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CAPILLARY
ELECTROPHORESIS
SUBMITTED BY: RAHUL PAL SUBMITTED TO: DR. YELE VIDHYASRILEKHA

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The differential movement or migration of ions by attraction or repulsion in an electric field. OR
it describes migration of charged particle or molecules under the influence of electric field.
Purpose of Electrophoresis:
 To determine number, amount and mobility of components in given sample or to separate
them.
 Separation of molecules of mixture on the basis of charge or size of molecule.
ELECTROPHORESIS

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THIN LAYER ELECTROPHORESIS
CELLULOSE ACETATE ELECTROPHORESIS
PAPER ELECTROPHORESIS
ZONE ELECTROPHORESIS
GEL ELECTROPHORESIS
TYPES OF ELECTROPHORESIS
ISOELECTRIC FOCUSING
IMMUNO ELECTROPHORESIS
ISOTACHOPHORESIS
FREE/MOVING BOUNDARY
ELECTROPHORESIS
CAPILLARY ELECTROPHORESIS

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INTRODUCTION: CE
DEFINITION: These kind of separation are facilitated by the use of high voltage, which may generate
electro-osmotic and electro-phoretic flow of buffer solution and ionic species, respective within the
capillary.
Electrophoretic Flow in which sample ions move under the influence of an applied voltage.
Electro-Osmotic Flow osmosis under the influence of an electric field, the speed of EOF can adjusted by changing the
buffer pH.
 Other Term: Separate the mixture components on the basis of molecular weight and charge.

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• Capillary tube is placed between two buffer reservoir, and an electric field is applied,
separation depends on electrophoretic mobility and electro-osmosis.
• Electrophoretic mobility: define as rate of migration (cm/sec) per unit field strength
(V/cm).
• Electrophoretic separation is measured by detector.
• Electrophoretic system; consists two electrodes of opposite charge (anode, cathode)
connected by a conducting medium called an electrolyte.
More efficient than gel electrophoresis.
CAPILLARY ELECTROPHORESIS: PRINCIPLE

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• It is also known as uncoated capillaries.
• The capillary tube is made up of fused highly purified
silica (pure).
• In silica, silinol group is present.
• When at acidic pH (pH =3) all the silinol group are protonated.
• At basic pH =9 the silinol group is completely deprotonated.
BARE SILICA CAPILLARIES

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• In this method, separation are carried out inside a capillary tube.
• The tube in CE is typically silica, which may be coated or uncoated.
• Uncoated silica lead to electroosmosis 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 and they
then travel in the same direction toward the negative electrode through a detector.
• The gel is filled in the capillary tube which is highly purified, gel may be made up of
polyacrylamide gel.

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• If an analyte has a migration rate faster than electro-osmosis, it may flow in the opposite
direction of the electro-osmotic flow. This is known as the “reverse polarity mode”.
• Changing the degree of de-protonation of the silica will alter electro-osmotic flow.
Analysis is done by injecting at the negative electrode.
• Using a neutral coating in the tube reduces electro-osmosis. While a positive coating will
reverse direction of flow toward the positive end.
• Charged molecules separated by their electrophoretic mobility in an alkaline buffer
(pH9.4)
CONT..

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• Simple
• Automated, easy to operation.
• Short analysis time.
• Low sample volume.
• High efficiency of separation.
• Ability to separate both charged and
non-charged molecules.
• Sensitivity and resolution limits.
• Aged, improperly stored blood samples-
degradation products.
ADVANTAGES AND DISADVANTAGES

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• A typical capillary electrophoresis system consists of:
• Buffer solution (like sodium dihydrogen phosphate NaH2PO4).
• Migration though the capillary is driven by applications
of a high-voltage current (5KV for 5 sec.)
• A sample introduction system/sample injection
(by pressure or vacuum, Voltage).
• A capillary tube with internal diameter of
10-100 micrometer, 20-100 cm length and a jacket of insulator is present outside the capillary tube.
INSTRUMENTATION

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• The ends of the capillary are dipped into reservoir filled with the electrolyte.
• The capillary tube is filled with running buffer, one end is dopped into the sample and a
electric field is applied to introduce the sample inside the capillary.
• A detector & Output device.
Some instruments include a temperature control device to ensure reproducible results. This
is because the separation of the sample depends on electrophoretic mobility and the
viscosity of solution decrease as the column temperature rises.

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Sample Injection:
 Hydrodynamic Injection: By applying high pressure or high voltage, by applying vacuum
and by gravitation.
 Electrokinetic Injection: By using electric supply.
Detectors:
Detector similar to those used in GC, HPLC.
In CE DAD (Diode Array Detector) is important detector, it can detect more than one
wavelength at the same time.
Alternative detector are UV detectors, IR, Fluorescence, laser include fluorescence, mass
spectrometers.

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Genetic Analysis (DNA Fingerprinting):
Analysis of DNA fragment length/pattern.
Analysis of single strand polymorphism.
Pharmaceuticals and
biopharmaceutical analysis
and applications.
Molecular Diagnosis and
Protein Characterization.
Infectious disease diagnosis.
Counter-ion analysis in drug
discovery.
APPLICATIONS:

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• Capillary electrophoresis is a technique with potential but currently has several problems,
i. Sensitive issues.
ii. Sample stacking problems.
iii. Lack data regarding and reproducibility of methods.
iv. No standardized method, determining appropriate test condition for unknown
sample.
CURRENT STATEMENTS: