Pre-lab: read the chapters in Zubrick as assigned on the lab syllabus, and look up the structures, physical property information, etc. for the compounds you will be working with (this includes the solvents as well as the analgesics).

In this experiment we will use TLC to identify the active ingredients present in a variety of over-the-counter analgesics. Determining the identity of the components will allow us to identify the brand of analgesic. You will be given reference samples of each of the components, and samples of the analgesics as unknowns. You will work in pairs, and each pair will have three unknowns to identify. The ones you will be working with are:
 

Commercial Analgesic	Active Ingredients
Anacin	aspirin and caffeine
Aspirin Free Excedrin	acetaminophen and caffeine
Bayer’s Aspirin	aspirin
Extra Strength Excedrin	acetaminophen, aspirin, and caffeine
Tylenol	acetaminophen

 

Theory:

The background theory of chromatography in general and thin-layer chromatography in specific is adequately explained in the reading you have done in Zubrick. The underlying principles of all chromatographic methods are identical. Essentially a compound to be analyzed or purified is dissolved in a mobile phase (gas or liquid) and allowed to travel through a stationary phase (liquid or solid). The two phases have different polarities. In TLC the less polar mobile phase (the eluent or eluting solvent) is an organic solvent and the stationary phase is a more polar solid such as silica gel (SiO₂) or alumina. The sample will distribute itself between the mobile and the stationary phases based on its relative affinity for the two phases. If a mixture of compounds is chromatographed, the individual components of the mixture will travel at different rates due to their different affinities for the two phases.

In TLC the sample is initially dissolved in an easily evaporated solvent and applied to a plate coated with the stationary phase using an applicator. The plate is placed in a developing tank containing the eluent, which may be a single solvent or a mixture. This eluting solvent will slowly travel up the plate. Once the solvent has moved almost to the top, the plate is removed and the height of the solvent front marked. The next step is to find the position of the sample spot(s) on the plate.

Chromatography originally got its name from the Greek word "chromatokis" meaning "suited for color" because early work involved the separation of colored pigments. However, the vast majority of organic compounds are colorless and must be visualized to enable analysis. Zubrick discusses a few of the methods available. We will use UV light to visualize the plates. Once the spots have been found they are circled lightly in pencil.

Now it is possible to measure the distances that the solvent and the individual components traveled from the baseline. We need to be able to report the results in a manner which allows comparison from one student to another and from one day to another. Since not every person will have the same length plate or will allow the solvent to travel the same distance, direct side by side comparison of plates is meaningless. We will standardize our results by calculating the retention factor (Rf). This is a ratio of the distance that a component has traveled from the baseline divided by the distance that the mobile phase has traveled from the baseline. The Rf is characteristic of a compound under a certain set of conditions – the same compound run on the same type of plate using the same eluting solvent should have the same Rf. However, even when performing a TLC under apparently identical circumstances, slight errors are involved. Moisture in the air, varying thickness of silica gel, amount of sample loaded on the plate, and other factors can all lead to variations. Therefore the acceptable experimental error on an Rf value is + 0.05. The best way to compare samples is to run them all on the same plate at the same time.

Our stationary phase is silica gel which is very polar. In our system, compounds with smaller Rf values are more polar because they have greater affinity for the polar stationary phase, and therefore, hang closer to the baseline. Conversely, compounds with larger Rf values are less polar because they have greater affinity for the non-polar mobile phase, and travel with it further up the plate. The organic solvents used as eluents are less polar relative to the silica gel, but there is still a range of polarities possible. When presented with an unknown sample of an unknown number of components, a scientist must test the sample in different polarity solvent systems in order to maximize the chances of separating all the components. Often there isn't one solvent system that separates all the components. Instead the unknown sample is run in different solvent systems that cleanly separate some of the components. In one solvent components A and B may separate but C and D overlap. In a different solvent, A and B overlap, but C and D do not. An analysis of more than one chromatogram may be necessary in order to identify all the components. In this experiment we will compare the results obtained in two different solvent systems.
 
 

Experimental Procedure:

Much of what is described below will be demonstrated before you are asked to do it.

Prepare spotters –you will need one for each solution, and they can be reused for the same solution. Since we need to use Bunsen burners to do this, we will get this done first before we get any flammable solvents out.

We will use two different eluting solvents (ethyl acetate and 25:1:1 ethyl acetate/ethanol/acetic acid), so each pair will need to make up two tanks, one with each solvent. Keep track of which one is which. As a developing tank use a 400 ml beaker. Cut a piece of filter paper to use as a wick, put in about 20 ml of the eluting solvent, and cover the beaker with a watch glass.

The reference compounds will be provided as solutions. Put a few drops of each one in a clean small test tube and stopper it. The unknowns will be distributed as solids. Each pair of students will sign out three unknowns from the stock room (take three clean small test tubes with you when you go). The majority of the tablet is inactive ingredients such as starch, lactose, and other substances that act as binders and permit rapid solution. You will have to add 40 drops of a 1:1 methylene chloride/95%ethanol solution to dissolve the active analgesic ingredient(s), but precipitate the inactive ingredients. Stopper the tube, shake well, then let the solution settle. Keep the test tubes stoppered.

Each pair will get two pre-cut silica gel-coated plastic-backed tlc plates. To prevent contamination do not touch the silica gel surface (the white side) with your fingers. Also be careful not to chip off the coating. You will spot solutions of each reference sample and each unknown on each plate, then run one plate in each solvent system. You may wish to practice your spotting procedure (see below) on a scrap piece of plate.

About 1 cm from the bottom of the plate, gently draw a pencil line (make sure it’s above the solvent level in the tank). You may make ticks underneath for each spot that you will apply. Using a spotter, make a small (~1mm) spot on the line. The smaller the initial spot the less it will diffuse during elution, and the more accurate the Rf measurement will be. If the initial spot is too large it may end up so diffuse that separate spots will overlap. One spot should be enough. However, if you do need to apply more material to the plate, let the solvent evaporate after applying the first spot, then apply another spot to the same location. Repeat the process several times if necessary. To check to see if you have applied enough material, see if you can detect a spot when you look at the plate in the UV light box.

Make sure your spots are above the solvent level in the tank, then carefully put the plate in the tank. Stand the plate up as straight as possible, don’t let it touch filter paper, and don’t disturb the tank. When the solvent front is high enough (~1 cm from top) remove the plate from the tank and mark the solvent height.

To visualize your spots check the plate in the UV light box using the short-wavelength lamp. Circle any spots and turn off the lamp when you are done. Make a sketch of each tlc plate in your notebook, and be sure you record which solvent system was used to run which plate. Calculate the Rf of each spot and record the Rf’s in your notebook. Determine the identity of each component in each unknown, and the identity (brand name) of each unknown.

Dispose of left-over solvents and solutions in the appropriate waste containers.

The report for this experiment will be a results section due in lab the week of Oct. 29.