Editors
TLC detection reactions

Please note, that only a part of these reagents are supplied by MACHEREY-NAGEL




4-Aminoantipyrine/potassium hexacyanoferrate(III) (Emerson reagent)
for detection of phenols and aryl amines

Solutions:

  1. 1 g Aminoantipyrine (4-aminophenazone) in 100 mL 80 % ethanol
  2. 4 g Potassium hexacyanoferrate(III) in 50 mL water, fill up to 100 mL with ethanol
Procedure:
  • Spray with solution 1
  • Dry 5 minutes with warm air
  • Spray with solution 2
  • Again dry 5 minutes with warm air
  • Place chromatogram in a chamber with vapour from 25 % ammonia solution, making sure that the layer does not contact the liquid

2-Aminoethyl diphenylborate see under Ethanolamine diphenylborate


Ammonium metavanadate, ammonium monovanadate see under Vanadium(V)/sulfuric acid reagent


Ammonium molybdate for detection of phosphoric acid derivatives

Solutions:

  1. 1 mol/L Perchloric acid in water – acetone (1:1, v/v)
  2. Ammonium molybdate solution: 5 g (NH4)6Mo7O24 · 4 H2O in 35 mL semi-concentrated nitric acid and 65 mL water
  3. Tin(II) chloride solution: 0.5 g SnCl2 · 2 H2O in 100 mL 0.5 mol/L hydrochloric acid
Procedure:
  • Dry developed chromatogram and heat to 60 °C
  • Hydrolyse di- and triphosphates by spraying perchloric acid (solution 1) onto the warm plate. After spraying 2 times, dry plate slowly at 50 °C; amidophosphates e.g., need not be decomposed.
  • In any case spray the still warm plate with ammonium molybdate solution (solution 2)
  • Then spray the still wet plate with tin(II) chloride solution (solution 3). Phosphates appear as blue to blue-green spots.
Polyphosphates can also be detected by dipping the plates in a solution of ammonium molybdate (1 g) dissolved in water (8 mL) and perchloric acid (3 mL, approx. 70 %), filled up to 100 mL with acetone. Then phosphates appear as yellow-green spots on a blue background. Also see Molybdenum blue reaction according to Dittmer and Lester.


Aniline phthalate spray ragent for detection of reducing sugars
  • Dry the developed chromatogram
  • Spray with aniline phthalate spray reagent
  • Briefly dry with hot air
  • Heat to 80–130 °C for 20–30 min
Resulting substance spots show different colors on an almost colorless background. Some spots give a fluorescence at 365 nm.
Aniline phthalate in 2-propanol – ethanol (1:1, v/v) is available under REF 814919.


Anisidine phthalate reagent for detection of carbohydrates
  • Spray with a solution of 1.23 g p-anisidine and 1.66 g phthalic acid in 100 mL 95 % ethanol 
Hexoses yield green, methylpentoses yellow-green, pentoses purple, uronic acids brown spots.


Boute reaction for detection of phenols
  • Dry and heat the developed chromatogram
  • Place hot plate for 3–10 minutes in a chamber with NO2 vapour (from conc. nitric acid)
  • Then treat with NH3 vapour (from conc. ammonia)

Bromine/carbon tetrachloride for detection of organothiophosphorus pesticides
  • Place chromatogram in a chamber with 10 % bromine/carbon tetrachloride without contact of the liquid

Bromocresol green for detection of acids
  • Dip chromatogram in a solution of 0.1 g bromocresol green in 500 mL ethanol and 5 mL 0.1 mol/L sodium hydroxide solution
Acids yield yellow spots on blue background.
Bromocresol green in 2-propanol is available under REF 814920.


Chloranil reagent for detection of phenols
  • Spray with a solution of 1 % tetrachloro-p-benzoquinone in benzene or toluene

Chlorine/o-tolidine
for detection of compounds forming chloroamines,
e.g. urea derivatives, carbamates, antibiotics

Solutions:

  1. 160 mg o-Tolidine in 30 mL glacial acetic acid, filled up to 500 mL with dist. water, plus 1 g potassium iodide
  2. Saturated solution of o-tolidine in 2 % acetic acid – 0.85 % potassium iodide solution (1:1, v/v)
Procedure A:
  • Place chromatogram 15–20 minutes in a chlorine atmosphere (e.g., 1.5 % potassium permanganate + 10 % hydrochloric acid)
  • Leave 5 minutes at ambient temperature until the chlorine is evaporated completely
  • Spray with solution 1
Procedure B (according to Greig and Leaback)
  • Spray with 2 % potassium hypochlorite solution in water
  • Leave 1–1.5 hours at ambient temperature
  • Spray with solution 2

