Capillary electrophoresis is an analytical technique that separates ions based on their electrophoretic mobility through thin capillary tubes when an electric field is applied. Separation occurs due to differences in electrophoretic mobility which depends on an ion's charge and size. Capillary electrophoresis uses high electric fields in thin capillaries to rapidly separate molecules in minutes versus hours for traditional electrophoresis. It has applications in sequencing DNA/RNA, analyzing pharmaceuticals, and separating proteins, peptides, and other biomolecules.

1. Presentation on -
Presented by
PURTY YAGYA SANI
M.Pharm 1st Sem
DEPARTMENT OF PHARMACOLOGY
Under the Supervision of-
Dr. SAVITA UPADHYAY
(Assoc. Prof. )
HYGIA INSTITUTE OF PHARMACEUTICAL EDUCATION &
RESEARCH LUCKNOW
Capillary electrophoresis

2. What is electrophoresis ?
 Anions (-) move toward the anode (+) & vice versa.
Charge and size
influence the
movement of
charged
particles, in
opposite ways.
 The differential movement or migration of ions by attraction or
repulsion in an electric field.

3. ▪ Separation of components in a mixture in an electric field depends
on velocity.
v = velocity of molecule
E = electric field
q = net charge on a molecule
f = friction coefficient
v = Eq/f

4. Types of electrophoresis
▪ Gel electrophoresis
▪ High resolution electrophoresis
▪ Capillary electrophoresis
▪ Isoelectric focussing
▪ Immunochemical
▪ 2-D electrophoresis
▪ Cellulose acetate electrophoresis at alkaline pH 8.5
▪ Globin chain electrophoresis

5. What is capillary electrophoresis ?
• Capillary electrophoresis is an analytical technique that separates
ions based on their electrophoretic mobility with the use of
an applied voltage.
• The electrophoretic mobility is dependent upon the charge of
the molecule, the viscosity, and the atom's radius.
• The rate at which the particle moves is directly proportional to
the applied electric field, the greater the field strength, the
faster the mobility.

6. •Proteins
•Peptides
•Amino acids
•Nucleic acids (RNA and DNA)
•Inorganic ions
•Organic bases
•Organic acids
•Whole cells
Types of Molecules that can be Separated by
Capillary Electrophoresis

7. Capillary electrophoresis: Principle
▪ Capillary tube is placed between two buffer reservoir, and an electric field is
applied, separation depends on electrophoretic mobility & electro-osmosis.
▪ Defined volume of sample is introduced into the capillary by replacing
one buffer reservoir with sample vial.
▪ Electrophoretic separation is measured by detector.

8. Basic Theory of CE
ELECTROOSMOTIC FLOW:
Electroosmotic flow is the
movement of the
separation buffer through
the silica capillary as a
results of the existence of
zeta potential at the
solvent /silica interface.
10-100µm
Fused silica capillary
• Inner diameter:10-100µm
• Length:40-100cm
Thin layer coated with
Polyimide (15-35µm thick)
Cross-section of a capillary

9. Fused silica capillary exposed with SiO-
The inside wall of the capillary is covered by silanol groups (SiOH)
that are deprotonated (SiO-) at pH > 2
SiO- attracts cations to the inside wall of the capillary.
The distribution of charge at the surface is described by the Stern
double-layer model and results in the zeta potential
INSIDE THE
CAPILLARY : ZETA
POTENTIAL

11. Electrophoretic mobility
Electrophoretic mobility is the solute's response to the applied
electrical field. As described earlier, cations move toward the
negatively charged cathode, anions move toward the positively charged
anode, and neutral species remain stationary.

12. µep α ionic charge on the analyte
µep α
Fricgtional retarding force of the analytes
1

16. • Fused-silica capillary
10-100 μm in internal diameter &40-
100 cm long
• Two electrode(platinum)
• High voltage supply (5 to 30 kv)
• Sample injector (by pressure or
vacuum)
• Detector
• Buffer solution (like sodium
dihydrogen phosphateNaH2 PO4)

17. • Hydrodynamic injection
By applying pressure
By applying vacuum.
By gravitation
• Electrokinetic injection
By using Electric supply

18. • Detectors similar to those used in GC, HPLC majority of
instruments have UV detectors available.
• The mass spectrometers is frequently used to give structural
information on the resolved peaks.
• Sensitive detectors are needed for small concentrations in
CE

19. All analytes travel the same
distance, but the migration time
(tm) for that distance is
measured.

20. Why Capillary electrophoresis ?
Normal electrophoresis requires more than an hour to run and
difficult to quantitate and automate.
Capillary electrophoresis is a technique in which electrophoresis is
carried out in very thin capillary tubes which is made up of fused
silica and it sometimes coated with polyimide.
 Capillaries rapidly dissipate heat and hence permit the use of high
electric fields typically 100 to 300 V per centimetre, which reduces
separation times to a few minutes.
It can sequence 750000 nucleotides per day.
 It can be automated.
Normal electrophoresis requires more than an hour to run and
difficult to quantitate and automate.
Capillary electrophoresis is a technique in which electrophoresis is
carried out in very thin capillary tubes which is made up of fused
silica and it sometimes coated with polyimide.
 Capillaries rapidly dissipate heat and hence permit the use of high
electric fields typically 100 to 300 V per centimetre, which reduces
separation times to a few minutes.
It can sequence 750000 nucleotides per day.
 It can be automated.
Higher electric fields result in high efficiency and narrow peaks
analyte migrates faster

22. Application
• Genetic analysis
• Analysis of pharmaceuticals
• For separating proteins and peptides
• For research in enzymology , immunology ,
cytology and toxicology.
• It is used in carbohydrate analysis for the
determination of post translational modification