In this laboratory practical, students are required to separate and purify unknown compounds of a given sample mixture using appropriate: Bachelor of Science in Analytical Chemistry Report, TARC, Malaysia

University Tunku Abdul Rahman College (TARC)
Subject Bachelor of Science in Analytical Chemistry

In this laboratory practical, students are required to separate and purify unknown compounds of a given sample mixture using appropriate chromatographic techniques. Students are expected to develop and critically analyze the TLC profile of the sample mixture and select the appropriate liquid chromatographic technique for the separation and purification of unknown compounds. The purified compounds are to be analyzed and their chemical structures will be established using spectroscopic techniques.


  • Draw a light pencil line about 0.5 cm from the end of a TLC plate.
  • Draw a small x along this line at approximately 0.5 cm from one edge. Label under the line with your pencil the sample that you will be placed at x.
  • Dissolve about 1-2 mg of the given mixture of compounds in about 2-3 mL of pure dichloromethane to prepare a clear solution for TLC analysis
  • Using capillary tubes, spot the above solution on the plate. Make each spot as small as possible, preferably not more than 3 mm in diameter.
  • Examine the plates under an ultraviolet lamp to see if enough amount of sample solution has been applied. If not, add more samples.
  • Take the eluting solvent into an appropriate beaker which serves as a developing chamber to a depth of approximately 1 cm.
  • Place the prepared TLC plate carefully in the developing chamber. Cover the beaker with a watch glass.
  • After the solvent has risen to about 1 cm from the top of the plate, remove the plate and mark the solvent front with a pencil.
  •  Let the plate dry in the fume hood.
  • Examine the plate under UV light. Outline the dark spots & coloured spots using a pencil.
  • The spots are then visualized by putting them in an iodine chamber. Remove the plate when a definite change in appearance takes place on the plate. Note which compounds are stained with iodine and to what intensity. The iodine stains will dissipate over time.
  • Record your observations to rationalize your selection of appropriate preparative chromatography for the isolation of pure compounds in your next step in the practical.

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