BSA CSP

The binding interactions between the solute and protein usually involves stereospedfic and nonstereospecific mechanisms. These mechanisms make the type V CSPs sensitive to the composition of the mobile phase, temperature, flow rate, and pH. These parameters can be adjusted to improve the chromatography and stereoselectivity of specific solutes on the AGP CSP (88,89), OVM CSP (90,91), BSA CSP (92,93), and HSA CSP (94). 

Human serum albumin and bovine serum albumin are closely related proteins and, consequently, the chromatographic properties of the CSPs based on these proteins are similar. The only difference between the two phases appears to be due to inherent differences in stereoselectivity between HSA and BSA. For example, on the HSA-CSP (S)-warfarin elutes before (K)-warfarin, whereas on the BSA CSP the opposite elution order is observed (85). This is consistent with the enantioselectivities of the native proteins (106). However, even though there are differences between the CSPs, the selectivity, mobile-phase effects, and chromatographic properties of the HSA CSP and BSA CSP are so similar that the two phases will be discussed together. 

Amim acids. Some aromatic amino acids such as kynurenin can be resolved on the BSA CSP without derivatization (92). However, most amino acids require precolumn derivatization of the amine moiety. The N-deriva-tives that have been used include acetyl, benzenesulphonyl, phthalimido, DANSYL, 2,4-dinitrophenyl, and 2,4,6-trinitrophenyl (85,92,107,108). 

Alcohols as mobile-phase modifiers. As with the other type V CSPs, the addition of an alcohol to the mobile phase appears to reduce the hydrophobic interactions between the solute and SA CSP, which results in lower k values and reduced a s(9-ll). An example of this phenomenon are the effects of ethanol, 1-propanol, and 1-butanoI on the retention and enantioselective resolution of N-benzoyI-D,L,alanine on the BSA CSP (111). The addition to a mobile phase composed of phosphate buffer (50 mM, pH 7.0) of 2% (v/v) of ethanol reduced the k of the last eluting enantiomer by 33% and the observed a by 6%. When ethanol was replaced by 1-propanol, the observed reductions were 67 and 40%, respectively, and when 1-butanol was the modifier, the observed reductions were 80 and 77%, respectively. In practice, 1-propanol appears to be the most commonly used alcoholic modifier. 

The purpose of this study is only intended to illustrate and evaluate the decision tree approach for CSP prediction using as attributes the 166 molecular keys publicly available in ISIS. This assay was carried out a CHIRBASE file of 3000 molecular structures corresponding to a list of samples resolved with an a value superior to 1.8. For each solute, we have picked in CHIRBASE the traded CSP providing the highest enantioselectivity. This procedure leads to a total selection of 18 CSPs commercially available under the following names Chiralpak AD [28], Chiral-AGP [40], Chiralpak AS [28], Resolvosil BSA-7 [41], Chiral-CBH [40], CTA-I (microcrystalline cellulose triacetate) [42], Chirobiotic T [43], Crownpak CR(-i-) [28], Cyclobond I [43], DNB-Leucine covalent [29], DNB-Phenylglycine covalent [29], Chiralcel OB [28], Chiralcel OD [28], Chiralcel OJ [28], Chiralpak OT(-i-) [28], Ultron-ES-OVM [44], Whelk-0 1 [29], (/ ,/ )-(3-Gem 1 [29]. 

The ability of proteins to form enantioselective interactions with a large variety of drugs is used in chiral affinity chromatography. Protein CSPs that are most frequently used for the enantioseparation of pharmaceuticals include bovine serum albumin (BSA), human serum albumin... 

The use of protein immobilised to the surface of a silica gel or to another support has been a very successful approach for the chiral separation of various pharmaceuticals. The AGP stationary phase has been shown to have the broadest enantiorecognition abilities while the BSA stationary phase is especially useful for aromatic compounds. " Table 9 shows some examples of separations that were obtained on the protein-type of CSPs. 

In 1973, Stewart and Doherty [9] resolved enantiomers of tryptophan on a column packed with BSA-succinoylaminoethyl-agarose in a discontinuous elution procedure. The mobile phase used was 0.1 M borate buffer (pH 9.2). The chromatograms of this classical research are shown in Figure 2. Several years later, this technique was applied for the chiral resolution of warfarin enantiomers [10]. In 1981, the enantiomers of tryptophan and warfarin racemates were resolved on various serum albumin CSPs [11,21,22]. The same method was used for the resolution of other drugs [12-14]. Allenmark et al. [23] studied the resolution of a series of active racemic sulfoxides on a BSA column using 0.08 M phosphate buffer (pH 5.8) as the eluting solvent. 

