Electrophoresis

1. PHARMACEUTICAL ANALYSIS 1
A
Seminar Presentation
On
ELECTROPHORESIS

2. Introduction
Principle
Electrophoretic Techniques
 Moving Boundary Electrophoresis
 Zone Electrophoresis
 Free Solution Method
 Gel Electrophoresis
 Paper Electrophoresis
 Immunoelectrophoresis
 Isoelectric Focusing
 Isotachophoresis
 Capillary Electrophoresis
Applications
References 2

3. It is a method based on differential rate of migration of charged
species in a buffer solution on application of dc electric field.
Developed by Swedish chemist Arne Tiselius for study of
serum proteins in 1930, was awarded the Noble prize in 1948.
It is been the principal method of separation of proteins
(enzymes, hormones, antibodies) and nucleic acids.
3

4.  Rate of migration (separation) depends upon e/m (charge to
mass) ratio
 The migration of particles or the rate of travel of particle, in
electrophoretic system depends on properties of the particles
as well as the instrumental system
1. Characteristic of particles
2. Property of electric field
3. Temperature
4. Nature of suspended medium
4

5. Mobility of particle is calculated by Strokes law:-
μ = Q/6πr η
where Q = charge on the particles
μ = mobility of particle
r = radius
η = viscosity of the medium
μ = Q/A π r2 η
where A has a value ranges 4 to 6 and is related to particle shape
5

6. The migration velocity v of an ion in cm/s in an electric field is
equal to the product of field strength E(V/cm) and the
electrophoretic mobility µe (cm2/V-s).
v=µe E
Electrophoretic mobility is prop.to ionic charge on the analyte
and inversely prop.to frictional retarding forces.
If two species differ in charge or frictional forces they will be
separated as they travel through buffer.
6

7. Neutral species are not separated.
The frictional retarding force is determined by the size and shape of
the ion and the viscosity of the medium.
For ions of the same size, greater the charge, the greater the driving
force and the faster the migration rate.
For ions of the same charge, the smaller the ion, the smaller the
frictional forces and faster the migration rate.
The ion’s charge-to-size ratio combines these two effects.
7

8.  Moving Boundary Electrophoresis
 Zone Electrophoresis
 Immunoelectrophoresis
 Isotachophoresis
 Capillary Electrophoresis
 Isoelectric Focusing
8

9. Principle: -
Method involves movement of charged particles in free moving
solution in absence of supporting medium.
9

10. Electro-Osmotic Flow : when a high potential is applied
across a capillary tube containing a buffer solution usually
results into migration of solvent toward the anode or cathode.
The cause of electro-osmotic flow is the electric double layer
that develops at the silica/solution interface.
In the presence of electro-osmosis, an ion’s velocity is the sum
of its migration velocity and the velocity of the electro-osmotic
flow.
10

11. Method:
The apparatus consists of a U shaped tube with provision for
introducing the cathode and anode electrodes into each of the
arms.
The sample solution is introduced and each arm is filled
carefully with a buffered solution.
If the sample consists of compounds with different mobilities,
their migration may be observed as several moving boundaries
11

12. 12
Initial
position
A +B +C
Substance
to be
separated
U tube
Buffer sol.
After
migration
A +B +C
C
B+C
A
A+B
+ve -ve
-ve+ve
Fig 1
Fig 2

13. Detection:-
Position of ions detected by measuring changes in refractive
index throughout the solution.
Advantages:-
Biologically active fractions can be recovered without using
denaturing agent.
As a reference method for measuring electro-mobility.
Gives information on isoelectric point and mobility of
compounds
13

14. Disadvantages:-
Complete separation is rarely achieved
Maintaining sharply defined boundaries (stabilization of
boundaries is needed)
Only fastest and slowest components can be separated in pure
form.
Not used for preparative and quantitative analysis.
Several problems are associated with the technique, including
stabilization of ion boundaries, boundaries anomalies, and the
need for specialized equipment
14

15. Zone electrophoresis makes use of a stabilizing medium to
minimize the problems associated with free-boundary
electrophoresis.
It involves migration of charged particles which are supported
on a relatively inert and homogenous solid or gel frame work.
Separated components are distributed into different zone in a
stabilizing media.
Make use of stabilizing media like paper, agar, cellulose,
starch, gels, polyacrylamide gels.
15

16. Types of supporting or stabilizing medium :
Free Solution Method
Gel Electrophoresis
Paper Electrophoresis
16

17. Rotating tube apparatus
Migration occurs in horizontal tube.
10 revolution / min
Micro syringe (small sample can be applied)
Profiles of zones are determined by scanning devices.
Advantages:-
Complete separation of electrophoretically different
components.
Small sample
17

18. 18
Cathode
Hydrogen
lamp motor
Electrophoretic
tubefilter
Sample
photomultiplier
Recorder
Seal
Free solution method

19. Electrophoresis in compact gels, which depends at least in part
on size-exclusion effects to achieve separation, is used
frequently for the separations of proteins and nucleic acids
The overall migration in these gels is a combination of
movement under the influence of the electric field and size
separation by the pores of the gel
It is carried out by using
Agar
Starch
Polyacrylamide
19

