The Separation of Dyes (Methyl orange and Methylene blue) by Column Chromatography using IV Tubing
Some important definitions/basic concepts:
Stationary phase: The phase to which the molecules have variable affinity by some reversible interaction and causes the molecules to stop or slow down. In this experiment, the stationary phase is aluminum oxide.
Mobile phase: The phase in chromatography that moves past the stationary phase, to which the molecules have variable affinity and causes the molecules to move. In this experiment, the mobile phase is ethanol and water.
Elute/Elution: To elute is to leach compounds off a stationary phase with a solvent. An elution is this process. We will use ethanol and water to selectively elute two dyes off of aluminum oxide.
In the most basic statement, all chromatography is a competition between the stationary phase and the mobile phase for the molecules being eluted. If the molecules have different polarities they will have different affinities for these phases and a separation can be achieved.
Procedure:
Obtain a column and associated tubing from your instructor. Please note that this entire idea came from your instructor sitting getting an IV and marveling at the systems devised for drug delivery.
The diagramed system will be assembled as followed.
1. Clamp the "column" and associated tubing to your monkey bars.
2. Close the flow controller on the tubing (roll it all the way down).
3. Add a small amount of glass wool to the bottom of the column and a tiny layer of sand.
4. Tap the column so that the sand becomes level.
5. Fill the column with ethanol, trying not to disturb the sand bed.
6. Measure out 4.2 grams of aluminum oxide.
7. Turn the flow controller on so that there is a slow drip.
8. Slowly add the aluminum oxide to the ethanol in the column. Allow it to settle and occasionally tap the column to release any trapped air.
9. When the column is filled, open the flow controller to a low setting and allow the ethanol to drop to a level such that it is just above the aluminum oxide.
10. Using a 3 mL syringe, gently add 0.5 mL of the dye mixture (found in the dispensing hood). Speak to TA/Instructor s about our new way of dispensing liquids.
11. Allow the dye mixture to percolate slowly (open the flow control to a low setting) into the column and stop the flow when it is just above the aluminum oxide bed.
12. Add some ethanol to the top of the column and allow this to percolate when it is just above the aluminum oxide bed.
13. Repeat step 12 two more times.
14. Fill up the area above the column with ethanol and attach the reservoir as indicated in the diagram, i.e., Cover the top of the column with the septum cap. Attach the 10 cc syringe to the reservoir tubing with needle. Clamp the reservoir above the column. Stick the needle in the septum cap of the column. Fill the reservoir with ethanol. .
15. Open the flow control to a moderate rate and allow the first dye to elute off the column into a container ( a 50 or 100 mL beaker). Fill the reservoir with more ethanol if need be to keep the flow going.
16. When the first dye appears to be nearly off, switch containers and collect an intermediate fraction.
17. When all of the first dye is eluted, fill the reservoir with distilled water and switch to a third container. Elute until the entire second dye is eluted.
