FACULTY OF MEDICINE AND HEALTH SCIENCES UNIVERSITI

PUTRA MALAYSIA

PRACTICAL 12: ION EXCHANGE CHROMATOGRAPHY

Lecturer’s name: Dr. Huzwah Khazaai

NAMES MATRICS NUMBER

1. Teea Yuan Xin 198815
2. Ng Sing Jie 196835
3. Crystal Arthini Arrumugam 198258
4. Muhammad Munir bin Ismail 196332
5. Nur Hidayah Bt Saubari 197559
6. Na'imah bt mohd rashid 199789
7. Siti syahnaz bt mohd rusli 199922
8. Wan Nur Tihani binti Wan Ahmad 197491
Jailani
9. Nivishaa Sagayarajan 199824
10. Fathin Dalila Bt Aminnuddin 199934
INTRODUCTION

Ion exchange chromatography is one of the most efficient methods for the separation

of charged particles. It works on almost any kind of charged molecule including large

proteins, small nucleotides, and amino acids. However, ion chromatography must be done in

conditions that are one unit away from the isoelectric point of a protein. Ion exchange

chromatography is most often performed in the form of column chromatography. However,

there are also thin-layer chromatographic methods that work basically based on the principle

of ion exchange. In the following, we will exclusively deal with column chromatographic

applications. Column materials used for ion exchange chromatography contain charged

groups covalently linked to the surface of an insoluble matrix. When suspended in an

aqueous solution, the charged groups of the matrix will be surrounded by ions of the opposite

charge. In this “ion cloud”, ions can be reversibly exchanged without changing the nature and

the properties of the matrix.

The charged groups of the matrix can be positively or negatively charged. A positively

charged matrix will bind negatively charged ions from the solution. Therefore, it is called an

anion exchanger. Cation exchanger matrices have negative charges.

One of the primary advantages for the use of ion chromatography is only one

interaction involved during the separation as opposed to other separation techniques;

therefore, ion chromatography may have higher matrix tolerance. Another advantage of ion

exchange, is the predictably of elution patterns (based on the presence of the ionizable group).

For example, when cation exchange chromatography is used, cations will elute out last.

Meanwhile, the negative charged molecules will elute out first. However, there are also

disadvantages involved when performing ion-exchange chromatography, such as constant

evolution with the technique which leads to the inconsistency from column to column. A

major limitation to this purification technique is that it is limited to ionizable group.

METHODOLOGY

Lysozyme

First, 8 collection test tubes were prepared. Then, layer of Equilibration buffer was drained.

0.2 ml of prepared lysozyme solution was pipetted gently, allowing the liquid to trickle down

column wall just above SEPHAROSE layer. After that, 2 ml Elution buffer was added to the

column by taking the same precaution as in previous step. All eluents were collected into a

tube labelled 1A. This step was repeated and the eluent was collected into tube 2A. Another 5

consecutive labelled tubes of eluents were collected in the same manner. 2ml Bradford

reagent was added to each test tubes. The solutions were then transferred to labelled plastic

cuvettes. Blank consisting of 2ml Elution buffer and 2 ml Bradford reagent were prepared

and then measured at absorbance 595 nm where the spectro machine was set to auto-zero.

Protein eluents were measured at same absorbance as well. A peak in the absorbance

measurement was identified to indicate release of lysozyme from the column. 2 ml Elution

buffer was again added to the column and the eluent was collected two more times. Wash

Protocol has been done and next protein was analysed.

Albumin

First, 8 collection test tubes were prepared. Then, layer of Equilibration buffer was drained.

0.2 ml of prepared albumine solution was pipetted gently, allowing the liquid to trickle down

column wall just above SEPHAROSE layer. After that, 2 ml Elution buffer was added to the

column by taking the same precaution as in previous step. All eluents were collected into a

tube labelled 1B. This step was repeated and the eluent was collected into tube 2B. Another 5

consecutive labelled tubes of eluents were collected in the same manner. 2ml Bradford

reagent was added to each test tubes. The solutions were then transferred to labelled plastic

cuvettes. Blank consisting of 2ml Elution buffer and 2 ml Bradford reagent were prepared
and then measured at absorbance 595 nm where the spectro machine was set to auto-zero.

Protein eluents were measured at same absorbance as well. A peak in the absorbance

measurement was identified to indicate release of albumin from the column. 2 ml Elution

buffer was again added to the column and the eluent was collected one more time. Wash

Protocol has been done and next protein was analysed.

Wash Protocol

Firstly, 2 ml Equilibration buffer was aliquoted onto the column. Then, the flow was

discarded. Steps are repeated. Next, 2 ml Elution buffer was aliquoted onto the column. Then,

the flow was discarded. Steps are repeated. Lastly, protein was loaded onto the cleaned

column.

Lysozyme and Albumin Mixture

First, 8 collection test tubes were prepared. 0.4 ml of prepared combination of lysosome and

albumin solution was pipetted gently, allowing the liquid to trickle down column wall just

above SEPHAROSE layer. After that, 2 ml Elution buffer was added to the column by taking

the same precaution as in previous step. All eluents were collected into a tube labelled 1C.

