35
Table 1: Resolution of the eight enantiomer pairs of tetrapeptide Tyr–Arg–Phe–Phe-NH2. Conditions: 10 mmol/l cyclodextrin in 0.100 mol/l phosphoric acid and 0.088 mol/l triethanolamine (pH 3.0). T = 30°C, UV detection at 200nm. From [[27]] with permission.
Configuration of
tetrapeptide DDLL / LLDD DLDD / LDLL DDDD / LLLL LDLD / DLDL DLLD / LDDL DDLD / LDLL LLLD / DDDL LDDD / DLLL
β-CD1) 2.3
0.7 1.1 1.1 0.9 0
1.0 3)
1) Capillary: 80.5 (72.0) cm x 50 µm I.D., V = 30 kV. 2) Capillary: 64.5 (56.0) cm x 50 µm I.D., V = 25 kV. 3) Not available at the time of analysis
to obtain chiral resolution. For example, although it is often stated that you need an aromatic group to resolve enantiomers with cyclodextrin as chiral selectors, it was demonstrated that some non-aromatic dipeptides could be separated with several different uncharged CDs [[18][13]
,[26] ].
A commonly made error is that too few selectors are tested during the initial screening phase. As demonstrated in Figure 5, it is very hard to predict separation. The 16 different forms of a tetrapeptide, sorted as eight enantiomeric pairs, resolved very differently in the same system. A screen of different CDs for separation of these tetrapeptide enantiomers gave different results for each CD (Table 1).
However, sometimes the highest resolution from an initial test will not necessarily lead to the optimum separation. For the enantiomeric separation of adrenaline, which can be present in local anaesthetic (LA) solutions, a screen of suitable selectors was performed. The aim was to obtain enantiomeric separation of adrenaline, preferably undisturbed by the presence of any of the LAs that could be present in the injection solution (Figure 6), in order to investigate racemisation of adrenaline during shelf life. The highest resolution for
adrenaline was obtained with HDAS-β-CD, but the best separations were with DM-β- CD. In the latter system, rac-adrenaline was
well separated and migrated before the LAs. The LAs are present in 1000x higher concentrations than L-adrenaline in injection solutions, and with the migration order in the
system using DM-β-CD the disturbance on adrenaline of the LAs was least. The example also demonstrates the importance of working with real samples in method development and validation.
Good Working Practice
To move on from a good separation to a robust and sensitive method, we need to adopt good working practices on top of fundamental knowledge and understanding of the technique and its modes of operation. While all aspects of CE good working practice cannot be discussed here, there are some important points worth highlighting.
Electrolyte System
For all CE methods, chiral or achiral, it is paramount to describe the electrolyte system unambiguously. This is best done by describing the exact concentrations for the electrolyte components. For example, “make a solution containing 100 mM phosphoric acid and 88 mM triethanolamine. Check the pH, which should be pH 3.0 ± 0.1” is to be preferred over “100 mM phosphoric acid adjusted to pH 3.0 with triethanolamine”. As long as the electrolyte is buffering (which it should) one drop more or less of the triethanolamine does not change the pH very much, but it does affect the ionic strength of the system. Better reproducibility is obtained when the ionic strength is controlled. Consequently, one should use appropriate volumetric glassware.
Although the above mentioned phosphate triethanolamine electrolyte still has some buffering capacity at pH 3.0, this sometimes is not sufficient [[18] pKa
values close to the pH of the buffer and
when the buffer capacity is low, problems can arise. Injection of a dipeptide in phosphate buffer pH 3.0 gave enantiomeric resolution, which disappeared with repeated injections. This problem was easily solved by exchanging phosphoric acid (pKa
2.15) to malonic acid
Table 2: Change in pH in phosphate buffer after applying the voltage in CE. 0.100 mol/l phosphoric acid adjusted to pH with NaOH. The rinse vial gives the original pH of the solution. Between injections, the capillary is rinsed from this vial, as to not vary the liquid levels in the inlet and outlet vials.
pH Rinse vial Anode Cathode pH 2.0 pH 2.5 pH 3.0 2.0 1.9 2.0 2.5 2.3 2.7 3.0 2.4 6.0
2.85) as buffering component. The change in pH due to the electrolysis of water in phosphoric acid - NaOH buffers after applying voltage is illustrated in Table 2. The best buffering capacity is obtained at as high buffer concentration as possible and with a pH within one pH unit from the pKa
(pKa [[28] , [29] ] of
the buffer protolyte. The buffering capacity steeply reduces the more the pH deviates from the pKa the buffer.
value of the weak acid in
M-β-CD1) 1.2
2.3 2.5 0
1.1 0 0 3)
DM-β-CD1) 3.6
6.2 7.4 0
4.1 2.7 1.4 3)
Resolution TM-β-CD1)
0 0 0
1.5 1.5 2.0 2.4 3)
γ-CD1) 0
0 0
0.7 1.2 0.4 0 3)
DM-β-CD + TM-β-CD2)
3.1 7.4 6.2 0.6 4.5 2.5 1.3 0
DM-β-CD2) 3.3
7.5 6.7 0
4.6 2.4 1.2 0.6
Temperature
The temperature control of a CE capillary is important. Temperature influences parameters such as the viscosity of the BGE and pH and pKa
values of buffers and ]. When the analytes have
analytes which influences the chiral as well as achiral equilibria in the BGE. For good repeatability and reproducibility, temperature therefore needs to be carefully controlled. In chiral systems, temperature may also influence the degree of complexation of the enantiomers with the chiral selector. Decreasing the temperature can either increase or decrease the resolution [[30]
].
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