Chemistry 439

TLC applications

Objectives:

    1. Learn how to use thin layer chromatographic equipment. 2. Learn how to screen field samples quickly for the analyte prior to other analyses. 3. Separate an ink sample into the different components.
Background:
Approximately 20 years ago, thin layer chromatography was the primary tool of law enforcement agencies as they attempted to identify the presence of illegal drugs in field samples they had collected. Confirmation was assumed if the Rf value of the unknown chemical matched that of a control substance in two or more solvent systems. However, chemists quickly made the point that this was not a reliable way to prove the presence of a specific illegal material in a sample. Positive results only increase the probability that the sample contains the analyte. However, thin layer chromatography can be used to prove absolutely that a sample does not contain the target analyte at a detectable concentration so, because it is fast, reliable and inexpensive, the process continues to be widely used.
Generally the parameter of interest is the Rf value as defined by the following equation:
Rf = cm the analyte moves / cm the solvent moves
Therefore, the analyst must know exactly where the sample was applied to the plate, how far the solvent noves up the plate and how far the analyte moves. This is done by marking the application line (where the sample is put to the plate) when the sample is applied, marking the solvent front when the run is stopped and finally marking the analyte spot as soon as it is developed.

Procedure for Ink:

    1. Place 7 ml of propyl alcohol and 3 ml of distilled water in each of the developing tanks (large test tube or bottle), mix well and close the top. While the systems are equilibrating, complete the following and then come back to the tanks.
    2. Obtain a sample of the printed material and of each ink pen to be tested.
    3. Cut out a few portions containing words out of each paper and place them in a test tube.
    4. Add a few drops of methanol and then pyridine (caution-foul oder) to extract the ink from the paper samples.
    5. Remove the paper and then place the tubes under a stream of nitrogen to evaporate the methanol and pyridine.
    6. While the solvent is evaporating, prepare (cut to size with a pair of scissors-do not touch with your fingers) the strips of the chromatographic material.
    7. Carefully apply a small drop of each of the concentrated ink samples to a chromatographic strip about 1.5-2.0 cm from the bottom of the strip. Allow the sample (ink) spot to dry and then place it in one of the developing tanks.
    8. Allow the strip to remain in the developing tank until the solvent has moved at least 10 cm (1 hour) above the spot where the ink was applied.
    9. Remove the strip, mark the solvent front and allow the strip to dry.
    10. Measure the distance each ink spot moved and calculate the Rf value for each compound in the ink.
    11. Compare the unknown ink with the known ink samples and determine if there is a match.
Procedure for Lipstick:
Follow the procedure above for ink with the following modifications.
    1. Use 5 ml of isoamyl alcohol, 3.5 ml of acetone and 3 ml of distilled water for the developing solvent.
    2. Examine the developed strips with a fluorescent lamp in addition to the visual inspection.
    3. Place the strips in a closed container containing some iodine crystals (in a small beaker). You may need to warm the iodine to cause it to sublime and complex any spots on the strip.
Report:
Describe how you might use this procedure to screen for illegal drugs, metabolites in blood or urine, toxic materials, etc. Can you think of ways to increase the sensitivity (hint-chemists have identified attomoles (10-18) of DNA adducts with this procedure) of the procedure.