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Application Note of Capillary Eletrochromatography (CEC)
Introduction of CEC Technology
 As chemical analysis in fields ranging from combinatorial chemistry to bio-technology to forensic science is pushed to samples with smaller volumes, the miniaturization of analytical instrumentation with high separation power and high detection sensitivity has become more prominent. For the past 10 to 15 years, analytical techniques such as capillary electrophoresis (CE) and micro high performance liquid chromatography (micro-HPLC) that use miniaturized columns have received considerable attention. The most common form of CE (free solution), in which a potential is applied to a buffer-filled capillary to generate electroosmotic flow (EOF), provides excellent efficiency because of the flat, plug-like flow profile of EOF as illustrated in Fig. 1. This technique is able to separate charged species via their different electrophoretic mobilities but is unable to resolve neutral components. Micro-HPLC, on the other hand, provides high selectivity in a wide range of applications, including analyses of mixtures containing neutral components, due to the variety of stationary phases that are available. However, because the mobile phase in micro-HPLC is driven through the column by pressure resulting in a parabolic flow profile (see Fig. 1), the column efficiency is typically lower.
As illustrated in Fig. 2, the high efficiency of CE can be combined with the high selectivity of micro-HPLC and the end result is a hybrid technique known as capillary electrochromatography (CEC). This technique utilizes columns that are similar to those used in micro-HPLC but the mobile phase is driven by an electric potential as in CE. The separation mechanism in CEC, therefore, is the result of the combination of chromatographic partitioning and electrophoretic migration. Practically, CEC can be performed in a CE instrument with a micro-HPLC column.
Fig. 1: Flow profile in CEC and micro-HPLC.
Fig. 2: CEC combines the strengths of two powerful analytical techniques - CE and micro-HPLC. 
Advantages of CEC
. 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.
Instrumentation of CEC
CEC is a state-of-the-art micro separation technology that combines micro-HPLC and capillary electrophoresis. It offers high efficiency, high selectivity, high resolution and fast speed. Multi-mechanism makes CEC an ideal technique for the analysis of complicated samples in biological and chemical fields.
In CEC, the retention mechanism is based on both chromatographic and electrophoresis separation. Due to the contribution of the dual mechanisms, CEC dramatically enhances the separation selectivity, compared to traditional HPLC and CE. Combined with on-column detection techniques, CEC could be readily coupled with UV, FLD, LIF, ECD, MS and other detectors. Currently, CEC technology is widely applied in various fields, including Pharmaceutical, Life Science, Toxicology, Forensic Medicine, Health Products, Chemical, Petrochemical, Environment, Chiral Compound, Food/Beverage, etc.
As the leader in nano and micro separations, Unimicro Technologies presented the world’s first dedicated pCEC system TriSep™-2000 and then developed the second generation pCEC system, TriSep™-2010GV. In 2004, Unimicro Technologies came up with the latest version, TriSep™-2100. Revolutionary design and engineering dramatically elevate the system performance, compared to the former models. Powerful technical support on application, brand-new coupling interface with various up-to-date detection technologies and patented EletroPak™ capillary columns make TriSep™-2100 an ideal tool for explorers in all research fields mentioned above.
Capillary Columns for CEC
ElectroPak™ capillary columns are packed columns, which can be easily coupled with CEC system, capillary HPLC system or capillary LC-MS system. Patented electro-packing technologies make ElectroPak™ capillary columns high efficiency, high resolution and high speed . These advantages will bring you more findings and new experiences in your analytical work.
The capillary columns are packed with various categories packing materials, such as Si, C18, C8, Phenyl, CN, Diol, Chiral materials, and so on. And several particle and pore sizes can be offered. They are available in diversified inside diameters, outside diameters and lengths.
High efficiency separation(500,000 plates/m) of 14 explosives in 7 min. See ref. Bailey, C. & Yan, C., Anal. Chem ., 1998 , 70, 3275
Applications of CEC
. HIGH RESOLUTION AND RAPID CEC ON 1.5μm NON-POROUS ODs
Fast separation on 1.5μm Non-porous ODS
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Figure 26. Fast separation of 16 EPA priority pollutants. Column: EP-100-20-1.5-C18 (1.5mm non-porous ODS, Micra Scientific, Inc., Northbrook, IL). Mobile phase: 70% CH 3 CN in 30% 2mM TRIS. Voltage: 55kV. Injection: 5kV/2s. Detection: LIF, ex: 257nm, em: 400nm. |
Electric Field Effect on Separation Speed
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Figure 27. Electrochromatograms showing the CEC separation of 5 PAHs using 1.5mm non-porous ODS particles. Column: EP-100-6.5-1.5-C18. Mobile phase: 70% CH3CN in 30% 2mM TRIS. Voltage: varied from 5 to 25kV. Injection: 5kV/2s. Detection: LIF, ex: 257nm, em: 400nm. |
High Resolution Separation on 1.5μm Non-porous ODS
