Gradient-CEC instrumentation

Fig. 2.10. Schematic diagram of the Agilent Technologies prototype gradient CEC instrument.

Fig. 2.11. Schematic drawing of the special inlet vial of the Agilent Technologies prototype gradient CEC instrument. Left graphic, pHPLC gradient mode right graphic, EOF driven gradient CEC mode.

Fig. 2.8. Right panel schematic diagram of instrument setup for EOF driven gradient CEC according to Lister et al. [39]. Left panel diagram of FIA CEC interface (Reproduced from Ref. [39] with permission of the publisher).

Fig. 8.2. Schematic illustration of a CEC instrument capable of isocratic and gradient elution and ESI-MS detection.

The possibility of switching between the LC and the CEC mode enables six modes of operations in one instrumental set-up isocratic and gradient CEC, isocratic and gradient p-LC, and isocratic and gradient pressure supported CEC (LC/CEC mixed... 

In both of these techniques progress has been somewhat limited by the lack of commercially available instruments. The results reported to date have all been obtained on home-made equipment that has been constructed from a combination of instruments. The injection method on the majority of the designs involves an injection port and therefore removes the ability to sample directly onto the column. It would be highly desirable to be able to make direct injections and perform gradient CEC in a more automated manner. There is also an opportunity to use the same CEC column to perform either p-LC, CEC or pCEC all on the same basic instrument platform. 

A CEC instrument basically consists of a system for injection (pressure driven or electrokinetic), a column in which the separation takes place, a detector and a high voltage supply (Fig. 16.1). The most commonly used detector so far has been UV with transmission through the capillary outside of the packed bed. Laser induced fluorescence detection has been employed in several studies. Also, mass-spectrometry has been used. Normally, isocratic CEC is performed, but approaches to gradient CEC have been reported [29]. However, special equipment must be employed in most cases. 

Unimicro Technologies Trisep-100 (Pleasanton, CA) Capillary array instrumentation CEC, pLC, CZE Gradient elution, several detectors... 

For isocratic mode of CEC separations, standard CE instrumentation is sufficient. This applies particularly for equipment that has the provision of column pressurization. In practice this is achieved by applying a gas under a pressure of 2-12 bar to both inlet and outlet vials. Column thermostating in CEC is regarded mandatory to avoid excessive radial temperature gradients within the capillary. In such instruments, sample is typically injected electrokinetically and alternatively by applying the external gas pressure to the sample vial. Detection occurs on-column i.e. directly through a non-packed section of the capillary following immediately the end of the bed. 

Peptide mapping represents one of the fundamental tasks in protein analysis. The fact that most of the applications of CEC are restricted to isocratic elution has considerably limited the practical applicability of this technique in the past, particularly in the analysis of biopolymers. Two approaches have been developed to generate continuous gradients, namely, the merging of two endo-osmotic flows [78] or the application of two separate HPLC pumps. The latter presents practically no complication with regard to instrumentation as the same equipment can be used for HPLC,... 

Novel instrumentation integrates capillary HPLC (cHPLC)-NMR, CE-NMR, and CEC-NMR and provides extreme versatility [29,49]. A number of CE-NMR and CEC-NMR applications performed with this integrated instrumentation have been described. In this system, saddle-type coils with 240-400-nL observe volumes increase the sensitivity and avoid current-induced magnetic field gradients. A mixture of lysine and histidine has been analyzed in a continuous-flow CE experiment with a detection limit of 336 ng (2.3 nmol) [29],... 

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