2.4- Dinitrophenylhydrazones separation

Dinitrophenylhydra2ones usually separate in well-formed crystals. These can be filtered at the pump, washed with a diluted sample of the acid in the reagent used, then with water, and then (when the solubility allows) with a small quantity of ethanol the dried specimen is then usually pure. It should, however, be recrystallised from a suitable solvent, a process which can usually be carried out with the dinitrophenylhydrazones of the simpler aldehydes and ketones. Many other hydrazones have a very low solubility in most solvents, and a recrystallisation which involves prolonged boiling with a large volume of solvent may be accompanied by partial decomposition, and with the ultimate deposition of a sample less pure than the above washed, dried and unrecrystal-lised sample. 

A) Phenylhydrazones and 2,4-Dinitrophenylhydrazones (see 2 abo e). The latter are often to be preferred to phenylhydrazones because (a) the phenylhydrazones may separate as syrups, and also may decompose in hot solvents cf. p. 257), ( ) the 2,4 dinitrophenylhydrazones are often formed in the cold or with only brief warming, are much less soluble, and have higher m.ps. (p. 263). 

The conversion of the compounds under investigation into coloured derivatives (e.g., the separation of carbonyl compounds by conversion into their 2 4-dinitrophenylhydrazones, etc. of hydrocarbons through their picrates of alcohols through their 3 5-dinitrobenzoates of glucose, fructose and other simple sugars through their p-phenylazobenzoyl esters). 

The experimental procedure to be followed depends upon the products of hydrolysis. If the alcohol and aldehyde are both soluble in water, the reaction product is divided into two parts. One portion is used for the characterisation of the aldehyde by the preparation of a suitable derivative e.g., the 2 4-dinitrophenylhydrazone, semicarbazone or di-medone compound—see Sections 111,70 and 111,74). The other portion is employed for the preparation of a 3 5-dinitrobenzoate, etc. (see Section 111,27) it is advisable first to concentrate the alcohol by dis tillation or to attempt to salt out the alcohol by the addition of solid potassium carbonate. If one of the hydrolysis products is insoluble in the reaction mixture, it is separated and characterised. If both the aldehyde and the alcohol are insoluble, they are removed from the aqueous layer separation is generally most simply effected with sodium bisulphite solution (compare Section Ill,74),but fractional distillation may sometimes be employed. 

Monocrotaline on alkaline hydrolysis yields retronecine and monocrotic acid, CjHijOg, b.p. 145-6°/18 mm., [a]p 0°, which forms a p-bromo-phenacylester, m.p. 78°,and a methyl ester, b.p. 94-6°/18 mm., characterised by a 2 4-dinitrophenylhydrazone, m.p. 95-6° see below). The acid gives the iodoform reaction and is oxidised by sodium hypobromite to a mixture of dl- and mcso-aa -dimethylsuccinic acids (I). These and other reactions show that monocrotic acid is a -dimethyllaevulic acid (II) and this has been eonfirmed by comparison with a synthetic specimen of the acid. The methyl ester of the synthetie acid forms a mixture of 2 4-dinitrophenylhydrazones, m.p. 108-9° and 121-2°, into which the analogous produet, m.p. 95-6°, first made from methyl monocrotate see above), has also been separated. 

When present in macro quantities, aldehydes and ketones can be determined by conversion to the 2,4-dinitrophenylhydrazone which can be collected and weighed. When present in smaller quantities (10 3M or less), although hydrazone formation takes place, it does not separate from methanol solution, but if alkali is added an intense red coloration develops the reagent itself only produces a slight yellow colour. Measurement of the absorbance of the red solution thus provides a method for quantitative determination. 

The dinitrophenylhydrazones were separated from the reaction mixture by thin-layer chromatography (silica gel G developed with benzene) and further purified by thin-layer chromatography on aluminum oxide G (petroleum ether-diethyl ether (96 to 4), silica gel G (chloroform), and silica gel G (diethyl ether)). In all cases, the specific activities of the dinitrophenylhydrazones remained constant over the course of the last two purifications. 

