CHEM 231: Organic Form and Function

Laboratory 1 Thin-layer chromatography

Specific Practical Goals  to master the technique of thin-layer chromatography (TLC)  to identify components of a mixture using TLC

The Premise Non-steroidal anti-inflammatory drugs (NSAIDs) represent an indispensable component in the arsenal of pain management, with global sales well over $5 billion, and much of this market in the US is made up of over-the- counter (OTC) pain and/or fever preparations. The history of NSAIDs as pharmaceuticals has followed and interesting, unusual, and serendipitous path. Today’s most recognizable OTC pain reliever, , was introduced in 1899 by Bayer, turning a compound known to the chemical community for a half-century into a commercial powerhouse. Some current Aspirin formulations (such as Excedrin and Anacin) contain caffeine, which is purported to result in faster and more effective headache relief. While Aspirin is often presumed to be the oldest synthetic pharmaceutical, it was actually preceded by acetanilide, which was put on the market in 1886 by Kalle & Co. under the trade name of Antifebrin. This substance was effective in reducing fever, and it was much less expensive than quinine, which was derived from plant extraction. Its one drawback was the tendency to induce cyanosis, which produced understandable consumer concern. Over the next several decades it became clear that this side-effect was caused by the toxicity of (phenylamine) liberated by hydrolysis in vitro. Blood serum studies also revealed that the chief metabolite of acetanilide was acetaminophen, which turned out to be the actual therapeutic compound. Acetaminophen was already known, but these results led to its full-fledged marketing as a pharmaceutical in the 1950s, and it quickly eclipsed acetanilide. In a post-apocalyptic landscape in which the legitimate OTC drug manufacturers have collapsed, you have obtained an mixture from a street vendor (not a recommended supplier of pharmaceuticals), who claims it is a combination of two common pain-relief products. Being a prudent citizen, you set about to identify the components using thin-layer chromatography.

Scheme 1. Common past and present components of NSAID preparations

Pre-lab Reading Technique Primer 1: Thin-layer chromatography

Safety Considerations You must abide by all laboratory safety rules. 4-Acetamidophenol [103-90-2]. Harmful if swallowed. Causes skin irritation. Causes serious eye irritation. May cause respiratory irritation. Toxic to aquatic life. Acetanilide [103-84-4]. Harmful if swallowed. Causes skin irritation. Causes serious eye irritation. May cause respiratory irritation. Harmful to aquatic life. Acetylsalicylic acid [50-78-2]. Harmful if swallowed. Causes skin irritation. Causes serious eye irritation. May cause respiratory irritation. Caffeine [58-08-2]. Harmful if swallowed. [64-17-5]. Highly flammable liquid and vapour. Causes skin and eye irritation. May cause respiratory irritation.

Procedural Overview Obtain a TLC plate and carefully draw a faint origin line with a pencil. Dissolve your unknown sample in 2-5 mL of ethanol and spot the solution on the origin line using a capillary tube. Evaporate the ethanol by briefly passing a stream of air over the TLC plate and then develop using the TLC solvent provided—about 6 mL of solvent is sufficient (Caution: do not allow the solvent to slosh over the origin line when placing the TLC plate in the chamber). When the solvent has risen to within 1 cm of the top of the plate, remove from the chamber and immediately mark the solvent front. Examine under UV light (Caution: do not look directly into the UV light; turn off the light when finished!). The TLC spots on the developed plate should be self-contained and tidy, as shown in Figure 1 (left plate). If the spots are large and oversaturated (right plate), this could be the result of applying too large a spot, too concentrated a solution, or both. If you encounter this, dilute your sample (the concentration difference between the two plates is about 50-fold) and try again.

Using the table of Rf values below, propose the two components in your mixture, then prepare a second TLC plate in which you place three spots side-by-side on the origin line (your mixture and each of the standards you believe to be present). Develop the plate as described above and confirm that the composition of your mixture is the one you proposed. Trace your TLCs (in actual size) in your notebook before discarding.

compound Rf acetaminophen 0.56 acetanilide 0.63 acetylsalicylic acid 0.44 caffeine 0.17

Table 1. Approximate Rf values of compounds (0.2% acetic acid in ethyl acetate)

Figure 1. Properly spotted (left) and overspotted (right) TLC plates