Chiral Bonded Ligand Exchanging Stationary Phases

The first chiral ligand exchanging polystyrene-type stationary phases were synthesized and tested by Rogozhin and Davankov as early as 1968, while analogous silica-bonded phases emerged a decade later. Even nowadays, cross-linked polystyrene resins remain popular in preparative chromatography, mainly due to their excellent chemical durability and high loading capacity. To prepare such chiral packings, spherical particles of styrene-divinylbenzene copolymers are usually subjected to chloromethyla-tion and the active chlorine atoms are then replaced by amino groups of chiral natural a-l-amino acids, in particular (S)-proline and (S)-hydroxyproline.

(According to modern stereochemical nomenclature, l- and d-a-amino acids belong to R and S configuration series, respectively, with a few exceptions, however.) The structure of a chiral selector immobilized in this way as well as its complex with a Cu(II) ion and an amino acid analyte, both of which bind to the selector during the process of LEC, can be represented by the following scheme:

) x = h, oh v______* = asymmetric carbon atom tate amino acid-type ligands, like histidine or allo-hydroxyproline, the opposite elution order of the enantiomers is observed, thus giving a values smaller than 1. Many other types of chiral selectors have been similarly immobilized on chloromethylated cross-linked polystyrene beads, with the result that cyclic amino acids as well as benzyl-substituted chiral pro-pan-1,2,-diamine display the highest selectivity and largest application range. Typical examples for resolving racemates of common a-amino acids on these phases are presented in Table 1.

Two additional polymeric matrixes, cross linked polyacrylamide and poly(glycidylmethacrylate), have also been shown to be useful for immobilizing chiral amino acid selectors. The mode of functionalizing of these resins, when they incorporate (S)-phenylalanine and (S)-proline as typical chiral selectors, can be represented by the following structures:

Here, the most important features of the ternary mixed-ligand bis(amino acidato)copper complex are:

• the formation of two five-membered chelate rings;

• the occupation of the four positions of the Cu(II) ion main coordination square by four electron-donating atoms of the ligands;

• the trans -arrangement of the two negatively charged carboxyl groups and the bi-substituted nitrogen atoms, respectively, which minimizes both the electrostatic and sterical repulsion of the ligands in the complex.

The resins incorporating (S)-proline and (S)-hy-droxyproline, when loaded with Cu(II) ions and eluted with ammonia solution, are usually found to retain a-amino acids of the opposite, (R)-configura-tion more effectively. In this case, the enantioselectiv-ity of the system, when expressed as the ratio of retention factors of the (R)- and (S)-enantiomers of the analyte, a = kR/kS, is larger than 1. With triden-

The first resin demonstrates higher affinity to (R)-isomers of all amino acids, without any exception, resolving all racemates with a selectivity of at least a = 1.3.

All the above polymeric ligand exchangers have been used successfully for both analytical and preparative resolution, e.g. for obtaining highly radioactive tritium-labelled optically active amino acids. In order to remove trace copper ions from enantiomers resolved in preparative experiments, the fractions of interest can be gravity filtered through a small additional column packed with silica, any chelating resin or even the same chiral resin, with a blue band of Cu(II) gradually forming at the top of the subsidiary column.

Silica-bonded chiral ligand exchangers were expected to demonstrate in analytical-scale separations still better column efficiency than polymer gels. Three types of bonded phases were suggested independently and simultaneously in 1979 by the groups of Foucault, Davankov and Guebitz, based on three different ways of activating the initial macroporous

Table 1 Enantioselectivity, a = kD/kL, of resolution of racemic amino acids on the Cu(II) forms of polystyrene resins which incorporate residues of L-proline, L-hydroxyproline, L-allo-hydroxyproline, L-azetidine carboxylic acid and A/1-benzyl-(fi)-propane-1,2-diamine. Mobile phases: aqueous ammonia solutions. Ambient temperature

Table 1 Enantioselectivity, a = kD/kL, of resolution of racemic amino acids on the Cu(II) forms of polystyrene resins which incorporate residues of L-proline, L-hydroxyproline, L-allo-hydroxyproline, L-azetidine carboxylic acid and A/1-benzyl-(fi)-propane-1,2-diamine. Mobile phases: aqueous ammonia solutions. Ambient temperature

