Proteomics/Protein Separations- Electrophoresis/Capillary Electrophoresis

1. Introduction to Electrophoresis
2. Gel Electrophoresis
3. One Dimensional Gel Electrophoresis
4. Two Dimensional Polyacrylamide Gel Electrophoresis(2D-PAGE)
5. Differential in Gel Electrophoresis(DIGE)
6. QPNC-PAGE
7. Capillary Electrophoresis
8. Timeline of Electrophoresis
9. Databases
10. Web Pages
11. Online Applications
12. Further Readings

Capillary electrophoresis (CE)

In Capillary electrophoresis, electrophoresis is conducted narrow-bore capillaries filled with buffer. The capillaries used in the electrophoresis instrument typically have internal diameter in the range 25 to 100 μm.

Procedure

The ends of a capillary are placed in separate buffer solutions and electrodes connected to a high-voltage power supply are placed in the buffers. One of the buffer reservoirs, usually at the anode, is replaced by sample. The sample is introduced into the capillary by capillary action, siphoning or pressure. When an electric current is passed across the capillary, molecules are separated. Separated analytes are detected directly through the capillary wall by UV or fluorometric detector connected near the opposite end.

Insrumentation of CE

Electrophoretic mobility

The separation by electrophoresis depends on differences in the migration velocity of ions or solutes through the given medium in the electric field. The electrophoretic migration velocity ($u_p$) of an analyte is:

$u_p={\mu }_{p}E$

Where E is the electric field strength and ${\mu }_p$ is the electrophoretic mobility.

The electrophoretic mobility is inversely proportional to frictional forces in the buffer and directly proportional to sample's the ionic charge. The forces of friction against an ion is dependent on size of the ion and the viscosity (η) of the medium. Analytes with different frictional forces or different charges will separate from one another when they move through a buffer. At a given pH, the electrophoretic mobility of an analyte is:

${\mu }_p=\frac{z}{6\pi \eta r}$

Where r is the radius of the analyte and z is the net charge of the analyte.

The differences in the ratio of charge to size of the analytes cause differences in electrophoretic mobility. Small and highly charged analyts have greater mobility, whereas large and low charged analytes have lower mobility.

Electrophoretic mobility is an indication of speed of a given analyte in a give medium. It is the balance of electrical force that acts in favor and the frictional force that acts against the motion. These two forces remain steady during electrophoresis; therefore electrophoresis mobility is a constant for a given ion under a given set of conditions. Based on this characteristic property of ion or solute, it can be separated using electrophoresis.

Electro osmotic Flow (EOF)

In capillary electrophoresis, an analyte's migration velocity also depends upon the electro osmotic flow (EOF)rate of the buffer. The EOF is the bulk flow of liquid through the capillary when current is applied. The capillary tube used for CE is typically an uncoated fused-silica tube and its internal surface has silanol groups which are easily ionizable and give a negative charge to the capillary wall. Therefore, when the capillary is filled with buffer positively charged ions from the buffer are attracted towards the negatively charged capillary. This creates electrical double layer and zeta potential( potential difference)close to the capillary wall. The electrical double layer includes a rigid layer of adsorbed ions and a diffuse layer. As the capillary wall surface increases the zeta potential decreases. When an electrical potential is applied across the capillary positive ions in the diffuse layer migrate towards the cathode carrying the bulk solution with them causing the EOF. At high pH, the silanols are extremely ionized and make large surface charge of the capillary wall. This increases zeta potential which in turn increases EOF. Thus EOF is very dependent on pH, being large at high pH.

The velocity of the electro osmotic flow ($u_o$) is:

$u_o={\mu }_{o}E$

where ${\mu }_o$ is the electro osmotic mobility, which is same as:

${\mu }_o=\frac{ϵ\zeta }{\eta }$

where ε is the relative permittivity of the buffer solution and ζ is the zeta potential of the capillary wall .

The electro osmotic flow of the buffer solution is usually higher than the electrophoretic flow of the analytes and therefore, at pH>7 buffer solution carries all analytes (both positive and negative)toward the cathode. Positively charged ions are carried faster while negatively charged analytes stay longer in the capillary. Therefore, migration velocity of a solute is a combination of both EOF mobility and electrophoretic mobility. The migration velocity (u) of an ion or solute in an electric field is thus $u_p+u_o=({\mu }_p+{\mu }_o)E$

Flow and dispersion

In other separation techniques like HPLC, separations are driven by pressure and that results in frictional forces in places where the mobile phase is in contact with solid surfaces. These frictional forces cause the velocity of mobile phase close to the wall to be zero while that in the center is large resulting in a parabolic flow profile in the capillary. This profile results in the solute zones being broadened as the move through the capillary, reducing the resolution of the separation. But in CE, since the driving force is EOF, the flow is flat which result much higher resolutions than comparative pressure driven techniques.

Visualization

The data output from CE is an electropherogram, which is a plot of migration time vs. detector response. The response by detector is usually concentration dependent fluorescence or UV-visible absorbance. Different peaks are shown in the electropherogram for separated analytes depending on different retention times. The electropherogram shows separation peaks of cationic, anionic and neutral solutes in the mixture.

Advantages

This is advantageous in many ways. The heating that takes place due to high voltage loads on slab gels can have a negative effect on the separation of the proteins and the use of capillaries lessens said heat build-up. Also, because the gel will not need to be handled, it can allow one to use liquid polymers for separation, and can be replaced between runs. Automation is also much more possible with this technique, lessening the time, and making reproducible results.

Next Section Timeline of Electrophoresis

References

• http://www.chemsoc.org/ExemplarChem/entries/2003/leeds_chromatography/chromatography/
• http://en.wikipedia.org/wiki/Capillary_electrophoresis
• http://www.beckmancoulter.com/resourcecenter/labresources/ce/cedefinitionmodes.asp
• http://www.rsc.org/pdf/books/capelectrosc.pdf