Chiral mobile phase additives enantioselectivity

Chiral mobile phase additives provide a more versatile and cost-effective approach for enantiomer separations in thin-layer chromatography. Typically, chemically bonded layers with cyclodextrin and its derivatives, bovine serum albumin, or macrocyclic glycopeptides are used as chiral additives in the reversed-phase mode [59,60,172-178]. For [5- and y-cyclodextrins and their derivatives, a 0.1 to 0.5 M aqueous methanol or acetonitrile solution of the chiral selector is used as the mobile phase. Bovine serum albumin is generally used at concentrations of 1-8 % (w/v) in an aqueous acetate buffer of pH 5 to 7 or in a 0.5 M acetic acid solution, in either case with from 3-40 % (v/v) propan-2-ol (or another aliphatic alcohol), added to control retention. Enantioselectivity usually increases with an increase in concentration of the chiral selector, and may be non existent at low concentrations of the chiral selector. 

In this mode of separation, active compounds that form ion pairs, metal complexes, inclusion complexes, or affinity complexes are added to the mobile phase to induce enantioselectivity to an achiral column. The addition of an active compound into the mobile phase contributes to a specific secondary chemical equilibrium with the target analyte. This affects the overall distribution of the analyte between the stationary and the mobile phases, affecting its retention and separation at the same time. The chiral mobile phase approach utilizes achiral stationary phases for the separation. Table 1 lists several common chiral additives and applications. 

Many ionic poly(saccharides), such as heparin, chondroitin sulfates, dextran sulfate, and natural poly(saccharides), such as dextran, dextrin, pullulan, and their charged derivatives have been used as mobile phase additives for the separation of different enantiomers. Figure 10.10 [191,192,205,206]. Dextrins were found to have a wide application range, thought to be due in part to their helical structures. Enantiomer-chiral selector complexes seem to be weaker than for cyclodextrins, and it has not been demonstrated that enantiomer separations obtained by the poly(saccharide) chiral selectors cannot be obtained using cyclodextrins. Natural poly(saccharides) are typically complex mixtures of homologues and isomers, with a composition that can vary for different sources, resulting in differences in enantioselectivity. 

M. Enquist and J. Hermansson, Influence of uncharged mobile phase additives on retention and enantioselectivity of chiral drugs using an al-acid glycoprotein column. J. Chromatogr., 519(1990) 271. 

The macrocyclic antibiotics were incorporated as mobile phase additives to determine the enantioselective effects on the chiral analytes. The resolution and the capacity factor of each analyte were assessed by a variation of the structure of the macrocyclic antibiotic and the mobile phase buffer pH. 

Lammerhofer and Lindner [62] reported on the enantiomer separation of deriva-tized amino acids on an ODS-packed capillary with a chiral quinine-derived selector as buffer additive in two different modes (i) in an electrophoretically dominated mode at high electrolyte concentration and (ii) in an electroosmotically dominated mode at a low electrolyte concentration. Enantiomer separation in the electrophoretically dominated mode (i) leads to high efficieny (about two to three times higher than in LC) but to a moderate enantioselectivity (about the same as in LC). In the electroosmotically dominated mode (ii) a higher enantioselectivity but a lower efficiency (even inferior to LC) occurs. The separations can also been performed in a non-aque-ous buffered mobile phase. Pressurization (8-10 bar) of the flow system on both ends of the separation capillary was applied. 

Several scientific reports about SFC indicate that the chromatographic retention mechanisms of charged analytes in the presence of suitable ionic modifiers involve ion-pairing [14]. Ion-pairing of sulfonates with ammoninm salt additives was effectively exploited to enhance the solvating power of the mobile phase [15] and sharpen analyte peaks [16], The use of ammonium acetate produced unique results (see Figure 15.1). Ion-pairing also explained enantioselectivity when chiral analytes were analyzed with packed columns in the presence of chiral connter ions [17]. An achiral IPR under SFC conditions played a crucial role in the enantioseparation of a variety of amines [18] for reason explained in Section 13.6. 

A different type of crown ether used to separate enantiomers is the one derived from 18-crown-6 tetracarboxylic acid, covalently immobilized on silica gel via reaction between 18-crown-6 tetracarboxylic acid and amino propyl silica gel [76]. The structure of 18-crown-6 tetra carboxylic is presented in Figure 22-18 [77]. The enantioselectivity on this chiral phase is improved by the addition of triethylamine into the mobile phase and operating at high methanol concentrations however, enhanced selectivity may come at the expense of greatly increased retention times [78]. 

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