Purification of Proteins using Ion Exchange

chromatography and Gel Filtration Chromatography

Maria Isabel B. Salvador
Institute of Chemistry, College of Science, University of the Philippines, Quezon City, Philippines
Abstract:
Ion exchange chromatography and Gel Filtration Chromatography are methods of protein purification and
separation. In this experiment both methods are used to purify extracted albumin solutions. Methods in gel
filtration are also used to estimate the molecular weights of albumin and casein by comparing the
chromatogram to ha calibration curve of standards.
Introduction
A crude protein extract obtained from a
sample must be purified to separate the target
protein from other proteins found in the sample.
The target protein is then subjected to further
testing usually to determine its sequence. The two
techniques used in this experiment are collectively
called methods of column chromatography.
Ion exchange chromatography separates
protein on the basis of charge. In the experiment
DEAE cellulose, an anion exchanger, was used as
the stationary phase. The ion exchange column
was used to purify crude albumin extract.
Gel filtration chromatography is a protein
separation technique. Sephadex G-100 was the
resin used. The sample was a protein extract
solution containing albumin and casein. Gel
filtration chromatography not only aims to separate
the two proteins but to also determine the
molecular weight of the proteins.
Experimental Details
The samples used in the experiment were 2mg/mL
bovine serum albumin and 5mg/mL extracted
albumin. The two samples were purified using a ion
exchange column and a gel filtration column.
Ion Exchange Chromatography
To prepare the gel slurry for the column, DEAE
cellulose was suspended then washed in 400mM,
400mM NaOH and water successively in order to
obtain a neutral pH. 5 mL column bed volume of
the gel slurry was then poured into the column. To
equilibrate the column, 5 washings of column
volumes of Tris-HCl buffer were done. The
samples were prepared in 1mL portions using TricHCl at pH 8 as a solvent. A pre-column was used
to separate any large molecules that are impurities
from the sample. The sample was then allowed to
pass through the ion exchange column thrice, the
flow-through was collected. After which the column
was washed with tris-HCl three times, the flow-

through was collected and labeled as washings.

The column was then washed with varying
concentrations of KCl (0.1 M, 0.2 M, 0.3M, 0.5 M
1.0 M and 6.0 M) which were labeled as fractions
1-12. The fractions and washings were subjected
to the Bradford Assay.
Gel Filtration Chromatography
Column preparation, gel preparation, and column
packing and equilibration were performed by the
instructor prior to the experiment. To determine the
void volume, blue dextran dissolved in equilibration
buffer was allowed to pass through the column. the
eluate was collected in 1.0mL fractions in 1.5 mL
tubes. The absorbance of each fraction was
obtained at 610 nm. The sum of the volumes from
the 1st fraction to the one with the highest
absorbance is the void volume (Vo). After which,
the Elution volume of the standard protein solution
and extracted albumin were obtained. The protein
samples were allowed to pass through the column,
the eluate was collected in 1.0 mL fractions. The
absorbance of the fraction were read at 280 nm.
The sum of the 1st fraction volume to the the one
with the highest absorbance is the elution volume
(Ve).
Results and Discussion
Ion Exchange Chromatography
Ion Exchange chromatography works on the
principle of the net charges of molecules. The resin
in an ion exchange column is positively or
negatively charged. The column is first equilibrated
with a buffer solution. Initially the resin is bound to
counterions, cation-exchange resin is bound to Na+
or K+ ions while anion-exchange resin is usually
bound to Cl- ions. When a protein sample is
passed through the column, the protein with a net
charge opposite to that of the resin exchanges
places with the counterions.
Proteins with a
neutral or same net charge pass through the
column and are eluted first. In order to obtain the

BSA Standard
A595 vs KCl Fractions

0.04

A595

0.02
0
0

10

-0.02
-0.04

Concentration of KCl (M)

Figure 1. Plot of the Absorbance of BSA Standard at

595 nm vs KCl fractions.

