TLC Stains Preparation

Why stain a TLC plate?

When a TLC plate has been developed it is required to identify the spots on the plate. If the compounds (components of spot) are colored then the spots can be easily seen by naked eye, but in many cases the compounds are colorless. Usually many of the organic compounds contain a chromophore and such compounds can be seen under a long or short range UV-lamp depending upon the chromophore present in the compound. The silica on the commercial TLC plates are impregnated with a fluorophor (exhibits fluorescence upon UV irradiation) so that they exhibit fluorescence when exposed to UV light. When a developed TLC plate is placed under UV lamp the UV active spots (compounds containing chromophores) absorb the UV light and they quench the fluorescence in that particular area so they appear dark colored spots. These UV active spots can marked with a pencil and the plate can be stained by using permanant and temporary stains. Often these stains are also helpful in identifying the functional group present in the compound. 

Many of the stains require heating of the plate for the visualisation of spots so make sure the type of TLC plate you are using. For example take the case of inexpensive polymer backed TLC plates. These can't be used for stains where heating is required for visulaising the spots. Glass and aluminium backed plates are suitable for all kind of stains including stains where heating is required to visualise the spots.

Temporary stains

Iodine is the only temperory stain available of now. This is one of the oldest methods for the visualization of organic compounds on TLC plates. Iodine has great affinity for unsaturated and aromatic compounds.

Preparation: To a glass bottle with cap (bottle size depends on how much stain you prepare)add 100 g of silica and 5 - 7 g of iodine crystals. Close the cap and shake 3 - 4 times such that iodine is dispersed over silica. Insert the TLC plate into the chamber and remove it after it develops light brown color over the entire plate. If the compound is iodine active then it apperas as dark brown spot. Circle the spots with a pencil as soon as they appear because iodine will not remain on TLC plate for longer time.

Permanant Stains

Permanant stains work by reacting with the chemical species and there by giving a characteristic color a compound which can be identified by visualization. In order for the stains to appear some times the plate needs to be heated with a hot gun. So use of aluminium or glass backed TLC plates is good.

P-Anisaldehyde Stain

This stain can be used for most of the functional groups. It is required to heat the TLC plate mildly to stain the plate. Different functional groups tend to show different color upon staining. As a hint of a track of which functional group shows which color may be kept for reference but this cannot be trusted fully.

Preparation:

Slowly add conc. H₂SO₄ (5.1 mL) to a solution of ethanol (133 mL) and water (7 mL). Cool the above solution to 5 to 10 °C and add p-Anisaldehyde (3.75 mL) in glacial acetic acid (1.6 mL). Stir the resulting solution to make it homogenous. The solution can be stored in a glass wide mouth jar with cap. Store away from direct sunlight.

2,4-Dinitrophenylhydrazine (2,4-DNP) Stain

This stain is mainly used for detection of aldehydes and ketones as they form hydrazones upon reacting with this stain and then apppear as yellow-orange spots. The TLC is dipped into the stain and removed for the stains to appear. No heating is required.

Preparation:

Dissolve 2,4-Dinitrophenylhydrazine (6 g) in 95%ethanol (100 mL) and to this add water (40 mL). Stir the resulting mixture to get a clear solution and slowly add conc. H₂SO₄ (60 mL) and stir to get a clear solution. The solution can be stored in widemouth glass bottle with cap and away from direct sunlight.

Bromocresol Stain

This stain is used for compounds whose pKa is approximately 5 and lower (like carboxylic acids or sulphonic acids). The spots appear as yellow spots on a blue background. and no heating is reuired for staining. Bromophenol can be substituted for bromocresol stain. This stain is also called as bromocresol green.

Preparation:

Dissolve bromocresol green (0.08 g) in ethanol (200 mL) to get a clear colorless solution. Slowly add 0.1 N NaOH dropwise until blue color just appears in the solution. The stain can be stored in widemouth glass bottles with cap and away from direct sunlight.

CAM (verghn's) Stain

This is a universal stain. It is required to heat the TLC plate upon dipping for the stains to appear. The spots appear as blue back spots on light background.

Preparation:

Slowly add conc. H₂SO₄ (80 mL) to water (720 mL) under stirring followed by ammonium molybdate (40 g) and ceric ammonium sulfate (1.6 g). Stir the resulting mixture to get a clear solution. During this preparation the order of addition is very important. The stain can be stored in widemouth glass bottles with cap and away from direct sun light.

Cericsulphate stain

This stain is veey effective for alkaloids. This stain should be sprayed on to the plate (not dipped) and then heated for the stains to appear as black spots on yellow-white background.

