Fall Organic Chemistry Experiment #4

Fall Organic Chemistry Experiment #4
Thin-Layer Chromatography (TLC)

Reading

From "The Student's Lab Companion: Laboratory Techniques for Organic Chemistry”, by John W. Lehman
Operation #18 Thin-Layer Chromatography p.104

Introduction

Chromatography is an efficient method for the separation of compounds distributed between two phases. These two phases are the stationary phase and the mobile phase. Do not confuse these phases with the two phases in extraction. Separation occurs because the compounds have different affinities (attraction) for the stationary phase and the mobile phase. Let us consider the following analogy.

Imagine that you and an alien are standing on the top of a mountain. The alien is covered from head to toe in super glue. You do not have the super glue on you. Now the mountain you are standing on is on Mars and is also made of a very sticky Martian material. Both of you decide that you want to get down off of the mountain and dive into a shallow olive oil slick running over the sticky surface of the mountain. Who will get there faster? If we think about the chemistry, we might say that each of you has a different affinity for the sticky material (stationary phase) on the mountain. In this case, the alien who is covered in glue will "stick" better to the sticky Martian stationary phase and has the higher affinity compared to you. On the other hand, you (without glue) will have a lower affinity for the sticky material (and, by default, a higher affinity for the olive oil mobile phase). Therefore, you should be able to slide down the mountain much faster.

The basic gist here is that depending upon the individual physical characteristics, molecules may have a relatively high or low affinity for either phase. Certain molecules move along readily with the mobile phase (like you in the analogy above), while others are held up by their affinity for the stationary phase (like the alien). All types of chromatography (column, gas, liquid, etc.) involve this same sort of separation scheme.

Thin-Layer Chromatography

In thin layer chromatography (TLC), a plastic or glass plate is coated with silica on one side. The silica functions as the stationary phase. The mobile phase is the solvent that moves up the plate by capillary action. The basic process involves applying a small drop of solution at one end of the plate (origin), immersing the same end of the plate in a developing solvent, letting the solvent run just about to the top of the plate, and visualizing the resulting chromatogram.

In the thin layer chromatography experiment you are doing today, some molecules will adhere to the plate (higher affinity for the stationary phase) and will not move very far up the plate. Others will move along more readily with the solvent (higher affinity for the mobile phase) and may move as far up the plate as the solvent front. In general, polar compounds, such as alcohols, amines, and carboxylic acids, stick tightly to the stationary phase and do not move far from the origin. As a result, a very polar developing solvent (mobile phase) is required to move polar compounds further up the plate. Nonpolar compounds have modest affinity for the plate and will move quickly up the plate in nonpolar solvents.

Designing the polarity of the developing solvent is a critical step in any TLC experiment. The solvent polarity plays a direct role in the efficiency of the separation of components (spots). The general rule of thumb is for the mobile phase to be polar enough to move the components up the plate away from the origin and nonpolar enough to trail behind the solvent front. Keep in mind that the lower the affinity of a compound for the silica and the higher the solubility in the developing solvent, the closer the compound will move to the solvent front. The silica is a constant in terms of polarity, but the mobile phase is not. We can adjust the polarity of the mobile phase. We can create the ideal solvent system by using a single solvent or by using a solvent mixture. Experience is the best teacher in this regard. It is useful to try a number of different single solvent systems along with some mixtures to achieve the desired separation. You may also consider the CRC Handbook of Chromatography which lists compounds and their separation efficiency in various solvent systems. Table 1 lists some common laboratory solvents in order of increasing polarity.

Table 1

Once you have designed a reasonable developing system, it is time to run the chromatogram and interpret the results. The results of a TLC experiment are measured by a quantity called the Rf value. This value corresponds to the relative distance a particular compound has moved up the plate. The Rf value is the ratio of the distance a compound moves from the origin to the distance the solvent front moves (Figure 1).

Figure 1

The Rf value is specific to the solvent conditions of the chromatogram. Normally, these conditions are difficult to reproduce. Therefore, if you wish to identify a particular compound, and if an authentic sample is available, the unknown is chromatographed on the same plate with the authentic sample by co-spotting the unknown and authentic compound on top of one another.

