FACULTY OF INDUSTRIAL SCIENCES AND TECHNOLOGY

BSB2452 ENZYME TECHNOLOGY LABORATORY

SEMESTER II 2021/2022

PRACTICAL 3

ION EXCHANGE PURIFICATION USING DEAE – CELLULOSE

(ANION EXCHANGER)

SECTION 01

PERSON IN CHARGE:

NAME MATRIC ID Part of contribution

NUR LIYANA BINTI ISMAIL SB20091 INTRODUCTION,

REFEERENCES

NUR AISYAH BINTI ZULKIFLI SB20093 OBJECTIVE,

CONCLUSION,
APPENDIX

MUHAMMAD HAMZAH BIN MUSTAFFA KAMAL SB20056 DISCUSSION

AIDA SYAFINI BINTI MOHD AKHIR SB20012 METHODOLOGY,

RESULT

Marks Distribution:

Bil. Lab report section Mark Marks obtained

distribution
1 Introduction 10
2 Objective(s) 5
3 Materials and Methods 10
4 Results and Answers to questions 20
5 Discussion 40
6 Conclusion 5
7 References 5
8 Appendix 5
TOTAL
1.0 INTRODUCTION
Ion exchange chromatography involves two primary steps, first the binding of a
protein to a charged resin and second the elution or displacement of the protein from the
charges of the resin. Critical to the former are the pH of the buffer used to equilibrate and
load the proteins onto the column. Factors that control the elution are pH or ionic strength.
Common ion exchangers include the positively charged anion exchanger - DEAE
(diethylaminoethyl) and the negatively charged cation exchanger - CM (carboxymethyl).

Chromatography is a technique that is used to separate components in a mixture based

on the molecules found in the mixture, in this case if it were to be protein as the target
molecule, the chromatography will be designed in a way to purify the protein as the target
molecule. The chromatography usually uses two phases which are stationary phase and
mobile phase. The stationary phase is substance used inside the chromatography which stays
fixed inside while the mobile phase is usually a condition where the solvent moves through
the chromatography or column. Chromatography uses physical and chemical properties such
as size, charge, hydrophobicity and binding affinity to separate the target molecule. In this
case, protein is our target molecule. Knowing that protein contain charged regions and are
usually polar in nature due to the ‘R’ group of amino acid. By using chromatography
technique which exploits the charged characteristics of protein to purify it can be a good way
of purifying the target protein from other non-target molecules. This chromatography is
known as ion-exchanged chromatography. Though it is important to include pH into
consideration, because if the pH of a solvent (buffer) used to be the target-protein’s
isoelectric point or pI value, the target-protein charges will be neutralized making it
problematic to purify through the stationary phase.

Ion exchange chromatography is known for its speciality to separates proteins based
on the charge of protein and the pH of the buffer used. In this case, the protein that was used
was Bovine Serum Albumin with pI value of ~4.7. The buffer that was used need to be either
above or below the isoelectric point of protein. This is to allow the separation or the binding
of the protein with the stationary phase to occur. This will also change the charge of the
protein. If the pH of the buffer is above the pI value it is called Anion exchange
chromatography. However, if it is below the pI value, then it is a cation exchange
chromatography. This is depending on the target proteins charge as if it is negatively charged,
it is called Anion exchange chromatography and if it is positively charged molecule is called
Cation exchanged chromatography.

In the stationary phase, we use resins as in this experiment, we used

Diethylaminoethyl (DEAE) which is an insoluble positively charge molecule. This is due to
protonated amine group. The function of DEAE in ion-exchange chromatography is to act as
a stationary phase resin to attract the negatively charged ions when the mobile phase is flow
through DEAE. After the stationary phase is set, the column is preequilibrated with buffer
before protein is applied. The negatively charged targeted proteins trapped to the positively
charged resin. To elute the targeted protein, high concentrations salt solution was used to
detach or disrupt the bonding of target protein with the resin, the high concentration salt
solution is usually high in concentration which will cooperate and break the bond of proteins
with weaker bonds toward the resin, this will then allow the target protein to elute out from
the column. This chromatography has different ion-exchange has different ion exchangers.

