Points to remember-
Ion |
Migrates towards |
Cation(+) |
Cathode |
Anion(-) |
Anode |
Purpose
To determine number , amount, mobility of components in a given sample OR to separate them |
To obtain information about the electrical double layers surrounding the particles |
Factors affecting electrophoretic mobility-
Sample |
Charge |
Higher the charge greater is electrophoretic mobility |
Buffer |
composition |
Type of buffer depend upon what sample is to run |
(charge to mass ratio) |
|
Charge depends upon pH |
|
|
e.g, formate, citrate, phosphate, EDTA, acetate pyridine, Tris, and barbitone etc |
|
Size |
Bigger the molecule greater the frictional force |
|
ionic strength |
between 0.05-0.1M |
|
|
Larger the particle slower the electrophoretic mobility |
|
|
Higher the strength of buffer more the current carried by buffer ions that affect electrophoretic run |
|
Shape |
Rounded molecules has lesser frictional and electrostatic retardation compared to sharp molecules |
|
pH |
Determines degree of ionization of organic compounds |
|
|
Globular and Fibrous proteins |
|
|
Amino acids shows charge based on surrounding pH |
Electric field |
|
rate of migration under unit potential gradient is referred to as mobility of the ion |
Medium |
led to electro-osmosis |
adsorption |
|
|
increase in potential gradient increases the rate of migration |
|
|
molecular sieving |
|
|
current (total charge carried per second to the electrode) in the solution placed between two electrodes is carried mainly by the buffer ions, only a small proportion being carried by the sample ions. An increase in the potential-difference therefore increases the current. |
Advantage over free electrophoresis
Microliters of sample are sufficient |
Sample components during their migration split up into as many different zones as it contains differently migrating components. each zone consisting of a single component which can be easily isolated |
stabilizing system does not allow the zones to disperse and spoil the separation |
Many detection schemes are Available And elution helps in further analysis |
Gel electrophoresis
Separation not only depends on the charge on the molecule but also on its size. |
Gels are porous and the size of the pores relative to that of the molecule determines whether the molecule will enter the pore and be retarded or will bypass it. |
Resolution of a sample is sharper and better in a gel than in any other type of medium. |
Electrophoretic Mobility in Gels
Molecular sieving action of the gels and its effect on the mobility of a macromolecule. |
The pore size thus the molecular sieving action and therefore the effect on electrÂophÂoretic mobility of a molecule are functions of gel concenÂtration |
Kr= C (R + r) |
Kr is the retardation coefficient. C is constant. R is the mean radius of the macromolecule and r is the radius of the gel fibers. |
Solubilizers
To study subunit composition of oligomeric proteins |
Solubilizers destabilize native structure of proteins by destroying charges that associate subunits together |
Urea - Disrupt hydrogen bonds At high concentration (3-12M). Also, dsDNA can be rendered into ssDNA by use of urea. |
SDS - SDS is an anionic detergent and disrupts macromolecules whose structure has been stabilized by hydrophobic associations. Also imparts a large negative charge to the denatured polypeptides |
beta mercaptoethanol - Disrupt Disulphide bridges. Separation of peptides in proteins Linked by disulphide bonds |
Types of Gel
Starch Gel, Agarose Gel, Acrylamide Gel and Agarose-Acrylamide Gel |
Starch Gel
High porosity starch gels are obtained by using 2% (w/v) starch and low porosity gels are obtained by adding 10-15% starch to the buffer. |
The pore size in a starch gel cannot be controlled and this is the biggest drawback of these gels. |
Difficult to prevent contamination of starch gels by microorganisms. |
Another disadvantage of starch gels is that upon staining to detect the separated components, the starch gel turns opaque making direct photoelectric determination impossible. |
the resolving power of starch gels is very high and can be matched only by polyacrylamide gels. One of their important applications is the analysis of isoenzyme patterns (zymograms). |
|
|
Principle
Any charged ion or molecule migrates when placed in an electric field. |
The rate of migration depend upon its net charge, size, shape and the applied electric current. |
Electrophoretic mobility (µ)
Electrophoretic mobility (µ) of an ion is used, which is the ratio of the velocity of the ion to field strength (v/E) |
Electrophoretic mobility v is directly proportional to the charge and inversely proportional to the viscosity of the medium, size and shape of the molecule |
