DEFINITIONCarbohydratesare defined aspolyhydroxy alcoholswith an aldehydeor ketoneas the functional group.CLASSIFICATIONCarbohydratesare classified according to thenumber of sugar molecules in themasmonosaccharides,oligosaccharidesand polysaccharides

Monosaccharide'sMonosaccharide'sare also called assimple sugars. Theyhaveonly onepotential sugar group. Theyconsist of a singlepolyhydroxy aldehydeor ketoneunit, and thuscannot behydrolyzed into simpler form. Theymaybesubdivided intodifferent groupsas follows:1.Dependingupon thenumber of carbon atomsthey possess, e.g

2.Dependingupon the functional groups:•Aldehyde (CHO) - Aldoses•Ketone (C=O) - Ketoses.

EXP.NO.7 Qualitative AnalysisOf Carbohydrates

OligosaccharidesOligosaccharidesconsist of a short chain of monosaccharide units(2 to 10 units), joined together by a characteristicbondcalled glycosidicbond. Oligosaccharidesare subdivided into different groupsbased on thenumber ofmonosaccharideunitspresent.

Disaccharidesareclassified as:•Reducingdisaccharides: In reducing disaccharidesone of the functional groupsisfree. e.g.Maltose, Lactose•Non- reducingdisaccharides: Non-reducingdisaccharidesdonot have free functional group. Thepotentialfunctional groupsare involved in glycosidic linkage. e.g. Sucrose, Trehalose

PolysaccharidesPolysaccharidesarecarbohydrateshavingmore than tenmonosaccharide units. They arealso called glycansorcomplex carbohydrates. They areclassified into two typesaccording to the typeof monosaccharideunitspresent.1. Homopolysaccharides:Madeup of repeated unitsof same typeof monosaccharideunits. e.g. Starch,glycogen, cellulose, inulin, dextrin, dextran.2. Heteropolysaccharides:Madeup of different typesof monosaccharideunitsand their derivatives. e.g. Agar,gum, pectins,

QualitativeAnalysisof CarbohydratesThevarioustests for carbohydratesare given below:1. Molisch test: specific test for carbohydrates2. Benedict’s test: specific test for reducingsugars3. Barfoed’s test: specific test for monosaccharide's4. Bial’s Test specific test for pentose's (5C sugars).5. Seliwanoff’s test: specific test for ketohexoses6. Osazone test: to differentiate the reducing sugarson thebasisof crystal formation7. Iodine test: specific test for polysaccharides

Classification Carbohydrate

2- Disaccharides

Maltose

Sucrose

Lactose:

The test results for different carbohydratesare summarized below:

The carbohydrates to be studied in the lab are:1.Glucose

• Glucose isamonosaccharide, an aldohexoseand a reducingsugar.2. Fructose

• Fructose isamonosaccharide, a ketohexose, and a reducingsugar., 3. Lactose

•Lactose isamilk sugar.•Composed ofβ- D glucose andβ- D galactose, Linked byβ1-4 glycosidic linkage..

4.MaltoseMaltose iscomposed of twomoleculesofαD glucose. Linked byα1-4 glycosidic linkage.5. Sucrose

•Sucrose iscomposed ofαD glucoseandβD fructose. Linked byαβ1, 2 glycosidic linkage.•In sucrose the linkage isbetween the functional groupsof glucose and fructose. Since, there isno freefunctional groupsucrose isnon-reducing. Onhydrolysisthe linkageiscleaved and it becomesa reducingsugar.. 6. Starch

•Starch isa plant polysaccharide.•Composed of amylose andamylopectin component.•Starch isa non-reducing sugar.

ReactionsOf Carbohydrates

Reaction with AlkaliesWeakAlkaliesWith weak alkali, the free functional group in monosaccharide getstautomerized and formsenediol, which is astrongreducingagent. Carbohydratessuch assucrose, which do not contain free functional groupsare not enolizedbyalkali and relatively stable in alkali solution.

StrongAlkaliesOnboilingwith strongalkali, thealdehydepolymerizes to form resin which is called caramelization. Thus, glucoseloses its reducingproperty

Reaction with AcidsWeakAcidsThesehavenoappreciable actionon sugars.StrongAcidsWith strongacids, sugar undergoesdehydration forming furfural. Thepentose'syield furfural and hexosesgive hydroxy-methyl furfural. Keto- hexoseslike fructose yield greater amount of furfural derivative than aldo- hexoseslike glucose.

TheMolisch-Test:-Procedure: -1- Add 1ml Sample.2- Add 2DropMolisch reagent. (10% α-naphthol in ethanol).3- Add 1ml H2SO4. is slowly added down the sides of the sloping test-tube, without mixing(Monosaccharide’s give a rapid positive test) A positive reaction is indicated by appearance of a purple red ringat theinterfacebetween theacid and test layers.

Principle:Concentrated sulphuricacid hydrolyses glycosidicbondsto give themonosaccharaides, which are thendehydrated tofurfural and itsderivatives. Theseproductsthen combinewith sulphonated naphthol to give a purple complex.

