Separating Effluent Components by Size
© Ellen Lamb and Kristi DeCourcy
Materials (for every 2 students):
BioGel P-30 column
0.3 ml sample solution
30 ml Loading Buffer (0.05 M imidazole buffer, pH 6.0)
test tubes and rack (to collect fractions)
Support stand and clamp
- Remove the stopper from the top of the column
- Place the column on the support stand.
- Place a beaker under the column to collect the effluent and remove the end cap from the bottom of the column. (The effluent is the solution that elutes, or exits from the column.)
- Allow the column to drain until it stops dripping.
- Add the sample to the top of the column bed using a transfer pipette.
- Allow the sample to enter the column.
- When the column has stopped dripping, add a small volume (~5-10 drops) of Loading Buffer and allow that to enter the column. When the column stops dripping, repeat with one more small volume of Loading Buffer.
- Add 5 ml of Loading Buffer to the column and let the column flow. You should be able to see the separation of the colored bands almost immediately.
- Replenish the buffer volume as necessary, making sure that the column never stops flowing.
- When the first colored band is about to drip from the column, switch to a test tube to collect the sample. This will be Fraction 1.
- After the first colored band has exited from the column, switch back to the original beaker to collect the effluent.
- When the second colored band is about to drip from the column, switch to a new test tube to collect the sample. This will be Fraction 2.
- Repeat steps 6 and 7 to collect fractions until all of the colored bands have exited from the column.
- Make sure there is buffer on top of the column, and put the end cap back on the bottom of the column.
- Make sure that there is at least 1 cm of buffer on top of the gel and replace the stopper in the top of the column.
Observations and Conclusions for Experiment 1
- Three different colored bands appear as the sample separates through the column. What are the colors of the bands?
- The _______ band comes off first, the ________ second, and the _______ last.
- In the space below, draw and label a diagram of how your column looked during the separation.
- The ______band is the largest molecule, the ______comes in second, and the ________ band is the smallest.