Capillary Electrophoresis
Capillary electrophoresis (CE) provides first principle insight into the solution charge of macromolecules. Electrophoretic mobility, effective charge and diffusion coefficient determinations provide novel measurements of hydrodynamic and electro-hydrodynamic properties. CE offers some advantages to MCE in valence determination:
- Speed of data acquisition
- Low sample volume required
- Automated data collection
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| An Animation of CE With permission from the University of Warsaw Department of Chemistry: http://www.chem.uw.edu.pl/people/AMyslinski/informator_07/zdchn.html |
Other Capillary electrophoresis
The primary focus of CE at CAMIS is for valence determination, (see reference at bottom); however, CE is a versatile instrument capable of many types of electrophoresis.
- Capillary zone electrophoresis (CZE)High field strengths are used to separate proteins introduced into the capillary by either electrokinetic or hydrodynamic means. Differences in charge-to-mass ratios of a heterogeneous solution lead to discrete zones which appear as peaks on the electropherogram. This free solution technique requires only minute amounts of sample, and multiple runs can easily be automated. Untreated fused silica capillaries used at physiological pH can produce fast separations (a few minutes) due to electroosmotic flow (EOF). Coated capillaries are used for more basic proteins to prevent adsorption on the capillary wall.
- Capillary gel electrophoresis (CGE)A UV transparent linear polymer is used as a molecular sieve analogous to traditional SDS-PAGE. Separation is based solely on size. Molecular weight determinations are made by comparison to standards.
- Capillary isoelectric focusing (CIEF)As with traditional isoelectric focusing, a pH gradient is created using ampholytes. Samples will migrate through this gradient until their net charge becomes zero. Once the sample components are separated into bands based on their prospective pIs, a low pressure is applied to move them past the detector window.
- Micellar electrokinetic capillary chromatography (MECC)Micelles formed with traditional surfactants are used to aid in separations. Cationic sample components are electrostatically attracted by these micelles and elute last as the EOF works against micelle migration. Migration of anionic components is faster than migration of neutral molecules, which are separated intermediately based on hydrophobicity.
- Isotachophoresis (ITP)A discontinuous buffer system is used to preconcentrate injection volumes that are 10 to 100 times the normal. Following a short focusing period, the sample is separated as in CZE.
CE reference:
Chase SF, Laue TM. The determination of protein valence by capillary electrophoresis. P/ACE Setter 2008;12:1-12. (PDF 3.24 MB)
