Questions Related to Size Exclusion Chromatography 1. There are two types of SEC, gel permeation(GPC) and gel filtration (GFC). GPC uses a hydrophobic column packing material and a non-aqueous mobile phase (organic solvent) to measure the molecular weight distribution. GFC uses a hydrophilic packing material and an aqueous mobile phase to separate, fractionate, or measure the molecular weight distribution of molecules soluble in water. a. If you needed to determine the molecular weight distribution of proteins, which on would you use? b. Give an example of a type of molecules that the other type of SEC would be useful for. 2. How do you think you could determine the MW of an unknown using SEC? 3. Explain the plot below. Each red line is data from a different column Shamadzu.com Questions Related to Affinity Chromatography 1. Affinity Chromatography (AC) separates molecules based on the reversible interaction between a target protein and the specific ligand attached to a chromatography matrix. The interaction can be specific (such as antibodies binding to protein A or protein G), or nonspecific (e.g., a histidine -tagged protein binging to metal ions). How do you think on would elute a sample in AC? (HINT: there are two ways) Questions Related to Ion Exchange Chromatography Define the following: 1. pI 2. pKa 3. ionic strength Answer the following: 1. There are two modes of IEC, Cation Exchange and Anion Exchange. Which mode uses a resin that has a positive charge? 2. Both modes of IEC dissolve the solute in a mobile phase that contains a salt (a buffer). a. If you were attempting to separate positive ions in a sample, would you want the buffer to be of high or low pH? b. Is this cation or anion exchange? 3. In IEC, samples are sometimes eluted off of the column with a salt gradient. Explain. 4. IN IEC, samples are sometimes eluted off of the column by varying pH. The pH of the buffer is decreased in order to elute the sample in which mode, cation or anion?