Source of protein

Crude enzyme extract

Extraction
Salt precipitation

Separation

Gel filtration Ion exchange

Purity &
characterization

Ion-exchange
Topic 4: Ion-Exchange Chromatography

Source of protein
What is the principle behind this
method?
What are the types of ion-exchange
Extraction chromatography available?
At which protein purification step is this
method a target?
Separation
Procedure and exploitation.
Can I use any ion-exchange resins and
Purity & buffers in together?
characterization
Think creatively

Ion-exchange
Principle of ion exchange chromatography

Proteins are charged molecules. At specific pH, it can

exist in anionic (-), cationic (+) or zwitterion (no net
charge) stage.

cationic pH =pI anionic

pH increase

In Ion exchange chromatography separation is based on

the charges carried by the protein molecules

Ion-exchange
Since proteins can have net positive or negative
charge, it becomes obvious that two forms of
ion-exchanger would be relevant

Negatively (anionic species) or positively (anionic

species) charged molecules can bind to functional groups
bearing the opposite charges.

Possible to have two types of ion exchangers

Anion exchanger (anion exchange chromatography)
Cation exchanger (cation exchange chromatography)

Ion-exchange
Anion exchange chromatography
Positively charged functional groups are bound to
the insoluble matrix

- Anion exchanger is positively

charge
+
-
+ -
+- Counter ions are negatively charged
+
+ +
-
-

Ion-exchange
Anion exchange chromatography

Negatively charged proteins can

- replace the counter ions and be
+ bound to the ion exchanger.
-
+ - Counter ions in the eluting buffer
+- can then exchange for the protein
+
+ species, thus releasing the proteins
+ from the ion exchanger
-
- Separation is based on the degree
of of binding strength of the proteins
to the ion exchanger

Ion-exchange
Ion exchangers – Functional groups

Counter ions
- +
+ -
- +
+ - - +
+- - + -
+
+ -
+ -
- +
- +

Anion exchanger Cation exchanger

Aminoethyl (AE-) Carboxymethyl (CM-)
Diethylaminoethyl (DEAE-) Phospho
Quaternary aminoethyl (QAE-) Sulphopropyl (SP-)

Ion-exchange
Topic 4: Ion-Exchange Chromatography

What is the principle behind this method?

What are the types of ion-exchange chromatography
available?
; At which protein purification step is this method a
target?
Procedure and exploitation.
Can I use any ion-exchange resins and buffers in
together?
Some recent development

Ion-exchange
 1

Ion-exchange chromatography can be used after salt

precipitation step or after gel-filtration.
1. Requires dialysis of sample prior to ion-exchange
2. Can be used directly after gel filtration
Ion-exchange
Topic 4: Ion-Exchange Chromatography

What is the principle behind this method?

What are the types of ion-exchange chromatography
available?
At which protein purification step is this method a target?
; Procedure and exploitation.
Can I use any ion-exchange resins and buffers in
together?
Some recent development

Ion-exchange
Set up for ion-exchange chromatography

Ion-exchange
 How?

Steps involved

Ion-exchange
Rationale behind steps

Steps Remarks
1. Sample application and Sample diffuses into ion-
adsorption and equilibration exchanger surface and matrix
Unbound proteins will be removed

2. Elution of column with specific Weakly bound proteins will be

buffers to achieve protein eluted first, followed by those
separation - which are tightly bound
salt gradient
ionic strength gradient

3. Regeneration of ion-exchangers Ion-exchanger can be reused

Ion-exchange
Which type of ion exchanger should I use?

Establish if you need to use an anion or cation

exchanger
Consider the strength and capacity of ion exchanger
Do you have any preference for matrix type?
Which buffer should I use with the selected ion
exchanger

Ion-exchange
Choosing your ion-exchanger: know
your proteins
Stability of proteins
ƒ stable below pI value, use cation-exchanger
ƒ stable above pI value, use anion-exchanger

Molecular size of proteins

ƒ <10,000 mw, use matrix of small pore size
ƒ 10,000-100,000 mw, use Sepharose equivalent grade

Other specific requirements

ƒ Inactivation of specific buffer types, then limit choice of ion-
exchanger

Ion-exchange
Important to consider the stability of proteins in choice of ion-
exchangers. Isoelectric focusing can be used to identify suitable
ion-exchanger type

pH=pI
pH>pI

pH<pI

Ion-exchange
What is the ideal condition (pH) for substance to
bind to ion-exchanger?

Ion-exchange
If too high a pH is chosen, binding of substance to ion-
exchanger becomes strong, and elution becomes
difficult and high salt concentrations may have to be
used

Ion-exchange
Strength and Capacity of Ion-exchangers

Strength
determined by functional group
strong or weak ion-exchangers -
reference to extent of ionisation
with pH
strong - complete ionisation over
a wide pH range
Example:
DEAE = weak ion-exchanger
SP = strong ion-exchanger
Capacity
quantitative measure of ion-
exchange ability to bind ions
depends on number of available
functional group
Information usually provided by
manufacturer

Ion-exchange
Ion-exchangers: Matrix available

Sephadex
Sepharose
Cellulose
Polyacrylic acid
Polystyrene

Ion-exchange
Choice of buffers. Use the correct buffers

Consideration
Buffer types
Cationic buffers with anion- DEAE: Tris, Imidazole, pyridine
exchangers
Anionic buffers with cation- CM: acetate, citrate, phosphate
exchangers
pH
1 unit + pI value of protein anion exchanger: 1 unit > pI
cation exchanger: 1 unit< pI
facilitate binding

ionic-strength
determined experimentally
for separation Salts used: NaCl, KCl

Ion-exchange
Ionic strength of buffer & elution types

To achieve good separation, choice of buffer ionic strength is as

important as choice of ion-exchangers
Initial strength: LOW ionic strength
Different approaches taken for different purposes
isocratic elution
ionic strength manipulated to elute or retain protein
gradient elution
increasing ionic strength (low to high)
continuous gradient
step gradient

Ion-exchange
Elution gradient and flow rates are important
factors in ion-exchange chromatography

Ion-exchange
Gradient steepness can affect resolution

Flow rate 8ml/h

Gradient 0 to 0.3M NaCl

Flow rate 8ml/h

Gradient 0 to 0.4M NaCl

Ion-exchange
Same elution gradient but different
flow rate

Flowrate 8 ml/h

Flowrate 20 ml/h

Ion-exchange
 Think creativity

You do not have to bind the protein of interest

to the ion-exchanger. It can be reversed.

You can always bind what you do not want to

the ion-exchanger. Sometimes this can be an
advantage.

Ion-exchange
Application. Using both anion and cation exchangers

Ion-exchange
 Crude enzyme extract

Salt precipitation

Dialysis step

Gel filtration Ion exchange

Concentration step
needed

Ion-exchange
 Summary

Know what is anion and cation exchange

chromatography
Know the availability of different types of ion-exchange
chromatography resins and how to apply them
Able to decide at which stage of protein isolation should
we use this method
Able to follow published procedure involving this
procedure

Ion-exchange