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Capillary Electrophoresis

Electrophoresis is an analytical separation technique. It is especially used in clinical and research fields, and molecular biology. It deals with the separation and characterization of molecules like proteins, nucleic acids, small organelles and viruses.

The base of this technique is that the different charged particles of sample migrate under the influence of applied electrical field with the different rate. The separation is based on the size and the rate of migration of particles in applied electric field. The polyacrylamide, agarose gel etc are used as support medium.

 

What is Capillary Electrophoresis?

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Capillary electrophoresis is an analytical separation technique of electrophoresis. The capillary electrophoresis is a high resolution separation technique. The separation of ions is based on the electrophoretic mobility of ions under an applied voltage. The mobility or the migration of ions depends on some factors which include charge of the ion, the viscosity, and the size of atom or its radius.

The rate of migration of particles is proportional to the applied electric field. So the mobility of ions increases with increasing the strength of field. Only the ions migrate while the neutral species remains unaffected. The smaller ions have high migration rate due to their smaller size and so they move with less friction.

The capillary electrophoresis is good and widely used due to the following advantage.
  • It is a high resolution non chromatography separation technique.
  • It has low operating cost and small amount of sample is required.
  • The capillary electrophoresis is easy method and can be used for small ions to macromolecules like proteins, peptides, chiral compounds, nucleic acid.

1. Theory of capillary electrophoresis


The electrophoresis involves the migration of ions and the opposite charged ions are attracted towards to the oppositely charged electrodes. Thus the movement of oppositely charged ions towards electrodes is the basic phenomena of capillary electrophoresis techniques.

2. Electrophoretic mobility


The ions are moved in the applied electric field. The mobility depends on the viscosity and the applied field. The ions feel a force under applied field which is equal to the product of net charge and the applied electric field strength.

So the electrophoretic mobility is equal to µEP = q / f = q / (6πηr)
  • where, q denotes the net charge and f is the force which is equal to 6πηr.
  • The force is given by stock law for spherical particle where η and r stands for viscosity of the solvent and the radius of the atom respectively.
  • The velocity of ion v = (µEP x E) where, E stands for the magnitude of applied electric field.
  • The attraction of ions increases with increasing the strength of applied field because their velocity is increased.

3. Electroosmotic flow (EOF)


The CE is used to separate the compounds contain similar structure. The separation is done successfully due to the EOF. The solution starts to move towards cathode as the field is applied across contains an electrolyte solution. This is due to high voltage.

As the buffer solution initiates the migration through the silica capillary, the capillary wall becomes negatively charged due to loss of proton from the SiOH groups and the formation of SiO- ions. Thus the cation attracts towards the capillary wall and there is double layer of cation formed on the wall. One is stationary and other is diffuse layer.

The inner stationary layer is not free to move like the outer diffusion layer which can move along the capillary. Thus the electro osmotic flow rate is shown by the below mentioned equation;

µEOF = ε/4πη *E*ζ
,

where
  • ε, η, and ζ demotes the dielectric constant and the viscosity of the solution and zeta potential respectively.
  • E is for the field strength.
  • The electrophoretic mobility and EOF works in opposite direction to give good resolution.
  • As the electrophoretic mobility is always higher than the electro osmotic flow, so negatively charged particles get easily separated.
  • The optimum conditions for EOF are low resistance of the solution, high zeta potential of the cation layer, buffer solution with basic pH up to 9 at which all SiOH get ionized.
The EOF is also known as plug-flow as it is done in a plug shape flow. This is due to even flow of molecule.

Capillary Electrophoresis Instruments

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The capillary electrophoresis instrument contains the following components.
  • Cathode (Negatively charged electrode)
  • Positively charged electrode anode
  • Power Supply for electric field
  • Buffer solution at the cathode and anodic end is called catholyte and anolyte respectively.
  • Capillary tube up to size 25mm to 100mm
  • A detector and output device
The high voltage power supply is connected to anode and cathode from each side. The electrodes induce the electric field. This induced electric field initiates the migration of ions of sample through the capillary tube. The capillary tube is made up with fused silica. Sometimes the polyamide coated capillary tube is also used.

The capillary tube is dipped in a vial which contains the electrode and buffer or electrolytic solution. It is necessary to flush the capillary before introducing the sample in to the column. The absorbance is measured with UV-VIS light. There is small window near the cathodic end of the capillary to pass light through the analyte. The photomultiplier tube presents at the cathodic end of the capillary for getting mass spectrum. The mass spectrum is used to measure the mass to charge ratio of the present ionic species.

