Enantiomeric analytes

Chiral resolution by HPLC can by divided into three categories (1) a direct resolution using a chiral stationary phase (CSP) (2) addition of a chiral agent to the mobile phase, which reacts with the enantiomeric analytes (chiral mobile phase additive method (CMPA)) (3) an indirect method that utilizes a precolumn diastereomer formation with a chiral derivatization reagent (Misl anova and Hutta, 2003). 

Silica-based material provides mechanical stability and high efficiency, while adsorbed polysaccharide helices offer chiral selectivity to the wide range of enantiomeric analytes. 

While Pasteur made the historical discovery, subsequent advances in the resolution of enantiomers by crystallization were based on empirical results. Several attempts to separate enantiomers using paper chromatography were met with unsystematic results. In 1952 Dalgliesh postulated that three points of simultaneous interaction between the enantiomeric analyte and the stationary phase are required for the separation of enantiomers [2]. 

For more hydrophobic analytes, the retention can be modulated by the addition of organic modifiers such as methanol in the mobile phase. However, there is not a linear relationship between the amount of the organic modifier in the mobile phase and the retention factor of the enantiomeric analytes, indicating multiple types of retention interactions [75]. 

Another type of CSP able to undergo charge transfer interaction is the one developed by Lindner s group [101], In order to determine the interactions between the quinine CSP and the enantiomeric analytes, a detailed computational study was undertaken of the interaction of this stationary phase with 3,5-dinitrobenzoyl derivatives of leucine (Figure 22-27) [102],... 

In Section 22.3 the main types of interactions occurring between the enantiomeric analytes and the stationary phase (hydrogen bonding, charge transfer, and inclusion complexes) was described. In the following section,... 

The mechanism of interaction between the enantiomeric analytes and the phenylcarbamates has been proposed based on chromatographic, computa-... 

Under reversed-phase elution conditions, the nature of organic modiher has a very important role on the separation. Thus, methanol proved to provide better enantioselectivity than acetonitrile for the separation of ergotamine on a vancomycin and teicoplanin stationary phase [129]. The buffer concentration also has an influence on the retention of the enantiomeric analytes. An increase in buffer concentration produces a decrease in the retention of the two enantiomers. It was found that in some cases the two stationary phases, teicoplanin and vancomycin, are complementary for a poor separation on vancomycin, the stationary phase produced a baseline separation on teicoplanin and vice versa [129]. 

In the case of ion-pair complexes between the chiral additive and the enantiomeric analytes, their interaction should be maximized by adjusting the mobile-phase polarity. Solvents of lower dielectric constant favor ion-pair formation. 

Method development for chiral separation is a multidisciplinary task. It requires knowledge of stereochemistry, organic chemistry, and separation techniques. Separation of enantiomers is not linked to a certain technique (i.e., GC, HPLC, etc.) but rather to an understanding of the specific interactions between the enantiomeric analytes and a certain chiral stationary phase. Knowing these types of relationships will enable one to easily understand the formation of transient diastereomeric complexes between enantiomers and a chiral stationary phase during a chromatographic separation as well as their stereochemical relationship within the complex. Once such dependencies are established, development of a method for the separation of enantiomers becomes an easy process. Based on such a relationship, chiral stationary phases can be divided in five categories [161] ... 

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. 

Separation of enantiomers is a technique driven mainly by the needs of pharmaceutical industry to produce drugs with controlled enantiomeric purity. Enantiomeric separation involves more than knowledge of chromatography it requires an in-depth assessment of the stereochemistry of enantiomeric analytes and chiral stationary phase, as well as the interactions involved therein. In this situation, chromatography is just a tool that helps to separate enantiomers. That is why this chapter presents the main types of interactions occurring between the selectands and the selectors. Understanding these relationships, chiral separation becomes a logical process and trial and error is minimized. 

CMP s advantages stem from the fact that it is cheaper, since it uses achiral stationary phases, and the chiral additive can be purchased at a low cost the approach is flexible, because after using a chiral additive, the chromatographic column can be washed out from the chiral additive and a new additive can be employed. On the other hand, the mechanism is difficult to predict due to the constant presence of a secondary chemical equilibrium in the column. Since the enantiomeric analytes are eluted out of the column as diastereomeric complexes, the detector response may be different for each complex. Also, the sample capacity is relatively small. 

Just as Pirkle does not have a monopoly on synthetic multiple-interaction CSP, similarly Regis do not have a monopoly on their commercialisation. Kromasil [C6] market a range of CSP based on the work of AUenmaik s research group [15] in which a derivatised tartramide chiral network polymer is covalently bonded to silica. As discussed later, these products are geared towards the preparative chiral LC market. In this market the advantage of all synthetic multiple-interaction CSP that the retention order of enantiomeric analytes may be switched by switching from the CSP based on the R-chiral selector to that based on the S-chiral selector or vice versa is particularly pertinent. This feature will ensure that there remains a future for synthetic multiple-interaction CSP even in the face of successftil developments of CSP with a broader spectrum of enantioselectivity. 

Chiral surfactants used in MEEKC have also been like dodecoxycarbonylvaline (DDCV) for the separation of different enantiomeric analytes [46,47]. Chiral polymeric surfactant has been also employed in the enantiomeric resolution of barbiturate, binaphthyl, and paveroUne [48],... 

The approach proposed by Schweitz, Nilsson and coworkers seems to be more promising for pseudoaffmity CEC. This approach establishes a continuous, macroporous MI stationary phase within the capillary. In the usual protocol, a mixture of MAA (functional monomer), TRIM (cross-linker) and the template (e.g., R-propranolol), is polymerized in situ by UV-initiated radical polymerization at a temperature of-20°C. The resulting capillaries have been used in enantioselective CEC for the separation of rac-propranolol. Baseline separations of the enantiomeric analytes could be demonstrated (Fig. 7.8) as well as separations of structurally related racemates. ... 

---