Free electrophoresis
carrier-free electrophoresis |
matrix-free electrophoretic separation technique |
used to quantitatively separate samples according to differences in charge or isoelectric point. |
Two Main Techniques- Microelectrophoresis and Moving Boundary Electrophoresis. |
Moving boundary electrophoresis
allows the charged species to migrate in a free moving solution in the absence of a supporting medium |
Samples are fractioned in a U shaped tube that has been filled with buffer |
An electrical field is applied by means of electrodes at the ends of the U tube and Separation takes place as a result of difference in mobilities |
Method was very popular for quantitative analysis of complex mixtures of macromolecules, especially proteins,e.g., those in blood plasma. |
Zone electrophoresis
separation technique employing stabilizing media |
electrophoresis in stabilized media |
Paper electrophoresis
Filter paper as a stabilizing medium is very popular for the study of normal and abnormal plasma proteins |
Paper of good quality should contain at least 95% of cellulose and should have only a very slight adsorption capacity. |
Chromatography paper is suitable for electrophoresis and needs no preparation other than to be cut to size. |
Two arrangements of paper in paper electrophoresis are horizontal and vertical |
Process
Filter paper |
Apparatus - Power pack and Electrophoresis cell |
Sample application |
Electrophoretic run |
Detection and Quantitative Assay
Fluorescence |
Ultraviolet absorption |
Staining |
Detection of enzymes in situ |
Applications of paper E-
Separating amino acids into acidic and basic |
Separation of enzymes in blood |
Protein separation in serum |
Studying SCA in blood |
|
|
History
Time |
Late 18th century |
- |
- |
1931 |
Scientist |
Faraday |
Johann Wilhelm Hittorf, Walther Nernst, and Friedrich Kohlrausch |
Friedrich Kohlrausch |
Arne Tiselius |
Experiment |
Laws of electrolysis |
To measure the properties and behavior of small ions moving through aqueous solutions under the influence of an electric field |
created equations for varying concentrations of charged particles moving through solution, including sharp moving boundaries of migrating particles |
Molecular separation and chemical analysis |
Problems
Generation of heat (of the electrophoretic medium) has following effects- |
increased rate of diffusion of sample and buffer ions leading to broadening of the separated samples |
thermal instability of samples that are rather sensitive to heat. |
formation of convection currents, which leads to mixing of separated samples |
decrease of buffer viscosity, and hence a reduction in the resistance of the medium |
Electroendosmosis
electroosmotic flow |
Cause- |
Due to the presence of charged groups on the surface of the support medium. |
E.g |
Eg: Carboxyl groups in paper, sulphate impurities in agarose, SiOH groups in capillary electrophoresis |
Points
1. Electroosmotic Flow (EOF): Movement of liquid in response to an electric field, toward the cathode. |
2. Zeta Potential: Surface charges on the gel form an electrical double layer, creating a zeta potential that drives ion flow. |
3. Cation Migration: Cations near the capillary wall migrate toward the cathode, dragging the solvent with them. |
4. Electric Field Setup: An anode (positive) and cathode (negative) are placed to create the electric field across the medium. |
5. Ion Separation: EOF aids in separating analytes; positive ions move quickly to the cathode, while negative ions are slowed. |
6. Applications: Used in capillary electrophoresis for separating molecules like DNA and proteins by charge and size. |
Microelectrophoresis
method for determination of zeta potentials |
apparatus-capillary cell, two chambers that include electrodes, and a means of observing the motion of particles. |
apparatus is filled with very dilute suspension and the chambers are closed. |
direct-current voltage is applied between electrodes in the respective chambers. |
One uses a microscope to determine the velocity of particles. |
Zeta potential values near to zero indicate that the particles in the mixture are likely to stick together when they collide, unless they also are stabilized by non-electrical factors. |
Particles having a negative zeta potential are expected to interact strongly with cationic additives |
In modern days this technique is applied only for measuring the zeta potentials of cells such as R.B.Cs, neutrophils, bacteria etc. |
Cellulose Acetate Electrophoresis
Advantages |
it is chemically pure |
cellulose strips are translucent |
very low content of glucose. |
Cellulose acetate is not very hydrophilic and thus holds very little buffer. |
|