BIOC 3622- Lab 2 (Size-Exclusion Chromatography)

What is chromatography?

Separating a mixture by passing it through a medium where components move at different rates.

What are the 3 main types of chromatography?

1. Size-Exclusion

2. Affinity

3. Ion Exchange

How are molecules separated in size-exclusion chromatography?

By size, shape, and molecular weight.

How are molecules separated in affinity chromatography?

By a specific binding interaction between a ligand and its binding partner.

How are molecules separated in ion exchange chromatography?

By their charge and attraction to the ion exchanger.

What are the 2 phases in size-exclusion chromatography?

1. Stationary phase

2. Mobile phase

What is the stationary phase in size-exclusion chromatography?

The porous packing material in the column.

What is the mobile phase in size-exclusion chromatography?

The solvent that flows through the column carrying the sample molecules.

What size molecules are filtered out faster in size-exclusion chromatography?

Larger molecules.

What is the exclusion limit in size-exclusion chromatography?

The upper size limit of molecules that will interact with the stationary phase.

For this lab, what is the exclusion limit?

60,000 daltons.

What are the 2 main applications of affinity chromatography?

1. Biological

2. Pharmaceutical

How does the target protein interact in affinity chromatography?

It binds to the stationary phase so the remaining proteins can be filtered out.

In this lab, what is the charge of the protein of interest?

Negative.

What kind of charged molecules filter out first in size-exclusion chromatography?

Positively charged molecules.

In this lab, what is the protein sample of interest used?

Bovine.

What is the purpose of examining bovine samples?

To prevent animal-based disease (e.g. Mad Cow Disease)

What is the purpose of examining hemoglobin?

To study and prevent sickle cell anemia.

What is the purpose of examining Vitamin B12?

To study the genetic explanation for Vitamin B12 deficiency.