Question

THIN LAYER CHROMATOGRAPHY OF ANALGESICS

Introduction:

Analgesics are compounds that relieve pain. They range from over the counter drugs such as aspirin, which is consumed at a rate of 40 million pounds per year, to prescription drugs like morphine and other related narcotics. In addition to aspirin, several other chemically similar compounds are widely used in nonprescription analgesic tablets. The most common non-aspirin pain relievers are acetaminophen and ibuprofen. Caffeine, a stimulant, is sometimes added to analgesic formulation to overcome drowsiness. In addition to the active ingredients, the tablets of these drugs also contain starch, lactose, and other substances that act as binders and permit rapid solution.

Over-the-counter analgesics typically contain one or more of the following active ingredients: acetaminophen, aspirin, caffeine, ibuprofen or naproxen. Thin layer chromatography (TLC) offers a simple method of analysis for these products.

Chromatography is a term that is widely used to describe a family of closely related separation methods. There are many separation methods, but the feature that distinguishes chromatography from other physical and chemical methods of separation is both a stationary and mobile phase; the sample to be separated interacts numerous times with these phases. The sample is carried through the system via the mobile phase, and the interactions that occur are due to the differences in the physical and chemical properties. These differing affinities govern the rate at which the individual components of the sample pass over the stationary phase under the influence of the mobile phase.

Thin layer chromatography (TLC) is one type of chromatography where the stationary phase is a thin layer of adsorbent particles attached to the solid plate. A small amount of sample is applied (spotted) near the bottom of the plate, and the plate is placed in the mobile phase. This solvent is drawn up by capillary action to a predetermined height. Each component, being different in chemical and physical composition and polarity, will interact with the stationary phase at a different time (retention time), thereby creating the individual bands on the plate. The retention time or retention factor (Rf) is used to characterize and compare components of various samples.

Rf = distance from origin to center of spot distance from origin to solvent front

Analysis of an analgesic by TLC.

You will perform TLC experiments using commercial TLC plates prepared by depositing a thin layer of silica gel onto a plastic sheet just like to ones you used last week. These are convenient for the student lab, but are less efficient than the glass-backed plates normally used in research laboratories. Great care should be taken to label the origin and the solvent front, as well as the source of each of the spots for future reference. Mark very lightly with a pencil on the coated side of the plates, being careful not to flake off the silica gel. Do not touch the plates on the active surface and make sure that the solvent front travels evenly up the plate, so as to result in a straight vertical elution of the samples. The application of very small amounts of the sample as distinct, tight spots on a horizontal origin line will provide the best separation and comparison data. Be especially careful not to contaminate your samples, since the detection method to be used can be very sensitive. Use forceps to carefully insert the plate into the developing jar without splashing solvent and to remove it after development. The plate should be squarely vertical during development to promote an even flow of the solvent up the plate. Note the appearance and position of the spots after developmentof the plate. During visualization under UV light, the spots should be lightly outlined with a pencil on the

C343 Spring 2020: TLC plate of analgesics

plate for later reference. Determine the Rf values of the compounds and record these in your lab notebook. Your AI will demonstrate these TLC techniques.

Prepare a table similar to the solvents table, but with the active ingredients of the following analgesics: Advil, Aleve, Anacin, Aspirin, Excedrin, Tylenol. Reference compounds: Naproxen, Ibuprofen, Acetaminophen, Acetylsalicylic acid, caffeine.

Analgesic (brand name)	Acetyl salicylic acid	Acetaminophen	Ibuprofen	Caffeine
Aspirin (Bayer)	325 mg
Anacin	500 mg			32 mg
Tylenol		325 mg
Advil (Motrin)			200 mg
Excedrin	250 mg	250 mg		65 mg

Table1. Commercial over-the-counter remedies and their ingredients

You will be given a sample of a commercial headache remedy (half a tablet of an unkown); your task is to identify the active components of this product. Among the common ingredients in over-the-counter preparations are aspirin, acetaminophen, and caffeine. Note which sample you are assigned in your notebook. Prepare a methylene chloride: ethanol (1:1) solution of the soluble components of your sample. The commercial samples contain various binders and other inert materials useful in the formulation of capsules or tablets. These are mostly insoluble in methylene chloride and can be allowed to settle to the bottom of the methylene chloride extract or, if necessary, they can be filtered from the extract.

Use pure known samples provided in the laboratory, as well as the caffeine sample you extracted from tea/coffee (if still around) as authentic TLC references. Examine the methylene chloride extract of the commercial product by TLC to determine which of these ingredients are clearly present in this sample, and which are not present. The use of a solvent mixture of 19:1 ethyl acetate/acetic acid is recommended as an eluting solvent for this TLC determination, 4:1 Ethyl acetate:hexane will also be used for visualizing the less polar compounds. Draw pictures of your TLC plates in your notebook and make TLC plates for your summary using the ChemDraw TLC tool, do not take pictures of your TLC, phones are prohibited in the lab.

In addition to the active ingredients, a number of inert materials are also listed on the label of these products. These might show up as unidentified spots on your TLC plates. Additional spots may also be visible which are derived from decomposition of the active ingredients as they stand in solution.

C343 Spring 2020: TLC plate of analgesics

Procedure:

1. Making your TLC unknown solution. Your AI will assign you an unknown (half a tablet). Make a solution for TLC analysis by taking a small spatula full of your crude mixture in 1:1 ethanol/dichloromethane and shake to help dissolve your components (4-6 mL). Individually create two TLC plates as shown in the picture. You should prepare TLC plates that look like these→

2. Prepping your plate. Draw a pencil line lightly on a
   TLC plate so as not to break through the silica gel
   on the plate. If you scrape the silica gel off the
   plate, it may lead to poor or no elution. Be
   organized and label your spots appropriately. Try
   writing single letter codes under the locations that
   you place your spots and record your labels in your
   notebook so that you do not forget how your results correlate. Remember you will want to write up your observations in the summary. You will analyze the Rfs of the known compounds and then find out what is in your unknown sample.

3. Spotting your plate. When you are ready to spot, it is best for samples to be applied as small (1-2 mm diameter) spots, spot twice waiting for solvent to evaporate between spotting. During development of the plate, spots tend to diffuse (i.e., broaden), so that the final spot is considerably larger than the original spot. The larger the final spot, the more difficult it is to resolveamixtureintocompletelyseparatespots. PlaceonlyasmallamountontheTLCplate, as too much will lead to tailing on the plate and compounds will bleed into each other (i.e., spots will overlap and this is undesirable). If you do have large tailing spots, you can add less to the plate or dilute your initial solution with more solvent. Regardless, calculate an Rf from the middle of the spot. Rinse your spotter between samples!

4. Running the TLC. Once your TLC plates are ready, run The TLC labelled 19:1 in 19:1 ethyl acetate/acetic acid and the 1:2 in Ethyl acetate: Hexanes. Record any observations or problems that arise while performing the experiment.

5. Eluting the plates. Be sure the elution chamber solvent level is below the spot level on the TLC plates. Too high a solvent level will wash the spots off the plate, preventing any meaningful analysis. The plate is placed into the chamber and the solvent is allowed to rise up to within 0.5 cm of the top of the silica gel coating, about 70% the height. Make sure you have a wicking filter paper in the chamber to saturate the atmosphere with solvent during elution.

6. Visualizing your plates. You will visualize your TLC under UV light. Most spots are clear under U.V. TLC plates are commonly stained after visualization under UV light, you will stain one of your TLC plates with PMA TLC stain, DO NOT STAIN YOUR TLC plates until you have visualize

C343 Spring 2020: TLC plate of analgesics

your Rfs.. Use a pencil to indicate the location of spots under UV light, most conjugated compounds appear as violet or purple spots under UV light, if the coating on the plates contains a UV absorber. Refer to the video about PMA staining posted in Canvas. The procedure is not complicated: wet the TLC in the stain, pat it dry moving it to one side, put it upwards on the heating plate until you see the color of the stain.

7. Recording your results. Sketch the TLC plate(s) you have developed in your lab notebook so that you can refer to them again in your summary below. Calculate the Rf values and enter them into your table remaining organized during lab. Do not do this after lab, as you will more likely make mistakes.

Questions:

1a) make a physical properties table (2)

1b ) (1) Explain how and why increasing polarity of the different eluting solvents would affect the

elution of the compounds

1. c) If a less polar TLC development solvent was used, how would that affect the Rf values of

   the reference compounds?

   a. (1)Why was it necessary to run a TLC experiment in a closed container and to have the

   1. interior vapor saturated with the solvent by using a wick?

   2. (2)What will be the result of applying too much compound to a TLC plate? Not enough?

   3. (2)What will be the appearance of a TLC plate if a solvent of too low of a polarity is used

      for its development? Too high of a polarity?

Answer 1

1(b) If the solvent is too polar or too non polar then in both the cases the spots of the compound will not move ftom their initial positions i.e. Rf = 0 i.e. the separation will be less. So an ideal solvent will be that which is neither too polar nor too non polar.

1(c) If a solvent is less polar then the componds having less polarity will move farther/faster as compared to the other compound which is having higher polarity. Also the eluting power of the solvent will be less.

a (1) The solvents used in TLC are quite volatile and evaporation of the solvent can change the Rf value . The interior vapour is saturated with the solvent by using a wick because if the stationary phase dry out prematurely then the components won't separate out properly and we will get incorrect results.

(2) If you use too much compound then the ink can with the eluting solvent and separation of uts components can give us lot of extra spots. The stop would show trailing.

(3) To explain the effect of polarity of solvent we will try to explain the answer of 1(b) .

In chromatography , stationary phases are usually polar wheras mobile phases are less polar or widely polar. So basily a non polar compound will travel farther. But the eluting power of solvents depend upon polarity.

When the solvent is highly polar , then all the components will move with solvent and thus the separation will be less. Similarly, if the solvent is highly non polar then all the compounds won't move much farther and again separation will ne less.