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1. MODERN PHARMACEUTICAL ANALYTICAL
TECHNIQUES
(MPC 101T)
TOPIC : ELECTROPHORESIS
RITIKA PAL
M.PHARM
FIRST SEMESTER
DEPARTMENT OF PHARMACEUTICAL
CHEMISRTY

2. INTRODUCTION
 Electrophoresis as an analytical tool was introduced
by the Swedish chemist Arne Tisel first in his doctoral
thesis in 1930, then later in a modified and improved
form (Tisel 1937).
Electrophoresis is one of the widely used techniques in
molecular biochemistry, microbiology, biomedical
research.
Electrophoresis is similar to other separation
techniques like chromatography but it differs in-terms
of the types of samples analyzed, the method used for
separation, principle used etc.

3. DEFINITION
Electrophoresis can be defined as electrophoresis is a
method of separation where in charged molecules
migrates in differential speeds in an applied electric
field."
The term Electrophoresis means :
Electro = electric field + Phoresis = migration.

4. PRINCIPLE
The charged molecules under the influence of electric
field migrate towards oppositely charged electrodes.
Those molecules with +ve charge move towards
cathode and -ve molecules move towards Anode.
The migration is due to charge on the molecules and
potential applied across the electrodes.
The sample under test is placed at one end of the
paper near one of electrodes.
When electricity is applied, the molecules start
moving to respective electrodes.

5. Anode Cathode
Movement of charged molecules under influence of an electric
field
But the movement is influenced by molecular weight and
charge on the molecule.
If charge on molecule is more, the migration is faster. If
charge on molecule is less, then migration is slower.
More molecular weight molecules migrate slowly, where as
smaller molecules move faster.
So when a mixture is placed on the electrophoresis paper
or agarose gel, different bands are seen along the paper after
the process.
µ = Q
6 πгη

6. Where,
µ = Electrophoretic mobility
Q = Charge of the molecule (esu)
r = Radius of the molecule (cm)
n= Viscosity of the medium
(poise)

7. TYPES OF ELECTROPHORESIS
Electrophoresis can be broadly divided into 2 types as:
1. Slab electrophoresis
2. Capillary electrophoresis

8. SLAB ELECTROPHORESIS
The slab method is the classical method which is
widely used for industrial scale.
It is slow, time consuming and bulky.
Yet it is the sole method available for separation of
proteins like enzymes, hormones, antibodies and
nucleotides like DNA and RNA.
This slab electrophoresis is further divided into 3 types
based on the principle used for separation.

9. a. Zone electrophoresis
In which charged particles are separated into different
zones or bands. This is of two types
1. Paper electrophoresis.
2. Gel electrophoresis.

10. b.Isoelectro-focusing
Here the isoeletric pH is set at different foci and hence
the molecules are immobilized to their isolectric
point.
They don't move towards electrodes but stay at a
specific isoelectric pH.
This is even more efficient to separate proteins and
from serum, 40 bands of protein can found.

11. c. Immuno electrophoresis
 This is the method with combination of principles of
both electrophoresis with immune reactions.
First the proteins are separated on to the
electrophoresis paper.
Then the antibodies are allowed to diffuse through the
paper and react with separated protein molecules in
bands.

12. FACTORS AFFECTING
ELECTROPHORETIC MOBILITY
 The following are the factors affecting migration of
ions in paper electrophoresis.
1) Charge of ions
The electrophoretic mobility is directly proportional
to the charge of the molecules, which means that
mobility of molecules is higher when its charge is
higher. Hence when a mixture is ions with X + and X
2+ are separated, the band of X 2+moves faster than
X+.

13. 2). Size of the ions
The electrophoretic mobility is inversely proportional to
the size of molecule ie. the mobility is more, when the size
is less and vice versa. The mobility also depends on the
shape of the molecules.
3). Viscosity of the medium
The electrophoretic mobility is inversely proportional
to the viscosity of the medium (buffer or other substance
used).

14. 4). Voltage applied
Higher the voltage applied faster the sepration and sharp
bands are obtained . However steps have to be taken to
prevent evaporation of the solvent / buffer , due to the heat
generated by high voltage .
5). pH of the buffer and ionic strength
The ionic strenght (IS) of the buffer used in paper
electrophoresis affects the migration velocity of the
compounds. Migration of compounds is inversely
propertional to the ionic strenght .

15. At low ionic strength ,migration is faster , but the sepration
bands apperar diffused usually ionic strengths (IS) of 0.05-0.5
is used in most separations.
6). Temperature
The electrophoretic mobility is directly proportional to the
temperature.

