Formation of Osazones

Phenylhydrazine is toxic avoid contact with it. If you spill some on your skin, rinse it off thoroughly with household bleach, dilute acetic acid, and then water. 

Preparation Sign in at www.cengage.com/login to answer Pre-Lab Exercises, access videos, and read the MSDSs for the chemicals used or produced in this procedure. Review Sections 2.9 and 2.17. 

Apparatus Test tubes, hot-water bath, apparatus for vacuum filtration. 

Setting Up Heat a large beaker of water to boiling. In each of three test tubes, separately dissolve 0.2-g portions of D-glucose. D-fructose. and sucrose in 4 mL of water. In a fourth test tube, place 4 mL of fhe solution that was used for rotation experiments for the hydrolysis of sucrose (Sec. 23.3). Add 0.5 mL of saturated sodium bisulfite solution to each test tube to suppress oxidation of the phenylhydrazine during the reaction this avoids contaminating the phenylosazone with tarry by-products. 

Reactions To each of the test tubes, add either 0.6 mL of glacial acetic acid, 0.6 g of sodium acetate, and 0.4 g of phenylhydrazine or 0.6 g of sodium acetate and 0.6 g of phenylhydrazine hydrochloride. Stir the solutions thoroughly, place the test tubes in the beaker of boiling water, and discontinue heating. Allow the test tubes to remain in the hot-water bath for 30 min. Remove fhe test tubes from the hot-water bath and cool the contents to room temperature.  

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Another reaction that is characteristic of a-hydroxy aldehydes or ketones, which has been found of value for the characterisation of sugars, is the formation of osazones with phenylhydrazine. This reagent reacts with either an aldose... 

The principal reactions of this class of compounds are summarized in Scheme 172. In most of these reactions the reactive nucleophilic center is the terminal NHj group, although the other exocyclic nitrogen may also be involved, as shown by acetylation, which yields 284 and 285. However, the structure of compound 281 is not the one proposed in a recent report (1582) that attributes the attack to the other exocyclic nitrogen. The formation of osazones (287) from sugars, 2-hydrazinothiazoles, and hydrazine has been reported (525, 531). 

An identical compound is formed from benzil by the action of phenylhydrazine, and from benzaldehyde phenylhydrazone by autoxida-tion (Busch). The formation of osazones from a-hydroxyketones (and a-hydroxyaldehydes) will be discussed later (p. 298). 

Historically, techniques such as the formation of osazones and the demonstration of fermentation have contributed significantly to the separation and identification of carbohydrates. Observation of the characteristic crystalline structure and melting point of the osazone derivative, prepared by reaction of the monosaccharide with phenylhydrazine, was used in identification. This method is not completely specific, however, because the reaction involves both carbon atoms 1 and 2 with the result that the three hexoses, glucose, fructose and mannose (Figure 9.19), will yield identical osazones owing to their common enediol form. 

Colorimetric Methods. The most frequently used colorimetric methods have been recently reviewed by Omaye et al. (5). Several methods of analyses are based upon the fact that ascorbic acid and dehydroascorbic acid possess certain chemical properties characteristic of sugars such as formation of osazones and conversion to furfural. Colorimetric determination of furfural, an aniline derivative, has been used to a limited extent for the estimation of ascorbic acid in certain materials. These methods have generally been found to be unsatisfactory... 

Sucrose likewise yields glucosazone identifiable as glucosazone formazan. This indicates that in sucrose it is the hydroxyl group on the first carbon atom of n-fructose which oxidizes to an aldehyde group. As has been observed by several authors (1, 7, 14), in the course of the formation of osazone the disaccharide bond 2, S) breaks up and glucosazone forms from both components of the disaccharide upon the addition of phenylhy drazine. 

L. Caglioti, G. Rosini, and F. Rossi, Azoalkenes as intermediate compounds in the formation of osazones from a-acetoxy ketones, J. Am.Chem. Soc., 88 (1966) 3865-3866. 

Formation of Osazones.—Osazones are readily formed from 3-deoxy-o-erythro-hexosuloae in the presence or absence of acid. However, the only crystalline osazones that have been obtained are the (4-nitrophenyl)osa-zone, the (2,4-dinitrophenyl)osazone, and the (2,5-dichlorophenyl)-osazone. The first two are identical with the respective osazones prepared from 3-deoxy-D-n 5o-hexose. The last two derivatives yield crystalline triacetates.The high melting points of many of these derivatives are not satisfactory for characterization, but their high optical rotations are a useful criterion of purity. ... 

The structure of 3-deoxy-D-erythro isomer, from the formation of osazones, of 5-(hydroxymethyl)-2-furaldehyde with acids, and of metasac-charinic acids with alkali. ... 

Many oxazoles react slowly at room temperature with 2,4-dinitrophenyl-hydrazine in hydrochloric acid to form hydrazones (223) by ring fission.84 160 On the other hand, refluxing results in the formation of osazones (224) of glyoxal derivatives, presumably via 223.877... 

Due to the presence of carbonyl, aldehyde or ketone radicals, sugars are capable of addition reactions with nucleophilic reagents such as phenylhydrazine (C6H5-NH-NH2). The addition of three phenylhydrazine molecules to an aldose (Figure 3.12) leads to the formation of osazone, a crystallized product with specific physicochemical characteristics, especially its melting point. This makes it possible to identify the corresponding sugar. 

The formation of osazones requires 3 moles of phenylhydrazine per mole of sugar but 1 mole is reduced during the reaction to yield 1 mole each of aniline and ammonia 221). To explain the formation of the aniline and ammonia, the following mechanism has been proposed HC=0 HC=N—NH—Ph... 

The formation of osazones from 2-methoxybutanone 225), 2-methoxy- 226) and 2-chlorocyclohexanone 2f, and 2-0-methyl and 2-amino sugars indicates the ready lability of the functional group alpha to the carbonyl function. In these cases, osazone formation may proceed by direct replacement of the alpha functional group by a phenylhydrazine molecule to give (III) without prior hydrolysis to give an alpha hydroxy compound. 

OSes which react with Fehling s solution. They give a precipitate of Cu oxide (cuprous oxide) due to their reducing power. Let s also recall the formation of osazones for the identification of oses. Oses react with phenylhydrazine in well-defined conditions to yield crystallized yellow phenylosazones poorly soluble in... 

The rate of formation of osazones and their solubility, which differ substantially from case to case, can also serve as characteristic features useful in the identification of sugars. For example, osazones of glucose and fructose precipitate shortly after the heating of the solution, while saccharose requires 20 — 30 min of heating. Osazones of lactose and maltose precipitate only after cooling. In addition, phenylosazones of D-glucose, D-fructose, and D-mannose (which are identical) are insoluble in cold acetone, in contrast to phenylosazones of xylose, arabinose, rhamnose, and fructose. 

Oxidation and Optical Activity Formation of Osazones Chain Extension of Aldoses Chain Shortening of Aldoses... 

The formation of osazones by treatment with phenyl hydrazine leads to liberation of the phosphate group when it is near the carbonyl. This occurs... 

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