Stain Recipes

Thin Layer Chromatography Stains and Recipes

Enhance the visualization of separated compounds using these popular stains.

Note: Stains highlighted in red require heat for activation.

Stain	Recipe	Used for the detection of:
UV Visualization	None required (TLC plates must contain a UV indicator).	Compounds with chromophore groups.
p-Anisaldehyde	Mix 15 mL acetic acid and 3.5 mL p-anisaldehyde into 350 mL ice-cold ethanol. Slowly add 50 mL of concentrated sulfuric acid dropwise over 60 minutes. Store at 0°C.	Allylic alcohols (green), phenols (violet), aldehydes, ketones, carbohydrates, and esters (blue/red). Alkenes, alkynes, and aromatic compounds are not detected.
Bromocresol Green	Dissolve 0.04 g bromocresol green in 100 mL ethanol. Add 0.1 M sodium hydroxide dropwise until pale blue.	Acidic groups with pKa < 5.
Cerium Molybdate (CAM or Hanessian’s stain)	Dissolve 0.5 g cerium ammonium sulfate dihydrate and 24 g ammonium molybdate tetrahydrate in water. Add 28 mL of sulfuric acid and stir for 1 hour.	Universal stain. More sensitive than PMA stain.
Cerium Sulfate	Prepare an aqueous solution with 10% cerium (IV) sulfate and 15% sulfuric acid.	General stain; effective for alkaloids.
2,4-Dinitrophenylhydrazine (DNP)	Dissolve 12 g DNP in 60 mL of sulfuric acid, 80 mL water, and 200 mL of 95% ethanol.	Specific for aldehydes and ketones.
Iodine Vapor	Place iodine crystals in a sealed chamber.	Good for alkanes, phosphines, and thiols.
Ninhydrin	Dissolve 0.3 g ninhydrin in 100 mL n-butanol. Add 3 mL acetic acid.	Specific stain for amino acids and primary amines. Secondary amines stain light yellow; tertiary amines do not stain.
Phosphomolybdic Acid (PMA)	Dissolve 10 g PMA in 100 mL of ethanol.	Universal stain (light-sensitive); does not detect some amines, amides, or oxidation-resistant aromatics.
Potassium Permanganate (KMnO₄)	Dissolve 1.5 g potassium permanganate, 10 g potassium carbonate, and 1.25 mL 10% sodium hydroxide in 200 mL water.	Universal stain; detects oxidizable compounds like alcohols, ethers, esters, and alkenes (brown-yellow spots). Reductive compounds like thiols appear white.