Gradient CEC

Robson et al. (1999) developed a simple interface for gradient CEC-HPLC. 

Gradient CEC system which includes two PU-980 HPLC pumps, flow rate range 1 pl/min to 10 ml/min, -30 or +30 kV. Electropak CEC columns. 

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).

This vial could be placed in two positions by the inlet lift. In the lower position of the lift, EOF driven isocratic and gradient CEC was possible. The solvent was delivered by the pump to the vial through the inner channel and removed via the outer channel using a pressure of nitrogen (right panel of Fig. 2.11). The resistance of the outlet restrictor was low. The pressure in the vial equaled the external gas pressure and was the same in the outlet vial. In this way, no hydraulic flow was generated and bubble formation was suppressed. The column continuously accepted the delivered solvent from the vial by electroosmotic flow. In this position, the flow delivered by the external pump must be low to avoid solvent overflow in the vial. 

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.

On-line CEC—NMR coupling is promising [72,73,74,75]. Of special interest is the coupling of gradient CEC with NMR. A Bruker AMX 600 NMR spectrometer was used to perform isocratic and gradient micro-LC—NMR and CEC—NMR. This hyphenation is presented in Fig. 2.22. 

The same experimental setup was used in the separation of metabolites of paracetamol from a human urine extract [73] and Thomapyrin, containing acetaminophen, caffeine, and acetylsalicylic acid [74], Compared to isocratic CEC—NMR, gradient CEC—NMR offers increased sample loading capacity because of preconcentration at the front of the column and higher separation efficiency together with a reduction in analysis time [75],... 

Fig. 6.12. Gradient CEC separation of derivatized neutral steroids. (Reprinted with permission [38], Copyright 2000 Elsevier). Conditions Column 35 cm (active length 25 cm) x 100 pm i.d., mobile phase gradient of acetonitrile-water-240 mmol/L phosphate buffer pH 3 from 35 60 5 to 65 30 50 in 15 min 600 V/cm injection 100 V/cm for 10 s. Peaks labeling reagent 1, progesterone 2, 11 P-hydroxyandrosterone 3, dehydroisoandrosterone and equiline 4, estrone 5, androsterone 6, 19-hydroxy-4-androsterone-3,17-dione 7, 5-a-androstan-17-one 8.

Fig. 8.24. Separation of a tryptic cytochrome C digest by pressure assisted gradient CEC. Column, 60 x 0.18 mm i.d. packed with 3 pm Vydac Cl8 eluents, (A) 0.07% trifluoroacetic acid in water, (B) 0.07% trifluoroacetic acid in acetonitrile gradient elution with 0-50% B in 20 min applied pressure, 9 MPa in (a), 5 MPa in (b) 7 MPa in (c) applied voltage, 0 kV in (a), 1 kV in (b), 0.6 kV in (c) Detection, nanospray ESI-MS, m/z 200-1500, 10 spectra/s sample, tryptic digest of 8 pmol bovine cytochrome C. (Reprinted with permission from ref. [45], Copyright [1997] American Chemical Society).

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... 

CEC. The separation performed by Fujimoto et al. was achieved on a polyacrylamide gel column in 17 min [90], Phenylthiohydantoin (PTH)-labeled amino acids were separated by Horvath s group by gradient CEC on a 3-pm Zorbax C18 column and, later, by employing an immobilized packed-bed capillary [91]. Alicea-Maldonado and Colon compared the separation of three dansylated amino acids by CE and CEC using a fluoropolymer as the stationary phase [92], Elution of positively charged amino acids after the EOF marker suggested the interaction of solutes with the fluoropolymer. 

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. 

---