34
May/June 2013
The high EOF decreases the time it takes for the analyte to reach the detector. The separation window for the analyte becomes rather small in case of an uncharged selector migrating with the EOF (Figure 4). One possibility to improve the separation window is to use a charged selector. Although there are alternative solutions to improve the separation window when there is no appropriate charged selector for the enantiomers at hand. An increase in the separation window can be obtained in the case of an uncharged chiral selector e.g., by using a coated capillary or by influencing the migration of the free enantiomer, such as with CD MEKC (explained in Figure 4) or with a dual CD system (explained in [[16]
, [17] Figure 5: Enantiomeric separation of the eight enantiomer pairs of tetrapeptide Tyr-Arg-Phe-Phe-NH2. BGE consisted of
100 mM phosphoric acid, 88 mM triethanolamine (pH 3.0) and 10 mM heptakis(2,6-di-O-methyl)-β-cyclodextrin. Reprinted from [40] with permission from Elsevier.
]). An
interaction or partition to an achiral micelle or with a charged CD, that not necessarily exhibit stereoselective interaction with the analyte, will promote a migration of the enantiomers delayed from the EOF. This delay increases the separation window.
For the separation of neutral analytes, the selector must be charged or the charge has to be induced on the enantiomers with CD- MEKC or a dual CD system similar as mentioned above for acidic enantiomers.
Figure 6: Selection of cyclodextrin for the enantiomeric separation of adrenaline in local anaesthetic solutions for injection. Sample: (1) 17 µg/ml adrenaline HCl, (2) 18 µg/ml lidocaine and (3) 15 µg/ml bupivacaine HCl. Conditions: BGE: 0.10 M phosphoric acid, 0.07 M triethanolamine (pH 2.5), cyclodextrin as in figure, 75 µm i.d. × 64.5 cm (56.0 cm to detector) fused- silica capillary, temperature 25 o
ramping over 30 s). Detection at 200 nm (4 nm bandwidth). DM-β-CD heptakis(2,6-di-O-methyl)-β-cyclodextrin; HP-β-CD hydroxypropyl-β-cyclodextrin; CM-β-CD Carboxymethyl-β-cyclodextrin; HDAS-β-CD heptakis-(2,3-diacetyl-6-sulphato)-β- cyclodextrin. Reprinted from [41] with permission from Elsevier.
for a typical starting situation. The strategy to select a starting point for a basic analyte is usually relatively simple. At a low pH, at least two pH units below a basic analyte’s pKa
, it is
mainly (≥ 99%) charged and at a low pH (i.e. below pH 4) also the electro-osmotic flow (EOF) is low. This means, that either a charged or an uncharged chiral selectors can be used. The advantage of using an uncharged selector is that it does not contribute to the ionic strength of the electrolyte and therefore does not increase the current and Joule heating. An uncharged selector migrates with the electro-osmotic flow and the separation window for the enantiomers is between the migration of the unaffected analyte, i.e. the migration velocity
of the enantiomeric analytes with no selector present, and the EOF (Figure 3). For basic analytes with a BGE at a low pH and slow EOF, the separation window is rather large. The advantage of a negatively charged selector is that it migrates slower than or in reverse direction of the EOF, which enlarges the separation window further. The disadvantage of a charged selector is that it can contribute considerately to the ionic strength of the system, resulting in (often too) high currents.
For acidic compounds, the strategy to obtain enantiomeric separation requires usually more attention. At a high pH, over 2 pH units over the pKa
, the analyte is charged but a high pH (i.e. pH ≥ 8) also gives a fast EOF. C, sample injection 30 mbar × 3 s, BGE injection 30 mbar × 3 s, applied voltage +24 kV (initial
Once a selector that gives enantioselective interaction for the chiral analytes is found, the separation can be optimised. An important factor affecting the separation is the concentration of the chiral selector, as the concentration will determine the equilibria between the selector and the enantiomers. Often pH, temperature, buffer composition or the magnitude of the EOF can also affect the enantiomeric separation so these parameters should be investigated as well, preferably in a multifactorial design. In the literature there are many examples both for method optimisation as well as robustness testing applying chemometrics [e.g. [12]
, [19] - [25] ]. An additional factor that
needs attention during robustness testing is the purity of the chiral selector. Often batch- to-batch variability results in a change in chiral resolution. The robustness test should include testing of selectors from different suppliers and batches and result in a system suitability test (SST) with appropriate requirements, such as resolution.
Pitfalls and Examples
It is key to find the right chiral selector. The most important point here is not to be prejudiced. Because of the high efficiency in CE, you need very little difference in selector interaction between the enantiomers in order
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60