16. i). PAPER ELECTROPHORESIS
It is a technique which employs a Whattman filter
paper No.1 which is moistened by a buffer and then
connected at two ends to two opposite charged
electrodes.
 Then sample is applied on to one end and let for
separation of components under electric gradients.
After separation, the paper is dried and stained to get
coloured bands.
 These coloured bands are recognized for the nature
of sample by comparing with the standard. For a
sample of serum, 5 bands of proteins can be seprated
by paper electrophoresis.

17. COMPONENTS OF PAPER
ELECTROPHORESIS
I ) . PAPER
In Paper electrophoresis, paper is used as the
supporting medium. Normally Whatman filter
paper (Grade 3MM or No.1) of suitable dimension
with a length so that both end of the strip of paper
touch the buffer solution, kept in the electrode
vessels. The paper to be used is washed with
distilled water followed by 0.1M HC1 or 0.01M EDTA
to remove impurities.

18. ii). ELECTRODES AND VOLTAGE TO BE APPLIED
The electrode in the form of a thin wire is made up of
carbon or platinum. A DC voltage of about 8-15V/cm length
of paper is normally applied.
Types of Paper Electrophoresis (PE) There are two types of
Paper electrophoresis based on the voltage applied,

19. i). Low voltage electrophoresis : The voltage across two
electrodes is about 100-300V, with acurrent of 0.4mAmp
per cm width or 1.5mAmp/ strip.
ii). High voltage electrophoresis : A potential of about
50-215V/cm (Total 10,000V/strip) isapplied across the
electrodes.

20. iii). Buffers : Buffers of different pH and ionic strength are
used in separation process. The pH of buffer to be used
depends upon the types of compounds to be separated. The
ionic strength (IS) of the buffer used in paper electrophoresis
affects the migration velocity of compounds. Migration of
compounds is inversely proportional to the ionic strength. At
low ionic strength, migration is faster, but the separated
bands appear diffused. Usually ionic strengths (IS) of 0.05 -
0.5 is used in most separations.

21. Example of buffers
1. Barbitone buffer (verona’ buffer) (0.07mole/liter ,pH
8.6),IS -0.05.
2. Iris-acetate buffer (0.07 mole/liter,pH 7.6).
3. Citrate buffer (0.07 mole/liter,pH3.0 or pH 6.8 )
 Other buffers of different pH and ionic strength can also
be
used for sepration ,based on the type of compounds.

22. Types of paper electrophoresis instruments
based on the design of the instrument
1. Horizontal paper electrophoresis
2. Vertical paper electrophoresis
3. Continuous paper electrophoresis
Horizontal and vertical types are used in analytical sacle,where
as continuous electrophoresis is used on a preparative scale(i.e A
Large quantity of sample mixture is seprated into individual com-
-Pounds and they are used for different purpose ). The principles
Of all types are same ,but the design of each instrument varies.

23. a). Horizontal paper
electrophoresis
 Buffer solution of known pH and ionic strength is filled in two
beakers / troughs.
 Whatman filter paper of suitable grade and convenient
dimensions are cut and immersed in buffer solution.
 10-20µl of sample solution is applied at the centre of the paper
and fixed in position.
 The transparent lid is closed for safety as well as to prevent
evaporation of buffer.
 A suitable potential is applied across two electrodes dipped in
buffer solution.
 The migration of ions takes place towards respective electrodes.
 Non-ionisable or neutral substances do not move. Hence
separation of compounds from the mixture takes place

25. b). Vertical paper electrophoresis
 The migration of ions takes place by gravity.
 After sufficient migration, the paper is taken out and
dried, to fix the spots or bands.
 Then the compounds or bands or spots can be visualized
like in paper chromatography.
 Quantitation of spots can also be carried out using
densitometer.

27. c). Continuous electrophoresis
In this type predetermined sample volume through a
valve device is applied continuously on the centre of
paper.
 Suitable Voltage causes migrations of samples and
compound separated as bands.
 Thus each band is made to fall down and pure
compounds are collected in separate containers.

29.  ADVANTAGES
1. The technique is easy to handle.
2. The cost of instrument is low because only paper and
buffer are used.
3. Number of samples can be useprated on a single paper at
a time.

DISADVANTADE
S
1. The techniques requries more separation time.
2. Use of high voltage may be dangerous so paper
precaution should be taken.

30. GEL ELECTROPHORESIS
Gel electrophoresis
There are several problems associated with the use of both
paper and cellulose acetate as support media in
electrophoresis . The introduction of gels as an alternative
overcame some of these problems and was developed from
their use in chromatography . Where in instead of paper ,a
gel made of agarose ,sodium dodecyl sulphate ,
polyacrylamide gels (pages), starch blocks or cellular
acetate strips are used as medium for separation .

32. TECHNIQUES OF GEL ELECTROPHORESIS
1. GELS
 A gel is a three-dimensional polymeric network with
a random structure.
 The gels used in electrophoresis are cross-linked
polymers that should be inert and not interact with
the molecules under study.
 It is particularly important that the material does
not have any ionisation groups.