20. The most frequently used techniques of polyacrylamide gel
electrophoresis (PAGE) Is the discontinuous buffer system
developed by Laemmli.
In this procedure sample is placed on a stacking gel with a low
level of cross linking and therefore a large pore size. During
movement through this gel, the sample is concentrated into a
narrow band and then deposited onto a separating gel that has a
higher cross linking and smaller pore size. The separation of
the solutes occurs in this phase.
20

21. In a special modification of this technique used for separation
of proteins, a detergent, such as sodium dodecylsulfate (SDS),
is introduced in the buffer.
This interacts with the proteins to produce particles of
consistent shape and uniform negative charge so that
separation occurs according to size alone.
This enables the simple determination of molecular weight
because the migration distance is proportional to the logarithm
of molecular weight, as in size exclusion chromatography.
21

22. One of the simplest procedures in electrophoresis involves
spotting a mixture of solutes in the middle of a paper strip,
moistening the paper with some electrolyte and placing it
between two sheets of glass. The ends of the paper strips are
immersed in beakers of electrolyte
Electrophoresis is allowed to continue for a period of several
hours.
22

23. 23

24. Enzymatic and immunological methods also have been used
to detect proteins following electrophoresis in gels.
Immunochemical methods add an additional dimension to
protein identification. Following electrophoresis in an agar
gel backed with a microscope slide, an antibody is placed
into a trough cut parallel to the direction of electrophoresis.
24

25. The antibody and electrophoretically separated antigens
diffuse towards each other resulting in precipitin arcs where
antigen antibody complexes form. This technique has been
referred to as immunoelectrophoresis.
25

26. Technique based on moving boundary electrophoresis
Amphoteric substances 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 point and is
immobilized here.
26

27. 27
reservoir
Strongly acidic
solution e.g
phosphoric acid
pH
gradient
Acidic pH
Isoelectric
pH
Basic pH
Strongly alkaline
solution e.g
tetranol amine

28. Advantage:-
In separation and characterization of proteins in one step.
High resolution – (identifying iso enzymes)
Application:-
Useful for microanalysis of proteins
Identifying isoenzymes
28

29. Based on principle of moving boundary electrophoresis.
Separation is achieved either horizontally or vertically.
Solution in which the separation takes place is normally an
aqueous medium, which contains sucrose to provide a higher
density to the solution.
Where the separation by Isoelectric focusing depends on the
existence of a pH gradient in the system, the technique of
Isotachophoresis depends on the development of a potential
gradient.
A leading electrolyte (e.g. Chloride) with a higher mobility
than the analytes, and a trailing electrolyte (e.g. Glycinate)
with a lower mobility are used
29

30. Instrumental version in which electrophoresis is carried out in a
capillary and separated species is eluted out from one end of
capillary.
The long length and small cross-sectional area of the capillary
results into high resistance to solution. Because power dissipation
is inversely prop.to resistance, much higher potential (20,000-
60,000 V) can be applied.
This leads to corresponding improvements in speed and resolution.
Plate counts range from 1,00,000-2,00,000.
Yields high-speed and high resolution separations on small sample
volumes (0.1-10nL)
30

31. Order of elution in a typical capillary electrophoretic
separation is first the fastest cation followed by successive
slower cations, then all the neutrals in a single zone, and finally
the slowest anion followed by successively faster anions.
It is possible to revert the direction by adding cationic
surfactant to buffer.
The surfactant adsorbs on the capillary wall and makes the wall
positively charged, buffer anions congregate near the wall and
are swept toward the cathode or positive electrode.
31

32. A buffer filled fused silica capillary of 10-100µm in internal
diameter and 40-100cm long, extends between two buffer
reservoirs that also hold platinum electrodes.
Sample introduction is performed at one end and detection at
the other.
The polarity of the high-voltage power supply can be as
indicated or can be reversed for rapid separation of anions.
Sample introduction and detection is tedious because of small
volume of capillary µL.
32

33. Most commonly used methods are :
1. Electro-kinetic injection –
one end of capillary and electrode are removed from buffer
compartment and placed in a small cup containing sample.
A potential is then applied for a measured time causing
sample to enter the capillary by ionic migration and electro-
osmotic flow. It discriminates slower moving ions relative to
more mobile ions.
33

34. 2. Pressure injection :
• End of capillary is placed in the small cup containing
sample and pressure difference is then used to drive the
sample solution into the capillary. Pressure difference can be
achieved by applying vacuum at detector end or by
elevating to sample end. No discrimination due to mobility
of ions but cannot be used for gel filled capillaries.
• For both the volume injected is controlled by duration of
injection.
• Microinjection tips are also been constructed for volumes
such as pL (used in study of amino acids and
neurotransmitters)
34

35. In capillary electrophoresis each ion migrates at a rate
determined by its electrophoretic mobility. Thus analyte
band passes through the detector at different rates, which
result in peak areas that are dependent on retention times.
 Absorbance methods
 Electrochemical methods
 Conductometry
 Amperometry
 Mass spectrometric methods
35

