Ion-exchange chromatography separates molecules on the basis of charge. The stationary phase contains resin beads with cationic or anionic functional groups that can bind positively or negatively charged molecules. Proteins either bind to the resin based on their net charge or pass through. Elution is achieved by changing pH or adding salts to compete for binding sites on the resin. Modern systems automate ion-exchange chromatography using gradients controlled by multiple pumps and fraction collectors.

1. Ion-Exchange Chromatography
Introduction to Chromatography: General Principles
Ion-Exchange Chromatography Principles
Ion-Exchange Chromatography Resins
Net Charge of Amino Acids, Peptides, and Proteins
Integrated and Automated Systems
Reading: N & B Ch. 5

2. Introduction
Chromatography literally means “color writing”.
Chromatography was invented by the Russian
botanist Mikhail Tsvet in 1900. He used it to
separate chlorophyll-containing extracts of plants.
Key idea is that molecules of interest interact
differentially with the stationary phase and a mobile
phase, and thus can be separated.

3. The Basic Principle

4. Partition Coefficient and Relative Mobility
Partition coefficient describes the affinity of a
compound for the stationary phase.
a or (Kav)= molecules adsorbed on stationary phase
molecules in stationary and mobile phase
Can have values between 0 and 1. Example, a
molecule with a = 0.4 will be 40% adsorbed on the
stationary phase.
Relative mobility or retention factor (Rf) describes
the affinity of a molecule for the mobile phase.
Rf = 1 – a (Recall Rf from TLC in Organic Chem)

5. Ion-Exchange Chromatography
Separates molecules on the basis of charge
Beads of the resin modified so that they contain
a cationic or anionic functional group that can be
positively charged, negatively charged, or neutral
depending on pH.
A solution that contains the species of interest is
applied to the column containing the resin, and the
sample either binds to the resin or passes through
the column. A gradient (e.g., salt or pH) can then be
used to elute the desired compound if the compound
adhered to the resin.

6. Ion-Exchange
Chromatography
Would the resin in
this example be
considered an
anion-exchange
resin or a cation-exchange
resin?

7. Ion Exchange Resins
Proteins with net negative charges (excess of negative
charges) adsorb to anion exchangers, while those with
net positive charges (excess of positive charges)
adsorb to cation exchangers. The strength of the
adsorption increases with increased net charge.

8. Ion-Exchange
Chromatography:
a closer look

9. Electrostatic Potential Map of the Surface of a Protein
http://www.expasy.ch/spdbv/images/1YDRsurf.jpg

10. Desorption
Two possibilities exist to
desorb sample molecules
from the ion exchanger:
1. Reducing the net charge
by changing pH.
2. Adding a competing ion
to "block" the charges on
the ion exchanger.

11. Principle of Ion-Exchange Chromatography
Fig. 5-13 (Ninfa & Ballou)

12. Principle of Ion-Exchange Chromatography cont.
Fig. 5-13 (Ninfa & Ballou)

13. What is the nature of the functional groups that are
covalently linked to the resin?
You will be using DE52, which
contains DEAE functional groups
attached to a cellulose matrix

14. Depending on the pKa value of the charged
ligand, the ion exchangers are divided into
strong and weak.
Strong ion exchangers are fully charged
over the total pH range normally
applicable to proteins and peptides.
With weak ion exchangers, the charge
displayed is a function of the eluent pH.

15. Examples
Strong anion exchangers
-CHN+(CH)trimethylaminoethyl TAM
233 -CHN+(CH)triethylaminoethyl TEAE
24253 Weak anion exchangers
-CHN+Haminoethyl AE
243 -CHN+(CH)diethylaminoethyl DEAE
24252 Strong cation exchangers
-SO- sulpho S
3
-CH2SO3
- sulphomethyl SM
Weak cation exchangers
-CH2COO- carboxymethyl Cadapted from N & MB Table 5-2

16. How Do We Know If “Our” Protein Is Going
to Bind the Ion-Exchange Resin That We Are
Using? – pH, pKa, pI & Buffers Revisited:
pH = -log[H+] (not strictly true but a useful, working
definition)
pH = pKa + log([basic form]/[acidic form]) [HH eq]
Isoelectric point (pI) is the pH at which a molecule
has a net charge of zero.
Buffers useful ±1 (or ±0.5) units above and below
their pKa

17. Deciding on the Charge of Our Protein:
We start by considering a simple, weak acid: RCOOH
Then, we consider a weak base: RNH2
Then, we will consider a compound that has both of
the above functional groups – i.e., an amino acid
Then, we will consider small peptides
Finally, we will extrapolate to a polypeptide – i.e., a
protein
This discussion will require some board work

19. Group pK
a
N-terminal
amino
8.0
C-terminal
carboxyl
3.1
Asp, Glu 4.0
Lys 10.
4
Arg 12.
5
The pKas of groups or side chains can and do vary
somewhat from what their values are in free amino
acids. The values in the table below are meant to be
approximate, but on average, fairly representative

20. Determination of pI for a Protein
http://ca.expasy.org/tools/pi_tool.html
http://emboss.sourceforge.net/
(and a number of other sites)
Or, experimentally determine pI by using
isoelectric focusing, a topic we will take up
when we discuss SDS PAGE.

21. Now that we understand the concept of pI (I hope),
we are in a better position to consider the choice of
ion exchanger
Pharmacia handbook

22. The pH vs. net
surface charge
curves for three
different proteins
are shown.
Schematic
chromatograms
for a CM and a
DEAE ion
exchanger are
shown at the top
and bottom,
respectively.

23. A Question to Ponder
Proteins are usually least soluble and often
precipitate at their isoelectric point.
WHY?

24. Considerations
Conditions used to purify a protein are often
determined empirically. You likely will choose which
resin to use on the basis of the pI of the protein (if it is
known or can be estimated). Then you need to decide
on the buffer, the salt, the steepness of the gradient,
etc… You may want to run some pilot experiments.
After you decide which resin you want to use,
you will then have to:
-swell (hydrate) of the resin -load the sample
-equilibrate with buffer -elute the sample
-pack or pour the column -locate the sample
-equilibrate the sample -determine purity

25. Gradients of a neutral
salt are formed by
mixing two eluents, one
containing a low
concentration of the
neutral salt (buffer A)
and one containing a
high concentration of
this salt (buffer B). But
for their salt contents,
the two eluents are
identical.
Chromatography
systems usually control
the gradient formation
by the use of two
pumps, one for buffer A
Gradient Elution
Amersham Biosciences and one for buffer B

26. A simple gradient maker:
high salt low salt
Fig. 5-14 (Ninfa & Ballou)
In our ion-exchange
chromatography
lab, we will not
use a gradient.
Rather, we will
use a step elution
in which we go from
low salt to high
salt in one step

28. Steep vs. shallow gradient elution

29. Steep vs. Shallow Gradient Elution – Another View
Amersham Biosciences
The distance
between
peaks is
controlled
by the
slope of
the gradient

30. A Bit More On Integrated and
Automated Chromatography Systems
HPLC
(High Performance or High-Pressure Liquid
Chromatography)
FPLC
(Fast Protein or Fine Performance Liquid
Chromatography)

31. Schematic
of an
automated
system (FPLC)
Low salt High salt
Pumps
Sample load
Fraction
collector
Sheehan, David (2003). Fast Protein Liquid Chromatography. 244. pp. 253.