Title: Ion Exchange Chromatography

Aim: To understand the chemistry behind ion exchange resins; to use ion exchange
chromatography to separate and purify biological compounds; to adequately use and construct a
calibration curve as a means to determine unknown quantities.

Theory: Separation in ion exchange chromatography depends upon the reversible adsorption of
charged solute molecules to immobilized ion exchange groups of opposite charge. Most ion
exchange experiments are performed in five main stages:
The first stage is equilibration in which the ion exchanger is brought to a starting state, in terms
of pH and ionic strength, which allows the binding of the desired solute molecules. The
exchanger groups are associated at this time with exchangeable counter-ions (usually simple
anions or cations, such as chloride or sodium).
The second stage is sample application and adsorption, in which solute molecules carrying the
appropriate charge displace counter-ions and bind reversibly to the gel. Unbound substances can
be washed out from the exchanger bed using starting buffer.
In the third stage, substances are removed from the column by changing to elution conditions
unfavourable for ionic bonding of the solute molecules. This normally involves increasing the
ionic strength of the eluting buffer or changing its pH.
The fourth and fifth stages are the removal from the column of substances not eluted under the
previous experimental conditions and re-equilibration at the starting conditions for the next
purification.
Separation is obtained since different substances have different degrees of interaction with the
ion exchanger due to differences in their charges, charge densities and distribution of charge on
their surfaces. These interactions can be controlled by varying conditions such as ionic strength
and pH. The differences in charge properties of biological compounds are often considerable,
and since ion exchange chromatography is capable of separating species with very minor
differences in properties, e.g. two proteins differing by only one charged amino acid, it is a very
powerful separation technique.
The fractionation of proteins by ion-exchange chromatography, which is what will be examined
in this lab, depends upon differences in the charge of different proteins. The charge of a protein
depends upon the number and type of ionisable amino acid side chain groups. Each ionisable
side chain group has a distinct pKa. Therefore the overall number of charges on a particular
protein at a particular pH will depend on the number and type of ionisable amino acid side chain
groups it contains. Since, by definition, different proteins have different amino acid
compositions, they will tend to have different charges at a given pH and so can be fractionated
on this basis.
For any one protein there will be a pH at which the overall number of negative charges equals
the number of positive charges and so it has no net charge. This is its isoelectric point (pI). At
this pH the protein will not bind to any ion-exchange resin. Below this pH the protein will have a
net positive charge and will bind to a cation exchanger, whilst above this pH it will have a net
negative charge and bind to an anion exchanger.

Procedure:

Materials

1) Anionic resin – Amberlite IR 120 (H)

2) Cationic resin – Amberjet 4400 (OH)
3) 0.1 M HCl
4) 0.1 M NaOH
5) 1.0 M NaOH
6) 1.0 M HCl
7) 0.8 M NaCl
8) 1.0 M NaCl
9) Chromatography column (20 x 0.75 cm) with tubing and clamp.
10) 50 mM Glycine – stock solution
11) Ninhydrin solution – freshly prepared (provided) – 2g of ninhydrin and 0.3g of
hydrindantin in 75mL of methylcellosolve. Add 25mL of 4.0 M acetate buffer pH 5.5.

This lab was divided into three parts, the first two of which dealt with an Anionic resin and a
Cationic resin respectively.

For the first part of the lab, the tip of the ion exchange column was first plugged with a small
piece of glass wool. The wool was not packed too lightly. Noting the exact weight, 3.0 ± 0.2g of
IR 120 H resin was placed into a 50 mL beaker to which water was added a little at a time in
order to make a slurry. This slurry was then transferred to carefully to the column. The clam of
the column was then released to allow excess water to flow through but it was ensured that the
liquid did not drain below the surface of the resin. The tip of the column was then clamped to
make sure that no air bubbles were trapped in the column.

The column was then primed by adding 4.0mL of 0.1 M HCl. The solution was allowed to
slowly pass into the column. The column was then washed with five 20mL portions of water to
remove excess HCl. The washings were then discarded.

2.0 mL of 1.0 M NaCl was then added to the column. An Erlenmeyer flask was then placed
under the column. The clamp was then slowly released and the NaCl solution was allowed to
pass into the column. 20mL of water was then gradually added and the elute was collected. The
elute was then titrated with 0.199 M NaOH and using methyl orange indicator.

The addition of 2.0mL of 1.0 M NaCl and elution with 20mL of water was repeated until five
additions of NaCl had passed through the column. 10mL of 1.0 M NaCl solution was then added
to the column. This was then elute with 40mL of water.
Part B of this lab dealt with an anion exchange resin. For this part of the lab, a column was first
prepared containing 3.0 ± 0.2g of amberjet 4400 resin. The column was then primed with 4.0mL
of 0.1 M NaOH followed by eight 20mL washings with water to remove excess NaOH. 3mL of
stock glycine solution was then placed in a test tube, to which was then added two drops of 1 M
NaOH to bring the pH above 9. Exactly 1mL of this alkaline solution was then added to the
column. The column was then washed with 20mL of water. The washings were discarded. It was
ensured that not more than 1mL of the alkaline glycine solution was added to the column since
overloading of the column would give erroneous results.

Three 15mL aliquots of 0.8 M NaCl were added to the column and three 15mL eluates were
collected. One drop of phenolphthalein indicator was added to each eluate and the pH was
adjusted to neutrality using acid or alkali. The volume of each eluate was then measured after pH
adjustment in a 25mL measuring cylinder. This volume was recorded.

From each eluate was then taken separately, 0.1mL and 0.3mL aliquots for quantitative glycine
determination. The eluates were saved until the experiment was completed. The volume of each
aliquot was then made up to 0.5mL with 0.8 M NaCl. 0.8mL of the prepared ninhydrin solution
was then added to the solution. The tubes were all mixed and placed in a boiling water bath for
20 minutes. The tubes were then cooled and 4mL of 50% aqueous n-propanol was added to each
tube. They were all then mixed thoroughly.

The tubes were allowed to stand for 10 minutes at room temperature and then the absorbance
was read at 540nm.

Part C of this lab dealt with finding values in order to plot a Calibration Curve for Glycine. For
this part of the lab, 100mL of 0.5 mM solution in 0.8 M NaCl was prepared using stock glycine
solution. A set of twelve test tubes were then labelled from numbers 1 – 12.

To test tube 1 was added 0.5mL of 0.8 M NaCl. To test tubes 3 and 4 were added 0.1mL of 0.5
mM glycine solution and 0.4mL of 0.8 M NaCl. To test tubes 5 and 6 were added 0.2mL of 0.5
mM glycine and 0.3mL of 0.8 M NaCl. To test tubes 7 and 8 were added 0.3mL of 0.5 mM of
glycine and 0.2mL of 0.8 M NaCl. To test tubes 9 and 10 were added 0.4mL of 0.5 mM glycine
and 0.1mL of 0.8 M NaOH. To test tubes 11 and 12 was added 0.5 mL of 0.5 mM of glycine.

To each of these test tubes 0.8mL of prepared ninhydrin solution was added. The tubes were all
mixed and placed in a boiling water bath for twenty minutes. The tubes were then allowed to
cool. 4mL of 50% aqueous n-propanol was then added to each tube which were all then mixed
thoroughly. The tubes were allowed to stand for ten minutes at room temperature after which
time their absorbencies were read at 540nm.

References:

http://sbio.uct.ac.za/Sbio/documentation/Ion_exchange_chromatography.pdf
David L. Nelson, Michael M. Cox. Lehninger Principles of Biochemistry. New York: W. H. Freeman and
Company, 2008.