Type IV CSPS

Bazylak [80] compared the resolution of underivatized primary and secondary amino alcohols by reverse phase HPLC. He used nickel(ll) chelates, 35, as a mobile phase additive. The coordination complexes prepared are depicted below where the substituents at stereocenters z and q were varied. 

Another report on ion-pair chromatography, where computed molecular descriptors from molecular modeling were nicely correlated with experimental separation factors, was published by Karlsson, Luthman, Pettersson and Hacksell [81]. They examined factors responsible for separation of aminotetralins on achiral stationary phases in the presence of the chiral additive N-benzyloxycarbonylglycyl-L-proline (L-ZGP), a protected peptide derivative. 

Using the MMX force field these authors first determine the distribution of conformational states accessible to the analytes and then evaluated the preferred conformations of L-ZGP in the neutral and ionic forms. Then the authors brought these components together to form the various diastereomeric complexes. Their 

Strategy was to implement a flexible docking scheme because it was felt that both molecules of the complex may change their conformations during association. 

These computational studies are comparable to those described in the section covering Type I CSPs. Experimentally the only difference between these separations and those above is that here the selectors are not stationary phases but rather are co-additives that form the diastereomeric complexes. Because no computational studies on type IV CSPs exist, molecular modeling of inorganic coordination complexes directed towards rationalizing enantioselective binding and chiral recognition presents itself as a ripe area for exploration. 

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Figure 1.12). It is beyond the scope of the present review article to discuss all of the findings of these studies in detail. However, in general, it can be stated that also these CSPs may reveal valuable complementary selectivity profiles for particular classes of chiral solutes. This may apply to an even greater extent to CSPs based on Sharpless phthalazine-derived cinchona alkaloids (see Figure 1.9) (type IV CSPs bottom) [38,59] and mutants thereof (N.M. Maier and W. Lindner, in preparation). They partly showed exceptionally high enantioselectivities for some specific applications (vide infra). 

Type IV CSPs are proteins immobilized primarily on silica. The sol-ute-CSP complexes are mainly due to ionic and hydrophobic interactions... 

The solutes resolved by the Type IV CSPs must be able to form coordination complexes with transition metal ions. This limits the classes of compounds to a-amino acids and mono- and dicarboxylic acids containing an ot hydroxyl moiety. The most common solutes resolved on these CSPs are underivatized amino acids, although a number of derivatized amino acids can be resolved, including W-acetyl, amide, N-carbamoyl, N-carbo-benzoxy, and hydantoin derivatives, as well as complex amino adds such as the biochemical modulator BSD (79). 

The Type IV CSPs are used with aqueous mobile phases that contain copper(II) sulfate (CS). The usual starting concentration of CS is 0.25 mM and retention times can be manipulated by increasing or decreasing the CS concentration an increase in CS concentration usually shortens retention, whereas a decrease has the opposite effect. Concentrations as low as 0.05 mM and as high as 20.0 mM have been used. 

Chromatographic retention on a type IV CSP can be manipulated by altering the pH of the mobile phase, adding mobile-phase modifiers, or changing the temperature. Examples of these effects are the following (1) The pH of the mobile phase can be varied between 3 and 7, and the lower the pH, the shorter the chromatographic retention. (2) The addition of methanol and acetonitrile usually increases retention, whereas sodium chloride has the opposite effect. (3) The chromatography is usually carried out at 50°C and a reduction in temperature increases the retention time. 

FIGURE 1.9 Selection of cinchonan carbamate CSPs that have been prepared in the course of selector optimization studies (type I prototype type II, O-9-linked thiol-silica supported prototype type III, C-ll-linked thiol-silica supported CSPs type IV, dimeric selectors). (Adapted from M. Lammerhofer and W. Lindner, J. Chromatogr. A, 741 33 (1996) W. Lindner et al., PCT/EP97/02888, US 6,313,247 B1 (1997) P. Franco et ah, J. Chromatogr. A, 869 111 (2000) C. Czerwenka et ah. Anal. Chem., 74 5658 (2002).)... 

