DETERMINATION OF LIPSTICK DYES BY THIN-LAYER CHROMATOGRAPHY

The examination of lipstick stains left on clothing, or cigarettes, may provide valuable clues as to the identity of a criminal suspect. We will use TLC to separate dyes that are impart to lipstick its color.

Thin layer chromatography utilizes a thin film of silica gel or alumina coated onto a glass or plastic strip. As in paper chromatography, this thin film is called the stationary phase. A mixture of the compounds to be separated is placed in a small spot at one end of a strip, and a liquid organic solvent (mobile phase) is passed over the spot. As the solvent moves up the strip, it carries with it the various components in the spot. Because each compound present has a different size, shape, and polarity, each compound will adhere to the stationary phase and dissolve in the solvent to a different extent. Thus, if two compounds are started at the same place and solvent passed over them, one compound will move along the strip faster than the other. After a period of time the flow of the movile phase is stopped; the strip is dried and then sprayed with a reagent that will produce colored spots, if the compounds are not colored. The distance the compound moves relative to the distance the mobile phase moves is a characteristic of that compound and is know as the R_f value.

Lipsticks are essentially composed of fats, oils, waxes, colorings, perfumes, and flavorings. The color of lipstick is due mainly to aluminum, calcium, or barium dyes dispersed in the lipstick preparation in concentrations on the order of 15 % to 20 %. By first extracting dyes from an object impregnated with lipstick, the dyes can then be separated and compared by thin layer chromatography.

CRIME SCENE

A wealthy playboy is found murdered in his penthouse suite. The evidence points to a fight with a jealous lover. A search of the premises unveils a napkin containing a lipstick stain. The police begin to question all female friends of the victim. They are asked to turn over to the police all of their lipsticks. These lipsticks along with the stained napkin, are sent to the crime laboratory for analysis. The police hope that by comparing the lipsticks to the stain left on the napkin, they may be able to determine a likely suspect. In the laboratory a chemist stains clean napkins with each lipstick. You will now proceed to make a comparison of the lipstick stains.

EQUIPMENT

1 beaker, 50 mL
1 lamp, UV (short wavelength, 254 nm)
2 Bottles, wide mouth, 4-oz (hold plates)
1 Medicine dropper
6 Bottles, wide mouth, 8-oz (sample holder)
1 pipet, disposable
1 rack. Funnel
2 Bottles, wide mouthed, 16-oz (developing tanks)
1 rack, test tube
Ruler, metric
1 Brush, test tube, small
1 pr scissors
1 cylinder, graduated 10 mL
1 silica gel TLC plate cut to fit inside 16 oz bottles
1 Florescent viewing box and/or a UV lamp (optional)
1 stand, ring
1 Funnel, powder
thumb tacks
1 Funnel, separatory, 250 mL
tubes, capillary melting
3 test tubes 10 cm
1 hair dryer, (optional)

REAGENTS

Acetone
Ammonium hydroxide
Isoamyl alcohol
Solvent mixture: hexane, chloroform, methanol, and water in the ratio 1:1:1:1. Mix thoroughly and pour into a separatory funnel. Recover and save the bottom layer. Discard the upper layer.

METHOD

Obtain three napkins, each marked with a lipstick stain. One napkin will be marked scene, and the other two are known lipsticks stains and are marked A and B.
Using scissors, cut out a stain about 1 cm long from each napkin. Use more stain if you wish and it is available.
Add 2 to 3 drops of solvent mixture to each of the three test tubes. If the solvent is completely absorbed by the tissue, add 1 to 2 drops more. You must have a small excess of dissolved stain to apply the TLC plate. Mark the tube containing the unknown stain "S". The other tubes are labeled "A" and "B" respectively.
Soak each stain for approximately 15 minutes with occasional agitation of the test tube.
Prepare a mixture of 5 mL of isoamyl alcohol, 3.5 mL of acetone, 3 of distilled water, and 0.5 mL of ammonium hydroxide to use as a solvent or mobile phase for the chromatography of the lipstick stains. Mix the solvent in a 50 mL beaker.
Into the small bottle to be used as a developing chamber, place enough of the developing solvent to produce a depth of approximately 1 cm.
Cap or cork the bottle, and let stand until you are ready to use it.
Using a capillary melting-point tube, apply the extracted unknown lipstick stain to the TLC plate. The applied spots should be very close to the solvent level, but not touching. Apply a very small spot of solvent mixture approximately 1.5 cm from the bottom of the plate. Let it dry thoroughly, (apply heat from a hair dryer if it is available), then apply a small spot again in the same place. Let dry thoroughly. Continue until the spot is dark in color.
Alongside of the unknown spot apply the extracted known stains in the same manner described in step 8. Be certain the spots are separated from each other.
Hold the spotted plate next to the bottle containing the solvent. Make certain that the level of solvent in the bottle will be below the samples on the TLC plate when it is placed inside the bottle. If the solvent is at too high a level, remove some solvent with a medicine dropper.
Place the TLC plate in the bottle. Let it dry, and compare the colored spots, if any are apparent, between the unknown stains. Also, view the plates under ultraviolet radiation. Compare the fluorescing spots.
Do any of the standard lipsticks compare to the unknown with respect to the number of spots and the distances they have moved up the plate?
Clean all the equipment, and return it to its proper place.

References

This experiment is based on a procedure reported by S. Angelos and W.R. Dietz at the conference of the Midwestern Association of Forensic Scientists, Indianapolis, Indiana, 1976.

Meloan, C. E.; James, R. E.; Saferstein, R. Lab Manual. Criminalistics: An Introduction to Forensic Science, Sixth Edition; Prentice Hall: Upper Saddle River, NJ, 1998.