Thin Layer Chromatography with Pigmented Bacteria
In TLC, solvent travels up a thin solid substrate via capillary action. Each species in the
sample is drawn up the substrate by the solvent. However, species affinity for the
substrate may differ and this affects the speed at which they travel. A species with a high
affinity for the substrate will bind and unbind many times, slowing its travel. In contrast, a
species with a low affinity for the substrate will travel quickly. For a given solvent,
substrate and species, the ratio of the distance travelled by the species compared to the
solvent is called the Rf value. TLC separates species on the basis of their Rf value. A
common reason for TLC failing is if the species have very similar Rf values, traveling very
close together. Adjusting the substrate and solvent properties can help this problem.
Materials and Equipment 1. Several colored bacterial colonies of your choice.
2. Lysis buffer
3. 10 L pipette and tweezers, filter paper.
4. Your solvent system: Petroleum ether, located in the hood.
5. A set of Silica gel 60 plates.
6. A TLC beaker or jar, also in the hood.
Note: Many impurities arise from improper sample handling. Gloves, clean surfaces,
properly cleaned glassware and tools (using organic and polar solvents) are all important.
Method 1. Suspend about 10l (a couple big strokes of a loop) of a bacterial colony in 4 l of
lysis buffer. Agitate vigorously with loop.
2. Resuspend the lysed cells in 25l acetone.
3. Repeat for your other samples, including a chlorophyll extract.
4. Draw (in pencil, with a ruler) a line 15 mm above one edge of the plate. Use a 10 l
pipette to place 1 L dots of each sample on the pencil line, about a centimeter
apart. Each sample dot should be as small as possible. It can help to apply several
small spots over each other to minimize spreading.
5. Give the solvent few minutes to evaporate. You will see the samples as spots on the
plate. 6. Fill the TLC beaker with your solvent up to about 5mm. Line the jar with filter paper
to help saturate the chamber. Cover tightly with foil while your plate dries.
7. Remove the foil and lower the TLC plate (spotted end first) into the solvent so the
bottom edge is covered to a depth of about 5mm. Be careful not to make a splash so
only the bottom 5mm is directly exposed to solvent. Cover the jar tightly. Before
your eyes, the solvent will climb up the plate via capillary action. The solvent makes
the white silica appear pale or almost transparent.
8. When the solvent line has almost reached the top of the plate (about 10mm from the
top), mark the solvent line and pull it out of the TLC chamber with tweezers and lay
it on a tissue under the hood to dry. Wait a few minutes for all the solvent to
evaporate.
9. Visualize the sample. You can photograph the plate with a ruler for scale to keep in
your records. Once the TLC plate is removed from the chamber, the spots may fade
so you must mark them with a pencil.
10. Calculate Rf values for all your spots and compare them to known chlorophyll a and
carotene from the spinach.
11. Clean up.