Ion-Exchange Chromatography

Lab 3: Ion-Exchange Chromatography

We now know how to analyze pure compounds, but what if we have a mixture? Spectrophometry becomes quite complex when dealing with multiple species of compounds at once. In order to purify a compound we can separate if from a mixture based on its intrinsic chemical properties. Remember that fluorescein is negatively charged at a pH above pKa of the carboxyl group. We can take advantage of this fact and use its attraction to positive charges to separate it from other molecules. In ion-exchange chromatography, we will use a stationary phase with a positive charge, allowing negatively charged molecules to bind and positively charged species to flow through. We can then disrupt this interaction and retrieve our now-purified molecule, and use spectrophotometric analysis of our purified fractions to determine how well we were able to separate our molecules.

Learning Objectives

1. To pour and run a chromatography column,
2. To relate previously established concepts of pK, and charge to the principles of ion-exchange chromatography, and
3. To further develop spectroscopic techniques in the analysis of proteins and molecules.

Materials

1. Spectrophotometer
2. Micropipettors
3. Parafilm®
4. Solutions of cytochrome c (from horse) SDS and fluorescein SDS
5. Chromatography column DE 52 anion-exchange resin (~10 mL of slurry) SDS in 100mM TrisCl, pH 8*
7.                    Elution Buffer (1.0 M NaCl in 10 mM)

Objective

To run an anion exchange column and separate a mixture of an organic dye and a protein on the basis of charge differences.

Protocol

Part I. Preparing the Column. Prepare you chromatography column by attaching fittings as instructed by your TA. Ensure that the bottom valve is closed and add 5 mL co water in the column. Indicate the 5mL mark with a Sharpie and add another ~20mL of water. Then, open the bottom valve to let the water flow out of the column until the level of liquid is about 1cm above the plastic frit at the bottom of the column, at which time you should again close the bottom valve. Your TA will provide you with a slurry containing the DE52 anion-exchange resin. You should gently rock the container of slurry to suspend the resin uniformly, and then you should add ~10 mL of the slurry to your column. Open the valve at the bottom of the column, allow the remaining buffer, which should be above the top of the resin bed. at this time switch the valve to the closed position and carefully add, in a manner that does not disturb the resin bed, 5 mL of 10mM TrisCl, pH 8 to the column. Switch the valve at the bottom of the column to the open position to allow the buffer to flow through the column. Repeat this process 4 more times so that a total of 5 column volumes pass through the column. Why is this done? After the 5 column volumes have passed through the resin, you can close the valve at the bottom of the column. The level of buffer should be ~1 cm above the level of the resin bed.