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Capillary Eletrochromatography (CEC)
CEC Advantages
. High Efficiency, High Resolution and High Selectivity
Because of the plug-like flow characteristic of the EOF driving the mobile phase, the column efficiency in CEC is much higher than that obtained with the same column driven at the same linear velocity using pressure-induced flow. This result is demonstrated in Figure 3. This high efficiency allows dramatic increases in resolution. Furthermore, because the flow in CEC is independent of the channel spacing between the particles in the column, longer columns (up to 100cm) containing very fine particles (down to 0.5mm) can be used without the increase in back pressure normally encountered in micro-HPLC.
Fig. 3: Comparison of peak broadening of thiourea between CEC and micro-HPLC. Column: 320mm i.d. × 25cm packed with 5mm ODS. Mobile phase: 80% CH3CN/20% 4mM sodium tetraborate ( pH 9.1). Detection: UV absorbance at 254nm .
Consequently, efficiencies up to 200,000 theoretical plates per column (up to 700,000 plates per meter!) as demonstrated in Figure 4 are achievable. (Efficiencies of 20,000 plates per column are typically obtained in micro-HPLC.) This amazingly high efficiency capability of CE combined with the high selectivity and versatility of micro-HPLC provide the analyst with an extremely powerful technique that can be used to tackle many challenging analytical problems. For example, although the identification of the 14 nitroaromatic and nitramine explosives (EPA Method 8330) and their degradation products is very important in forensic and environmental applications, a complete separation of these structurally similar compounds using an isocratic HPLC system has proven to be a challenge.
Fig. 4: High efficiency CEC separation of 4 PAHs on 1.5mm non-porous ODS (Micra Scientific, Inc., Northbrook, IL). Column: 100mm i.d. × 28cm packed length. Mobile phase: 70% CH3CN/30% 4mM sodium tetraborate (pH 9.1). Voltage: 20kV. Injection: 5kV/2s. Detection: on-column, laser-induced fluorescence (LIF), ex: 257nm, em: 400nm. Sample: a) Fluoranthene, b) Benz-[a]anthracene, c)Banzo[k]fluoranthene, and d) Benzo[ghi]perylene.
The analysis usually requires solvent gradient elution for 20 minutes to resolve the 14 components. However, CEC provides a significantly better solution than that attainable by HPLC. As shown in Figure 5 , a baseline separation is achieved for all of the 14 compounds in 7 minutes under isocratic conditions, featuring efficiencies of over 500,000 theoretical plates per meter.
 Fig. 5: CEC separation of 14 explosive compounds. Column: 75mm i.d. × 17cm packed with 1.5mm non-porous ODS. Mobile phase: 15% CH3OH/85% 10mM MES (pH 8.5). Voltage: 12kV. Injection: 1kV/1s. Detection: UV Absorbance at 254nm. 
. Fast Speed
The high resolving power of the CEC columns packed with small particles (e.g., 1.5mm) alleviates the need to use longer columns. This characteristic leads to the capability of performing ultra-fast CEC separations using shorter columns (<10cm) with relatively high efficiencies. Fig. 6 shows a rapid separation of a test mixture of 5 PAHs on a 1.5mm non-porous ODS phase (Micra Scientific Inc., Northbrook, IL). Note that the linear velocity of the EOF is about 20mm/s under an electric field of 2,800V/cm.
. Economically Attractive and Environmentally Friendly
Because of the small internal diameter of the capillary columns used in CEC, both solvent flow (~100nL/min) and sample size (~nL) are reduced by a factor of ~10,000 times compared to conventional HPLC. This dramatically reduced consumption of solvent and sample makes CEC economically attractive and environmentally friendly.
Fig. 6: Rapid CEC separation of 5 PAHs. Column: 100mm i.d.× 6.5cm packed with 1.5mm non-porous ODS. Mobile phase: 70% CH3CN/30% 2mM TRIS (pH 9). Voltage: 28kV. Injection: 1kV/1s. Detection: LIF, ex: 257nm, em: 400nm.
. Mass Spectrometry (MS) Compatibility
CEC is easily adaptable to MS. (e.g., using electro-spray for added versatility in identifying sample components as shown in Figure 7). Courtesy of Jianmei Ding and Prof. Paul Vouros, Northeastern University, Boston, MA, USA. 
Fig. 7: CEC-MS analysis of a reaction mixture of anti benzo[g]chrysene 11,12-dihydrodiol 13,14-epoxide with calf thymus DNA. Column: 75mm i.d. × 20cm packed with 3mm ODS. Mobile phase: 29% CH3CN/71% 5mM NH4OAc (pH 6.5). Voltage: 14.5kV. Injection: 1kV/1s. ESI voltage: 2.5kV. Sheath liquid (0.5ml/min): 75% CH3OH/24% H2O/1% acetic acid. A to D: extracted single ion electrochromatograms for m/z 480 (N7-dG adduct), 607 (unknown), 580 (isomeric deoxyadenosine adducts) and 596 (isomeric deoxyguanosine adducts), respectively. (E): C and D combined. |
