Phenylhydrazine Reaction

Phenylhydrazine Reaction

• This is a test for reducing carbohydrate, reducing disaccharides and many monosaccharides to be identified with the formation of osazone crystals

• Used to differentiate simple sugars by their varied forms of osazones and their rates of osazone formation

Procedure

• Fill round bottoms of 6 test tubes with phenyhydrazine mixture.

• Add 5 ml of the different sugars to each corresponding test tube.

Procedure

• Shake vigorously to insure complete solution• Loosely place a cotton stopper• Place immediately in a boiling water bath• Record time

Procedure

• Observe tube carefully for a distinct precipitate to form. Record time

• Remove tube from the water bath• *If no precipitate forms after 40 minutes of

heating, remove tube and add 4ml distilled water. (causes immediate precipitation of osazones)

Procedure

• Tube then returned to boiling water bath for few minutes until osazones redissolves.

• Allow water bath and test tubes to cool slowly to room temperature.

• Examine crystals under the microscope

Sample pictures showing crystals

After 3 minutes After 8 minutes

http://jchemed.chem.wisc.edu/JCESoft/CCA/CCA5/MAIN/1ORGANIC/ORG18/TRAM18/C/PICTURE.HTM?5

Mullikin’s Figures

Sugar Time (minutes)

Glucose 4-5

Fructose 2

Sucrose 30-35 (after hydrolysis)

Maltose Osazone soluble in hot water

Lactose Osazone soluble in hot water

Principle

Principle1. Free C=O groups of sugars react with

phenylhydrazine to form phenylhydrazones.

fructose

+

phenylhydrazone

phenylhydrazine

Principle1. Free C=O groups of sugars react with

phenylhydrazine to form phenylhydrazones.

2. With excess phenylhydrazine, the C adjacent to C=O reacts with phenylhydrazine to form yellow compounds called OSAZONES.

phenylhydrazone

+

osazone

phenylhydrazine

Results

Glucose Fructose

MaltoseLactose

Galactose Sucrose

No crystallization will be observed until hydrolysis of the sucrose occurs, which will yield both fructosazones and glucosazones