Thin Layer Chromatography Troubleshooting Guide
This guide highlights and resolves common challenges encountered during thin layer chromatography.
| Issue | Possible Cause | Solution |
| No spots visible | Compound is not UV-active or not retained | Use a stain to visualize |
| Poor separation | Incorrect solvent system or overloading | Optimize solvent polarity and reduce sample concentration |
| Smudged or tailing spots | Overloading or impure solvent | Reduce sample load or use high-purity solvents |
| Solvent front uneven | Plate not level in the chamber | Ensure the TLC plate is completely vertical |
| Weak UV fluorescence | Compound concentration is too low | Spot a higher concentration or use a more sensitive stain |
| There are multiple spots on the TLC | The compound may be degrading on the TLC plate | Perform a two-dimensional (2D) TLC to confirm. Refer to next page for the procedure |
Two-Dimensional (2D) TLC: A Tool for Checking Compound Stability
2D TLC is a fast, reliable method to assess compound stability, separating compounds in two dimensions to identify degradation products and optimize purification.
Steps for Running a 2D TLC
- • Prepare a square TLC plate: Cut a TLC plate into a square shape, approximately 7 x 7 cm.
- • Spot the sample: Apply your sample solution to one corner of the plate, leaving a 0.5 - 1 cm margin from each edge.
- • First elution: Place the plate in a TLC chamber with an eluent that gives an Rf value around 0.5 for your compound. Allow the solvent to rise, then remove the plate and let it dry.
- • Second elution: Rotate the plate 90 degrees so the lane of spots is now at the bottom. Using the same solvent system, elute the plate in this new direction. Allow the plate to dry completely.
Analyze the Results
- • Stable compounds: Appear along the diagonal of the plate.
- • Decomposing compounds: Appear below the diagonal, indicating degradation on silica gel.