Once the run has been completed the spots are visualized. There are a variety of visualization methods that can be used. The most common method is to use a plate impregnated with a fluorescent material that allows spots to be visualized under UV light. Compounds with conjugated bond systems (e.g. benzene) will show up nicely because they absorb UV light. Another method is to place the plate in a chamber containing iodine crystals. The spots will turn dark brown due to a weak electronic interaction of the iodine with the compound on the plate. A final method involves dipping the plate in a solution of phosphomolybdic acid. Molybdenum (+6) will oxidize most organic compounds to yield a permanently colored spot.

Thin layer chromatography is a simple, rapid method for analyzing mixtures of compounds. As a result, TLC is routinely used in the organic chemistry laboratory for a variety of functions. The two most common applications include using TLC to test the fractions collected from a (column) chromatography experiment and using TLC to monitor the course of a chemical reaction. We will see examples of these two applications throughout the year.

Procedure

Before you arrive in lab, you should generate a drawing the describes the alien scenario at the beginning of this experiment. We will share these during pre-lab. As usual, work in pairs. During today's lab period, you will perform two separate TLC experiments.

Experiment A (determing the identity of the common analgesic from experiment #3): Prepare and test the standard analgesics caffeine, salicylamide, acetaminophen, ibuprofen, and aspirin using Thin Layer Chromatography analysis (TLC) in order to VERIFY the identity of your common analgesic from Experiment #3.

To prepare caffeine, salicylamide, acetaminophen, ibuprofen, and aspirin for TLC analysis separately crush up one of each tablet or obtain a small amount of the solid material. Then, in a small vial add just enough ethanol to create a suspension. The tablet will not completely dissolve in the ethanol. Once completed, you can run your TLC experiment using a polar mobile phase. You will have to design the mobile phase using the solvents available in the lab (see list above). Don’t be afraid to use a mixed solvent system (e.g. two solvents). You might want to split this up amongst all teams in the lab to find the best possible mobile phase solvent system. The guidelines for performing a TLC run are shown below.

Guidelines:

Use a pencil when marking the TLC plate.

When handling the plate, take care not to touch it with your hands before you run the plate in the solvent. The coating on the TLC plate is of an absorbent nature and can absorb oils off of your fingers thereby contaminating the plate. Contamination is less likely when you use forceps.

Once you have obtained the pre-cut TLC plate make sure it fits into the developing chamber, meaning, it can be easily placed inside and taken out without resistance from touching the sides of the glass.

The plate is not to touch the paper liner in the chamber. Check the diagram on the blackboard for a correct positioning of the plate vs. the paper liner.

Check to make sure you do not have too much or too little solvent in the chamber before you place the plate inside. The amount of solvent in the chamber should never exceed the line on which your samples have been spotted. If this happens you will not receive a clean separation.

Watch the solvent run up the plate to make certain that the solvent front does not run off the plate, sometimes taking the spotted sample with it.

Once you have spotted and developed the test solutions, you can visualize the spots using a hand-held UV lamp. Circle the spots and calculate the Rf values. Be sure to prepare a sample of your unknown common analgesic in the same manner as the standards (caffeine, salicylamide, acetaminophen, ibuprofen, and aspirin). Add enough ethanol to make a suspension. Not all of the solid material will dissolve. Spot the TLC plate and develop using the same solvent system. Calculate the Rf values and identify your unknown common analgesic.

Experiment B (designing a mobile phase to monitor the progress of a reaction): You will need to develop a mobile phase that can be used to visualize the results from a chemical reaction that we are working on in the Knoerzer Research Group. During the past two summers, we performed a series of Mitsunobu reactions in order to couple two alcohols together in an SN2 fashion to generate an ether. This reaction is shown below.

In this reaction, we employ triphenylphosphine (PPh3) and the starting phenol which can both be visualized under UV light. At the end of the reaction, we should have the ether product and triphenylphosphine oxide as a side product. In order to monitor the progress of the reaction, we need to develop a TLC system that will allow us to see each spot with an appreciably different Rf value. Previous efforts have indicated that some combination of ethyl acetate and hexane should work. Your job is to determine the optimum ratio of solvents. You will have samples of PPh3, the starting phenol (you will not be able to find safety information on this compound), and an authentic reaction mixture containing numerous components (most notably, the expected ether product, OPPh3, and leftover starting phenol and PPh3). Each will be dissolved in a small amount of CH2Cl2 in a vial. Once developed, you should analyze in the same fashion as experiment A.