Protein Stationary Phases

Table 1 Physical properties of proteins

Protein

Molecularmass Carbohydrate Isoelectric Origin (kDa) composition (%) point

Albumins BSA HSA

66 66

Bovine serum Human serum

Enzymes Trypsin a-Chymotrypsin

Pepsin

Lysozyme

25 34 14

10.1 Bovine pancreas

8.1-8.6 Bovine pancreas

< 1 Porcine stomach

10.5-11.0 Egg white

Glycoproteins arAcid glycoprotein 44

Cellobiohydrolase I 60

Ovoglycoprotein 30

Avidin 68

Ovotransferrin 77

Flavoprotein 32-36

45 6

Human or bovine serum

Fungus

Egg white

Egg white

Egg white

Egg white or yolk phases is that the eluent pH is limited to the ranges 2-8. However, in a strong acidic or alkaline solution, a protein sometimes suffers from denaturation. The separation of enantiomers on a protein-based stationary phase is generally attained using an eluent whose pH is between 3 and 8. Thus, the limitation of eluent pH ranges originated from silica-based materials is no problem for the use of protein-based stationary phases. Figure 1 shows the typical preparation method for protein-based HPLC chiral stationary phases; in part A the method includes activation of porous aminopropylsilica gels by N,N'-disuc-cinimidylcarbonate (DSC), binding of a protein and blocking of the activated amino groups. In this case, a side chain amino group(s) of a protein such as lysine and arginine and/or an N-terminal amino group could be used for binding the protein to the activated gel. On the other hand, using water-soluble carbo-diimide and N-hydroxysulfosuccinimide (HSSI), the carboxyl group of a protein can be bound to aminopropylsilica gels, as shown in Figure 1B.

Further, proteins can be bound to aminopropyl-silica gels using glutaraldehyde as a cross-linker, resulting in cross-linking by Schiff-base formation. The resulted imino functions are reduced by using sodium cyanoborohydride. Glycerylpropylsilica gels activated with 1,1'-carbonyldiimidazole have been used for the preparation of chiral stationary phases. According to the two methods described above, an amino group of a protein is used to bind to the derivatized silica gels. In addition, a protein can be physically adsorbed on to porous silica gels. The disadvantage of the adsorption method is that the adsorbed protein can be eluted, and it is better to bind a protein covalently to the base materials in order to avoid losses. The chiral recognition properties of a bound or adsorbed protein may be different from those of the protein in solution because of blocking of functional groups and/or conformational changes. The bound protein is often more stable to the changes of eluent pH and eluent composition compared to the protein in solution.

Retention and Enantioselectivity of Solutes on Protein-based Stationary Phases, and Optimization of Resolution

Table 2 shows the influence of eluent pH on the retention and enantioselectivity of various solutes on AGP-based chiral stationary phases. The retention factor of a basic solute, metoprolol, increased with an increase in the eluent pH. The decrease in the retention factor of an acidic solute, 2-phenoxypropionic acid, is ascribable to ion exclusion in addition to ionic repulsion between the carboxyl group of the 2-phenoxypropionic acid and the negatively charged AGP with an increase in eluent pH. The retention of basic solutes should be due to electrostatic interactions with the positively charged solutes and the negatively charged protein, in addition to hydrophobic interactions. Although the retention factor of an uncharged solute (ethotoin, hexobarbital) shows almost no pH dependence, a slight increase is observed with increasing eluent pH. This increase might be

Figure 1 Synthesis scheme for the preparation of protein-based stationary phases: (A) via an amino group of a protein; (B) via a carboxyl group of a protein.

due to changes in the binding properties of the pro- retention and enantioselectivity of various solutes tein resulting from conformational changes. Table 3 on AGP-based chiral stationary phases. With an inshows the influence of the 2-propanol content on crease in the 2-propanol content, the retention and

Table 2 Influence of eluent pH on retention of enantioselectivity of various solutes on AGP-based chiral stationary phase

Solute

pH4.

5

pH5.

5

pH6.

5

pH 7.5

k1

a

k1

a

k1

a

k1

a

2-Phenoxypropionic acid

8.55

1.59

1.77

1.57

0.32

1.48

Ethotoin

4.06

3.82

3.87

4.19

3.82

4.59

4.13

5.06

Metoprolol

0.40

1.25

2.20

1.29

9.23

1.42

22.5

1.48

Hexobarbital

9.39

1.44

9.47

1.47

10.3

1.66

11.6

2.10

Mobile phase, 0.01 mol L~1 phosphate buffer; k is the retention factor of the first eluted enantiomer; a is enantio separation factor = k2/k1, where k2 is the retention factor of the second eluted enantiomer. (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.)

Mobile phase, 0.01 mol L~1 phosphate buffer; k is the retention factor of the first eluted enantiomer; a is enantio separation factor = k2/k1, where k2 is the retention factor of the second eluted enantiomer. (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.)

Table 3 Influence of 2-propanol on the retentivity and enantioselectivity of various solutes on AGP-based chiral stationary phase

Disopyramide 8.51 3.70 3.62 3.37 1.77 3.20

Chlorpheniramine 11.2 2.34 7.35 1.71 4.59 1.38

Mepensolate 6.35 1.54 2.65 1.40 1.42 1.38 1.00 1.21

Mepivacaine 26.0 1.36 10.7 1.31 4.42 1.33 2.48 1.36 1.58 1.35

Bupivacaine 18.6 1.70 8.84 1.72 5.01 1.74

Mobile phase, 2-propanol in phosphate buffer, pH 7.2; k, retention factor of the first eluted enamtiomer; a, enantioseparation factor = k2/k1, where k2 is the retention factor of the second eluted enantiomer. (Reproduced with permission from Hermansson J (1989) Enantiomeric separation of drugs and related compounds based on their interaction with a1-acid glycoprotein. Trends In Analytical Chemistry 8: 251.)

enantioselectivity of solutes are decreased. These results suggest that hydrophobic and electrostatic interactions play an important role in the retention and enantioselectivity of racemic solutes on AGP-based columns. Further, the hydrogen bonding properties of the organic modifier influence enantioselectivity to a large extent. As shown in Figure 2, verapamil enan-tiomers are not resolved on the AGP-based column using 1-propanol as an organic modifier, but are resolved using acetonitrile.

Similar retentive and enantioselective properties are observed with other protein-based stationary

Figure 2 Influence of the nature of the organic modifier on the enantioselectivity of verapamil on an AGP-based column. HPLC conditions: column, Chiral-AGP (4.0 mm i.d.x100mm); eluent: (A) 10% acetonitrile in 0.01 mol L~1 phosphate buffer, pH 7.0; (B) 4% 1-propanol 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.

phases. As described above, hydrophobic, electrostatic and hydrogen bonding interactions play an important role in chiral recognition of solutes on these phases. Thus, enantioseparations of solutes can be optimized by changing eluent pH, and the type and content of the uncharged organic modifier. Sometimes, charged modifiers such as N,N-dimethyl-octylamine and octanoic acid are used for the enan-tioseparation of a charged solute. Figure 3 shows a scheme for the optimization procedure for ovomucoid-based stationary phases.

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