Albumin extract
A595 vs KCl Fractions

0.06
0.04
0.02
A595

bound proteins the eluent is changed to a buffer

solution or salt solution. The buffer to be used must
have a pH that removes the net charge of the
protein while the salt solution will outcompete the
bound proteins for the limited binding space on the
column. The target protein then elutes and is
collected.
Buffers
used
in
ion
exchange
chromatography are selected based on their pH
and ionic strength in order to optimize the
interaction of the resin with the proteins. The pH of
the buffer is maximized while minimizing the ionic
strength. The pH of the buffer should be in
between the pI of the protein and the pKa of either
positive or negatively charged groups. The ionic
strength of the buffer is minimized in order to avoid
any of the buffering species to interact with the
charged resin.
The resin used for the experiment is
DEAE-cellulose. This resin is a weak anion
exchanger containing a diethylaminoethyl group
that is positively charged at neutral pH. The
counter ion of DEAE-cellulose is Cl-.The resin is
swelled in order to expose the functional groups in
preparation for ionic exchange. In the experiment
DEAE-cellulose was washed in 400mM HCl and
400mM NaOH successively. This was done in
order to keep the resin in a pH between the ranges
of 5-9 in order to maintain the resins charge.
Proper column packing is done to avoid
unevenness in flow rate. The column was
equilibrated by washing the column 5 times with
Tris-HCl buffer. Equilibration was done in order to
associate the exchangeable groups with counter
ions. After equilibrium is achieved and the sample
is added the positively charged protein displaces
the counter ions bound to DEAE-cellulose. The
column must be regenerated and equilibrated for
re-use. Regeneration was done by washing the
column with 6M KCl solution. The Cl- ions will
displace the protein bound to DEAE-cellulose. The
column was then re-equilibrated with Tris-HCl
buffer. Ion exchange columns are short and narrow
in order to maximize particle collision.
The samples used in the experiment were
2mg/mL bovine serum albumin and 2mg/mL
extracted albumin both dissolved in Tris-HCl buffer
at pH 8. Tris-HCl buffer was used as a solvent to
ensure uniformity in pH of the sample and the
column. The pH of the samples and the column
must be equal in order for the desired charges to
be maintained.

-0.02

-0.04
-0.06
Concentration of KCl (M)
Figure 4. Plot of the Absorbance of Albumin extract at
595 nm vs KCl fractions.

According to the graph of the BSA standard (Figure

1.) the protein eluted out of the column at 0.3 M
KCl. The graph of the Albumin extracts reports that
the protein eluted out of the column at 6M KCl. The
two graphs have a large difference in the salt
concentration from which the column eluted.
Gel Filtration Chromatography
Gel Filtration is a kind of column chromatography
that separates proteins based on their molecular
size. The gel resin is composed of a carbohydrate
polymer and a polyacrylamide. These polymers
have a cross-link structure that produces the pores
in the resin. The pore size may be adjusted by
controlling the extent of cross-linking. Proteins with
smaller molecular weights are trapped in the pores
and are eluted last.
Gel
Filtration
Chromatography
advantageously separates protein based on their
molecular weight. It is also used to roughly

determine the molecular weights of the sample

based on a set of protein standards. However gel
filtration cannot be used to separate proteins with
close molecular weights.
The resin used in the experiment in
Sephadex G-100. Sephadex is composed of a
highly cross-linked agarose matrix. Sephadex G
can be used at pH levels 3-12. The number
preceding the G notation varies with the function
of Sephadex. G-100 is specifically used for
molecular weight determination of proteins.
The buffer used in the experiment is 0.1 M
buffer at pH 7. The pH and ionic strength of the
buffer has no direct affect on the resolution
obtained. However buffer composition is capable of
altering the shape, denaturing or dissociating the
proteins of interest.
An ideal flow rate allows times for
molecules to diffuse in and out of the resin in order
to achieve separation. In order to maximize
resolution a long column with a low flow rate is
used. A high flow rate ensures faster separation
however there is loss of resolution in separation.
Column packing is optimized in order to
obtain narrow symmetrical peaks during elution.
The column resin was tightly packed in order to
ensure a consistent rate flow. The volume of the
column bed usually varies with varying sample
volumes to be applied.

There are two advantages of using blue dextran.

Its high molecular weight makes it too large to
penetrate the gel matrix. Blue dextran is also
convenient to use because of its blue color.

Elution Volume Determination

0.6
0.5

0.4
0.3
0.2
0.1
0
-0.1

20
40
Fraction number

60

80

Figure 5. Plot for Elution Volume Determination of

the standard protein solution.
To determine elution volume (Ve) the standard
protein solution was allowed to pass through the
column. The five protein standards used were
BSA, alcohol anhydrase, carbonic anhydrase,
cytochrome c, and -amylase. The five peaks in
the graph show at which fraction each protein
standard eluted. The Ve is the summation of the
volumes of the fractions until the highest peak.

