Analysis of a Commercial Pain Medication: An Introduction to Separations

Objective. To develop a method to effectively separate four compounds commonly found in over-the-counter pain medications. You will use your optimized method to determine the composition of a commercially available product.

 

Background. What is stuff made of? This fundamental question, even when clumsily worded, is a cornerstone of the science of chemistry and provides us with the context of our first lab. As you will see over the term (or terms, if you continue with the subject), there are many different ways this question can be answered. For now, we will have to be content with the following: most matter that we encounter on a routine basis is composed of fundamental particles called molecules, of which millions of different types are known. Each molecule of the same type is identical to others of its kind and distinct from those of other kinds. A large collection of identical molecules is called a compound, such as water, sucrose (aka table sugar), and linoleic acid, the focus of much of the first chapter of your text.

Most matter that you encounter, however, consists of mixtures of different compounds rather than pure ones. Yes, you can find examples of pure compounds (such as a packet of sugar), but they are rarely found in nature in that state. Sucrose, which you can buy in any grocery store, must be purified from natural sources such as sugar cane via processes that remove materials such as cellulose (the woody part of the stalk which is indigestible), moisture, and various impurities. You are a mixture of compounds, as is virtually all the food you eat and things you use.

One of the more common challenges in chemistry is to determine the specific types of compounds that comprise a mixture. This usually requires the physical separation of the various types of molecules, “sorting” them by type and then determining what compound they correspond to. That’s what you will do in this experiment.

A host of technologies have been developed to physically separate different compounds from one another, including filtration, distillation, and crystallization, some of which you may already be familiar with. Today’s lab focuses on another tool: chromatography. The word itself literally translates to “color writing”, reflecting the origins of the techniques which involved the separation of plant pigments (this lab is, sadly, devoid of pretty colors). There are many different forms of chromatography, but they are all intended to separate molecules from each other on the basis of their physical properties, usually their polarity, that is, the extent to which local positive and negative charges exist on the molecular surface. Polar molecules have relatively large charges and nonpolar molecules are more electrically neutral over their surfaces.

How does chromatography work? Let’s imagine the following scenario: you have two compounds, A and B. Furthermore, let’s say that A is more polar than B. If you place a small amount of the mixture on a surface that has a lot of static electrical charges on it, which compound will “stick” to the surface more strongly: A (the more polar one) or B (the nonpolar one)? If you said “A”, congratulations!, you have the necessary insight to see the underlying principle of most forms of chromatography. The electrostatic attraction between the charges on molecule A and the charged surface will make it adhere more strongly to the surface. In contrast, B, lacking any charges, will not be as attracted to the surface. 

The fact that one compound has a stronger affinity for the surface you placed it on means that the other compound can be more easily removed. How? If you “wash” the surface with a liquid, the compound with the lower affinity for the surface will be preferentially pulled away from the initial position. In practice, the surface is washed by allowing a solvent to move vertically upward by capillary action as illustrated below. In the far left image below, 3 spots of different sizes of the mixture are placed toward the bottom of the support (called a “plate”) along the line drawn. The plate is then placed in a beaker containing a small amount of liquid solvent (colored light blue in the second image). Immediately after, a lid is placed over the top of the container to prevent the solvent that is moving upwards from evaporating (the 3rd through 5th images). Finally, the plate is removed from the container, the maximum extent of the solvent migration up the plate, called the “solvent front” is immediately marked in pencil, and then the solvent is allowed to evaporate from the surface (6th image).

The images above show how the A/B mixture are separated as the solvent carries one of them upward more effectively (B) than the other (A). The movement is quantified simply by measuring the distance each spot moves (labeled as “2” and “3” on the rightmost figure) from the original point of application (“1”) relative to the distance the solvent itself (“4”). The movement is often expressed using an R_f value, defined simply as the ratio of the distance traveled by a given component relative to the solvent; it can have any value between 0 and 1.

For example, if B moves 4.6 cm and the solvent moves 5.8 cm, the R_f value is:

\[ R_f = \frac{4.6 cm}{5.8 cm} = 0.79 \nonumber \]

Similarly, if A moves 1.9 cm, its R_f value would be 0.33.

