Thin Layer Chromatography

Identifying Drug Components

Introduction:

A person overdosed on an over the counter analgesic drug. It is our job to identify the drug so that the emergency room personnel can apply the appropriate treatment. Unlike most of the materials we encounter on a daily basis, most analgesic drug preparations contain only one or two active ingredients from the following: aspirin, acetaminophen, ibuprofen, salicylamide, and caffeine (Figure 1).

Figure 1

In order to identify the components of the unknown drug thin layer chromatography is used and the retention factor (Rf) values (Equation 1) of the unknowns are compared to the Rf values of the active ingredients in known analgesic drugs. Chromatography is a chemical method used to separate and or to analyze complex mixtures. The components to be separated are distributed between two phases: a stationary phase and a mobile phase. The separation occurs due to the differences in polarity and solubility of the mixture components.

Equation 1 Rf=distance traveled by spotdistance traveled by solvent

A hypothesis will be formed after examining the thin layer chromatography (TLC) plate under ultraviolet light. Other visualization methods are used to either support or disprove the tentative hypothesis.

Procedure/Observations:

Materials: mason jar , paper wick,watch glass, 10 mL graduated cylinder, stirring rod, filter tip pipet, small test tube, silica gel TLC plate, micropipet, mechanical pencil, tweezers, gloves, UV light, ethyl acetate/acetic acid (200:1), ethanol/ dichloromethane (1:1), unknown drug, aspirin solution, acetaminophen solution, ibuprofen solution, salicylamide solution, caffeine solution

**Avoid contact with both ethyl acetate/acetic acid (200:1) and ethanol/ dichloromethane (1:1) and their vapors by using gloves and the fume hood.

Prepare a mason jar as the developing chamber for the TLC paper with ethyl acetate/acetic acid as the developing solvent. Fill the chamber to a depth of about 2mm solvent. Cover it and set aside. Prepare the TLC plate by drawing a line 1.5 cm from the bottom with a mechanical pencil. This will be your start line.

Obtain a small amount (about ¼ tablets worth) of unknown drug A and bring it over to your fume hood. Transfer the powder to a 10 mL graduated cylinder and add 2.5 mL of ethanol/ dichloromethane. Try and dissolve as much of the unknown into the solvent using a stirring rod. You will notice that almost all of the powder will dissolve. With a filter tip pipet, transfer the solution into a small test tube.

With a micropipet spot the unknown solution and the other known solutions onto the TLC plate. It is advised to practice spotting before you actually spot your TLC plate for best results. Keep track of what spots went where. For best results, the spots should be 1.5 cm apart.

Develop the plate in the chamber created by placing the bottom of the plate down in the developing solvent with tweezers. Seal the lid and allow it to sit until the solvent has risen to a line 1cm from the top of the TLC plate. Remove the plate after this point and let it sit to dry.

Once dry observe the TLC plate under UV light. Outline, with pencil, the spots of the known and unknown solutions and mark the center of greatest intensity for each spot. At this point we noticed that our spots were really close together and hard to differentiate. It is extremely important to place the spots 1.5 cm apart from each other. Calculate the Rf values (Equation 1) for each spot and record the data in a table. Identify the unknown given the Rf values and other visualization techniques, if performed.

Results:Unknown A

Drug

First Rf Value

Second Rf Value

acetaminophen

0.21

aspirin

0.29

0.49

caffeine

0.09

ibuprofen

0.09

0.33

salicylamide

0.09

0.58

unknown A

0.09

Table