The document discusses iso-electric focusing, a technique for protein separation based on iso-electric points, which was discovered by H. Sevennson. It outlines the principle, instrumentation, advantages, and disadvantages of the method, and highlights its applications in various fields, including enzymology and forensic genetics. The document emphasizes that iso-electric focusing is the first step in 2D gel electrophoresis for protein separation.

1.  SUBMITTED BY:- MOHAMMAD JAVED
 M.PHARM 1ST SEM
 PHARMACEUTICAL CHEMISTRY
 SUBJECT:- MODERN PHARMACEUTICAL ANALYTICAL
TECHNIQUE
 TOPIC:- ISO-ELECTRIC FOCUSING.

2. CONTENTS
1. Introduction
2. Graphical representation
3. Principle
4. Instrumentation
5. Factor affecting seperation
6. Advantage and disadvantage
7. Summary
8. Reference

3. INTRODUCTION
• It was first discovered by H.Sevennson, Sweden.(1)
• We use this technique as protein separation on the basis of Iso-elelectric point( p.I).
• It is also called electro-focusing.
• It is a technique for separating different molecule in their iso-electric point. It is a type of
zone electrophoresis performed by protein.(2)
• Iso-electric point(pI):- The pH at which net electric charge on protein becomes zero.
• Below p.I- positive charge(acidic)
• Above p.I- negative charge(basic)
• Protein moves towards the electrode with opposite charge.

4. 
 Graphical representation of iso-electric point of Alanine

5. PRINCIPLE
• It is performed in pH gradient.
• The proteins are amphoteric molecules with acidic and basic buffering group.
• In acidic environment the basic group becomes positively charged while in Basic
environment the acidic group becomes negatively charged.
• Proteins are positively charged in solution at pH value below pI,migrate towards
cathode.
• Proteins are negatively charged in solution at pH value above pI, migrate towards
anode.
• At pH=pI the protein have no charge and stop.(3)

7. INSTRUMENTATION
 Focusing Tray : These are the trays that hold the gel media,
i.e. IPG strips on which proteins will migrate.
 IPG Strip : IPG(immobilized pH gradient) Strips are
commercially available gel media having a stable pH
gradient of mostly 3 - 10. These are formed by casting
polyacrylamide gels using acrylamide buffers on a plastic
backing. These are more stable and reproducible.
 Wick : Wicks are placed over the ends of the gel to collect
salts and proteins/peptides that are outside of the pI range
of the IPG strip.
 Electrodes : There are two electrodes of opposite charge are
attached at the opposite end of the focusing tray which will
provide the necessary electric field.
 Cup Holder : Samples are mixed with ampholytes and
loaded in the Cups so that it can migrate.(2)

8. FACTOR AFFECTING SEPERATION
i. pH
ii. Intensity of electric field
iii. Temperature
iv. Starting and find voltage
v. Time
vi. Mode of ramping

9. ADVANTAGE AND DISADVANTAGE
 Advantages:
 Proteins that as by little 0.001 pH unit can be separated.
 I.E.F is a powerful analytical tool for separation of protein.
 Disadvantage:-
 Inconsistency
 Limited stability of solution
 Inadequate purity of application as a standard.

10. SUMMARY
• Food and agricultural industries.
• In enzymology, immunology, membrane bio chemistry.
• Forensic and human genetic lab.
• I.E.F is the 1st step in 2D gel electrophoresis and then furthur separated by SDS-
PAGE.
• It is used to separate different protein/peptide molecule on the basis of the pI and
charge value.

11. REFERENCE
1. Rilbe, H. (1973, June). HISTORICAL AND THEORETICAL ASPECTS OF
ISOELECTRIC FOCUSING. Annals of the New York Academy of Sciences,
209(1 Isoelectric F), 11–22.
2. Mahadik k, Sathiyanarayan L:, Instrumental Method of analysis: Nirali
publication, August 2020; Page no,: 13.4.3
3. Pergande, M., & Cologna, S. (2017, January 25). Isoelectric Point
Separations of Peptides and Proteins. Proteomes, 5(4), 4.
https://doi.org/10.3390/proteomes5010004