2,4-Dinitrophenylhydrazine, collection

Reagents. Methanol. This must be free from aldehydes and ketones if necessary reflux 1L of the purest material available for 2 hours with 5 g of 2,4-dinitrophenylhydrazine and five drops of concentrated hydrochloric acid. Then distil the methanol through a fractionating column and collect the fraction boiling at 64.5-65.5 °C. 

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. Filter 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 solid separates within 10 minutes, dilute the solution carefully with 2N sulphuric acid. Collect 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. 

A mixture of 20 g (0.1 mol) of aluminum isopropoxide, 0.1 mol of an aldehyde or a ketone and 100 ml of dry isopropyl alcohol is placed in a 250 ml flask surmounted by an efficient column fitted with a column head providing for variable reflux. The mixture is heated in an oil bath or by a heating mantle until the by-product of the reaction - acetone - starts distilling. The reflux ratio is adjusted so that the temperature in the column head is kept at about 55° (b.p. of acetone) and acetone only is collected while the rest of the condensate, mainly isopropyl cohol (b.p. 82°), flows down to the reaction flask. When no more acetone is noticeable in the condensate based on the test for acetone by 2,4-dinitrophenylhydrazine the reflux regulating stopcock is opened and most of the isopropyl alcohol is distilled off through the column. The residue in the distilling flask is cooled, treated with 200 ml of 7% hydrochloric acid and extracted with benzene the benzene extract is washed with water, dried and either distilled if the product of the reduction is volatile or evaporated in vacuo in the case of non-volatile or solid products. Yields of the alcohols are 80-90%. 

The procedures used to determine ambient carbonyl concentrations involve a collection step with silica or C18 cartridges impregnated with 2,4-dinitrophenylhydrazine. Contamination is inevitable with this system, and blanks must be used to compensate for the degree of contamination. Selection of the appropriate blank values to subtract is a difficult and uncertain process. Consequently, development of a gas chromatographic system that will resolve and respond to the low-molecular-weight aldehydes and ketones is needed. The mercuric oxide and atomic emission detectors should provide adequate response for the carbonyls. 

Some commercial samples of precipitated manganese dioxide may be active enough for use directly in an oxidation process. To assess the activity of a sample of manganese dioxide, dissolve 0.25 g of pure cinnamyl alcohol in 50 ml of dry light petroleum (b.p. 40-60 °C) and shake the solution at room temperature for 2 hours with 2g of the sample of manganese dioxide (previously dried over phosphoric oxide). Filter, remove the solvent by evaporation and treat the residue with an excess of 2,4-dinitrophenylhydrazine sulphate in methanolt (Section 9.6.13, p. 1257). Collect the cinnamaldehyde 2,4-dinitrophenyl-hydrazone and crystallise it from ethyl acetate. An active dioxide should give a yield of the derivative, m.p. 255 °C (decomp.), in excess of 0.35 g (60%). 

To an ice-cold mixture of 1.0 ml of concentrated sulphuric acid and 5 ml of saturated aqueous potassium dichromate solution, add 2 ml of the alcohol or its concentrated aqueous solution. If the alcohol is not miscible with the reagent, shake the reaction mixture vigorously. After 5 minutes, dilute with an equal volume of water, distil and collect the first few ml of the aqueous distillate in a test tube cooled in ice. (Aldehydes and ketones are volatile in steam.) Test a portion of the distillate for a carbonyl compound with 2,4-dinitrophenylhydrazine reagent (p. 1218). If a solid derivative is obtained, indicating that the compound was a primary or secondary alcohol, test a further portion with SchifFs reagent (p. 1291) to distinguish between the two possibilities. The derivative may be recrystallised the m.p. may give a preliminary indication of the identity of the alcohol. 

Transfer about 0.2 g of d-camphor, accurately weighed, to a 300-ml glass stoppered flask, and dissolve in 25 ml of aldehyde-free ethanol. Add slowly 75 ml of 2,U-dinitrophenylhydrazine TS while shaking, and heat for U hours on a water bath under a reflux condenser. Cool, add 100 ml of diluted sulfuric acid (1 50), and allow to stand for 2h hours. Collect the precipitate so obtained on a glass filter (G3), wash the residue on the filter with cold water until the washings become neutral, dry for 2 hours at 105° and weigh as the 2,U-dinitrophenylhydrazone of d-camphor (C1gH2(-)N 0 332.36). ... 

