Spray Reagents For the thin layer chromatography of drug extracts No. 1 Anisaldehyde-acetic acid reagent (AA) 0.5 ml Anisaldehyde mixed with 10 ml of 98% acetic acid. The TLC plate is sprayed with 5-10 ml, then heated at 120 0 C for 7-10 min in a drying cabinet. Detection 0/ petasin/isopetasin. The plate may be sprayed afterwards with conc. sulphuric acid and evaluated in vis. or UV-365 nm. NO.2 Anisaldehyde-sulphuric acid reagent (AS) 0.5 ml Anisaldehyde is mixed with 10 ml glacial acetic acid, followed by 85 ml metha­ nol and 5 ml concetrated sulphuric acid, in that order. The re agent has only limited stability, and is no longer useable when the colour has turned to red-violet. The TLC plate is sprayed with ab out 10 ml, heated at 100° C for 5-10 min, then evaluated in vis. or UV-365 nm. Detection 0/ essential oils, pungent principles, bitter principles, saponins, etc. NO.3 Antimony(lII) chloride reagent (SbCI 3) 20% solution of antimony(III) chloride in chloroform. The TLC plate must be sprayed with 15-20 ml reagent, then heated for 5-6 min at 100° C. Evaluation in vis. or UV-365 nm. Detection 0/ cardiac glycosides, saponins, visnagin (Ammi visnagae /ructus), etc. NO.4 Benzidine re agent (BZ) 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 0/ aucubin (Plantaginis folium) NO.5 Berlin blue reagent (BB) A freshly prepared solution of 10 g iron(III) chloride and 0.5 g potassium hexacyano­ ferrate in 100 ml water. The plate is sprayed with 5-8 ml and evaluated in vis. Detection 0/ arbutin. NO.6 Blood reagent (BL) 10 ml of 3.6% sodium citrate are added to 90 ml fresh bovine blood. 2 ml of this mixture are mixed with 30 ml phosphate buffer pH 7.4*. The plate is sprayed in a horizontal position. * Phosphate buffer pH 7.4 25.00 ml potassium dihydrogen phosphate solution (27.281 g potassium dihydrogen phosphate dissolved in CO2 -free water (double distilled) and volume adjusted to 1,000 ml) mixed with 39.34 ml of 0.1 M sodium hydroxide, and volume made up to 100 ml with CO2 -free, double distilled water. Detection 0/ saponins; white zones are formed against the reddish background of the plate. Haemolysis may be immediate, or it may not occur until the plate has been warmed. Boroxyl reagent. See Natural products-polyethyleneglycol reagent No. 28. 299 NO.7 Chloramine-trichloroacetic acid reagent (CTA) 10 ml freshly prepared 3% aqueous chloramine T solution (syn. sodium sulphamide chloride, or sodium tosy1chloramide) is mixed with 40 ml of 25% ethanolic trichloro­ acetic acid. The plate is sprayed with 10-15 ml, heated at 100° C for 5-10 min and evaluated in UV-365 nm. Detection of cardiac glycosides. NO.8 Dichloroquinonechloroimide reagent (DCC) 1 % methanolic solution of 2,6-dichloroquinonechloroimide. The plate is sprayed with 5-10 ml, then immediately exposed to ammonia vapour. Detection of arbutin (DAB 8), capsaicin (DAB 8). NO.9 Dinitrophenylhydrazine reagent (DNPH) 0.1 g of 2,4-dinitrophenylhydrazine is dissolved in 100 ml methanol, followed by the addition of 1 ml of 36% hydrochloric acid. After spraying with about 10 ml, the plate is evaluated immediately in vis. Detection of ketones and aldehydes. No. 10 DNPH-acetic acid-hydrochloric acid reagent 0.2 g of 2,4-dinitrophenylhydrazine in a solvent mixture consisting of 40 ml 98% acetic acid, 40 ml 25% hydrochloric acid and 20 ml methanol. The plate is sprayed with 10 ml and evaluated in vis. It is then heated at 100° C for 5-10 min and evaluated again in vis. (Ph. Eur. III). Detection of valepotriates (Valerianae radix). Chromogenic dien es react without warming. Remarks: Dienes can also be detected with HCL-AA reagent (No. 32). No. 11 Dragendorffreagents (DRG) Detection of alkaloids, heterocyclic nitrogen compounds, quarternary amines. No. 11A Dragendorffreagent (Ph. Eur. I, p. 139) 0.85 g basic bismuth nitrate is dissolved in 40 ml water and 10 ml glacial acetic acid, followed by addition of 8 g potassium iodide dissolved in 20 ml water. No. 11 B Dragendorff reagent Rl (Ph. Eur. III, p. 105) 100 g tartaric acid are dissolved in 400 ml water. 