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"; Thin Layer Chromatography (TLC) Lab Report - Chem Homework Help
Thin layer Chromatography (TLC) Lab Report





Solvent mix that gave best separation: 1:1 Ethyl acetate: Hexane

Solvent travelled =4.10 cm

Calculated Rr


0.52 cm

Acetylsalicylic acid



0.15 cm


3.65 cm


0.49 cm


Measured melting

temperature range




Acetylsalicylic acid






Measured melting

temperature range


Unknown analgesic



1. What is the purpose of the filter paper in the developing chamber?

2. Why is a pencil, and a not a pen used to mark the TLC plate?

3. What would happen if the sample applied is too concentrated? What if it is too dilute?

4. What precautions do you need to take when developing the plate?

5. What would happen if the solvent level is about the level of the spots?

6. How do you properly record information obtained from a TLC plate?

7. Are Rf values unique to a compound?

8. What happens to the Rf values as you increase the polarity of the developing solvent mixture?

9. How do you visualize the spots on the UV plate?

10. Show the calculations for Rf and identify the unknown analgesic.

Identifying an Unknown Analgesic
by Melting Temperature and
Thin-Layer Chromatography

Thin-layer chromatography (TLC) is an important technique in organic chemistry. TLC uses the different affinities a compound has for the mobile and stationary phases to achieve separation of mixtures of organic compounds. TLC can also be used to identify compounds by comparison with known samples, check the relative purity of a compound, and monitor the progress of a reaction.

In thin-layer chromatography, the stationary phase is the adsorbent (usually silica or alumina) coated on a sheet of glass, metal, or plastic. The sample is applied as a spot near the bottom of the plate. The TLC plate is then placed in a developing chamber containing a shallow layer of solvent where the mobile phase (solvent) slowly rises by capillary action.

Under a given set of conditions, a specific compound will travel a fixed unique distance relative to the solvent front. Different compounds generally move at different rates. As a result, if the sample is a mixture of compounds it will separate into a series of spots at varying distances up the plate (see Figure 1). If the sample is pure, then only one spot will result. A UV light source is used to visualize the spots on the TLC plate. Under UV light, the chemical deposits will appear as dark spots against a bright background.

TLC separation results are expressed in terms of Rf (retention factor) values. The Rf is a ratio calculated by dividing the distance traveled by the sample by the distance traveled by the solvent at the end of the experiment.

Figure 1 Example TLC plate at the end of the experiment

In this experiment, TLC and melting temperature will be used to identify the active ingredients in an unknown over-the-counter medicine tablet. The tablet you will test contains one or more of the following: acetylsalicylic acid (aspirin), acetaminophen (the active ingredient in Tylenol®), caffeine (ingredient in Excedrin®), and/or ibuprofen (the active ingredient in Advil®). The structures of these compounds are shown here.


Acetylsalicylic acid Acetaminophen Caffeine Ibuprofen

Figure 2 Structures of analgesics


In this experiment, you will

  • Calculated the Rf values of acetylsalicylic acid, acetaminophen, caffeine, and ibuprofen.
  • Determine the melting temperature of each of these analgesics.
  • Identify the solvent system for good separation.
  • Use TLC and melting temperature to identify your unknown analgesic.


Part I Thin-Layer Chromatography

acetaminophen in ethyl acetate

10 mL graduated cylinder

acetylsalicylic acid in ethyl acetate

UV lamp (shortwave)

caffeine in ethyl acetate

ethyl acetate

ibuprofen in ethyl acetate


three TLC plates (5 10 cm)

spotting capillary tubes

three 400 mL beakers

five 10 75 mm test tubes

three 9 cm watch glasses

test tube rack

cotton plug

unknown sample

disposable Pasteur pipets and bulb

mortar and pestle


filter paper



Parts II and III Melting Temperature

LabQuest or computer interface


LabQuest App or Logger Pro

acetylsalicylic acid

Vernier Melt Station


glass capillary tubes, one end closed


tissues (preferably lint-free)

unknown sample

mortar and pestle



Part I Thin-Layer Chromatography

1. Obtain and wear goggles. Protect your arms and hands by wearing a long-sleeve lab coat and gloves. Conduct this reaction in a fume hood

2. Obtain your unknown analgesic sample and dissolve approximately 50 mg in 2 mL of ethyl acetate. Filter the solution through a pipet containing a cotton plug into a test tube.

3. Transfer approximately 12 mL of each pure compound to test tubes. Be sure to label each test tube. Solutions of the pure compounds will be available in an ethyl acetate solution.

4. Prepare three development chambers using the 400 mL beakers and watch glasses.

Label the chambers 13. These will contain ethyl acetate, hexane, and 1:1 ethyl acetate/hexane, respectively.

