Thin-layer chromatography is used widely in the undergraduate organic laboratory^1-4. The technique often is illustrated with an early experiment involving the analysis of a mixture of colored substances such as those extracted from paprika or spinach. For colorless compounds, a step to make spots visible after development of the plate is needed.

Most Common Recommendations

• Commercially available plates can be used that incorporate a fluorescent dye in the adsorbent layer and make many substances visible when viewed with ultraviolet light.
• A TLC plate can be inserted into a jar containing iodine crystals to produce brown spots due to absorption of iodine from the vapor phase into the organic material.

Both methods, however, involve disadvantages. The UV method works only for UV active substances, and the iodine method is somewhat more general, but the spots produced tend to be fuzzy and indefinite, precluding fine resolution. We have found that these problems may be ameliorated largely by including "spray reagents" among the visualization methods available to students.

Reagents

• 2,4-Dinitrophenylhydrazine (used to detect aldehydes and ketones)
• Potassium permanganate (detection of alcohols, alkenes, and other oxidizable compounds)
• Phosphomolybdic acid (non-specialized reagent)

The spots resulting from the use of these solutions usually are well-defined and often appear in characteristic colors. These solutions are applied to the TLC plate with any of several types of spraying devices available from major chemical companies. This step, however, has several objectionable features that have discouraged the use of these reagents in most student laboratories.

Objectionable Features

• The process is wasteful. Only a small fraction of the solution ever reaches the plate.
• Unless scrupulously maintained, sprayers tend to clog and splatter, resulting in an uneven coating.
• Most serious is the fact that most of the reagent is released into the atmosphere to remain as a noxious contaminant.

Employing a Dipping Technique Avoids Disadvantages

We have avoided these disadvantages by employing a dipping technique with the reagents. The solutions are stored in plastic centrifuge tubes of proper size (a few centimeters longer and a few millimeters wider than the TLC plate). After development, the plate is held at the solvent front end with forceps, submerged in reagent up to the solvent front, and immediately removed. Contact time with the solution should be kept to a minimum. Spots usually begin to emerge almost immediately but become more definite over a period of up to one hour. In some cases (e.g., after the phosphomolybdic acid dip) spot development requires the application of mild heat such as hot air from a hair dryer. A large number of general and specialized visualization reagents are listed in an easily available chemical-biochemical catalog⁵. Use of such reagents with the dip technique should lead to optimal results with a minimum of cost, mess, wastage, and environmental contamination.

Materials and Procedures

The TLC plates are 2.5x7.5-cm plastic sheets with a silica gel coating containing a fluorescent dye (J. T. Baker catalog number IB2-F). The dip solution containers are 50 mL polypropylene centrifuge tubes, 2.8 x 11.5 cm, with screw cap (Fisher Scientific Company catalog number 05-538-55). The phosphomolybdic acid reagent is purchased most economically as a 20% solution in ethanol (Aldrich Chemical Company, catalog number 31,927-9). The permanganate dip was prepared as an aqueous solution, 1% in KMnO₄, and 2% in K₂CO₃. Brady's reagent⁶ was employed for the 2,4-dinitrophenyhydrazine solution.