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Thin Layer Chromatography stains
TLC Stains

TLC Staining procedure

TLC Stain Recipe

Stain chemistry / physics UV light excites a fluorescent additive in TLC plate. Compounds screen some of the UV, making fluorescence weaker. Sometimes, visible fluorescence is exited by UV making a spot brighter, and so is colored differently than the background Oxidative stain. Strong heating required.


254nm UV light

254nm UV lamp with filter. Darker spots on light green if plates with VU indicator are used. Occasionally brighter spots (typically blue)

A compound must have a chromophore. Even then, this technique is NOT sensitive for isolated alkenes and benzyl ethers. Fluorescence is often excited in polyconjugated compounds (benzophenone, antracene and so on).

Permanganate stain


3g 10g

The most universal stain. Harsh oxidizer. Any oxidizable compound such as alcohol, ether, ester, alkene, alkyne, alkyl aromatic, ketone, carboxylic acid, amine, amide and

Alkanes and pyridine won't show-up at all. there will be smears and streaks. This stain is NOT compatible with elutants containing triethylamine . Put a TLC Different absorption of brown colored iodine on clean silica as opposed to silica loaded with analytes. heat uniformly) until the pink color of plate just starts to become yellowish. . If overheated all plate goes brown. Thiols will almost all the rest will show up as brownyellow spots (MnO2). Works in about 50% of all cases. Dip in stain briefly.the entire plate will became yellow before spots develop.. Material develops as yellow-brown spots. Strongly reductive compounds such as thiols. If all residual elutant is not removed. phosphines and even dienes will show up as white spots (Mn+2) BEFORE heating. Let it sit for 30 min to equilibrate. Do not overheat! The plate must be hot. wipe up excess (soak with a paper towel from the side) and heat up with a heat gun on high setting (move the plate. Spots may be darker or lighter than background. Prepare a well-closed chamber with a few crystals of elemental iodine.Water 300mL Carefully dry the plate from elutant with a heat gun.. Works with alkanes! Thiols and phosphines will immediately show up as white spots. but touchable (5060C). Iodine chamber Hit and miss stain.

Some amines. PMA (CAS:1202657-2) oxidize to disulfides: 2R-SH + I2 = R-S-S-R + 2HI PMA stain (Phosphomoly bdic acid stain) Oxidative stain. amides and oxidation-resistant aromatics won't show up. Pick the best moment to circle spots . Fairly universal stain.5g Procedure same as above 1g Very universal stain. . More sensitive than PMA stain (above). keep the plate for 2 sec to 10 min inside (trial and error) and then take the plate out.plate inside.they might soon disappear. Stain solution is somewhat light-sensitive. Cerium Ammonium Molybdate stain) Oxidative stain. dark green spots called Keggin structure) which are appear on light green green or blue colored background 10g Ammonium molybdate tetrahydrate (CAS:12054-85-2) Cerium ammonium sulfate Expensive. Ethanol 100mL H3[P(Mo3O10)4] * xH2O is Mo6+ compound. light yellow. Upon formed (molybdenum blue. 2. Upon Application technique is exposure to reducing organics the same as for Mo5+ and Mo4+ compounds are Permanganate stain. Strong heating required. CAM stain (Hanessian's Stain. Strong heating required. Watch the color change. so heating.

alkynes and aromatic compounds will NOT show up. esters like alkylphtalates (blue/red spots) and some other compounds. phenols (violet spots) amines. Probably something along the following lines: 4mL 5mL 2mL 150mL Application technique same as for the Permanganate stain. Works for allylic alcohols (green spots). Chemistry of this stain is unknown. aldehydes. Upon heating spots of varying Not as universal as oxidative stains. carbohydrates. dark blue spots appear on light blue background p-Anisaldehyde (CAS:123-11-5) Sulfuric Acid pAnisaldehyde Stain Acetic Acid Ethanol Some heating required. Alkenes. ketones. Upon heating.dihydrate(CAS:1037 8-47-9) 10m L 90m L Sulfuric acid Water Application technique same as for the Permanganate stain. .

color appear on light pink background ninhydrin (CAS:485-47-2) Acetic Acid Ninhydrin Stain Acetone Gentle warming required. Stains can be applied by spraying or by dipping of a plate into solution. 2004. especially if heating is required for visualization.(An interesting review:Friedman. J. 385406. Still. Specific stain for amino acids (see group TLC below) and primary amines. Secondary amines stains light yellow which is difficult to see. Food Chem. permangantate stain is perfect for most not-too-polar organic compounds while acetone-based nynhydrin stain is excellent for amino acids. let acetone evaporate and then gently warm up the plate. a visualizing technique is needed to observe TLC results. the right the stain solution SHOULD NOT dissolve analyte spots.1g 100mL Dip plate into stain. if a compound that must be analyzed is volatile. it may evaporate before the stain visualizes it. The latter is by far more convenient. If analyte solubility in stain solution is inevitable. Blue-pink color is due to the 0. and then immediately wipe off an excess of stain. 52.5mL formation of Ruhemann's purple: 0. there will be some artificial tails added to spots. try to dip the plate as quickly as possible. Agric. do not forget. Also. The table below represents a few of these techniques: . Usually a light pink coloration may develop on a background. For example. Tertiary amines do not stain. in order to work.) See ninhydrin stain colors for all aminoacids! For colorless compounds. However. M.