2.0 OBJECTIVES

1. To understand the mechanism of ion-exchange chromatography.

2. To purify bovine serum albumin (BSA)
3. To analyze the purified BSA with spectrophotometer.
4. To measure the amount of BSA being purified.
3.0 METHODOLOGY

Firstly, 10ml column was prepared by using the glass column. The pump and the adaptor
were attached. Secondly, the flow rate was adjusted to 1-2 ml/ minute. Thirdly, 10 ml of Tris-
HCL buffer was used for washed the column. It should be same buffer to a height of 2cm to
avoid air bubbles and dry. Next, 1ml of protein was added into the column by using the
pipette. Other than that, 25ml of Tris-HCL buffer were added into the column and 5ml x 5
fractions were collect. Moreover, 25ml of 50mM NaCl were added into the column and 5ml x
5 fractions were collected to measure the absorbance. Furthermore, 25ml of 100mM NaCl
were added into the column and 5ml x 5 fraction was collect. Lastly, all the fraction were put
into the cuvette to analyse the absorbance at wavelength 600nm.
4.0 RESULT

OD600nm (optical density at 600nm wavelength) readings for the purification of BSA using
anion exchange chromatography. [blank absorbance: 0.000 A]

Washing with Tris-HCL buffer (25ml of Tris-HCL buffer)

Sample OD600nm
1. -0.004
2. 0.039
3. 0.004
4. 0.007
5. 0.002
Blank 0.000
Table 1: absorbance reading for five samples of 5mL by Tris-HCl buffer washing

Washing with 50mM NaCl solution (25ml of NaCl)

Sample OD600nm
1. -0.001
2. -0.004
3. -0.004
4. -0.003
5. -0.000
Blank 0.000
Table 2: absorbance reading for five samples of 5mL by 50mM NaCl solution washing

Washing with 100mM NaCl solution (25ml of NaCl)

Sample OD600nm
1. 0.010
2. 0.002
3. 0.016
4. 0.001
5. 0.005
Blank 0.000
Table 3: absorbance reading for 5 samples of 5mL by 100mM NaCl solution washing
Graph 1: the relationship of absorbance and types of buffers washing
 5.0 DISCUSSION

Proteins are made up of thousands or hundreds of smaller components known as

amino acids that are linked together in long chains. There are 20 different types of amino
acids that can be combined to form a protein. Because amino acids had acidic and basic sides
of the chain, the protein's surface was generally charged. The pH of the surrounding solution
can be changed to achieve this. Ion exchange chromatography (IEX) is a type of
chromatographic separation that is based on the protein's net charge. Proteins and other
biomolecules that have a negative charge will attach to the resins. The ionic groups of the
columns are covalently attached to a gel matrix, and small amounts of counterions in the
buffer protect them. The charged molecules exchange with the weakly bound counter ions
and attach to the solid support when a sample is introduced to the column. Cation and anion
exchange chromatography are the two forms of IEX. The different amino acid residues that
are capable of hydrogen ion ionization will determine the net charge of the proteins at any
given pH. Ion exchange chromatography (IEX) separates ions and polar molecules based on
their surface charge, which differs widely amongst proteins. Cation exchange
chromatography is a kind of ion exchange chromatography (IEX) that uses the net surface
charge of molecules to separate them. Cation exchange chromatography, in particular, uses a
negatively charged ion exchange resin with an affinity for molecules with net positive surface
charges. Anion exchange chromatography (AEC) is a kind of ion exchange chromatography
(IEX) that is used to separate molecules depending on their net surface charge. Anion
exchange chromatography, in particular, uses a positively charged ion exchange resin with an
affinity for molecules with net negative surface charges.

In this experiment, we use the anion-exchange chromatography technique to discover

and purify a specific protein discovered in bovine serum albumin (BSA) solution. The
negative charge of the purified protein molecule contrasts with the positively charged
stationary phase of DEAE, which is made up of weak, basic, and insoluble anion exchange
resins connected to cellulose. DEAE, or diethylaminomethyl, is a positively charged chemical
that keeps column charge density constant over a pH range of 5 to 9. Cellulose-based resins
with wide pores that segregate big molecules, help in protein binding, and have a low
hydrophobic character are covalently connected together. While tris-HCL buffer is used for
mobile equilibration, sodium chloride (NaCl) salt buffer is used for elution, which has no
effect on the target protein's structure. Covalently coupled together are cellulose-based resins
with large pores that may segregate large molecules, help in protein binding, and have a low
hydrophobic character. While tris-HCL buffer is used for equilibration, sodium chlorine salt
buffer is used for elution, which has no effect on the target protein structure.

