Conducting Capillary Electrophoresis

Clever variations of electrophoresis allow us to separate neutral molecules as well as ions, to separate optical isomers, and to lower detection limits by up to 106. Adaptations of electrophoresis provide a foundation for new technology called analysis on a chip. In the future, drug discovery and clinical diagnosis will depend on small chips carrying out unprecedented numbers of operations with unprecedented speed. 

The inside capillary wall controls the electroosmotic velocity and provides undesired adsorption sites for multiply charged molecules, such as proteins. A fused-silica capillary should be prepared for its first use by washing for 15 min each ( 20 column volumes) with 1 M NaOH and 0.1 M NaOH, followed by run buffer ( —20 mM buffer). For subsequent use at high pH, wash for 10 s with 0.1 M NaOH, followed by deionized water and then by at least 5 min with run buffer.28 If the capillary is being run with pH 2.5 phosphate buffer, wash between runs with 1 M phosphoric acid, deionized water, and run buffer.29 When changing buffers, allow at least 5 min of flow for equilibration. For the pH range 4-6, at which equilibration of the wall with buffer is very slow, the capillary needs frequent regeneration with 

Silica wall with bound negative charges 

1 M NaOH if migration times become erratic. Buffer in both reservoirs should be replaced periodically because ions become depleted and electrolysis raises the pH at the cathode and lowers the pH at the anode. The capillary inlet should be 2 mm away from and below the electrode to minimize entry of electrolytically generated acid or base into the column.30 Stored capillaries should be filled with distilled water. 

Example of covalent coating that helps prevent protein from sticking to the capillary and provides reproducible migration times  

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Figure 6.11 Separation of carboxylic acids (10 mJW each) by suppressed conductivity capillary electrophoresis. Conditions capillary, 60 cm X 75 jum I.D. fused silica voltage, +24 kV detection, suppressed conductivity using 15 mJV sulfuric acid as regenerant. Peaks (ppm) 1, quinic (1.92) 2, benzoic (1.44) 3, lactic (0.90) 4, acetic (0.60) 5, phthalic (1.66) 6, formic (0.46) 7, succinic (1.18) 8, malic (1.34) 9, tartaric (1.50) 10, fumaric (1.16) 11, maleic (1.16) 12, malonic (1.04) 13, citric (1.92) 14, isocitric (1.92) 15, cis-aconitic (1.74) 16, oxalic (0.90). (Reprinted from Ref. 63 with permission.)...

In capillary electrophoresis the conducting buffer is retained within a capillary tube whose inner diameter is typically 25-75 pm. Samples are injected into one end of the capillary tube. As the sample migrates through the capillary, its components separate and elute from the column at different times. The resulting electrophero-gram looks similar to the chromatograms obtained in GG or HPLG and provides... 

Detectors Most of the detectors used in HPLC also find use in capillary electrophoresis. Among the more common detectors are those based on the absorption of UV/Vis radiation, fluorescence, conductivity, amperometry, and mass spectrometry. Whenever possible, detection is done on-column before the solutes elute from the capillary tube and additional band broadening occurs. 

Limits of detection become a problem in capillary electrophoresis because the amounts of analyte that can be loaded into a capillary are extremely small. In a 20 p.m capillary, for example, there is 0.03 P-L/cm capillary length. This is 1/100 to 1/1000 of the volume typically loaded onto polyacrylamide or agarose gels. For trace analysis, a very small number of molecules may actually exist in the capillary after loading. To detect these small amounts of components, some on-line detectors have been developed which use conductivity, laser Doppler effects, or narrowly focused lasers (qv) to detect either absorbance or duorescence (47,48). The conductivity detector claims detection limits down to lO molecules. The laser absorbance detector has been used to measure some of the components in a single human cell (see Trace AND RESIDUE ANALYSIS). 

A.J. Zemann, Conductivity detection in capillary electrophoresis. TrAC Trends in Analytical Chemistry 20 (2001) 346-354. 

J. Muzikar, T. van de Goor, B. Gas and E. Kenndler, Extension of the application range of UV-absorbing organic solvents in capillary electrophoresis by the use of a contactless conductivity detector. J. Chromatogr.A 924 (2001) 147-154. 

Fused silica capillaries are almost universally used in capillary electrophoresis. The inner diameter of fused silica capillaries varies from 20 to 200 pm, and the outer diameter varies from 150 to 360 pm. Selection of the capillary inner diameter is a compromise between resolution, sensitivity, and capacity. Best resolution is achieved by reducing the capillary diameter to maximize heat dissipation. Best sensitivity and sample load capacity are achieved with large internal diameters. A capillary internal diameter of 50 pm is optimal for most applications, but diameters of 75 to 100 pm may be needed for high sensitivity or for micropreparative applications. However, capillary diameters above 75 pm exhibit poor heat dissipation and may require use of low-conductivity buffers and low field strengths to avoid excessive Joule heating. 

