Human serum albumin, stationary phase

Proteins. A chiral stationary phase with immobilized a -acid glycoprotein on silica beads was introduced by Hermansson in 1983 [18, 19]. Several other proteins such as chicken egg albumin (ovalbumin), human serum albumin, and cellohy-drolase were also used later for the preparation of commercial CSPs. Their selectivity is believed to occur as a result of excess of dispersive forces acting on the more retained enantiomer [17]. These separation media often exhibit only modest loading capacity. 

There is a wide variety of commercially available chiral stationary phases and mobile phase additives.32 34 Preparative scale separations have been performed on the gram scale.32 Many stationary phases are based on chiral polymers such as cellulose or methacrylate, proteins such as human serum albumin or acid glycoprotein, Pirkle-type phases (often based on amino acids), or cyclodextrins. A typical application of a Pirkle phase column was the use of a N-(3,5-dinitrobenzyl)-a-amino phosphonate to synthesize several functionalized chiral stationary phases to separate enantiomers of... 

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. 

T. A. G. Noctor and I. W. Wainer, The use of displacement chromatography to alter retention and enantioselectivity on a human serum albumin-based HPLC chiral stationary phase A mini review, J. Liquid Chromatogr., 16 183 (1993). 

Silica-base stationary phases have also been employed for enantiomeric separations in CEC [6,72-81]. In the initial work on chiral CEC, commercially available HPLC materials were utilized, including cyclodextrins [6,74,81] and protein-type selectors [73,75,80] such as human serum albumin [75] and ai-acid glycoprotein [73]. Fig. 4.9, for example, depicts the structure of a cyclodextrin-base stationary phase used in CEC and the separation of mephobarbital enantiomers by capillary LC and CEC in a capillary column packed with such a phase. The column operated in the CEC mode affords higher separation efficiency than in the capillary LC mode. Other enantiomeric selectors are also use in CEC, including the silica-linked or silica-coated macrocyclic antibiotics vancomycin [82,83] and teicoplanin [84], cyclodextrin-base polymer coated silicas [72,78], and weak anion-exchage type chiral phases [85]. Relatively high separation efficiency and excellent resolution for a variety of compounds have also been achieved using columns packed with naproxen-derived and Whelk-0 chiral stationary phases linked to 3 pm silica particles [79]. Fig. 4.10 shows the... 

Machtejevas E and Maruska A. A new approach to human serum albumin chiral stationary phase synthesis and its use in capillary liquid chromatography and capillary electrochromatography. J. Sep. Sci. 2002 25 1303-1309. 

T. A. G. Noctor, G. F61ix, and I. W. Warner, Stereochemical resolution of enantiomeric 2-aryl propionic acid non-steroidal anti-inflammatory drugs on a human serum albumin based high-performance liquid chromatography chiral stationary phase, Chtmiatogra ia, 31 55 (1991). 

E. Domenici, C. Bertucd, F Salvadori, and I. W. Wainer, Use of a human serum albumin-based chiral stationary phase for high performance liquid chromatography for the investigation of protein binding Detection of the allosteric interaction between warfarin and benzodiazepine binding sites, /. Pharm. Set., 80 164 (1991). 

I. W. Wainer, Synthesis and chromatographic properties of an HPLC chiral stationary phase based upon immobilized human serum albumin, Oirrnia-tograjitia, 29 171 (1990). 

Andrisano, V. Bertucci, C. Cavrini, V. Recatini, M. Cavalh, A. Veroli, L. Fehx, G. Wainer, I.W. Stereoselective binding of 2,3-suhstituted 3-hyrox3fpropionic acids on an immobilized human serum albumin chiral stationary phase. Stereo-chemical characterisation and quantitative structure-retention relationship study. J. Chromatogr., A 2000, 876, 75-86. 

Kaliszan, R., Noctor, T.A. and Wainer, I.W. (1992b). Quantitative Structure-Enantioselective Retention Relationships for the Chromatography of 1,4 Benzodiazepines on a Human Serum Albumin Based HPLC Chiral Stationary Phase An Approach to the Computational Prediction of Retention and Enantioselectivity. Chromatographia, 33,546-550. 

Domenici, E., Bertucci, C., Salvador , P., Felix, G., Cahagne, I., Motellier, S., Wainer, I. W. Synthesis and chromatographic properties of an HPLC chiral stationary phase based upon human serum albumin, Chromatographia, 1990, 29, 170-176. 

Kaliszan, R Noctor, TA. and Wainer, I.W. (1992) Quantitative structure-enantioselective retention relationships for the chromatography of 1,4-benzodiazepines on a human serum albumin based HPLC chiral stationary phase an approach to the computational prediction of retention and enantioselectivity. Chromatographia, 33, 546-550. 

Hayball, P.J. Holman, J.W. Nation, R.L. Influence of octanoic acid on the reversible protein binding of ketorolac enantiomers to human serum albumin (HSA) comparative liquid chromatographic studies using a HSA chiral stationary phase. J.ChromatognB, 1994, 662, 128-133 [chiral]... 

There are three commonly used protein based LC chiral stationary phases, the ttj-acid glycoprotein phase (CHIRAL-AGP), cellobio-hydrolase (CHIRAL-CBH) and human serum albumin (CHIRAL-... 

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... 

Fig. 3 Typical plots of (a) total plate height (//tot) and (b) the plate height contribution due to stationary phase mass transfer (//s) for injections of D-tryptophan at various flow rates onto an immobilized human serum albumin column. Symbols u, linear velocity kf, retention factor.

Fig. 9 Separations of five standard protein samples by mixer-settler HSCCC. Experimental conditions are as follows apparatus type-J coil planet centrifuge with 10 cm revolution radius column a mixer-settler spiral disk assembly consisting of eight barricaded disks with a 160 ml capacity A, solvent system 12.5% (w/w) PEG-1000 and 12.5% (w/w) K2HPO4 mobile phase lower phase sample five proteins each 5-6 mg in 1 ml of each phase, cytochrome c (K = 0.02), myoglobin (K = 0.59), ovalbumin (K = 1.26), lysozyme (K = 1.69), and bovine serum albumin (K = 1.95) flow rate 0.25 ml/min rpm 800 detection 280 nm stationary phase retention 52% B, solvent system PEGIOOO/K2HPO4/KH2PO4/H2O (16 8.3 4.2 71.5, w/w) sample cytochrome c (5 mg, K = 0.035), human serum albumin (20 mg, K = 0.4), 3-lactoglobulin (20 mg, K = 0.69), a-chymotrypsin (20 mg, K = 1.2), and trypsinogen (20 mg, = 2.1) in 2 ml of each phase flow rate 0.5 ml/min rpm 1000 stationary phase retention 53.6%.

Vandenbosch C, Massart D, Lindner W (1992) Evaluation of six chiral stationary phases in LC for their selectivity towards drug enantiomers. J Pharm Biomed Anal 10 895-908 Sugio S, Kashima A, Mochizuki S, Noda M, Kobayashi K (1999) Crystal stmcture of human serum albumin at 2.5 A resolution. Protein Eng 12 439-446... 

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