8.5 g basic bismuth nitrate are added and the solution is shaken for 2 h. 200 ml of 40% potassium iodide are then added, and the solution is shaken vigorously. After standing 24 h, the solution is filtered. Diluted Dragendorff reagent (Ph. Eur. III, p. 105) 50 ml Dragendorff reagent Rl are added to a solution of 100 g tartaric acid in 500 ml water. No. 11 C Dragendorff reagent with tartaric acid Solution A: 17 g bismuth subnitrate and 200 g tartaric acid in 800 ml water. Solution B: 160 g potassium iodide in 400 ml water. A + B = stock solution Spray reagent: 50 ml stock solution + 500 ml water + 100 g tartaric acid. No. 11 D Dragendorff reagent with hydrochloric acid (modified) Solution A: 0.3 g bismuth sub nitrate, 1 ml of 25% HCl, 5 ml water. Solution B: 3 g potassium iodide in 5 ml water. Spray reagent: 5 ml A + 5 ml B + 5 ml of 12.5% HCI + 100 ml water. 300 No. HE Dragendorffreagent according to Vaguifalvi A mixture of 2.6 g bismuth carbonate, 7 g dried sodium iodide and 25 ml glacial acetic acid is heated to boiling for a few minutes. After standing overnight, the precipitated sodium acetate is removed by filtration. 20 ml of the c1ear red filtrate are mixed with 80 ml ethyl acetate to give the stock solution. Spray reagent: 2 ml stock solution, 5 ml glacial acetic and and 12 ml ethyl acetate. Detection limit: approx. 1 Ilg. After the plate has been dried (i.e. the smell of acetic acid is no longer detectable), it can be sprayed again with 5% ethanolic sulphuric acid, ihereby giving a ten-fold increase in sensitivity. No. HF Dragendorffreagent,Jollowed by sodium nitrite or H Z S04 After treatment with any type of Dragendorff reagent, the plate may be additionally sprayed with 5% aqueous sodium nitrite or with 5% ethanolic sulphuric acid, thereby intensifying the coloured zones. No. 12 Fast blue salt reagent (FBS) 0.5 g fast blue salt B is dissolved in 100 ml water. (Fast blue salt B=3,3'-dimethoxy­ biphenyl-4-4' -bise diazonium)-dichloride) The plate is sprayed with 6-8 ml, dried and inspected in vis. Spraying may be repeated, using 0.1 M NaOH, followed again by inspection in vis. Detection of bitter principles /rom hops, and phenolic compounds in general. No. 13 Fast red salt reagent (FRS) 0.5% aqueous solution of fast red salt B (=diazotized 5-nitro-2-aminoanisole). The plate is sprayed with 10 ml, followed immediately by either 0.1 M NaOH or exposure to ammonia vapour. Detection 0/ amarogentin. No. 14 Iron(III) chloride reagent (FeCI3) 10% aqueous solution. Spray with 5-10 ml and evaluate in vis. Detection %leuropein; carnosolic acid (Salviae folium, Rosmarini folium); hop bitter principles. No. 15 EP reagent according to Stahl (EP) 0.25 g of 4-dimethylaminobenzaldehyde is dissolved in a mixture of 45 ml of 98% acetic acid, 5 ml of 85% o-phosphoric acid and 45 ml water, followed by 50 ml conc. sulphuric acid with cooling. The sprayed plate is evaluated immediately in vis., or after heating. Detection 0/ azulenes (Matricariae flos) ; the natural blue of the azulenes is intensified by EP reagent. After warming at 100° C for 3-5 min, proazulene gives a blue-green (vis.). No. 16 Acetic anhydride-sulphuric acid reagent Liebermann-Burchard reagent (LB) 5 ml acetic anhydride and 5 ml conc. sulphuric acid are added carefully to 50 ml absolute ethanol, while cooling in ice. The reagent must be freshly prepared. The sprayed plate is warmed at 100° C for 5-10 min, then inspected in UV- 365 nm. Detection oftriterpenes, steroids (saponins, bitter principles). No. 17 Iodine-chloroform reagent (I/CHCI3) 0.5% Iodine in chloroform. 301 The sprayed plate is warmed at 60° C for ab out 5 min. It may be evaluated immediately, or after standing for 10 min at room temperature. Detection o/Ipecacuanha alkaloids. No. 18 Iodine re agent About 10 g solid iodine are spread on the bottom of a chromatography tank. Com­ pounds containing conjugated double bonds give yellow-brown (vis.) zones on expo­ sure to the atmosphere of iodine vapour. No. 19 Iodoplatinate re agent (IP) 0.3 g hydrogen hexachloroplatinate (IV) hydrate is dissolved in 100 ml water and mixed with 100 ml of 6% potassium iodide solution. The plate is sprayed with 10 ml and evaluated in vis. Detection o/nitrogen-containing compounds, e.g. alkaloids (blue-violet). For the detec­ tion of Cinchona alkaloids, the plate is first sprayed with 5% ethanolic H ZS04 (No. 34), and then with IP reagent. No.20 Iodine-hydrochloric aicd reagent (I/HCl) 1 g potassium iodide and 1 g iodine are dissolved in 100 ml ethanol (soln. I). 25 ml of 25% HCI are mixed with 25 ml of 96% ethanol (soln. Ir). The plate is first sprayed with 5 ml soln. 1, followed by 5 ml soln. Ir. Dark brown (vis.) zones are produced. Detection o/the purine derivatives, caffeine, theophylline and theobromine. No.21 Potassium hydroxide (KOH) 5% or 10% Ethanolic potassium hydroxide (Bornträger reaction) The plate is sprayed with 10 ml and evaluated in vis.