Performing Thin Layer Chromatography (TLC)

Draw a pencil line approximately 1 cm from the bottom of the TLC plate, being careful not to scratch the silica gel layer. Mark four evenly-spaced dots on this baseline and label them A, B, A+B, and U for the four samples to be tested.

Use microcapillary tubes to pick up each sample via capillary action, then gently tap the tube tip onto each corresponding dot three times to deposit a small sample spot. Use a fresh tube for each sample to prevent cross-contamination and ensure spots remain separated.

Pour approximately 5 ml of the solvent system (95% ethyl acetate and 5% acetic acid) into the glass TLC chamber, then cap it for a few minutes to allow saturation of the chamber atmosphere.

Using tweezers, carefully place the spotted TLC plate into the chamber ensuring the solvent level remains below the baseline. Immediately cap the chamber and monitor as the solvent travels upward, carrying and separating the samples by polarity.

Remove the plate from the chamber when the solvent front approaches near the top, then immediately draw a pencil line marking the solvent front before it becomes invisible. Allow the entire plate to dry completely before proceeding to analysis.

Place the dried plate under a UV lamp to visualize compounds containing pi electron systems. Circle the visible sample spots with pencil to mark their final positions on the plate.

Measure the distance from the baseline to the center of each spot and the distance to the solvent front using a ruler. Divide the first distance by the second to calculate the retention factor (RF) value for each sample, then compare these values to known standards for compound identification.