7


respective N-2,4-dinitrophenyl derivatives prior to separation. Employing O-9-(tert- butylcarbamoyl)quinine as chiral counterion allowed development of a NACE PTF protocol capable of simultaneously separating all eight phosphonic acid stereoisomers of interest. Again, reversal of enantiomer elution order could be conveniently effected by the use of the pseudoenantiomeric O-9-(tert- butylcarbamoyl) quinidine selector to facilitate sensitive peak detection and quantification. The achieved separations are depicted in Figure 6.


2.3.3. Capillary Electrochromatography


Cinchona carbamate-type selectors have been successfully implemented as chiral recognition elements for capillary electrochromatographic (CEC) separation techniques [33, 34]


. CEC represents a hybrid


Figure 6. Capillary electrophoretic separation of the stereoisomers of 1-amino-2-hydroxypropane phosphonic acid and 2- amino-1-hydroxypropane phosphonic acid after derivatization with Sanger’s reagent as N-2,4-dinitrophenyl derivatives by nonaqueous CE with O-9-(tertbutylcarbamoyl)quinine (a) and O-9-(tert-butylcarbamoyl) quinidine (b) as counterions. Note the reversal of elution order induced upon switching the pseudoenantiomeric counterions. Experimental conditions: Fused-silica


capillary, 50 µm i.d., 45.5 cm total length, 37 cm to detection window; background electrolyte, 100 mM acetic acid and 12.5 mM triethylamine in ethanol-methanol (60:40, v/v); selector solution, 10 mM counterion in background electrolyte; partial-filling technique, filling of the selector solution with 50 mbar for 5 min (corresponds to ca. 30 cm selector plug length); injection, 50 mbar for 5 s; applied voltage, −25 kV (plain background electrolyte at both inlet and outlet electrode vessels); temperature, 15°C. Reproduced from [32] with permission.


separation technique combining the favorablemethodological aspects of HPLC and CE. In contrast to HPLC, CEC utilizes electro-osmotic flow (EOF) phenomena rather than pressure-driven transport to effect analytemigration through a chromatographic bed, leading to a plug-like instead of a parabolic flow profile, with the benefit of tremendously enhanced separation efficiencies. Additional enhancements in performancemay result fromEOF-induced pore flow phenomena, leading to a total improvements in plate numbers by a factor up to 10 as compared to HPLC.


Particularly appealing results could be achieved with CEC phases obtained by in- situ integration of polymerizable versions of quinine carbamate-type selectors in polymethacrylate-type monoliths [35, 36]


. These


Figure 7. Capillary electrochromatographic separations of DNP-Val (a), Bz-Leu (b), and Fenoprop (c) enantiomers on a 150-mm- long quinidine-functionalized chiral monolith. Conditions: polymerization mixture, chiral monomer 8 wt %, 2-hydroxyethyl methacrylate 28 wt %, ethylene dimethacrylate 4 wt %, 1-dodecanol 30 wt %, and cyclohexanol 30 wt %; UV-initiated polymerization for 16 h at room temperature; pore diameter, 1097 nm; capillary column, 335 mm (250-mm active length) _ 0.1 mm i.d.; EOF marker, acetone; mobile phase, 0.6 mol/L acetic acid and 6 mmol/L triethylamine in 80:20 mixture of acetonitrile and methanol; separation temperature, 50 °C; voltage, -25 kV. Reproduced from [36] with permission.


counterions has preferentially been carried out in nonaqueous mode (NACE) to address solubility issues and exploit the tendency for enhanced ion pair formation in these media. Generally, countercurrent [30, 31] filling techniques (PFT) [32]


and partial- have been


employed to avoid detection issues associated with the inherently strong UV


absorbance of cinchona carbamates. The exceptional resolution power of this technique has been exploited for the separation of a complex mixture of phosphonic acid stereoisomers obtained by aminolysis of fosfomycin [32]


.


To facilitate sensitive detection, the UV- transparent analytes were converted into the


phases were generated by copolymerization O-9-[2-(methacryloyloxy)ethylcarbamoyl]- 10,11-dihydroquinine with a hydrophilic co- monomer and a crosslinking agents with optimized binary porogenic solvent mixture within the confines of fused-silica capillaries. Comprehensive optimization of all experimental parameters gave rise to robust synthesis protocols, allowing the fabrication of CEC phases with excellently reproducible performance characteristics in terms of retention time, enantioselectivity and column efficiency. As is evident from the chromatograms depicted in Figure 7, the resulting enantioselective capillary columns produced enantiomer separations with extremely good performance in the CEC mode for chiral acidic analytes. Separation efficiencies in excess of 100,000 plates/meter could be routinely achieved for a variety of amino acid derivatives (with chromophoric and fluorophoric labels) as well as other chiral acids such as 2- aryloxycarboxylic acids.


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