Transcript OSAZONE - mStudyGroup.com

PRACTICAL NO
PHENYL HYDRAZINE TEST
OSAZONE TEST
TO IDENTIFY REDUCING SUGAR IN A GIVEN
SOLUTION.
• This test is for reducing carbohydrates.
• Reducung disaccharides and many monosaccharides
can be identified with the formation of characteristic
Osazone crystals’
• Hence this test is also known as Osazone formation
test.
REAGENTS:
• Phenyl hydrazine hydrochloride
• Sodium acetate
• Glacial acetic acid
• Osazone mixture is prepared by mixing thoroughly one
part of phenyl hydrazine hydrochloride and two parts of
sodium acetate by weight.
PRINCIPAL:
• When the reducing carbohydrates are treated with phenyl hydrazine at
100 C and pH 4.3 a series of reaction takes place resulting in the
formation of osazones of respective carbohydrates. The general
reaction is
• Glucose + 3 phenylhydrazine molecules Glucosazone + NH3 +H2O
• A constant temperature is provided by the boiling water bath .
• A constant pH is provided by the buffer pair of sodium acetate and
acetic acid.
• The reaction involves only first and second carbon atoms of the
reducing carbohydrate.
• Since glucose and fructose differ only with respect to
the 1st and 2nd carbon atoms they form the same
osazones. Mannose will also form the same osazone.
Galactose differs from glucose at carbon number
4,therefore its crystal shape is different.
• Sucrose and starch do not form crystals, because they
are non reducing sugars. They can form crystals only if
they are first hydrolyzed into their components.
PROCEDURE
• Take 5ml each of glucose,fructose,galactose,maltose and
lactose solutions in correspondingly label test tubes.
• Add about 0.3gm of osazone mixture and 3 drops of glacial
acetic acid in all the test tubes.
• Put all test tubes in boiling water bath.
• Observe each test tube after every 5 minutes till crystals
appear.
• Note in each test tube how much time (in minutes) is being
taken by the crystals to appear.
• If no crystal appears after 30 minutes,remove these tubes from
the water bath and allow them to cool spontaneously.
• Observe after every 5 minutes till crystals appear upto 25 min.
• With the help of a glass rod,take out some crystals on a glass
slide, cover them with a cover slip and observe under a
microscope.
INTERPRETATION
• Yellow crystals will appear in the tubes containing glucose and
fructose within 10 min of heating.
• Galactose gives crystals within 20 min of heating.
• Maltose gives crystals within 10-15 min of cooling.
• Lactose gives crystals within 20-25 min of cooling.
• Generally monosaccharides give crystals on heating and all
disaccharides give crystals on cooling.
• The osazones of different carbohydrates differ in their time of
formation,solubility,melting point and crystalline structure.
• Carbohydrates osazone
solubility
shape of crystals
glucose
glucosazone
insoluble
bundle of grass
mannose
mannosazone insoluble
bundle of grass
fructose
fructosazone
insoluble
bundle of grass
galactose
galactosazone insoluble
needle shaped
maltose
maltosazone
soluble
sunflower shaped
lactose
lactosazone
soluble
puff ball shaped
PRECAUTIONS
• Reagents should be taken in correct proportion.
• Cooling should always be spontaneous,preferably in
the water bath itself.
• Before boiling it should be noted that the solution is not
too concentrated,because crystals will not be formed in
very concentrated solution.