetic resonance spectroscopy. Phytochem Anal 9.T0... 

K Shimada, K Hirakata. Retention behavior of derivatized amino acids and dipeptides in high-performance liquid chromatography using cyclodextrin as a mobile phase additive. J Liq Chromatogr 15 1763-1771, 1992. 

In 1978, Harada et al. [17] used polymerized CD with gel support for the chiral resolution of mandelic acid and its derivatives. Later Zsadon et al. [18-21] used cyclodextrin-based CSPs for the chiral resolution of indole alkaloids, with aqueous buffers as the mobile phases. Today CD-based CSPs have a good reputation. In separate studies, Fujimura [22] and Kawaguchi [23] and their colleagues resolved the enantiomers of aromatic compounds in the reversed-phase mode. Armstrong et al. [29,30,33,34,41,44 46,48,54-63] carried out extensive and remarkable work on the chiral resolution of various racemic compounds using CD-based CSPs. 

A Guide to Using Cyclodextrin Bonded Phases for Liquid Chromatography, Advanced Separation Technologies, Inc., Whippany, NJ (1996). 

Because the steric effect contributes to the complex formation between guest and host, the chiral resolution on these CSPs is affected by the structures of the analytes. Amino acids, amino alcohols, and derivatives of amines are the best classes for studying the effect of analyte structures on the chiral resolution. The effect of analyte structures on the chiral resolution may be obtained from the work of Hyun et al. [47,48]. The authors studied the chiral resolution of amino alcohols, amides, amino esters, and amino carbonyls. The effects of the substituents on the chiral resolution of some racemic compounds are shown in Table 6. A perusal of this table indicates the dominant effect of steric interactions on chiral resolution. Furthermore, an improved resolution of the racemic compounds, having phenyl moieties as the substituents, may be observed from this Table 6. ft may be the result of the presence of n—n interactions between the CCE and racemates. Generally, the resolution decreases with the addition of bulky groups, which may be caused by the steric effects. In addition, some anions have been used as the mobile phase additives for the improvement of the chiral resolution of amino acids [76]. Recently, Machida et al. [69] reported the use of some mobile phase additives for the improvement of chiral resolution. They observed an improvement in the chiral resolution of some hydrophobic amino compound using cyclodextrins and cations as mobile phase additives. 

The chiral resolution using cyclodextrins as mobile phase additives is also controlled by a number of chromatographic parameters as in case of CSPs. The chiral resolution occurred by the formation of diastereoisomeric inclusion complex formation and, hence, the composition of the mobile phase, pH, concentration of cyclodextrins, and temperature are the most important controlling parameters. 

TABLE 1 Chiral Resolution of Some Racemic Compounds Using Cyclodextrins as the Mobile Phase Additives... 

In LC, cyclodextrins have been used in both the mobile and stationary phases, although the latter application predominates using bonded phases. Both normal and reversed phase separations are possible. With polar mobile phases, the cyclodextrin cavity hosts and retains hydrophobic analytes but when large concentrations of organic solvent are used in the mobile phase, solvent molecules occupy the cavity—inclusion of analytes is suppressed and hydrogen bonding and dipole-dipole interactions with analytes predominate. Applications to electrochromatography have also been described. [96]... 

The above phases represent the most common phases used in solving nearly all of the frequently encountered application problems. There are many other stationary phases which are produced to tune the phase polarity for specific applications. In addition to these phases, there are liquid crystalline, chiral, cyclodextrin, polymers such as polystyrene, divinylben-zene, molecular sieves, and alumina, which are designed for specific separation problems. The chemistry of fused silica deactivation and stationary-phase application, bonding, and cross-linking has been reviewed in detail [3,4]. 

K. Shimada, K. Mitamura, M. Morita, and K. Hirahata, Separation of the diastereomers of baclofen by HPLC using cyclodextrin as a mobile phase additive, J. Liquid Chromatogr., 76 3311 (1993). 

Minority Access for Research careers Program, National Institute of Health, Grant Number 5F31GHL1689 is acknowledged. The authors are grateful to Dr. Thanas Beesley of ASTEC for the beta-cyclodextrin silica stationary phases used in this study. 

Silica-base stationary phases have also been employed for enantiomeric separations in CEC [6,72-81]. In the initial work on chiral CEC, commercially available HPLC materials were utilized, including cyclodextrins [6,74,81] and protein-type selectors [73,75,80] such as human serum albumin [75] and ai-acid glycoprotein [73]. Fig. 4.9, for example, depicts the structure of a cyclodextrin-base stationary phase used in CEC and the separation of mephobarbital enantiomers by capillary LC and CEC in a capillary column packed with such a phase. The column operated in the CEC mode affords higher separation efficiency than in the capillary LC mode. Other enantiomeric selectors are also use in CEC, including the silica-linked or silica-coated macrocyclic antibiotics vancomycin [82,83] and teicoplanin [84], cyclodextrin-base polymer coated silicas [72,78], and weak anion-exchage type chiral phases [85]. Relatively high separation efficiency and excellent resolution for a variety of compounds have also been achieved using columns packed with naproxen-derived and Whelk-0 chiral stationary phases linked to 3 pm silica particles [79]. Fig. 4.10 shows the... 

In CE a successful method for enantiomer separation is the addition of a chiral selector to the mobile phase. This practice can be transferred to p-CEC by using a packing bed which consists of bare silica or ODS (octadecylsilica) and a mobile phase containing a chiral additive. At the first time, Lelievre et al. added hydroxypropyl-[5-cyclodextrin to the mobile phase using an ODS packed capillary. The enantiomer separation of chlorthalidone with a resolution Rs of 1.4 was feasible [41]. Deng et al. [59] used an ODS-packed column and (3-cyclodextrin as a mobile phase additive. A theoretical model for the enantiomer separation of salsolinol was developed and compared with the experimental data. For pressure supported CEC, very high pressure (about 100 bar) was applied to the inlet vial so that the mobile phase was mainly driven by the applied pressure. 

Five dihydropyridine enantiomers (including nimodipine) were separated by HPLC using cyclodextrin as the chiral selector [25]. Columns containing (5-cyclodextrin covalently bonded to 5 pm silica (25 cm x 4 mm i.d.), or of (R)- or (S)-naphthyl ethyl carbomyl (1-cyclodextrin covalently bonded to 5 pm silica (25 cm x 4.6 mm i.d.), were used. The mobile phases (flow rate of 0.8 mL/min) were mixtures of ethanol or acetonitrile, with 0.1% triethylamine, adjusted to pH 5 with acetic acid. Detection was at 239 nm. 

The selectivity of cyclodextrins toward the various molecules is not high enough to attain complete (or acceptable) separations by one-step operations. Enrichment, of one component or partial separation of various components of a mixture can be attained relatively easily. However using cyclodextrins in multistep processes, i.e. the various chromatographic techniques, very effective separations can be achieved. Particularly in RP-HPLC the application of immobilized CDs and CDs dissolved in the mobile phase became one of the most promising methods. 

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