Capillary Electrophoresis

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
AUTONOMOUS
NAAC &
NBA (UG)
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
NAAC &
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
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
17
Capillary isoelectric focussing

18. RIPER
AUTONOMOUS
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
AUTONOMOUS
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