A capillary electrophoresis is a technique that is used in laboratories to separate macromolecules. This technique is mainly used for DNA sequencing, to identify proteins, and to analyze the structure of polymers. This technique involves the use of an electric field to move charged molecules through a small tube (called a capillary) with a gel matrix. The movement of these molecules can be monitored by an optical detector that reads the light emitted by markers at different positions along the tube. some types are: Capillary Zone electrophoresis (CZE). Capillary gel electrophoresis (CGE). Capillary isoelectric focusing (CIEF). and (CITP).

1. CAPILLARY
ELECTROPHORESIS

2. What is
Electrophoresis?
• Separation of solutes based on
different rates of migration
though an electric field through
background electrolyte [running
buffer].
• ▪ Anions (-) move toward the
anode (+) & vice versa.
• Charge and size influence the
movement of charged particles,
in opposite ways.

3. CAPILLARY
ELECTROPHORESIS
The tube in CE is typically silica, which may be coated or
uncoated.
▪ Uncoated silica led to electro-osmosis when run at
neutral or basic pH due to de-protonation of silanol
groups.
▪ In “normal polarity mode,” a sample with many types
of ions can be injected (at the + end), and they then
travel in the same direction toward the negative
electrode through a detector.
▪ Observed mobility will be the sum of inherent electro-
osmosis plus electrophoretic mobility. ▪ These affect
time, efficiency, and separation.

4. CAPILLARY
ELECTROPHORESIS
Using narrow bore tubes, CE removes the Joule
heating effect, which decreases band broadening,
giving faster separations than gel.
▪ CE uses tubes 20-100mm diameter and 20-100 cm
in length.
▪ CE is used with/without gel. Longitudinal diffusion is
the main source of band-broadening.
▪ Higher electric fields result in high efficiency and
narrow peaks (analyte migrates faster).

5. 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 analyst is
introduced into the capillary by replacing
one buffer reservoir with sample vial.
▪ Electrophoretic separation is measured
by detector.

6. THEORY
Electrophoretic Flow
• It is the process in which sample ions
move under the influence of an
applied voltage.
• The ion undergoes a force that is
equal to the sample of the
Electrophoretic mobility and the
electric field strength.
• The flow of ions is toward the
opposite charged electrode. µEP =
q/6ηπr & VEPF = µEP.E Where, µEP =
Electrophoretic Mobility. q = Charge on
ions. η = Viscosity. r = Radius. E =
Electric field strength. VEPF = Velocity.

7. INSTRUMENTATION
• A typical capillary
electrophoresis system
consists of a:
• Buffer solution
• High-voltage power supply,
• A sample.
• A capillary tube.
• A detector.
• Output device.

8. Capillary Zone electrophoresis (CZE).
 Capillary gel electrophoresis (CGE).
 Capillary isoelectric focusing (CIEF).
Capillary isotachophoresis (CITP).
Modes Of CE

9. Capillary Zone
electrophoresis (CZE)
i. CZE analytes move in the EOF but
separate into bands because of
differences in their electrophoretic
mobilities, µ.
ii. Differences in µ make each analytics'
overall migration velocity slightly
different, and difference in migration
velocity = separation.
iii. µ's are roughly a function of
analyte charge and frictional and size
differences.

10. Capillary gel
electrophoresis
(CGE)
i. CGE is the
adaptation of
traditional gel
electrophoresis into
the capillary .
ii. CGE uses
separation based on
the difference in
solute size as a
particle migrate
through the gel.
iii. Gels prevent the
capillary walls from
absorbing then
solute.

11. Capillary Isoelectric Focusing (CIEF)
i. CIEF is a technique commonly used to separate peptides and
proteins.
ii. These molecule are called zwitterionic compounds.
iii. So, each molecule has a specific isoelectric point (pI).
iv. If pH = pI then molecule become a neutral.

12. Genetic Analysis.
Analysis of Pharmaceuticals.
Pharmaceuticals with Chiral Centers (Enantiomers).
Counter-ion analysis in drug discovery.
Protein Characterization.
Applications

13. Advantages
. Offers new selectivity, an alternative to HPLC.
. Easy and predictable selectivity. • High separation efficiency
(millions of theoretical plates).
. Small sample required (1-10 ml).
. Fast separations (1 to 45 min).
. Can be automated.
. Easily coupled to MS.

14. Aged , improperly stored blood samples –
degradation products.
Abnormal Hb – use other means of
identification.
Migration of Hb variant close to HbA –
underestimation of Hb A & variant +
overestimation of HbA2.
Sensitivity & resolution limits.
Can not do preparative scale separations. •
Low concentrations and large volumes
difficult.
DISADVANTAGES

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