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Thin-Layer Chromatography

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  1. Preparation of Solutions

    Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.

    • Before getting started, put on a lab coat, safety glasses, and nitrile gloves. You'll be working with hexane, ethanol, and ethyl acetate, which all permeate thin nitrile gloves within minutes, so change your gloves promptly if you get any solvent on them.
    • Prepare ~1 mg/mL solutions of butyl phenyl ether, phenol, benzoic acid, aspirin, caffeine, and acetaminophen. Use an analytical balance or a micropipette to measure approximately 10 mg or 10.7 μL of butyl phenyl ether into a 20-mL glass vial. Note: Butyl phenyl ether is a liquid at room temperature and should be handled in a fume hood.
    • Measure 10 mL of ethanol, add it to the vial, and label it ‘1 mg/mL butyl phenyl ether in ethanol’.
    • Prepare the other five solutions, starting with phenol. Tare a 20-mL glass vial and measure 10 mg of phenol into it.
    • Then, add 10 mL of ethanol to the vial and stir the mixture with a glass rod until the phenol has completely dissolved.
    • Cap the vial and label it ‘1 mg/mL phenol in ethanol’. Repeat this process to prepare 1 mg/mL solutions of benzoic acid, aspirin, caffeine, and acetaminophen. Store the vials in the instructor's hood or a common workspace.
    • Prepare the three unknown solutions of commercially available analgesics in ethanol. Tablet formulations can vary, so you might want to weigh the tablets beforehand to estimate the weight percent of the active ingredients.
    • Use a small mortar and pestle to crush an acetaminophen tablet to powder. Measure 10 – 15 mg of the powder into a 20-mL glass vial.
    • Then, add 10 mL of absolute ethanol to the vial and stir the mixture with a clean glass rod for 1 – 3 min. It's fine if some solid remains at the bottom of the vial.
    • Cap the vial and label it ‘Unknown 1’. Discard the remaining powder and thoroughly clean and dry the mortar and pestle.
    • Use 10 – 15 mg of a crushed aspirin tablet to make the unknown 2 solution and 30 – 40 mg of a crushed tablet containing a mixture of acetaminophen, aspirin, and caffeine to make the unknown 3 solution in the same way. Store the unknown solutions with the known solutions.
    • Make the solvent mixtures that the students will use as eluents. Prepare 100 mL of a mixture of 60% hexane and 40% ethyl acetate. Measure 60 mL of hexane and pour it into a 250- or 400-mL beaker. Add 40 mL of ethyl acetate and stir the mixture until it appears homogeneous.
    • Label a 150-mL bottle ‘60% hexane, 40% ethyl acetate (v/v)’ and use a funnel to transfer the solution. Cap the bottle before setting it aside.
    • Make 100 mL of a mixture of 90% ethyl acetate and 10% hexane. Measure 10 mL of hexane and pour it into a 250- or 400-mL beaker. Then, add 90 mL of ethyl acetate and stir the mixture well.
    • Label a 150-mL bottle as ‘90% ethyl acetate, 10% hexane (v/v)’. Transfer the mixture to the bottle and cap it tightly. Store the solvents and the solvent mixtures in a flammables cabinet.
  2. Preparation of the Laboratory
    • Prepare or obtain waste containers for glass, iodine, and non-halogenated organic waste. Ensure that every lab sink has a supply of paper towels.
    • Plug in and test a small 254-nm UV lamp in a shared area.
    • Set up a hotplate in a central fume hood, label a 500-mL beaker ‘iodine chamber’, and place approximately 1 – 2 g of iodine in the beaker. Cover it with a large watch glass and set it on the hotplate.
    • Set out the following glassware and equipment at each student lab station (we suggest that students work in pairs):
       3    50-mL glass screw-top jars
       3    10-mL graduated cylinders
       1    Pencil
       1    Labeling pen
       1    Pair of tweezers
       1    Small ruler with metric increments
    • Label a 150-mL bottle as ‘100% hexane’, pour about 100 mL of hexane into the bottle, and tightly cap it. Put away the stock bottle of hexane and place the mixtures of hexane and ethyl acetate in the solvent hood with the bottle of pure hexane.
    • Arrange the six known solutions and the three unknown solutions on a shared bench.
    • Place containers of disposable glass capillaries, medium-sized filter papers, and TLC plates nearby.
    • At the beginning of the lab, randomly assign an unknown compound to each student group.
    • Once some students are about halfway through the first development, heat the iodine to 110 °C on the hotplate.
    • After the lab, let the beaker cool, and dispose of the iodine waste appropriately.

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What is thin-layer chromatography?

Thin-layer chromatography (TLC) is a technique used to separate organic compounds based on differences in their polarities.

What are the three components of TLC?

The three components of the TLC system are the stationary phase, the mobile phase or solvent, and the solute.

In general, which phase will less-polar.......

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  • 50-mL screw-top glass jar with solvent-resistant cap (inner diameter of 4 cm)
  • 10-mL glass graduated cylinder
  • Pencil
  • Labeling pen
  • Metal tweezers (10 or 12 cm)
  • Small ruler with mm and cm increments
  • Plastic/glass backed normal-phase silica gel analytic TLC plate with fluorescent indicator at 253 nm (2.5 cm x 6.5 cm)
  • Filter paper
  • Disposable glass capillary (+ extra)
  • Phenol
    20 mg
  • Benzoic acid
    20 mg
  • Butoxybenzene/butyl phenyl ether
    20 mg
  • Acetylsalicylic acid/aspirin
    20 mg
  • Caffeine
    20 mg
  • Acetaminophen/paracetamol
    20 mg
  • Ethanol (denatured)
    200 mL
  • Hexane
    300 mL
  • Ethyl acetate (98% HPLC grade)
    200 mL
  • OTC tablet (325 mg acetaminophen)
  • OTC tablet (325 mg acetylsalicylic acid)
  • OTC tablet (250 mg acetaminophen, 250 mg acetylsalicylic acid, 65 mg caffeine)
  • Iodine crystals
    2 g
  • Stirring hotplate
  • Mortar and pestle
  • 10-mL graduated cylinder
  • 100-mL graduated cylinder
  • 250- or 400-mL glass beaker
  • 150-mL glass bottle with solvent-resistant cap
  • 20-mL glass vials with solvent-resistant cap
  • Glass stirring rod
  • Glass funnel
  • 20- or 100-μL micropipette with pipette tips
  • 500-mL glass beaker
  • 100-mm watch glass
  • Analytical balance
  • Weighing boats
  • Spatulas
  • Labeling tape
    1 roll
  • Lab wipes
    -1 Dependent on lab size
  • 254-nm UV lamp (at least 1)
    -1 Dependent on lab size

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