Void Volume Determination

0.14
0.12

0.1

A610

0.08
log (MW)

0.06
0.04
0.02
0

-0.02 0

Calibration Curve

B-Amylase

20

40

Fraction number
Figure 3. Plot for Void Volume Determination.
The void volume (Vo) was determined by
allowing blue dextran solution to pass through the
column. The absorbance of each fraction was read
at 610 nm. The fraction with the highest
absorbance is fraction 10 with an absorbance of
0.122. The sum of the volumes from fraction 1-10
was obtained to determine Vo=5 mL. Blue dextrain
has a molecular weight of about 2 million Da.

60

BSA

alcohol
anyhydra
se

4
3

y=-0.16x + 5.76

carbonic
anyhydrase
cytochrome C

2
1
0
2.4

3.6

5.8
Ve/Vo

10.2

Figure 6. Calibration curve of protein standards

from highest molecular weight to lowest molecular
weight.
The calibration curve (Figure 6.) is a plot of log
(MW) of protein standards vs Ve/ Vo. The equation
of the line of the calibration curve was obtained.

Sample (Casein and Albumin)

2.5

A280

2
1.5
1
0.5
0
-0.5 0

10

20

30

40

Fraction Number
Figure 7. Plot for Elution Volume Determination of
the extracted protein solution.
The elution volumes of the extracted protein
solution containing casein and albumin was
obtained in order to determine the molecular
weights of casein and albumin. The ratio between
elution volume and void volume was obtained. The
ratio was then plugged into the equation of the line
of the calibration curve. Figure 8. shows the
calculated molecular weights in comparison with
molecular weights found in literature.
Protein

Calculated

Literature

Casein

24,354.55

22,380

Albumin

35,455.28

33,80040,500

Figure 8.

Calculation of the molecular weights from Ve/Vo

shows which protein eluted first. Albumin eluted at
fraction # 37 while casein eluted at fraction # 42.
Albumin has a higher computed molecular weight
compared to that of casein and based on the
principles of gel filtration chromatography, will elute
first.
Conclusion and Recommendation
Ion exchange chromatography purifies
protein on the basis of net charge. In the
experiment two-setups were performed. The first
setup allowed bovine serum albumin (BSA) to pass
through the column while the second used the
albumin protein extract. The two setup yielded very
different results. BSA eluted out of the column at
0.3 M KCl while extracted albumin eluted at 6 M
KCl.
Gel Filtration Chromatography is a
convenient protein purification method that

50

separates protein on the basis of molecular weight.

In the experiment a extracted protein solution
containing albumin and casein where allowed to
pass through the gel filter. Albumin eluted out of
the column first with a molecular weight of
35,455.28 Da while casein had a molecular weight
24,354.55 Da.
Gel Filtration Chromatography is not
recommended for fractionation of crude protein
extracts. Crude protein extracts contain may
contain more than five proteins. Theoretically, note
more than five to six proteins can be separated
from each other by gel filtration. This is because
the effective fractionation volume is slightly higher
than half of the total column volume.
A possible source of error in both methods
chromatography is errors in column preparation.
Not swelling the resin and packing the column bed
tight enough may have an effect of the flow rate of
the column which in turn will have an effect on
resolution.
References:
(1)Harvard Apparatus. Guide to Ion-Exchange
Chromatography.www.harvardapparatus.com.
Availablehttps://www.harvardapparatus.com/media
/harvard/pdf/Ion%20Exchange%20Chroma%20Spi
nColumn%20Guide.pdf
(2) Amersham Pharmacia Biotech. Ion Exchange
Chromatography Principles and Methods.
(3)Bernhart, F.W. Molecular Weight of Egg
Albumin. Department of Biochemistry, Tulane
University, New Orleans.
(4)
Worthington
Biochemistry
Corporation.
Amylase, Beta.
(5) Harlow and Lane, Proteins used as molecularweight standars.
(6) Amersham Biosciences. Gel Filtration
Principles
and
Methods.
Available
http://kirschner.med.harvard.edu/files/protocols/GE
_gelfiltration.pdf.