Laboratory Activity: Teacher Notes Continued
- Give a brief introduction to the TLC techniques (see Major Chemical Concept).
- Instruct students on importance of small spots. If spots are too large, tailing and poor separation will occur.
- Explain why the spotted mixture should be above the solvent level. (If it is below, it will dissolve in the solvent and not separate.)
- Remind students not to move the jar during the development time to avoid splashing solvent on the plate.
- Explain to students how to calculate R f values. Tell them to get their best estimate for the center of each developed spot.
Walk around laboratory and ask students if they understand what is happening. Discuss how to calculate R f values. Caution students about inhaling solvent vapor.
Anticipated Student Results
- Typical experimental results are:
- Salicylamide and acetaminophen produce dark spots. Caffeine produces a light spot. Aspirin does not produce a spot.
- Yes. The approximate order of migration is salicylamide, followed by acetaminophen, caffeine, then aspirin.
- Yes. The iodine permanently darkens some of the spots.
- Thedifferencesinthe rateofmovementof substancesdependuponsolubility of the substance and adsorption properties of the backing on the plate. Because the solvent is typically nonpolar and the backing is strongly polar, the backing holds a polar substance more tightly than a nonpolar substance. These polar substances move considerably more slowly than nonpolar substances in the nonpolar solvent.
Assessing Laboratory Learning
- List the steps in the determination of the number of components in a mixture by TLC. [Spot the plate, develop the chromatogram, visualize the spots, calculate R f values, compare with suspected knowns.]
- How is an R f value determined? [R f is the distance the substance travels divided by the distance the solvent travels.]
- Two substances have R f values of 0.3 and 0.5, respectively. Which substance is carriedfartherbya polarsolvent?Whichsubstance islesspolar?[Substance with R f = 0.5 is the polar substance and is carried better by the polar solvent. Substance with R f = 0.3 is less polar.]
- Name two ways of visualization of spots. [Ultraviolet light and iodine vapor. Discuss with the students other possibilities for visualization. Lead them to think about color reactions, where the spots can be developed by spraying the TLCplatewithreagents thatreactwiththe samplestogivea colororavisible spot. For example, ninhydrin sprayed on hydrolyzed protein samples yields a purple color and conc. sulfuric acid dehydrates most organic compounds to yield yellow to brown (carbon) spots (see Demonstration 3).]
- Identifythestationaryand mobilephasesinTLC. [Solidadsorbentandsolvent, respectively.]
- Using the following chromatogram, decide which substances are identical? Which mixtures are identical? [Identical substances: 1a, 2a, and 4a; 1b, 3b, and 4b; 1c and 4c. Identical mixtures: 1 and 4.]