Ovomucoid separation

Proteias, amino acids bonded through peptide linkages to form macromolecular biopolymers, used as chiral stationary phases for hplc iaclude bovine and human semm albumin, a -acid glycoproteia, ovomucoid, avidin, and ceUobiohydrolase. The bovine semm albumin column is marketed under the name Resolvosil and can be obtained from Phenomenex. The human semm albumin column can be obtained from Alltech Associates, Advanced Separation Technologies, Inc., and J. T. Baker. The a -acid glycoproteia and ceUobiohydrolase can be obtained from Advanced Separation Technologies, Inc. or J. T. Baker, Inc. 

Proteins present in whole egg, yolk, and albumen (egg white) are excellent sources of nutrients, and they possess valuable functional properties. Shell eggs consist of 8-11% shell, 56-61% albumen, and 27-32% yolk. The solid content of albumen is about 11-13%, depending on the strain and on the age of the hens, and the solid content of yolk is about 52-53.5%. Albumen solids contain mainly protein, whereas lipids are the major constituents of egg yolk (92). Yolk can be separated by centrifugation into sedimented granules and a supernatant, plasma. The granules contain the major part of the yolk proteins. The main proteins in albumen and yolk are ovalbumin, ovotransferin, lysozyme, ovomucoid, ovomucin, and immunoglobulin Y (93). 

Two HPLC methods for the determination of enantiomers of donepezil HC1 in rat plasma have been developed [36].The first method involves chiral separation of donepezil HC1 on an ovomucoid-bonded column, and native fluorescence detection of donepezil HC1 with excitation at 318 nm and emission at 390 nm. The fluorometric detection is without interference from background components and is about five times more sensitive than UV detection at 271 nm. The method was applied to monitoring the racemization of each enantiomer of donepezil HC1 in buffer solutions and in rat plasma. The second method involves separation of donepezil HC1 from background components of rat plasma on an a chiral column, collection of the donepezil HC1 fraction into a sample loop, concentration to a trap column, transfer of donepezil HC1 to a chiral column, resolution of the enantiomers of donepezil HC1 on the chiral column, and fluorometric detection of the enantiomers of donepezil HC1 with excitation at 318 nm and emission at 390 nm. The detection limits of donepezil HC1 and each enantiomer of donepezil HC1 were 1 ng/ml, respectively, with a 200 (A injection of deproteinized plasma samples. 

Balmer et al. [60] separated the two enantiomers of omeprazole on three different stationary phases with immobilized protein, viz, Chiral-AGP with a-1 acid glycoprotein, Ultron ES-OVM with ovomucoid, and BSA-DSC with BSA cross-linked into 3-aminopropyl silica using N-suc-cinimidyl carbonate. The mobile phase (1 ml/min) was phosphate buffer solution with 3—10% 2-propanol as the organic modifier. The enantiomers of omeprazole were separated on Chiralpak AD, an amylose-based chiral stationary phase, with ethanol-hexane (1 4) as mobile phase (1 ml/min). 

J. Haginaka, J. Wakai, K. Takahashi, H. Yasuda, and T. Katagi, Chiral separation of propranolol and its ester derivatives in ovomucoid-bonded silica Influence of pH, ionic strenght and organic modifier on retention, enantioselectivity and enantiomeric elution order, Chromatographia, 29 581 (1990). 

K. M. Kirkland, K. L. Neilson, D. A. Me Combs, and J. J. DeStef-ano, Optimized HPLC separations of racemic drugs using ovomucoid protein-based chiral column, LC-GC, 10 322 (1992). 

Since ovomucoid is a glycoprotein the solutes that can be stereo-chemically resolved on the OVM CSP are similar to the basic, neutral, and acidic compounds separated on the AGP CSE The major differences between the two CSPs appears to be quantitative i.e the magnitude of the chiral separation), rather than qualitative. The two CSPs have been recently compared and a representative series of successfully resolved solutes is contained in this article (102). 

It is well known in biochemistry that many proteins, especially enzymes but also transport proteins such as albumin, show high enantioselectivities in their interactions with small chiral molecules. It is possible to bind proteins to silica and to obtain a valuable class of CSPs that is mainly suited for the separation of chiral drugs. Several protein phases are commercially available albumins, a-acid glycoprotein, ovomucoid, avidin, cellobiohydrolase I, and pepsin. They differ in their chromatographic and enantioselective properties which is not a surprise because their biological functions and their size, shape or isoelectric point are not identical at all. 

Duck ovomucoid inhibits two trypsins simultaneously. It was not possible to separate the fast and intermediate amino groups. 

Rosell, G. Camacho, A Parra, P. Direct enantiomeric separation of cts-( )diltiazem in plasma by high-performance liquid chromatography with ovomucoid column. J.Chromatogr., 1993, 619, 87-92... 

Fujima, H. Wada, H. Miwa, T. Haginaka, J. Chiral separation of lorazepam on ovomucoid-bonded columns Peak coalescence due to racemization. J.Liq.Chromatogr., 1993, 16, 879-891 [chiral column temp 7]... 

The distinct nature of these multiple binding sites are shown by (a) the simultaneous binding of more than one mol/mol of a given protease or of two or more different proteases (b) amino acid sequence work (69) and (c) by fragmentation of the inhibitor molecule into separate, active parts Cyanogen bromide has been used effectively to cleave the inhibitor at methionine residues to give active fragments. Examples include the Bowman-Birk soybean inhibitor (73), turkey ovomucoid (86), the three isoinhibitors of Brazilian pink beans (77), potato inhibitor Ila (60), and potato inhibitor Ilb (62) ... 

Tamai, G. Edani, M. Imai, H. Chiral separation and determination of propranolol enantiomers in rat or mouse blood and tissue by column switching high performance liquid chromatography with ovomucoid bonded stationary phase. Biomed. Chromatogr. 1990, 4, 157-160. 

Protein encapsulation has also been used to prepare sol-gel columns for CEC separations [7]. This involved incorporating a protein that has a chiral center such as bovine semm albumin or ovomucoid. Because these proteins have a chiral center, it is possible to use the hybrid protein/sol-gel column to separate enantiomers. In these separations, the amount of protein that... 

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