33. Unlike those used in chromatography, the gels used for
electrophoresis are not granular.
Instead, they are formed into continuous columns or
slabs, which give them very different properties compared
to chromatography gels.
An electrophoresis gel consists of a network of polymer
molecules surrounded and penetrated by buffer.
The spaces between the gel molecules are the pores of
the gel.
 When an external voltage is applied, charged sample
molecules migrate towards the appropriate electrode.
A molecule moving through a continuous gel
experiences a frictional resistance to its movement which
is related to the relative sizes of the gel pores and the
radius of the molecule.

34.  A molecule smaller than the average pore size
of the gel will be able to move relatively easily
through the gel, whereas a molecule larger than
the average pore size will b e held back by a
resistance that depends on its radius.
 If a molecule is very much larger than the gel
pores it may not be able to enter the gel at all.
 A continuous gel therefore experts sieving
effects on the passage of molecules through it.

36.  ADVANTAGES
1. The separation is more efficient than paper type as the
rate of running of molecules is slow and area of
separation is larger by thickness.
2. The method is more effective than paper and for
instance from serum eamples, 15 proteins bands can be
isolated .

DISADVANTAGES
1. The technique requires more separation time.
2. It is costly than paper electrophoresis.
3. Use of high voltage may be dangerous so proper
precautions should be taken

37. CAPILLARY ELECTROPHORESIS
 As the name indicates, here the process of separation takes
place inside a capillary tube.
 The capillary electrophoresis is an advanced method of
electrophoresis.
 This was developedwith intent to minimize the time taken
for separation and analysis in slab electrophoresis.
 High performance capillary electrophoresis (HPCE) or
simply called as capillary electrophoresis (CE) employing a
narrow bore fused silica capillary tube, has proved to be
very powerful separation technique. Another new
separation technique is Capillary electrochromatography
(CEC).

38. PRINCIPLE
 In Capillary Electrophoresis a capillary is filled with a conductive
fluid at a certain pH value.
 This is the buffer solution in which the sample will be separated.
A sample is introduced in the capillary, either by pressure
injection or by electrokinetic injection.
 A high voltage is generated over the capillary and due to this
electric field (up to more the sample components move
(migrate) through the capillary at different speeds.
 Positive components migrate to the negative electrode, negative
components migrate to the positive electrode.
 When you look at the capillary at a certain place with a detector
you will first see the fast components pass, and later on the
slower components.

39. INSTRUMENTATION

40. WORKING
A). MOBILITY
 Mobility of ion is dependable on size and charge.
 The size is a combination of the sample component and the shield of
water that is bound to the component.
 Even a small ion can be big due to a large water shield. In general, the
bigger the the slower it will migrate through the buffer.
 The charge of ions can be strongly dependent on pH value.
 That is the reason why a buffer at certain pH is used for separations.
 By changing the pH of a buffer system, the migration of the different
components can be altered to achieve the best separation.
 In general the best pH for a separation is between the pK values of the
sample components.

41. B) ELECTRO OSMATIC FLOW
(EOF)
In most applications the capillary that is being used is made of bare
fused silica.
This material has at its surface silanol groups (Si-O-H).
These groups are slightly acidic. In buffers at higher pH value there
are a lot of negative charges at the capillary wall
(Si- O-).
In the buffer fluid positive charges will be present because of the
law of electrical neutrality.
When a high voltage is generated over the capillary, these positive
charges will start to migrate through the capillary towards the
negative electrode.
They will drag along the buffer fluid with them. This flow is called
the Electro Osmotic Flow (EOF). It is well possible to calculate a
mobility of this EOF.
The higher the pH, the more negative charges on the capillary wall
and the more positive charges in the fluid.
This will generate a stronger EOF.

42. C). SEPARATION
The positive charges are all located close to the capillary wall, and
there is no pressure force in the middle of the capillary, the flow
profile of the EOF is completely flat.
This will cause no peak broadening like the parabolic flow profile in
HPLC and GC, and that is one of the reasons why such a high
resolution can be achieved in CE.
As mentioned before the EOF is towards the negative electrode.
This flow drags along neutral components (which would not
migrate without any fluid flow), and even positive components,
whose mobility is lower than the mobility of the EOF, will migrate
towards the negative electrode.
In this way in one run negative, neutral and (slow) positive
components can be separated and detected.

43. ADVANTAGES
1. Capillary electrohporesis requires small sample in the range if 0.1 to
10 ml while slab method requires in pl range.
2. It yields high speed and high resolution separations.
3. The separated components which exit from one end of capillary, are
immediately analysed by detectors fixed at the end of tubes.
DISADVANTAGES
It is time-consuming, expensive and technical skilled procedure
required.

46. THANK YOU