Capillary electrophoresis.pptx

CAPILLARY ELECTROPHORESIS
Elizabeth Philip

INTRODUCTION
Capillary electrophoresis is an analytical technique that separates charged particles using
electricity and a very small tube called “capillary”.
Popularized by Jorgenson and Lukacs in the late 1980’s.
Capillary electrophoresis is used most predominately because it gives faster results and provides
high resolution separation.
The rate at which the particle moves is directly proportional to the applied electric field--the
greater the field strength, the faster the mobility.
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.

INSTRUMENTATION
• Commercially available CE instruments
consist of:
i. Buffer solution(like sodium dihydrogen
phosphate, 𝑁𝑎𝐻2𝑃𝑂4)
ii. A high voltage power supply(5 to 30
kv)
iii. A capillary tube with internal diameter
of 10 – 100 mm & 20 – 100 cm length
iv. A sample introduction system/ sample
injector(by pressure or vacuum)
v. A detector
vi. Output device

 Capillary:
 A capillary is a thin tube and for CE typically used is fused silica capillaries.
 Fused silica capillary has silanol groups extending out on the inner surface of
capillary.
 Mostly used inner diameters are 10 to 100 mm.
 Common capillary lengths for a CE separation are 20-100 cm.
 On the outside, the capillary is covered with a protective coating, usually polyimide.
 On the inside, you can also use coatings, so it is important to distinguish between
them.

PRINCIPLE
Separation of ions is based on their electro-osmotic flow and electro-phoretic mobility with the
use of an applied voltage.
1. Electroosmotic flow:
In CS – due to deprotonation of silanol groups
the inner surface of capillary is negatively charged.
The cations from the buffer gets tightly adsorbed
on the inner surface of capillary forming a fixed
layer.
The negativity of the silanol group is partially
compensated by the anions; the fixed layer will
attract more number of cations from the buffer
solution forming the diffused layer.
Next layer forms the bulk solution where the
number of cation is equal to number of anions.
Occurs when the buffer moves through the capillary in response to the applied electrical
field.

When voltage is applied across the capillary, cations in the diffuse layer will get
attracted towards cathode dragging the bulk solution with them this is known as
Electro osmotic flow.

2. Electrophoretic mobility:
 Defined as the rate of migration of particles(cm/sec) per unit field
strength(volts/cm).
 It is the solute’s response to the applied electrical field in which cations move
toward the negatively charged cathode, anions move toward the positively charged
anode, and neutral species remain stationary.
 The electrophoretic mobility depends
upon the charge and atom radius and
the viscosity of the medium.

OPERATION
• A small volume of sample is injected into
one end of the capillary.
• When high voltage(typically 10 -30kV) is
applied sample ions move towards the
electrode with the opposite charge.
• Sample ions in the capillary tube passes
through a detector which is connected to a
computer
• The plot of detector response generated by
the computer is called the
electropherogram
• Capillary tube is placed between two buffer reservoirs, filled with the electrolyte.
• These reservoirs are connected with platinum electrodes.

Sample injection:
In capillary electrophoresis, a small amount of sample solution is injected from the inlet end of a
capillary and a high voltage is applied between the two ends of the capillary, both of which are
immersed in electrolyte. Satisfactory reproducibility of the injection volume is required for
quantitative analysis.
The most common modes of sample injection in CE is generally classified into two categories,
hydrodynamic injection and electrokinetic injection.

b) Electrokinetic injection:
No special exclusive devices or regulation mechanisms are required for
electrokinetic injection.
The inlet end of a capillary is placed in a sample vessel instead of a buffer and a
high voltage is applied, that is for electromigration to take place.
The electrokinetically injected volume depends on the strength and direction of
the applied voltage and injection time, but also on the capillary length, capillary
diameter and electro osmotic mobility.
a) Hydrodynamic injection:
Accomplished by application of pressure at one end of the capillary.
Involves injection by gravity flow, pressure and vacuum suction.
Advantage: the quantity of individual analytes injected is constant, independent of
their own mobility, in contrast to electrokinetic injection.
When the inlet end of the capillary immersed in the sample solution is placed at a
higher position than the outlet end in the container of buffer, gravity flow generates
sample injection.

 Detector:
 Present at the end of the capillary.
 Normally, the detector used to perform the process is an ultraviolet-visible absorption type.
 Other types of detectors used are diode array detector, fluorescence detector, electrochemical detector, mass
spectrometer detector.
 The detector sends the data to an output device such as an integrator or computer.
 The data sent from the detector is displayed as an electropherogram.

DETECTION OF SOLUTE AND ELECTROPHEROGRAM
• When electrolytes of buffer solution hit the detector we get the base line in the electropherogram and when
solute molecules hit the detector we get the signal.

Absorbanc
e
 All solute molecules travel
the same distance but the
migration time, 𝑡𝑚 for that
distance is measured.
 Migration time is helpful
for identifying the solute
(qualitative parameter).
 Peak height is used to
determine the amount of
solute in the sample
(quantitative parameter).

TYPES OF CE
Capillary zone electrophoresis (CZE): separation based on size and charge
difference between solutes.
Capillary gel electrophoresis (CGE): separation by sieving of solutes through a
gel network.
Capillary electrochromatography (CEC): separation involves both
electrophoretic and chromatographic procedures.
Capillary isoelectric focusing (CIEF): separation of peptides and proteins
(zwitterionic compounds) within a pH gradient.

APPLICATIONS
1. Genetic analysis: DNA fragment analysis
2. Analysis of pharmaceuticals: separation of drugs of the mixture for the
quality control of medicines.
3. Protein characterization

REFERENCES
1. Biophysical chemistry: Principles and techniques- Upadhyay
2. https://www.slideshare.net/Santoshi10/capillary-electrophoresis-
116225446
3. Kitagishi, K. (1997). Sample injection. In: Shintani, H., Polonský, J.
(eds) Handbook of Capillary Electrophoresis Applications. Springer,
Dordrecht. https://doi.org/10.1007/978-94-009-1561-9_2
4. https://www.priyamstudycentre.com/2021/09/capillary-
electrophoresis.html
5. https://blog.sepscience.com/pharmascience/the-capillary-
electrophoresis-ce-capillary

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