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Thin Layer Chromatography

Making TLC slides

VOCABULARY TO LEARN:

“tare” = to zero a balance, in other words to remove the weight of the container from the measurement

 

  1. Set out two microscope slides onto a paper towel.
  2. Use a sharpie to label the slides with your initials and today’s date.
  3. Turn the slides over so the ink is facing down.
  4. Place a weigh boat on a top loading balance and tare the balance.
  5. Measure 1 g of Plaster of Paris using a plastic spoon to remove the powder from the stock container.
  6. In a new weigh boat, measure 1 g of Cornstarch or powder.
  7. Mix the Plaster of Paris with the Cornstarch in a single weigh boat.
  8. Using a transfer pipet, add 3 ml of water to the powders.
  9. Quickly mix the water into the powders to form a smooth slurry. You can use the transfer pipet as a mixing rod or to pipet the slurry up and down.
  10. Use the transfer pipet to move the slurry from weigh boat to the surface of the microscope slide you prepared, using the transfer pipet to smooth the surface and remove bubbles.
  11. Allow the slides to sit on the bench until they have dried, approximately 30’-one hour.

Vitamin A stock

For experiments that need a stock of Vitamin A, you will have to snip the end of a capsule off, and squeeze the oily liquid into an eppendorf tube. There will likely be around 100 ul of solution that you can then pipet as needed. Be sure to label your eppendorf tube to note the contents of the tube and the date.

Yeast Extraction

  1. Mix the starting liquid overnight culture so it’s homogeneous. This can be done by swirling the culture if it’s in a flask, by inverting if the cap is tight, by flicking a small tube, or by vortexing if there is a vortex available.
  2. Place a tip on your pipetman and move 500 ul of the culture to an eppendorf tube.
  3. Using an eppendorf tube with end ½ trimmed off at the 100 ul mark, measure 100 ul of small glass beads and add them to the 500 ul of yeast you measured out.
  4. Close the eppendorf tube.
  5. Vortex the mixture of yeast and glass beads, keeping the tube on the vortex at full speed for 15 seconds and then letting the solution “rest” for 15 seconds. This rest is needed to keep the temperature of the yeast/beads mixture from overheating.
  6. Repeat the vortexing and resting series a total of 4 times so the yeast will have been mixed with the beads for a full minute.
  7. Allow the beads to sink to the bottom of the solution and then remove the solution of yeast extract from the top, using a pipetman and tip and a new eppendorf tube.
  8. Label the eppendorf tube that has your yeast extract with its contents, your initials and today’s date.

Thin Layer Chromatography

  1. Begin by putting on gloves to protect the TLC sheets from any oils or pigments on your hands
  2. Use a microscope slide to measure and a pencil to mark on the white side of the TLC sheet the size of the piece you’ll need to cut.
  3. Cut the sheet to size.
  4. Mark the starting line for your materials by measuring 1.5 cm from one of the narrow ends and lightly drawing a line across the white surface of the TLC slide. Do not dig deeply into the material on the surface.
  5. Spot 3 ul of Vitamin A about 1/3 of the way across the line you drew at the bottom of the slide.
  6. Spot 3 ul of Yeast extract about 2/3 of the way across the line you drew at the bottom of the slide.
  7. Allow the spots to dry while you make up the solvent that you’ll use to carry the vitamins up the TLC slide. Mix 9 ml of water with 500 ul EtOH and 500 ul Isopropanol. These can be mixed directly in a 50 ml conical tube. Label the tube with the contents, your initials and today’s date.
  8. Lean the TLC slide in the 50 ml conical tube, keeping the upper edge of the solvent below the line where you’ve spotted the vitamins. The idea is that the solvent must pass through the vitamins to carry them along as the solvent gets wicked up the slide.
  9. Replace the lid on the 50 ml conical tube and allow the TLC to proceed undisturbed for 20 or 30 minutes.
  10. Using a gloved hand, remove the slide from the conical tube.
  11. Allow the solvent to evaporate and then visualize the extent and the intensity of the materials that have moved along the slide, using a UV lamp to visualize the vitamin.
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