ISOTACHOPHORESIS & IEF(Iso Electric Focusing) By J.Rajesh (256213886012) MALLA REDDY COLLEGE OF PHARMACY
Transcript
1. ISOTACHOPHORESIS & IEF(Iso Electric Focusing) By
J.Rajesh (256213886012) MALLA REDDY COLLEGE OF PHARMACY
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
5. INSTRUMENTATION:
6. Principle of regulation counter flow of electrolytes
7. APPLICATIONS:- Fast separation technique minute quantity of
sample sufficiennt simple to perform sample pretretment not
necessary separation of small ions upto 1000Da is possible
BIOMEDICAL 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
8. INTERPRETATION: calibration curve obtained from a set of
standard isotachopherograms Qulitative-step height
Quantitative-Zone length
9. 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.
10. Principle of IEF:
11. 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
12. 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.
13. One of the best method for protein seperation or
purification can be easily done if a isoetectric point of a protein
is known
14. 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
(pI), which is necessary to separate 2 neighbouring bands: