2.4- Dinitrophenylhydrazone chromatography

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). 

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. 

Many organic chemicals are analyzed by RPC. These include various arylhydroxylamines as the N-hydroxyurea derivative with methyl isocyanate (614) alkyl- and alkoxy-disubstituted azoxybenzenes (6t5), n-alkyl-4-nitrophenylcarbonate esters ranging in length from methyl to octyl (616), 4-nitrophenol in the presence of 4-nitrophenyl phosphate (617), ben-zilic acid, and benactyzine-HCI using ion-pair chromatography (618), as well as aniline and its various metabolites (619), stereoisomers of 4,4 -dihydroxyhydrobenzoin (620), and aldehydes and ketones as the 2,4-dinitrophenylhydrazones (621). The technique has also been used to analyze propellants and hydrazine and 1,1-dimethylhydrazine were quantitita-vely determined (622, 623). 

More definite evidence for the transient existence of the un-cyclized l-(jS-aminoethyl)-3,4-benzoquinones has been obtained recently by Kodja and Bouchilloux,77 78 who noted that a transient yellow color (Amax ca. 385 mp) was occasionally observed during the enzymic oxidations of catecholamines (particularly in unbuffered systems at low temperatures). This phenomenon was probably due to the formation of the transient o-quinones. (The absorption maximum of o-benzoquinone, the effective chromophore of the open-chain quinones, is known to occur at ca. 390 mp.79) An absorption maximum at 390 mp is characteristic of the formation of the dopa-quinone chromophore during oxidation of small C -terminal tyrosine peptides in the presence of tyrosinase.37 48 Similar spectroscopic features were observed when the oxidations were carried out with lead dioxide in sulfuric acid solutions (pH> 1). If the initial oxidation was carried out for a short period of time, it was possible to regenerate the original catecholamines by reduction (e.g. with sodium bisulfite, potassium iodide, and zinc powder) and to show that the 385 mp peak disappeared.77,78 Kodja and Bouchilloux were also able to identify 2,4-dinitrophenylhydrazones of several of the intermediate non-cyclized quinones by paper chromatography and spectroscopy (Amax n weakly acid solution ca. 350 mp with a shoulder at ca. 410 mp).77,78... 

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

RETENTION TIMES OF 2,4-DINITROPHENYLHYDRAZONES OBTAINED WITH LIQUID-SOLID CHROMATOGRAPHY... 

Liquid-liquid partition chromatography. The stationary phase consists of 1% tris(2-cyanoethoxy)propane (TCEP) on Zipax support. The mobile phase, hexane, is saturated with TCEP prior to use. A pre-column consisting of 30% TCEP on Gas-Chrom Q is used in order to prevent stripping of the liquid phase from the analytical column. The detector is set at 254 nm for monitoring the column effluent. The retention times of a number of 2,4-dinitrophenylhydrazones obtained with this system are given in Table 4.12. Gradient elution may be made with Permaphase ETH as stationary material and a gradient of hexane-chloroform. The limit of detection is ca. 5 ng per injection. 

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. 

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. 

A procedure developed by Mattox and Kendall for the liberation of cortisone acetate from the C3 2,4-dinitrophenylhydrazone utilizes a mixture of pyruvic acid, acetic acid, chloroform, and hydrogen bromide and affords the parent compound in 80% yield. For comparison. DePuy° prepared a mixture of 1 g. of A -cholestene-S-one 2,4-DNP with 100 ml. of chloroform and 100 ml. of the usual levulinic-hydrochloric acid and heated it under reflux for 3 hrs. Chromatography afforded pure A -cholestene-3-one in high yield. 

It also has been used as a spray reagent and for quantitation in paper and thin layer chromatography Section 6.5). It should be noted that A - 4-3-keto steroids react much less readily with isonicotinic acid hydrazide than do A4-3-keto steroidsThe 2,4-dinitrophenylhydrazone has also been described4. 

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,... 

The milk and cream were centrifuged, and the fat fraction was removed. The fat was extracted with carbonyl free hexane, and the fat extract was immediately reacted with 2,4-dinitrophenylhydrazine. The carbonyl compounds formed respective 2,4-dinitrophenylhydrazones (2,4-DNPH). The hydrazones were separated into monocarbonyls and then further purified using thin layer chromatography (18). Alkanal 2,4-DNPH were analyzed by gas chromatography using a 1.83 m glass column (i.d. 4 mm) packed with 2% OV-1 on acid washed Chromosorb W ( 180-200 mesh) (18,19). ... 

Other workers separate by paper chromatography the amino acids obtained by catalytic hydrogenolysis of the 2,4-dinitrophenylhydrazones of the a-keto acids (H19, K19, T24). 

Paper electrophoresis provides a good way of separating the 2,4-dinitrophenylhydrazones (B18, N6, T2, VI, Z3) and gives results which can be compared to those obtained by paper chromatography. 

Kuwata, K., Uebori, M., and Yamaski, Y., Determination of aliphatic and aromatic aldehydes in polluted airs as their 2,4-dinitrophenylhydrazones by high performance hquid chromatography,... 

Carbonyl products were identified by paper chromatography of the 2,4-dinitrophenylhydrazones (20). The irradiated N-acetylalanine showed only pyruvic acid and acetaldehyde. These were determined quantitatively by the method of Johnson and Scholes (13) with minor modifications. Chloride ion was determined by the method of Luce et al. (15) after Hayon and Allen (12). 

Buyske, D.A., L.H. Owen, P. Wilder Jr, and M.E. Hobbs Chromatography of the 2,4-dinitrophenylhydrazones of some aldehydes and ketones in tobacco smoke Anal. Chem. 28(1956)910-913. 

Donzel, M. The separation by gas chromatography of 2,4-dinitrophenylhydrazones from carbonyl compounds of cigarette smoke. Application to the determination... 

Seligman, R.B. and M.D. Edmonds Chromatography of 2,4-dinitrophenylhydrazone derivatives 9th Tobacco Chemists Research Conference, Program Booklet and Abstracts, Vol. 9, Paper No. 8, 1955, pp. 4—5. 

Fung, K. and D. Grosjean. 1981. Determination of nanogram amounts of carbonyls as 2,4-dinitrophenylhydrazones by high performance liquid chromatography. Anal. Chem. 53 168-171. 

Two new alkaloids of the flindersine group, zanthophylline (13 R = Me) and desmethylzanthophylline (13 R = H), were obtained from Zanthoxylum mono-phyllum by chromatography of the chloroform extract of the plant. The structure of zanthophylline was indicated by and n.m.r. spectroscopy and by hydrolysis to 8-methoxyflindersine (2 R = OMe) and formaldehyde (identified as its 2,4-dinitrophenylhydrazone derivative). When aqueous acid was used in the isolation procedure, the only alkaloid obtained was 8-methoxyflindersine, and this is apparently an artefact formed by hydrolysis of zanthophylline. Desmethylzanthophylline is only a minor constituent of Z. monophyllum although the alkaloid has not been fully investigated, its mass spectrum is in accord with structure (13 R = H), and the presence of N-acetoxymethyl and hydroxy-groups is confirmed by the appropriate i.r. absorptions. 

S. Takeda, S. Wakida, M. Yamane and K. Higashi, Analysis of lower aliphatic-aldehydes in water by micellar electrokinetic chromatography with derivatization to 2,4-dinitrophenylhydrazones, Electrophoresis, 15,1332-1334, 1994. 

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