FACULTY OF INDUSTRIAL SCIENCES AND TECHNOLOGY

BSB2452 ENZYME TECHNOLOGY LABORATORY

SEMESTER II 23/24

Practical 03: Ion Exchange Purification using DEAE-cellulose

Section: 02G

GROUP 01

MEMBERS CELINE CHONG XIU YING SB21051

SRI SARVESHAA A/L SRIRUPAN SB21070

KUMUTHA A/P ARAMUGGAM SB21103

NABILA BALQIS BINTI RIMUL SB22001

REPORT OUTLINE MARKS PERSON IN-CHARGE

Introduction 10 CELINE CHONG XIU YING

Objectives 5 CELINE CHONG XIU YING

Methodology 10 NABILA BALQIS

Results 20 NABILA BALQIS

Discussion 40 SRI SARVESHAA

Conclusion 10 SRI SARVESHAA

References 3 KUMUTHA

Appendices 2 KUMUTHA
Introduction

One essential and frequently used chromatographic method in the field of molecular
biology and biochemistry is ion exchange purification. The underlying idea of this technique
is to use the charge properties of biomolecules to selectively capture and separate them.
Diethylaminoethyl (DEAE)-cellulose has become an increasingly prevalent choice for
purification procedures among the several ion exchange resins that are available. In order to
demonstrate the methodology, the purification of bovine serum albumin (BSA) in a pH 7.4
Tris-HCl buffer and subsequently in both 50mM and 100mM high salt buffer will be considered
as an example of the process. The examples provided will show the versatility and applicability
of DEAE-cellulose chromatography in the extraction of biomolecules under various
experimental setups.

Bovine Serum Albumin, commonly referred to as BSA, a versatile and commonly

found protein. Because of its remarkable qualities, including its high solubility, stability, and
non-specific binding capacity, BSA is an essential component of many laboratory applications.
Researchers frequently utilise BSA as a blocking agent in immunoassays, a stabilising agent in
enzymes, and as a standard for protein quantification assays (Thermo Fisher Scientific, 2021).

Diethylaminoethyl cellulose, or DEAE-cellulose, is made up of a matrix containing

positively charged diethylaminoethyl groups. This makes it an ideal substrate for interacting
with biomolecules that include negatively charged functional groups, including carboxylate or
phosphate moieties. The reversible adsorption of these biomolecules onto the DEAE-cellulose
matrix underlies the technique's selectivity, wherein biomolecules are differentially retained
based on their net charge (Chromatography Online, 2021). In order to demonstrate the
application of this method, investigate a situation where purification of bovine serum albumin
(BSA), a widely used protein standard, is necessary. The procedure can be carried out in a
physiologically regulated setting by using a pH 7.4 Tris-HCl buffer. DEAE-cellulose will allow
the selective binding and purification of BSA based on its net charge, thereby isolating it from
other components in the sample (Sarwar et al., 2018).

Moreover, DEAE-cellulose chromatography's adaptability is shown when, for example,

particular purification objectives necessitate high salinity conditions. To efficiently disrupt the
ionic interactions between the target biomolecule and the DEAE-cellulose matrix, a high salt
buffer can be applied for eluting or washing the biomolecule. This demonstrates the
adaptability of the technique to diverse experimental conditions, making it a valuable asset in
the purification toolbox (Chromatography Online, 2021).
 DEAE-cellulose chromatography has benefits beyond its capacity to separate
biomolecules according to charge. It is a cost-effective approach that strikes a compromise
between affordability and effectiveness, making it especially desirable in industrial and
research contexts. Moreover, the method can be scaled up for large-scale purification
processes, making it suitable for biopharmaceutical production, where high-purity
biomolecules are essential (Chromatography Online, 2021).

Objectives

1. To acquire knowledge of performing column chromatography

2. To acquire knowledge of and skill in ion-exchange purification.
3. To isolate and purify biomolecules in an effective and selective method according to
their charge properties.
Methodology

Apparatus

BSA, 50mM sodium chloride, 100mM sodium chloride, Tris-HCl buffer pH 7.4, DEAE-
cellulose, glass column, pump and adaptor, micropipette, cuvette, spectrophotometer,
measuring cylinders, glass rod, test tubes, beakers, retort stand, timer and dropper.

