Isoelectric Focussing
MODERN PHARMACEUTICAL ANALYSIS AND
TECHNIQUE
Seminar
on
Isoelectric Focussing
Topic: Introduction, Principle, Instrumentation, Working and
application of Isoelectric Focussing
Presented By: Ranjitha P Nandeesh
Dept. of Pharmacology Dept. of pharmaceutics
I M. Pharm I M. Pharma
Submitted To: Dr. Somashekar P L
Copy To: Mr/Ms
Government College of Pharmacy, Bangalore
P. Kalingarao Road, Subbaiah Circle, Mission Road
Bengaluru, Karnataka
560 027
Ranjitha P and Nandeesh (2021-22), Govt College of Pharmacy, Bangalore 1
Isoelectric Focussing
ISOELECTRIC FOCUSING
Definition
Isoelectric focussing also known as electro focusing. It is a technique for separating
different molecules by difference in their isoelectric point. It is a type of zone
electrophoresis usually performed on proteins in a gel.
Isoelectric pH
It is the pH at which a molecule carries no net electrical charge or is electrically neutral
in the statistical mean. The pH at which the net charge on the protein is zero. For a
protein with many basic amino acids, the pH will be high, while for an acidic protein,
the pH will be low. The proteins with below isoelectric pH are positive and with above
are negative.
Separation of proteins at the isoelectric point is called isoelectric focusing. Separation
occurs on the basis of the positive or negative groups present on the molecule.
Isoelectric technique is based on moving boundary electrophoresis. Amphoteric
substance such as amino acids and peptides are separated in a specially designed
vertical column down to which there is both Ph and voltage gradient. Each compound
migrates towards the region in the column. where the pH corresponds to that of its
isoelectric points and immobilised here. Isoelectric focussing is a modification or
variation of zone electrophoresis.
Principle involved in Isoelectric focusing
Here proteins are focussed at their isoelectric pH, where they carry no net charge.
They are neutral. The electrophoresis separation method which separates amphoteric
molecules such as proteins and peptides according to their charges as defined by pKa
values of proton accepting sites within a molecules, substance which are initially at
pH regions above their isoelectric points will be negatively charged and will migrate
towards the anode until they reach isoelectric points and becomes stationary the
anode upper end of the column is connected to a reservoir containing an acidic
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solution like phosphoric acid and cathode (lower end is connected to reservoir
containing alkaline solution like sodium hydroxide).
On opening the two reservoir valves the two solution are allowed to diffuse into the
column from their respective ends setting up a pH gradient between the acidic anode
and alkaline cathode.
The valves are then closed and the current is switched on causing the carrier
ampholytes to migrate until they reach the pH regions where they have no net charge.
then they will remain stationary at these points. Therefore isoelectric pH for different
components varies from one another, they can be easily resolved based on their
isoelectric point resulting in well defined protein bands.
Theory involved in isoelectric focusing
IEF also known as electro focusing is a technique for separating charged molecules
usually proteins or peptides on the basis of their isoelectric point i.e. the pH at which
the molecule has no charge. IEF works because in an electric field molecule in a pH
gradient will migrate towards their pH.
An amphiprotic compound is a species that in solution is capable of both donating
and accepting a proton. A typical amino acid such as glycine is an amphiprotic
compound.
The amino acid in the product bearing a both positive and negative charge is called a
zwitter ion.
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Isoelectric Focussing
The zwitterion of an amino acid containing as it does a positive and a negative charge
has no tendency to migrate to an electric field but the singly charged anionic and
cationic species are attracted to oppositely charged electrodes. No net migration of the
amino acid occurs in an electric field when the pH of the solvent is such that the
concentrations of the anionic and cationic are identical. The pH at which no net
migration occurs is called the isoelectric point and is an important physical constant
for characterizing amino acids. The isoelectric point is readily related to the ionisation
constants for the species. An amphiprotic compound is a species that in solution is
capable of both donating and accepting a proton. A typical amino acid such as glycine
is an amphiprotic compound. When glycine is dissolved in water,
NH2CH2COOH NH3+CH2COO-
NH3+CH2COO- +H20 NH2CH2COO- + H30+
[𝐻3𝑂+][𝑁𝐻2𝐶𝐻2𝐶𝑂𝑂−]
Ka = =2×10-10
[𝑁𝐻3𝐶𝐻2𝐶𝑂𝑂−]
NH3+CH2COO- + H2O NH3+CH2COOH + OH-
Kb = [ OH-][NH3+CH2COOH] =2×10-12
[NH3+CH2COO-]
The first reaction constitutes a kind of internal acid base reaction and is analogous to
the reaction one would observe between a simple carboxylic acid and an amine. The
typical aliphatic amine has a base dissociation constant of 10-4 to 10-5 and many
carboxylic acids have acid dissociation constants of about the same magnitude. The
result is that the first reaction proceeds far to the right with the product far to the right,
with the product or products being the predominant species in the solution.
