TLC Visualization Reagents Part 5

Dear Friends,
I am  posting this last and concluding post for my collection of a list of several derivatizing agents/ visualization indicators. I hope you found it helpful and informative...................... 

Rhodamine B

For detection of a wide variety of compounds

Dry the developed chromatogram and spray with 0.025-0.25% ethanolic rhodamine B reagent.Results: In most cases red-violet zones with an intense fluorescence at 365nm 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 ammonia atmosphere, the detection sensitivity can be improved. 

Rhodamine 6 G

For detection of lipids

Spray plate with a solution of 1mg rhodamine 6 G in 100ml acetoneInspect at long wavelength UV.

Silver nitrate / hydrogen peroxide

For detection of halogenated hydrocarbons

Spray plate with a solution of 0.1g silver nitrate in 1ml water, add 10ml 2-phenoxyethanol, fill to 200ml with acetone and add 1 drop hydrogen peroxide (30% solution)Irradiate with unfiltered UV light until optimal contrast is obtained. For alumina plates, about 50 minutes, for silica gel plates about 15 minutes

Results: dark spots are formed. 

Sodium azide

For detection of antibiotics (penicillins and cephlosporins)

Solution I: 0.5% solution of soluble starchSolution II: 3.5% sodium azide in 0.1N iodine solutionProcedure: spray with 1), dry, spray with 2)

Results: Detects penicillin, sensitivity 0.2μg. Penicillin and penicilloic acids are also detected by starch-iodineiodine reagents, as are cephalosporins. 

Sodium 1,2-napthaquinone-4-sulfonate (NZS reagent)

For detection of thiazide drugs, basic drugs with primary amino groups

Solution I: 0.1N NaOHSolution II: saturated solution of reagent in 1:1 ethanol: waterSpray with I, then II.

Results: Thiazide drugs appear as orange spots within 15min; basic drugs with primary amino groups also react but barbiturates do not. 

Sodium nitrite / hydrochloric acid

For detection of indoles and thiazoles

Spray plate with a freshly made solution of 1g sodium nitrite in 100ml hydrochloric acid, 1mol/L and heat at 100°C.

Results: Indoles turn red and thiazole derivatives turn light green. 

Sodium nitroprusside / hydrogen peroxide

For detection of guanidine, urea, thiourea and derivatives, creatine and creatinine.

Spray with a solution made up of 2ml 5% aqueous sodium nitroprusside, 1ml 10% aqueous sodium hydroxide and 5ml 3% aqueous hydrogen peroxide and dilute with 15ml water. This solution can be stored several days in the refrigerator. 

Sodium nitroprussate / potassium hexacyanoferrate (III)

For the detection of aliphatic nitrogen compounds, cyanamide, guanidine, urea, thiourea and derivatives, creatine, and creatinine.

Spray with a solution of 1 volume part each of 10% aqueous sodium hydroxide, 10% sodium nitroprussate, and 10% potassium hexacyanoferrate (III) with 3 vol. parts water. Let the solution stand at least 20min at ambient temperature before use. Stored in the refrigerator, it is stable for several weeks.

Procedure: For use, mix the reagent solution with an equal part of acetone and spray. 

Tetracyanoethylene - TCNE reagent

For detection of aromatic hydrocarbons and heterocycles, aromatic amines, and phenols

Spray with a solution of 0.5-1.0g tetracyanoethylene in dichloromethane or toluene.

Results: Aromatic hydrocarbons show various colors, some of them for a brief time. Also try heating at 100°C for a short time. 

Tetranitrodiphenyl

For detection of cardiac glycosides

Spray solution I: Saturated solution of 2,3’,4,4’-tetranitrodiphenyl in toluene

Spray solution II: 10% potassium hydroxide solution in 50% aqueous methanol

Spray with I, dry at room temperature, then spray with II.

Results: Blue spots are observed. 

Tetrazolium blue

For detection of corticosteroids and other reducing compounds.

Spray with a freshly prepared 1:1 mix of a) 0.5% methanolic tetrazolium blue solution and 6M NaOH in water or methanol/water (1:1).

Results: violet spots are observed at room temperature or with slight warming. 

Thymol / sulfuric acid

For detection of sugars

Spray with a solution of 0.5g thymol in 95ml ethanol, and add 5ml 97% sulfuric acid with caution.Heat 15-20min at 120°C

Results: Sugars show pink spots. 

Tin (IV) chloride

For detection of triterpenes, sterols, steroids, phenols, and polyphenols

Spray with a solution of 10ml tin (IV) chloride in 160ml equal volumes of chloroform and glacial acetic acid.Heat the layer for 5-10min at 100°C and inspect in visible and long wavelength UV light. 

o-Tolidine, diazotized

For detection of phenols

Tolidine solution – fill up 5g o-tolidine and 14ml conc. hydrochloric acid to 100ml water

Nitrate solution – 10% aqueous sodium nitrate solution prepared fresh

Spray solution – mix 20ml tolidine solution and 20ml nitrate 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. 

p-Toluenesulfonic acid

For detection of steroids, flavonoids and catechins

Spray with a solution of 20% p-toluenesulfonic acid in chloroform and heat a few minutes at 100°C.Inspect under long wavelength UV light. 

Trichloroacetic acid

For detection of steroids, digitalis glycosides, veratrum alkaloids and vitamin D

Spray solution I: 25% solution of trichloroacetic acid in chloroform.

