chromatofocusing chromatography

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)