(Disaccharidesand polysaccharides react slower but Monosaccharidesgive a rapid positive test.)

Barfoed's-Test:-

Thistest isused to distinguish betweenmonosaccharideand disaccharides. Barfoed'stest issimilar to Benedict’s test inusingcupper ionsasan oxidizingagent to form a carboxylic acid and a reddish precipitate of cuprousoxidewithin threeminutes. However, the test medium isweakly acidic, therefore, only reducingmonosaccharide’s give positive result usingBarfoed'sreagent.

PreparingReagent :Barfoed'sreagent consistsof a 0.33molar solution of neutral copper acetate in 1% acetic acid solution.

Procedure:1-Add 0.5ml of TheSample.2-Add 1ml of Barfoed reagent.3-Boil 3-5min in Water Bath.

Seliwanoff’s -Test:-Thistest isused for identification of keto-hexosesor to distinguish between ketosesand aldoses. Fructoseand sucrose(A disaccharide consistingof fructose and glucose) are two commonsugars that give a positive test

Procedure: -1-Add 2Drop Sample to 2.5ml Seliwanoff’s Reagent. 0.5 g resorcinol per liter 10% HCl2-Warm 1-2min inWater Bath.3-Thered Color within 2min indicates positiveResult Ketoses (Fructose , Sucrose).two commonsugars that give a positive test Ketoses (Fructose , Sucrose)

Bials -Test:-Bial’s Test is to determine thepresence of pentoses(5C sugars). Thecomponentsof this reagent are resorcinol, HCl,and ferric chloride. In this test, thepentose isdehydrated to form furfural and thesolution turnsbluish and aprecipitate may form. Perform this test with riboseandglucose.

Procedure:-1-Add 0.5ml Sample to 1.5ml of BialsReagent.2-Heat Boiling in Water Bath.3- TheFormation of a Blue-Green Color positiveResult for pentose.

compounds(pentosans) will give a positive test

Benedict's -Test:-(also called Benedict'ssolution): isa chemical reagent named after an American chemist, StanleyRossiter Benedict .Benedict'sreagent isused asa test for thepresenceof reducingsugars. Thisincludesall monosaccharidesandmanydisaccharides, including lactoseandmaltose. Evenmoregenerally, Benedict'stest will detect thepresenceof aldehydes ,and alpha-hydroxyl -ketones, including those that occur in certain ketoses. Thus, although theketose fructose isnot strictlya reducing sugar, it isan alpha-hydroxyl -ketone, and givesa positive test because it isconverted to thealdosesglucose bythebase in thereagent

Procedure:-1- Add 2ml Sample in Test Tube.2- Add 1ml Benedict’s Reagent.3- Boil 10-15min inWater Bath

Principle of Benedict’s TestWhen Benedict’s solution and simple carbohydrates are heated, the solution changes to orange red/ brick red. This reactionis caused by the reducing property of simple carbohydrates. The copper (II) ions in the Benedict’s solution are reduced toCopper (I) ions, which causes the color change.The red copper(I) oxide formed is insoluble in water and is precipitated out of solution. This accounts for the precipitateformed. As the concentration of reducing sugar increases, the nearer the final colour is to brick-red and the greater theprecipitate formed. Sometimes a brick red solid, copper oxide, precipitates out of the solution and collects at the bottom ofthe test tube.

Color of the precipitate depends on the concentration of sugar present .

Iodine -Test:-Starch:Plantsstore glucoseas thepolysaccharide starch. Thecereal grains (wheat, rice, corn, oats, barley) aswell as tuberssuch aspotatoesare rich in starch.Starch can beseparated into two fractions--amyloseand amylopectin.Natural starchesaremixtures of amylose (10-20%) andamylopectin (80-90%).Amylose formsa colloidal dispersion in hot water whereasamylopectin is completely insoluble. Thestructureof amyloseconsistsof longpolymer chainsof glucose unitsconnected by an alphaacetal linkage.

Chemical Test for Starch or Iodine:

Amylose in starch is responsible for the formation of a deep blue color in thepresence of iodine. Theiodinemoleculeslipsinside of theamylose coil.Iodine -KI Reagent: Iodine isnot very soluble in water, therefore the iodine reagent ismadeby dissolving iodine inwater in thepresenceof potassium iodide.Starch Test: Add Iodine-KI reagent to a solutionor directly on a potato or other materialssuch asbread, crackers, orflour. A blue-black color results if starch ispresent. If starch amylose isnot present, then thecolor will stay orange oryellow.

Reagents:i. 1% Iodine solution ( which contain potassium iodide and water).ii. Original solution (O.S) containing a carbohydratePrinciple:Starch, glycogen and higher dextrin's on reaction with dilute iodine solution form colored compounds.Procedure:1. Take 2 ml of the starch solution (original solution) in a test tube.2. Add 1-2 drops of the diluted iodine solution.3. Observe the production of a blue colour.4. The blue color produced disappears on heating, and it reappears on cooling the solution.Observation And Result:Starch is responsible for the formationof, a deepblue color in thepresenceof iodine.