Nitrous fumes reagent

The dried chromatograms are first sprayed homogeneously with reagent 1, then dried for 2 to 3 min in the air and for ca. 10 min at 60 C in a drying cupboard until the pyridine has completely evaporated. Afterwards they are briefly sprayed with a little hydrochloric acid (25%) and exposed to nitrous fumes (reagent 2) for several minutes. Finally after removal of the excess nitrous fumes in a stream of cold air the chromatograms are sprayed with reagent 3. 

Note Note that the diazotization of primary aromatic amines can also be achieved by placing the chromatogram for 3 — 5 min in a twin-trough chamber containing nitrous fumes (fume cupboard ). The fumes are produced in the empty trough of the chamber by addition of 25% hydrochloric acid to a 20% sodium nitrite solution [2, 4], iV-(l-Naphthyl)ethylenediamine can be replaced in the reagent by a- or -naphthol [10, 14], but this reduces the sensitivity of detection [2]. Spray solutions Ila and lib can also be used as dipping solutions. 

The dried chromatograms are first treated with nitrous fumes for about 5 min. The excess nitrous fumes are then removed in a stream of cold air (ca. S min) and the TLC/HPTLC plates are sprayed homogeneously with reagent 2. 

Nitmus fumes Mix a solution of 1 g sodium nitrite in 5 ml water with 5 ml fuming hydrochloric acid in one trough of a twin-trough chamber to generate nitrous fumes. The reagent should always be made up fresh. 

It can be advantageous to heat the chromatogram to 160 °C for 15 min before treating with nitrous fumes and to place it in the reagent chamber while still hot [1]. Heating to 260 °C has even been recommended for the purpose of reducing the fluorescent background [14], whereby the layer is previously immersed in 1 percent Ludox solution (silidc acid sol) to increase its stability [2]. The fluorescence of the substances detected usually remains stable for at least 2 weeks [2]. 

The earliest oxidations were effected with nitrous fumes and later with mercuric oxide and isoamyl nitrite.74 Lead tetraacetate in acetic acid is in many cases the reagent of choice, but the removal of by-products can present some difficulties.75 IV-Haloimides and amides in alcoholic solutions have been reported to yield essentially pure tetrazolium salts76 and have been found specially useful in the preparation of heteroaryl-substituted tetrazolium salt.77,78 The novel formazans 49 have been successfully oxidized to 50 using 7V-chloro succinimide (Eq. II).79 tert-Butyl hypo-... 

Nitric acid (concentrated). The commercial concentrated nitric acid is a water-like solution with a density of 1-42 gem-3. It contains 69-5% (w/w) HN03 or 0-99 g HN03 per millilitre. It is approx. 15-6 molar. Nitrous fumes make the somewhat decomposed reagent reddish-brown. It should be handled with utmost care, wearing gloves and eye protection. 

Standard stock copper solution, dissolve 0.1 g copper in 15 ml 1 + 4 nitric acid, (use B.D.H. lead-free reagent for foodstuff analysis grade nitric acid), boil gently to expel nitrous fumes, cool and dilute to 100 cm with distilled water. 

To 90 ml of a suitable concentration of mercury in neutral or acetic acid solution add 5 0 ml of N nitric acid from a pipette, mix thoroughly, add 3 0 ml of /)-dimethylaminobenzylidine-rhodanine reagent (0 04 g shaken with 200 ml of ethanol, left overnight and filtered), adjust the volume to 100 ml and mix. Compare with standards prepared by adding suitable known amounts of standard mercuric nitrate solution (0-5 g mercury in 5 ml of concentrated nitric acid, dilute, boil to remove nitrous fumes and dilute to 250 ml make further dilutions so that 1 ml 0 0001 g Hg) to similar amounts of nitric acid and reagents as used in the test solution. Develop the colour in both test solution and standards simultaneously, allow to stand for five minutes and compare. 

Detection - Expose plate to nitrous acid fumes (add several drops of HCI to ca 5 grams of sodium nitrite in a convenient container) followed by spraying with 0.1% Marshall s reagent in methanol. 

---