In this slide contains types, factors, instrumentation, advantage, disadvantage of Capillary Electrophoresis. Presented by: HARSHAVARDHAN NAIDU (Department of pharmacology). RIPER, anantapur

1. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
A seminar as a part of curricular requirement
for I year M. Pharm. I semester
Presented by
A. Harsha Vardan Naidu (20L81S0107)
M. Pharmacy
Department of Pharmacology
Under the guidance of
Dr. P. Ramalingam, M.Pharm, Ph. D
Director- R & D Division
Professor of Pharmaceutical Analysis and Medicinal Chemistry

2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2
1. Electrophoresis
2. Factors affecting electrophoresis
3. Types of electrophoresis
4. Capillary electrophoresis
5. Electro-osmotic flow
6. Instrumentation
7. Modes of capillary electrophoresis
8. Advantages
9. Disadvantages
10. Applications
11. Reference
Contents

3. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3
• Electrophoresis is a physical method of analysis.
• It may be defined as the migration of charged particles through
a solution, under the influence of an external electric field.
Electrophoresis

4. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4

5. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5
I. Zone electrophoresis
• Paper electrophoresis
• Cellulose acetate electrophoresis
• Gel electrophoresis
• Thin layer electrophoresis
II. Free/Moving boundary electrophoresis
• Capillary electrophoresis
• Isotachophoresis
• Isoelectric focusing
• Immuno electrophoresis
Types of electrophoresis

6. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6
• In this method, separation are carried out inside a capillary tube.
• The capillary tube has a diameter of 50 μm to facilitate
temperature control.
• The length of the capillary is typically 20–50 cm.
• The ends of the capillary are dipped into reservoirs filled with the
electrolyte.
• The capillary is filled with running buffer, one end is dipped into
the sample, and an electric field is applied to introduce the
sample inside the capillary.
• Migration through the capillary is driven by application of a
high-voltage current (5KV for 5 sec).
Capillary electrophoresis

7. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7
Capillary electrophoresis

8. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
8
• Due to usage of high electric field, osmosis may occur.
• The speed of EOF can be adjusted by changing the buffer pH.
• Bulk movement of solute occurs because of EOF.
• EOF is sufficient to sweep all the neutral molecules.
Electro-osmotic flow (EOF)

9. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9
Electro-osmotic flow

10. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10
Instrumentation
A typical capillary electrophoresis system consists of:
• A buffer solution (Sodium dihydrogen phosphate, Sodium
tetraborate).
• A high voltage supply (5 – 30 KV).
• A sample introduction system or sample injection (by
pressure/vaccum).
• A capillary tube with internal diameter of 10-100mm and a length of
20-100cms.
• A detector and an output device.
• Some instruments include temperature control device, to ensure
reproducible results.

11. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11
Sample Injection:
• Hydrodynamic injection: by applying pressure/ by applying vacuum/
by gravitation.
• Electro kinetic injection: by using electric supply.
Detectors:
• Majorly UV detectors are used.
• Others include IR detector, fluorescence detector, conductivity
detector.
• The mass spectrometers are used to give structural information of
the resolved peak.

12. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 12
Capillary electrophoresis

13. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 13
Modes of capillary electrophoresis
• Capillary zone electrophoresis
• Capillary gel electrophoresis
• Capillary isoelectric focusing
• Capillary isotachophoresis
• Micellar electro kinetic chromatography(MECC/MEKC)
• Non-aqueous capillary electrophoresis

14. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 14
• In CZE, the analytes move in the EOF but separate into bands
because of differences in their electrophoretic mobilities.
• Difference in mobility will cause separation.
Capillary zone electrophoresis (CZE)

15. RIPER
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NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15
• CGE is the adaptation of traditional gel electrophoresis into
capillary.
• Separation is based on the difference in solute size, as the
particle migrates through the gel.
• Gel prevents the capillary wall from absorbing the solute.
Capillary gel electrophoresis (CGE)

16. RIPER
AUTONOMOUS
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NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
16
• When a pH gradient is applied across the capillary, and a voltage is
applied from positive voltage at low pH to negative voltage at high
pH.
• Components will migrate to the pH value that equals their pI value.
• At lower pH value, the components are positively charged, at
higher pH values the components are negatively charged.
• In this way, each component migrates to a different position in the
capillary.
• When pressure is applied, the complete pH gradient moves through
the capillary, and subsequently the components will pass the
detection window.
Capillary isoelectric focussing:

17. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 17
Capillary isoelectric focussing

18. RIPER
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NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 18
• In this technique two kinds of
buffers are used.
• One buffer with high mobility as a
leading buffer and one buffer with
very low mobility as a terminating
buffer.
• The mobility of the sample
components must be between the
leading and terminating.
• In the stable situation, all
components migrate through the
capillary at the same velocity.
Capillary isotachophoresis

19. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 19
• Sample components in the buffer solution will be divided over the
micelles and the buffer solution, depending on their affinity to the
micelles.
• When the migration speed of the buffer differs from the speed of the
micelles, it is possible to separate different components on the fact
that there is a different affinity for the micelles.
Micellar electro kinetic chromatography

20. RIPER
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NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 20
• In this technique, components that are insoluble in water are
separated, using organic solvents.
• The viscosity and dielectric constants of organic solvents affect both
sample ion mobility and the level of electro-osmotic flow.
Non-aqueous capillary electrophoresis

21. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 21
Advantages:
• Offers a new selectivity, an alternative to HPLC.
• Easy and predictable selectivity.
• High separation efficiency (more theoretical plates are formed).
• Small amount of sample is required.
• Time required for separation is less (1-45mins).
• Easily coupled to MS.
Disadvantages:
• Cannot be used for preparative scale separations.
• Sample may stick to capillary walls.
• Inconsistent retention time.

22. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 22
• DNA fingerprinting.
• Diagnosis of hereditary disorders and prenatal testing.
• Diagnosis of infectious diseases.
• Analysis of pharmaceuticals.
• Protein characterization.
• Counter-ion analysis in drug discovery.
Applications

23. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 23
1. Robert L. C. Voeten. Capillary Electrophoresis: Trends and recent
advances. NCBI. 2018; 90(3): 1464-1481.
2. Matthew Geiger, Amy L. Hogerton. Capillary electrophoresis. ACS
publications. 2012; 84(2): 577-596.
3. F. Robert. Capillary electrophoresis: Principle and applications.
PubMed. 1991; 49(3): 137-148.
4. Juliet Precissi. Capillary electrophoresis. LibreTexts. Aug 16, 2020.
References

24. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 24
THANK YOU