ppt. by GULAM RAFEY &SAURAV,ON ELECTROPHORESIS

1. ELECTROPHORESIS
Beckmann column
chromatography instrument
Guided by By
Mr. Saurabh Pandey Saurav Ghoshal
Gulam Rafey
M.Pharm (Pharmaceutics)
PSIT
1

2. Electrophoresis
2
The term literally means “migration with
electricity.”
It involves the separation of components of a
sample by the differential rate of migration of
ions by attraction or repulsion in an applied dc
electric field.
The technique was first developed by Arne
Tiselius in 1930s for the study of serum proteins.

3. Why Electrophoresis
3
The process has exhibited unparalleled
resolution in the separation of charged species.
Electrophoresis may separate between the
polynucleotides which may differ by may be a single
or just a few nucleotides.
The Human Genome Project was possible only
as a result of electrophoresis.

4. Types of Electrophoretic techniques
4
Electrophoretic techniques may be of two types
A) Electrophoresis
-Slab electrophoresis-TLC
-Capillary electrophoresis-Column
B) Electrochromatography

5. Principle
5
The principle involved in both slab and capillary
electrophoresis are same which involves
electrophoretic mobility of the ions.
It has been found that the migration velocity
(v)(cms-1)of a molecule in an electric field is given by
v=µeE
where E=strength of electric field(Vcm-1)
µe=electrophoretic mobility (cm2V-1s-1)

6. 6
The value of (E) depends upon
 The charge of the analyte ion
 The frictional retarding forces
which includes
Size and shape of the ion
Viscosity of the medium in which migration
occurs

7. Plates
7
The plate count in electrophoresis is given by the formula
N=µeV/2D
Where V=applied voltage
D=diffusion coefficent of the solute(cm2s-1)
Because the capillary has a small cross sectional area and
longer length when compared with that of a slab and
hence has much higher resistance when compared to that
of a slab.
Thus in the capillary format much higher voltages
may be applied than in the case of slab which is of greater
advantage.

8. Electroosmotic Flow (EOF)
8
 An important feature of CE is the bulk flow of liquid
through the capillary this is called the electroosmotic flow
and is caused as follows.
 Silica capillary walls contain surface silanol(Si-OH) groups
and these are ionized at pH values higher than 3 and the wall
becomes (-)vely charged.
 Wall attracts cations and double-layer forms
 Cations in the diffuse layer are attracted towards cathode
and migrate in that direction as they are in solution the
buffer fluid is also dragged along with them.
 The direction of flow of EOF may be reversed by treatment
with cetyl trimethylammonium bromide.

9. Formation of double layer for electroosmotic flow
9

10. Net result of electrophoretic and electroosmotic
flow
10

11. Flow Profile in Electroosmotic flow
11
A key feature of EOF is that it has flat flow profile, which is shown in Figure
alongside the parabolic flow profile generated by an external pump, as used for
HPLC.
EOF has a flat profile because its driving force (ie., charge on the capillary wall)
is uniformly distributed along the capillary, which means that no pressure drops
are encountered and the flow velocity is uniform across the capillary.
In HPLC, in which frictional forces at
the column walls cause a pressure drop across
the column, yielding a parabolic or laminar
flow profile.
The flat profile of EOF is important because it minimizes zone
broadening, leading to high separation efficiencies that allow separations
on the basis of mobility differences as small as 0.05 %.

12. Slab electrophoresis
12
1. porous layer 2-10 cm long - paper, cellulose acetate,
polymer gel soaked in electrolyte buffer
2. Slow
3. simple but difficult to automate
4. poor quantitation
5. large quantities (mL)
6. large cross-sectional area, short length leading to low
electrical resistance, high currents
7. Vmax=500 V
8. N=100-1000 low resolution

13. Slab Electrophoresis
13

14. Slab Electrophoresis
14

15. Capillary Electrophoresis
15
 Electrophoresis in buffer filled, narrow-bore
capillaries.
 Each capillary is about 25-100 μm in internal
diameter.
 Small cross-sectional area, long length leading to
high resistance, low currents
 Vmax=20-100 kV
 N=100,000-10,000,000 high resolution

16. Contd...
16
 When a voltage is applied to the solution, the
molecules move through the solution towards the
electrode of opposite charge.
 Depending on the charge, the molecules move
through at different speeds.
Thus separation is achieved.
 A suitable detector is then used to detect the solute
as it comes out from the end of the capillary.
 The data obtained are analyzed by a computer and
represented graphically.

17. Line Diagram of instrumentation
17

18. Instrumentation
18

19. Instrumentation - Capillary
19
The length varies but normally 30-100 cm.
Internal diameter (10-100µm).
Small bore and thickness of the silica play a role.
Using a smaller internal diameter helps prevent Joule
Heating, heating due to voltage.

