Chiral selectors proteins used

Capillary electrophoresis employing chiral selectors has been shown to be a useful analytical method to separate enantiomers. Conventionally, instrumental chiral separations have been achieved by gas chromatography and by high performance liquid chromatography.127 In recent years, there has been considerable activity in the separation and characterization of racemic pharmaceuticals by high performance capillary electrophoresis, with particular interest paid to using this technique in modem pharmaceutical analytical laboratories.128 130 The most frequently used chiral selectors in CE are cyclodextrins, crown ethers, chiral surfactants, bile acids, and protein-filled... 

Different classifications for the chiral CSPs have been described. They are based on the chemical structure of the chiral selectors and on the chiral recognition mechanism involved. In this chapter we will use a classification based mainly on the chemical structure of the selectors. The selectors are classified in three groups (i) CSPs with low-molecular-weight selectors, such as Pirkle type CSPs, ionic and ligand exchange CSPs, (ii) CSPs with macrocyclic selectors, such as CDs, crown-ethers and macrocyclic antibiotics, and (iii) CSPs with macromolecular selectors, such as polysaccharides, synthetic polymers, molecular imprinted polymers and proteins. These different types of CSPs, frequently used for the analysis of chiral pharmaceuticals, are discussed in more detail later. 

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. 

TABLE 9 Examples of the Chiral Separation of Drugs Using Proteins as Chiral Selectors... 

An extremely important aspect in pharmaceutical research is the determination of drug optical purity. The most frequently applied technique for chiral separations in CZE remains the so-called dynamic mode where resolution of enantiomers is carried out by adding a chiral selector directly into the BGE for in situ formation of diastereomeric derivatives. Various additives, such as cyclodextrins (CD), chiral crown ethers, proteins, antibiotics, bile salts, chiral micelles, and ergot alkaloids, are reported as chiral selectors in the literature, but CDs are by far the selectors most widely used in chiral CE. 

Brush-type, proteins, CDs, natural molecular imprint-based polymers (MIP), and macrocyclic antibiotics have been immobilized as chiral selectors on packed-CEC columns. Zheng and Shamsi demonstrated the possibility of using chiral CEC—ESI/MS with a commercially packed column for the determination of warfarin enantiomers in human plasma using coumachlor as an internal standard (IS). Robustness of this chiral CEC capillary was recently improved by a novel procedure and applied for the simultaneous enantiosepara-tion of height /1-blockers with multimodal CSP using different combinations of vancomycin and teicoplanin, as presented in Figure 5. ... 

HSA and BSA were used as chiral selectors to separate ofloxazin, propranolol, and verapamil enantiomers. With ketoprofen and warfarin as displacers, preferred binding sites at both proteins were discussed. 

J Haginaka. Enantiomer separation of drugs by capillary electrophoresis using proteins as chiral selectors. J Chromatogr A 875 235-254, 2000. 

Although all proteins are complex in structure and chiral in nature, some of them could achieve the status of a chiral selector in liquid chromatography. The complex structures of proteins are the result of the different intramolecular hydrogen-bonding, disulfide bridges, and other types of bonding. All of the proteins used for chiral resolution in liquid chromatography are obtained from animals except for cellobiohydrolase-I. The structures and properties of some of the most commonly used proteins as chiral selectors are discussed herein. 

Ovotransferrin is also obtained from the white portion of a chicken egg and has been used as a chiral selector in liquid chromatography. This protein is also called conalbumin. It is a metal ion (iron, copper, manganese, and zinc) binding protein of molecular mass 70,000-78,000 and with an isoelectric point of 6.1-6.6. This protein is sensitive to acids and heat. 

This protein is extracted from pancreatic tissues. This protein occurs in a-form and /1-form, but the a-form is used as chiral selector in liquid chromatography. The molecular mass is 25,000, with an isoelectric point of 8.1-8.6. It is inhibited by metal ions. The protein is useful for chiral resolution of amino acids and amino esters. 

As discussed earlier, the proteins used as chiral selectors in affinity chromatography cannot be used under the high-pressure HPLC with a variety of mobile phases therefore, these proteins were immobilized with some solid support such as hydroxyethylmethacrylate, polystyrene-divinylbenzene, polyethylene fibers, and silica gel [14,15]. Avariety of techniques have been used for the immobiliza-... 

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