Overview
This lecture explains how to perform thin layer chromatography (TLC), including setting up, spotting, developing, visualizing, and interpreting results.
Equipment and Chamber Preparation
- TLC requires a developing chamber, often a beaker with a watch glass lid.
- Add 3–4 mL of chosen solvent (developing or eluting solvent) to the chamber.
- Place trimmed filter paper inside the chamber to saturate with solvent vapor.
Preparing and Spotting the TLC Plate
- Select an undamaged end of the TLC plate for use.
- Use a pencil (never pen/ink) to lightly mark a baseline above the solvent level.
- Dissolve samples (starting material and product) in solvent.
- Use capillary tubes to spot small amounts of each sample on the baseline.
- Use UV light to check that spots are the right size and not overloaded.
Developing the Plate
- Handle the plate by the edges to avoid contaminating the silica gel.
- Insert the plate into the chamber so that the solvent is below the baseline.
- Allow the solvent to rise until about 1 cm from the top of the plate.
- Remove the plate and immediately mark the solvent front with a pencil.
Visualization and Results Interpretation
- Visualize the plate under UV light and circle visible spots.
- Visualize with iodine, which reacts with polar or pi bonds for further spot identification.
- The RF value (retention factor) is calculated as spot distance from baseline divided by solvent front distance.
- Higher RF means less polar; lower RF means more polar (adheres more to silica gel).
Key Terms & Definitions
- TLC (Thin Layer Chromatography) — a technique for separating and identifying compounds.
- Developing/Eluting Solvent — the liquid that moves up the TLC plate, carrying samples.
- Silica Gel — a polar stationary phase coating on TLC plates.
- Baseline — the starting line where samples are applied.
- RF Value (Retention Factor) — (distance moved by spot) ÷ (distance moved by solvent front).
Action Items / Next Steps
- Practice setting up a TLC chamber and preparing/sampling a TLC plate.
- Calculate RF values for different compounds using measured distances.
- Review polarity concepts as they relate to TLC movement.