2.4- Dinitrophenylhydrazones paper chromatography

In the analysis of mixtures, or during the identification of small amounts of 2,4-dinitrophenylhydrazones, paper chromatography can be used successfully (35). For preparative separation of 2,4-dinitrophenyl-hydrazones column chromatography on alumina or calcium sulfate is suitable (36). 

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... 

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,... 

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. 

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). 

The solv ent migration rate can be accelerated through centrifugal force. The plate and layer are rotated in a horizontal plane and solvent introduced at the axis of rotation. Rosmus, PAVLf EK and Deyl [585], who had previously studied centrifugal paper chromatography in detail, have tried out the technique with TLC. They showed that the Stahl test mixture and mixtures of 2,4-dinitrophenylhydrazones could be separated in 5—10 min on silica gel G layers of 20 cm diameter, using 500 revs/min. 

For paper chromatography of 2,4-dinitrophenylhydrazones of aliphatic and alicyclic aldehydes and ketones the solvent systems dimethylforma-... 

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. 

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. 

The coloured p-nitro- and 2,4-dinitrophenylhydrazones are ideal derivatives for the separation and characterization of carbonyls by paper, thin-layer and column chromatography. The oximes can easily be revealed on the thin-layer plates by spraying with solutions of copper(Il) chloride or copper(II) acetate (alcoholic) or iron(III) chloride [21,22]. For the visualization of carbonyl compounds by means of fluorogenic labeling by Schiff base formation with reagents such as dansyl-hydrazine, we refer the reader to Chapter 9. 

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