Place 0.2 g of the unknown sample in a test tube and add 0.4 g of phenylhydrazine hydrochloride, 0.6 g of crystallized sodium acetate, and 4 mL of distilled water. Place the test tube in a beaker of boiling water. Note the time that the test tube was immersed and the time of the precipitation. After 20 min, remove the test tube from the hot water bath and set it aside to cool. A small amount of the liquid and solid is poured on a watch glass. Tip the watch glass from side to side to spread out the crystals, and absorb some of the mother liquid with a piece of filter paper, taking care not to crush or break up the clumps of crystals. Examine the crystals under a low-power microscope (about 80-100×), and compare with photomicrographs. The formation of tarry products due to oxidation of the phenylhydrazine may be prevented by the addition of 0.5 mL of saturated sodium bisulfite solution. This should be done before heating if it is desired to isolate the osazone and determine its melting point.
Carbohydrates and 1,2-diols give a positive test.
The times required for the formation of the osazones can be a valuable aid in distinguishing among various sugars. The following figures are the times required for the osazone to precipitate from the hot solution: fructose, 2 min; glucose, 4-5 min; xylose, 7 min; arabinose, 10 min; galactose, 15-19 min; raffinose, 60 min; lactose, osazone soluble in hot water; maltose, osazone soluble in hot water; mannose, 0.5 min (hydrazone); sucrose, 30 min (owing to hydrolysis and formation of glucosazone).
Osazone formation involves hydrazone formation at C-1 of an aldose (or C-2 of a ketose) and oxidation of C-2 (or C-1) of an alcohol group to a ketone (or an aldehyde). The new carbonyl group is also converted to a hydrazone. It has been suggested that the reaction stops here (rather than further oxidation at C-3, etc.) because of hydrogen-bonding stabilization of the osazone.
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