Type IV includes chiral phases that usually interact with the enantiomeric analytes through the formation of metal complexes. There are usually used to separate amino acid enantiomers. These types of phases are also called ligand exchange phases. The transient diastereomeric complexes are ternary metal complexes between a transitional metal (usually Cu +), an amino acid enantiomeric analyte, and another compound immobilized on the CSP which is able to undergo complexation with the transitional metal (see also the ligand exchange section. Section 22.5). The two enantiomers are separated based on the difference in the stability constant of the two diastereomeric species. The mobile phases used to separate such enantiomeric analytes are usually aqueous solutions of copper (II) salts such as copper sulfate or copper acetate. To modulate the retention, several parameters—such as the pH of the mobile phase, the concentration of the copper ion, or the addition of an organic modifier such as acetonitrile or methanol in the mobile phase—can be varied. 

The PO mode is a specific elution condition in HPLC enantiomer separation, which has received remarkable popularity especially for macrocyclic antibiotics CSPs and cyclodextrin-based CSPs. It is also applicable and often preferred over RP and NP modes for the separation of chiral acids on the cinchonan carbamate-type CSPs. The beneficial characteristics of the PO mode may arise from (i) the offset of nonspecific hydrophobic interactions, (ii) the faster elution speed, (iii) sometimes enhanced enan-tioselectivities, (iv) favorable peak shapes due to improved diffusive mass transfer in the intraparticulate pores, and last but not least, (v) less stress to the column, which may extend the column lifetime. Hence, it is rational to start separation attempts with such elution conditions. Typical eluents are composed of methanol, acetonitrile (ACN), or methanol-acetonitrile mixtures and to account for the ion-exchange retention mechanism the addition of a competitor acid that acts also as counterion (e.g., 0.5-2% glacial acetic acid or 0.1% formic acid) is required. A good choice for initial tests turned out to be a mobile phase being composed of methanol-glacial acetic acid-ammonium acetate (98 2 0.5 v/v/w). 

In a similar manner, NP conditions (chloroform-based eluents) could be adopted for the separation of iV-tert-butoxycarbonyl-proline (Boc-Pro) enantiomers on a hybrid urea-linked epiquinine-calixarene type CSP and an acidic displacer such as acetic acid promoted elution [42]. Since Ink vs. ln[CH3COOH] dependencies gave straight lines, it may be concluded that this may be attributed to an ion-exchange process still existing in the NP mode. Acids cannot be eluted within reasonable run time without adding an acidic displacer. The practical relevance of the NP mode has to be seen in its much wider solvent choice, which may greatly extend the fiexibility in the course of method development. 

Replacement of the carbamate group with isosteric functionalities such as an IV-methyl carbamate, urea, or amide group clearly confirmed the favorable qualities of the carbamate group [57], While the introduction of a urea group, as in case of iV-9-(tert-butylcarbamoyl)-9-desoxy-9-aminoquinine selector, instead of carbamate functionality turned out to be virtually equivalent in terms of enantiorecognition capabilities [57,58], the enantiomer separation potential was severely lost on iV-methylation of the carbamate group, like in 0-9-(N-me hy -N-tert-butylcarbamoyl)quinine [32,58], or its replacement by an amide, such as in case of Af-9-(pivaloyl)-9-desoxy-9-aminoquinine selector [57,58], For example, enantioselectivities dropped for DNB-alanine from 8.1 for the carbamate-type CSP, over 6.6 for thein-ea-type CSP, to 1.7 for the amide-type CSP, and 1.3 for the A -methyl... 

Since the introduction of CSPs based on macrocyclic antibiotics by Armstrong in 1994 [278], they have gained much interest owing to their (i) broad spectrum of applicability, (ii) complementary activity of the different types of macrocyclic antibiotics, (iii) multiple modes of operation (normal-phase, reversed-phase, polar-otganic phase modes) with complementary enantioselectivities in each mode, and (iv) the ability to separate the enantiomers of underivatized a- and P-amino acids. 

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