FIGURE 8 Effect of buffer (ionic) strength on enantioresolution of (a) enantiomers of C6H5C0NHCHRC02H, where R is ( ) -CH2OH, ( ) -CH3 and ( ) -CH3 on the BSA-based CSP, (b) iV-benzoyl alanine on BSA-based CSP, and (c) ketoprofen on avidin CSP with phosphate buffer as the main component of the mobile phases in all of the studies. (From Refs. 28, 32, and 70.)... 

As in case of other CSPs, the chiral resolution is also effected on protein phases by the structures of the racemic compounds. Allenmark and co-workers [23] resolved several sulfoxides on the BSA-based CSP. These sulfoxides contain different structures with various groups such as methyl, methoxy, and acetate. We analyzed the results of this study and, generally, it was found that the separation factor decreased by introducing larger groups in the sulfoxides. This behavior may be the result of a steric effect the author did not consider in the discussion... 

TABLE 3 Effect of Substituents on the Chiral Resolution of Barbiturates (Fig. 11) on the BSA-Based CSP Using Phosphate Buffer (50mM, pH 5.8)-l-Propanol (94 6, v/v) as the Mobile... 

Figure 10.3 Electrophysiological response of the CHC sensillum to nestmate or non-nestmate CHCs with or without CjapCSP. Left column Recordings to stimulus solutions of 10 mM NaCI, 10 mM NaCI plus CSP and 10 mM NaCI plus bovine serum albumin (BSA). Middle column Recordings to non-nestmate CHCs dissolved in the same stimulus solutions. Right column Recordings to nestmate CHCs dissolved in the same stimulus solutions.

A number of proteins are commercially available as CSPs including a-acid glycoproteins (AGP, the major plasma binding protein for basic drugs), human serum albumin (HSA, the major plasma binding protein for weakly acidic drugs), bovine serum albumin (BSA), ovomucoid (OVM), and cellobiohydro-lase (CBH) [12]. The proteins are bonded to silica and utilized in reversed-phase mode with an aqueous buffer/organic modifier eluent. Mobile-phase... 

The use of a convective macroporous polymer as an alternative support material instead of silica for the preparation of protein-based CSPs has successfully been demonstrated by Hofstetter et al. [221]. Enantioseparation was performed using a polymeric flow-through-type chromatographic support (POROS-EP, 20 pm polymer particles with epoxy functionalities) and covalently bound BSA as chiral SO. Using flow rates of up to 10 ml/min, rapid enantiomer separation of acidic compounds, including a variety of amino acid derivatives and drugs, could be achieved within a few minutes at medium efficiencies, typical for protein chiral stationary phases (Fig. 9.13). 

The ability of proteins to stereoselectively bind small molecules has been used to develop a series of commerdally available protein-based CSPs (the type V HPLC CSPs), including phases that contain immobilized AGP (84), HSA (85), BSA (86), and ovomucoid (OVM) (87) (see Table 5). All these CSPs are useful in the HPLC resolution of enantiomeric compounds and appear to have an extremely wide range of applications, and the AGP CSP seems to have the broadest utility of any of the current CSPs (9-11). However, although the type V CSPs display high enantioselectivities, they also have low capadties due to the relatively small amounts of the chiral selector that can be immobilized per g silica. Thus, these CSPs are useful... 

CSPs incorporating BSA have been successfully employed to separate the enantiomers of acidic and neutral compounds [161-163], such as N-acylated amino acids, aromatic amino acids and sulfoxides. Retention and enantioselectivity can be optimized by adjusting pH, ionic strength and organic modifier content of the mobile phase. 

HSA bears structural and functional resemblance to BSA, and HSA-type CSPs [164] also show similar enantioselective binding preferences for acidic and neutral drug molecules, such as 2-aryloxypropanoic acids [165], warfarin [166] and benzodiazepines [167]. The chiral recognition mechanism of HSA has been the subject of a number of investigations [168], which revealed that enantioselective binding occurs primarily at two well-defined hydrophobic sites. Acidic drugs have been shown to bind preferentially to the so-called warfarin-azapropazone (site I) and neutral drugs to the indol-benzodiazepine site (site II). 

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