Guided By:

Dr. Shaik Abdul Rahaman

Department of Pharmaceutical Analysis

Presented By:

K. Santoshi Sravya
Y17MPH0857
Department of Pharmaceutical Analysis
 What Is Electrophoresis?

The differential movement or migration of ions by attraction or repulsion

in an electric field.
OR
It describes migration of charged particles or molecules under the
influence of electric field.
 Purpose For Carrying Out Electrophoresis:

1. To determine the number, amount and mobility of components in a

given sample or to separate them.

2. To obtain information about the electrical double layers surrounding

the particles.

3. Determination of molecular weight of proteins and DNA sequence.

 Types Of Electrophoresis

Zone Moving Boundary

Electrophoresis Electrophoresis
 Capillary Electrophoresis

Definition:

These kind of separations are facilitated by the use of high voltages, which may
generate electro-osmotic and electro-phoretic flow of buffer solutions and ionic
species, respectively within the capillary.

Principle:

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 i.e. the greater the field
strength, the faster the mobility & vice versa.
 Cont…

Neutral species are not affected, only ions move with the electric field. If two ions
are the same size, the one with greater charge will move the fastest. For ions of the
same charge, the smaller particle has less friction and overall faster migration rate.
Capillary electrophoresis is used most predominately because it gives faster results
and provides high resolution separation. It is a useful technique because there is a
large range of detection methods available.
Theory:

1. 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.
 2. Electroosmotic Flow

• Osmosis under the influence of an electric field.

•The speed of EOF can be adjusted by changing the buffer pH.
• Bulk movement of solutes is caused by EOF.
•EOF is usually sufficient to sweep all +ve, neutral, -ve species towards the same end.

μEOF = C.ƺ/4πη
&
VEOF= μEOF.E

Where,
μEOF =Electro osmotic mobility.
C= Dielectric constant
E= Electric field strength.
ƺ= Zeta potential.
η= Viscosity.
VEOF=Velocity.

The velocity of ions is sum of velocity of EOF and EPF:

VTotal=VEOF+VEPF
Instrumentation:

A typical capillary electrophoresis system consists of a:

• Buffer solution (like sodium dihydrogen phosphate,NaH2 PO4).

• High-voltage power supply (5 to 30 kv).
• A sample introduction system / sample injection (by pressure or vacuum).
• A capillary tube with internal diameter of 10-100mm & 20-100cm length.
• 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 the
electrophoretic mobility and the viscosity of the solutions decreases as the column
temperature rises.
 Sample Injection

Hydrodynamic injection: By applying pressure, by applying vacuum and by gravitation

Electrokinetic injection: By using Electric supply.

Detectors

• Detectors similar to those used in GC,HPLC.

• Majority of instruments have UV detectors available.
• Alternative detector modes include commercially available IR, fluorescence, laser
induced fluorescence, conductivity and indirect detection.
• The mass spectrometers is frequently used to give structural information on the resolved
peaks.
Modes Of CE:

 Capillary Zone electrophoresis (CZE).

 Capillary gel electrophoresis (CGE).
 Capillary isoelectric focusing (CIEF).
Capillary isotachophoresis (CITP).

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 analytic's 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.
 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.
 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.

Capillary Isotachophoresis (CITP)

CITP is a focusing technique based on the migration of the sample

components between leading and terminating electrolytes.
Applications:

• Genetic Analysis.
• Analysis of Pharmaceuticals.
• Pharmaceuticals with Chiral Centers (Enantiomers).
• Counter-ion analysis in drug discovery.
• Protein Characterisation.
 Genetic Analysis

DNA Fragment Analysis:

•Detector: IR light detectors.

•Genotyping data , DNA fragment sizes and amounts and identifies alleles represented
by specific DNA fragments.
•For fragment analysis, alleles in each sample can be labeled using PCR (Polymerase
Chain Reaction) amplification with primers that are tagged with a dye.
•Four IR dyes are used, three separate dyes
for multiplexing, and the fourth dye is
used for the size standard.
• The y axis shows the dye signal output
and the x axis shows the fragment size.
The red dye is the size marker, while the
blue and green dyes are attached to PCR
products.
 Analysis of Pharmaceuticals

Separation of drugs of the cocktail, for the quality control of AIDS medicines:

A mixture of drugs known as "cocktail" with a hundred-fold potency increase in

comparison to the monotherapy.
20 mol L-1 in 1:1 Methanol;Acetonitrile containing 10mol L-1 Sodium Dodecyl
Sulphate as Mobile Phase.
Detected at 220nm.
 Pharmaceuticals with Chiral Centers (Enantiomers)

Enantiomeric Separation of Sibutramine:

•Detector: Diode Array Detector.

•Capillary of 30 cm length (22 cm effective length) × 50 μm inner diameter &
temperature of 15 ºC.
•Detection at 220 nm.
•50mol L-1phosphate running buffer, pH 4.5.
•The differences between the pharmacokinetic and pharmacological properties of the
sibutramine enantiomers, R-sibutramine decreases body weight and food uptake,
while S-sibutramine increases body weight and food uptake.
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.

Disadvantages:

• Can not do preparative scale separations.

• Low concentrations and large volumes difficult.
Conclusion:

• 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 conditions for
unknown sample.

• Capillary electrophoresis is not suitable for producing independently conclusive

results.
Reference:

1.http://dx.doi.org/10.1590/S0103-50532003000200016 Journal of the Brazillian

Chemical Society. Print version ISSN 0103-5053On-line version ISSN 1678-4790.
2.http://articles.extension.org eXtension.
3.http://analytics.scielo.org SciELO Analytics.
4.www.public.asu.edu Capillarory Electrophoresis.
5.https://www.sciencedirect.com Capillary Electrophoresis & Electrophoresis.
6.http://www.springer.com Modes of Capillary Electrophoresis.