Ion-Exchange Chromatography

1. Preparing the Sample and the Column

1. In this demonstration, a mixture of 2 proteins will be separated on a cation-exchange column: hemoglobin and cytochrome C. Add 0.2 mL equilibration buffer (pH 8.1) to the protein sample and vortex to mix thoroughly. Centrifuge for 2 min to remove any froth.
2. Place the cation-exchange column in a test tube for 5 min to allow resin to settle. Clamp the test tube with the column onto a ring stand to make sure it is upright.
3. Open the top cap of the column, and then the bottom cap. Allow the buffer in the column to drip out under gravity into the test tube.
4. Wash the column twice with a column-volume (in this case the column volume is 0.3 mL) of equilibration buffer. Let the first column-volume (0.3 mL) of equilibration buffer drip out into the waste vial before adding the second column-volume. This step lets the column equilibrate with the equilibration buffer. Add the equilibration buffer slowly in this step to not disturb the resin.

2. Running a Protein Sample Through a Cation-Exchange Column

1. Put the column in a 2 mL centrifuge tube labeled "Unbound 1". Carefully load 0.1 mL of the protein sample onto the top of the column.
2. Once the sample has been loaded onto the top of the column, wash it with 0.3 mL of equilibration buffer by adding the buffer at the top of the column and allowing it to drip all the way through. Repeat this step 4x (for a total of 5 washes) and collect each wash in a separate, 2-mL centrifuge tube. Label the tubes as "Unbound 1-5".
3. For the last 2 washes, centrifuge for 10 s at 1,000 x g to make sure all unbound species come off the column. Any sample that binds to the column should not be in these washes, but sample that does not bind will wash out unretained. Centrifuging provides more force to wash unbound materials off the column.
4. Put the column in a new 2-mL centrifuge collection tube labeled "Bound 1". Put 0.3 mL of elution buffer (high salt, pH 5.5) on top the column. Centrifuge the resulting eluent for 10 s at 1,000 x g.
5. Repeat the elution step 2 more times by placing the column in a new centrifuge tube, adding 0.3 mL, and centrifuging for 10 s. Label these "Bound 2 and 3".
6. Record any color changes or observations about the fractions. Hemoglobin is a colored protein that looks reddish-brown while cytochrome C is reddish colored.

3. Results: Cation-exchange of Hemoglobin and Cytochrome C

1. Cytochrome C has a pI of 10.5 and hemoglobin a pI of 6.8. Thus, when a pH 8.1 equilibration buffer is used, hemoglobin does not bind to the column because it is negatively charged. Cytochrome C is positively charged at this pH and does bind to the column because the pH < pI.
2. Hemoglobin is observed in the unbound fractions because it is not bound to the column.
3. Cytochrome C is observed in bound fractions, because it was bound to the cation exchange column.

Figure 1. Picture of unbound fraction (hemoglobin) and bound fraction (cytochrome C).