2.4- Dinitrophenylhydrazine,

Suspend 35 g. of finely-powdered hydrazine sulphate in 125 ml. of hot water contained in a 400 ml. beaker, and add, with stirring, 118 g. of crystallised sodium acetate or 85 g. of potassium acetate. Boil the mixture for 5 minutes, cool to about 70°, add 80 ml. of rectified spirit, filter at the pump and wash with 80 ml. of hot rectified spirit. Keep the filtered hydrazine solution for the next stage in the preparation. 

Equip a 1 Utre three-necked flask or a 1 litre bolt- head flask with a reflux condenser and a mercury-sealed stirrer. Dissolve 50-5 g. of commercial 2 4-dinitro-l-chlorobenzene in 250 ml. of rectified spirit in the flask, add the hydrazine solution, and reflux the mixture with stirring for an hour. Most of the condensation product separates during the first 10 minutes. Cool, filter with suction, and wash with 50 ml. of warm (60°) rectified spirit to remove unchanged dinitrochlorobenzene, and then with 50 ml. of hot water. The resulting 2 4-dinitrophenylhydrazine (30 g.) melts at 191-192° (decomp.), and is pure enough for most purposes. Distil oflF half the alcohol from the filtrate and thus obtain a less pure second crop (about 12 g.) recrystallise this from n-butyl alcohol (30 ml. per gram). If pure 2 4-dinitrophenylhydrazine is required, recrystallise the total yield from n-butyl alcohol or from dioxan (10 ml. per gram) this melts at 200° (decomp.). 

The following alternative method of preparation is recommended. Dissolve 50 g. of purified 2 4-dinitrochlorobenzene (1) in 100 ml. of triethylene glycol (gentle warming nia be necessary alternatively, 125 ml. of warm diethylene glycol may be used) in a 600 ml. beaker and cool, with mechanical stirring, in an ice bath to 15-18°. Place 15 ml. of commercial 60-65 per cent, hydrazine solution in a small separatory funnel supported over the beaker. Add the hydrazine solution to the stirred solution in the beaker at such a rate that the temperature is maintained between 15° and 20° (20-30 minutes). When 

2 4-Dinitroaniline. This preparation is another illustration of the mobile character of the chlorine atom in 2 4-dinitro-l-chlorobenzene  

2 4-dinitro-1-chlorobenzene (1) in a 250 ml. bolt-head flask, and fit it with a reflux condenser and inlet tube (at least 2 cm. diameter in order to prevent clogging) which terminates just above the surface of the reaction mixture. Half immerse the flask in an oil bath. Pass ammonia gas (from a cylinder) through a bubble counter, which contains a solution of 

Thirty-five grams (0.27 mole) of hydrazine sulfate (Coll. Vol. 1, 302) is suspended in 125 cc. of hot water in a 400-cc. beaker and stirred by hand during the addition of 85 g. (o.87 mole) of potassium acetate (Note 1). The mixture is boiled five minutes and then cooled to about 70° 75 cc. of alcohol is added, and the solid is filtered with suction and washed with 75 cc. of hot alcohol. The filtered hydrazine solution is saved for the next step. 

In a i-l. flask fitted with a stirrer and reflux condenser, 50.5 g. (0.25 mole) of technical 2,4-dinitrochlorobenzene is dissolved in 250 cc. of alcohol the hydrazine solution is added, and the mixture is refluxed with stirring for an hour. Most of the product separates during the first ten minutes (Note 2) it is cooled well, filtered, and washed once with 50 cc. of warm alcohol (6o°) to remove unchanged halide and then with 50 cc. of hot water. The solid weighs 30 g. and melts at 190-192° with evolution of gas (Note 3) it is pure enough for most purposes. By distilling half the alcohol from the filtrate a less pure second crop is obtained this is recrystallized from w-butyl alcohol (30 cc. per g.) (Note 4). The total yield is 40-42 g. (80-85 Per cent of the theoretical amount) (Notes 5-10). 

An equivalent amount of sodium acetate may be substituted for the potassium acetate. 

The melting point is not sharp in the capillary tube the sample shrinks about io° below the melting point. 

For recrystallization, w-butyl alcohol is the best solvent in spite of the large amount required, but tetralin, pyridine, or dioxane (10 cc. per g.) may be used where large quantities are involved. Fortunately, most of the material as prepared does not need further purification. 

Other Names (2,4-Dmitrophenyl)hydrazine l-(2,4-Dinitrophenyl)hydrazine l-Hydrazino-2,4-dinitrobenzene DNPH NSC 5709 CA Index Name Hydrazine, (2,4-dinitrophenyl)-CAS Registry Number 119-26-6 Merck Index Number 3283 Chemical Structure 

Chemical/Dye Class Miscellaneous Molecular Formula C6H6N4O4 Molecular Weight 198.14 pH Range 11.0-12.5 

Boiling Point (Calcd.) 378.6 + 32.0°C Pressure 760 Torr Synthesis Synthetic methodsi-  

Major Applications Fuel cells, detecting microorganisms, antibacterial agent, treatment of acute and chronic hepatitis, gynecopathy, cardiovascular diseases, cerebrovascular diseases Safety/Toxicity Bladder toxicity, carcinogenicity, genotoxicity, mutagenicity  

Three-step synthesis of 2,4-dinitrophenylhydrazine from benzene. J. Chem. Educ. 1965, 42, 267. 

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Dinitrophenylhydrazine is a very important reagent for the identification of aldehydes and ketones (pp. 342, 346). It is readily prepared from chloro-2,4-dinitrobenzene (I). In the latter compound the chlorine is very reactive in... 