Figure 1: shows the setup of the experiment

Procedures

Initiation of the Procedure:

1. Commencement of the procedure was undertaken by the meticulous preparation of a

50-millilitre (ml) column. This entailed the affixation of an adaptor to a glass column
and the placement of a layer of cotton at the lower extremity of the column using a
glass rod.

2. The subsequent step featured the meticulous packing of the DEAE cellulose, which is
the designated ion exchange adsorbent, into the column.

3. Diligent care was taken to ensure a thorough and comprehensive rinsing of the 50 ml
column with a Tris-HCl buffer solution, maintaining a pH of 7.4, and meticulous
precautions were observed to prevent the formation of any undesirable air bubbles.

4. This was succeeded by the deliberate act of manipulating a valve to augment the flow
of the Tris-HCl pH 7.4 buffer solution within the column, thereby expediting the rapid
sedimentation of the DEAE-cellulose at the subaqueous portion of the column.
 5. Following the sedimentation process, it was prudent to withdraw the glass rod as the
DEAE cellulose had unequivocally reached a state of complete sedimentation at the
column's nadir.

6. Furthermore, an exhaustive column washing procedure was undertaken, involving the

usage of a voluminous quantity, equivalent to one column volume, amounting to 50
ml of the Tris-HCl buffer solution with a pH of 7.4.

7. Subsequently, an indispensable step in the process necessitated the calibration and

establishment of the column's flow rate, pegged at a constant and deliberate pace of 2
ml per minute. To prevent desiccation, it was imperative to perpetuate the flow while
vigilantly introducing supplementary buffers until the column was rendered eminently
ready for operational use.

Washing Phase:

8. Subsequent to these initial steps, it was obligatory to introduce a precisely calculated

quantity, representing one-half the volume, totaling 25 ml, of the Tris-HCl buffer
solution with a pH of 7.4 into the column. Moreover, it was imperative to meticulously
collect the effluent in the form of 5 ml fractions throughout the course of this phase.

Absorbance Reading:

9. To ascertain the degree of the procedure's progress and outcomes, a meticulous and
precise measurement of absorbance at a wavelength of 280 nanometers was conducted.
It is noteworthy that the Tris-HCl buffer solution, retaining its pH of 7.4, served as the
designated blank for the absorbance reading.

Elution Process:

10. Upon the consummation of the preceding phases, the subsequent step entailed the
addition of one-half of the predetermined volume, specifically 25 ml, of a 50-
millimolar (mM) sodium chloride solution into the column. As before, a thorough and
exhaustive collection of effluent was maintained in the form of 5 ml fractions.

Absorbance Reading (Continued):

 11. It is pertinent to note that a meticulous measurement of absorbance at the
aforementioned wavelength of 280 nanometers was once again diligently conducted.
In line with the previous step, the Tris-HCl buffer solution, consistent with its pH of
7.4, remained the designated blank for the absorbance reading.

Subsequent Elution Steps:

12. In the ensuing step, a final one-half of the stipulated volume, constituting 25 ml, of a
100-millimolar (mM) sodium chloride solution was introduced into the column.
Similar to the previous phases, effluent was scrupulously collected in the form of 5 ml
fractions.

13. Thereafter, a meticulous measurement of absorbance at the predefined wavelength of

280 nanometers was diligently undertaken. The Tris-HCl buffer solution, retaining its
pH of 7.4, continued to serve as the designated blank for this final absorbance reading.

