This presentation will give you idea about one of the mostly used Electrophoresis technique which is Capillary Electrophoresis. It covers one part of syllabus. It will help you to learn about the new aspects in CE and also to upgrade your knowledge.
1. 1
Dr. Rajendra Gode Institute of Pharmacy, Amravati
Capillary Electrophoresis
Presented by
Shraddha S. Raut
M. Pharm (Pharmacology)
Guided by
Dr. Pramod V. Burakale
M. Pharm, Phd
2. Contents :
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Introduction
Objectives
Types of Electrophoresis
Capillary Electrophoresis
Principle
Instrumentation
Working Conditions
Factors affecting separation
Advantages and Disadvantages
Applications
3. CAPILLARY ELECTROPHORESIS 3
Introduction
Electrophoresis is the phenomenon of migration of charged particles under the
influence of applied electrical field.
• Charged particles in solution travels at different rate towards oppositely charged electrodes.
• The concept of electrophoresis was discovered in 1807 by Ruess.
Anode
Cathode
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+
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+
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4. Objectives
CAPILLARY ELECTROPHORESIS 4
1) To determine the amount and mobility of component
2) To separate the component based on their size and charge
3) To obtain information about electrical double layers surrounding the particles
4) To separate mixture of biomacromolecules by using electric current
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5. Types of Electrophoresis
CAPILLARY ELECTROPHORESIS 5
Electrophoresis
Zone Electrophoresis
• Capillary Electrophoresis
• Immunoelectrophoresis
• Isoelectric Focusing
• Isotachophoresis
Moving Boundary Electrophoresis
• Paper Electrophoresis
• Gel Electrophoresis
• Cellulose acetate Electrophoresis
• Thin Layer Electrophoresis
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6. Capillary Electrophoresis
CAPILLARY ELECTROPHORESIS 6
• Capillary Electrophoresis is a liquid-based separation technique in which charged species
are separated with the help of electrical field applied in buffer solution.
• It is a superior form of electrophoresis.
• It uses narrow bore capillary as a separation channel.
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7. CAPILLARY ELECTROPHORESIS 7
Principle
• Capillary Electrophoresis separates ions based on their Electrophoretic mobility with
the use of an applied voltage.
• Electrophoretic mobility and Electro-osmotic force plays very important role.
• Because velocity of migration is given by –
analyte’s electrophoretic mobility inside capillary
buffer’s electroosmotic flow inside capillary
• Electrophoretic mobility depend on charge of molecule
size and viscosity of medium
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8. CAPILLARY ELECTROPHORESIS 8
Conti……
• Electrophoretic mobility : Response of solute to the electric field is called as
electrophoretic mobility. Charged solute particles will move towards oppositely charged
electrodes.
Rate at which particle moves is directly proportional to applied electric field.
Greater the field strength, Faster the mobility
• Electro-osmotic flow : When buffer solution will generate electrical field and flows
through capillary after applying the high voltage power supply is called as electro-osmotic
flow.
Anions will move in opposite direction of the electro-osmotic flow with less velocity
cations will move in same direction of the flow with higher velocity.
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9. Fig. Ionization of Silanol into Silanate
Conti……
Electro-osmotic flow generation
CAPILLARY ELECTROPHORESIS 9
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12. CAPILLARY ELECTROPHORESIS 12
Capillary - These are made up of fused silica with external
coating of polyimide. The length of capillaries varies from
25-100 cm and inner diameter from 50-75 microns.
Conti……
Power Packs - It is used to supply constant high voltage in range of 10-30 kV. It generates
current between 20-100 mA.
Thermostatic system – It is required to maintain constant
temperature inside the capillary for which the air or liquid
coolant is used.
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13. CAPILLARY ELECTROPHORESIS 13
Conti……
Sample Injector - Two types of injectors are commonly used to introduce the sample in
capillary. These are hydrodynamic injection and electrokinetic injection.
Buffer Reservoirs - CE requires two buffer reservoirs, one is source reservoir and another
is destination reservoir. Ex: Phosphate buffer, Zwitterionic buffers, etc.
Electrodes - one anodic and one cathodic electrode
Detector - For monitoring the amount of substance that passes through the capillary at
given time. UV detector is mostly used. However several other detectors are also used like
fluorescence, mass spectrophotometer, etc.
Integrated system – convert the response of detector into readable format.
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14. CAPILLARY ELECTROPHORESIS 14
Instrumental setup
• It consists of a high voltage power supply which is connected to an electrode.
• Each side of capillary tube is dipped in a reservoir containing the electrode and
electrolytic solution or buffer.
• There is usually a small window near the cathodic end of the capillary where the
detector is connected.
• And Integrated system or computer is their for construction of a graph.
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15. Working condition
Dip one end of the capillary in destination reservoir
Introduce the sample solution in capillary tube
Then dip that end in the Source reservoir
Switch on the power, ions will starts migrating as both the electrodes are connected with power supply
Electro-osmotic flow is generated which pumps the solute ions towards the detector
Detectors measures the response
Computer constructs an Electropherogram
CAPILLARY ELECTROPHORESIS 15
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17. CAPILLARY ELECTROPHORESIS 17
Factors affecting separation
The Capillary Surface
Sample
Buffer
pH
Additives
Temperature
Electrical field
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18. CAPILLARY ELECTROPHORESIS 18
Advantages
• Cost of maintenance is less.
• High voltage can be applied.
• Very high level automation is possible.
• Small sample is required.
Disadvantages
• Cannot do preparative scale separation.
• Changing capillaries is challenging.
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