Preparation:

Dissolve Ceric sulfate (8 g) in 15% sulfuricacid (aq) (100 mL) and stir the resulting solution to get a clear solution. the solution can be stored in widemouth glass bottle with cap and away from direct sunlight.

Dragendorff's Stain 

This stain is useful for unreactive amines (especially for carbamate protected amines), alkaloids. The compounds appear as orange spots. Heating is not required for the TLC plate to stain.

Preparation:

Dragendorff's stain is unstable it should be prepared every 2 weeks by mixing the below described solutions.

Solution-1 Dissolve bismuthyl nitrate (3.4 g) in 20% aq acetic acid (200 mL).

Solution-2 Dissolve potassium iodide (40 g) in DI water (200 mL).

Dragendorff's stain is prepared by mixing solution-1 (1 mL), solution-2 (1 mL) to a solution of glacial acetic acid (4 mL) in DI water (14 mL). The resulting solution needs to be stored in glass bottles in refrigerator and tightly capped as it is unstable (~2 weeks life time). The individual solutions-1,2 can be stored for longer periods. 

Ferric chloride Stain

This stain is used for phenols. No heating is rewuired for staining the TLC plates.

Preparation:

Dissolve ferric (lll) chloride (1 g) in a mixture of methanol (50 mL) and DI water (50 mL). Stir the above mixture to get a homogenous solution. This stain can be stored at rt in tightly capped wide mouth glass bottle and store away from light.

Hanessian's (Ceric Molybdate) stain

This is highly sensitive (even minor impurities or constituents may show significant color) stain. the TLC plate should be dipped into this stain and heated vigorously for plate to stain. The sopts would appear as dar blue on light blue or green background. One cannot distinguish functional groups based upon color of spots but it is good to keep a track of color variations with functional groups. This will be useful to correlate while doing similar chemistry.

Preparation:

Dissolve ammonium molybdate (6 g), ceric ammonium molybdate (0.25 g) in DI water (120 mL). To the above solution add slowly add conc. sulfuric acid (7.5 mL) and stir to get a clear solution. The solution can be stored in tightly sealed glass bottle covered with aluminium foil (stain is photo sensitive). The stain tends to fade away after a few days so it is a good idea to circle the spots upon staining.

Iodoplatinate Stain

This works like potassium permangenate stain. Usually it is not required to heat the TLC plate dipped in stain but if heated the spots appear faster (less visibility). With no heating the spots appear slow (greater visibilty). This stain is also called PIP (potassium iodoplatinate) stain

Preparation:

Dissolve hexachloroplatinate(alpha) (0.5 g) and potassium iodide (10 g) in DI water (295 mL). To the above mixture add conc. HCl (27 mL). Stir the mixture 4 hours at 0 °C. Store the stain in wide mouth glass bottle in refrigerator at 0 °C. This stain decoposes over period of time.

KMnO₄ (Potassium permanganate) Stain

This stain is useful for functional groups which are easily oxidisable. Some functional groups which are detected are diols, C=C, reactive methylene, phenol, thiol, phosphine etc. One good thing is that tertiary amines can be easily detected by this stain also good for weinreb amides. It is a good idead to circle the developed spots as thestains tends to fade away in few days. Usually heating is not required for the stains to develop but in some cases mild heating may be done for the spots to appear. The spots appear as yellow spots on purple background.

Preparation:

Dissolve KMnO4 (1.5 g), K2CO3 (10 g) in DI water (200 mL). To this add 10% NaOH (1.25 mL) and stir to get a clear solution. It would take sometime for the solution to get clear. The solution can be stored in widemouth glass bottles with cap at rt and away from direct sunlight. The stain is stable for abut 2 - 3 months.

Morin Hydrate Stain

This a general stain which is fluorescently active. Morin is a hydroxy flavone.

Preparation:

Dissolve Morin hydrate (100 mg) in methanol (100 g) and stir to get a clear solution. The solution can be stored at rt in widemouth glass bottle with cap.

Ninhydrin Stain

This excellent stain for amino acids. Never spill this stain onto your hands because they turn your hands blue. Primary amine show blue color at rt, Boc protected amines show up as brown spots only upon heating (Boc grop falls off) and sometimes secondary amines are detected but not very clear.

Preparation:

Dissolve Ninhydrin (1.5 g) in n-butanol (100 mL) and then add glacial acetic acid (3 mL). Ethanol can be used in place of butanol. The solution can be stored in wide mouth glass bottle with cap. This can stored at rt and away from direct sunlight.