Concluding Wash:

14. In conclusion, the protocol prescribed a comprehensive washing procedure for the
column, involving the utilisation of a 25 ml volume of the Tris-HCI buffer solution,
maintaining a pH of 7.4. This procedure was of paramount importance, as it was
deemed essential for eluting proteins using a high salt buffer wash, encompassing a
volume equivalent to that of the column, effectively removing tightly bound proteins
and regenerating the resin for subsequent use. It was further underscored that the
column should be subjected to an additional 25 ml wash with the Tris-HCI buffer
solution with a pH of 7.4 upon the successful completion of the experiment.
 Figure 2: shows the summary of the experiment steps

Results

Washing buffer
Absorbance
reading at
280 nm (A) Tris-HCl Buffer 50 mM NaCl 100 mM NaCl

Blank 0.000 0.000 0.000

Test tube 1 -0.030 -0.027 -0.029

Test tube 2 -0.008 -0.007 -0.008

Test tube 3 -0.012 -0.013 -0.011

Test tube 4 0.096 0.107 0.104

Test tube 5 0.098 0.101 0.096

Graph
Figure 3: shows the graph of Tris-HCl Buffer, 50mM Sodium Chloride and 100mM Sodium
Chloride with absorbance reading at 280 nm(A)
Discussion

In this experiment, we learned how to perform ion exchange chromatography. Ion

exchange chromatography, sometimes referred to as ion chromatography, is a method for
separating ions and polar molecules according to their affinity for ion exchangers. Anionic and
cationic exchangers are two types of exchangers that may be employed in this process.
Negatively charged groups are owned by cationic exchangers, and charged cations will surely
be drawn to them. Because acidic groups ionize these exchangers and cause negative costs,
they are also known as "acidic ion exchange" materials. Businesses have surely been charged
by anionic exchangers to attract negatively charged anions. They are sometimes referred to as
"Basic ion exchange" materials.

Positively charged anion-exchange chromatography is the separation method used in

this study. Using an ion-exchange resin containing positively charged groups, such as diethyl-
amino ethyl groups (DEAE), anion-exchange chromatography is a technique that separates
compounds according to their charge. An ion exchange resin that is positively charged and has
an affinity for molecules with a net negative surface charge is specifically used in anion
exchange chromatography. Large proteins, nucleotides, amino acids, and other compounds can
be separated using anion exchange chromatography for both analytical and preparative
applications. Changes in pH are another way that many chromatographs influence separations.
Lowering the pH of the mobile phase buffer in anion exchange chromatography causes the
molecule to become more protonated and more positively (and negatively) charged. The
molecule is eluted from the column because of the protein's inability to establish ionic
connections with the positively charged solid support.

Ion exchange chromatography has two main steps: the binding of a protein to a
positively charged resin and the protein's displacement from the resin's charges. Initially, the
column was filled with cotton bits to prevent the DEAE-cellulose from flowing out of the
bottom and from combining with other solutions. After that, run a Tris-HCL buffer over the
column. Since it will return the polymer beads to their initial state, it is a crucial step. This is
the cellulose's condition prior to ion exchange. For this experiment, the flow rate must be kept
constant at 1-2 ml/minute. We must give the protein plenty of time to interact with the resin. It
is preferable to have a lower retention time. The purpose of this step is to stop protein leakage
from the column. After that, the glass column was filled with 1 ml of BSA. In this experiment,
the protein mixture was called BSA.
 Based on the results, it can be concluded that the DEAE-cellulose resin was effective
in separating and purifying the target molecules. The negative absorbance readings for test tube
1(-0.030A, -0.027A, -0.029A), test tube 2(-0.008A, -0.007A, -0.008A) and test tube 3(-0.012,
-0.013, -0.011) for Tris-HCl buffer, 50 mM NaCl, and 100 mM NaCl respectively indicate that
the resin was able to bind to and remove unwanted molecules from the sample. The positive
absorbance readings for test tubes 4(0.096A,0.107A,0.104A) and 5 (0.098A,0.101A,0.096A)
for Tris-HCl buffer, 50 mM NaCl, and 100 mM NaCl respectively indicate that the target
molecules were successfully eluted from the resin.