The amino acid product in the first reaction bearing both positive and a negative
charge is called a zwitterion. As shown by the equilibrium constants for the second
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Isoelectric Focussing
and third reactions the zwitter ion of glycine is stronger as an acid than as a base. Thus
an aqueous solution of glycine is somewhat acidic. For glycine at the isoelectric point,
[NH2CH2COO-] = [NH3CH2COOH]
If we divide Ka by Kb and substitute this equality, we obtain at the isoelectric point
Ka\Kb=[ H30+]iso\[OH-]iso
Substituting Kw\[H3O+]iso for [OH-]iso in the above equation and rearranging yields
[H30+]iso= √𝐾𝑎𝐾𝑤\𝐾𝑏
We can convert the above equation in to the iso electric Ph by taking the negative
logarithm on both sides. Thus, pH can be expressed as
pH=( pKa+pKw-pKb)\2
For glycine,
pka=-log(2×10-10)=9.7,pkb=11.7 and pkw=14.0
Thus pH =(9.7+14.0-11.7)\2=6.O
Hence isoelectric point for glycine occurs at a pH of 6
Instrumentation and working;
In isoelectric separation of amphiprotic species, the separation is performed in a buffer
mixture that continuously varies in pH along its length. This pH gradient is prepared
from a mixture of several different ampholytes in an aqueous solution. Ampholytes
are amphoteric compounds usually containing carboxylic and amino groups.
Ampholyte mixtures having different pH ranges can be prepared or available from
several commercial sources.
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Isoelectric Focussing
To perform an isoelectric focusing experiment in a capillary tube, the analyte mixture
is dissolved in a dilute solution of the ampholytes which is then transferred to the
tube. One end of the capillary is then inserted in a solution of strong base, such as
sodium hydroxide, that also holds the cathode.
The other end of the tube is immersed in a solution of a strong acid such as phosphoric
acid that also holds the anode. When the electric field is applied, hydrogen ions begin
to migrate from the anode compartment toward the cathode. Hydroxide ions from the
cathode begin to move in the opposite direction. If a component of the ampholyte or
the analyte has a net negative charge,
it migrates towards the positive anode. As it migrates it passes into continuously
lower pH regions, where progressive protonation of the species occurs, which lowers
its negative charge. Ultimately, it reaches the pH where its net charge is zero (its
isoelectric point). Migration of the species then ceases. This process goes on for each
ampholyte species and ultimately provides a continuous pH gradient throughout the
tube. Analyte ions also migrate until they reach their isoelectric points. These
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Isoelectric Focussing
processes then result in the separation of each analyte into a narrow band that is
located at the pH of its isoelectric point Very sharp focussing is realised in such
systems. Note that isoelectric focussing separations are based on differences in
equilibrium properties of the analytes (Ka, Kb) rather than on differences in rates of
migration. Once each analyte has migrated to a region where it is neutral, the positions
of the bands become constant and no longer change with time.
Modification of the focused bands
To detect the focused bands in a capillary isoelectric focusing separation, it is
necessary to move or mobilise the contents of the capillary so that the bands pass the
detector located at one end. This mobilisation can be accomplished by applying a
pressure difference just as for sample loading or by simply changing the solution in
the electrode compartment. During the focusing step equal numbers of H+ and OH-
ions enter opposite ends of the capillary so the pH gradient remains stable. Suppose
that sodium chloride is added to the sodium hydroxide solution after focussing is
finished. Now both Cl- ions and OH- ions migrate in to the cathode end of the column
and the sum of these two concentrations is balanced by H+ entering the opposite end.
That means that there is now less OH- than H+ flowing into the capillary. The pH
decreases at the cathode end. The pH gradient is no longer stable. It moves towards
the cathode end and along with it the focussed bands. The bands that pass the detector
first are the ones corresponding to proteins with the most alkaline isoelectric points.
