TLC Visualization Reagents Part 4

Dear Friends,
I am going to post in this week my collection of a list of several derivatizing agents/ visualization indicators which will prove helpful to you also.continued from last post...................... 

Lead tetraacetate / 2,7-dichlorofluorescein

For detection of vicinal diols, glycosides and phenols, e.g., sugar acids

Solution I: 2% (w/v) lead tetraacetate in glacial acetic acidSolution II: 1% (w/v) 2,7-dichlorofluorescein in ethanolMix 5ml of each solution 1 and 2, fill to 200ml with dry toluene. This reagent solution is stable for only about 2hours. 

Manganese / salicylaldehyde

For detection of organothiophosphorus pesticides

Solution I: dissolve 100mg manganese chloride (MnCl2.4H2O) in 100ml 80% alcoholSolution II: dissolve 1.3g 2-hydrozine quinoline in the lowest possible volume of hot ethanol. Dissolve 1 gsalicyaldehyde in 5ml ethanol and add 1-2 drops glacial acetic acid. Combine both solutions and reflux 30 minutes. The crystals of salicyl-2-aldehyde –2-quinolinehydrazone precipitated during the cooling are recrystallized from ethanol. For solution dissolve 50mg of the salicylate derivative in 100ml ethanol

Spray with a mixture of equal volumes of solutions 1 and 2. 

Mercury (II) chloride / diphenylcarbazone

For detection of barbituratesSolution I: 2% ethanolic mercury (II) chlorideSolution II: 0.2% ethanolic diphenlycarbazoneMix freshly before use in equal parts

Results: Pink spots on a violet background 

Mercury (II) chloride / dithizone

For detection of barbiturates

Spray with a freshly prepared 1:1 mixture of 1-2% mercury (II) chloride in ethanol and 0.1-0.2% dithizone in ethanol.

View under 360nm UV light 

4-Methoxybenzaldehyde / sulfuric acid / ethanol

For detection erythromycin and metabolites

Spray with 4-methocybenzaldehyde/sulfuric acid/ethanol (1:1:9)Heat 1 minute at 100 C 

Methyl yellow

For detection of chlorinated insecticides and antimicrobial compounds

Spray dried plate with a solution of 0.1g methyl yellow (N,N-dimethyl-4-phenylazoaniline) in 70ml ethanol, add 25ml water and fill to 100ml with ethanol. Dry at ambient temperatureIrradiate 5 min with UV light without a filter

Results: Red spots on a yellow background 

Molybdenum blue reaction according to Dittmer and Lester

For detection of phospholipids and phosphoric acid derivatives

Solution I: Boil 40.11g MoO3 in 1 liter 25N sulfuric acid for 3-4 hours until the molybdenum oxide is completely dissolved. Let the light yellow solution slowly cool to ambient temperature overnight. The solution will turn light blue.Solution II: Boil 1.78 g molybdenum powder and 500ml of solution I for 15min, cool and decant from the remaining residue.For preparation of the spray reagent, add equal volumes of solutions I and II to 4.5 volume parts water.

A dark green solution is formed.Solutions I and II are stable for several months when stored in the dark. The spray reagent has to be prepared weekly.  

Ninhydrin

For detection of amino acids, amines, amino sugars.

Spray with a solution of 0.2g ninhydrin in 100ml ethanol and heat to 110 C until spots appear.

Results: reddish spots appear 

Ninhydrin / cadmium acetate

For detection of amino acids and heterocyclic amines

Dissolve 1g ninhydrin and 2.5g cadmium acetate in 10ml glacial acetic acid and fill to 500ml with ethanol.Spray and heat 20min at 120 C

Results: Red, pink, or purple spots are seen. 

Ninhydrin / pyridine / glacial acetic acid

For detection of peptidesSpray with a 1% ninhydrin in pyridine/glacial acetic acid (5:1, v/v)Heat 5 min at 100 C 

Nitric acid / ethanol

For detection of amines and alkaloids

Spray with a solution of 50 drops 65% nitric acid in 100ml ethanol (higher acid concentrations are also possible).If necessary, heat to 120 C for some time. 

