Preparative enantiomeric separations

Figure 21-7. Preparative enantiomeric separation of 3-benzyloxycarbonyl-2-f-bntyloxazoUdinone on Chiralcel-OD (50 cm x 5 cm) mobile phase hexane/2-propanol = 8/2 (V/V), 50mL/min injection amounts 2g (hatched area) and 3g. (Reprint from reference 50, with permission.)...

While native CF6 showed only limited enantioselectivity to a few compounds, derivatized CF6s appeared to be versatile chiral selectors and can be tuned for the best HPLC separation of different types of compounds. Aliphatic-derivatized cyclofructans operating in the polar organic mode provide best enantiomeric separations for primary amines. On the other hand, extensively aromatic-derivatized CF6 were able to separate a variety of different classes of enantiomers. Cyclofructans are the newest types of chiral selectors, and their further development and their applications in both analytical and preparative enantiomeric separations are not yet fully explored and are expected to grow significantly in the future. 

Although some applications for preparative-scale separations have already been reported [132] and the first commercial systems are being developed [137, 138], examples in the field of the resolution of enantiomers are still rare. The first preparative chiral separation published was performed with a CSP derived from (S -N-(3,5-dinitrobenzoyl)tyrosine covalently bonded to y-mercaptopropyl silica gel [21]. A productivity of 510 mg/h with an enantiomeric excess higher than 95% was achieved for 6 (Fig. 1-3). 

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). 

Packed column SFC has also been applied to preparative-scale separations [42], In comparison to preparative LC, SFC offers reduced solvent consumption and easier product recovery [43]. Whatley [44] described the preparative-scale resolution of potassium channel blockers. Increased resolution in SFC improved peak symmetry and allowed higher sample throughput when compared to LC. The enhanced resolution obtained in SFC also increases the enantiomeric purity of the fractions collected. Currently, the major obstacle to widespread use of preparative SFC has been the cost and complexity of the instrumentation. 

Enantiomeric separations have proven to be one of the most successful applications of packed column SEC. Despite initial reluctance, many analysts now use SEC routinely for both analytical and preparative chiral separations. Additional studies of chiral recognition in SEC and continued improvements in instrumentation will ensure a prominent role for SEC in chiral separations methodology in the future. 

Additionally, the inj ected matrix must also be miscible with the solvents used in the separations. For normal phase mode separations, all water must be removed from the injected matrix. Since many of the complex matrixes, such as plasma, urine, and other biological fluids contain a large amount of water, this requires more time consuming sample preparation. However, water can be injected into a polar organic or reverse phase mode separation. Even within the same mode, mobile phases that are very different can cause large disturbances in the baseline. Oda et al., (1991) solved this problem by inserting a dilution tube followed by a trap column in order to dilute the mobile phase used on the achiral column. Following the dilution tube, a trap column was used to reconcentrate the analyte of interest before the enantiomeric separation. 

Because plasma and urine are both aqueous matrixes, reverse-phase or polar organic mode enantiomeric separations are usually preferred as these approaches usually requires less elaborate sample preparation. Protein-, cyclodextrin-, and macrocyclic glycopeptide-based chiral stationary phases are the most commonly employed CSPs in the reverse phase mode. Also reverse phase and polar organic mode are more compatible mobile phases for mass spectrometers using electrospray ionization. Normal phase enantiomeric separations require more sample preparation (usually with at least one evaporation-to-dryness step). Therefore, normal phase CSPs are only used when a satisfactory enantiomeric separation cannot be obtained in reverse phase or polar organic mode. 

With the increased popularity of LC-MS, the problem of overlapping enantiomer peaks from other amino acids has largely been resolved. The mass spectrometer can act as an additional dimension of separation (based on mass to charge ratio). Thus, only amino acids having the same mass-to-charge ratio must be separated achirally (see Desai and Armstrong, 2004). This additional dimension of separation also has implications for the applications in the matrices discussed previously. With the ability of the mass spectrometer to discriminate on the basis of mass, this lessens the need for complete achiral separation. For example, an LC-MS method was recently developed to study the pharmacokinetics of theanine enantiomers in rat plasma and urine without an achiral separation before the enantiomeric separation (Desai et al., 2005). In such matrices, proteins must still be removed by appropriate sample preparation. 

Klein, R., Tatischeff, I., Tham, G., Mano, N. (1994). Chiral lumazines preparation, properties, enantiomeric separation. Chirality 6, 564—571. 

Each entry is an average of data obtained from two separate experiments using different microsomal preparations. Enantiomeric composition was determined by CD spectral data (18) and by CSP- HPLC (19,20). 

Additional enantiomerically pure 4,5-dihydroisoxazoles are prepared by separation of racemic compounds via chiral sulfoxides5-8, or by microbial reduction of 5-acetyl-4,5-di-hydroisoxazoles 34. 

Capillary gas chromatography (GC) using modified cyclodextrins as chiral stationary phases is the preferred method for the separation of volatile enantiomers. Fused-silica capillary columns coated with several alkyl or aryl a-cyclo-dextrin, -cyclodextrin and y-cyclodextrin derivatives are suitable to separate most of the volatile chiral compounds. Multidimensional GC (MDGC)-mass spectrometry (MS) allows the separation of essential oil components on an achiral normal phase column and through heart-cutting techniques, the separated components are led to a chiral column for enantiomeric separation. The mass detector ensures the correct identification of the separated components [73]. Preparative chiral GC is suitable for the isolation of enantiomers [5, 73]. 

For the enantiomeric separation of propanolol, MIP monoliths have been rendered porous by the addition of isooctane in toluene at 2%. The poor solvent content is a crucial parameter for controlling the porosity of the MIP monolith, a higher concentration of poor solvent leading to a more porous but also more fragile material. Actually, a combination of these two techniques, where the selection of the poor solvent and the timing of polymerisation is optimised, can also be employed for the preparation of preformed imprinted monoliths [166, 167],... 

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-... 

As the Diels-Alder cyclic adduct 33 has been recognized as the most accessible starting compound for the synthesis of racemic pseudo-sugars, a resolution of 33 has been attempted to prepare enantiomeric pseudo-sugars, starting from an optically active antipode of 33. It has been revealed that 33 was readily separated into the enantiomers by using optically active a-methylbenzylamines as resolution reagents. 

Key Words Preparative-scale GC, enantiomeric separation, chiral stationary phases, SMB-GC, nitrogen invertomer, inhalation anesthetics, terpenoids, flavours,... 

Figure 2. Left Preparative-scale enantiomeric separation of N-TFA-alanine tert,-butyl ester on N-TFA-(L)-valine-(L)-valine cyclohexyl ester (experimental conditions cf, text). Right ORD spectra of the two isolated fractions corresponding to the marked areas (Gil-Av and Feibush, 1967).

The presence of water was subsequently identified as being essential for the performance of the above set-up and for maintaining high enantiose-lectivity (Lindstrom et al., 1990). The preparative-scale enantiomeric separation of racemic camphene with a separation factor as high as a — 3.7 (measured at 30°C on an analytical column) was subsequently achieved at 45°C on a 2.1 m x 4 mm (i.d.) column containing a-cyclodextrin hydrate in formamide (1 5, w/w) coated on Chromosorb W (AW, 45-60 mesh) by saturating the carrier gas (helium) with water vapour (Lindstrom et al., 1990). 

The preparative-scale enantiomeric separations of enflurane, isoflurane and desflurane (cf. Figure 16) by gas chromatography on modified cyclo-dextrins have been achieved. The separation was aided by unprecedentedly large separation factors a of the enantiomers. Two approaches were followed. 

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