FACULTY OF ENGINEERING TECHNOLOGY

Lab 01

SIZE EXCLUSION CHROMATOGRAPHY (SEC)

BTP 3243
PROCESS BIOTECHNOLOGY TECHNIQUES

Lab Objectives
By the end of this lab, students should be able to:
1. Perform size exclusion chromatography in protein separation. (Level in Bloom
Taxonomy: P5)

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1.0 Introduction
2.0 Material and Apparatus
2.1 Methodology

3.0 Result
The results from the observation of the colour change of the fractions were shown in Figure 1
and summarized in Table 1.

Figure 1: Colour change of the solutions.

Table 1: Summary of colour change of the solutions.

Tube
Colour

10

Brown

Dark

Brown

Light

Very

Pink

Dark

Pink

Light

Colour-

brown

light

pink

less

Brown

pink

brown

3.1 Discussion
The column beads in this experiment allowed the separation of molecules with molecular
weight smaller than 60000 daltons (Bio-Rad). Molecules smaller than 60000 daltons will be
trapped for some times in the pores of the column beads therefore will be eluted from the
column slower. While for the molecules larger than 60000 daltons, they cannot enter the
pores of the beads thus they will be eluted faster. In short, in the size exclusion
chromatography, the components that are eluted faster are the molecules with bigger size
while those eluted slower are the molecules with smaller size. The results can be investigated
by observing the colour change of the solutions in each fraction and by the study of the
molecular size of each single components in the sample.

The molecular weight of the heamoglobin is around 64,500 daltons (Wuthrich, 1968). The
molecular weight of Vitamin B12 is 1355.388 daltons (Vitamin B12, 2016). As the molecular
size of the heamoglobin is bigger, the molecules of haemoglobin will be eluted first and
followed by molecules of Vitamin B12. The colour of the haemoglobin is brown due to the
iron-containing haem group within the haemoglobin. The colour of the Vitamin B12 is pink.
By referring to Figure 1 and Table 1, the colour changed from brown to pink had proven that
the Vitamin B12 was eluted after haemoglobin.
Tube 1 was used to develop a mobile phase for the components in protein mix to travel
through the gel in the sizing column by flowing 250L of buffer (Bio-Rad). As for the Tube 2
to Tube 10, they were used to test the colour of the solution changed from brown to pink and
lastly colourless. The brown colour of the solution in Tube 1 was actually the colour of the
mixture of buffer and little amount of heamoglobin. After some times, the components in
protein mix which were heamoglobin and Vitamin B12 will be seperated. The intensity of the
of the colour changed was based on the concentration of the molecules eluted.
The solutions colour in Tube 2 to Tube 5 are brown in colour. However, the intensity of the
brown colour decreases with the number of tube. This was because at the early stage of the
separation, greater amount of haemoglobin molecules exit from the column. At the later
stages, the amount of the haemoglobin decreased until there was no more haemoglobin inside
the column. At Tube 2, the haemoglobin concentration is the highest therefore the intensity of
brown is the highest. The colour of the solution changed from very light brown to pink from
Tube 6 to Tube 9. Same as Tube 2-5, the intensity of pink decreased due to the decreased in
the concentration of Vitamin B12 molecules. Lastly, in Tube 10 the solution turns colourless
beacause all the protein mix had been eluted.
4.0 Conclusion

5.0 References
Bio-Rad. Size Exclusion Chromatography. Dalam R. Mardigian, Instruction Manual
(hlm. 2-14).
Vitamin B12. (2016, October 15). Didapatkan October 21, 2016, daripada
Pubchem: https://pubchem.ncbi.nlm.nih.gov/compound/5311498#section=Top

Wuthrich, K. (1968). Proton Magnetic Resonance Studies of Human

Cyanomethemoglobin. Proceedings of NAtional Acedemy of Sciences of the
United States of America , 1200.