DDQ reagent (dichlorodicyanobenzoquinone) for detection of phenols
  • Spray with a solution of 2 % 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in benzene or toluene

Dichlorofluorescein reagent for detection of sweeteners saccharin and cyclamate
  • Spray with a 0.2 % solution of dichlorofluorescein in 96 % ethanol
  • Dry with warm air
  • If necessary spray with water
  • View under UV at 360 nm
2´,7´-Dichlorofluorescein in 2-propanol for detection of lipides is available under REF 814921.


Dichlorofluorescein/fluorescein sodium salt for detection of N-substituted barbiturates
  • Spray with a 0.1 % ethanolic solution of dichlorofluorescein
  • Then spray with a 0.1 % ethanolic solution of the 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
  • Heat 10 minutes to 110 °C
  • Treat with ammonia vapour

4-(Dimethylamino)-benzaldehyde/hydrochloric acid reagent (Ehrlich's reagent)
for detection of amines, indole derivatives
  • Spray with a solution of 1 % 4-(dimethylamino)-benzaldehyde in conc. hydrochloric acid – methanol (1:1, v/v)
  • Heat plates 20 min to 50 °C
4-(Dimethylamino)-benzaldehyde in 2-propanol for detection of terpenes, sugars and steroids is available under REF 814922.


Dithizone for detection of metal ions

Solution:

  • Dissolve 20 mg dithizone in 100 mL acetone, store in a brown bottle in a refrigerator.
Procedure:
  • Spray with dithizone solution
  • Spray with 25 % ammonia solution

Dittmer and Lester see Molybdenum blue reaction


Dragendorff reagent for detection of nitrogen compounds, alkaloids, antiarrhythmic drugs, surfactants

Solutions:
  1. 1.7 g Basic bismuth nitrate and 20 g tartaric acid in 80 mL water
  2. 16 g Potassium iodide in 40 mL water
Stock solution:
  • Mix equal volumes of solutions 1 and 2; stable several weaks in a refrigerator
Procedure:
  • Spray with a solution of 10 g tartaric acid, 50 mL water and 5 mL stock solution
Reagent according to Dragendorff-Munier in water is available under REF 814402.


Ethanolamine diphenylborate (flavone reagent acc. to Neu), e.g. for flavonoids
  • Spray with a 1 % solution of ethanolamine diphenylborate in methanol
  • Spray with a 5 % ethanolic solution of polyethylene glycol for fluorescence stabilization
  • Irradiate 2 minutes with intense UV at 365 nm
  • View under UV at 365 nm

Ehrlich's reagent see 4-(Dimethylamino)-benzaldehyde


Emerson reagent see 4-Aminoantipyrine/potassium hexacyanoferrate(III)


Fast Blue B reagent for detection of cannabinoids, phenols, tanning agents, amines which can be coupled
  • Spray with a solution of 0.5 g Fast Blue B [tetraazotized di-o-anisidine] in acetone – water (9:1, v/v), always prepared fresh
  • Spray with 0.1 mol/L sodium hydroxide solution

Flavone reagent acc. to Neu see under Ethanolamine diphenylborate


Fluorescamine for detection of primary and secondary amines, peptides and sulfonamides, e.g. nitrosamines after photolysis
  • Spray plate with a solution of 0.1 mg/mL 4-phenyl-spiro[furan-2(3H),1-phthalan]-3,3-dione in acetone, prepared fresh daily
  • For stabilization of fluorescence at 366 nm spray with 10 g triethylamine, filled up to 100 mL with dichloromethane

Formaldehyde/sulfuric acid for detection of alkaloids, aromatic hydrocarbons, e.g. antihypertensive drugs
  • Spray with a solution of 37 % formaldehyde in conc. sulfuric acid (1:10)

Gibb's reagent for detection of phenols. For further applications see 2,6-Dichloroquinone-4-chloroimide.
  • Spray with a solution of 2 % 2,6-dibromo-N-chloro-p-benzoquinone imine in benzene or methanol

