Ion-exchange Chromatography and Gel Filtration

Chromatography
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ABSTRACT
Ion-exchange chromatography and gel filtration chromatography were performed in order to understand the
principles behind the way they purify proteins. The main difference the two chromatographys possess is the basis
in which they separate their proteins. IEC separates based on charge and GFC separates on the basis of molecule
size, but both determine through absorbance readings and fractionation of the sample. For IEC the protein was
obtained in fraction 12, and for GFC the molecular weight was estimated for albumin and casein were 74,182 Da
and 44,355 Da, respectively.

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INTRODUCTION
Proteins are purified in many ways on the basis of
such characteristics as solubility, size, charge, and
specific binding affinity. Protein purification occurs in a
series of processes that aims to isolate a protein from
the biological tissue or microbial culture. [1] The
protein mixtures are subjected to series of separations
based on a different property in order to obtain the
pure protein.
In this experiment ion-exchange chromatography and
gel filtration chromatography were used to purify the
albumin extracted from a previous experiment.
However, gel filtration was not done, rather it was only
discussed due to lack of resources. The main
difference between the two column chromatographys
is their way of separating the proteins from other
substances. Ion-exchange chromatography separates
the proteins based on their charge, and gel filtration
chromatography separates them based on their size.
Ion-exchange chromatography makes use of the
positive and negative charges different proteins have.
Buffers are used to control the ionization of different
proteins and the net charge of the protein itself.
During chromatography, proteins that have the same
charge as the stationary phase will be eluted out while
those with the opposite charge will bond with the
charged resin. [2] In gel filtration chromatography, the
size of the molecules limits them from passing through
the pores of the gel matrix. Large molecules are
eluted out faster because they cannot pass through
the pores of the gel. The smaller molecules that can fit
through gel pores, which slows down its elution. [3]
Gel filtration is often used for estimation of the
molecular weight of proteins. Although, GFC is limited
to very stable samples only, and limits the researcher
to analysing only one sample at a time.

The objective of this experiment is to understand the

principles behind these two types of chromatographic
set-ups and understand further their ability to purify
proteins, specifically albumin.
EXPERIMENTAL DETAILS
The experiments were performed in two separate
days, first the ion-exchange chromatography and the
gel filtration came second.
For the first experiment the equipment used were the
following: an IEC column, micropipettes, pasteur
pipettes, measuring pipettes, test tubes, a quartz
cuvette and a UV-Vis spectrophotometer.
The experiment started with the preparation of the ionexchange column. The resin, DEAE cellulose, was
allowed to settle in 400 mM HCl after being stirred for
about 30 minutes. The solution with the gel was then
washed for 30 minutes with 400 mM NaOH and then
with water to reach a neutral pH of 7. The gel was
then suspended in a graduated cylinder with water,
90% of which was allowed to settle. Swelling the resin
minimises the cross linking in the resin allowing small
ions to diffuse in and out. The top layer of the solution
was decanted to rid of fine particulates that could clog
the column. The resin was then placed in the column
and the flow rate was fixed to 29 drops per minute and
washed with 5 column volumes of the Tris-HCl buffer
to equilibrate. The equilibration gives the column the
proper pH and ionic strength needed for the binding
the proteins to the resin.
The sample was loaded into the column by adding a 1
mL portion of the extracted albumin with 2 mg/mL
protein and was allowed to pass through with Tris-HCl
pH 8.0 buffer as the solvent three times. The flowthrough from the sample was pooled and labelled.
Then the column was washed with Tris-HCl buffer of
portions three times the bed volume, collected and
labelled as washings. For the elution of the absorbed
proteins the column was washed twice each with

equal bed volumes of the following concentrations of

KCl: 0.05 M, 0.1 M, 0.2 M, 0.3 M, 0.5 M and 1 M KCl.
The portions were all labelled according to their order
of elution as fractions 1-12 and plated on a 96-well
plate. Then to finish, two extra fractions were collected
using distilled water as the eluent. The regeneration
process of the column was done by washing the
column five times the bed volume with distilled water
and then re-equilibrated by the buffer at pH 8 for
future use.
The albumin was detected using the UV-vis
spectrophotometer and the Bradford assay. For the
Bradford Assay where 40uL of the Bradford reagent,
135 uL of distilled water and 25uL of the fractions
were plated. The absorbance was read at 595 nm,
and plotted to determine which fraction had the
highest absorbance.
For the second part of the experiment gel filtration
chromatography was discussed in class. The
equipment to be used are the following: micropipettes,
quartz cuvettes, measuring pipette, Eppendorf tubes,
GFC column and the UV-Vis spectrophotometer.
For the set up the GFC column should be set on an
iron stand, and calibrated by 1 mL increments of
distilled water using a micropipette. The water should
then be drained slowly through a controlled rate flow
which which will be stopped when the column is about
a third full. Sephadex G-150 will then be swelled in
distilled water over 4 hours and allowed to settle. Any
excess water should be decanted before the gel is
suspended in 4 volumes of the equilibration buffer.

