Thin-Layer Chromatography (TLC)

Handle the TLC plate only by its edges or with tweezers to avoid contaminating the silica gel surface. Use a ruler and pencil to lightly draw a horizontal line (the origin) about 1 cm from the bottom edge, then mark vertical reference points spaced 1 cm apart for the four lanes (A, B, C, D).

Using clean capillary applicators for each solution to prevent cross-contamination, spot standard X in lanes A and C, standard Y in lanes B and C, and the unknown sample in lane D. Allow spots to dry completely between applications.

Pour a pre-measured volume of solvent into the developing chamber and cover with a lid for several minutes to saturate the atmosphere with solvent vapors. Quickly place the spotted plate into the chamber with the origin above the solvent surface, then allow the solvent to ascend the plate until it reaches approximately 1 cm from the top.

Remove the plate from the developing chamber once the solvent front nears the top. Immediately mark the solvent front with a pencil to indicate the boundary between the wet and dry silica.

Place the developed plate under a UV light source to visualize the separated components. The silica with fluorescent indicator will glow while most compounds appear as dark spots; outline all visible spots with a pencil to record their positions for analysis under visible light.

Compare the position and number of spots in the unknown lane (D) with the reference standards in lanes A, B, and the mixture in lane C. Index the references by confirming which compound corresponds to each spot height to determine the identity of the unknown.

Measure the distance from the origin to the center of each spot and from the origin to the solvent front in millimeters. Calculate RF values using the formula: RF = (distance moved by spot) / (distance moved by solvent), reporting values to two decimal places. Compare the RF value of the unknown to the RF values of the standards to confirm compound identity.