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Isoelectric Focussing
Some examples for proteins with their isoelectric pointprocedure involved in
isoelectric focusing:
Isoelectric focusing involves adding of an ampholyte solution into immobilised ph
gradient gels. These gels are made up of acrylamide gel matrix co polymerised with
results in completely stable gradients is obtained by the continuous change in the ratio
of immobilines. An immobiline is a weak acids or base defined by its pka value.
A protein that is in a pH region below its isoelectric points will be positively charged
and so will migrate towards the cathode (negatively charged electrode). As it migrates
through a gradient of increasing pH, however the proteins overall charge will
decrease until the protein reaches the pH region that corresponds to its principle. At
this point it has no net charge and so migration ceases (as there is no electrical
attraction towards their electrodes). As a result the proteins becomes focused into
sharp stationary bands with each protein positioned at a point in the pH gradient
corresponding to its principle. The technique is capable of extremely high resolution
with proteins differing by a single charge being fractionated into separate bands.
Separation based on their relative content of acidic and basic residues,
Protein is introduced into an immobilised pH gradient gel composed of
polyacrylamide, starch or agarose where a pH gradient has been established. Gels
with large pores are usually used in this process to eliminate any effects or artefacts
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in the principle caused by differing migration rates for proteins of different sizes.
Isoelectric focusing can resolve protein that differ in the principle values by as little as
0.01
Isoelectric focusing is the first step in two-dimensional gel electrophoresis in which
proteins are first separated by their principles and then further separated by molecular
weight.
Factors affecting isoelectric focusing
1.Establishment of pH gradients:
Stable, linear pH gradients are the keys to successful isoelectric focusing.
Establishment of such gradient is accomplished in two ways with two different types
of molecules, carrier ampholytes and acryl amido buffers carrier ampholytes are
mixture of molecules containing multiple aliphatic amino and carboxylate groups.
The use of carrier ampholytes is the most common and simplest means for forming
pH gradient acryl amido buffers are derivatives of acrylamide containing both
reactive double bonds and buffering groups.
2.Gels for isoelectric focusing:
As an analytical tool. Isoelectric focusing is carried out in large pore polyacrylamide
gels which serves mainly as anti-convective matrices, polyacrylamide isoelectric
focusing gels are polymerised with an interior system including riboflavin for photo
polymerisation. The effects of resolution are very high, high electric fields also results
in shortened run times.
3.Protein solubilisation for isoelectric focusing:
Is that some protein tends to precipitate carrier ampholytes sometimes help overcome
precipitation and they are usually included in the sample solutions for ipg strips in
addition, non-ionic detergents or urea are often included in isoelectric focusing to
minimise protein precipitation.
Advantages of isoelectric focusing:
1.As spreading of bands is minimised due to the application of the applied field and
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the pH gradient, high resolution can be achieved.
2.Protein that differ by as little as 0.001 pH units can be separated.
3.IEFs greatest advantage is its high resolution, resulting in greater separation of
solutes. IEF of serum proteins result in many more bands, these bands are sharper
because each pH region is very narrow.
4.Performing IEF is easier because the placement of sample application is not
important. The sample and ampholytes can be mixed before application, the
ampholytes will migrate, create the gradient and then the proteins separate and
migrate.
5.Some isoenzymes and variant haemoglobins in prenatal screening are separated
with IEF. Detection of oligoclonal bands in gamma-globulin is a newer use of IEF.IEF
is commonly used as one of the separations in two-dimensional electrophoresis.
Disadvantages of isoelectric focusing:
1.Limited stability of solutions.
2.Carrier ampholytes generally are used in relatively high concentration, a high
voltage power source(upto 2000v) is necessary and power is in the vicinity of 2 to 50w.
3.Proteins usually tend to precipitate therefore solubilising agents are added.
4.Inadequate purity for application as a standard.
5.High volume of rehydration solution needs
Applications
1.Mainly used for separating proteins and peptides.
2.Preparative scale procedure for the isolation of purified fractions.
3.Used in clinical, forensic and human genetic laboratories for the separation and
identification of serum protein by the food and agricultural industries.
4.Used for research in enzymology, membrane bio chemistry, microbiology and
immunology.
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Isoelectric Focussing
References:
1. Fundamentals of Analytical Chemistry, 7th edition, Thomson cole book
publishing by SKOOG WEST HOLLER
2. Righetti.PG,2011 isoelectric focusing theory,methodology and application
3. Analytical chemistry by khupkar
4. Internet sources(images)
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