A Practical Investigation of Thin-Layer Chromatography

Lab 2: Thin-Layer Chromatography

Abstract:

In the TLC lab, TLC plates are used to experimentally approximate molecular weight and compare weights through Rf values. Different molecular components of each solution tested have a general distance that they will travel on the plate that depends on how heavy they are. Heavier molecules will not diffuse as far through the TLC plate as lighter molecules.

Skills Acquired:

In this lab session, I learned how to properly prepare over the counter analgesics for mixing in an acetone solution.

Procedure:

First, we prepared two TLC chambers by measuring out approximately 5 mL of the eluent solution and putting it in the chamber with some folded filter paper. Then we marked two TLC plates 1 cm above the bottom forming the baseline. Micropipets were then used to apply three different solutions to one TLC plate and two to another by dipping the micropipet in the solution tube and then by pressing it against the TLC plate for about 3 seconds. After all solutions were applied to the TLC plates, we checked both plates under short wave UV radiation to make sure that we had an appreciable amount. This instance, and all other instances involving handling of the TLC plates was done in such a way that avoided physical contact with the gel coated side. Once we checked each plate and confirmed the presence of each solution we were testing, we put them in the TLC chambers, making sure that the filter paper did not touch any side of the TLC plate.

While waiting for the basic analgesic TLC plates to develop, we prepared the over the counter drug solutions. This was done by taking a pinch of each pre ground drug with metal tongs and adding it to a test tube and then adding acetone to dissolve the powdered drugs. The test tubes with the over the counter drugs were held firmly and tapped to increase the rate of dissolution.

After waiting approximately five minutes for the TLC plates that were in the TLC chambers to develop, we checked the progression of the solvent up each plate. If the solvent was around 1 cm from the top of the plate, we took it out for examination. Finished plates were analyzed with short wave UV radiation and spots were marked with a pencil on the surface of the plate. The process in the first and third paragraphs were repeated with the plates prepared and mentioned in paragraph two with the only difference being that each plate had three tested solutions and one extra plate had to be prepared and tested due to an error where the Alleve solution did not appear after development in the TLC chamber.

After each TLC plate was developed and marked, measurements were taken to calculate the Rf value of each spot in the solutions, and each plate was sketched. To calculate the Rf value, the total distance covered by the solvent was recorded (cm) from the drawn baseline and used as the denominator for each plate. Each separate spot was then given an approximate middle point in which we measured to from the baseline (cm). With the spot distance as the numerator and the solvent distance as the denominator, we calculated the Rf values and were then able to compare between each tested solution.

Data:

Analgesics Rf Values

Aspirin Naproxen Acetaminophen Ibuprofen Caffeine

0.88 0.73 0.35 0.64 0.10

Table 1

Over the Counter Drugs Rf Values

Excedrin Anacin Bayer Tylenol Advil Aleve

0.63 0.63 0.68 0.98 0.76 0.68

0.36 0.11 0.11 0.39 0.37 0.49

0.11

Table 2

Conclusion:

When the Rf values of the analgesics and the over the counter drugs are compared, the component analgesics of each drug can be semi accurately judged as shown in Table 3

Excedrin Anacin Bayer Tylenol Advil Aleve

Ibuprofen Ibuprofen Naproxen or Ibuprofen Aspirin Naproxen Naproxen or Ibuprofen

Acetaminophen Caffeine Caffeine Acetaminophen Acetaminophen Ibuprofen or Acetaminophen

Caffeine

Table 3

As the data suggests, TLC can only provide an approximation due to its highly qualitative nature. A Google search shows that some of the data led conclusions are either wrong or ambiguous.

Error Analysis:

During the experiment we encountered two errors:

We had to discard our first TLC plate due to aspirin not being visible under the short wave UV radiation.

We created a new plate for the first three analgesics after observing this and this did not have any effect on the data.

Aleve had to be retested on its own TLC plate due to a perceived absence of it on its original plate.

Aleve was very problematic to test since it did not show up on the first TLC plate we tested it on. On the plate where we individually tested it, it showed very vague results which resulted in very vague data for it. In the future we could test multiple applications of any solutions that do not show up the first time.