20. Capillaries (Column)
20
The capillaries are normally made of fused silica as in
the case of Gas chromatography Open Columns.
The column is coated with polyimide to improve the
durability.
Glass and Teflon have also been used but silica
provides best functionality.
These may be made hydrophobic by the incorporation
of alkyl groups which would shield the silanol groups
and thus reduce electroosmotic flow.

21. Injection
21
1.Remove anode end capillary from buffer.
2.Place end of capillary in sample.
3.Injection volume ranges from 5-50nl. While values
as low as 100pl have been reported.
3.Apply field for short time (electrokinetic
injection)discriminates against low migration rate
analytes or apply pressure for short time (pressure
injection) or place the sample chamber at a higher
level (Siphoning).
4.Replace in buffer.

22. Detector
22
 UV/Visible absorption
 Fluorescence
 Radiometric (for radioactive substances)
 Mass Spectrometry
 Electrochemical
 Conductivity
 Amperometry
 Potentiometry
 Indirect Detection

23. Detectors used and their limits of detection
23
Type of detector LOD (attomoles)
Absorption 1-1000
Fluorescene 1-0.01
Mass 1-0.01
Conductivity 100
Potentiometry 1
Amperometry 0.1

24. Detector Cells
24
a) 3 mm Z cell
b) 150 µm bubble cell
c) Multireflection cell

25. Variants of CE
25
Capillary Zone Electrophoresis
Capillary Gel Electrophoresis
Capillary Isoelectric Focusing
Capillary Isotachophoresis
Micellar Electrokinetic Chromatography

26. Capillary Zone Electrophoresis
26
In this the buffer composition is constant
throughout the region of the separation.
Here the anions/cations when allowed to migrate in
the field towards the anode and cathode respectively
tend to be separated into bands which may be separate or
may overlap depending upon the migration velocity.
The method may be used for both ions as well as
derivatized molecules.
Frequently used for separation of mixtures of
cations and anions. Synthetic herbicides, Pesticides,
pharmaceuticals may be separated and analyzed after
derivatization.

27. Zone Electrophoresis
27

28. Capillary Gel Electrophoresis
28
It is generally performed in porous gel formed by a polymer matrix.
The polymer used is normally Polyacrylamide ( monomer Acrylamide
CH2=CH-CO-NH2)
The gel used adds an additional component of sieving (size) into the
separation process.
Other gels used include
Agarose
Polyethylene Glycol
Methyl Cellulose
Polyvinyl Alcohol
Dextran
Polydimethylacrylamide, etc..
The method finds most frequent application in DNA sequencing.
The columns used may be of two types
 Low viscosity gels-replaceable
 High viscosity gels-fixed

29. Sieving Action
29

30. Capillary Isotachophoresis
30
In this process all analyte bands ultimately migrate at the
same velocity.
In this the sample is injected between two buffers , a
leading one containing ions of higher mobility than any of the
analyte ions and a terminating one with ions of mobilty lower
than the analyte ions.
At the beginning of the separation procedure all the ions
migrate at the different velocities and then as there is
separation the faster moving electrons experience a weaker
electric field as compared to that of the slow moving electrons
and thus are retarded. As this process of retardation based on
charge differences reaches equilibrium each of the bands are
separated on the basis of their migration rates.
Markers may be added between bands to separate them.

31. Isotachophoresis
31

32. Capillary Isoelectric Focussing
32
 In this technique the separation of amphiprotic substances can be
performed in a buffer soln. whose pH varies along its length. In this
procedure one end of the capillary bearing the cathode is dipped in
a soln. of strong base (NaOH) and the end bearing anode is dipped
into a soln. of a strong acid (H3PO4).
+
 When the process starts the H ions migrate towards the cathode
-
and the OH migrates towards the anode, thus establishing a pH
gradient, with higher pH towards anode.
 The analyte in combination with some other ampholytes (contain
carboxylic and amine gp.) is also introduced in the sample and these
migrate on the basis of their charge and during migration they may
be protonated or lose proton depending on the direction of
migration due to the existing pH gradient, the migration continues
till the pH region where the ion has reached is equal to the pI and at
this point the analyte or ampholytes becomes neutral. Once this
occurs several sharp bands are formed and no further migration
occurs.

33. Contd..
33
The bands may then be mobilized by adding NaCl to
-
base end and then this leads to less migration of OH
and then the pH gradient is disturbed leading to
migration of the soln. towards the cathode leading to
the detection of the bands.

