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, …

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

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.

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)

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