The document provides an overview of electrophoresis, including: 1) Electrophoresis is a separation technique where charged molecules migrate at different rates in an electric field, allowing separation. 2) It is used to separate biological substances like proteins, nucleic acids, and amino acids. 3) There are different electrophoresis methods including capillary electrophoresis, which uses narrow capillaries, and zone electrophoresis using papers, gels, or thin layers to support molecules. 4) Factors like each molecule's charge, size, and the electric field strength determine their movement during electrophoresis.

1. ELECTROPHORESIS
Sudheer kumar kamarapu
Assistant professor
Sri Shivani college of Pharmacy
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2. CONTENTS
 Introduction
 Principle And Theory
 Factors
 Classification
 Capillary electrophoresis
 Zone Electrophoresis
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3. INTRODUCTION
 The word electrophoresis is derived from a Greek word, which
means borne by electricity.
 It is a separation technique in which the components are
separated due to their varying behaviour under the influence
of applied electric field.
 It is defined as the migration of charged molecules under the
influence of external electric field.
 The major requirement of the component to be subjected to
electrophoresis is that the component should be charged.
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4. INTRODUCTION (cont..,)
 It is mostly used for the separation of complex biological
substances such as:
 Proteins
 Polysaccharides
 Nucleic acids
 Peptides
 Aminoacids
 Oligosaccharides
 Nucleosides
 Organic acids
 Small anions and cations in body fluids
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5. Cont...
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 Science
 Other Disciplines
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6. PRINCIPLE
 This technique is mainly used for separation of complex
mixtures of biological substances which possess ionisable
functional groups.
 Therefore they can be made to exist as electrically charged
species, either as cation/anion.
 Molecules with similar charges will have different m/e ratio.
 This forms basis for differential migration when these ions in
solution are subjected to an electric field.
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7. PRINCIPLE (cont..,)
 Therefore electrophoresis can be applied to any mixture in which the
components carry a charge & have differential mobilities in an electrical
field.
 The migration in an electrophoretic system depends on properties of
particle as well as instrumental system
 Based on Stoke’s law the mobility of particle (µ) can be calculated from
µ = Q/π r η
Where,
Q= charge of particle
µ= mobility of ion
r= radius of particle in cm
η = viscosity of medium
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8. TECHNIQUES OF ELECTROPHORESIS
 It can be carried out by using either:
1. Low voltage
2. High voltage
Low voltage electrophoresis:
• It consists of two compartments to hold the buffer &
electrodes & a suitable carrier for support medium, such
that its ends are in contact with buffer compartments
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10. TECHNIQUES OF ELECTROPHORESIS
(cont..,)
• The design of carrier depends on the medium
• The medium doesn’t dip into electrode compartments, but
into separate compartments connected by wicks with anode
& cathode cells
• The apparatus is enclosed to avoid evaporation
• LVE can be used in principle to separate any ionic substances
• Its main application is examination of biological and clinical
specimens for aminoacids and proteins
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11. TECHNIQUES OF ELECTROPHORESIS
(cont..,)
High voltage electrophoresis:
• The construction of apparatus is similar to that of LVE except
that it contains additional cooling system.
• It was found that much reduced analysis time could be
achieved by using high voltage gradient
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12. CLASSIFICATION
A. Capillary electrophoresis
B. Zone electrophoresis
1. Paper electrophoresis
2. Cellulose acetate electrophoresis
3. Thin layer electrophoresis
4. Gel electrophoresis
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13. Capillary
Electrophoresis
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14.  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 cations
(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).
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15.  Definition: The differential movement for migration of
ions by attraction or repulsion in an electric field.
 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
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16. Capillary Electrophoresis – The Basics
Of Instrumentation
 Electrophoresis in a buffer filled, narrow-bore
capillaries
 Each capillary is about 25-100 μm in internal
diameter
 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
 Separation is achieved
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17. Basics cont.
 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
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18. Equipment
 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
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19. Equipment Cont….
