Separation, enantiomeric resolution

Recently, two examples of the separation of enantiomers using CCC have been published (Fig. 1-2). The complete enantiomeric separation of commercial d,l-kynurenine (2) with bovine serum albumin (BSA) as a chiral selector in an aqueous-aqueous polymer phase system was achieved within 3.5 h [128]. Moreover, the chiral resolution of 100 mg of an estrogen receptor partial agonist (7-DMO, 3) was performed using a sulfated (3-cyclodextrin [129, 130], while previous attempts with unsubstituted cyclodextrin were not successful [124]. The same authors described the partial resolution of a glucose-6-phosphatase inhibitor (4) with a Whelk-0 derivative as chiral selector (5) [129]. 

Enantiomeric separations have become increasingly important, especially in the pharmaceutical and agricultural industries as optical isomers often possess different biological properties. The analysis and preparation of a pure enantiomer usually involves its resolution from the antipode. Among all the chiral separation techniques, HPLC has proven to be the most convenient, reproducible and widely applicable method. Most of the HPLC methods employ a chiral selector as the chiral stationary phase (CSP). 

The high diffusivity and low viscosity of sub- and supercritical fluids make them particularly attractive eluents for enantiomeric separations. Mourier et al. first exploited sub- and supercritical eluents for the separation of phosphine oxides on a brush-type chiral stationary phase [28]. They compared analysis time and resolution per unit time for separations performed by LC and SFC. Although selectivity (a) was comparable in LC and SFC for the compounds studied, resolution was consistently... 

Mixing the additive in the eluent used as a mobile phase can also modify the chromatographic system (dynamic modification), but the use of modified adsorbents has led to an improvement of resolution. Example works include that by Armstrong and Zhou [11], who used a macrocyclic antibiotic as the chiral selector for enantiomeric separations of acids, racemic drugs, and dansyl amino acid on biphenyl-bonded silica. 

The resolution of enantiomers by liquid chromatography using chiral stationary phases is based on the formation of reversible diastereomeric complexes of different stability between the sample and stationary phase. Since the formation of the complexes is strongly dependent on the structure of the sample, there are no universal chiral stationary phases. The specific advantages of TLC for enantiomeric separations result from its low cost, convenience and speed (10,97,98). The main limitation, particularly with respect to column liquid chromatography, is the small number of phases currently available. 

A chiral GC column is able to separate enantiomers of epoxy pheromones in the Type II class, but the applications are very limited as follows a custom-made column packed with a p-cyclodextrin derivative as a liquid phase for the stereochemical identification of natural 3,4- and 6,7-epoxydienes [73, 74] and a commercialized column of an a-cyclodextrin type (Chiraldex A-PH) for the 3,4-epoxydiene [71] (See Table 3). The resolution abilities of chiral HPLC columns have been examined in detail, as shown in Table 7 and Fig. 14 [75,76, 179]. The Chiralpak AD column operated under a normal-phase condition separates well two enantiomers of 9,10-epoxydienes, 6,7-epoxymonoenes and 9,10-epoxymonoenes. Another normal-phase column, the Chiralpak AS column, is suitable for the resolution of the 3,4-epoxydienes. The Chiralcel OJ-R column operated under a reversed-phase condition sufficiently accomplishes enantiomeric separation of the 6,7-epoxydienes and 6,7-epoxymonoenes. 

Enantioselective separation by supercritical fluid chromatography (SFC) has been a field of great progress since the first demonstration of a chiral separation by SFC in the 1980s. The unique properties of supercritical fluids make packed column SFC the most favorable choice for fast enantiomeric separation among all of the separation techniques. In this chapter, the effect of chiral stationary phases, modifiers, and additives on enantioseparation are discussed in terms of speed and resolution in SFC. Fundamental considerations and thermodynamic aspects are also presented. 