The flow rate was set to 1 to 2 ml/min, which is within the optimal range for an ion-
exchange column. The flow rate of a buffer through a resin is referred to as the flow rate. The
flow rate, also known as the residence time of a specific column at a certain flow rate,
determines the number of times proteins may interact with the column resin. The resolution
and capacity might be affected by the flow rate. This means that the longer the residence
time, the better the resin's capacity and resolution. The ion exchange adsorbent packed into
the glass column is pre-equilibrated in tris-HCL buffer before running the BSA protein
mixture by anion-exchange chromatography, as stated earlier, with the purpose of adjusting
the protein charge to become negatively charged. The pH of the BSA protein has been
determined to be 4.7 pH units. The isoelectric point's pH value, also known as the pH value,
is defined as the pH value at which all charged molecules in the protein solution have a net
charge of zero. Ion exchange is normally done at least one pH unit distant from the protein of
interest's pI to ensure that it is charged. When the pH reaches the pI, the molecule is
negatively charged. When the pH reaches the pI, the molecule becomes negatively charged,
requiring the use of an anion exchange resin, and vice versa. When flow rates increase,
however, pressure on the resin increases until the backpressure reaches lit, causing the
column to crush and the separation efficiency to decrease. Slower flow rates, on the other
hand, may give better resolution and capacity, but at the expense of degraded protein activity
in the chromatographic system.

The BSA protein mixture was then put into the column for ion exchange and ion
exclusion, where it was exposed to ion exchange and ion exclusion. The former occurred
when positively charged ions in the stationary phase were attached to their opposite charge
exchangeable ions (target protein), whereas the latter occurred when analytes of similar
charge repel the ions on the stationary phase's functional group, as in the case of the target
protein. Coulomb's law states that electrostatic forces cause interactions between ions in the
solute and oppositely charged ligands on the matrix in ion-exchange chromatography. If the
charges on both ions are the same, the force is repulsive; however, if the charges are
different, the force is attracting. After the buffer has passed through the column, it is washed
to remove any unbound contaminants as well as any uncharged or positively charged proteins
that may have collected on the column during the buffer's passage. Chromatography is done
in a positive mode, which means that the washing phase helps remove unwanted compounds
while the protein of interest is kept in a negative charge throughout the procedure. Washing
the chromatographic system with 2 column volumes of 100 ml of equilibration buffer and
collecting 5 ml fractions. In this step, utilise the same buffer, Tris-HCl, with the same pH to
help the target protein stay bound to the stationary phase. Because the total volume of Tris-
HCl equilibration buffer is 100 ml, and 5 ml fractions were obtained each time, there are 15
fractions. A spectrophotometer calibrated to wavelength 660nm was used to determine the
absorbance value of all fractions from the washing process. Samples 3 to 5 show absorbance
values of 0.004A, 0.007A, and 0.002A, respectively, according to the findings. This means
that the impurities have been thoroughly removed.

The second step involves elution of the required negatively charged protein with
gradually increasing salt concentration (linear salt gradient). The elution procedure employed
two NaCl salt buffer concentration levels: 50mM and 100mM. The initial eluent was a 25mL
50mM NaCl buffer, and protein with a low net charge was washed away. Following that, the
column was rinsed with additional 25mL of 100mM NaCl buffer. In addition, 5ml fractions
were taken from each of the ten fractions, for a total of 100 fractions collected. The
absorbance was measured with a spectrophotometer set at 660nm. According on the results of
the elution with 50mM salt banquet, sample 1 has -0.001A, sample 2 has -0.004A, sample 3
has -0.004A, sample 4 has -0.003A, and sample 5 has -0.000A. This section has all of the
negative value for the absorbance readings.

This is due to the reference's absorbance value is higher than the sample's, sample
measurements. The sample and reference have been mixed up, or the sample is very dilute
and close to the absorbance of the reference. In the meanwhile, the absorbance readings for
sample 1 was 0.010A, sample 2 was 0.002A, sample 3 was 0.016A, sample 4 was 0.005A,
and sample 5 was 0.005A for the elution with 100mM salt buffer. This indicated that all of
the strongly bound target negatively charged protein was clotted out and properly purified,
however there are some inconsistencies in the absorbance measurement from what we can
observed. The stationary phase resin had been regenerated and was ready for use in the
subsequent step.
 In practise, protein purification is followed by 1D SDS-PAGE electrophoresis to
ensure that the protein purified contains the required protein. In this experiment, protein
purification is terminated at ion-exchange chromatography. 1D electrophoresis is a method
for separating proteins with molecular sizes ranging from 10 to 300 kilodaltons depending on
their molecular weight (kDa). Only one band will appear on the 1D SDS-PAGE result,
showing that the target protein was successfully purified. To ensure the precision and
consistency of the findings obtained during the experiment, we must take a few precautions
step to ensure that a consistent flow rate in the range of 1-2mV/min is maintained throughout
the experiment.