 Detector
 UV/Visible absorption
 Fluorescence
 Radiometric (for radioactive
substances)
 Mass Spec.
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21. Capillary Electrophoresis Apparatus
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22. The Electropherogram
 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.
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23. ZONE
Electrophoresis
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24. ZONE ELECTROPHORESIS
 It involves migration of charged particles, which are supported on
relatively inert and homogeneous solid or gel framework.
 In this method the separated components are distributed into
discrete zones on stabilizing media.
 The zones are heterogeneous and physically separated from one
another.
 It is classified based on supporting material used.
They are:
1. Paper electrophoresis
2. Cellulose acetate electrophoresis
3. Thin layer electrophoresis
4. Gel electrophoresis
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25. Principle :
Basically a supporting media is saturated with
buffer solution and a small amount of sample solution is
applied as narrow band.
On application of potential difference between the ends
of strip, each component migrates at a rate determined by its
electrophoretic mobility.
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26. ADVANTAGES AND DISADVANTAGES
 Useful in biochemical investigation.
 Very small quantity of samples can be analysed.
 Useful to study both simple and complex mixtures equally.
 Equipment cost is low and maintenance is easy.
 Unsuitable for accurate mobility and isoelectric point
determination.
 Complications such as capillary flow, electro osmosis,
adsorption and molecular sieving are introduced.
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27. GENERAL METHOD OF OPERATION
 Saturation of medium: the supporting medium other than gel
must be saturated with a buffer so that it can conduct current.
 Sample application: sample is applied as spot or streak.
 Electrophoretic separation: the power is switched on at
required voltage.
 After completion of separation the power is switched off
before supporting media is removed.
 Removal of supporting medium: paper, cellulose acetate strips
and thin layer plate are removed and air dried or in oven. The
gels are removed by forcing water from hypodermic syringe.
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28. INSTRUMENTATION
1. Electrophoretic chamber: It contains buffer solution.
2. Electrodes : Ag/AgCl reversible electrodes can be used.
3. Diffusion barriers: The electrode should be separated from
the electrophoretic bed by a barrier such as gel, filter paper,
sponge.
4. Supporting media: It should have low resistance to electric
current, inert to sample, electrolyte and developing
reagents.
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29. PAPER ELECTROPHORESIS
 One of the simplest process in electrophoresis involves
spotting a mixture of solute in middle of paper , moistening
the paper with some electrolyte and placing it between two
sheets of glass.
 The ends of paper strip extending beyond glass plate are
immersed in beakers of electrolyte.
 A potential of 5V/cm of paper length is placed from a DC
source.
 It is allowed to continue for a period of several hours.
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30. ADVANTAGES AND DISADVANTAGES
 It is economical and also easy to use.
 Some compounds such as proteins can not be adequately
resolved.
 There are three types of paper electrophoresis:
1. Horizontal
2. Vertical and
3. Continuous
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33. CELLULOSE ACETATE
ELECTROPHORESIS
1. Cellulose acetate strips, which are used widely in clinical
laboratories produce excellent separations of 7 to 9 protein
fractions in a few hours.
2. this material is exceedingly fine and homogeneous, and
little tailing is encountered due to adsorption.
3. It is especially useful for separating alpha immunoglobulins
from albumin.
4. It contains 2 to 3 acetyl groups per glucose unit and its
absorption capacity is less than that of paper.
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34. GEL ELECTROPHORESIS
The separation here is brought about through molecular sieving
technique, based on molecular size of substances
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35. THIN LAYER ELECTROPHORESIS
1. Electrophoretic studies can be also carried out on thin
layers of silica, keisulghur,alumina.
2. The studies with these materials offer advantages of
speed and resolution when compared with paper.
3. They have greatest application in combined
electrophoretic-chromatography studies in two-
dimensional study of proteins and nucleic acid
hydrolysates.
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36. sudheerkumar kamarapu
THANK
YOU
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