Place a piece of filter paper against the side of the beaker. The filter paper will help saturate the beaker with solvent vapors.

Fill each beaker with 510 mL of the developing solvent and cover with the watch glass.

5. Prepare the TLC plates.

  1. Obtain three TLC plates and number the plates 13. Handle them carefully and by the edges so that the adsorbent does not flake off.

Using a pencil (NOT an ink pen), lightly draw a line across the plate, approximately 1 cm from the bottom. Across this line, evenly mark five places indicating the location where the sample will be spotted, making sure they are not too close to the edge of the plate (see Figure 1). Do this for all three TLC plates.

Under each mark, lightly label each spot starting left to right as Ac, As, C, I, and U. Repeat this step on the other TLC plates.

Dip one end of a spotting tube into the ethyl acetate solution containing acetaminophen. Capillary action will draw the liquid into the tube.

Lightly tap the tube on the mark for acetaminophen on all three TLC plates. Only a small amount of sample needs to be delivered. The spot should be 12 mm in diameter.

Repeat Steps de for the aspirin, caffeine, ibuprofen, and unknown sample on all three TLC plates.

6. Place each of the TLC plate in the chamber and cover with the watch glass. The solvent level must not be above the spots on the plate or your samples will dissolve into the solvent.

7. When the solvent has risen to within 1 cm from the top of the plate, remove the plate from the chamber and with a pencil, gently draw a line to mark the position of the final solvent front.

8. After the plate has dried, observe the TLC plate under a UV lamp. Lightly outline the spots with the pencil. CAUTION: Do not allow skin or eyes to come in contact with UV light. Wear gloves, a lab coat, and UV resistant eye protection.

9. Identify the solvent mixture that gave the best separation and use that TLC plate to calculate the Rf value for each spot. Record the values in your data table.

Part II Test the Melting Temperature of Analgesic Standards

10. Obtain a small amount of acetaminophen. The solid should be in a powdered form. If it is not, use a mortar and pestle to carefully grind the solid to a powder. Pack a capillary tube 34 mm (~1/8 inch) deep with your sample.

11. Check the control dial on the Melt Station to confirm that it is in the Off position. Connect the Melt Station power supply to a powered electrical outlet.

12. Connect the Melt Station to LabQuest or a computer interface. Choose New from the File menu of the data-collection program.

13. Carefully insert the capillary tube of solid into one of the sample holders of the Melt Station.

14. Begin collecting melting temperature data using the Melt Station.

15. Adjust the control dial in order to determine the approximate melting temperature range for the sample. When the temperature is within approximately 10ºC of the lowest possible melting temperature of your sample, turn the control knob to a temperature setting corresponding to the expected melting temperature provided in the table below.


Melting temperature range



Acetylsalicylic acid (aspirin)






16. When finished, stop data collection and turn the dial to the Fan/Cooling setting. Record the melting temperature range in your data table.

17. Store the run and collect a second run, if desired.

18. Prepare a new sample and repeat Steps 1417 for samples of aspirin, caffeine, and ibuprofen.

Part III Test the Melting Temperature of Unknown Sample

19. Obtain a small amount of your unknown analgesic compound. The solid should be in a powdered form. If it is not, use a mortar and pestle to carefully grind the solid to a powder. Pack a capillary tube 34 mm (~1/8 inch) deep with your sample.

20. In the first trial, you will want to observe the melting process and make a rough estimate of the melting temperature of your unknown sample.

21. When you have determined the approximate melting temperature range for the sample, stop data collection and turn the dial to the Fan/Cooling setting. Record the melting temperature range in your data table.

22. Now that you have a rough idea of the melting temperature, a more accurate determination of the melting temperature can be made. Prepare a sample in a capillary tube and determine the melting temperature of the sample.

23. When finished, stop data collection and turn the dial to the Fan/Cooling setting. Record the melting temperature range in your data table.

24. At the end of the experiment turn the control dial on the Melt Station to Off. Dispose of the capillary tubes as directed by your instructor.

25. Complete the Data Analysis section before exiting Logger Pro or LabQuest App. Print a copy of your graph and/or save your data, as directed by your instructor.


Part I Thin-Layer Chromatography

Solvent mixture that gave
the best separation


Calculated Rf



Acetylsalicylic acid








Part II Melting Temperature of Analgesic Standards


Measured melting temperature range



Acetylsalicylic acid






Part III Melting Temperature of Unknown Sample


Measured melting temperature range

Unknown analgesic



1. Draw your TLC plate. Label each analgesic and show the calculation of the Rf value.

2. Based on your melting temperature data and the Rf value of the unknown, identify your unknown analgesic.

3. What would happen if the solvent level is above the level of the spots?

4. How do you properly record information obtained from a TLC?

5. Are Rf values unique to a compound?

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