The identification and quantification of potentially cytotoxic carbonyl compounds (e.g. aldehydes such as pentanal, hexanal, traw-2-octenal and 4-hydroxy-/mAW-2-nonenal, and ketones such as propan- and hexan-2-ones) also serves as a useful marker of the oxidative deterioration of PUFAs in isolated biological samples and chemical model systems. One method developed utilizes HPLC coupled with spectrophotometric detection and involves precolumn derivatization of peroxidized PUFA-derived aldehydes and alternative carbonyl compounds with 2,4-DNPH followed by separation of the resulting chromophoric 2,4-dinitrophenylhydrazones on a reversed-phase column and spectrophotometric detection at a wavelength of378 nm. This method has a relatively high level of sensitivity, and has been successfully applied to the analysis of such products in rat hepatocytes and rat liver microsomal suspensions stimulated with carbon tetrachloride or ADP-iron complexes (Poli etui., 1985). 

The ozonides of choline and ethanolamine phosphatides and triglycerides can be subjected to reduction with triphenylphosphine to yield the corresponding core aldehydes, and further derivatized to the 2,4-dinitrophenylhydrazones (DNP). The core aldehydes and their DNP derivatives can be separated by HPLC and characterized by various techniques, including EI-MS and TS-MS of positive and negative ions . See also Section VHI.E. 

Acetylnaphthalene [941-98-0] M 170.1, m 10.5°, b 93-95°/0.1mm, 167°/12mm, 302°/atm, d 01.12. If the NMR spectrum indicates the presence of impurities, probably 2-acetylnaphthalene, convert the substance to its picrate by dissolving in benzene or EtOH and adding excess of satd picric acid in these solvents until separation of picrates is complete. Recryst the picrate till m is 118°. Decompose the picrate with dil NaOH and extract with EtzO. Dry the extract (Na2S04), filter, evap and dist. The 2,4-dinitrophenylhydrazone crysts from EtOH and has m 259°. [A 380 95 7977 JACS 61 3438 7939],... 

Derivatization is a procedure in which analyte is chemically modified to make it easier to detect or separate. For example, formaldehyde and other aldehydes and ketones in air, breath, or cigarette smoke25 can be trapped and derivatized by passing air through a tiny cartridge containing 0.35 g of silica coated with 0.3 wt% 2,4-dinitrophenylhydrazine. Carbonyls are converted into the 2,4-dinitrophenylhydrazone derivative, which is eluted with 5 mL of acetonitrile and analyzed by HPLC. The products are readily detected by their strong ultraviolet absorbance near 360 nm. 

The analysis of keto steroids as their 2,4-dinitrophenylhydrazone (DNPH) derivatives by TLC [30] and HPLC [31,32] is a sensitive and reliable method for the determination of these compounds in urine and in other biological fluids. The derivatives are easily separated by TLC or HPLC and can be detected in quantities as low as 1 ng. Several variations of the reaction procedure may be used. Two of these are described below. 

Conditions for the separation of 2,4-dinitrophenylhydrazones by liquid-solid [69] and liquid-liquid chromatography [70] are described below. 

Separation of 2,4-dinitrophenylhydrazones. The solutions are prepared by dissolving 10 mg of each of the 2,4-dinitrophenylhydrazones of acetone, butan-2-one and hexan-3-one (or hexan-2-one) in 0.5 ml of ethyl acetate. Prepare a flexible silica gel sheet of dimensions 20 x 5 cm in the manner already described and apply c. 0.5 pi of each of the three solutions to give the marker spots of a diameter of between 2 and 3 mm. A mixed spot is conveniently obtained by loading sequentially to the same area further 0.5 pi aliquot portions of each of the solutions and allowing the solvent to evaporate completely between each addition. 

The GC separation of 2,4-dinitrophenylhydrazones has been studied by a number of workers [52-55], Non-polar stationary phases of the SE-30 and SF-96 type were utilized for this purpose at temperatures of 200—250°C, mostly with temperature programming. Retention indices of the 2,4-DNPHs of some carbonyl compounds on OV-type stationary phases are presented in Table 5.5. Using columns with a higher separation efficiency, some 2,4-DNPHs provide two peaks. The discussion of whether these artifacts are caused by thermal decomposition or syn-anti isomerization of the derivatives seems to favour the latter. The ratio of the areas of the peaks of the two derivatives depends on the polar-... 

