Chromatography
Chromatography remains a popular laboratory topic because the samples are familiar and colorful, and the technique is used in modern science laboratories. Although the standard chromatography exercise is the separation of plant pigments, many other samples can be used, including marker pen inks (Hall 1993), food dyes (O'Neill 1990), and transition metal ions (Beran 1994).Acid-base indicators perform particularly well as chromatography samples because they are colorful, inexpensive, and commercially available as ready-to-use mixtures. Bogen universal indicator solution, for example, indicates the pH of solutions over the range of 4 to 10 and contains the indicators phenolphthalein, bromothymol blue, and methyl red. The green mixture separates on thin-layer chromatography plates to give 3 well-resolved spots that can be identified by their characteristic color changes (Fig. 2).
To separate the indicators, obtain a thin-layer chromatography plate and place it on a flat surface, plastic side down. Lightly, so as not to scrape off the white solid, draw a line with a pencil approximately 1 cm from the narrow end of the plate. Touch the end of a capillary pipet to a solution of Bogen universal indicator. While keeping your finger on the other end of the pipet, deposit a small drop of sample to the center of the pencil line. Set the plate aside and let the plate absorb the sample drop.
To make the chromatography solvent, mix 5 mL of ethyl acetate and 10 mL of hexane (or petroleum ether) and pour this solution into a 250-mL beaker. When the sample has been absorbed onto the plate, place the plate in the beaker with the pencil-marked end in the solvent. Make sure that the solvent does not touch the sample spot, or it will dissolve the sample off the plate.
Figure 2 Left, a spot of Bogen universal indicator. Right, the indicator separated into its 3 components (shown after treatment with base). The blue spot is bromothymol blue, the red spot is phenolphthalein, and the yellow spot is methyl red.
Cover the beaker with plastic film (for example, Parafilm®), and let the solvent wick up the plate. When the solvent has run 80% of the plate's height, remove the plate from the beaker and note the colors of the spots. Since the plate surface is acidic, the colors are the acid forms of the indicators. To see the base colors of the indicators, dip the plate in a 0.1 M sodium hydroxide bath for several seconds. From these color changes, each spot can be identified as one of the 3 indicators in Bogen universal indicator solution. Reinforce the acid-base chemistry of indicators by drawing the chemical structures of the acid and base forms of phenolphthalein. These structures can be found in most general chemistry texts.
Analgesic mixtures can also be separated on thin-layer chromatography plates (Elder 1995). You can compare standard samples of pure analgesics with commercially available pain relievers and then identify the active ingredients. The exercise requires an ultraviolet light source to visualize the spots on the plate.