of the set up is obtained and the steps repeated for

the extracted albumin.
RESULTS AND DISCUSSION
In IEC the buffer used must have an appropriate pH
for binding. Substances often dissociate if the pH is
too close to the isoelectric point of the protein. The
ideal buffer has a pH only 1 above the isoelectric point
for an anion exchanger and 1 pH below the for a
cation exchanger. The buffer solutions must also have
appropriate ionic strengths and minimal interaction
with the resin. [4] The buffer used in this experiment
was the Tris-HCl buffer because it fit into the criteria
needed for a buffer in IEC.
The matrix is a mother important consideration for
IEC, it is where the charged groups are covalently
bound. Usually it is composed of inorganic
compounds, synthetic resins, or polysaccharides. It is
important that the matrix is the opposite charge of the
sample being purified so that they may bind to each

While the stopcock is opened, 10-15 mL column

volume of the gel slurry should be dispensed and
allowed to settle. The buffer will then be allowed to
flow until there approximately only 1 cm level of it is
left above the gel bed, then the stopcock can be
closed. The column should equilibrated by running 2
column volumes of the buffer, replenishing it every
time as required.
For the determination of the void volume, 1 mg blue
dextran shall be dissolved in 1 mL equilibration buffer
in a 1.5 mL tube. The column bed should be drained
until the buffer level is 1 cm above the gel bed. Then
the dextran solution is applied onto the column, as
close to the column as possible. The buffer solution
will then be drained enough so that the dextran
solution is above the gel bed. The buffer should be
added to the column until its 1 cm high. The column is
allowed to flow while the eluate is collected in 1.0 mL
fractions using labelled 1.5 mL tubes.After the dextran
is eluted from the column, and column flow is stopped
the fraction with the highest absorbance was
determined. Then the absorbance of the fraction
should be read at 610 nm.
The standard protein solution should then be applied
on the column using the equivalent amounts of
volume as the dextran run. The protein solutions are
allowed to enter the top of the gel bed. The eluates
will be collected in 1 mL fractions and their
absorbance measured at 280 nm. The elution volume

other. [4] The resin used in this experiment was DEAE

cellulose, an anion-exchanger, because it is a suitable
matrix for the purification of biological samples and
because the it is suitable to bind with albumin.
The fractions in the ion-exchange chromatography
were plated in a 96-well plate and the Bradford
reagent was added to the fractions and analyzed
through a UV-vis spectrophotometer. Figure 1 is the
graph obtained from plotting the absorbances
obtained vs. the fraction number read.
Figure 1. Plot of Fraction Number vs.
Absorbance Readings obtained in Bradford Assay

From the graph it is seen that fraction 12 had the

highest average absorbance reading, it can be then
be inferred that this had the highest protein

concentration. This information relays that the protein

was eluted out in fraction 12.
Factors that could possibly affect separation of the
ions in IEC are the resin bead size, the size and
geometry of the column, the density of the exchange
sites, and the chemistry of the ionic strength of the
sample solution and concentration of the mobile
phase. The flow rate of the mobile phase is
determined by the size of the resin beads; finer
grained resins provide more exchange sites for better
separation and the fluid will move faster when there
are coarser resin beads because of the larger interbead spaces which will lead to poor separations. [5]
For GFC the type of buffer used does not depend on
the ionic strength or the pH. The only consideration for
the buffer is that is does not cause inactivation or
precipitation of the proteins. The buffer used for GFC
was phosphate buffer. The stationary phase in GFC is
composed of a porous matrix. The beads must have a
defined pore size range. The resin used in this
experiment was Sephadex G-150. After it was swelled
and the column was equilibrated with phosphate
buffer, blue dextran was added to the column to
determine the void volume. Blue dextran was used in
the experiment because the blue dye allows us to see
when it is eluted out. [5]
The column size for GFC is very important for the
data. The column must be able to minimise dilution
and maximise the separation of the molecules. A large
column will dilute the sample and a small column will
not successfully separate the proteins. For maximum
resolution in the fractionation of the sample, long
columns are recommended. [3]
After the determination of the void volume using blue
dextran, the standard protein solution was placed into
the column and collected as it eluted out. The
fractions were plated and analyzed in a UV-vis
spectrophotometer for their absorbance at 280 nm.
This was done for the determination of the elution
volume of each of the standard proteins in the
solution. The standard proteins used are shown in
Table 1. And the results from the absorbances
obtained are shown in Figure 2.
Table 1. Molecular Weight of Standards Used

