33
stationary phases in LC [4]
. With CE it is
possible to screen multiple chiral selectors to a much lower cost compared to the cost for the purchase of one or more chiral columns, which reduce the expenses for the method development especially for novel compounds. Furthermore, the low solvent and solute consumption makes CE a more environmentally friendly as well as a relatively cheap technique. Finally, the high efficiency in CE means that a rather small mobility difference between enantiomers can be sufficient for their separation. So a chiral selector in CE is often more generally applicable, i.e. resolves the enantiomers of more compounds, than in LC [c.f. [5]
, [6] ]. Figure 2: Starting point for chiral CE method development Chiral CE
Chiral CE separation is achieved by adding a chiral selector to the BGE. The enantiomers form fast, reversible equilibria with the selector. This means that part of the time an enantiomer migrates free in the BGE, and part of the time it migrates as an enantiomer- selector complex. The apparent mobilities of the enantiomers will change depending on the strength of the complex (i.e. the equilibrium constants) and the mobility of the enantiomer-selector complex. In order to separate the enantiomers, a few requirements need to be met. First of all, either the enantiomers or the selector have to be charged. Furthermore, the enantiomers of a chiral molecule need to have different affinities for the chiral selector, and/or the enantiomer-selector complexes need to differ in mobilities [[7]
- [9] ]. A typical example
of a chiral separation is shown in Figure 1. The top trace shows an achiral CE separation of five local anaesthetic (LA) drug molecules.
In the bottom trace, 10 mM dimethyl-β- cyclodextrin is added as the chiral selector to the BGE and the enantiomers of all LAs are efficiently separated [[10]
, [27] ]. For the local
anaesthetic ropivacaine, marked 3, this system was further developed into a quality control method [[11]
, [12] been taken up in the
Ph.Eur. and USP [[13]
] and the method has , [14]
].
Cyclodextrins (CDs) are commonly used as chiral selectors, as there is a wide variety of CDs available and they absorb little UV light at the common detection ranges. This makes CDs rather popular, although the disadvantages are that they are poorly characterised and only exist in one absolute configuration. The latter means that one cannot change the order of migration of enantiomers in a system by exchanging the chiral selector from one configuration to its
Figure 4: Separation window for an acidic compound at a high-pH electrophoresis buffer (fast EOF) with an uncharged chiral selector in chiral CE (top) and CE-MEKC (bottom). The neutral chiral selector migrates with the EOF. In the top trace, that means that the separation window is between the mobility of the compound free in solution and the EOF. For the acidic analyte in this example that gives a narrow separation window. As a neutral CD was the only chiral selector that resulted in some enantiomeric separation (the charged CDs were unsuccessful), the separation window was enlarged by using micelles. When micelles were added to the electrophoresis buffer, micellar electrokinetic chromatography (MEKC) took place. The analyte of interest had some affinity for the micelle and migrated later than in free solution. When the neutral CD, which migrated with the EOF, was added, the separation window was enlarged and the enantiomeric separation of this compound was successful.
mirror image. The poor characterisation can give supplier or batch-to-batch reproducibility issues [e.g. [11]
, [15] , [18] ]. Other
chiral selectors that have been used are e.g., chiral crown ethers, optically active micelles, bile salts, macrocyclic antibiotics, proteins and ion-pair selectors.
Method Development
In CE, the analyte needs to carry charge, either by itself or by complexation with e.g. a chiral selector. In chiral CE, different starting points are applied for the enantiomeric separation of basic, acidic or neutral analytes. Figure 2 sketches our experience
Figure 3: Separation window for a basic chiral compound in a BGE at low pH with low EOF and with a neutral chiral selector. The separation window is between the mobility of the chiral selector (which is with the EOF) and the free mobility of the compound (which is the mobility in BGE without chiral selector).
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