Spray reagents for detection of herbal ingredients in TLC

Spray reagents for detection of herbal ingredients in TLC

Acetic anhydride reagent

The TLC plate is sprayed with 10 ml acetic anhydride, heated at 150°C for about 30 min and then inspected in UV-365 nm. 

- Detection of ginkgolides.

Anisaldehyde-acetic acid reagent

0.5 ml anisaldehyde is mixed with 10 ml glacial acetic acid.The plate is sprayed with 5-10 ml and then heated at 120°C for 7-10 min.

- Detection of petasin/isopetasin.

Anisaldehyde-sulphuric acid reagent

0.5 ml anisaldehyde is mixed with 10 ml glacial acetic acid, followed by 85 ml methanol and 5 ml

concentrated sulphuric acid, in that order.The TLC plate is sprayed with about 10 ml, heated at 100°C for 5-10 min, then evaluated in vis or UV-365 nm.The reagent has only limited stability and is no longer useable when the colour has turned to red-violet

-Detection of terpenoids, propylpropanoids, pungent and bitter principles, saponins.

Antimony-lll- chloride reagent (SbCl3)

20 per cent solution of antimony-lll chloride in chloroform (or ethanol)

The TLC plate must be sprayed with 15-20 ml of the reagent and then heated for 5-6 min at 110°C

Evaluation in vis. or UV-365 nm.

- Detection of cardiac glycosides, saponins.

Bartons reagent

(a) 1 g potassium hexacyanoferrate (III) in 100 ml water.

(b) 2 g iron-Ill-chloride in 100 ml water.

The TLC plate is sprayed with a 1.1 mixture of (a) and (b). Evaluation in vis. or UV - 365 nm

- Detection of gingerols (Zingiberis rhizoma).

Benzidine reagent

0.5 g benzidine is dissolved in 10 ml glacial acetic acid and the volume adjusted to 100 ml with ethanol. Evaluation in vis.

- Detection of aucubin (Plantaginis folium). 

Berlinblue reagent

A freshly prepared solution of 10 g iron-lll-chloride and 0.5 g potassiumn hexasyandterrae in 100 ml water.The plate is sprayed with 5-8 ml. Evaluation in vis.

- Detection of arbutin.

Blood reagent

10 ml of 3.6 per cent sodium citrate solution is added to 90 ml fresh bovine blood, 2 ml blood is mixed with 30 ml phosphate buffer pH 7.4. The plate is sprayed in a horizontal position. Phosphate buffer pH 7.4 : 20.00 ml potassium dihydrogen phosphate solution (27.281 g potassium dihydrogen phosphate dissolved in double-distilled, CO2- free water and volume adjusted to 10.00ml) mixed with 3934 ml 0.1M

sodium hydroxide, and volume made up to 100 ml. with CO2-free, double-distilled water.

- Detection of saponins: White zones are formed against the reddish background of the plate.Hemolysis may be immediate or may occur when the plate has been dried under slight warming.

Chloramine-trichloroacetic acid reagent

10 ml freshly prepared 3 per cent aqueous chloramine T solution (syn.sodium sulphamide chloride or sodium tosylchloramide) is mixed with 40 ml 25 per cent ethanolic trichloroacetic acid.BThe plate is sprayed with 10 ml, heated at 100°C for 5-10 min; evaluated in UV - 365 nm.

Detection of cardiac glycosides.

Dichloroquinone chloroimide = Gibb's reagent

0.5 percent methanolic solution of 2,6-dichloroquinone chloroimide.The plate is sprayed with 10 ml, then immediately exposed to ammonia vapour.

Detection a arbutin, capsaicin.

Dinitrophenylhydrazine reagent

0.1 g 2,4-dinitrophenylhydrazine is dissolved in 100 ml methanol, followed by the addition of 1 ml of 36 percent hydrochloric acid. After spraying with about 10 ml, the plate is evaluated immediately in vis.

-Detection of ketones and aldehydes.

DNPH-acetic acid- hydrochloric acid reagent

0.2 g 2,4-dinitrophenylhydrazine in a solvent mixture consisting of 40 ml glacial acetic acid (98 per cent), 40 ml hydrochloric acid (25 per cent) and 20 ml methanol.The plate is sprayed with 10 ml and evaluated in vis. It is then heated at 100ºC for 5-10 min and evaluated again in vis.

- Detection of valepotriates (Valeriana).Chromogenic dienes react without warming. Dienes can also be detected with HCI-AA reagent.

Dragendorff reagent

Solution (a) : Dissolve 0.85 g basic bismuth nitrate in 10 ml glacial acetic acid and 40 ml water under heating. If necessary, filter.

Solution (b): Dissolve 8 g potassium iodide in 30 ml water.

Stock solution : (a) + (b) are mixed 1:1.

Spray reagent : 1 ml stock solution is mixed with 2 ml glacial acetic acid and 10 ml water.

-Detection of alkaloids, heterocyclic nitrogen compounds.

Dragendorff reagent, followed by sodium nitrite or H2SO4

After treatment with Dragendorff reagent, the plate may be additionally sprayed with 10 per cent aqueous sodium nitrite or with 10 per cent ethanolic sulphuric acid, thereby intensifying the coloured zones.

( NaNO2,, dark brown; → H2SO4, bright orange).

EP reagent

0.25 g 4-dimethylamino benzaldehyde is dissolved in a mixture of 45 ml 98 percent acetic acid, 5 ml 85 percent o-phosphoric acid and 45 ml water, followed by 50 ml concentrated sulphuric acid (under cooling with ice). The sprayed plate is evaluted in vis.

- Detection of proazulene (Matricariae flos); After heating at 100ºC for 5 - 10 min, proazulene gives a blue-green colour (vis). The blue colour of azulene is intensified by EP reagent.

Fast blue salt reagent

0.5 g fast blue salt B is dissolved in 100 ml water. (Fast blue B = 3,3' - dimethoxybiphenyl-4,4'-bis

(diazoniu)-dichloride).The plate is sprayed with 6-8 ml, dried and inspected in vis. Spraying may be repeated, using 10 percent ethanolic NaOH, followed again by inspection in vis.

- Detection of phenolic compounds.

Fast red salt reagent

0.5 per cent aqueous solution of fast red salt B (= diazotized 5-nitro-2-aminoanisole). The plate is spraed with 10 ml, followed immediately by either 10 per cent ethanolic NaOH or exposure to ammonia vapour.

- Detection of amarogentin.

Hydrochloric acid - glacial acetic acid reagent

Eight parts of concentrated hydrochloric acid and two parts of glacial acetic acid are mixed. After spraying, the plate is heated at 110°C for 10 min. Evalution in vis. or in UV-365 nm.

- Detection of valepotriates with diene structure (Halazuchrome reaction).