TLC Visualization Reagents
This is a brief selection of the many available TLC visualization reagents. Below each title is the type of compounds or structure which can be detected with the specific reagent. When beginning work with these reagents, acquire any MSDS (material safety data sheets) to see if there are any extra precautions needed in safely using them.
Before spraying, plates should be well dried in the hood of residual solvents and components. Amines and organic acids used in the mobile phases may adversely affect the visualization reaction being attempted. If heating to remove these components is done, consideration should be given so that loss of components or their decomposition is avoided (by lowering the temperature or using a shorter time in the oven).
Always spray any of these reagents onto plates in a well ventilated hood while wearing safety glasses. Also apply moderate amounts to the plate so it always appears dull and flat (if it looks wet, you have sprayed too much). You can always overspray to enhance the detection.
Aluminium chloride
For flavonoids
Spray plate with a 1% ethanolic solution of aluminum chloride.
Results: Yellow fluorescence in long wavelength UV light (360nm)
4-Aminoantipyrine/potassium hexacyanferrate (III) (Emerson reaction) – check something missing
(Emerson reagent) for the detection of phenols and arylamines
Solution I: 1g aminoantipyrine (4-aminophenazone) in 100ml 80% ethanol
Solution II: 4g potassium hexacyanoferrate (III) in 20ml water, fill to 100ml with ethanol
Procedure:
Spray with solution I
Dry 5 minutes with warm air
Place chromatogram in a chamber with vapor from 25% ammonium solution, making sure that the layer does not contact the liquid.
Results: Red-orange to salmon pink spots
2-Aminoethyl diphenylborate, see Ethanolamine diphenylborate
Ammonium metavanadate, ammonium monovanadate, see Vanadium(V)sulfuric acid reagent
For detection of phosphoric acid derivatives
Solution I: 1M perchloric acid in water/acetone (1:1)
Solution II: ammonium molybdate soln: 5g (NH4)6Mo7O24.4 H2O in 35ml semi-conc. Nitric acid and 65ml water.
Solution III: Tin(II) chloride soln: 0.5g SnCl2.2 H2O in 100ml 0.5M hydrochloric acid:
Dry developed chromatogram and heat to 60 C
Hydrolyse di- and triphosphates by spraying perchloric acid (solution I) onto the warm plate. After spraying 2 times, dry plate slowly at 50 C. Amidophosphates might not be decomposed.
In any case, spray the still warm plate with ammonium molybdate solution (solution II)
Then spray the still wet plate with tin (II) chloride solution (solution III)
Results: Phosphates appear as blue to blue-green spots. Polyphosphates can also be detected by dipping the plates in a solution of ammonium molybdate (1g) dissolved in water (8ml) and perchloric acid (3ml, ca. 70%),
filled up to 100ml with acetone. Then phosphates appear as yellow-green spots on a blue background. Also see Molybdenum blue reaction according to Dittmer and Lester.
For the detection of reducing sugars
Dry the developed chromatogram
Spray with 0.93g aniline and 1.66g o-phthalic acid dissolved in 100ml n-butanol saturated with water.
Briefly dry with hot air, then heat to 105 C for 10 minutes
Results: Substance spots show different colors on an almost colorless background. Some spots give fluorescence at 365nm.
and heat to 105°C until maximum visualization of spots. The background might be brightened by water vapor.
Results: Lichen constituents, phenols, terpenes, sugars, and steroids turn violet, blue, red, grey or green.
For detection of sugars
Spray with a solution of freshly prepared 1ml p-anisaldehyde, 1ml 97% sulfuric acid in 18ml ethanol and heat at 110°C.
Results: Sugar phenylhydrazones produce green-yellow spots in 3 min. Sugars will produce blue, green, violet spots in 10min. Also detects digitalis glycosides.
For detection of carbohydrates / sugars
Mix a solution of 3% p-anisidine hydrochloride in n-butanol
Spray and heat at 100°C for 2-10min.
For detection of carbohydrates and reducing sugars
Spray with a solution of 1.23 g p-anisidine and 1.66g phthalic acid in 100ml 95% ethanol.
