1. Column Chromatography
Chromatography is commonly used in biotechnology to purify biological molecules from complex mixtures.
3 basic types
Gel filtration or size exclusion chromatography (SEC)
Filters by size using resin beads with pockets
Affinity chromatography
A complimentary biomolecule binds to the molecule of interest
Once the protein is captured another buffer is added to release the protein for purification
Ion exchange chromatography
The resin beads are given a charge to attract or repel charged molecules
+ beads called anion exchange
- beads called cation exchange (ea. named for the charge of the molecule it traps)
No matter what type there are 2 phases present
Mobile phase: solvent and molecules
Stationary phase: paper, resin beads, filter, …

2. Size Exclusion Chromatography (SEC)
Column Design:
Traps & Sieves
Resin beads trap smaller molecules in pores (holes) while letting larger molecules pass
The kit that we are using has an exclusion limit of 60,000 daltons (Mass of 1 amu)
The exclusion limit is the largest molecule that will be trapped
Fractionation is the process of separating the molecules
A buffer is the solution that suspends the molecules in the mobile phase and moves through the column

3. Molecules – 4 Structural levels
primary - sequence of amino acids
secondary - b pleat or a helix
tertiary - 3D structure aided by the formation of H-bonds, Van der Waals interactions (hydrophobic attraction), & disulfide bridges (-S-S-)
quaternary - 2 or more polypeptides wound into one macromolecule.

4. Hemoglobin (Hb)
Hemoglobin is the oxygen carrying molecule in red blood cells. It is rich in iron that when oxidized (attached to oxygen) causes it to have a rust color (reddish brown)

5. Vitamin B12
Vitamin B12 is an essential amino acid used as a cofactor in various biochemical pathways