Hydroxylamine/iron(III) chloride for detection of amides, lactones, carboxylic acid esters and anhydrides

Solutions:

  1. Mix 1 volume part of 7 g hydroxylammonium chloride in 100 mL methanol with 1 volume part of a solution of 7.2 g potassium hydroxide in 100 mL methanol. Filter from precipitated potassium chloride.
  2. 2 % solution of iron(III) chloride in 1 % aqueous hydrochloric acid
Procedure:
  • Spray airdried plate first with solution 1, then with solution 2
Iron(III) chloride in water for detection of acetylsalicylic acid / paracetamol is available under REF 814403.


Iodine compounds
  • Detection by decomposition under UV
  • Dry plates at 100 °C
  • After cooling spray with little 50 % acetic acid
  • Irradiate some minutes with unfiltered UV light
Iodine compounds show weakly violet to brown spots. The color can be enhanced by spraying to transparency with 10 % acetic acid and irradiation with UV light (sudden appearance of blue spots)


Iodine vapour, relatively unspecific universal reagent for many organic compounds
  • Charge chamber with some crystals of iodine
  • Place developed, dried chromatogram in iodine atmosphere

Iodoplatinate reagent for detection of organic nitrogen compounds, alkaloids, e.g. cocaine metabolites
  • Spray with a freshly prepared mixture of 3 mL hexachloroplatinic(IV) acid solution (10 %) in 97 mL water
    and 100 mL 6 % aqueous potassium iodide solution

Iron(III) chloride/potassium hexacyanoferrate/sodium arsenate (acc. to Patterson and Clements)
for detection of iodine compounds, e.g. thyroid gland hormones

Solutions:
  1. 2.7 % Iron(III) chloride hexahydrate in 2 mol/L hydrochloric acid
  2. 3.5 % Potassium hexacyanoferrate in water
  3. Dissolve 3.8 g arsenic trioxide in 25 mL 2 mol/L sodium hydroxide solution heating slightly, cool to 5 °C
    and add 50 mL 1 mol/L sulfuric acid, fill up to 100 mL with water
Procedure:
  • Immediately before use mix 5 mL solution 1, 5 mL solution 2 and 1 mL solution 3; spray onto the dry layer
  • Dry carefully (temperature below 50 °C)
  • Cover with glass plate and leave 15 minutes in the dark
Iron(III) chloride in water for detection of acetylsalicylic acid / paracetamol is available under REF 814403.

Potassium hexacyanoferrate(III) in water for detection of acetylsalicylic acid / paracetamol is available under REF 814404.


Lead tetraacetate/dichlorofluorescein
for detection of vicinal diols, glycosides and phenols, e.g. sugar acids

Solutions:

  1. 2 % (w/v) Lead tetraacetate in glacial acetic acid
  2. 1 % (w/v) 2´,7´-Dichlorofluorescein in ethanol
Procedure:
  • Mix 5 mL each of solution 1 and 2 and fill up to 200 mL with dry benzene or toluene. This reagent solution is stable for only about 2 hours.

Manganese/salicylaldehyde for detection of organothiophosphorus pesticides

Solutions:

  1. Dissolve 100 mg manganese chloride (MnCl2 · 4 H2O) in 100 mL 80 % ethanol
  2. Dissolve 1.3 g 2-hydrazine quinoline in the lowest possible volume of hot ethanol. Dissolve 1 g salicylaldehyde in 5 mL ethanol and add 1–2 drops glacial acetic acid. Combine both solutions and reflux 30 min. The crystals of salicyl-2-aldehyde 2-quinolylhydrazone precipitated during cooling are recrystallized from ethanol. For solution 2 dissolve 50 mg of the salicyl derivative in 100 mL ethanol.
Procedure:
  • Spray with a mixture of equal volumes of solutions 1 and 2

Mandelin's reagent see under Vanadium(V)/sulfuric acid reagent


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 UV light at 360 nm

4-Methoxybenzaldehyde/sulfuric acid/ethanol for detection of erythromycin and metabolites
  • Spray with 4-methoxybenzaldehyde/sulfuric acid/ethanol (1:1:9, v/v/v)
  • Heat 1 min to 110 °C

Methyl yellow for detection of chlorinated insecticides and antimicrobial compounds

Solution:

  • Dissolve 0.1 g methyl yellow N,N-dimethyl-4-phenylazoaniline) in 70 mL ethanol, add 25 mL water
    and fill up to 100 mL with ethanol
Procedure:
  • Spray with reagent solution
  • Dry at ambient temperature
  • Irradiate 5 min with UV light (without filter)

Molybdatophosphoric acid
for detection of reducing substances, e.g. alcohols, bile acids, lipids, fatty acids, steroids
  • Spray with a solution of 250 mg molybdatophosphoric acid in 50 mL ethanol
  • Heat to 120 °C until spots appear (heatgun)
  • If necessary treat with ammonia vapour
Molybdatophosphoric acid in ethanol is available under REF 814302.


Molybdenum blue reaction according to Dittmer and Lester
for detection of phospholipids and phosphoric acid derivatives

Solutions:

  1. Boil 40.11 g MoO3 in 1 L 12.5 mol/L sulfuric acid for 3–4 hours until the molybdenum oxide is completely dissolved. Let the light yellow solution slowly cool to ambient temperature over night, the solution will turn light blue.
  2. Boil 1.78 g molybdenum powder and 500 mL of solution 1 for 15 min, cool and decant from the remaining residue.
For preparation of the spray reagent add equal volumes of solutions 1 and 2 to 4.5 volume parts water. A dark green solution is formed. Solutions 1 and 2 are stable for several months when stored in the dark, the spray reagent has to be prepared about weekly.


Ninhydrin for detection of amino acids, amines, aminosugars
  • Spray with a solution of 0.2 g ninhydrin in 100 mL ethanol
  • Heat to 110 °C until reddish spots appear
Ninhydrin in ethanol is available under REF 814203.


Ninhydrin/cadmium acetate for detection of amino acids, and heterocyclic amines
  • Dissolve 1 g ninhydrin and 2.5 g cadmium acetate in 10 mL glacial acetic acid and fill up to 500 mL with ethanol
  • Spray and heat 20 minutes to 120 °C

Ninhydrin/pyridine/glacial acetic acid for detection of peptides
  • Spray with 1 % ninhydrin in pyridine – glacial acetic acid (5:1, v/v)
  • Heat 5 minutes to 100 °C

Nitric acid/ethanol for detection of amines, and alkaloids
  • Spray with a solution of 50 drops 65 % nitric acid in 100 mL ethanol (higher acid concentrations are also possible)
  • If necessary heat to 120 °C for some time

Patterson and Clements see under Iron(III) chloride/potassium hexacyanoferrate/sodium arsenate


Phenylhydrazine sulfonate for detection of some antimicrobial compounds

Solutions:
  1. Dissolve 3.5 g phenylhydrazine 4-sulfonic acid hemihydrate in 10 mL water and 20 mL 1 mol/L sodium hydroxide solution
  2. Mix 30 mL 2 mol/L sodium hydroxide solution with 40 mL acetone
The spray reagents have to be prepared fresh every time.

Procedure:

  • Wet chromatogram evenly with spray solution 1
  • After airdrying the plate shake spray solution 2 and spray

Phosphomolybdic acid
see under Molybdatophosphoric acid


Pinacryptol yellow for detection of sweeteners, surfactants

Solution:
  • Dissolve 100 mg pinacryptol yellow in 100 mL hot water or ethanol
Procedure:
  • Spray with reagent solution
  • View under UV light

Potassium dichromate/sulfuric acid (chromosulfuric acid), universal visualization reagent for organic compounds (e.g. alcohols, also for bile acids, lipids)
  • Spray with a solution of 5 g potassium dichromate in 100 mL conc. sulfuric acid
  • If necessary heat plate to 150 °C

Potassium permanganate/sulfuric acid, universal reagent for organic compounds, e.g. fatty acid derivatives
  • Spray with a solution of 1.6 % potassium permanganate in conc. sulfuric acid
    (Beware of explosions during dissolution!)
  • Heat plates 15 to 20 minutes to 180 °C

Pyrocatechol violet reagent for detection of organotin compounds
  • Completely decompose metalorganic compounds by irradiation of the developed plates with UV light
  • Dip plates in a solution of 1 g pyrocatechol violet in 1000 mL ethanol (99.5%)

Rhodamin B spray reagent for detection of lipids
  • Dry the developed chromatogram
  • Spray with rhodamin B spray reagent
In most cases red-violet zones with an intense fluorescence at 365 nm develop on a pink background. RP phases are less suited, because in this case the environment of the spots also forms an intense color. By placing the sprayed chromatograms into an NH3 atmosphere the detection sensitivity can be improved.
Rhodamin B in ethanol is available under REF 814923.


