Chiral phases bovine serum albumin

Chiral mobile phase additives provide a more versatile and cost-effective approach for enantiomer separations in thin-layer chromatography. Typically, chemically bonded layers with cyclodextrin and its derivatives, bovine serum albumin, or macrocyclic glycopeptides are used as chiral additives in the reversed-phase mode [59,60,172-178]. For [5- and y-cyclodextrins and their derivatives, a 0.1 to 0.5 M aqueous methanol or acetonitrile solution of the chiral selector is used as the mobile phase. Bovine serum albumin is generally used at concentrations of 1-8 % (w/v) in an aqueous acetate buffer of pH 5 to 7 or in a 0.5 M acetic acid solution, in either case with from 3-40 % (v/v) propan-2-ol (or another aliphatic alcohol), added to control retention. Enantioselectivity usually increases with an increase in concentration of the chiral selector, and may be non existent at low concentrations of the chiral selector. 

Chiral stationary phases that are currently available can be classified into those containing cavities (cellulose derivatives, cyclodextrins, synthetic polymers, crown ethers, and chiral imprinted gels), affinity phases (bovine serum albumin, human serum albumin, a-glycoprotein, enzymes), multiple hydrogen-bond phases, Ti-donor and Ti-acceptor phases, and chiral ligand exchange phases. This classification scheme was used in a review that gave numerous pharmaceutical examples of separation by... 

F W. Wainer and R. M. Stifhn, Direct resolution of the stereoisomers of leucovorin and 5-methylteti ahydrofolate using a bovine serum albumin liigh-performance liquid cliromatographic chiral stationary phase coupled to an acliiral phenyl column , 7. Chromatogr. 424 158-162 (1988). 

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]. 

Membranes offer a format for interaction of an analyte with a stationary phase alternative to the familiar column. For certain kinds of separations, particularly preparative separations involving strong adsorption, the membrane format is extremely useful. A 5 x 4 mm hollow-fiber membrane layered with the protein bovine serum albumin was used for the chiral separation of the amino acid tryptophan, with a separation factor of up to 6.6.62 Diethey-laminoethyl-derivatized membrane disks were used for high-speed ion exchange separations of oligonucleotides.63 Sulfonated membranes were used for peptide separations, and reversed-phase separations of peptides, steroids, and aromatic hydrocarbons were accomplished on C18-derivatized membranes. 

Chiral stationary phases for the separation of enantiomers (optically active isomers) are becoming increasingly important. Among the first types to be synthesized were chiral amino acids ionically or covalently bound to amino-propyl silica and named Pirkle phases after their originator. The ionic form is susceptable to hydrolysis and can be used only in normal phase HPLC whereas the more stable covalent type can be used in reverse phase separations but is less stereoselective. Polymeric phases based on chiral peptides such as bovine serum albumin or a -acid glycoproteins bonded to... 

Although similar efforts have been devoted to related polymer systems (Overberger and Cho, 1968 Overberger and Dixon, 1977 Okamoto, 1978), large enantioselectivity has not been observed. Goldberg et al. (1978) conducted borohydride reduction of phenyl ketones in micelles of the chiral surfactant [44]. The result was disappointing, since the maximal enantioselectivity was only 1.66% for phenyl propyl ketone. A much better optical yield was reported when this reaction was carried out under phase-transfer conditions (Masse and Parayre, 1976). The cholic acid micelle and bovine serum albumin exhibited the relatively high enantioselectivity in the reduction of trifluoroacetophenone (Baba ef al., 1978). 

Proteins, amino acids bonded through peptide linkages to form macromolecular biopolymers, used as chiral stationary phases for hplc include bovine and human serum albumin, OL-acid glycoprotein, ovomucoid, avidin, and cellobiohydrolase. The bovine serum albumin column is marketed under the name Resolvosil and can be obtained from Phenomenex. The human serum albumin column can be obtained from Alltech Associates, Advanced Separation Technologies, Inc., and J. T. Baker. The a1-acid glycoprotein and cellobiohydrolase can be obtained from Advanced Separation Technologies, Inc. or J. T. Baker, Inc. 

Other chiral immobilised stationary phases are available commercially, for example covalently bonded bovine serum albumin or chemically bonded l-hydroxyproline-Cu2 complexes to wide-bore Nucleosil (both from Macherey-Nagel). 

Chiral Stationary Phases for HPLC Name Bovine serum albumin (covalently fixed to silica gel)... 

Examples of optimizations in HPLC using the simplex approach can be found in [28,84]. In [28] the mobile phase composition for the chiral separation of (6/ )- and (65)-leucovorin on a BSA (bovine serum albumin) stationary phase is optimized by means of a variable-size simplex. Three factors were examined, the pH of the mobile phase buffer, the ionic strength of the buffer and the percentage of 1-propanol in the mobile phase. Table 6.19 shows the experimental origin, the initial step size and the acceptable limits for the factors. The criterion optimized is the valley-to-peak ratio (Section 6.2). The points selected and the results are pre.sented in Table 6.20 and... 