The graph presented in the lab report shows the absorbance reading at 280 nm (A) for
Tris-HCl buffer, 50 mM NaCl, and 100 mM NaCl. The graph shows that the absorbance
readings increased with increasing salt concentration. This is consistent with the principle of
ion exchange chromatography, where the binding of charged molecules to the ion exchange
resin is dependent on the salt concentration in the buffer solution. As the salt concentration
increases, the ionic strength of the buffer solution increases, which disrupts the electrostatic
interactions between the charged molecules and the ion exchange resin, leading to their elution
from the resin.The trend observed in the graph is important in the interpretation of the results
obtained from the experiment. The positive absorbance readings for test tubes 4 and 5, which
were eluted with 50 mM and 100 mM NaCl, respectively, indicate that the target molecules
were successfully eluted from the resin. The negative absorbance readings for test tubes 1 to 3,
which were washed with Tris-HCl buffer, indicate that the resin was able to bind to and remove
unwanted molecules from the sample.

Overall, the results of the experiment demonstrate the effectiveness of DEAE-cellulose

for ion exchange purification. The experiment also highlights the importance of selecting the
appropriate ion exchange resin based on the charge of the target molecules and the pH range
over which they are stable.
Conclusion

In conclusion, the experiment effectively illustrated the DEAE-cellulose ion exchange

purification process. The experiment's outcomes demonstrated how flexible and useful DEAE-
cellulose chromatography is for extracting biomolecules in a range of experimental
configurations. The experiment also demonstrated how crucial it is to choose the right ion
exchange resin depending on the target molecules' charge and the pH range across which they
are stable.In short, the experiment effectively addressed its goals of learning how to do column
chromatography, learning about and developing proficiency in ion-exchange purification, and
isolating and purifying biomolecules in a technique that is efficient and selective based on their
charge characteristics. The work also showed how DEAE-cellulose chromatography may be
adjusted to a variety of experimental setups, which makes it an invaluable tool in the
purification toolbox.
References

1. Chromatography Online. (2021). Ion-Exchange Chromatography Principles and

Methods. https://www.chromatographyonline.com/view/ion-exchange-
chromatography-principles-and-methods
2. Sarwar, G., Aziz, M. A., Siddique, M. H., & Din, A. U. (2018). Overview of Protein
Purification Techniques. In Protein Purification: Advanced Techniques (pp. 1-17).
CRC Press.
3. Thermo Fisher Scientific. (2021). Bovine Serum Albumin (BSA).
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-
biology-learning-center/protein-biology-resource-library/pierce-protein-
methods/technical-notes/bovine-serum-albumin-bsa.html
4. Cummins, P. M., Rochfort, K. D., & O’Connor, B. F. (2017). Ion-Exchange
Chromatography: Basic Principles and Application. In Methods in Molecular Biology
(Vol. 1485, pp. 209–223). Springer New York.
5. Marques, J., das Neves, G. B., Ungri, A. M., de Souza Franco, C., Galdino, N. A. de
L., Ribeiro, B. G., Borges, G. K., & Miletti, L. C. (2023). Comparative study of three
novel ion exchange resins with DEAE-cellulose for the purification of Trypanosoma
evansi. Analytical Biochemistry, 676(115226), 115226.
https://doi.org/10.1016/j.ab.2023.115226
6. Co, W. H. (n.d.). Guide t Ion-Exchang Chromatograph. Harvardapparatus.com.
Retrieved November 6, 2023, from
https://www.harvardapparatus.com/media/harvard/pdf/Ion%20Exchange%20Chroma
%20SpinColumn%20Guide.
7. Quimby, B., & To. (2022, September 23). Deae-cellulose: CAS 9013-34-7: SCBT -
Santa Cruz Biotechnology. SCBT. https://www.scbt.com/p/deae-cellulose-9013-34-7/
8. Zagorodni, A. A. (2007). Ion Exchange Purification and separation. Ion Exchange
Materials, 263–281. https://doi.org/10.1016/b978-008044552-6/50013-7
Appendices

Figure 4: 50 ml of Tris-HCI buffer pH7.4 was measured.

Figure 5: The column was washed with Tris-HCI buffer

 Figure 6: The flowrate of the column was set up to 2ml/min

Figure 7: The column was never let to dry. More buffer was added until the column was
ready to use.
Figure 8: 5 ml of fractions were collected after the addition of sodium chloride into the
column.