To apply a screening approach to proactive method development, analyses of selectivity samples under a variety of mobile phase conditions are conducted on different HPLC columns. HPLC columns should be as orthogonaT as possible and variations in solvent composition should be designed to maximize the probability of selectivity differences. Alternate separation techniques, such as ion exchange chromatography (IC), supercritical fluid chromatography (SFC), or capillary electrophoresis (CE) may also be used to obtain orthogonality. 

Timerbaev, A. R., and Hirokawa, T. (2006). Recent advances of transient isotachophoresis-capillary electrophoresis in the analysis of small ions from high-conductivity matrices. Electrophoresis 27, 323-340. 

Zemann, A. J. (2003). Capacity coupled contactless conductivity detection In capillary electrophoresis. Electrophoresis 24, 2125—2137. 

Kuban, P., and Hauser, P. C. (2004). Contactless conductivity detection In capillary electrophoresis a review. Electroanalysis 16, 2009—2021. 

Williams, R. C., and Boucher, R. J. (2000). Analysis of potassium counter ion and inorganic cation impurities in pharmaceutical drug substance by capillary electrophoresis with conductivity detection. J. Pharm. Biomed. Anal. 22, 115 — 122. 

The recent introduction of non-aqueous media extends the applicability of CE. Different selectivity, enhanced efficiency, reduced analysis time, lower Joule heating, and better solubility or stability of some compounds in organic solvent than in water are the main reasons for the success of non-aqueous capillary electrophoresis (NACE). Several solvent properties must be considered in selecting the appropriate separation medium (see Chapter 2) dielectric constant, viscosity, dissociation constant, polarity, autoprotolysis constant, electrical conductivity, volatility, and solvation ability. Commonly used solvents in NACE separations include acetonitrile (ACN) short-chain alcohols such as methanol (MeOH), ethanol (EtOH), isopropanol (i-PrOH) amides [formamide (FA), N-methylformamide (NMF), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA)] and dimethylsulfoxide (DMSO). Since NACE—UV may present a lack of sensitivity due to the strong UV absorbance of some solvents at low wavelengths (e.g., formamides), the on-line coupling of NACE... 

Nilsson, S., Wetterhall, M., Bergquist, J., Nyholm, L., and Markides, K. E. (2001). A simple and robust conductive graphite coating for sheathless electrospray emitters used in capillary electrophoresis/mass spectrometry. Rapid Commun. Mass Spectrom. 15, 1997-2000. 

Unlike capillary electrophoresis, wherein absorbance detection is probably the most commonly utilized technique, absorbance detection on lab-on-a-chip devices has seen only a handful of applications. This can be attributed to the extremely small microchannel depths evident on microchip devices, which are typically on the order of 10 pm. These extremely small channel depths result in absorbance pathlengths that seriously limit the sensitivity of absorbance-based techniques. The Collins group has shown, however, that by capitalizing on low conductivity non-aqueous buffer systems, microchannel depths can be increased to as much as 100 pm without seeing detrimental Joule heating effects that would otherwise compromise separation efficiencies in such a large cross-sectional microchannel [38],... 

Wang, J., M. Pumera, and G. Collins. A chip-based capillary electrophoresis-contactless conductivity microsystem for fast measurements of low-explosive ionic components. Analyst 127, 719-723 (2002). 

Wang, J., G. Chen, and A. Muck, Jr. Movable contactless conductivity detector for microchip capillary electrophoresis. Anal. Chem. 75, 4475-4479 (2003). 

Bioluminescence detector Charge-coupled device Contactless-conductivity detector Capillary electrophoresis Capillary electrophoresis-Electrochemistry Collision-induced dissociation Chemiluminescence detector Sodium chlorate-nitrobenzene Commercial off-the-shelf (U.S. Army) Cold Regions Research and Development Center Croatian Mine Action Center Council of Scientific and Industrial Research,... 

Guijt RM, Evenhuis CJ, Macka M, Haddad PR. Conductivity detection for conventional and miniaturised capillary electrophoresis systems. Electrophoresis 25, 4032-4057, 2004. 

Zemann AJ, Schnell E, Volgger D, Bonn GK. Contactless conductivity detection for capillary electrophoresis. Analytical Chemistry 70, 563-567, 1998. 

Borissova, M., Gorbatsova, J., Ebber, A., Kaljurand, M., Koel, M., and Vaher, M., Non-aqueous capillary electrophoresis using contactless conductivity detection and ionic liquids as background electrolytes in acetonitrile. Electrophoresis, 28, 3600-3605,2007. 

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