Capillary Electrophoresis Instrument

In some capillary instruments also contains temperature control device because the separation of the sample depend electrophoretic mobility and the viscosity which decreases with increasing the column temperature.

Injection methods


Samples can be injected through the two methods. One is Electro kinetic injection and other is Hydrodynamic Injection.

1. Electrokinetic injection
  • The electrokinetic injection method is used for analyzing the small concentration of ions.
  • In the electrokinetic method, the capillary is placed between catholyte at one end and the anolyte at another end.
  • The anolyte contains the sample.
  • The EOF moves from one end to another end of the capillary in the presence of electric field ad the ions also start to move from buffer solution to capillary.
2. Hydrodynamic injection
  • It works under applied pressure at one end of the capillary or in vacuum.
  • Thus there is pressure difference between the two ends of the capillary which initiate the movement of liquid into the capillary.

Separation Methods


The electrophoresis separation techniques are classified into two main systems. One is continues and other is discontinues. Continues system is further divided into kinetic with constant electrolyte composition and steady-state with vary electrolyte composition. So the kinetic system includes the below four type of capillary electrophoresis methods.
  • Capillary zone electrophoresis
  • Capillary gel electrophoresis
  • Micellar electrokinetic capillary chromatography
  • Capillary electrochromatography
The steady state includes the Capillary isoelectric focusing (CIEF). Discontinues system involves the Capillary isotachophoresis (CITP) separation method.

Capillary Zone Electrophoresis (CZE)

  • This is commonly used separation technique which is based on the differences in electrophoretic mobility at which the velocity of ion depends.
  • The electroosmotic velocity is adjusted by pH, viscosity, field, dielectric constant of buffer solution.
  • The silanol group gets ionized which form the double layer on the capillary wall.
  • Thus the cation of outer layer migrates towards the cathode under the applied electric field while anions get attracted to positively charged anode.
  • Thus the cation fist separate than the neutral species and the anions separate in the last as they have low charge to mass ratio than other.

Capillary Electrophoresis System

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The gel is used to separate the ions in the CGE. The separation method is based on the solute size difference. The use of gel reduces the diffusion and absorption of solute on the capillary walls and limits the heat transfer. This technique is used for the separation of protein and for the DNA sequencing samples. It is very sensitive method and small amount of sample is required for the separation.

For protein the SDA-PAGG is used. The SDA-PAGE method is based on electrophoretic mobility. This is sodium dodecyl sulfate-polyacrylamide gel electrophoresis which is used for the separation of protein. The SDA is a type of anionic detergent which binds to the polypeptides chains of protein and gives them negative charge. Thus it makes the partition of proteins.
  1. The DNA fragments are separated by size.
  2. The agarose gel is used for separation of large DNA molecule and the polyacrylamide gel is used for the small DNA fragments.
  3. The gel is dipped in the buffer solution.
  4. Tris-Borate or Tris-Acetate EDTA solutions are used as buffer solution.
  5. The migration of DNA depends on the size of fragments.
  6. The smaller fragments move faster than the bigger one.
  7. The separation of DNA fragments are detected with the use of dyes which binds the molecules.

1. Micellar Electrokinetic Capillary Chromatography (MEKC)

Micelles are aggregates which form by addition of a surfactant to the solution. These are not formed below the critical micelle concentration. This technique is based on the partition of solute between the solvent and micelles. The micelles have two parts. One is hydrophobic molecules and another is hydrophilic molecules.

The hydrophobic molecules present in the micelles and not move while the hydrophilic molecules have tendency to quick move. Thus for the separation, the micelles are first produced and then the polar negatively charged surfaces of micelles easily attract towards the positively charged anode and the separation occurs. The separation technique also affects with the pH, concentration of surfactants etc.

2. Capillary Electrochromatography (CEC)

This technique is similar to column chromatography. The CEC and CZE are similar in the plug-type flow. The mobile phase liquid passes through the silica gel.

3. Capillary Isoelectric Focusing (CIEF)

Like the CGE, the CIEF is also used for the separation of peptides and proteins. The peptides and proteins are zwitter ionic compounds which contain both positive and negative charges and a particular isoelectric point. The charge on the molecules depends on the attached functional groups and pH of the environment.

When the pH is equal to isoelectric point of molecule then there is no net charge on the molecule. But when the pH is below and above the isoelectric point then the molecule contains positive and negative charge respectively. Thus the pH factor is the base of separation. The anodic ends are present in the acidic solution at low pH while the cathodic is in basic solution at high pH.
4. Capillary Isotachorphoresis (CITP)

In the CITP, the migration of analyte occurs in consecutive zones whose length is measured to detect the quantity of sample.
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