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Thin-Layer Chromatography of Drosophila melanogaster Eye Pigments
In this lab, you will be working with many familiar and simple supplies. The only item which may be new to you is the thin-layer chromatography plate (TLC plate). One side of this plate is plastic and is used for support of the material on the other side. The other side is made of an absorbent, white material. This white side is the important 'functional' side. It is significant to note that this white side can easily be damaged by scratching the material from the plastic, supportive side.
Chromatography is a technique used to separate solids (or solid-like) materials from liquids. In our lab, the solid-like material is the ground-up fly eye pigments. (You remember that pigments are the chemicals that give color to living things.) The liquid portion is the solvent (probably acetone).
In TLC, the sample (the eye pigments) is applied to the plate as a small spot (less than 1/4" in diameter) near the base of the plate. This technique is called spotting. During the spotting process, you drop repeated samples onto the plate, letting each drop completely dry before putting on the next one. In our lab, however, we will carefully and gently smash the fly directly on to the white side of the TLC plate. We will attempt to get the eye pigments to directly absorb into the white side.
After we have the pigments on the TLC plate, we will placed them into a solvent (while under the fume hood). During the next 30-40 minutes, the pigments will become separated based on the differences in the rates at which the individual components (pigments) of the mixture advance upwards on the plate. Remember that the final eye color of the fly is the sum of many
individual pigments with different concentrations of each.
The pigments from the fly eye dissolve into the solvent (acetone). The solvent is absorbed by the white material (cellulose) and as it does so, you will notice the line of absorption move up towards the top of the TLC plate. This cellulose material is much like that in a paper towel ("Downy - The Quicker Picker Uper"). Depending on how well each pigment dissolves into the solvent, the individual pigments will travel along with the solvent, being deposited on the plate at the point where it is no longer soluble in the solvent.
In addition, TLC is influenced by molecular size. The pigments that are bigger (and thus, heavier) will not be able to travel up the plate as far as the smaller, lighter pigments.
The result of chromatography is a TLC plate with "lanes" of separated pigments, some more visible and showing more bands of color than others. In this lab, we will view the TLC plate under ultra-violet light. This will enable us to see pigments that are not necessarily in the visible-light spectrum.
SAFETY CONCERNS/LAB TECHNIQUES
1. Hold the TLC plate by the sides like a photograph.
2. Avoid gouging the TLC plate when making your Xs and when squashing your flies.
3. Use a pencil when making your Xs. Pen will run.
4. Gently handle the flies with a paintbrush.
5. It is important that you squish both the head and the body on the Xs.
6. Be sure to squish four flies of the same type on each X, as
7. Check the solvent level before placing your TLC plate in the separation chamber. The flies should be above the level of the solvent.
8. Place the TLC plate in the separation chamber carefully. Do not drop it in.
9. Do not let the solvent reach the top of the TLC plate. It should come to within 1 cm.
10. Remember to mark the solvent front before it dries. It will be difficult to see once it dries.
11. Keep the separation chambers in the fume hood at all times.
12. Let the TLC plate dry completely before putting it in the foil.
13. Wear goggles when using the UV illuminator.
14. Use the foil shield when using the UV illuminator.