Iso

1. Dr. Rakesh Kumar Marwaha,
Department of Pharmaceutical Sciences,
M.D.U. Rohtak

2. CAPILLARY ELECTROPHORESIS
 The separation technique
that is capable of separating
minute quantities of subs in
a relatively short time with
high resolution is called CE.
 Capillaries are typically of 50 µm
inner diameter and 0.5 to 1 m in
length.
 Due to electro osmotic flow, all
sample components migrate
towards the negative electrode.
 The capillary can also be filled with
a gel, which eliminates the electro
osmotic flow. Separation is
accomplished as in conventional
gel electrophoresis but the capillary
allows higher resolution, greater
sensitivity, and on-line detection.

3. Common Modes of CE in
Analytical Chemistry
 Capillary Zone electrophoresis (CZE)
 Capillary gel electrophoresis (CGE)
 Capillary electrochromatography (CEC)
 Capillary isoelectric focusing (CIEF)
 Capillary isotachophoresis (CIT)

4. ISOELECTRIC FOCUSING
 Electrophoretic method that separates proteins
according to the iso-electric points
 Separation is achieved by applying a potential
difference across a gel that contain a pH gradient
 Isoelectric focusing requires solid support such
as agarose gel and polyacrylamide gel

5. ESTABLIHING THE PH GRADIENT
 Isoelectric focusing gels contain synthetic buffers
called ampholytes that smooth the pH gradients.
 Ampholytes are complex mixtures of synthetic
polyamino-polycarboxylic acids
 Commercially available ampholytes are-
 BIO-LYTE
 PHARMALYTE

6. PROPERTIES OF AMPHOLYTE
 Should have a certain buffering capacity at their
pl
 Should have conductance at their pI
 Should have low molecular weight so that
macromolecules can be separated from them
easily
 Should be soluble in water
 Should have low light absorbance at 280nm

7. RESOLUTION DEPENDS ON
I. The pH gradient,
II. The thickness of the gel
III. Time of electrophoresis,
IV. The applied voltage,
V. Diffusion of the protein into the gel

8. CAPILLARY ISOELECTRIC
FOCUSSING
 In this technique the separation of amphiprotic
substance can be performed in a buffer solution.In this
procedure one end of the capillary bearing the cathode
is dipped in a solution of strong base(NAOH) and the
end bearing anode is dipped into a solution of strong
acid(H3PO4).
 When the process starts the H+ ions migrate
towards the cathode and OH- ions migrates
towards the anode, thus a pH gradient ,with higher
pH towards anode.

9. The analyte in combination with some other
ampholyte(contain carboxyl and amine group) is
also introduced in the sample and these migrate on
the basis of their charge and during migration they
may be protonated or lose proton depending on the
migration due to the existing pH gradient, the
migration continues till the pH region where the
ion has reached is equal to the pl and at this point
the analyte become neutral.

10. PREPARATION OF IEF GEL
Gel is polymerised
Second glass plate is placed on first
Mixture is poured over a glass plate which
contain spacer
Carrier ampholytes (suitable pH) and
riboflavin mixed with acrylamide solution

11. Ampholytes form a pH gradient between
anode and cathode
Potential difference is applied
Electrode wicks are laid along the long length
of each side of the gel
After the gel has set glass plates are prised
apart
This takes 2-3 hr

12. Become stationary when they reaches isoelectric point
Proteins having positive charge will migrates towards the cathode.
negatively charged protein will migrates towards anode
Voltage is again applied for 30 min
Samples applied by laying on gel filter paper soaked in
the sample
The power is then turned off

14. Destained
Gel is stained with Coomasie Brilliant Blue
This precipitaes the proteins and allows smaller
ampholytes to be washed out
The gel is washed with trichloroacetic acid

15. APPLICATION OF IEF
 Widely used for separation and identification of
serum proteins
 Used by the agriculture and food industries,
forensic and human genetics lab
 For research in enzymology, immunology and
membrane biochemistry

16. ISOTACHOPHORESIS
 Isotachophoresis means migration with the same
speed and it depends on the potential difference.
 Characteristics of ITP:
 Migration of all ions with the same speed
 Separation of components as an "Ion train"
 Zone sharpening effect
 Concentration regulating effect

17. PRINCIPLE OF
ISOTACHOPHORESIS
 The separation compartment is divided into 3
unequal parts
i. One part of the anode compartment and the
separation column is filled with the leading
electrolyte.
ii. A second part is formed by a compartment into
which the mixture of substances to be reported
is intoduced.
iii. The third part consist of the compartment filled
with a terminating electrolyte

18.  The leading electrolyte contains anions with
effective mobility higher than that of any off
anion in the sample mixture
 The terminating electrolyte contain an anion with
an effective mobility lower than that of any anion
in sample mixture

20. PROCEDURE :- On application of electric field, the
ionized sample migrates
between leading electrolyte with a high mobility
and a terminating ion with a low mobility, all of
them migrating with the same speed
 The different components are separated
according to their electrophoretic mobilities and
form stacks: the substance with the highest
mobility directly follows the leading ion, the one
with the lowest mobility migrates directly in front
of the terminating electrolyte.

21.  In an anionic separation, the leading electrolyte
will be at the anodal, and the terminating
electrolyte at the cathodal side. The sample is
applied between the two. The system also
contains a common cationic counter-ion.
 A practical example: Chloride is the leading ion,
Glycine is the terminating ion, Tris is the counter
ion.

22. APPLICATION
 For the determination of important analyte in food.
 Determination of organometallic compounds.
 Application of the nanotechnology.
 Application in algebra.