Amino acids separation by thin layer chromatography

Separation of Amino Acids by Thin Layer Chromatography

This week we will learn to separate and identify the amino acids in a mixture by thin .


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Read the theory, procedure, watchon how the experiment is performed and how the unknown is identified, then answer the following questions and upload the document in assignments.


For background information please also refer to




1. Describe in detail Thin layer chromatographic experiment. Example: the theory behind it, how you would prepare the materials to spot on the plate with different mobile amino acids and unknown and how TLC Plate is developed and the reasoning behind which solvent/ solvent mixture should be used, along how to correctly identify of the unknown.(5pts)















2. Calculate the Rf value if a solute travelled 5 cm from the base spot and the solvent front is 10 cm from the origin?






3. In a TLC experiment using a 70:30 mixture of Petroleum ether and ethyl acetate, a student noted the development of spots in the origin, what can you suggest about this observation?






4. Why is it necessary to run TLC in a closed container and have the chamber saturated with solvent vapor?




5. Why must the spot be applied to the TLC plate above the level of development solvent?



6. What will be the result of adding too much sample to the TLC plate?



7. Why must a pencil be used for drawing the line and spotting?




8. How will the Rf value of a compound be affected if the developing solvent is allowed to run off the top of the TLC plate (e.g. the TLC plate is allowed to remain in the developing chamber after the solvent front has reached the top of the plate.)? Explain. Be specific.




9. Describe what an iodine vapor chamber is and explain how does it work? (Hint: Background information showing spots up chemically talks about two and Iodine crystals)




10. Consider the following compounds when answering the questions below. Assume these compounds can be separated by TLC.

a. Arrange the above compounds in order of increasing Rf in a on their structure and how they adsorption and separation on TLC plate. (Hint: Background information document: What separates the compounds as a chromatogram develops?)




11. A TLC plate is spotted with each of the three compounds. The plate is : ethyl acetate (5:95) to give the chromatogram shown below. How could you change the solvent system to give better separation of these three compounds?





12. Arrange the following in order of increasing Rfon thin-layer chromatography analysis plate based on their structure and how they adsorp and separate on TLC plate: Octanoic acid, Butyraldehyde, 2-Octanone, Decane, and 1-pentanol. (Hint: Background information document: What separates the compounds as a chromatogram develops?)





13. You are trying to determine TLC solvent system which will separate the compounds X, Yand Z. you ran a TLC plate using hexane/ ethyl acetate 95:5 as the eluting solvent and obtained the chromatogram below. How would you change the solvent system to give better separtion of these three compounds?










14.A student conducted an experiment using the silica gel TLC plate shown below by spotting a sample of MidolTM, caffeine, and acetaminophen on the plate and eluting with petroleum ether: chloroform (9:1, v/v).

Solvent front

















a. What are the Rf values of acetaminophen and of caffeine, respectively? (Use a ruler and directly calculate Rf values based on distance travelled by solvent and compounds)



b. Based on this TLC analysis, what are the ingredients in a tablet of MidolTM?


c. Which one is more polar, caffeine or acetaminophen?



d. Another student accidently used MidolPM in his experiment and observed only one spot. Speculate as to which spot was absent and explain.




15.Consider the following plate of compounds, A, B, and C developed in Hexanes:

a. Determine the Rf values of compounds A, B, C run on a silica gel TLC plate using hexanes as solvent






2. Which compound, A, B, or C is most polar?




3. What would you expect to happen to Rf values if you of hexanes as the eluting solvent





4. How would the Rf values change if the eluted with hexanes using alumina TLC plates?

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