Standard Proteins
B-Amylase

Molecular Weight
200000

Alcohol Anhydrase

150000

Cytochrome C

124000

BSA

66000

Carbonic Anhydrase

29000

Figure 2. Absorbance Values Obtained in the Elution

of Standard Proteins

The heaviest molecules elute out first because they

are unable to fit through the pores of resin unlike the
smaller molecules. The elution volumes were
determined based on the molecular weights of
standard proteins used.
The albumin and casein mixture was poured into the
column to determine the elution volumes of the
unknown proteins. The fractions obtained were plated
the absorbance was read at 280 nm. The results are
shown in Figure 3.
Figure 3. Chromatogram

Figure 3 is the plot of the absorbance versus the

fraction number from the unknown protein elution. The
lit value obtained for egg albumin was 33,800-40,500
Da [6] and the value obtained for casein was
75,000-100,000 Da. [7] The expected range at which
the protein sample would elute out was towards the
latter half of the fractions, which is seen in the
chromatogram in Figure 3.
Figure 4. Calibration Curve from Standard Protein

Elution

Figure 4 shows the calibration curve obtained from the

given data. Which is the plot of the log MW vs the Ve/
Vo of the fractions. From the graph the linear equation
was used to determine the estimated molecular
weight of albumin and casein. The value calculated for
albumin was 74,182 Da and for casein was 44,355
Da.

Molecular mass estimation or determination of

unknown proteins can be done by comparing the Ve/
Vo of the protein in question and the Ve/Vo from the
protein standards. The Ve/Vo is independent of the
column size and the protein concentration. However,
unreliable molecular weights may be obtained as it
forms a complex with the gel. [6]
The data obtained from the experiment may not be
accurate due to errors during the experiment.
CONCULSION AND RECOMMENDATION

[5] Hedhammer, Karlstrom, & Hober. (n.d.). Retrieved

September 5, 2015, from http://www.biotech.kth.se/
courses/gru/courselist/BB2040_ENG/
ChromMethods.pdf [7]
[6] Bernhart, F.W., (1940). Molecular Weight of Egg
Albumin. (p.189). The Journal of Biological Chemistry.
Tulane University
[7] Carpenter, D.C., (1931). The Molecular Weight of
Casein. J. Am. Chem. Soc., 53 (5), pp 18121826.

Ion-exchange chromatography and gel filtration

chromatography were done to purify and separate
albumin from contaminants.
The resin used in IEC was DEAE cellulose. The
positive binding sites allowed the albumin to bind to it.
Twelve fractions of the protein were eluted with
different concentration of KCl solution used as an
eluent. The fractions were plated and analyzed
through a spectroscophotometer. Fraction 12 was
recognised to have the highest absorbance value,
meaning it had the highest protein concentration. This
was concluded to be the fraction in which albumin
eluted out.
In GFC the gel matrix used was Sephadex-150
because of its chemical and physical stability. The
expected results from the chromatogram were
obtained, with the protein to being detected towards
the end of the elutions. The molecular weights of
albumin and casein were computed using the
equation obtained from the calibration curve. The
weights obtained for albumin and casein were 74,182
Da and 44,355 Da, respectively.
However, all of the results obtained from both
experiments have possible errors taken from the set
up, the calculations and the calibrations. Careful
execution and analysis must be done for better
results.
REFERENCES:
[1] Campbell, M., Farrell, S. (2012). Biochemistry. 7th
edition.
[2] Guide to Ion Exchange Chromatography. (p. 2).
Harvard Apparatus. Retrieved from https://
www.harvardapparatus.com/downloads/Ion
%20Exchange%20Chroma%20SpinColumn
%20Guide.
pdf
APPENDIX
[3] Guide to Gel Filtration and Size Exclusion
Chromatography. Harvard Apparatus. Retrieved from
http://www.harvardapparatus.com/guide+for+gel
+filtration.pdf
[4] Chromatography: Principles and Methods. (p. 12,
69. 76-77). BioProcess Media. Retrieved from http://
labs.mcb.harvard.edu/Gaudet/Resources_Files/
GEHealthcare_chromatography/Don't%20move/
18111421AA.pdf

Table 1. Average Absorbance Readings at 595 nm

for Each Fraction

Table 1. Ve/Vo and log MW obtained for Albumin

and Casein Using Linear Equation