Besides the conventional factors causing peak broadening in LC. another source for lower efficiency in LC with chiral stationary phases may be the existence of at least two different adsorption sites (the stereoselective and non-stereoselective ones) that may considerably differ in their adsorption kinetics (heterogeneous mass transfer kinetics) and thus cause peak broadening and tailing. These factors have been investigated and modelled by Fomstedt et al.. e.g. for protein type CSPs [75,91-9.3] and their contribution was determined for different analytes and different type of CSPs (bovine serum albumin, cellobiohydrolase 1, tris(4-methylbenzoyl) cellulose) [94j. In a recent study, these authors reported on the adsorption isotherms as well as selective and nonselective contributions of propranolol enantiomers on a cellobiohydrolase 1 CSP in dependence of the mobile phase pH [95[. 

A CSP consists of a chiral selector, which either alone constitutes the stationary phase or which has been immobilised to a solid phase. The chiral selector is a low molecular weight compound or a polymer, either synthetic or natural. A broad range of CSPs has been developed. Examples of CSPs that have been used successfully include polysaccharides, such as cellulose and its derivatives [6] and cyclodex-trins [7], and proteins, e.g. bovine serum albumin, aj-acid glycoprotein, cellulase, trypsin and a-chymotrypsin [8]. Several different synthetic polymers have also proven to be useful CSPs, for example the Blaschke-type CSPs (polyacrylamides and polymethacrylamides) [9] and the Pirkle-type CSPs [10]. 

This isotherm model has been used successfully to accoimt for the adsorption behavior of numerous compounds, particularly (but not only) pairs of enantiomers on different chiral stationary phases. For example, Zhou et ah [28] foimd that the competitive isotherms of two homologous peptides, kallidin and bradyki-nine are well described by the bi-Langmuir model (see Figure 4.3). However, most examples of applications of the bi-Langmuir isotherm are found with enantiomers. lire N-benzoyl derivatives of several amino acids were separated on bovine serum albumin immobilized on silica [26]. Figure 4.25c compares the competitive isotherms measured by frontal analysis with the racemic (1 1) mixture of N-benzoyl-D and L-alanine, and with the single-component isotherms of these compounds determined by ECP [29]. Charton et al. foimd that the competitive adsorption isotherms of the enantiomers of ketoprofen on cellulose tris-(4-methyl benzoate) are well accounted for by a bi-Langmuir isotherm [30]. Fornstedt et al. obtained the same results for several jS-blockers (amino-alcohols) on immobilized Cel-7A, a protein [31,32]. 

In the former case [32], the production rate of 99% pme enantiomers from the racemic mixture of R- and S-2-phenylbutyric acid was maximized as a function of the sample size and the mobile phase composition. The calculations were based on the column performance and the equilibrium isotherms of the two components (bi-Langmuir isotherms. Chapter 3). The separation was performed on immobilized bovine serum albumin, a chiral stationary phase, using water-methanol solution as the mobile phase. The retention times decrease with increasing methanol content, but so does the separation factor. For this reason, the optimum retention factor is around 3. Calculated production rates agree well with those measured (Table 18.4). The recovery yield is lower than predicted. 

Immobilised protein columns. A further group of chiral stationary phases has been developed by immobilising naturally occurring asymmetrical peptides, e.g. bovine serum albumin, ai-acid glycoproteins, ovomucoid and... 

Columns packed with silica-bound proteins have been devised bovine serum albumin-derived columns ( J.) are available from Macherey Nagel. These CSPs show a fairly extensive scope of action, although the nature of the chiral recognition processes employed is still vague. Owing to the low concentration of active sites, these columns require quite small samples (0.5 - 5 nmol). Mobile phase variation is possible (over a limited range) and alters the chromatographic behavior of the enantiomers markedly. 

Nakagawa et al. studied the chiral discrimination in the transport of ketoprofen and ibuprofen esters through an aqueous phase mediated by various serum albumins [41]. Serum albumins that act as carriers discriminated between enantiomers of alkyl esters of ketoprofen and ibuprofen in transport in the O/W/O (oil/water/oil) system using a U-shaped cell. The transport rate and the preferred enantiomer of the esters were substantially affected by pH, temperature, and species of albumin. Among five serum albumins studied, bovine serum albumin (BSA) showed the largest rate constant, and rat serum albumin (RSA) manifested the highest enantio-selectivity. Regarding enantiomer selectivity in overall transport, it is anticipated that the ester uptake step plays an important role for BSA, whereas the ester release is the key step for RSA. 

Enantioselective Reductions. NaBH4 has been employed with less success than LiAULt or BH3 in enantioselective ketone reductions. Low to moderate ee values have been obtained in the asymmetric reduction of ketones with chiral phase-transfer catalysts, chiral crown ethers, -cyclodextrin, and bovine serum albumin. On the other hand, good results have been realized in the reduction of propiophenone with NaBH4 in the presence of isobutyric acid and of diisopropylidene-D-glucofuranose (ee = 85%), " or in the reduction of cr-keto esters and -keto esters with NaBHa-L-tartaric acid (ee >86%). ... 

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