Results: Hexoses, green; pentoses, red-violet – sensitivity 0.5ug; methylpentoses, yellow-green; uronic acids, brown – sensitivity 0.1-0.2ug.
Antimony (III) chloride
For detection of flavonoids
Spray with a 10% solution of antimony (III) chloride in chloroform
Results: Fluorescing spots in long wavelength light (360nm).
Antimony (III) chloride
For detection of vitamins A & D, carotenoids, steroids, sapogenins, steroid glycosides, terpenes
Spray with a solution of 25g antimony (III) chloride in 75ml chloroform (generally a saturated solution of antiomony (III) chloride in chloroform or carbon tetrachloride is used).
Heat 10min at 100C, view under long wavelength light (360nm).
For detection of organothiophosphorous pesticides
Place chromatogram in a chamber with a 10% bromine and tetrachloride without contact with the liquid.
For detection of organic acids
Dip chromatogram in a solution of 0.1g bromocresol green in 500ml ethanol and 5ml 0.1M NaOH
Results: Acids yield yellow spots on a blue background.
Bromthymol Blue
For detection of lipids and phospholipids
Reagent: 0.1% bromthymol blue in 10% aqueous ethanol made just alkaline with NH4OH
Spray dried plate.
Results: Compounds above produce blue-green colors; sensitivity 0.1-1µg.
For detection of phenols
Spray with a solution of 1% tetrachloro-p-benzoquinone in toluene
Chlorine / o-tolidine
For detection of of compounds forming chloroamines, e.g., urea derivatives, carbamated, antibiotics
Solution I: 160mg o-tolidine in 30ml glacial acetic acid, filled to 500ml with distilled water, plus 1g KI solution
Solution II: saturated solution of o-tolidine in 2% acetic acid/0.85% KI solution (1:1, v/v)
Procedure A
Place chromatogram 15-20min in a chlorine atmosphere (e.g., Potassium permanganate +10% Hydrochloric acid)
Leave 5 minutes at ambient temperature until the chlorine is evaporated completely (spray corner of plate to insure no blue color is seen, showing complete absence of chlorine).
Spray with solution I
Procedure B
Spray with 2% potassium hypochlorite solution in water
Leave 1-1.5hr at ambient temperature
Spray with solution II
Copper sulfate / phosphoric acid
Used as a charring reagent for polymer bound TLC plates (the newer hard layer plates)
Spray with a solution of 10% copper (II) sulfate in 10% phosphoric acid
Heat 5-30min at 110°C
Results: 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.
Chromosulfuric acid
See under Potassium dichromate / sulfuric acid
DDQ Reagent (Dichlorodicyanobenzoquinone)
For detection of phenols
Spray with a solution of 2% 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in toluene
For the detection of sweeteners saccharine & cyclamate
Spray with a 0.2% solution of dichlorofluorescein in 96% ethanol
Dry with warm air; if necessary, spray with water
View under 360nm UV light
For detection of N-substituted barbiturates
Spray with a 0.1% ethanolic solution of dichlorofluorescein
Then spray with a 0.1% ethanolic solution of fluorescein sodium salt
2,6-Dichloroquinone –4- chloroimide
For detection of antioxidants, phenols, primary and secondary aliphatic amines, secondary and tertiary aromatic amines, aromatic hydrocarbons, pharmaceuticals, phenoxyacetic acid herbicides, etc
Spray with a freshly prepared 0.5-2% solution of 2,6-dichloroquinone-4-chloroimide in ethanol (reagent stable for 3 weeks if refrigerated).
Heat 10min at 110 C; treat with ammonia vapor
p-Dimethylaminobenzaldehyde
For detection of sulfonamides
Spray with a solution of 1% p-dimethylaminobenzaldehyde in 5% hydrochloric acid; add 5% ethanol
Detects sulfonamides
p-Dimethylaminobenzaldehyde / hydrochloric acid reagent (Ehrlich’s reagent)
For detection of amines, indole derivatives
Spray with a solution of 1% p-dimethylaminobenzaldehyde in conc. hydrochloric acid/methanol (2:2)
Heat plates for 20min at 50 C
2,4-Dinitrophenylhydrazine
For detection of aldehydes and ketones
Spray plate with solution of 0.4 g 2,4-DNPH in 100ml 2N hydrochloric acid, add 1ml ethanol
Results: Yellow-red spots will be seen.