Collection of the GC effluent and subsequent MS analysis allowed assignment of a possible molecular formula as Ci9H280. Since boar taint can be eliminated by castration of male pigs, attention was focused on the testosterone and androsterone family of compounds as possible candidates. When the crude volatiles were treated with 2,4-dinitrophenylhydrazine, the boar taint odor was completely removed, implicating a ketone functionality for the lone oxygen atom. Anecdotal information implicated several androstene derivatives, including 47, which was described as having an intense, urine-like odor. 143 An authentic sample of 47 was prepared, and comparison of the GC and MS properties allowed the definitive structural identification of the boar taint compound. 

To 10 mL of the stock solution of 2,4-dinitrophenylhydrazine in phosphoric acid add about 0.1 g of the compound to be tested. Ten milliliters of the 0.1 M solution contains 1 millimole (0.001 mole) of the reagent. If the compound to be tested has a molecular weight of 100 then 0.1 g is 1 millimole. Warm the reaction mixture for a few minutes in a water bath and then let crystallization proceed. Collect the product by suction filtration (Fig. 1), wash the crystals with a large amount of water to remove all phosphoric acid, press a piece of moist litmus paper on to the crystals, and if they are acidic wash them with more water. Press the product as dry as possible between sheets of filter paper and recrystallize from ethanol. Occasionally a high-molecular-weight derivative won t dissolve in a reasonable quantity (20 mL) of ethanol. In that case cool the hot suspension and isolate the crystals by suction filtration. The boiling ethanol treatment removes impurities so that an accurate melting point can be obtained on the isolated material. 

Cleaning Up The filtrate from the preparation of the 2,4-dinitrophenylhydrazone should have very little 2,4-dinitrophenylhydrazine in it, so after dilution with water and neutralization with sodium carbonate it can be flushed down the drain. Similarly the mother liquor from crystallization of the phenylhydrazone should have very little product in it and so should be diluted and flushed down the drain. If solid material is detected, it should be collected by suction filtration, the filtrate flushed down the drain, and the filter paper placed in the solid hazardous waste container since hydrazines are toxic. 

Deluca (929) described a 2,4-dinitrophenylhydrazine procedure for the collection of carbonyl compounds in ETS. 

In annular denuder sampling, potassium iodide (20 g KI in 10 ml of deionized water + 10 ml of 10% glycerol in methanol), phenoxazine (0.4 g in 20 ml of acetone), and 4-allyl-2-methoxyphenol (eugenol, 10% in methanol) are used as absorbents. The third absorbent releases formaldehyde by ozonolysis, which is collected with another downstream denuder coated with 2,4-dinitrophenylhydrazine (0.5% in acetonitrile containing 1% phosphoric acid). 

Place 2 mL of a solution of 2,4-dinitrophenylhydrazine (prepared as described for the classification test in Experiment 52D) in a test tube and add 0.15 g of the unknown compound. If the unknown is a solid, it should be dissolved in the minimum amount of 95% ethanol or 1,2-dimethoxyethane before it is added. If crystallization is not immediate, gently warm the solution for a minute in a hot water bath (90°C) and then set it aside to crystallize. Collect the product by vacuum filtration. 

The separation by TLC of the 2,4-dinitrophenylhydrazone (DNP) of dehydroascorbic acid is extremely specific and has been employed for detection and determination of vitamin C in food and feeding stuffs, fats, fruit juices, wines and bacteria etc. In the method of Stroheckeb et al. [129, 130] the ascorbic acid in the extracts is oxidised with 2,6-dichlorophenol-indophenol (Vuilleumieb and Nobile [139] use bromine for this) the dehydroascorbic acid formed is then reacted for 3 hours at 70 C with 2,4-dinitrophenylhydrazine in the presence of a little trichloroacetic acid and thiourea [131]. After cooling 10 min in ice, the red or red-brown precipitate is collected on a sintered glass filter, washed with water and dissolved in ethyl acetate or acetone the solution is evaporated and the residue taken up in acetone. 0.1—1.0 ml (20 to 50 [xg vitamin C) of this solution is applied as a band to air dried silica gel H layers and chromatographed with chloroform-ethyl acetate (50 + 50) or, better, with chloroform-ethyl acetate-acetic acid (60 + 35 -f 5). The red DNP of the dehydroascorbic acid is thus clearly separated... 

If a precipitate is not obtained within 15 minutes, dilute the solution very carefully with 2N sulphuric acid. It is advisable to collect and wash the derivative and determine its melting point to ensure that it is the 2,4-dinitrophenylhydrazone of the test substance, and not 2,4-dinitrophenylhydrazine (m.p. 194 ) or its sulphate (m.p. 185°). (Dinitrophenylhydrazine sulphate is stable only in the presence of excess of sulphuric acid injudicious dilution of the reagent or addition of a basic compound promotes hydrolysis and the precipitation of 2,4-dinitrophenylhydrazine.)... 

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