34. Capillary Isoelectric Focussing
34
Theoretical
separation of
compounds with
varying pI

35. Micellar Electrokinetic Chromatography
35
• Earlier, neutral molecules were not considered to be separated by electrophoresis
• In 1984, Terabe and his group reported a technique that enabled capillary
electrophoresis (CE) instrumentation to be used in the separation of neutral (as well
as ionic) species=MEKC
MEKC is primarily used for separation of neutral analytes but is also used for
separation of ionic analytes
• In this system, micelles are described as “pseudo-stationary phase” and the buffer as
“mobile phase”=system for partitioning of analytes
• When micelles are not present, neutral molecules will migrate with the
electroosmotic flow and no separation will occur.
• MEKC has the capability of separating, within a single run, anionic, neutral, and
cationic species.
• The micellar solution is divided into 2 flow preferences. The aqueous buffer has a
strong flow toward the cathode (EOF) and the micelles are slowed down because
they have anionic character and due to electrophoretic mobility they are attracted
towards the anode.

36. Contd..
36
• The EOF is much stronger than the electrophoretic mobility, the solution is all
headed toward the cathode, but the micelles head at a much slower rate
because they are attracted to the anode on the opposite side.
• The negatively charged species are repelled by the anionic micelle while the
positively charged species are attracted to the micelle; because the micelle
elutes later than the buffer, so do the positively charged species which are
attracted to it
 The more negatively charged species - more repelled by micelle than the less
negatively charged species (elute earlier)
 The more positively charged species - more attracted (strong electrostatic
interaction) to the micelle than the less positively charged species (elute later
with the micelle)
 The neutral species migrate between the two extremes and their separation is
based on hydrophobicity

37. PACKED COLUMN
ELECTROCHROMATOGRAPHY
37
 Electrochromatography is a hybrid of HPLC & CE
that factor offers some of the best features of the two
methods. Like HPLC & MEKC, it is applicable to the
separation of neutral species or charged species.
 In electrochromatography, a polar solvent is usually
driven by electroosmotic flow through a capillary
packed with a reversed-phase HPLC packing.
Separation depend on distribution of the analyte
species between the mobile phase and the liquid
stationary phase held on the packing.

38. Field Flow Fractionation
38
This separation technique involves the separation of
the components of a mixture during its flow through
a ribbon like channel by the application of a thermal,
electrical, centrifugal or flow field.
As the components migrate through the channel
the channel the applied field in perpendicular
direction leads to the accumulation of the particles at
the bottom known as accumulation wall.

39. Field Flow Fractionation
39

40. Contd..
40
The ribbon like channel has following dimensions
 Length 25-100 cm
 Width 1-3 cm
 Thickness 50-500 µm
Depending on the type of applied field it may be of four
types:
Thermal
Electrical
Sedimentation
Flow
Application- Mainly for particles of high mol. mass and
neutral particles.

41. Applications
41
The vast applications of electrophoresis include
 Vaccine analysis.
 Protein and DNA analysis are also important
electrophoresis applications.
 It has allowed researchers to map and see the differences
in the genetic code of species on earth.
 Electrophoretic DNA analysis also provides a reliable tool
in forensic investigations.
 Determination of impurities.
 Chiral Analysis.
 Analysis of carbohydrates and other macromolecules.
 Analysis of inorganic anions/metal ions.

42. Vaccine Analysis
42
Vaccine analysis is one of the many important
applications of electrophoresis. There are several
vaccines that have been purified, processed and
analyzed through electrophoresis, such as the
influenza vaccine, hepatitis vaccine and polio
vaccine.
Manufacturers such as Wyeth, Merck and Sanofi-
Aventis have been using electrophoresis as an
effective vaccine analysis method.

43. DNA Analysis
43
Electrophoresis is one way of analyzing DNA, which is the unique code of
every individual.
Through electrophoresis, specific DNA sequences can be analyzed,
isolated and cloned. The analyzed DNA may be used in forensic investigations
and paternity tests.
For this wells are formed at one end of an agarose gel for the loading of
the DNA sample. The slab is then placed horizontally into the electrophoresis
buffer chamber The DNA migrates in bands toward the positive electrode. The
smaller molecules pass through the matrix more rapidly than the larger ones,
which are restricted. The DNA bands are then stained using a fluorescent dye
such as ethidium bromide. The stained gel is then viewed directly under
ultraviolet light and photographed.
Another technique for detection involves the use of different fluorescent
dyes which tend to bind to the bases of nucleotides and thus give unique staining
profile

44. Protein Analysis
44
Electrophoresis has advanced our understanding on
the structure and function of proteins. These
molecules are needed by our body cells and may be
analyzed, for instance, by getting blood and urine
samples. Then through electrophoresis, the amount
of proteins in your blood or in your urine is
measured and compared to established normal
values---lower or higher than the normal levels
usually indicates a disease.

45. Assay of Drugs
45
Atropine sulphate i.v. soln.
Codeine phosphate syrup
Ketamine HCl i.v.soln.
Phenylepherine HCl i.v. soln.
have sucessfully been assayed by this process.

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