Spray solution II – for vitamin D – 1% trichloroacetic acid in chloroform.

Spray solution III – for digitalis glycosides – dissolve 3.3g trichloroacetic acid in 10ml chloroform and add 1-2 drops hydrogen peroxide.

After applying the appropriate solution, heat 5-10min at 120°C.Inspect the spots in daylight and in long wavelength UV light.

Trifluoroacetic acid

For detection of steroidsSpray with a solution of 1% trifluoroacetic acid in chloroform and heat 5min at 120°C

Triethanolamine

For enhancement of fluorescence species20% solution of reagent in isopropanol 

Ultraviolet Light - fluorescence

For detection of various compounds which have native fluorescence.

With a plate containing a fluorescent indicator (F254), examine the dried chromatogram under 360nm UVlight.With a TLC plate containing no fluorescent indicator, examine the dried chromatogram under both 254 and 360nm UV light. 

Ultraviolet Light - quenching

For detection of various compounds which absorb 254nm UV light; quenching of TLC plate fluorescence.

Examine a fluorescent indicator (F254) containing plate after dried, under 254nm UV light.Compounds absorbing this wavelength light will appear as dark spots against a green or white fluorescence caused by the UV activation of the fluorescent indicator in the plate. 

Urea / hydrochloric acid

For detection of sugars

Spray with a solution of 5g urea in 20ml 2M hydrochloric acid, with 100ml ethanol added and heat to 100°C.

Results: Ketoses and oligosaccharides containing ketoses turn blue. 

Vanadium (V) / sulfuric acid

For detection of carbohydrates, glycols, reducing carboxylic acids, steroids, antioxidants, vitamins, phenols,aromatic amines, antihistaminesa)

Ammonium monovanadate (ammonium metavanadate) /sulfuric acid reagent

Spray with a solution of 1.2g ammonium monovanadate in 95ml water and 5ml conc. sulfuric acid or for beta blockers: spray with saturated solution of ammonium monovanadate in conc. sulfuric acid.

b) Vanadium pentoxide / sulfuric acid reagentSpray with a solution of 1.82g vanadium pentoxide in 30ml 1M sodium carbonate, sonicate to achieve complete dissolution, after cooling add 46ml 2.5M sulfuric acid and fill to 100ml with acetonitrile. 

Vanillin / potassium hydroxide

For detection of amines and amino acids

Spray with a solution of 1g vanillin in 50ml 2-propanol and dry 10min at 110 CThen spray with 1ml 1M potassium hydroxide solution filled up to 100ml with ethanol and dry 10 min at 110oC View at 365nm UV

Results: After spraying with a) ornithine will be seen as a bright green-yellow fluorescence, lysine as a greenyellow fluorescence. After spraying with b) ornithine will show a salmon color that will fade; proline, hydroxyproline, pipecolinic (pipecolic) acid and sarcosine will turn red after a few hours. Glycine will produce a green brown spot, and other amino acids will turn faint brown. 

Vanillin / phosphoric acid

Used as a charring reagent for polymer bound TLC plates (the newer hard layer plates)

Spray plate with 1g vanillin in 100ml 50% aqueous H3PO4 

The charring visualization reagents given below can only be used with glass TLC plates in which a G (gypsum) binder has been used in the formulation to hold the silica gel on the plate: 

Potassium dichromate / sulfuric acid (chromosulfuric acid)

Universal visualization reagent for organic compounds (e.g., alcohols, also for bile acids, lipids) – note: do not use this reagent on polymer bound TLC plates since it will char the binder; use only with G (gypsum) binder plates.

Spray with a solution of 5g potassium dichromate in 100ml conc. Sulfuric acid.If necessary heat plate to 150 C

Results: Brown, grey, or black spots result 

Potassium permanganate / sulfuric acid

Universal reagent for organic compounds, e.g., fatty acid derivativesNote: Do not use this reagent on polymer bound TLC plates since it will char the binder; use only with G (gypsum) binder plates.

Spray with a solution of 1.6% potassium permanganate in conc. sulfuric acid (make this solution very carefully, and slowly to prevent any accidents).Heat plate for 15-20min at 180 C 

Vanillin / sulfuric acid

For detection of steroids, Note, this reagent can only be used with G (gypsum) binder plates since it will char the polymer binders in the harder layer plates.

Spray with a 1% solution of vanillin in conc. sulfuric acid and at 120 C until maximum color formation. Another formulation of this reagent: 0.5g vanillin in 100ml sulfuric acid/ethanol (40:10). 

TLC Texts with Visualization Reagent Information:

Thin Layer Chromatography, 2nd edition, E. Stahl, ed, Springer-Verlag, NY, 1969

TLC Reagents & Detection Methods – Physical & Chemical Detection Methods: Fundamentals, Reagents,I, Vol 1a, H. Jork, W. Funk, W. Fishcer, & H. Wimmer, Wiley, NY, 1990

Practice of Thin Layer Chromatography, 3rd edition, J.C. Touchstone, Wiley-Interscience, NY, 1992

TLC Reagents & Detection Methods – Physical & Chemical Detection Methods: Activation Reactions, Reagent Sequences, Reagents, II, Vol 1b, H. Jork, W. Funk, W. Fishcer, & H. Wimmer, Wiley, NY, 1994