Protein-based CSPs

In the following studies, the same computational steps have been used in a straightforward manner as before to compare pairs of CSP. The molecule datasets employed in these studies are the same as that used above. In addition, two protein-based CSP were also compared ... 

Armstrong et al. ° first introduced chiral stationary phases based on macrocyclic antibiotics. Vancomycin, ristocetin A, teicoplanin, avoparcin, rifamycin B and thiostrepton are used as chiral selectors. They posses a broad enantiorecognition range, similar to protein based CSPs. However, CSPs based on macrocyclic antibiotics show higher stability and capacities.Underivatized amino acids, N-derivatized amino-acids, acidic compounds, neutrals, amides, esters and amines can be separated.The first four of the above-mentioned chiral selectors appear to have the largest enantiorecognition range.The selectors can also be derivatized to obtain different enantioselectivities. 

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... 

An interesting extension of enantioselective chromatography on protein based CSPs in LC has been made15-17 using proteins to show qualitative and quantitative aspects of ligand-biopolymer interactions this includes chiral aspects. 

Protein based CSPs and chiral columns14 operate in reversed-phase mode and have found extensive use for analytical scale resolutions of chiral drugs and other chemicals. Protolytic... 

TABLE 2 Enantiomeric Resolution of Different Racemic Compounds Using Protein-Based CSPs... 

One of the advantages of protein-based CSPs is that chiral chromatography is carried out under the reversed-phase mode that is, aqueous mobile phases are used frequently and, therefore, there is a great chance to optimize the chiral resolution. The most important parameters to be optimized are the composition of... 

The degree of separation of the two enantiomers obviously plays an important part in the CSP selection. Another equally important parameter is the loading capacity of the stationary phase. The higher the loading capacity, the greater the amount of material that can be separated (44). For example the polysaccharide-based CSPs have a saturation capacity of 5-100 mg/g of CSP this is clearly dependent on the type of racemate that is be-ingresolved. On the other hand, protein-based CSPs have lower saturation capacities, of the order 0.1-0.2 mg/g of CSP. 

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... 

As with the other protein-based CSPs, initial studies indicate a relationship between the structure of the protein and chromatographic properties of the OVM CSP (101). When the sialic acid residues, were enzymatically removed from the protein, the capadty factors (k ) of the enantiomers of an addic solute (ketoprofen) were reduced, whereas the k s of the enantiomers of a basic solute (chlorpheniramine) were un-... 

The type-I sites have, in protein based CSPs, identical behavior toward the two enantiomers, and cannot distinguish between them. Many columns contain mostly type-I sites. On type-I sites all possible molecular interactions, between the analyte molecules and atoms or groups of atoms belonging to the adsorbent surface, take place. These interactions can originate from the nonchiral parts of the protein and/or from the adsorbent (silica) matrix. The energies of each interaction on type-I sites are small. The other type of adsorption sites have, in protein based CSPs, much higher adsorption energy and are enantioselective (chiral). These sites, type-II sites, are responsible for the enantiomeric separations. On most CSPs the type-II sites are relatively few. 

Proteins such as bovine serum albumin, immobilized to silica, achieve enantiomer separation primarily via hydrophobic and electrostatic interactions. Although the protein-based CSP columns have low capacity and preparative use is impossible, these phases offer the analyst the convenience of being able to resolve a broad spectrum of analytes with a single column. 

Some enzymes are well known to show high chiral recognitions and have been used as asymmetric catalysts in organic synthesis. A few proteins, such as bovine serum albumin, a i-acid glycoprotein, and ovomucoid, have also been used as CSPs for HPLC [32-34]. These CSPs can resolve many chiral drugs. Protein-based CSPs are often not stable because they change their conformation depending on the conditions such as solvents and temperature. The proteins contain many different... 

The loading capability of a CSP is related to the available number of selector-analyte interaction sites. For example, protein-based CSPs are very vulnerable to overloading. Due to the high molecular mass of protein selectors, the number of sites available in a protein column is very limited. Since protein selectors generally interact enantioselectively with only one analyte at a time, there is no way... 

It is true that the unambiguous elucidation of chiral recognition mechanisms on various protein-based CSPs is challenging and often difficult since precise information about the tertiary and quaternary stmctures of proteins is not always available. Multiple stereo-specific sites may be involved in chiral recognition process. However, it is encouraging to see the progresses that have been made in this field in recent years [17, 95—102]. 

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