Discussion

The absorption spectrum of Cytochrome C, which is known for its function in the mitochondria

as an important participant in the life-supporting ATP synthesis, ranges from 400nm to 600nm

and consists of three peaks in its reduced form at 550nm, 531nm and 414nm. It also has two

peaks in its oxidized form at 530nm and 408nm. The absorption spectrum for riboflavin, which

is a soluble vitamin B, ranges from 310nm to 510nm with an absorption peak at 440nm.

From Figure 1, the superficial velocity of the first molecule in the size exclusion chromatography

was calculated to be 2.78 cm/min. Similarly, the superficial velocity for the second molecule was

1.36 cm/min. The size exclusion chromatography is based simply on the physical dimensions of

the molecules. Smaller molecules (smaller hydrodynamic radius) access a bigger fraction of the

gel and therefore are retained in the stationary phase. Consequently, larger molecules travel

through the gel faster than smaller ones do. Following this concept, Figure 1 shows that molecule

A was larger than molecule B. A two-component separation was calculated to be 1.86 for the

SEC. Since 1.86 > 1.5, this indicated that the separation was fully resolved. During the

experiment, it was also clearly visible how the protein was traveling through the column and the

separation was definitive.

The ion-exchange chromatography is based on the interactions between the charged regions on a

given molecule with a changed-group supported on a beaded resin. A peak at around 400nm can

be observed in Figure 3. From literature, it is known that Cytochrome C has an absorbance peak

at 414nm. It is safe to assume that Cytochrome C came out first from the ion exchange column.

A slight increase in the absorbance can be observed around 450nm. From scientific literature, it

is known that riboflavin has an absorbance peak at 440nm. It can be assumed that riboflavin
came out second from the column however in lower amounts. Figure 2 shows that the fractions

from 2 to 7 had large absorbance and fractions 9, 11 and 12 also had high absorbance. Fractions

2 to 7 are related to Cytochrome C which came out first and fractions 9, 11 and 12 could be

related riboflavin. There is not enough evidence to conclude whether the other molecules came

out of the column since no other peaks were observed from the absorbance spectra graph.

The ion-exchange chromatography functions with the isoelectric point of molecules. The

isoelectric point (pI) is defined as the pH at which a molecule has no net charge. From scientific

literature, it was found that the pI of Cytochrome C was of 9.6 and that of riboflavin of 6. The

elution buffer in this experiment had a pH of 8.0. The pH of the elution buffer is lower than that

of Cytochrome C (9.6), the protein therefore carried a positive net charge. Thus, as assumption

that a negatively-charged cation exchange resin was chosen can be made. The positively charged

molecule (Cytochrome C) bound to the negatively charged surface. Unbound molecules were

washed with the running buffer. After that, the elution buffer was used to overburden the resin

with counter-ions which removed the bound molecules from the column.

The results of this experiment could have been faltered due to human error. From Figure 3, it is

clear that the absorbance for fractions 10 and 11 were not taken correctly. It is also visible in

Figure 2 that fractions 10 and 11 had abnormal absorbance values compared to the rest of the

fractions. This could have been due to the presence of fingerprints on the cuvettes which would

have prevented the spectrophotometer from taking the correct values for the absorbance by

preventing light from passing through the cuvettes correctly. Due to the error for these 2

fractions, the identification of the proteins could have also been wronged.
References

https://www.researchgate.net/figure/UV-spectra-of-cytochrome-c-typical-spectra-of-cytochrome-

c-with-three-absorbance-peaks-of_fig7_51873414#:~:text=Attribution%203.0%20Unported-,UV

%2Dspectra%20of%20cytochrome%20c%2Dtypical%20spectra%20of%20cytochrome

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https://www.bio-rad.com/fr-ca/applications-technologies/ion-exchange-chromatography?

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