Enzymebased Stationary Phase

Trypsin and a-chymotrypsin are a family of serine proteases. Trypsin-based stationary phases can resolve O-, N,O-derivatized amino acids which are substrates of the enzyme. This means that chiral separations are due to the activity of the enzyme, and that the chiral recognition site is on the enzyme activity site. a-Chymotrypsin stationary phases can resolve amino acids and amino acid derivatives.

When the eluent of pH 7 is continuously delivered, the pepsin-based stationary phases lose their chiral recognition properties. This result reveals that the immobilized pepsin is irreversibly denatured above pH 7. Thus, the use of an eluent with pH less than 6 is

Figure 5 Chromatograms of lorazepam enantiomers on (A) BSA-based and (B) BSA-fragment-based columns. HPLC conditions: column, 2.1 mm i.d. x 100 mm; eluent, 50 mmol L~1 phosphate buffer (pH 7.5) containing 4% 1-propanol; flow rate, 0.2 mL min~1. (Reproduced with permission from Haginaka J and Kanasugi K (1995) Enantioselectivity of bovine serum albumin-bonded columns produced with isolated protein fragments. Jour-nalofChromatographyA 694: 71.)

Figure 6 Enantioseparations of (A) homochlorcyclizine, (B) verapamil, (C) alprenolol and (D) oxazepam on a pepsin-based column. HPLC conditions: column, 4.6 mm i.d. x 100 mm; eluent, 20mmolL~1 phosphate buffer (pH 5.1) containing (A) 5 and (B) 10% acetonitrile and (C) and (D) 5% ethanol; flow rate, 0.8mLmin~1. (Reproduced with permission from Haginaka J, Miyano Y, Saizen Y et al. (1995) Separation of enantiomers on a pepsin-bonded column. Journal of Chromatography A 708: 161.)

Figure 5 Chromatograms of lorazepam enantiomers on (A) BSA-based and (B) BSA-fragment-based columns. HPLC conditions: column, 2.1 mm i.d. x 100 mm; eluent, 50 mmol L~1 phosphate buffer (pH 7.5) containing 4% 1-propanol; flow rate, 0.2 mL min~1. (Reproduced with permission from Haginaka J and Kanasugi K (1995) Enantioselectivity of bovine serum albumin-bonded columns produced with isolated protein fragments. Jour-nalofChromatographyA 694: 71.)

Figure 6 Enantioseparations of (A) homochlorcyclizine, (B) verapamil, (C) alprenolol and (D) oxazepam on a pepsin-based column. HPLC conditions: column, 4.6 mm i.d. x 100 mm; eluent, 20mmolL~1 phosphate buffer (pH 5.1) containing (A) 5 and (B) 10% acetonitrile and (C) and (D) 5% ethanol; flow rate, 0.8mLmin~1. (Reproduced with permission from Haginaka J, Miyano Y, Saizen Y et al. (1995) Separation of enantiomers on a pepsin-bonded column. Journal of Chromatography A 708: 161.)

recommended. By using a mixture of phosphate buffer and organic modifier as an eluent, cationic (especially ft-blocking agents) and neutral enantiomers are resolved, while no resolution of acidic enantiomers is observed. Figure 6 shows enantioseparations of

Figure 7 Enantioseparations of (A) metoprolol and (B) pindolol on an AGP-based column. HPLC conditions: column, Chiral-AGP (4.0 mm i.d. x 100 mm); eluent (A) 3.8% ethanol in 0.01 mol L~1 phosphate buffer, pH 7.0; (B) 15% methanol in 0.01 mol L~1 phosphate buffer, pH 7.0. (Reproduced with permission from Hermansson J (1989) Enantiomeric separation of drugs and related compounds based on their interaction with aracid glycoprotein. Trends in Analytical Chemistry 8: 251.)

Figure 7 Enantioseparations of (A) metoprolol and (B) pindolol on an AGP-based column. HPLC conditions: column, Chiral-AGP (4.0 mm i.d. x 100 mm); eluent (A) 3.8% ethanol in 0.01 mol L~1 phosphate buffer, pH 7.0; (B) 15% methanol in 0.01 mol L~1 phosphate buffer, pH 7.0. (Reproduced with permission from Hermansson J (1989) Enantiomeric separation of drugs and related compounds based on their interaction with aracid glycoprotein. Trends in Analytical Chemistry 8: 251.)

homochlorcyclizine, verapamil, alprenolol and oxazepam on a pepsin-based column. Also, lysozyme-based stationary phases showed chiral recognition ability to cationic and neutral solutes but no resolution of anionic solutes.

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