Additionally, we must guarantee that the equilibrium buffer of Tris-HCl is added to

the glass column to a height of 2 cm before loading the protein sample in order to avoid the
formation of air bubbles during the separation process. To avoid the chromatographic system
from being disrupted, for example, the use of a glass rod to help in the addition of solution to
the column is required. Since the Tris-buffers are sensitive to temperature, we must ensure
that the PH 7.8 is maintained at room temperature. Before loading the protein, we need to add
buffer of Tris-HCl equilibrium to the glass columns to a height of 2 cm to avoid the creation
of air bubbles during the separation process.
6.0 CONCLUSION

In conclusion, Ion Exchange Chromatography is a popular purification method of

proteins, peptides, nucleic acids, and other charged biomolecules, preferred for its high
resolving power, high protein binding capacity, versatility with different types of ion
exchangers, versatility with composition of buffers and pH, straightforward separation
principle (primarily according to differences in charges), and ease of performance. Ion
exchange chromatography is a technique often used in protein purification, water analysis,
and quality control and it can be used for large proteins, small nucleotides and amino acids.
The principle of ion exchange chromatography was that, charged molecules bonded
electrostatically to oppositely charge groups that have been bound covalently on the matrix.
From the printout there was one distinct peak corresponding to the protein. From the printout
there was one distinct peak corresponding to the protein.

At that peak the LDH was pure. Ion exchange chromatography occurred due to
electrostatic attraction between buffer-dissolved charged proteins and oppositely charged
binding sites on a solid ion exchange adsorbent. Graph of the Absorbance plotted versus
fractions number at 600 nm. The fractions collected were assayed for total protein. The
values were plotted against the fraction number which is called elution profile. The peak on
the graph indicated where the fractions are that contain protein. Enzyme activity was
determined by performing an enzyme assay on each fraction that contain protein

In this experiment, a 10ml column was prepared using a glass column. The pump and
adaptor were connected. The flow rate was then reduced to 1-2 ml/min. Finally, the column
was washed with 10 ml of Tris-HCL buffer. To avoid air bubbles and dry, use the same
buffer up to a height of 2cm. The pipette was then used to add 1ml of protein to the column.
In addition, 25ml of Tris-HCL buffer was added to the column, and 5ml x 5 fractions were
collected. In addition, 25ml of 50mM NaCl was added to the column, and 5ml x 5 fractions
were collected to determine absorbance.
 There are precaution steps to avoid the error that can affect the accuracy of result in
this experiment. First, handle the samples carefully to avoid contamination for high
sensitivity analysis. Next, as a stationary phase in the ion separation column, a packing
material modified with ion-exchange groups is used. As a result, it has greater
hydrophobicity, which can lead to the accumulation of hydrophobic components from the
sample, which can have negative consequences. Moreover, Handle the column with care, as
hitting it or exposing it to sudden pressure changes or high pressures can cause deterioration.
Precipitation should be avoided in the column. Because tris-buffers are temperature sensitive,
they must be adjusted to pH 7.8 at room temperature.
 7.0 REFERENCES

1. Bahadir, O. (2013). Ion-Exchange Chromatography and Its Applications. Column

Chromatography. https://doi.org/10.5772/55744
2. Experiment #5 Ion Exchange Chromatography. (n.d.).
http://www2.chem.uic.edu/min/chem455/docs/Lab_5.pdf

3. Ion Exchange Chromatography. (2022). Bio-Rad Laboratories. Retrieved 2 April

2022, from https://www.bio-rad.com/en-my/applications-technologies/ion-exchange-
chromatography?ID=MWHAY9ESH
4. Susha Cheriyedath, M. (2016). How Does Ion Exchange Chromatography Work?.
Retrieved 2 April 2022, from https://www.news-medical.net/life-sciences/How-Does-
Ion-Exchange-Chromatography-Work.aspx
5. Acikara, O. B. (2013, April 10). Ion-exhange chromatography and Its Applications.
IntechOpen. https://www.intechopen.com/chapters/44033
6. NCBI ION EXCHANGE. (2013). ION EXCHANGE CHROMATOGRAPHY.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4795180/
8.0 APPENDIX

A 10ml column was prepared using a glass

column

Pipette to add 1ml of protein to the column

25ml of Tris-HCL buffer was added to the
column,5ml x 5 fractions were collected. In
addition, 25ml of 50mM NaCl was added to
the column, and 5ml x 5 fractions were
collected.

Transfer collected sample to the cuvette to

determine the absorbance using centrifuge.