This step was repeated and the eluent was collected into tube 2C. Another 5 consecutive

labelled tubes of eluents were collected in the same manner. 2ml Bradford reagent was added

to each test tubes. The solutions were then transferred to labelled plastic cuvettes. Blank

consisting of 2ml Elution buffer and 2 ml Bradford reagent were prepared and then measured

at absorbance 595 nm where the spectro machine was set to auto-zero. Protein eluents were

measured at same absorbance as well. Two peaks in the absorbance measurement was

identified to indicate release of lysozyme and albumin from the column. 2 ml Elution buffer

was again added to the column and the eluent was collected two more times.
DISCUSSION

Ion exchange chromatography is used for protein purification and to separate molecules

according to the charge density, charge distribution and the size of the molecule. In this

experiment, we wanted to separate two groups of protein that are differently charged at the

same level of pH.

Based on the results, we can see that resin selection occurring in the column have help in

separating the lysozyme and albumin mixture from each other. This is important as it proves

that the diethyl aminoethyl is positively charged at pH 7.2, which attracts the negative charge

from albumin at the same pH level.

There were some errors though while doing the experiment, firstly from a lack of correct

pipetting skill, which may result in the final result being wrong. Besides, some of the errors

in the results can also came from incorrect labelling of the 2 mL mark on the test tubes,

therefore the measurement were inaccurate each time.

Besides, we did not clean the glass cuvette wall properly after each spectrometry, which

may lead to inefficiency and inaccuracy in getting the final reading. Besides, we may have

also accidentally use the same pipette tip to measure two different effluents without proper

cleaning, which may contaminate either effluents, and affect the final result.

Some precautionary steps are necessary to prevent this errors again, including assuring

that the experiment was repeated again for a more accurate result. However, we were

experimenting under a time constraint and could not afford to repeat the experiment more

than once. Then, we have to correctly label the measurement of solutions or effluents we

need to ensure no error in measurements occur again.

Next, we need to use proper cleaning method to ensure more accurate result, which is

where the Wash Protocol comes in. Finally, we have to properly separate our apparatus from
the start, to ensure we do not cause mishaps like using the same pipette tip for two different

effluents again.

RESULT

Lysozyme
Test tube Absorbance
1 2 3 Average
1A 0.273 0.245 0.246 0.243
2A 0.194 0.195 0.217 0.202
3A 0.193 0.198 0.198 0.196
4A 0.065 0.059 0.06 0.061
5A 0.211 0.217 0.218 0.215
6A 0.076 0.081 0.077 0.078
7A 0.008 0.009 0.01 0.009

Absorbance
0.3

0.25

0.2

0.15

0.1

0.05

0
1A 2A 3A 4A 5A 6A 7A

Graph 1 Absorbance of Lysoszyme

According to the graph we constructed, the initial absorbance reading is 0.243

whereas the final reading is 0.009. Basically, the absorbance of lysozyme keeps
fluctuating throughout the experiment. In fact, we suppose to get a graph which
increase gradually at first until it reaches a peak and decrease consistently until a
reading above 0.
Albumin
Test tube Absorbance
1 2 3 Average
1A 0.225 0.225 0.241 0.230
2A 0.202 0.202 0.218 0.207
3A 0.211 0.217 0.212 0.213
4A 0.222 0.210 0.232 0.221
5A 0.209 0.197 0.219 0208
6A 0.010 0.012 0.005 0.009

Absorbance
0.25

0.2

0.15

0.1

0.05

0
1B 2B 3B 4B 5B 6B

Graph 2 Absorbance of Albumin

Based on graph 2 that we get from the second experiment, the absorbance is decreasing at

first which is 0.230, keeping constant for few readings and finally decreasing sharply to 0.009.

In fact, we suppose to obtain a graph which increase gradually at first until it reaches a peak

and decrease consistently until a reading above 0.

Lysozyme and Albumin Mixture
Test tube Absorbance
1 2 3 Average
1A 0.268 0.293 0.275 0.279
2A 0.266 0.265 0.271 0.267
3A 0.232 0.231 0.237 0.233
4A 0.049 0.048 0.063 0.053
5A 0.255 0.257 0.258 0.257
6A 0.298 0.282 0.288 0.289
7A 0.300 0.284 0.290 0.291

Absorbance
0.35

0.3

0.25

0.2

0.15

0.1

0.05

0
1C 2C 3C 4C 5C 6C 7C

Graph 3 Absorbance of Lysozyme and Albumin Mixture

Based on the graph 3, the absorbance of lysozyme and albumin mixture keeps declining from

0.279 until 0.053. Later, the absorbance starts to increase until 0.291. In fact, we suppose to

get two graphs with two peaks itself. The first graph which comes first should be graph of

lysozyme and the second graph should be graph of albumin. This is because the ion exchange

chromatography for lysozyme is faster than albumin one.

QUESTIONS

1. What evidence do you have that the ion exchange column separated the two proteins?

Describe what is happening inside the column(s).

Since proteins have difference characteristic features as size, shape, net charge, stationary

phase used, and binding capacity, each one of these characteristic components can be purified

using chromatographic methods. On a column (stationary phase) firstly the sample to be

separated, then wash buffer (mobile phase) are applied. Their flow through inside column

material placed on a fiberglass support is ensured. The samples are accumulated at the bottom

of the device in a time, and volume-dependent manner

2. What information have you gained about the characteristics of lysozyme and albumin?

The protein that left the columns before the other was oppositely charged to the resin. The

lysozyme is oppositely charged to resin and albumin only left the column when the elution

buffer was added

3. What result might you have expected if you used a resin with opposite charge (cation)?

Albumin would exit the columns in the first couple trials while lysozyme would only leave

when the elution buffer is added.