For detection of glycosides, glycolipids
Reagent: 10ml 10% diphenylamine in ethanol, 100ml HCl and 80ml glacial acetic acid
Spray lightly, cover plate with another glass plate, heat 30-40min at 110°C until positive areas appear
Results: Glycolipids produce blue spots.
s-Diphenylcarbazone
For detection of barbiturates
Spary with a solution of 0.1% s-diphenylcarbazone in 95% ethanol
Results: Barbiturates will produce purple spots
2,2’-Diphenylpicrylhydrazyl
For detection of aldehydes and ketones
Reagent: dissolve 15mg of 2,2’-DPPH in 25ml chloroform
Spray, heat 5-10min at 110°C;
Results: Yellow spots on a purple background will be seen.
For detection of heavy metal ions
Dissolve 20mg dithizone in 100ml acetone, store in a brown bottle in a refrigerator
Procedure:
Spray with dithizone solution
Spray with 25% ammonia solution
See Molybdenum blue reaction
For detection of nitrogen compounds, alkaloids, antiarrhythmic drugs, surfactants
Solution 1) 1.7g basic bismuth nitrate and 20g tartaric acid in 80ml water
Solution 2) 16 g potassium iodide in 40ml water
Stock solution (stable for several weeks in a refrigerator):
Mix equal volumes of solutions 1 and 2
Procedure:
Spray with a solution of 10g tartaric acid, 50ml water and 5ml stock solution
Ethanolamine diphenylborate (flavone reagent according to Neu)
For detection of flavonoids
Spray with a 1% solution of ethanolamine diphenylborate in methanol
Spray with a 5% ethanolic solution of polyyethylene glycol for fluorescence stabilization
Irradiate 2 minutes with intense 365nm UV light
View under 365nm UV light
See p-Dimethylaminobenzaldehyde
See 4-aminoantipyrine/potassium hexa-cyanoferrate (III)
For detection of cannabinoids, phenols, tanning agents, amines with can be coupled
Spray with a solution of 0.5g Fast Blue B (tetraazotized di-o-anisidine) in acetone/water (9:1, v/v), always prepared fresh
Then overspray with 0.1M sodium hydroxide solution
Results: Cannabinoids turn dark red/purple in color
Ferric Chloride / sulfuric acid
Used as a charring reagent for polymer bound TLC plates (the newer hard layer plates)
Spray with a solution of 2g FeCl3 in 83ml n-butanol and 15ml conc. sulfuric acid.
Heat 5-30min at 110°C
Results: 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.
See ethanolamine diphenyl borate
For detection of primary and secondary amines, peptides, sulfonamides, e.g., nitrosoamines after photolysis
Spray plate with a solution of 0.1mg/ml 4-phenyl-spiro[furan-2(3H),1-phthalan]-3,3-dione in acetone prepared fresh daily
For stabilization of fluorescence at 366nm spray with 10g triethylamine, brought to 100ml with dichloromethane.
Fluorescent Indicator
For detection of compounds which absorb UV light
Some TLC plates when manufactured have an inorganic fluorescent indicator added to the slurry poured to make the final plates. This type of indicator will not dissolve or elute off. They are activated at 254nm or 360nm (see recommendations of the manufacturer for that type of plate).
Results: When activated the fluorescent indicator will turn a green or white (depending on the indicator added) and the compounds appear as dark spots or shadows against this background. If viewing at other than the activation wavelength, the compounds might also have some fluorescence of their own, so various colors against a dark background would be seen.
For detection of alkaloids, aromatic hydrocarbons, e.g., antihypertensive drugs
Spray with a solution of 37% formaldehyde in conc. sulfuric acid (1:10) immediately after taking the plate from the developing chamber. Heating is not necessary.
Results: various colored spots.