Orcinol (Bials reagent)

For detection of glycosides, glycolipids

Reagent: dissolve 0.1g orcinol in 40.7ml conc. HCl, add 1ml 1% ferric (111) chloride, and dilute to 10ml

Spray and heat at 80°C for 90 minutes.

Results: Glycolipids produce violet spots.  

Paraffin oil

For enhancement of fluorescence spots – more stable and greater intensity1% paraffin oil in hexaneSpray evenly over the TLC plate

Results: spots should be more stable (no fading with time) for scanning and are of greater intensity 

m-Phenylenediamine

For detection of reducing sugars

Spray with a solution of 3.6g m-phenylenediamine dihydrochloride in 100ml 70% ethanol and heat briefly at 105°C

Results: Intensely fluorescence colors in UV light (wavelength not specified, so check 254 and 366nm). 

o-Phenylenediamine – trichloroacetic acid

For detection of alpha-keto acids

Spray with a solution of 0.05g 1,2-phenylenediamine in 100ml 10% aqueous trichloroactic acid and heat plate at 100°C for no more than 2 minutes.

Results: Green fluorescence spots in long wavelength UV light. 

p-Phenylenediamine – phthalic acid

For detection of conjugated 3-ketosteroids

Spray with a solution of 0.9 p-phenylenediamine and 1.6g phthalic acid in 100ml 1-butanol saturated with waterand heat plate at 100-110°C

Results: Yellow to orange spots 

Phenylhydrazine sulfonate

For detection of some antimicrobial compounds

Solution I: dissolve 3.5g phenylhydrazine 4-sulfonic acid hemihydrate in 10ml water and 20ml 1N NaOH solutionSolution II: mix 30ml 1N sodium hydroxide solution with 40ml acetoneThe spray reagents have to be prepared fresh each time.Procedure:Wet chromatogram evenly with spray solution 1After air drying the plate, shake spray solution 2 and spray plate. Phosphoric acidFor detection of sterols, steroids, and bile acidsSpray heavily until the layer appears transparent with a solution of 85% phosphoric acid with water (1:1, v/v)Then heat 10-15minutes at 120°C

Results: Sterols, steroids and bile acids and bile acids produce various colors under visible and UV light. 

Phosphoric acid – bromine

For detection of digitalis glycosides

Spray solution I: 10% aqueous phosphoric acid solutionSpray solution II: Mix 2ml saturated aqueous potassium bromide, 2ml saturated solution aqueous potassium bromate and 2ml 25% hydrochloric acid.Procedure: Spray plate with I and heat 12 min at 120°C.Results: Digitalis glycosides of the series B, D, and E show blue fluorescence in long wavelength UV lightProcedure continued: Heat the plate again at 120°C and spray lightly with II

Results: Glycosides of the series A show orange, of the series C show grey-green to grey-blue fluorescence in UV light. 

Phosphomolydbic acid

For detection of reducing substances, e.g, alcohols, bile acids, lipids, fatty acids, steroidsAlso used as a charring reagent for polymer bound TLC plates (the newer hard layer plates)

Spray with a solution of 250mg molybdatophosphoric acid in 50ml ethanolHeat to 120°C until spots appear (oven or heat gun)If necessary, treat with ammonia vapors to remove some background coloration.The reagent solution is stable for only 10 days even in the dark.

Results when using as a charring reagent: View frequently (every 5-10min) to see if colored or fluorescent spots (at 254 and 360nm) can be seen. Charring can be continued until spots are brown, grey or black. 

Phosphotungstic acid

For detection of cholesterol and its esters, reducing compounds, lipids, sterols, and steroids

Spray with 20% phosphotungstic acid in ethanol, heat at 110°C for 5-15min or until maximum visualization of the spots occurs

Results: Cholesterol, esters will produce red spots.

Pinacryptol yellow

For detection of sweetners, surfactants, alkyl- and arylsulfonic acids

Dissolve 100mg pinacryptol yellow in 100ml hot water or ethanol (or some combination for polymer bound plates)Spray with reagent solution

Results: Yellow to orange fluorescence spots under long wavelength UV light (366nm)