Molisch’s test:

PrincipleAll carbohydrates when treated with conc. sulphuric acid undergodehydration to give furfural compounds. These compounds condensewith Alpha-napthol to form colored compounds.Molisch test is given by sugars with at least five carbons because itinvolves furfural derivatives,which are five carbon compounds.

Benedict’s Test:All Reducing sugars give positive benedict's test. Reducing sugars havea free aldehyde or keto group.

PrincipleGlucose (R-CHO) + 2Cu2+

+ 2H2O--------->Gluconic acid ( R-COOH) + Cu2O + 4H+

The principle of Benedict's test is that when reducing sugars areheated in the presence of an alkali(pH 10.6), they get converted topowerful reducing compounds known as enediols. Enediols reduce thecupric ions (Cu2+) present in the Benedict's reagent to cuprous ions(Cu+) which get precipitated as insoluble red copper(I) oxide.The color of the obtained solution gives an idea about the quantity ofsugar present in the solution, hence the test is semi-quantitative.

Carbohydrates giving positive Benedict ’ s test:Glucose, Fructose, Galactose,Ribose, Glucuronic acid,Lactose, MaltoseNote: Sucrose with no free reducing group give negative test.

Non-Carbohydrates giving positive Benedict ’ s test:High concentration of Uric acid , Creatinine and KetonesHomogentisic acid (solution turns black due to black colored oxidizedhomogentisic acid)Vitamin C (even without Boiling)Certain drugs like aspirin, cephalosporins

StarchesStarches do not react or react very poorly with Benedict's reagent, dueto the relatively small number of reducing sugar moieties, which occuronly at the ends of carbohydrate chains.

Different concentration of glucose gives different color of solution withBenedict’s test, depending on amount of precipitate and residual cupricsulphate.Grade Color of Reaction MixtureApproximate Glucoseconcentration+ Green 0.5-1 gm%++ Yellow 1-1.5 gm%+++ Orange 1.5-2 gm%++++ Red >2 gm%Benedict’s test is frequently used to detect glucose in urine. Althoughglucose is most frequent reducing substance present in urine, in somepatient positive Benedict’s test may be due to non-glucose reducingsubstances listed above. This phenomenon may be called falsepositive result.Then test specific for glucose should be performed to confirm presence of glucose in urine.

Fehling's test: Same Principle as Benedict's as sir told u.

Barfoed’s Test:Principle: This test is based on the same principle as Benedict’s test. But, the test medium is acidic. In acidic medium(pH 4.6) monosaccharides reactfaster than disaccharide. Monosaccharides react fast within 1-2 minutesbut disaccharides take longer i.e. 7-12 minutes.

RCHO + 2Cu2+ + 2H2O --------------------->RCOOH + Cu2O↓ + 4H+

Seliwanoff’s TestSeliwanoff’s test is a chemical test which distinguishes between aldoseand ketose sugars. This test is based on the fact that, when heated,ketoses are more rapidly dehydrated than aldoses.

PrincipleKetohexoses like fructose on treatment with HCl form 5-hydroxymethylfurfural, which on condensation with resorcinol gives a cherry red complex.Sucrose is hydrolyzed into glucose and fructose when boiled in acidicmedium of Seliwanoff’s reagent. Fructose, present in hydrolysate givespositive Seliwanoff’s test.

Inversion Test:

PrincipleWhen sucrose is boiled with conc. HCl, It is hydrolyzed into itsconstituent monosaccharides i.e. fructose and glucose. The hydrolyzedglucose and fructose give Benedict’s test. Fructose gives seliwanoff’stest.Sucrose is dextrorotatory. The optical rotation changes fromdextrorotatory to levorotatory on hydrolysis, since fructose causes amuch greater levorotation than the dextrorotation caused by glucose.This is known as inversion. The resultant hydrolysate is called invertsugar, which is sweeter than sucrose because fructose is sweeter thansucrose.

Osazone formation:

All reducing sugars will form osazones with excess of phenylhydrazine when kept at boiling temperature. Osazones are insoluble.They have characteristic crystal form.

Glucose, fructose and mannose ----> Needle shaped osazone

Lactose ----> Cotton ball, powder puff

Maltose -----> sunflower shaped or star shaped crystals