1. TLC Plates Common adsorbents for TLC are silica gel, alumina, and cellulose. TLC plates are commercially available with a variety of properties. Choose a TLC plate and cut it down to an appropriate size (approximately 5 cm x 5 cm is sufficient for most applications). For glass-backed TLC plates, score the glass using a ruler and a glass cutter, then carefully break along the line. 2. Spotting Dissolve the sample in a suitable solvent to make an approximately 1% solution. If possible the solvent should be nonpolar. Column chromatography fractions and other dilute solutions may be used without dilution if the solute is present at a concentration between 0.2% and 2.0%. Mark the baseline with a pencil about 1.0 cm from the bottom of the plate. Position the spots at least 1.0 cm from the edge of the plate and label them appropriately. Spots may be applied using a glass capillary. To spot the TLC plate, dip the capillary into the solution to draw in a small quantity of liquid. Gently touch the tip to the desired location on the TLC plate and remove it immediately. Alternatively, apply spots with a microliter syringe by delivering approximately 1 μL of solution for each application. Spots may be applied successively at each location, taking care not to disturb the surface of the adsorbent with the spotter. Allow solvent to dry in between applications. 3. Choosing a Developing Solvent It is preferable to use the least polar solvent possible for good separation. Common solvents for TLC include hexane, ethyl acetate, dichloromethane, and methanol (Table 1). A convenient way to find an appropriate mobile phase is to spot the TLC plate with a sample. Apply enough solvent directly to the spot to form a circle of solvent 1–2 cm in diameter. Mark the circumference of the circle. Upon visualization an appropriate solvent will show well-separated rings, with the outermost ring about 50% of the distance from the center to the solvent front. It may be necessary to adjust the polarity of the mobile phase by choosing two miscible solvents and testing them in varying proportions. Examples of common mixtures are hexanes with ethyl acetate and dichloromethane with methanol. 4. Development Place the spotted TLC plate in a developing chamber containing the appropriate developing solvent. The solvent line should be below the baseline marked on the TLC plate. The developing chamber can be a jar with a lid, or a beaker covered with aluminum foil or plastic film. Use the smallest container available that will accommodate the TLC plate. Do not allow the solvent to reach the top edge of the plate. When the solvent front (the boundary where the wet part of the adsorbent ends) is 5–10 mm from the top the plate, remove the TLC plate from the developing chamber and mark the solvent front with a pencil before the solvent dries. 5. Visualization Colored spots may be visualized immediately and marked with a pencil. Often, spots are not visible and therefore must be visualized by some other method. Often, the TLC adsorbent contains a fluorescent indicator. Spots may be visualized using a hand-held ultraviolet (UV) lamp. Compounds that quench fluorescence will appear as dark spots when the plate is irradiated with short-wave (254 nm) UV light. Fluorescent compounds will produce bright spots when irradiated with UV light of an appropriate wavelength. Mark the center of each spot with a pencil. Spots may also be visualized by applying a visualizing reagent or stain to the TLC plate. The visualizing reagent may be applied by dipping the plate into the reagent, or by wiping the plate with a cotton ball saturated with a noncorrosive reagent. A 20% solution of phosphomolybdic acid in ethanol is useful for the visualization of most organic compounds. The spots appear upon heating of the plate with a heat gun or in an oven. Other reagents may be used for visualization of specific classes of compounds. For example, ninhydrin reagent is used for amino acid visualization, and 2,4-dinitrophenylhydrazine for aldehydes and ketones. 6. Analysis The retardation factor (Rf) is the ratio of the distance a compound travels up a TLC plate to the distance the solvent travels. This may be determined by measuring the distance from the baseline to the center of the spot and dividing the value by the distance from the baseline to the solvent front. The Rf of a compound is characteristic of its physical properties and dependent on factors such as temperature, sample size, and thickness and activity of the adsorbent, temperature, and sample size. To confirm that an unknown is identical to a known compound, a standard should be spotted on the same TLC plate. Identical substances will have the same characteristic Rf.