Rubeanic acid for detection of heavy metal ions
  • Spray with a solution of 0.5 % rubeanic acid in ethanol
  • Dry briefly
  • Spray with 25 % ammonia solution or place in a chamber with ammonia vapour
Rubeanic acid in ethanol is available under REF 814206.


Silver nitrate/hydrogen peroxide for detection of halogenated hydrocarbons

Solution:

  • Dissolve 0.1 g silver nitrate in 1 mL water, add 10 mL 2-phenoxyethanol, fill up to 200 mL with acetone and add 1 drop hydrogen peroxide (30 % solution)
Procedure:
  • spray plate with reagent solution
  • irradiate with unfiltered UV light until optimal contrast is obtained
Luckow [Fresenius Z. Anal. Chem. 294 (1979) 288] uses silver nitrate/phenoxyethanol/acetone without addition of hydrogen peroxide for detection of the pesticide ioxynil (3,5-diiodo-4-hydroxybenzonitrile).


Sodium nitroprussiate/potassium hexacyanoferrate(III) for detection of aliphatic nitrogen compounds

Solution:

  • Mix 1 volume part each of 10 % aqueous sodium hydroxide solution, 10 % sodium nitroprussiate solution and 10 % potassium hexacyanoferrate(III) solution with 3 volume parts water. Let the solution stand at least 20 min at ambient temperature before use. Stored in a refrigerator, it is stable for several weaks.
Procedure:
  • For use mix the reagent solution with an equal volume of acetone and spray

TCNE reagent (tetracyanoethylene)
for detection of aromatic hydrocarbons and heterocycles, aromatic amines and phenols
  • Spray with a solution of 0.5–1 g tetracyanoethylene in dichloromethane or benzene

TNF reagent (trinitrofluorenone) for detection of phenols
  • Spray with a solution of 2 % 2,4,7-trinitrofluorenone in benzene or toluene

o-Tolidine/diazotized for detection of phenols

Solutions:

  1. Tolidine solution: fill up 5 g o-tolidine and 14 mL conc. hydrochloric acid to 100 mL with water
  2. Nitrite solution: 10 % aqueous sodium nitrite solution, prepared fresh

Procedure:

  • Mix 20 mL tolidine solution and 20 mL nitrite solution at 0 °C, stirring constantly. The spray solution is stable for about 2–3 hours.
After spraying it can take several hours until colored spots are formed!


Triethanolamine, 20 % in isopropanol for enhancement of fluorescence


Vanadium(V)/sulfuric acid reagent for detection of carbohydrates, glycols, reducing carboxylic acids, steroids, antioxidants, vitamins, phenols, aromatic amines

a) Ammonium monovanadate (ammonium metavanadate)/sulfuric acid reagent
  • Spray with a solution of 1.2 g ammonium monovanadate in 95 mL water + 5 mL conc. sulfuric acid
  • For β-blockers: spray with a saturated solution of ammonium monovanadate in conc. sulfuric acid
b) Vanadium pentoxide/sulfuric acid reagent
  • Spray with a solution of 1.82 g vanandium pentoxide in 30 mL 1 mol/L sodium carbonate, sonicate to achieve complete dissolution, after cooling add 46 mL 1.25 mol/L sulfuric acid and fill up to 100 mL with acetonitrile

Vanillin reagent for detection of amines and amino acids
  • Spray with a solution of 1 g vanillin in 50 mL 2-propanol
  • Dry 10 minutes at 110 °C
  • Spray with 1 mL 1 mol/L potassium hydroxide solution, filled up to 100 mL with ethanol
  • Dry 10 minutes at 110 °C
  • View under UV at 365 nm

Vanillin/sulfuric acid reagent for detection of steroids
  • Spray with a 1 % solution of vanillin in conc. sulfuric acid
  • Heat 5 minutes to 105 °C

top of page


Copyright 2014 by MACHEREY-NAGEL GmbH & Co. KG   Imprint / Impressum Privacy StatementTerms Of Use