On the other hand, the two nitro groups make 2,4-dinitrophenylhydrazine a very weak base, and it has therefore to be used in reasonably concentrated acid solution. 

Precautions, (i) The above tests must be carried out with discretion. If the substance is only moderately soluble in the solvent selected, and a comparatively large volume of the latter is required, the consequent dilution of the acid in the reagent may cause the separation of the free 2,4 dinitrophenylhydrazine (although this is more likely to happen with Reagent B than with A). Furthermore, if the compound under investigation should have basic properties, the neutralisation of part of the acid in the reagent may have the same result. 

Dinitrophenylhydrazones, To a few drops of formalin, add a few drops of 2,4-dinitrophenylhydrazine reagent A (p. 261) a yellow precipitate is produced in the cold. Acetaldehyde gives an orange-coloured precipitate. Dissolve a few drops of benzaldehyde or salicylal-dehyde in 2 ml. of methanol and then add a few drops of the reagent B an orange-coloured precipitate is obtained. In each case filter and recrystallise from ethanol. Take the m.p. (M.ps., p. 539-540.)... 

K. Treat with 2,4-dinitrophenylhydrazine reagent (pp. 263, 334). Yellow or orange-yellow precipitates given by most aldehydes and ketones. 

Suspend 0 25 g. of 2 4-dinitrophenylhydrazine in 5 ml. of methanol and add 0-4 0-5 ml. of concentrated sulphuric acid cautiously. FUter the warm solution and add a solution of 0 1-0-2 g. of the carbonyl compound in a small volume of methanol or of ether. If no sohd separate within 10 minutes, dUute the solution carefuUy with 2N sulphuric acid. CoUect the solid by suction filtration and wash it with a little methanol. RecrystaUise the derivative from alcohol, dUute alcohol, alcohol with ethyl acetate or chloroform or acetone, acetic acid, dioxan, nitromethane, nitrobenzene or xylene. 

This method cannot be applied to polynitro amines, since these are so weakly basic that they can be diazotised only under special conditions in strongly acidic solutions. In such cases use may, however, be made of the mobility oonfared upon halogen atoms by the presence of nitro groups in the orlko and para positions. Thus the valuable reagent 2 4-dinitrophenylhydrazine is readily prepared by the condensation of 2 4-dinitrochlorobenzene with hydrazine ... 

Method 1. Suspend 2-0 of 2 4-dinitrophenylhydrazine in 100 ml. of mothanol add cautiously and slowly 4-0 ml. of concentrated sulphuric acid. The mixture becomes warm and the solid usually dissolves completely. Filter, if necessary. 

Method 2. Dissolve 0-25 g. of 2 4-dinitrophenylhydrazine in a mixture of 42 ml. of concentrated hydrochloric acid and 50 ml. of water by warming on a water bath dilute the cold solution to 250 ml. with distilled water. This reagent is more suitable for water-soluble aldehydes and ketones since alcohol is absent. 

If an unknown compound gives a positive test with the 2 4-dinitrophenylhydrazine reagent, it then becomes necessary to decide whether it is an aldehyde or a ketone. Although the dimedone reagent (Section 111,70,2) reacts only with aldehydes, it is hardly satisfactory for routine use in class reactions. It is much simpler to make use of three other reagents given below, the preparation and properties of which have already been described (Section 111,70). 

Because of the time and expense involved, biological assays are used primarily for research purposes. The first chemical method for assaying L-ascorbic acid was the titration with 2,6-dichlorophenolindophenol solution (76). This method is not appHcable in the presence of a variety of interfering substances, eg, reduced metal ions, sulfites, tannins, or colored dyes. This 2,6-dichlorophenolindophenol method and other chemical and physiochemical methods are based on the reducing character of L-ascorbic acid (77). Colorimetric reactions with metal ions as weU as other redox systems, eg, potassium hexacyanoferrate(III), methylene blue, chloramine, etc, have been used for the assay, but they are unspecific because of interferences from a large number of reducing substances contained in foods and natural products (78). These methods have been used extensively in fish research (79). A specific photometric method for the assay of vitamin C in biological samples is based on the oxidation of ascorbic acid to dehydroascorbic acid with 2,4-dinitrophenylhydrazine (80). In the microfluorometric method, ascorbic acid is oxidized to dehydroascorbic acid in the presence of charcoal. The oxidized form is reacted with o-phenylenediamine to produce a fluorescent compound that is detected with an excitation maximum of ca 350 nm and an emission maximum of ca 430 nm (81). 

Interaction of formaldehyde with 2,4-dinitrophenylhydrazine in acid media causes 2,4-dinitrophenylhydrazone (DNPhydrazone) formaldehyde formation. Gas-chromatographic analysis of 2,4-DNP-hydrazone formaldehyde toluene extract with an electron holding detector makes it possible to detect it at the level of 0,001 mg/dm. Phenol is detected in the form of tribromphenol yield, the hexane extract of which undergoes chromatography with an electron holding detector which provides the level of phenol detection of 0.001 mg/dm (the limit of quantitative detection). 

MATTOX KENDALL Dehydrohaiogenation Dehydrohaiogenatton of a haloketones with 2,4 dinitrophenylhydrazine or LiCI DMF... 

Cyclohexanol [108-93-0] M 100.2, m 25.2", b 161.1", d 0.946, n 1.466, n s 1.437, n 1.462. Refluxed with freshly ignited CaO, or dried with Na2C03, then fractionally distd. Redistd from Na. Further purified by fractional crystn from the melt in dry air. Peroxides and aldehydes can be removed by prior washing with ferrous sulfate and water, followed by distillation under nitrogen from 2,4-dinitrophenylhydrazine, using a short fractionating column water distils as the azeotrope. Dry cyclohexanol is very hygroscopic. 

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