The example above is idealized and much simpler than most separations. Real mixtures are usually messier in that there are more than two components, some of which can have similar R_f values. This results in overlapping spots that can make it difficult to tell if more than one compound is present. To address this problem, the solvent used can be altered. By making the solvent more or less polar, its ability to carry compounds will change. This is because the movement of each compound is the manifestation of a sort of tug-of-war between the solvent (trying to pull the compound off the surface) and the surface (trying to hang on to the molecules). As the solvent becomes more polar, it becomes more competitive with a charged surface and tends to move compounds more effectively (giving them higher R_f values). This can be helpful because it may result in better separation of compounds.

The example above describes a specific type of chromatography called thin-layer chromatography (TLC), but other types also exist, with gas chromatography (GC) being one of the most widely used instrumental methods in science today. Biologists use related techniques to examine proteins that are expressed in cells. All types of chromatography have the following in common:

• A mobile phase (also called an eluent): the material that carries the components of a mixture away from the origin; and

• A stationary phase (or support): the material that the sample is initially applied to.

To effect a particular separation, chemists will often adjust both the mobile phase and the stationary phase. In this lab, you will only adjust the composition of the mobile phase, determining what combination of two specific solvent provides the best conditions to separate four compounds that are often found in pain medication.
 

 

Analysis of Pain Medication

You will use TLC to determine the components of a commercially available pain medication. Most such products contain some or all of the following compounds:

• Aspirin

• Ibuprofen

• Acetaminophen

• Caffeine

To determine which (if any) of these compounds are in the unknown you need to characterize each of them and then compare those results to the product mixture. You must first determine what conditions are best-suited to separate these four compounds from each other. Specifically, you will determine the optimal mixture of two solvents:

• hexane, which is very non-polar, and 

• ethyl acetate, which is moderately polar. 

You will do so by performing three TLC different separations using different solvent mixtures. The polarity of the eluent depends on its composition so by using different solvent mixtures you will see how polarity affects the separation. While you will only do three separations of the test compounds, the entire class will do a total of eleven different solvent mixtures, ranging from pure hexane to pure ethyl acetate, allowing you to see a much more detailed picture of the effect of eluent polarity on the separation. The data from the class will be pooled and used to prepare a graph of each compound’s R_f values as a function of eluent composition, from least polar to most polar. Each pair of students will interpret the graph to determine the optimal solvent mixture with which to analyze the unknown.

 

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Procedure 

Part 1: Method Development

1. Prepare three TLC plates as demonstrated in lab; spot the plates with solutions of the four test compounds in a straight line, evenly spaced, roughly 1 cm from the bottom (see figure at right). You will get better results if the spots are small. Make two small pencil marks to indicate the position of the line where the compounds were spotted. In your notebook, make sure you record which spot corresponds with which compound.

2. After the spots dry (it will only take a few seconds), place one of the plates in a beaker containing a small amount of one of your assigned solvent blends (about 0.5 cm in depth); cover the beaker with a watch glass and develop the plate. 

3. Repeat the above with the other two other solvent mixtures that you were assigned.

4. When the solvent reaches to within about 2 cm of the top of the plate, remove the plate and mark the solvent front with a pencil. 

5. Allow the solvent to evaporate and then examine the plate under the ultra-violet (UV) lamp. Use a pencil to circle the spots while the plate is under the UV lamp. 

6. Calculate the R_f values for each compound and enter them in the spreadsheet as described in lab. You should enter a total of 12 values, four for each solvent mixture you used.

 

Part 2: Sample Analysis

1. Using the pooled class data, decide what solvent mixture is likely to give you the best results in separating a mixture of all four compounds.

2. Spot a TLC plate with the solution of pain medication. Make three spots, all of the same solution. This will allow you to see the reproducibility of the technique.

3. Develop your plate using the solvent mixture you identified in Step 1.

4. Examine the developed plate under the UV light and circle the spots that are present. Measure their R_f values and identify which of the four test compounds are present based on those values.

 

Discussion. 

In your notebook, write a brief explanation of the method you developed and the logic employed. Briefly explain the results of your analysis and indicate whether any compounds are present in the pain medication that were not among the four test compounds.