Chiral Ligand Exchanging Mobile Phases

In the technique described in the previous section, the chiral selector permanently resides on the surface of

Figure 3 Enantiomeric separation of racemic amino acids and hydroxy acids with a chiral coating of A/-(2-naphthalenesulfonyl)-L-phenylalanine on porous graphite. Conditions: column PGC 94F (5 ^m, 50 x 4.6 mm), eluent 2.0 mM Cu(CH3COO)2, pH 5.6, flow rate 0.5 mL min~\ UV 254 nm, temperature 30°C. (Reprinted with permission from Knox JH and Wan Q-H (1995) Chromatographia 40: 9-14.)

Figure 3 Enantiomeric separation of racemic amino acids and hydroxy acids with a chiral coating of A/-(2-naphthalenesulfonyl)-L-phenylalanine on porous graphite. Conditions: column PGC 94F (5 ^m, 50 x 4.6 mm), eluent 2.0 mM Cu(CH3COO)2, pH 5.6, flow rate 0.5 mL min~\ UV 254 nm, temperature 30°C. (Reprinted with permission from Knox JH and Wan Q-H (1995) Chromatographia 40: 9-14.)

the column packing. The coating is deposited prior to the chromatographic experiments and no eluents should be used which cause a leakage of the selector. A different approach, which was independently suggested in 1979 by the groups of Karger and Lindner and Hare and Gil-Av, consists in using chiral selectors which predominantly reside in the mobile phase. In this case, the selector complex has to be continuously introduced into the column. Analytes to be resolved in such a chiral eluant form mixed-ligand complexes with the selector, which are diastereomeric in their structure, and, therefore, may be differently retained through the interaction with the column packing. A great advantage of this technique is that many different chiral selectors can be easily tested with respect to the analyte to be resolved. Another advantage is that smaller ligands are usually applied as the eluent dopants, which results in enhanced rates of ligand exchange and better efficiency of the columns. A disadvantage is the loss of the chiral selector and problems of separating the selector from the enantio-mers resolved in the case of preparative experiments. It should be emphasized also that the two enantio-mers separated in the achiral column enter the de tector cell with the chiral eluent in the form of ternary complexes. These complexes are diastereomeric and can significantly differ in molar extinction. Indeed, a case of enantioselective fluorescence quenching of dansylamino acids by a chiral Cu(II) complex of l-phenylalanylamide in aqueous solutions has been reported, thus requiring two calibration curves for a quantitative determination of the two enantiomers.

In combination with Cu(II), Zn(II), Ni(II) and some other transition metal ions, many chiral selectors have been shown to function successfully in reversed phase systems as chiral dopants. They mainly belong to the following classes of chelating compounds:

• unsubstituted l-a-amino acids (proline, phenylalanine, isoleucine, histidine, etc.);

• N-alky-l-amino acids (N-benzyl-proline, N,N-di-propyl-alanine, N,N-dimethyl-valine);

• l-a-amino acid amides (valine amide, phenyl-alanine amide, prolyloctylamide, aspartyl-alkylamide);

• esters of l-a-amino acids (histidine methyl ester);

• peptides (prolyl-valine, aspartylphenylalanine methyl ester or aspartame);

• chiral diamine (N,N,N',N'-tetramethyl-(R)-pro-panediamine-1,2-(l)-alkyl-4-octyl-diethylene-triamine);

• derivatives of (R,R)-tartaric acid (tartaric acid mono-n-octylamide);

• nucleotides (adenosine diphosphate, ^-nicotinam-ide, adenine dinucleotide and, in particular, fiavine adenine dinucleotide).

Mainly racemic a-amino acids and their numerous derivatives, a-hydroxy acids and amino alcohols are successfully resolved. Less common, but nevertheless successful, are resolution of substituted pterins, phenylhydantoins and ornithine analogues.

Depending on the hydrophobicity of the analyte and chiral selector applied, the content of the organic modifier in the chiral mobile phase has to be adjusted in such a manner that the retention of enantiomers remains in the desired time window. In addition to this parameter, retention (and enantioselectivity) in LEC would slightly decrease with the rise in the ionic strength of the eluent and temperature of the column. Conversely, rising pH and concentration of the chiral additive in the mobile phase cause a clear increase in retention and a less marked increase in enantiomeric resolution.

Under normal phase conditions, with bare silica gel as the packing, copper complexes of N,N,N',N'-tetramethyl-(R)-propane-1,2-diamine can be used to resolve amino acids and mandelic acid. Bis(l-prolinato)copper is efficient even in combination with sulfonated polystyrene cation exchangers, which additionally demonstrates the versatility of the chiral mobile phase approach.

The high efficiency of the method is exemplified by Figure 4.

mers. This approach is useful in analysing enan-tiomeric impurities in chiral compounds, where it is always advisable to have the trace component eluting before the major enantiomer.

Finally, it is worth mentioning that the chiral mobile phase mode of LEC is very convenient to prove the reciprocity relations in chiral recognition, where the selector is exchanged for the analyte and vice versa. The enantioselectivity of the system should remain the same, unless some additional factors interfere with the essential interactions within the dia-stereomeric sorption complexes.

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