Molisch's Test (procedure)
In a dry test tube take 2 ml of the solution and add 6 drops of 1% a napthol in ethanol. Mix and add 1 ml of H2SO4 along the sides of the test tube. (positive = a violet ring is formed at the junction of the two liquids)
Molisch's Test (reagent)
alpha napthol in 95% ethanol
Molisch Test (principle)
Its a general test for carbohydrates. Concentrated sulfuric acid dehydrates the carbohydrate to for furfural and its derivatives. The furfural derivative then condense with alpha napthol to produce colored condensation products
pentose --> furfural --> colored products
hexose --> hydroxy methyl furfural --> colored products
Molisch's Test Note
Only carbs of greater than five C atoms will answer.
Green color is renders test negative.
Benedict's Test (procedure)
To 5ml of benedict's reagent, add 8 drops of sugar solution. Mix well. Heat over a direct flame for about 2 minutes. (positive = browninsh greenish red)
Benedict's Test (reagent)
a. Copper sulphate : furnishes cupric ions
b. sodium carbonate : provides alaline medium which is condusive for the reaction
c. sodim citrate : keeps cupric ions in solution by forming cupric sodium citrate complexes.
Benedict's Test (principle)
sugars having a free aldehyde or keto group are called reducing sugars. Reducing sugars tautomerize in mild alkali medium to form enediols which are powerful reducing agents. On heating, cupric ions from the reagent form cuprous ions. An unstable cuprous hydroxide is formed which precipitates cuprous oxide. The sugar oxidized forms various acids like aldonic and saccharic acids.
Benedict's Test (applied importance)
Benedict's test is used to detect reducing sugars in urine. Used to detect and monitor the progress of diabetes from the bedside.
Barfoed's Test (procedure)
To 5ml of Barfoed's reagent, add 10 drops of sugar solution. mix and heat for 1 minute. (positive = red scum)
Barfoed's Test (reagent)
copper acetate in glatial acetic acid
Barfoed's Test (principle)
on heating, monosaccharides reduce the cupric ions from the reagent to form the red scum of cuprous oxide. the mild acidic medium prevents disaccharides like lactose and maltose from answering the test.
Benedict's Test (note)
prolonged heating may cause even the disaccharides to reduce the cupric ions because disaccharides hydrolyze to form monosaccharides.
Seliwanoff's Test (procedure)
To 5ml of the reagent add 10 drops of sugar solution. Boil the solution on a direct flame for 30 seconds. (positive : a cherry red color is seen if a keto sugar is present)
Seliwanoff's Test (reagent)
Resorcinol in 33% HCl
Seliwanoff's Test (principle)
on heating with seliwanoff's reagent, ketoses get dehydrated by the HCl to form furfurals. This condenses with resorcinol to form the red colored complexes.
Seliwanoff's Test (note)
if the heating exceeds 30 seconds aldoses may also answer the test.
Osazone Test (procedure)
2ml of the sugar solution is added to 1ml of Phenyl Hydrazine mixture, heated in a boiling water bath for 15 minutes and then allowed to cool by keeping it in the test tube rack. The crystals are examined under a microscope.
Osazone Test (reagent)
Osazone Test (principle)
Phenyl hydrazine reacts with the reducing sugars to form derivatives called phenyl hydrazones. Phenyl hydrazones react with another molecule of phenyl hydrazine to form osazones. These osazones have characteristic crystalline shape which ca be seen under the microscope.
The reaction with phenyl hydrazine takes place in two stages. It first reacts with the carbonyl group to form phenyl hydrazones. In the next stages, there is simple oxidation followed by addition of second phenyl hydrazine to form osazone.
Osazone formed by
glucose: needle like crystals
fructose: needle like crystals
maltose: sunflower shaped crystals
lactose: hedgehog shaped crystals