Polysaccharide-based chiral columns

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. 

The use of polysaccharide-based CSPs instead of protein-based CSPs often increases the peak efficiency and facilifafes faster separafions. Papini ef al. [159] recently developed a method for the enantioseparation of lorazepam and on a Chiralpak OD-R column and an enzymatic hydrolysis was used to determine the amount of the glucoronide metabolite of lorazepam present. The separation was performed in 7 min with an LOQ of 1 and 10 ng/mL for lorazepam in plasma and urine, respectively. Another relatively fast separation for chiral analysis was published by Lausecker and Eischer [188]. They developed a method for determination of the drug candidate R483 within... 

CSPs (CTA-I and CTA-II) have inverse selectivity for Troger s base and trans-1,2-diphenyloxirane racemates. These characteristics of CTA CSPs are responsible for good chiral resolution of small cychc carbonyl compounds [42]. In 2001 Aboul-Enein and Ah [63] observed the reversed order of elution of nebivolol on a Chiralpak AD column when ethanol and 2-propanol were used separately as the mobile phases. Table 1 presents selectivity data for the polysaccharide-based CSPs. Okamoto et al. [42] observed that the introduction of a methyl group at the para position of cellulose tribenzoate results in a dramatic shift of the structural selectivity toward aromatic compounds with larger skeletons, and its selectivity was rather similar to that of cellulose tricinnamate. 

Temperature is also an important parameter for controlling the resolution of enantiomers in HPLC. The enthalpy and entropy control of chiral resolution on antibiotic CSPs is similar to the case of polysaccharide-based CSPs (Chapter 2). Armstrong et al. [1] have studied the effect of temperature on the resolution behavior of proglumide, 5-methyl-5-phenylhydantoin and A-carbamyl-D-pheny-lalanine on the vancomycin column. The experiments were carried out from 0°C to 45°C. These results are given in Table 6 for three chiral compounds. It has been observed that the values of k, a, and Rs for the three studied molecules have decreased with the increase in temperature, indicating the enhancement of chiral resolution at low temperature. In another work, the same workers [22] have also studied the effect of temperature on the resolution of certain amino acid derivatives on the teicoplanin chiral stationary phase. They further observed poor resolution at ambient temperature, whereas the resolution increased at low... 

Cass et al. [66] used a polysaccharide-based column on multimodal elution for the separation of the enantiomers of omeprazole in human plasma. Amylose tris (3,5-dimethylphenylcarbamate) coated onto APS-Hypersil (5 /im particle size and 120 A pore size) was used under normal, reversed-phase, and polar-organic conditions for the enantioseparation of six racemates of different classes. The chiral stationary phase was not altered when going from one mobile phase to another. All compounds were enantioresolved within the elution modes with excellent selectivity factor. The separation of the enantiomers of omeprazole in human plasma in the polar-organic mode of elution is described. 

Chiral separation by HPLC is a practically useful method not only for determining optical purity but also for obtaining optical isomers, and numerous CSPs are presently on the market. In order to achieve the efficient resolution of chiral compounds, we have to choose a suitable chiral column and eluent. The polysaccharide-based CSPs have a high chiral recognition ability and offer a high possibility for the successful resolution of racemates including aliphatic and aromatic compounds with or without functional groups under normal and reversed-phase conditions. 

Yashima, E., Yamamoto, C., and Okamoto, Y. (1998) Polysaccharide-based chiral LC columns, Synlett, 344-360. 

Modern polysaccharide columns are based on cellulose or amylose derivatives coated onto silica. Chiral discrimination and applications have been extensively documented, but the mechanism of chiral discrimination is not yet fully understood. Whereas numerous phases are available within this subset, orthogonality can generally be obtained from a set of three or four columns as a first approach to method development. A typical choice of columns would be to try a set of different amylase (Chiralpak AD and AS) and cellulose (Chiralcel OD or OJ) columns and defer more extensive method development to the subset of samples not separated by these columns. The columns specified are run in the normal-phase mode and, accordingly, mobile phases are typically mixtures of hexane with small amounts of isopropanol or ethanol to control retention. However, selectivity is changed by different polar modifiers. Tailing may be minimized by the addition of 10-50 mM trifluoroacetic acid (TFA) or triethylamine (TEA). Analogue of the columns specified (AD-R, AS-R, OD-R, and OD-J) are available for reversed-phase separation. 

FIGURE 144 (A) Achiral- and (B) chiral-method screen strategies. (A) Platform 1 (HPLC-1) uses two different mobile phases, one is acidic (mobile phase 1) and another is neutral-to-basic (mobile phase 2) to screen on two columns, a classic reverse-phase C-18 and a special polar group-embedded C-18 (AQ). Platform-2 (HPLC-2) uses one mobile phase (pH is usually acidic) to screen four different columns of wide range of polarity (PFP fluorinated, and Phen phenyl-hexyl phases). Platform-3 (SFC) uses three different mobile phases and five different columns (PYD 2-ethylpyridine, BENZ benzamide). Platform-4 (CE) uses two different mobile phases. (B) AD, OJ, OD, AS are different polysaccharide-based chiral stationary phases. 

TABLE I Polysaccharide-Based Chiral Columns Commonly Used in Pharmaceutical HPLC Method Development Screening Systems... 

The most popular CSPs selected to be included in the reported chiral column screening schemes are four polysaccharide-based phases, including cellulose tris(3,5-dimethylphenylcarbamate), cellulose tris-4-methylbenzoate, amylose... 

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