Esther Juárez

Chromatography of Analysis of Analgesic Drugs


In this experiment, a thin-layer chromatography (TLC) was used to determine the composition of various over-the-counter analgesics and compounds. (Acetaminophen, Acetylsalicylic Acid, Acetylsalicylic Acid and Caffeine). Chromatography is a technique that we used to separate and identify individual components in a mixture. These chromatography techniques focused on the fact that components of a mixture tend to move at different speeds along the flat surface from the paper used to differentiate them from each other. The different rates of movement were the results of differing attractions of the components to the coating material compared to the tendency of the components to remain in the moving fluid. The results obtained were compared using a Co-Spot method to ensure their identity.


Thin-layer chromatography is one of the easiest techniques available to the many chromatography techniques to identify compounds. A thin layer of a suitable solid substance is coated on a sheet of glass or plastic. A very small sample of the mixture to be analyzed is “spotted” onto the sheet. By immersing one edge of the sheet in an appropriate liquid developing solvent, the solvent is drawn up the sheet by capillary action, and the compounds of interest are carried along at different traveling rates, effectively separating the components. This is commonly called “developing” the plate. After the plate has been developed, it is examined under ultraviolet (UV) light, which allows you to note the location of the spots. Experimental conditions in TLC include the solvent system used to dissolve the compounds, the absorbent coated onto the TLC plate, the thickness of the absorbent layer, and the relative amount of the material spotted onto the plate. Under an established set of experimental conditions, a given compound always travels a fixed distance relative to the distance the solvent front travels. This ratio of the distance the compound travels to the distance the solvent front travels is called the Rf value. The symbol Rf stands for “retardation factor,” or “ratio-to-front,” and it is expressed as a decimal fraction. The equation is shown below:

In this experiment, each pair was given two commercially prepared TLC plates with a flexible backing and a silica gel coating with a fluorescent indicator. On these TLC plates, spot solutions were prepared from commercial analgesic tablets. Including an unknown for identification. The crushed tablets were dissolved in an ethyl acetate-ethanol-acetic acid solvent mixture. On each plate, a standard reference mixture was placed, which contains the four standard compounds (Mix- located on the hood for safety reasons) often used in analgesic formulations dissolved in the same solvent mixture.

The standard 4 compounds used in these plates are:

  • Acetaminophen (Ace)
  • Acetylsalicylic Acid (Asp)
  • Acetylsalicylic Acids (Ace)
  • Caffeine (Caf)
  • Mix (All combined to look for matching spots)
  • Unknown (U) – Compound to analyze in this experiment

Preparing the TLC Plates

TLC plates were obtained. These plates identify with a shiny flexible backing, which is not supposed to be bent as it could damage the plates for analysis. The plates were handled carefully, and only by the edges, or the absorbents would have flaked off. Using a pencil (not a pen), on the non-shiny side, the group lightly drew a line across the plate about 1.0 cm from the bottom (as shown in the introduction). When marking the plate, we made sure not to disrupt the absorbent with the pencil. In order to spot the respective substances, we marked 6 spots on the line for the respective substances. Using a centimeter ruler, we marked off 6 points about 0.75cm apart on the line. These were the points at which the samples were spotted. Another plate was made to compare the ensure results obtained called the ‘Co-Spot Method’. We repeated procedure from above to make the second reference plate. Using a centimeter ruler, we lightly marked off three points 1.5 cm apart on the line. These were the points at which the other samples were spotted.

Preparation of Analgesics

We obtain half a tablet of four different analgesics. The group took each analgesic and and crushed it in order to dissolve it. Transferred each crushed half-tablet to a DRY, labeled 50.0 mL beaker. Using a graduated cylinder, add 5.0 mL of the Solution Solvent, which was an ethyl acetate-ethanol-acetic acid mixture, to each of the crushed half-tablets, and swirl to mix. Then heat each of them gently for a few minutes on a hot plate set at low temperature. Since analgesics usually contain an insoluble binder, they did not dissolve in its entirety. Some of them also contained inorganic buffering agents or coatings that are insoluble in this solvent mixture. After heating the samples, we swirl them again to mix and then allowed the insoluble particles to settle.

