2. ISOTACHOPHORESIS
Introduction:
ITP-Isotachophoresis
iso=equal
tacho=speed
phoresis=migration
Defination:-
ITP is a technique in analytical chemistry used for
selective separation and concentration of ionic analytes. it is a
form of electrophoresis charged analytes are separated based
on ionic mobility a factor which tells how fast an ion migrates
through an electric field.
3. Principle:
Under the influence of an electricfield(E) charged
particles move at a velocity is
V=M*E
Mobility is different for differnt molecules.
ITP
- Cationic
- Anionic
carried out in discontinuous electrolyte system
4. Stadia during an ITP separation of a mix of two analytes.
The "self-sharpening effect" in ITP: due to a
difference in electrical field, an ion will move faster
when it comes in the previous zone, and slower
when it comes in the next zone. Therefore it will
return to its "own" zone. Below the corresponding
electrical field for each zone
10. IEF(Iso Electric Focusing)
• Isoelectric focusing (IEF), also known
as electrofocusing, is a technique for separating
different molecules by differences in theirisoelectric
point (pI). It is a type of zone electrophoresis, usually
performed on proteins in a gel, that takes advantage
of the fact that overall charge on the molecule of
interest is a function of the pH of its surroundings.
12. • Separation of basis of pI (not Mw)
• Requires very high voltages (5000V)
• Requires a long period of time (10h)
• Presence of a pH gradient is critical
• Degree of resolution determined by slope of
• pH gradient and electric field strength
• Uses ampholytes or IPG(strips) to establish
• pH gradient
13. Determination ofIsoelectrical point
IEF is a method of protein seperation it is determine the
iso electric point pH gradient. The Isoelectric point
denotes pl which pH is known net charge and protein
become immobile an electrical field.
14. One of the best method for protein seperation or
purification can be easily done if a isoetectric
point of a protein is known
15. THEORETICAL ASPECTS :
The pH gradient forces a protein to remain in its isoelectric point position, thus
concentrating it ; this concentrating effect is called "focusing". Increasing the applied
voltage or reducing the sample load result in improved separation of bands. The applied
voltage is limited by the heat generated, which must be dissipated. The use of thin gels
and an efficient cooling plate controlled by a thermostatic circulator prevents the
burning of the gel whilst allowing sharp focusing. The separation is estimated by
determining the minimum pI difference (ΔpI), which is necessary to separate 2
neighbouring bands:
