THIN LAYER CHROMATOGRAPHY OF ANALGESICS

Introduction:

Analgesics are compounds that relieve pain. They range from overthe counter drugs such as aspirin, which is consumed at a rate of40 million pounds per year, to prescription drugs like morphine andother related narcotics. In addition to aspirin, several otherchemically similar compounds are widely used in nonprescriptionanalgesic tablets. The most common non-aspirin pain relievers areacetaminophen and ibuprofen. Caffeine, a stimulant, is sometimesadded to analgesic formulation to overcome drowsiness. In additionto the active ingredients, the tablets of these drugs also containstarch, lactose, and other substances that act as binders andpermit rapid solution.

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

Chromatography is a term that is widely used to describe afamily of closely related separation methods. There are manyseparation methods, but the feature that distinguisheschromatography from other physical and chemical methods ofseparation is both a stationary and mobile phase; the sample to beseparated interacts numerous times with these phases. The sample iscarried through the system via the mobile phase, and theinteractions that occur are due to the differences in the physicaland chemical properties. These differing affinities govern the rateat which the individual components of the sample pass over thestationary phase under the influence of the mobile phase.

Thin layer chromatography (TLC) is one type of chromatographywhere the stationary phase is a thin layer of adsorbent particlesattached to the solid plate. A small amount of sample is applied(spotted) near the bottom of the plate, and the plate is placed inthe mobile phase. This solvent is drawn up by capillary action to apredetermined height. Each component, being different in chemicaland physical composition and polarity, will interact with thestationary phase at a different time (retention time), therebycreating the individual bands on the plate. The retention time orretention factor (Rf) is used to characterize and comparecomponents of various samples.

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

Analysis of an analgesic by TLC.

You will perform TLC experiments using commercial TLC platesprepared by depositing a thin layer of silica gel onto a plasticsheet just like to ones you used last week. These are convenientfor the student lab, but are less efficient than the glass-backedplates normally used in research laboratories. Great care should betaken to label the origin and the solvent front, as well as thesource of each of the spots for future reference. Mark very lightlywith a pencil on the coated side of the plates, being careful notto flake off the silica gel. Do not touch the plates on the activesurface and make sure that the solvent front travels evenly up theplate, so as to result in a straight vertical elution of thesamples. The application of very small amounts of the sample asdistinct, tight spots on a horizontal origin line will provide thebest separation and comparison data. Be especially careful not tocontaminate your samples, since the detection method to be used canbe very sensitive. Use forceps to carefully insert the plate intothe developing jar without splashing solvent and to remove it afterdevelopment. The plate should be squarely vertical duringdevelopment to promote an even flow of the solvent up the plate.Note the appearance and position of the spots after developmentofthe plate. During visualization under UV light, the spots should belightly outlined with a pencil on the

C343 Spring 2020: TLC plate of analgesics

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

Prepare a table similar to the solvents table, but with theactive 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 theiringredients

You will be given a sample of a commercial headache remedy (halfa tablet of an unkown); your task is to identify the activecomponents of this product. Among the common ingredients inover-the-counter preparations are aspirin, acetaminophen, andcaffeine. Note which sample you are assigned in your notebook.Prepare a methylene chloride: ethanol (1:1) solution of the solublecomponents of your sample. The commercial samples contain variousbinders and other inert materials useful in the formulation ofcapsules or tablets. These are mostly insoluble in methylenechloride and can be allowed to settle to the bottom of themethylene chloride extract or, if necessary, they can be filteredfrom the extract.

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

In addition to the active ingredients, a number of inertmaterials are also listed on the label of these products. Thesemight show up as unidentified spots on your TLC plates. Additionalspots may also be visible which are derived from decomposition ofthe 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 anunknown (half a tablet). Make a solution for TLC analysis by takinga small spatula full of your crude mixture in 1:1ethanol/dichloromethane and shake to help dissolve your components(4-6 mL). Individually create two TLC plates as shown in thepicture. You should prepare TLC plates that look like these→

1. 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 thesummary. You will analyze the Rfs of the known compounds and thenfind out what is in your unknown sample.

2. Spotting your plate. When you are ready to spot, it is best forsamples to be applied as small (1-2 mm diameter) spots, spot twicewaiting for solvent to evaporate between spotting. Duringdevelopment of the plate, spots tend to diffuse (i.e., broaden), sothat the final spot is considerably larger than the original spot.The larger the final spot, the more difficult it is toresolveamixtureintocompletelyseparatespots.PlaceonlyasmallamountontheTLCplate, as too much will lead totailing on the plate and compounds will bleed into each other(i.e., spots will overlap and this is undesirable). If you do havelarge tailing spots, you can add less to the plate or dilute yourinitial solution with more solvent. Regardless, calculate an Rffrom the middle of the spot. Rinse your spotter betweensamples!

3. Running the TLC. Once your TLC plates are ready, run The TLClabelled 19:1 in 19:1 ethyl acetate/acetic acid and the 1:2 inEthyl acetate: Hexanes. Record any observations or problems thatarise while performing the experiment.

4. Eluting the plates. Be sure the elution chamber solvent level isbelow the spot level on the TLC plates. Too high a solvent levelwill wash the spots off the plate, preventing any meaningfulanalysis. The plate is placed into the chamber and the solvent isallowed to rise up to within 0.5 cm of the top of the silica gelcoating, about 70% the height. Make sure you have a wicking filterpaper in the chamber to saturate the atmosphere with solvent duringelution.

5. Visualizing your plates. You will visualize your TLC under UVlight. Most spots are clear under U.V. TLC plates are commonlystained after visualization under UV light, you will stain one ofyour TLC plates with PMA TLC stain, DO NOT STAIN YOUR TLC platesuntil you have visualize

C343 Spring 2020: TLC plate of analgesics

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

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

Questions:

1a) make a physical properties table (2)

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

elution of the compounds

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

   the reference compounds?

   a. (1)Why was it necessary to run a TLC experiment in a closedcontainer 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 aTLC plate? Not enough?

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

      for its development? Too high of a polarity?