Chromatofocusing is a technique that separates proteins based on their isoelectric point (pI). It uses buffers like Polybuffer 74 or 96 to generate a linear pH gradient within an ion exchange column containing beads like PBE 94. Proteins are separated as they elute from the column based on their net charge, with more acidic proteins eluting first when the pH is below their pI and more basic proteins eluting later when the pH exceeds their pI. Chromatofocusing can resolve small differences in pI and is useful for analytical separations and purification of proteins.

1. by
Amlan Barai
13I300002
Department of Bio-Science & Bio-Engineering
Indian Institute of Technology Bombay

2. pH and Protein
pH=-log[H+]
So,
A protein at pH3 have more [H+] in its environment
than a protein at pH9

3. pKa
• pKa is the pH at which any group donates half of its ionisable proton
pKa=-logKa
• pKa tells us how acidic (or not) a given hydrogen atom in a
molecule is. The stronger the acid, the lower its pKa; the
stronger the base, the higher its pKa

4. pH
pH
2.34
1
Amlan Barai, 13i300002, BSBE, IIT Bombay

5. pH
9.69
6
Amlan Barai, 13i300002, BSBE, IIT Bombay

6. Increasing the
at pKa1
=2.34
pH
Amlan Barai, 13i300002, BSBE, IIT Bombay

7. Decreasing the
at pKa2
=9.69
pH
Amlan Barai, 13i300002, BSBE, IIT Bombay

8. The Midpoint : pI or Isoelectric Point
Amlan Barai, 13i300002, BSBE, IIT Bombay

9. So pI or Isoelectric Point
is simply the pH at which the net charge is Zero.

10. Calculation of pI:the pKa & pI of Amino
Acids
Table: Modified from Lehninger Principle of Biochemistry, 5th ed. P-73.

11. pI of Protein
above pI: -ve
Below pI:+ve

12. So the Bottom line
• Proteins and Amino Acids are Amphoteric molecules with positively
and negatively charged groups, where their dissociation depends on
the H+ ion concentration of the surrounding environment.
• Molecules at pH above its pI have net –ve charge & Below its pI have
net +ve charge.

13. Chromatofocusing
• Separating on the basis of pI (Isoelectric Point)
• First Discovered in 1978 by Sluyterman And His Colleagues

14. above pI: -ve
Below pI:+ve
Ref: http://macromol.sbcs.qmul.ac.uk/oldsite/expertise/CF3.jpg

15. The Elution profile of two Proteins
above pI: -ve
Below pI:+ve
9
8
Ref(modified from): Chromatofocusing- Douglas D Frey,Chittoor R Narahari, Ronald C
Bates, Encyclopedia Of Life Sciences /&2001 Nature Publishing Group / www.els.ne

16. Focusing in chromato“focusing”
Amlan Barai, 13I300002, BSBE, IIT Bombay.
pH<8
pH=8(=pI)
pH>8
+
-
pH<8
pH=8(=pI)
pH>8
pH<8
pH=8(=pI)
pH>8
+
-
pH<8
pH=8(=pI)
pH>8
-
Elution with Gradually increasing
pH
+
+
+
+
+
+
+
+
+
+
+
above pI: -ve
Below pI:+ve

17. Focusing in chromato“focusing”
above pI: -ve
Below pI:+ve

18. Chromatofocusing: The Assembly

19. The pH Gradient
The self generated gradient
5
pH5
pH9

20. Buffers used for chromatofocusing
Polybuffer 74
• Polybuffer 74 forms a linear pH gradients from pH 7 to 4.
• Use Polybuffer 74 for any pH gradient between 7 and 4.
• Developed for chromatofocusing, form linear pH gradients.
• Mixtures of selected amphoteric buffering substances of different pI
and pKa values.
• Resolves pI differences of 0.04 pH units.
Ref:GE Healthcare(http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/products/AlternativeProductStructure_17391/17071201)
& Amersham Pharmacia Biotechnology.

21. Buffers used for chromatofocusing
Polybuffer 96
• Polybuffer 96 forms a linear pH gradients from pH 9 to 6.
• Developed for chromatofocusing, form linear pH gradients.
• Mixtures of selected amphoteric buffering substances of different pI
and pKa values.
• Resolves pI differences of 0.04 pH units.
• Use Polybuffer 96 for pH gradients that should begin above pH 7.
Ref:GE Healthcare(http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/products/AlternativeProductStructure_17391/17071201)
& Amersham Pharmacia Biotechnology.

22. Beads for chromatofocusing
PBE 94 (Polybuffer exchange94)
• PBE 94 is a bead-formed exchanger gel (Sepharose®). Charged groups
has coupled with them via ether linkage.
• It has an even capacity over a wide pH range.
• It is developed specifically for chromatofocusing with Polybuffer™
• Highly stable can even function in presence of 8M Urea and at 120°C.
Ref:GE Healthcare(http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/products/AlternativeProductStructure_17391/17071201)
& Amersham Pharmacia Biotechnology.

23. Application
• Separating proteins according to isoelectric point (pI)
• It is a powerful method for high resolution, since it can resolve very
small differences in pI (down to 0.02-0.05 pH units) and thus
separate very similar proteins.
• Used for analytical separations.

24. Applied for
• Separation of two isoforms of the proteinb2-macroglobulin that
differ by a single amino acid residue(Odani H, Oyama R, Titani K, Ogawa H and Saito A
(1990)Biochemical and Biophysical Research Communications)
• Purification and concentration of proteins produced by Haemophilus
influenzaefor use in proteome analysis (Fountoulakis M, Langen H, Gray C and Takacs B
(1998)Journal of Chromatography A806: 279–291)
• Preparative-scale separation and purification of the peptides
thymosinb4 and thymosinb9 from bovine tissue (Roboti A, Livaniou E, Evangelatos
GPet al. (1994)Journal of Chromatography A662:27–34)
• Separation of cortisol–bovine serum albumin conjugates (Giraudi G and
Baggiani C (1990)Analyst115: 1531–1534)

25. Reference
• Protein Liquid Chromatography-edited by Michael Kastner
Chapter7-Chromatofocusing:Richard Lukacin and Wolfgan R. Deppert
(http://books.google.co.in/books?id=3WhftkdNpxYC&dq=chromatofocusing+principle)
• Chromatofocusing- Douglas D Frey,Chittoor R Narahari, Ronald C Bates, Encyclopedia Of Life
Sciences /&2001 Nature Publishing Group / www.els.ne
• Amersham Pharmacia Biotechnology (1987)Chromatofocusing with Polybuffer and PBE.
Uppsala: Amersham.
• Chromatofocusing :L . A . Ae . Sluyterman and J . Wijdenes Isoelectric Focusing On Ion-exchange
Columns
Journal of Chromatography, 150 (1978) 31-44Q Elsevier Scientific Publishing Company,
Amsterdam - Printed in The Netherlands
• GEHealthcare(http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSci
ences/products/AlternativeProductStructure_17391/17071201)