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Figure 28. Baseline CEC separation of 16 PAHs on 1.5mm non-porous ODS. Column: EP-100-20-1.5-C18. Mobile phase: 65% CH 3 CN in 35% 2mM TRIS. Voltage: 29kV. Injection: 5kV/2s. Detection: LIF, ex: 257nm. em: 400nm. |
Effect of Linear Velocity on Efficiency
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Figure 29. Effect of linear velocity (EOF) on the height equivalent to a theoretical plate (HETP) for three PAHs. Column: EP-100-6.5-1.5-C18. Mobile phase: 70% CH 3 CN in 30% 2mM TRIS. Injection: 5kV/2s. Detection: LIF, ex: 257nm, em: 400nm. |
. APPLICATIONS
Separation of Chiral Compounds on Silica
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Figure 30. CEC separation of synephrine enanthiomers. Column: EP-75-20-3-SI (3mm silica). Mobile phase: 10 mM TRIS (pH adjusted to 3.12 using H3PO4) with 14 mM hydroxypropyl-b-cyclodextrin as a mobile phase additive. Voltage: 15kV. Injection: 1kV/1s. Detection: UV at 214nm. Temperature: 25EC. CE instrument: Beckman P/ACE 2000. |
Separation of Basic Compounds on Silica
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Figure 31. CEC separation of basic compounds on silica stationary phase. Column: EP-75-20-3-SI (3mm silica). Mobile phase: 80% CH 3 CN/20% 10mM TRIS (pH adjusted to 8.29 using HCl). Voltage: 20kV. Injection: 5kV/5s. Detection: UV at 214nm. Temperature: 25EC. CE instrument: Beckman P/ACE 2000. Sample: 1. aniline, 2. cocaine hydrochloride, 3. berberine hydrochloride, 4. thebaine, 5. jatrorrhizine hydrochloride, 6. ephedrine hydrochloride, and 7. codeine phosphate. |
Separation of 25 aromatic Compounds
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Figure 32. CEC separation of a mixture of nitrogen containing aromatics and PAHs. Column: EP-75-30-3-C18. Mobile phase: 70% CH 3 CN/30% 4mM sodium tetraborate. (pH 9). Voltage: 20kV. Injection: 5kV/5s. Detection: LIF, ex: 257nm, em: 400nm. |
Separation of 14 Explosives
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Figure 33. Rapid CEC separation of explosives on 1.5mm non-porous ODS (Micra Scientific, Inc.). Column: EP-75-25-1.5-C18. Mobile phase: 7.5% CH 3 OH/7.5% isopropanol/85% 10mM MES/5mM SDS. Voltage: 11kV. Injection: 2kV/1s. Detection: UV at 254nm. |
Considerations of CEC
. RUN TO RUN REPRODUCIBILITY
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Figure 18. Run to Run Reproducibility test in CEC. Column: EP-75-20-3-C18. Mobile phase: 80% CH 3 CN/20% 5mM phosphate (pH 6.5). Voltage: 10kV. Injection: 5kV/5s. Detection: 254nm. Temperature: 20EC. CE instrument: Beckman P/ACE 2000.
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Figure 19. Four different electrochromatograms taken with the same column during the period of 1 week. Column: EP-75-33-3-C18. Mobile phase: 80% CH 3 CN/20% 4mM sodium tetraborate (pH 9). Voltage: 15kV. Injection: 5kV/5s. Detection: LIF, ex: 257nm, em: 400nm. Sample: 1. naphthalene, 2. acenaphthylene, 3. acenaphthene, 4. fluorene, 5. phenanthrene, 6. anthracene, 7. benzo[b]fluoranthene, 8. pyrene, 9. benz[a]anthracene, 10. chrysene, 11. benzo[b]fluoranthene, 12. benzo[k]fluoranthene, 13. benzo[a]pyrene, 14. dibenz[a,h]anthracene, 15. benzo[ghi]perylene, and 16. indeno[1,2,3-cd]pyrene. |
. DAY TO DAY COLUMN EFFICIENCY
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Figure 20. Variations of day to day column efficiency in CEC over a period of two weeks. Column: EP-75-20-3-C18. Mobile phase: 80% CH 3 CN/20% 5mM phosphate (pH 6.5). Voltage: 10kV. Injection: 5kV/5s. Detection: 254nm. Temperature: 20EC. CE instrument: Beckman P/ACE 2000. |
. MOBILE PHASE EFFECTS IN CEC
ORGANIC MODIFIER EFFECT ON EOF
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Figure 21. Effects of organic modifier (CH3CN) on EOF in CEC. Column: EP-50-25-3-C18. Mobile phase: 55% to 95% CH3CN in 1mM TRIS (pH 9). Voltage: 20kV. Injection: 5kV/2s. Detection: fluorescence, ex: 254nm. em: 370nm. |
. ORGANIC MODIFIER EFFICT ON SELECTIVITY
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Figure 22. Retention order of three compounds can be completely reversed by changing organic modifier content in the mobile phase. Column: EP-75-20-3-CN (cyano column). Voltage: 10kV. Injection: 2.5kV/3s. Detection: UV at 254nm. Sample: 1. thiourea, 2. benzyl alcohol, and 3. naphthalene. |
. BUFFER EFFECT ON EOF
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Figure 23. Buffer effects in CEC. Column: EP-50-25-3-C18. Mobile phase: 70% CH 3 CN in 30% 1mM TRIS, sodium tetraborate and MES, respectively. Voltage: 20kV. Injection: 5kV/2s. Detection: fluorescence, ex: 254nm, em: 370nm. Sample: 1. EOF marker, 2. fluorene, 3. anthracene |
. GRADIENT CEC APPARATUS
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Figure 24. Schematic of gradient CEC apparatus with LIF detection. The electroosmotic flows in capillary A and capillary B are regulated by computer-controlled voltages. Results are shown in Figure 25. |
. RESULTS OF GRADIENT CEC
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Figure 25. Electrochromatograms showing the comparison of isocratic and gradient elution for the separation of the 16 PAHs. Column: EP-75-26-3-C18. Voltage: 20kV for the isocratic separations. Injection: 5kV/5s. Detection: LIF, ex: 257nm, em: 400nm. Sample: the same as in Figure 19 |
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