Note that not all enantioseparations in SFC are better than in HPLC [34], Bernal et al. [62] described the enantiomeric separation of several pharmaceutical-related compounds on a polysaccharide-based column using HPLC and SFC. They showed that most of the separations obtained by SFC are better, in terms of resolution and analysis time, than the separations obtained by HPLC. However, one compound could not be resolved using SFC, but LC provided baseline resolution. 

From the method development and robustness point of view, the temperature is a parameter that controls equilibria such as pK and enantiomer—chiral selector complexation, or induces structural changes in, e.g., proteins.For chiral separations, generally a lower temperature results in better enantioseparation, but even the opposite has been observed. Sometimes a raise in temperature does not so much affect the enantiomeric separation, but increases the resolution between an enantiomer and a matrix component. ... 

Konig, W.A. 1993. Collection of enantiomeric separation factors obtained by capillary gas chromatography on chiral stationary phases. J. High Resolut. Chromatogr. 16 569-586. 

Chiral resolution is a matter of great importance. For example, the food and beverage industry is increasingly concerned with enantiomeric separations, because they can affect flavor, fragrance, and nutrition and can be used to monitor fermentation or product adulteration. Separations are carried out mainly on chiral stationary phases (CSPs). 

Ligand exchange has proved to be very successful in the separation of several enantiomers. Davankov and Rogozhin (41) used chiral copper complexes bonded to silica. The enantiomeric separation is based essentially on the formation of diastereomeric mixed complexes with different thermodynamic stabilities. It is generally accepted that chiral discrimination proceeds via the substitution of one ligand in the coordination sphere of the metal ion. Ligand exchange technique is especially effective for the enantiomeric resolution of aminoacids, aminoacids derivatives, and hydroxy acids (42). 

In sub-FC, a detailed study of the influence of mobile phase additives on the chiral resolution of isoxazoline-based Ilb/IIIb receptor antagonists was carried out by Blackwell [145] on Chiralcel OD-H CSPs. The different mobile phase additives used were acetic acid, trifluoroacetic acid, formic acid, water, triethylamine, triethanolamine, n-hexylamine, trimethyl phosphate, and tri-w-butyl phosphate. In general, n-hexylamine and tri-/ -butyl phosphate mobile phase additives resulted in better resolution. The chiral separation of four 1,3-dioxolane derivatives on an amylose-based column has been described [151]. The effects of mobile phase composition, temperature, and pressure have been investigated. The nature of the modifier is the main parameter it has the highest impact on chiral resolution and is more important than the polarity of the mobile phase. Therefore, the organic modifier that gave the best enantiomeric separation was different for each compound. 

The CSPs prepared by the molecular imprint technique have also been used for chiral resolution by CEC [98-100]. Lin et al. [91] synthesized L-aromatic amino acid-imprinted polymers using azobisnitriles with either photoinitiators or thermal initiators at temperatures ranging from 4°C to 60° C. Methacrylic acid (MAA) was used as the functional monomer and ethylene glycol dimethacrylate (EDMA) was used as the cross-linker. The resulting polymers were ground and sieved to a particle size less than 10 pm, filled into the capillary columns, and used for enantiomeric separations of some amino acids at different temperatures. The relationships of separation factor and column temperatures were demonstrated to be linear between the logarithm of the separation factors and the inverse of the absolute temperature (Fig. 24). The authors also compared the obtained chiral resolution with the chiral resolution achieved by HPLC and reported the best resolution on CEC. The chromatograms of the chiral resolution of dl-... 

Owing to the high thermal and long-term stability, amide phases bonded to polysiloxane are regarded as sensitive materials for the sensoric approach to enantiomeric separation. An important factor for the resolution of the stationary material is the number of dimethylpolysiloxane units between the chiral moieties. More than 200 different amide phases have been synthesised and applied for the discrimination of amino acids, lactate esters and many other substances [10]. 

Enantiomeric self-resolution through complexation with a hydrogen-bonding guest solvent has interesting potential as a means of separation. Unfortunately, little prediction of new examples seems possible with our current level of understanding. 

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