ELECTROPHORESIS

Principle:-

1. Biological molecules such as aminoacids, peptides, proteins, nucleotides &

nucleic acids possess ionizable groups and are made to exist as electrically
charged species either as cations or anions (+ve or −ve). Even carbohydrates
can be given weak charges by derivatization as borates or phosphates.
2. Molecules having same charges will have different charge/mass ratio (due to
different molecular weight).
These combination forms the basis of electrophoresis.
In electrophoresis, cation move towards cathode & anion move towards
anode at rates which depend upon the balance between impelling force of
electric field & frictional effect and electrostatic retarding effect between
sample and supporting medium.
To maintain a constant state of ionization, buffer is

Factors affecting migration rate in electrophoresis:-

These are 4 different factors that are affecting the electrophoretic mobility of
the sample. They are: (1) sample (2) electric field (3) buffer (4) supporting medium.

SAMPLE:-

a) Charge: Rate of migration increases with increase in net charge. The

magnitude of charge is pH dependent.
b) Size: Rate of migration decreases for larger molecules. This is due to
increased frictional & electrostatic forces which are exertd by the
surrounding medium.
c) Shape: Molecules having similar charge but different shape (such as
fibrous & globular proteins) exhibit different migration rate due to different
effects of frictional and electrostatic forces.

K. VINOTH KALAISELVEN., Academia.edu/ Asst. Prof., Islamiah College, VNB. Page 1

ELECTRIC FIELD:-

According to Ohms law,

I = V/R

i.e,

Current (Ampere) = Voltage (V) / Rsistance (R)

In electrophoresis, when a given voltage is applied between the electrodes, the

current passed will be determined by resistance exerted by the medium.

a) Voltage: Rate of migration under unit potential gradient is mobility of ions

[increase in potential gradient (voltage) leads to increase in the rate of
migration].
b) Current: Current in solution between two electrodes are conducted mainly
by buffer & small portions by sample ions.

If, no. of ions per m3 = ‘n’,

Area of each electrode = ‘A’ m2

Potential gradient = ‘V/I’ Volts m-1

Mobility of ions bearing charge Couloumbs towards one electrode = ‘α’

Then,

Speed of migration = α V/I m sec-1

Total charge per second conveyed to that electrode by ions (current) =nAα
V q/I couloumbs sec-1(or) amperes.

Increase in Voltage leads to increase in total charge per second conveyed

towards electrode (current). Distance migrated by ions is proportional to Hme.

c) Resistance: Electrphoretic medium exerts a resistance to a current flow

which is a function of the medium (the buffer & its concentration).

Rate of migration, total charge per second conveyed towards electrode is

inversely proportional to resistance.

1/R = n A α q/I

Increase in temperature causes decrease in resistance. And hence, increase in

rate of migration & increase in total charge per second conveyed towards electrode.

K. VINOTH KALAISELVEN., Academia.edu/ Asst. Prof., Islamiah College, VNB. Page 2

BUFFER:-

Buffer determines & stabilizes the pH of supporting medium & also affects of
migration rate of compounds in a number of ways.

a) Composition:

Buffers commonly used are acetate, barbiburate, citrate, EDTA formate,

phosphate, and pyridine. Buffers used should not bind with the compounds to be
separated as this may alter the rate of migration.

b) Ionic strength:

As ionic strength of buffer increases,

 Prorportion of current carried by buffer increases &

 Proportion of current carried by sample decreases and hence showing
decrease in sample rate of migration.

High ionic strength,

 Also increases overall current & hence heat is produced.

As ionic strength of buffer deccreases,

 Prorportion of current carried by buffer decreases &

 Proportion of current carried by sample increases and hence showing
increase in sample rate of migration.

Low ionic strength,

 Decreases overall current & hence heat is produced.

But the disadvantage is diffusion & loss of resolution.

The ionic strength is usually selected as 0.05 to 0.1 m.

C) pH: pH has got little effect on fully ionized compounds such as inorganic salts and
for organic compounds. pH determines the extent of ionization. Ionization of organic
acid increases as pH increases & ionization of organic base decreases as pH increases.
Therefore, degree of ionization is pH dependent. Both effects can be applied to
compounds such as amino acids which has both acidic & basic properties.

pH ACIDIC IOSOELECTRIC BASIC

Ionic form Cation Zwitter ion Anion
Migration Cathode Stationary Anode
towards

K. VINOTH KALAISELVEN., Academia.edu/ Asst. Prof., Islamiah College, VNB. Page 3

SUPPORTING MEDIUM:-

Although supporting medium is inert, its precise composition may cause

adsorption, electro osmosis and molecular sieving each of which may influence the
migration rate of compounds.

a) Adsorption:

Adsorption is retention of the sample

molecules by supporting medium.
Adsorption causes tailing of sample so that
it moves in the shape of a ‘comet’ rather
than as a distinct compact brands.
Adsorption reduces both the rate &
resolution of separation.
b) Electro–Endo osmosis:

Electro osmosis is due to the presence

of charged groups on the surface of
supporting medium.
Example:
Paper – Carboxyl group,
Agarose – sulphate group,
Glass wall – Silanol.
Above the value of 3, these charged groups will ionize & generates
negatively charged sites. These ionized groups create an electrical double layer
or region at supporting medium & electrolyte interface.
When voltage is applied cations, in electrolytenear suppoting medium
migrate towards cathode pulling electrolyte near supporting medium migrate
towards cathode pulling electrolyte solution with them. This creates a net
electro osmotic flow towards the cathode.
This electro osmosis will accelerate the movement of cations, but retard
anion movement.
c) Molecular sieving:
 Gels have sieve like structure.
 In agar, starch & polyacryl amide gels, the movement of large molecules
are hindered by decreasing the pore size, since all molecules have to
transverse through pores.
 If sephadex gel is used, small molecules are tightly held by pores and
large molecules are excluded by small pores causing movement outside
the spores.

K. VINOTH KALAISELVEN., Academia.edu/ Asst. Prof., Islamiah College, VNB. Page 4