Thin Layer Chromatography Lab

Materials:

Lettuce
Carrot
Cyanobacteria (dense)
Alumina
Acetone
Pentane
Chloroform
Eppendorf tubes
Micropipet and tips (1000 pl, 200 pl)
Disposable, plastic blue pestles (fitting #5 tubes)
140 ml beaker
thin layer chromatography sheets
centrifuge

Procedure:

1. Crush the lettuce and carrot each in an eppendorf tube using the disposable, plastic pestles.  Continue grinding them until you see a liquid form.  Only a small amount of the lettuce and the carrot are needed.
2. Set aside the eppendorf tubes containing the lettuce and carrots, you will use them later.
3. Make a solution of 2:1 (acetone: pentane).
4. Add 1.5 mL of the acetone: pentane solution to each of the eppendorf tubes that contain the carrots and lettuce.
5. Continue to crush each mixture.  Set them aside for later use.
6. Take 100 mL of the cyanobacteria and place it in a tube to be centrifuged.
7. After centrifuging, add 0.6 mL of the acetone: pentane solution.
8. Transfer the cyanobacteria to an eppendorf tube and crush the mixture with the disposable, plastic pestle.
9. Measure up 1 cm from the bottom of the chromatography paper and draw a line with a pencil.
10. Label 3 points on the line-equidistant.
11. Place 5 microliters of sample onto one of the 3 points.  Allow the sample to draw and apply 5 microliters again. Repeat this procedure 8 more times.
12. Repeat step 11 for the other two samples (carrots and lettuce).
13. Make a 3:1:1 solution (pentane:acetone:chloroform).
14. Place this solution into the 140 mL beaker-you should place enough of the solution into the beaker so it’s below the pencil mark you made on the paper.
15. Place the paper into the beaker.
16. Watch carefully, the events will occur quickly,
17. Measure the distance of each band of color moves up and away from the drawn line and the determine the proportional distances.  Measure these after the solution has moved approximately 5 cm up the paper.

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