Spotting the TLC Plates

The group obtained from the chemistry hood four capillary micropipettes to spot the plates with the four solutions of analgesics prepared above. The chemistry professor demonstrated proper set up for the plates to be analyzed. This procedure was repeated for every compound (total of six spots). The standard reference mixture contains acetaminophen, aspirin, caffeine and acetylsalicylic acid.

Developing the TLC Plates

After spotting the plates, the group obtained a jar to be used as a development chamber. Using a graduated cylinder, we measure about 10.0-15.0 mL of Development Solvent (0.5% glacial acetic acid in ethyl acetate) and poured it into the jar. Making sure it didn’t go above the reference line or the samples would have been dissolved off the plate into the solution instead of developing. When the solvent front had risen to a level about 0.5 cm off from the top of the plates, we removed the plate from the jar and, using a pencil, marked the position of each solvent front. Set the plate on a piece of paper towel in your bench-top hood to dry. We saved the developing solvent into the jar for our ‘Co-Spot Method’. The overall purpose of the development of a second plate is to have a plate with all Rf values for comparison along with the unknown analgesic to be analyzed.

Observing the TLC Sample & Reference Plates: UV visualization

We then, observed the reference plate under the UV lamp. We also, sketched the spots on the plate using the ruler; we measured, in centimeters, the distance traveled by each spot on both sample plates as well as the distance traveled by the solvent front. (to get our own Rf values). See Lab Notebook. Also, these values were used to compare the identity of the unknown compound.

Iodine Analysis

This was performed after the UV visualization was performed. We placed the second plate called the “Co-Spot Method” in the jar located on the hood (pre-heated). The yellow spots appeared, which were used to ensure the result already established.  Below a picture shows these results.Results and Conclusions

Results and Conclusions 

Below, pictures from the actual results obtained performing this experiment.

P.1 This reference plate shows the results obtained after looking at the plate under UV light. The spots localized were sketched and our first matching spot to the unknown compound showed similarities to acetyl salicylic acid underlined in this picture. The Ref line showed that every compound was included in the reference mixture.

P.2 This reference plate shows the yellow/brownish spots that appeared after the plates were placed into the jar containing the iodine crystals and after the UV visualization was performed. The one on the left is the ‘Co-spot Method’, which contained:

  • Unknown: Acetylsalicylic Acid
  • Known: Ibuprofen

Note: The one on the right side is the first reference plate.

Contained: the 6 mentioned Ref compounds.

Results: According to data obtained the unknown was Acetylsalicylic acid.

Explanation: If the unknown compound would have been ibuprofen then the first unknown would have matched up, but it didn’t. Ibuprofen and acetylsalicylic acid were used in the first plate to compare similar results.


With this lab experiment, a thin-layer chromatography (TLC) was used to determine the composition of various over-the-counter analgesics, and how they will be identified using chromatography techniques (Acetaminophen, Acetylsalicylic Acid, Caffeine, and Acetylsalicylic Acid). These chromatography techniques focused on of the fact that components of a mixture tend to move at different speeds along the flat surface from the paper used to differentiate them from each other. We identified one of the compounds as Acetylsalicylic Acid.


Introduction to Organic Laboratory Techniques: A Microscale Approach, 4th Edition. “Introduction to Organic Laboratory Techniques.” Google Books. N.p., n.d. Web. 17 Sept. 2012.

“Search Avantor Materials.” Search Avantor Materials. N.p., n.d. Web. 17 Sept.

Springer, Joseph, Ph.D. “TLC Analysis of Analgesic Drugs.” CHM235 Glendale, Arizona. Sept. 17. 2012. Glendale Community College. Laboratory and Lecture Notes.


Glendale Community College, Arizona

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