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
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.
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
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)
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.
Would the resin in
this example be
resin or a cation-exchange
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.
Electrostatic Potential Map of the Surface of a Protein
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.
Principle of Ion-Exchange Chromatography
Fig. 5-13 (Ninfa & Ballou)
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
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.
Strong anion exchangers
233 -CHN+(CH)triethylaminoethyl TEAE
24253 Weak anion exchangers
243 -CHN+(CH)diethylaminoethyl DEAE
24252 Strong cation exchangers
-SO- sulpho S
- sulphomethyl SM
Weak cation exchangers
-CH2COO- carboxymethyl Cadapted from N & MB Table 5-2
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
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
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
This discussion will require some board work
Asp, Glu 4.0
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
Determination of pI for a Protein
(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.
Now that we understand the concept of pI (I hope),
we are in a better position to consider the choice of
The pH vs. net
curves for three
for a CM and a
shown at the top
A Question to Ponder
Proteins are usually least soluble and often
precipitate at their isoelectric point.
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
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
systems usually control
the gradient formation
by the use of two
pumps, one for buffer A
Amersham Biosciences and one for buffer B
A simple gradient maker:
high salt low salt
Fig. 5-14 (Ninfa & Ballou)
In our ion-exchange
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