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Luis Pinacho-Rios

Biotechnology 2

Husselstein

September 14, 2022

Size Exclusion Chromatography

Overview:

In the lab performed we separated the proteins in a protein mix that contained

Hemoglobin and Vitamin B12. We separated the proteins by preparing a size exclusion

chromatography. To be able to analyze the sizes we collected drops of the proteins in different

fractions.

Results:
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Postlab Focus Questions:

1. Hemoglobin and Vitamin B12 were separated via size exclusion chromatography. In our

results we can see a clear color transition from brown to pink. Thus, the proteins were

successfully separated.

2. Column fractions 1-3 appear to contain Hemoglobin because there is a tint of brown in

the fractions. Column fractions 4-9 appear to contain Vitamin B12 because of the tint of

pink in the fractions.

3. Based on our results, we can conclude that Hemoglobin was the larger protein because

the protein exited the resin in the earlier fractions. Vitamin B12 was the smaller protein

because it appeared in the later fractions.

4. The resin used in this activity was small. Based on this fact we can assume that the

Vitamin B12 molecule was smaller than 60,000 Da since it was caught in the resin and

exited the resin in the later fractions. Whereas the Hemoglobin molecule was larger than

60,000 Da because it was excluded from the resin since it exited the column in the earlier

fractions.

Additional Questions:

1. In order to separate mixtures we have taken advantage of the following chemical

properties: size, shape, polarity, charge, and density. In the electrophoresis lab we

separate mixtures based on size, charge, and shape. In the kool aid chromatography lab

we separate mixtures based on polarity. In labs that involve centrifuging, mixtures were

separated based on size and density.

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2. In the real world protein purification is necessary to isolate different proteins from any

factors that may alter its state. That way the desired protein can be retrieved from a group

of proteins for analysis. Similar to how we retrieved Hemoglobin and Vitamin B12 from

our protein mix, except that in the real world the desired proteins are analyzed for

different purposes. Such as the making of an antibody, clinical studies, protein doses in

our food, and etc.