ISOTACHOPHORESIS & IEF(Iso Electric Focusing)

By J.Rajesh

(256213886012)MALLA REDDY COLLEGE OF PHARMACY

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.

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 - Anioniccarried out in discontinuous electrolyte system

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

INSTRUMENTATION:

Principle of regulation counter flow of electrolytes

APPLICATIONS:-

• Fast separation technique • minute quantity of sample sufficiennt• simple to perform• sample pretretment not necessary• separation of small ions upto 1000Da is possibleBIOMEDICAL FIELD:-• Protein analysis• serum proteins, lipoproteins,CSF proteins• forensic analysis:-• human blood from bovine and ovine• purine& pyramidine analysis -metabolism disorders• nucleotide analysis-ATP,ADP,AMP,cAMP,NADH,IMP.• Amino acid analysis• phenylalanine-phenylketonuria• various metabolic disorers• peptide analysis • food analysis • environmental analysis i.e., analysis for air,water,soil

Qulitative-step heightQuantitative-Zone length

INTERPRETATION:calibration curve obtained from a set of standard isotachopherograms

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.

Principle of IEF:

• 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

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.

One of the best method for protein seperation or purification can be easily done if a isoetectric point of a protein is known

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: