Pharmaceutical Analysis- III

Electrophoresis and Capillary Electrophoresis

Presented by
Dr. Subhash Chander (M.S., Ph.D)
Assistant Professor,
School o f Pharmacy, MAU
1
 INTRODUCTION
Electrophoresis is a method used to separate charged
particles from one another based on differences in their
migration speed. In electrophoresis, two electrodes are
immersed in two separate chambers in such a way that
charged particles can migrate from one chamber to the other
By using an electric power supply, electric potential (E) is
generated between the two electrodes.
Capillary Electrophoresis (CE): 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 analyte is introduced in to the

capillary by replacing one buffer reservoir with sample
vial.

▪ Electrophoretic separation is measured by detector.

 CE uses capillary tubes having 20-100 mm diameter and
20-100 cm in length.
 CE is used with/without gel.
 Higher electric fields result in high efficiency and narrow
peaks (analyte migrates faster).
 Types of Molecules that can be Separated
by Capillary Electrophoresis
 Proteins
 Peptides
 Amino acids
 Nucleic acids (RNA and
DNA)
 Inorganic ions
 Organic bases
 Organic acids
 Whole cells
Separation of components in Capillary
Electrophoresis
Movement of charged component (Migration Velocity)
depends upon two types of motilities:

1. Electrophoretic Mobility
2. Electroosmosis Mobility
Electrophoretic Mobility
It depends on its charge and how much it is attracted or
repelled by the voltage. It is inversely proportional to
the frictional drag force that resists movement. As the
radius of the molecule increases frictional force also
increases

So, overall electrophoretic mobility is based on size and

charge.
Electroosmotic mobility
It the mobility of ions induced by the movement of the
ions generally originates form buffers or medium.

This is crucial parameter play important role in

movement of ions.

The net movement of charged component depends

upon the Electrophoretic Mobility and Electroosmosis
Mobility.

The concept of electroosmosis mobility can be

explained with the help capillary filled with silica.
Cont..
 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
Excess cations in the diffuse Stern double layer flow
towards the cathode, exceeding the opposite flow towards
the anode
Separation Mechanism Cont…
Effect of Electrophoresis and Electroosmosis
Separation Mechanism Cont…
Effect of Electrophoresis and Electroosmosis
The Electropherogram
 Application of CE
1. Capillary electrophoresis may be used for the
simultaneous determination of the ions NH4+,, Na+, K+,
Mg2+ and Ca2+ in saliva.
2. It is used for separation of blood proteins and enzymes.
3. It is used in pharmaceutical industry for detecting
bacterial/microbial contamination quickly.
4. One of the main application of CE in forensic science is
the development of methods for amplification and
detection of DNA fragments using polymerase chain
reaction
5. Another application of CE in forensic is ink analysis.
 Application of CE
6. Hemoglobin electrophoresis gives an estimation about
the abnormal Hb.
7. It also useful in determination of DNA sequence.
8. It is used for analysis of human serum proteins.
9. It is also used for analysis of immunological products.
 Advantages of CE
 Offers high selectivity, an alternative to HPLC
 Easy and predictable selectivity
 High separation efficiency (105 to 106 theoretical plates)
 Small sample sizes is required (1-10 ul)
 Fast separations (1 to 45 min)
 Can be automated
 Easily coupled to MS
 It consumes limited quantities of reagents.
 It is applicable to a wider selection of analytes compared to
other analytical separation techniques.
 Disadvantages of CE
 Cannot do preparative scale separations
 Creates problems if compound is Sticky in nature or difficult to
dissolve
 Reproducibility problems
THANKS