Capillary electrophoresis

Published by Pratik Parikh
Master of Pharmacy (Pharmaceutical Biotechnology)

1. Capillary Electrophoresis
PRESENTED BY :
PRATIK UMESH PARIKH
MASTER OF PHARMACY
(PHARMACEUTICAL BIOTECHNOLOGY)
PRESENTED BY : PRATIK UMESH PARIKH

2. Contents :
Introduction of CE
Principle of CE
ElectroStatic & Electroosmotic forces
Electrophoretic Mobility
Mode of operation Capillary Electrophoresis
Instrumentation of CE
Advantages & Disadvantages
Application of CE
Reference
PRESENTED BY : PRATIK UMESH PARIKH

3. Introduction of CE:
o Capillary electrophoresis is separation technique
o Capillary electrophoresis is a superior form of electrophoresis
o Due to use of narrow bore capillary it reduces the problem of overheating
o It is mainly used for separation and analysis of biological samples in Diagnostic and
clinical Labs
o Separation of solute molecules is based on difference in trait of migration that is
electrophoretic mobility under the influence of electric field in buffer solution
o Here electrophoretic mobility and electro cosmetic force plays very important role
o Finally we can say that rate of migration depends on charge and mass of solute
molecules and viscosity of medium
PRESENTED BY : PRATIK UMESH PARIKH

4. • The principle behind electrophoresis is that charged molecules will migrate
toward the opposite pole and separate from each other based on physical
characteristics.
• Capillary electrophoresis has grown to become a collection of a range of
separation techniques which involve the application of high voltages across
buffer filled capillaries to achieve separations
PRESENTED BY : PRATIK UMESH PARIKH
Principle of CE:

5. What is electro-static force and how it works
 Fused silica capillary has silanol groups extending out on the inner surface of
capillary generally in capillary electrophoresis buffer solution use has PH close to
9.





Which means most of the silanol groups are reactive and deprotonated having
negative charge.
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6. What is electro-osmotic force and how it works
 Cross section of fused silica capillary – due to the deprotonation of silanol
groups the inner surface of capillary negatively charged.
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7. When voltage is applied across the capillary contains in diffuse layer will get
attach towards cathode dragging the bulk solution with them this is known as
electroosmotic flow
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8. Electrophoretic mobility:
Electrophoretic mobility is defined as the rate of migration (cm/sec) per unit field
strength (volts/cm).




1. Net charge on the particles (molecule)
2. Size and shape of the particle (molecule)
3. viscosity of medium
PRESENTED BY : PRATIK UMESH PARIKH

9. 1.Net charge on the particles (molecule) - If molecule have higher charge
greater will be electrophoretic mobility charge depends on pH of medium
2. Size and shape of the particle (molecule)- Large particles have less
electrophoretic mobility than small particles. Molecules having round shape
will have more electrophoretic mobility than sharp shapesviscosity of
medium



3.If medium is more viscous the electrophoretic mobility will be less
PRESENTED BY : PRATIK UMESH PARIKH

10. Total velocity of Solute :
Total velocity of solute = electro-osmotic flow + electrophoretic mobility

Solute with net positive charge will move faster then electro osmotic flow
Solute with no net charge will more at the same speed of electro osmotic flow
Solute with net negative charge will move slow than electro osmotic flow

PRESENTED BY : PRATIK UMESH PARIKH

11. Mode of Operation :
Common method of operation of capillary electrophoresis in analytical
chemistry

1. Capillary zone electrophoresis (CZE)
2. capillary gel electrophoresis (CGE)
3. capillary isoelectric focusing (CIEF)
4. capillary isotachophoresis (CITP)
5. Micellar electrokinetic capillary chromatography (MEKC)
PRESENTED BY : PRATIK UMESH PARIKH

12. Instrumentation of CE:
PRESENTED BY : PRATIK UMESH PARIKH
 Capillary electrophoresis instrument has two buffers reservoirs filled with buffer solution
 A fused silica capillary extend between these two buffer reservoirs
 Cathode and anode our place in the buffer solution in a separate reservoir this electrodes are
connected to High Voltage power supply High Voltage about 10000 volt is used in this technique
 Sample can be introduced at one end of the capillary and detector is present near the other
end of the capillary

13. Capillary used CE is a 20 - 100 um in diameter and 20 - 100 cm in length this capillary
maybe coated with support medium or uncoated fused silica has a polyimide coating on
outer side for protection

Sample injection in CE
Pressure differential method - pressure is applied across the capillary while it is dipped
into the sample solution

Electrokinetic injection - voltage is applied and ions are allowed to to migrate into the
capillary (ions migrate due to the charge on them)

Detectors used in CE -
1. UV visible detector
2. Diode array detector
3. Fluorescence detector
4. Electrochemical detector
5. Mass spectrometer detector
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14. PRESENTED BY : PRATIK UMESH PARIKH

15. Detection of Solute:
All solute molecule Travels same distance but the migration time for that distance
is measured
Migration time is helpful for identifying the solute.
And peak area for peak height is used to determine amount of solute in sample.
PRESENTED BY : PRATIK UMESH PARIKH

16. Advantages & Disadvantages of CE:
Advantages:
High efficiency of separation
Short analysis time
Simple and automated technique
Very less amount of sample is required

Disadvantages:
Simple molecules May stick to the wall of capillary
The result obtained are not much reproducible
PRESENTED BY : PRATIK UMESH PARIKH

17. Application of CE:
Applications:
Capillary electrophoresis is mostly used in Diagnostic and clinical Labs
It used for abnormal HB detection and characterization
It used for immunotyping
It is used for analysis of DNA
It used in carbohydrate analysis for the determination of post-translational
modifications
Capillary electrophoresis used in analysis of Pharmaceuticals
PRESENTED BY : PRATIK UMESH PARIKH

18. Reference :
1.Watson G.David,pharmaceutical analysis,2nd edi.2005,Churchill
Livingstone,Pno.333-353.
2.Frank A. Settle,Handbook of Instrumental Techniques for Analytical
chemistry,1st edi.,2004,Pearson education,Pno.165.
3.http://www.ceandcec.com/presentation.htm
4.http://www.hbc.ukans.edu/CBAR/Electrochrom.htm
5.https://www.slideshare.net/sandhyatalla/capillary-electrophoresis-final-ppt

PRESENTED BY : PRATIK UMESH PARIKH

19. PRESENTED BY : PRATIK UMESH PARIKH