Laboratory Activity: Teacher Notes

Activity 1: Drug Identification

Major Chemical Concept

TLC is a separation technique. Specifically, substances in a mixture partition between a solid and liquid phase. A thin film of silica or alumina coated on a glass or plastic strip constitutes the solid phase. This thin film is called the stationary phase. The liquid phase is the solvent that ascends the solid layer by capillary action. The liquid phase is called the mobile phase. When a TLC sheet is spotted with a mixture of substances and placed in a solvent, the solvent (mobile phase) moves up the sheet and carries with it the various components of the spot. Because the molecules of each compound present have a different size, shape, and polarity, each compound will adhere to the stationary phase and dissolve in the solvent to a different degree. The stationary phase is typically polar and will hold polar compounds more tightly than nonpolar compounds (they will move more slowly). Less polar compounds will dissolve in the nonpolar mobile phase and move along the sheet faster. Once the flow of the mobile phase is stopped, the sheet is dried. The various spots, if not colored, can be made visible by either ultraviolet light or iodine vapor. The distance the compound moves relative to the distance the mobile phase moves is characteristic of that compound and is called the R f value.

Level

This laboratory activity can be used in a general chemistry course.

Expected Student Background

Students should have an understanding of "like dissolves like." A polar substance is more likely to dissolve in a polar solvent rather than a nonpolar solvent. However, it will not be obvious from the structural formulas of the substances used in this activity why one substance is more polar than another.

Time

The activity may take two or three 50-min periods. During the first period students should practice spotting filter paper and TLC sheets. The developing chambers should be set up and the solvent added. A discussion of TLC could be given. During the second period, students develop the sheets containing the knowns, perform the visualization tests, and calculate the R f values. In the third period, they develop the sheets containing the reference mixture and one or two unknowns, perform visualization tests, calculate R f values, and identify the compounds in their unknown. If only two days can be devoted to this activity, students should work in pairs, one student obtaining the chromatogram of the knowns and reference mixture of knowns, and the other obtaining the TLC of the reference mixture and the unknown(s).

Safety

Read the Safety Considerations in the Student Version. Caution students to avoid breathing methylene chloride. It is toxic and can have a narcotic effect in high concentrations. Ethyl acetate is flammable, and its vapors can be irritating but can be minimized by keeping the developing chamber closed unless a plate needs to be inserted or removed. Iodine vapors are irritating. Students should not look at the ultraviolet light.

Materials (For 24 students working in pairs)

4 Small bottles (25-mL size) containing the "known" compounds in solvent (50:50 v/v methylene chloride and absolute ethanol).

4-Hydroxyacetanilide (acetaminophen) (1 g per 20 mL solvent)

Caffeine (1 g per 20 mL solvent) Salicylamide (1 g per 20 mL solvent)

Acetylsalicylic acid (0.5 g per 20 mL solvent)

1 Small bottle, 20 mL of a combined standard reference mixture of the four compounds at the same concentration. (1 g of each of the above in 20 mL of solvent. Stir to dissolve.)

Methylene chloride, 100 mL

Ethanol, 95%, 100 mL

Ethyl acetate, 100 mL

Eastman Chromatogram Sheets with Fluorescent indicator (Eastman No. 13181, box of 20 sheets). Each sheet is cut into six 10 x 6.6 cm sheets. Thus four large sheets will suffice.

A hand-held ultraviolet lamp. Preferred: 4 watt long and short wavelength ultraviolet light. (Model Mineralight UVSL-25 available from Ultraviolet Products, Inc., 5100 Walnut Grove Avenue, San Gabriel, CA 91778 or Model UVGL-25 from Central Scientific Company, 11222 Melrose Avenue, Franklin Park, IL 60131-1364)

Thin-wall, open-end capillary tubing (1 mm) for micropipets; alternatively, use toothpicks

Filter paper, 12 squares for liners

24 Wide mouth, clear screw cap jars (Mason jars will work), 16 oz, or 600-mL beaker with aluminum foil cover also work well

Iodine, several crystals

Advance Preparation

Prepare the solutions of the knowns before the laboratory period. Spot the TLC in one area of the laboratory. Have students come to a table of knowns rather than taking the knowns to their individual desks. Keep development chambers at individual desks. Place the ultraviolet lamp in a darkened hood, darkened area of the laboratory room, or in a large upside-down cardboard box with appropriate holes cut for viewing.

Prepare about 10 micropipets as shown in Figure 4.

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