Racemic amino acid

RPro

RHyp

RaHyp

RAzCA

RBzpn

Alanine

1.08

1.04

1.04

1.06

0.70

Aminobutyric acid

1.17

1.22

1.18

1.29

0.49

Norvaline

1.34

1.65

1.42

1.24

0.48

Norleucine

1.54

2.20

1.46

1.40

0.49

Valine

1.29

1.61

1.58

1.76

0.31

Isovaline

-

1.25

-

-

-

Leucine

1.27

1.70

1.54

1.24

0.49

Isoleucine

1.50

1.89

1.74

1.68

0.62

Serine

1.09

1.29

1.24

2.15

0.62

Threonine

1.38

1.52

1.48

0.78

0.44

Allothreonine

1.55

1.45

-

-

-

Homoserine

-

1.25

-

-

-

Methionine

1.04

1.22

1.52

1.29

0.60

Asparagine

1.18

1.17

1.20

1.44

0.70

Glutamine

1.20

1.50

1.40

1.25

-

Phenylglycine

1.67

2.22

1.78

1.38

0.49

Phenylalanine

1.61

2.89

3.10

1.86

0.52

a-Phenyl a-alanine

-

1.07

-

-

0.38

Tyrosine

2.46

2.23

2.36

1.78

-

Phenylserine

-

1.82

-

-

-

Proline

4.05

3.95

1.84

2.48

0.47

Hydroxyproline

3.85

3.17

1.63

2.25

-

Allo-hydroxyproline

0.43

0.61

1.48

1.46

-

Azetidine carboxylic acid

-

2.25

-

-

-

Ornithine

1.0

1.0

1.20

1.0

1.0

Lysine

1.10

1.22

1.33

1.06

1.0

Histidine

0.37

0.36

1.32

0.56

0.85

Tryptophan

1.40

1.77

1.10

1.13

-

Aspartic acid

0.91

1.0

0.81

0.88

1.0

Glutamic acid

0.62

0.82

0.69

0.77

-

acids, dipeptides, some ft-amino acids, etc. Amino alcohols do not form stable chelates with Cu(II) ions, but are easily resolved after conversion into Schiff bases or, better still, after reaction with bromoacetic acid, which converts the amino group into a chelating glycine moiety.

The silica-bonded phases, however, still require elevated temperatures (usually 50°C) to generate sufficient column plate numbers. In addition only neutral or weakly acidic aqueous-organic eluents, in particular, in the pH range between 6.0 and 4.0 can be applied. Therefore, a much more hydrolytically stable packing was introduced by multiple-point binding of chloromethylated polystyrene chains to the silica matrix, followed by substituting the active chlorine atoms of the polymer for chiral selectors. This type of polymer-bonded phase can safely be used at elevated temperatures, and display high resolving power towards numerous racemates, as exemplified in Figure 1. In LEC, the elevated temperature does not noticeably affect the enantioselectivity of the column.

microparticulate silica gel, namely, by first grafting silica with aminopropyl, chloroalkyl, and 3-glycidoxypropyl groups, respectively:

Whereas the aminopropyl spacer binds a chiral amino acid-type selector via the carboxyl group, the other two functions easily react with the amino group of the selector. The epoxy-activated and l-proline incorporating silica was the first chiral-bonded stationary phase on the market (Chiral ProCu, Serva, Heidelberg, Germany). Silica-bonded ligand exchangers have been used successfully to resolve racemates of numerous amino acids, hydroxy acids, N-dansyl-and N-alkyl-amino acids, a-trifiuoromethyl-a-amino

Figure 1 Enantiomeric resolution of a mixture of eight DL-amino acids with a chiral stationary phase, polystyrene grafted on silica gel and substituted with L-Hypro. Conditions: column, 250 x 4 mm i.d, dp 5 |im: eluent, 0.5 mM copper acetate, 10 mM ammonium acetate, pH 4.5, acetonitrile 30%; flow rate, 0.7 mL min~1; temperature, 75°C; UV 254 nm. (Reprinted with permission from Davankov VA (1989) Chapter 15, Figure 5, p. 464. In: Krstuloviz AM (ed.) Chiral Separations by HPLC. Applications to Pharmaceutical Compounds, pp. 446-475. Chichester, England: Ellis Horwood.)

Figure 1 Enantiomeric resolution of a mixture of eight DL-amino acids with a chiral stationary phase, polystyrene grafted on silica gel and substituted with L-Hypro. Conditions: column, 250 x 4 mm i.d, dp 5 |im: eluent, 0.5 mM copper acetate, 10 mM ammonium acetate, pH 4.5, acetonitrile 30%; flow rate, 0.7 mL min~1; temperature, 75°C; UV 254 nm. (Reprinted with permission from Davankov VA (1989) Chapter 15, Figure 5, p. 464. In: Krstuloviz AM (ed.) Chiral Separations by HPLC. Applications to Pharmaceutical Compounds, pp. 446-475. Chichester, England: Ellis Horwood.)

When dealing with complicated analyte matrixes, it is often useful to combine in sequence a chiral column with a conventional achiral one, in order to supplement the resolution of enantiomeric pairs in the chiral column with the selectivity towards different analytes of the second column. Thus, chiral ligand exchanging bonded phases are easily compatible with achiral reversed phase columns, since both columns can be operated in a gradient mode with aqueous-organic eluents which contain about 0.1 mM copper acetate and about 0.25 M ammonium acetate.

Several silica-bonded and polymeric chiral ligand exchangers are commercially available, e.g. Chiral-Si 100 L-ProCu, Chiral-Si 100 L-ValCu, Chiral-Si 100 L-HyProCu (Serva, Heidelberg, Germany), Nucleosil Chiral-1 (Macherey-Nagel, Dueren, Germany),

Chiralpak WH and WM (Daicel, Japan), TSK gel Enantio L1 (Toso, Japan), MCI gel CRS 10W (Japan), Chirosolve l-proline, Chirosolve l-valine (JPS Chimie, Switzerland). Any scientific evaluation of results obtained by using these phases is, however, complicated, since the exact structure of immobilized chiral ligands on many commercially available chiral stationary phases is not specified by the manufacturers.

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