Presented by

Guided by Ms.Arpana
Mr. Obulesu M Pancholi
Lect. Deparment of M.sc
1. Introduction
2. Electrophoresis-an overview
3. Types of electrophoresis
4. Why separation technique?
5. What is capillary electrophoresis?
6. Why capillary electrophoresis?
7. Operation of a capillary electrophoresis
8. Modes of capillary electrophoresis
9. Capillary electrophoresis - The basics of instrumentation
10. Equipment
11. Basics
12. Electroosmotic Flow
13. Electrooosmotic Mobility
14. Flow profile in capillary electrophoresis
15. The electropherogram
16. Applications
17. Advantages & Disadvantage
18. Summary
19. Conclusion
20. References
 As in the emerging era separation of different
pharmaceutical components are important issue so for
that purpose different techniques are used.
 Among that one of the most efficiently used
separation technique is capillary Electrophoresis.
 This describes the basic theoretical concepts and
principles of capillary electrophoresis (CE).
 The depth of discussion should provide enough
background to understand the basic operation of CE
instruments and the principles by which CE separates
analytes.
 Definition : The differential movement for migration of
ions by attraction or repulsion in an electric field.
 This technique was pioneered by A.Tiselius in 1937.
 Separation of components of a mixture using an electric
field
V=Eq/f
v = velocity of molecule
E = electric field
q = net charge of molecule
f = friction coefficient
 Different forms of electrophoresis are used for each of
these factors independently or in combination.
 Routine Electrophoresis
 High Resolution Electrophoresis (HRE)
 Agarose gel electrophoresis (AGE)
 Polyacrylamide gel Electrophoresis (PAGE)
 Isoelectric Focusing (IEF)
 Capillary Electrophoresis (CE)
 Two-Dimensional Electrophoresis
 Immunochemical Electrophoresis
 Immunofixation Electrophoresis
 Electro immunoassay Electrophoresis
 Pulsed Field Electrophoresis
 Separation Science has grown to become one of
the most useful scientific tools.
 Used extensively in diagnostic and clinical
science.
 Separation Science is relied upon by scientists,
physicians, law enforcement officials and the
general public to provide quantified information
about our health, our food, our environment, our
products we use and in almost every aspect of our
modern society
Separation Scientists work in many
different disciplines, some of these include:
 Analytical Chemistry
 Biochemistry
 Biotechnology
 Forensic Science
 Food Science
 Clinical Science
 Neuro-Science
 Medical Research and Production
 Pharmaceutical and Nutraceutical
Science
 Other Disciplines
 In practical terms, a positive (anode) and negative
(cathode) electrode are placed in a solution
containing ions.
 Then, when a voltage is applied across the
electrodes, solute ions of different charge, i.e.,
anions (negative) and cat ions (positive), will move
through the solution towards the electrode of
opposite charge.
 Capillary electrophoresis, then, is the technique of
performing electrophoresis in buffer-filled, narrow-
bore capillaries, normally from 25 to 100 pm in
internal diameter (ID).
 In 1980, Jorgensen and Lukacs popularized the
use of capillary electrophoresis.
 It has many advantages over conventional
electrophoresis techniques this advantages are as
follow
- very high level automation is possible
- fast analysis times
- detection of separated peaks is done online
- heat generated inside the capillary is
effectively dissipated through the walls of the
capillary, therefore high voltages can be applied
 Free solution capillary electrophoresis ; Separation
based on size and charge differences.
 Micellar Electrokinetic capillary electrophoresis ;
Separation of neutral compounds using surfactant
micelles
 Capillary gel electrophoresis ; Separation by sieving
of solutes through a gel network.
 Capillary Isoelectric focusing ; Separation of
zwitter ionic solutes within a pH gradient
 Capillary electrochromatography ; Separation
involves both electrophoretic and chromatographic
procedures.
 Typically, a capillary connects two buffer
reservoirs.
 The buffer reservoirs have electrodes dipped into
them.
 The capillary passes through a detector.
 When sample is loaded onto this capillary and
voltage applied across electrodes, electrophoretic
separation takes place.
 Operation of a CE system involves application of a
high voltage (typically 10-30 kV) across a narrow
bore (25-100 mm) capillary.
 Commercially available CE instrument consist
of
- an electrolyte filled capillary which passes
through the optical centre of a detector.
- a sample injector.
- a high voltage power supply.
- an auto sampler
 The entire instrument is computer control
 Voltages used fall in the range of 10-30 kV
 This translate in currents in the range of 20-
200 mA
Capillary tube
- Varied length but
normally 25-50 cm
- Small bore and
thickness of the silica play
a role
- Using a smaller internal
diameter and thicker walls
help prevent Joule
Heating, heating due to
voltage.
Detector
- UV/Visible
absorption
- Fluorescence
- Radiometric (for
radioactive
substances)
- Mass Spec.
 A photocathode is
then used to measure
the absorbencies of the
molecules as they pass
through the solution
 The absorbencies are
analyzed by a computer
and they are
represented graphically
 A vitally important
feature of CE is the
bulk flow of liquid
through the capillary.
 This is called the
electroosmotic flow and
is caused as follows.
Stern’s model of the double-layer charge distribution at a
negatively charged capillary wall leading to the generation of a
zeta potential and EOF
Zeta Potential
 The change in
potential across a
double layer
 Proportional to the
charge on the capillary
walls and to the
thickness of the
double layer.
 Both pH and ion
strength affect the
mobility
 A further 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 %.
The data output from CE is
presented in the form of an
electropherogram, which is
analogous to a chromatogram.
 An electropherogram is a plot
of migration time vs. detector
response.
The detector response is usually
concentration dependent, such as
UV-visible absorbance or
fluorescence.
The appearance of a typical
electropherogram is shown in
Figure for the separation of a
three component mixture of
cationic, neutral and anionic solutes.
 CE finds numerous applications in biotechnology &
particular in pharmaceutical industry
 CE plays an important role in ensuring a constant product
quality of therapeutic glycoprotein
 CE is of major use in the isolation of the correct optical
isomer
 Analysis of carbohydrates
 Analysis of inorganic anions/metal ions
 DNA profiling
 Protein identification
 This technique has the potential to be a far better
analytical technique compared to other conventional technique
and can even detector those performance enhancing drugs
Advantages Disadvantages
- Fast - Cannot identify
- Small Sample neutral species
- Relatively - Joule Heating
inexpensive - Cannot discern shape
- Automated
1. CE is based on the principles of electrophoresis.
2. The speed of movement or migration of solutes in CE
is determined by their size and charge.
3. Small, highly charged solutes will migrate more
quickly than large, less charged solutes.
4. Bulk movement of solutes is caused by EOF.
5. The speed of EOF can be adjusted by changing the
buffer pH used.
6. The flow profile of EOF is flat, yielding high
separation efficiencies.
7. The data output from CE is called an
electropherogram.
 It is the most efficient separation technique
available for the analysis of both large and small
molecules.
 DNA Profiling, protein identification, inorganic
metals and ions can be detected easily by this
method.
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R.C Dubey ; “Text book of Biotechnology” : 4th edition : S.

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