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.