electrophoresis- separation by charge migration

ELECTROPHORESIS

BINDU KSHTRIYA

ELECTROPHORESIS• Electrophoresis is a technique in which ions in solution are

separated based on their differences in size and charge.

• When a high voltage is applied to the solution positive ion migrates toward the negative electrode and negative ions

migrates to the positive electrode.

GENERAL PURPOSE OF ELECTROPHORESIS

• Determine the number, amount and mobility of a components in a given sample or to separate them.

• To obtain information about the electrical double layers surrounding the particles.

• Determination of molecular weight of proteins and DNA sequencing.

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

• The movement of charged particle in an electric field is expressed in terms of

electrophoretic mobility denoted by µ.

where

µ = v/E or µ = q/f

FACTORS AFFECTING ELECTROPHORETIC SEPARATION:

A. ANALYTE- nature of the compound affects the separation in many ways-

1. CHARGE– higher the charge, greater is the electrophoretic mobility.

2. SIZE-bigger the molecule greater are the frictional and electrostatic forces exerted on it by the medium. Consequently, larger particles have smaller electrophoretic mobility compared to smaller particles

3. SHAPE- rounded molecules elicit lesser friction compared to sharper molecules.

e.g. globular protein move faster than fibrous protein.

B.ELECTRIC FIELD- according to Ohm’s law the current flowing through the solution is given by

I=V/R

where I= current

V= voltage

R= resistance

1. VOLTAGE: regulates the current , rate of migration is proportional to voltage applied.

2. CURRENT: rate of migration is directly proportional to the current.

3. RESISTANCE: rate of migration is inversely proportional to resistance.

C.BUFFER- the nature of buffer used as an electrolyte is extremely important and it influences the success of the separation.

pH

The product of net charge & the field strength provides the total motive power for electrophoretic separation.

Since the net charge carried by each species in the solution is pH dependent, therefore, rate of migration of ion is affected by the pH of the medium.

D.TEMPERATURE- the mobility of ions increases with increase in temperature upto certain level, but higher level of temperature causes evaporation of solvent from bed.

TYPES OF ELECTROPHORESIS

FREE SOLUTION ELECTROPHORESIS

• In this, the sample solution is introduced as a bond at the bottom of U tube that has been filled with unstabilised buffer solution.

• The sample are usually injected into the bottom of the U tube through a capillary tube side arm.

• An electric field is applied and separation takes place as result of differences in mobilities.

MOVING BOUNDARY ELECTROPHORESIS

• U shaped tube is filled with buffer.

• Sample is injected at the bottom of the U-tube through a capillary

• An electrical field is then applied.

• Separation takes place as a result of differences in mobilities.

• Different fractions obtained are located by optical measurement using optical system i.e. by measuring difference in Refractive indices.

ADVANTAGES-• Greater sensitivity.

• Better separation.

• Direct method to separate solute from buffer solution.

• Concentration as low as 0.05 mg/ml can be detected.

DISADVANTAGES-• High equipment cost .

• Optical system required.

APPLICATIONS-• For analytical purpose –Protein mixture.

• To study colliding dispersion.

ZONE ELECTROPHORESIS

The separation is carried out in a stabilizing medium or supporting medium.

Types of supporting medium used :

• Filter Paper

• Cellulose Acetate

• Starch powder

• Starch gel

• Cellulose powder

• Agar gel

• Agarose gel

PAPER ELECTROPHORESIS

It is the form of electrophoresis that is carried

out on filter paper. This technique is useful for

separation of small charged molecules such

as amino acids and small proteins.

FILTER PAPER : It is the stabilizing medium. We can use what-man filter paper, cellulose acetate filter paper or chromatography paper.

APPARATUS : Power pack, electrophoretic cell that contains electrodes, buffer reservoirs, support for paper, transparent insulating cover

SAMPLE INJECTION

The sample may be applied as a spot(about 0.5 cm in diameter)or as a uniform streak.

ELECTROPHORETIC RUN :

The current is switched on after the sample has been applied to the paper and the paper has been equilibrated with the buffer. The types of buffer used depends upon the type of separation. Once removed, the paper is dried in vacuum oven.

DETECTION AND QUANTITATIVE ASSAY:

Fluorescence, ultraviolet absorption or radioactivity are exploited for detection.

CELLULOSE ACETATE ELECTROPHORESIS

Cellulose acetate is more homogeneous and stabilizes fluid more

efficiently, it gives better resolution than the paper.

Other advantages:

➢ Free of surfaces charges

➢ Can be made transparent for densitometry by treating

it with plasticizing agent

➢ Smaller sample capacity than paper

Strip of electrophoresis

Electrophoresis

Major protein components

separate into discrete

zones

Densitometer tracing density of

zone to the amount of protein

GEL ELECTROPHORESIS

Involves the use of a

gelatinous material such as

Agarose, polyacrylamide,

starch as the matrix.

The gel acts as a support

medium for the sample.

AGAROSE ELECTROPHORESIS

▪A highly purified uncharged polysaccharide

derived from agar.

▪Used to separate macromolecules such as

nucleic acids, large proteins and protein complexes.

▪It is prepared by dissolving 0.5% Agarose in

boiling water and allowing it to cool to 40°C.

▪It is fragile because of the formation of weak

hydrogen bonds and hydrophobic bonds

POLYACRYLAMIDE GEL

▪ Components: Acrylamide monomers, Ammonium

persulphate, Tetramethylenediamine (TEMED)

Etc.

▪These free radicals activate acrylamide monomers

inducing them to react with other acrylamide

monomers forming long chains.

SDS-POLYACRYLAMIDE GELELECTROPHORESIS

• SDS (also called lauryl sulfate) - anionic detergent

• Molecules in solution with SDS have a net negative

charge within a wide pH range.

• A polypeptide chain binds amounts of SDS in

proportion to its relative molecular mass.

• The negative charges on SDS destroy most of the

complex structure of proteins, and are strongly

attracted toward an anode (positively-charged

electrode) in an electric field.

ADVANTAGES OF ZONE ELECTROPHORESIS

▪ SIMPLE AND INEXPENSIVE.

▪ HIGH RESOLUTION POWER.

▪ SMALL QUANTITY OF SAMPLE CAN BE ANALYSED.

▪ DETERMINATION OF LOW MOLECULAR WEIGHT SUBSTANCES.

ZONE ELECTROPHORESIS

A D V A N T A G E S :

▪ Simple and inexpensive.

▪ High resolution power.

▪ Small quantity of sample

can be analyzed.

▪ DISADVANTAGES:

Less accurate.

Excessive spreading of zones.

Absorption of solute takes place.

APPLICATIONS OF ZONEELECTROPHORESIS

▪ In clinical ,diagnosis, analysis of serum, urine and

other body fluids.

▪ Convenient method of separating the inorganic ions.

▪ Desirable to measure the special kinds of proteins.

▪ For the study of chemical coagulation of water.

CONTINUOUS ELECTROPHORESIS

• A thin sheet of filter paper is usually used as a supporting

medium.

• Buffer solution is made to flow over the bed at a uniform

rate.

• Sample to be separated is applied in small stream that

• flows in the line across the bed in the same direction as

a buffer.

• Potential difference is applied across the electrodes, ions

• will migrate at different rates, and the components of the

• sample are carried out down by the flow of

buffer solution.

• Separation occurs at vertical direction.

APPLICATIONS OF CONTINUOUS ELECTROPHORESIS

▪ Determination of variety of charged species.

▪ Determination of low molecular ions, membranes, and

cells.

▪ Optimization for the fast and efficient separation.

THANK YOU

BINDU KSHTRIYA