36. Absorbance methods : both fluorescence and absorbance
detectors are used but more common are absorbance detectors.
To keep the detection volume to nL or small it is carried on-
column done by taking a small section of capillary and
removing the polyimide coating from exterior by burning,
dissolution, or scraping.
This reduces the detection path lengths small with respect to
conc so small that it cannot be measured.
36

37.  For increasing the sensitivity three main techniques are used
–
 Bending the capillary to ‘Z’ shape
 Bubble formation near the end of capillary.
 Reflection of radiation (reflective coating of silver)
 Indirect absorbance detection done by incorporation of ionic
chromophore in the buffer solution which makes the
detector receive constant signal and when analyte displaces
some of these the detector signal decreases.
37

38. Fluorescence detection –
Laser based instrumentation is used in order to focus the
excitation radiation on the small capillary and to achieve the
low detection limits available from intense sources.
Laser fluorescence detection have allowed detection up to
zeptomoles.
38

39. Mass Spectrometric Detection :
Small flow rates- 1 µL/min from electrophoretic capillaries
makes it feasible to couple the effluent to the ionization source
of a mass spectrometer.
Sample introduction/ionization interface currently used is
electrospray or fast atom bombardment is also used.
Widely used for detection of large molecules such as proteins,
DNA fragments and peptides.
Detection limits few tens of femtomoles for mol. Wt. 1,00,000
or more.
39

40.  Capillary electrophoretic separations are performed in
several ways called modes.
 These modes include-
 Capillary zone electrophoresis (CZE)
 Capillary gel electrophoresis (CGE)
 Capillary isoelectric focusing (CIEF)
 Capillary isotachophoresis (CITP)
40

41. 41

42. Buffer concentration is constant throughout the region of
separation.
Applied potential causes the different ionic components to
migrate according to its own mobility and separate into zones.
These zones may be completely resolved (buffer between
each) or partially overlapped.
Best for small ions as analyte move in the same direction as the
electro-osmotic flow.
For cations wall is untreated.
42

43. For anions the electro-osmotic flow is usually reversed by
treating walls of capillary with an alkyl ammonium salt- cetyl
triethylammonium bromide.
+ly charged ammonium ions get attached to –ly charged silica
surface and create a –ly charged double layer of solution,
which is attracted towards anode thus reversing the electro-
osmotic flow.
43

44. Applications of CZE :
Separation of molecular species-
Synthetic herbicides
Pesticides-allizarin, methyl xanthine.
Pharmaceuticals such as anti-inflammatory drugs- naproxen,
ibuprofen, tolmetin.
Proteins, amino acids and carbohydrates.
 Separation of smaller ions.
Advantages of CZE :
Lower equipment and maintenance cost.
Smaller sample size.
Greater speed and better resolution.
44

45. CGE performed in a porous gel matrix, the pores of which
contain a buffer mixture in which the separation is carried out.
This porous gel medium provides a molecular sieving action
that retards the migration of analyte species to various extents
depending upon the pore size of the analyte ions.
Most common type of gel used is a polyacrylamide (CH2=CH-
CO-NH2) polymer and cross-linking agent.
45

46. The pore size depends upon ratio of monomer to cross-linking
agent.
Increase in the amount of cross-linking agent results in smaller
pore size.
Other gels used are:
 Agarose- polysaccharide extracted from marine algae.
 Methyl cellulose
 Polyethylene glycol
46

47. Applications
Separation of macromolecules such as proteins
differing in their sizes.
e.g. lactalbumin, carbonic anhydrase, ovalbumin,
BSA, phosphorylase B.
Separation of DNA fragments and oligonucleotides
that have substantially the same charge but differ in
their sizes.
47

48. Used to amphiprotic species.
An amphiprotic compound is a species capable of both
donating and accepting a proton.
When glycine is dissolved in water 3 equilibria operate-
The AA product bearing a +ve and a –ve charge is called a
zwitterion (no migration).
48

49. No net migration occurs in an electric field when the pH of the
solvent is such that the conc of anionic and cationic forms are
identical.
The pH at which no net migration occurs is called isoelectric
point (pI) and is an important physical constant for
characterizing amino acids.
The pI is related to the ionisation constant of the species. Thus
for glycine Ka and Kb are-
49

50. Electrophoresis is employed in biochemical and clinical field.
In the study of protein mixtures
Antigen antibody reactions
In fractioning protein.
In analysis of lipoprotein
Hemoglobin
In combination with autoradiography
Separation of organic acid, alkaloids, carbohydrates, amino
acids, alcohols, phenols, nucleic acids, insulin.
In food industry
50

51. Skoog,15th
edition, 778-795.
www.google.com/electrophoresis
www.ce-resources.com
Notes of Dr. M.N.Noolvi sir
Pharmaceutical analysis, by “P.Parimoo”
Introduction to chemical analysis, by “H.K.Kaur”
Pharmaceutical analysis, by “Robert.D.Braun”
Instrumental methods of chemical analysis, by
“B.K.Sharma”
51

52. 52
Thank
you…