Formaldehyde / phosphoric acid
For detection of steroid alkaloids, steroid sapogenins and phenothiazine derivatives
Spray with a solution of 0.03g formaldehyde in 100ml of 85% phosphoric acid with stirring at room temperature. The reagent is stable for several weeks.
For detection of carbamate esters
Spray solution I: 1% solution of furfural in acetone
Spray solution II: 10% solution of sulfuric acid in acetone
Spray plate with I, then II.
Spray 0.1% gentian violet (crystal violet) in methanol onto plate and place in a tank containing bromine vapor.
Results: lipids produce blue spots on a yellow background.
For detection of phenols. For further applications see 2,6-dichloroquinone-4-chloroimide
Spray with a solution of 3% 2,6-dibromo-N-chloro-p-benzoquinone imine in toluene or methanol.
For detection of amides, lactones, carboxylic acid esters and anhydrides
Solution 1) Mix 1 vol part of 7g hydroxylammonium chloride in 100ml methanol w 1 vol part of a solution of 7.2 g potassium hydroxide in 100ml methanol. Filter from precipitated potassium chloride.
Solution 2) 2% solution of iron (III) chloride in 1% aqueous hydrochloride acid
Spray air dried plate first with solution 1, then with solution 2
Detection by decomposition under UV
Dry plates at 100 C
After cooling spray with a small amount of 50% acetic acid
Irradiate some minutes with unfiltered UV light.
Results: Iodine compounds show weakly violet to brown spots. The color can be enhanced by spraying with 10% acetic acid and irradiation with UV light (sudden appearance of blue spots).
Relatively unspecific universal reagent for many organic compounds
Charge chamber with some crystals of iodine
Place developed, dried chromatogram in iodine vapor
Results: spots turn tan-brown in color
For detection of organic nitrogen compounds, alkaloids, e.g., cocaine metabolites
Spray with a freshly prepared mixture of 3ml hexachloroplatinic (IV) acid solution (10%) in 97ml/min water and 100ml aqueous potassium iodide solution.
Note - use 5% ethanol or methanol in water to prepare these solutions for the new polymer bound TLC plates.
Iron (III) chloride / potassium hexacyanoferrate / sodium arsenate (according to Patterson & Clements)
For detection of iodine compounds, e..g., thyroid gland hormones
Solution I: 2.7% iron (III) chloride hexahydrate in 2N hydrochloric acid
Solution II: 3.5% potassium hexacyanoferrate in water
Solution III: dissolve 3.8g arsenic trioxide in 25ml 2N sodium hydroxide solution heating slightly, cool to 5 C, and add 50ml 2N sulfuric acid, fill to 200ml with water
Immediately before use mix 5ml solution I, 5ml solution II and 1ml solution III.
Spray onto the dry layer and dry carefully (temp below 50 C)
Cover with glass plate and leave 15min in the dark
Results: Iodine containing compound show light blue spots on a yellowish background.
For detection of vicinal diols, glycosides and phenols, e.g., sugar acids
Solution I: 2% (w/v) lead tetraacetate in glacial acetic acid
Solution II: 1% (w/v) 2,7-dichlorofluorescein in ethanol
Mix 5ml of each solution 1 and 2, fill to 200ml with dry toluene. This reagent solution is stable for only about 2 hours.
For detection of organothiophosphorus pesticides
Solution I: dissolve 100mg manganese chloride (MnCl2.4H2O) in 100ml 80% alcohol
Solution II: dissolve 1.3g 2-hydrozine quinoline in the lowest possible volume of hot ethanol. Dissolve 1 g salicyaldehyde 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.
See Vanadium(V) / sulfuric acid reagent
For detection of barbiturates
Solution I: 2% ethanolic mercury (II) chloride
Solution II: 0.2% ethanolic diphenlycarbazone
Mix freshly before use in equal parts
Results: Pink spots on a violet background
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
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 temperature
Irradiate 5 min with UV light without a filter
Results: Red spots on a yellow background
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.
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
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.
For detection of peptides
Spray with a 1% ninhydrin in pyridine/glacial acetic acid (5:1, v/v)
Heat 5 min at 100 C
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.
Orcinaol (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. <