The disadvantage of normal phase HPLC for the separation of carbonyl dinitrophenylhydrazones is the difficulty in maintaining reproducible retention times due to absorption of water from the solvent and interaction with the active groups on the column (Fig. 5.10). To overcome this problem, a cyano-propyl bonded phase (Exsil 100, 5 /im Chromtech, U.K.) column may be used for isolation of hy-droxyalkenals and a mixture of alkanals and alk-2-enals from rat liver microsomes. The less polar carbonyls can be separated under isocratic conditions as follows. 

After the separation of carbonyl dinitrophenylhydrazones into classes or into polar and non-polar fractions, the samples can be re-dissolved in a small volume of methanol and the amount present calculated approximately from the absorbance maxima at 365-370 nm using an average molar absorption coefficient of 26000 (Esterbauer, 1982). Alternatively, the amount of carbonyls within individual classes can be calculated from the molar absorptivities shown in Table 5.1. 

The 2,4-dinitrophenylhydrazone derivatives of a-ketoisovaleric acid and a-keto-jS-methylvaleric acid are separated from 2,4-dinitrophenylhydrazine by chromatography at room temperature on a Zorbax Cjg column (4.6 mm x 250 mm). Solvent A was 25% acetonitrile in water containing 0.1% triethyla-mine (v/v) and adjusted to pH 4.5 with acetic acid. Solvent B was acetonitrile. A linear gradient from 20 to 50% B was made within 20 minutes. The effluent was monitored at 254 nm. 

Gates and Helg(102) established earlier that /3-thebainone (47, R = H) (B/C trans) could be readily inverted under both acid (HOAc) and base (NaOEt) conditions to give predominantly dihydrothebainone (48, R. = H) (B/C cis), which was separated from the mixture in a pure yield of 60%. In addition, /3-thebainone 2,4-dinitrophenylhydrazone is subject to quantitative conversion to the B/C cis isomer in warm acetic acid within 6 h. 

Colorometric procedures involving reaction of aldehydes with hydrazines, semicarbazide, or piperidine/nitroprusside solutions are also non-specific and lack sensitivity (15, 35, 36). Schmidt et al. (33) have proposed an HPLC method for analyzing the 2,4-dinitrophenylhydrazone (DNPH) derivatives of specific aldehydes. This procedure allows for a number of ddehydes to be separated and measured simultaneously, however, HPLC methods in general suffer from poor resolving power and may have low sensitivity (37). In addition, hydrazine derivatizations are often performed under acidic conditions for maximal reactivity these conditions would not provide quantitative information on total aldehyde content. 

Several representative compounds from which abnormal products had been previously isolated in ozonizations carried out by other investigators were selected to test the above theory. Ozonizations were carried out in pure methanolic solutions at about —20° C. and in each case the yield of peroxide, based on the ozone consumed, was determined quantitatively. Overozonization was avoided, inasmuch as excess ozone is known to cause side reactions or decomposition of the peroxides (5). The peroxides thus formed were reduced with aqueous sodium bisulfite and the carbonyl compounds isolated as their 2,4-dinitrophenylhydrazones, which were separated quantitatively by chromatography through a mixture (2 to 1) of silica gel and Celite. In no case were any of the abnormal products found. The results are summarized in Table I. 

Special techniques have been used for the measurement of individual steroids. For blood progesterone Harbert et al. (H4) obtained an initial separation on an alumina column, but final purification and assay was achieved by further alumina chromatography after preparing the bis-dinitrophenylhydrazone. The method of Short (S16) involving paper chromatography and assay by ultraviolet absorption, with Allen corrections for impurity, has been used by Greig et al. (G5) and Aitken et al. (A3). Cortisol has been measured in urine and blood by fluorimetry and isotope dilution (U3, U4), and in blood by double isotope dilution derivative techniques together with cortisone (H9) and also with cortisone,... 

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