Carbohydrates Identification Tests PDF

Determination Of An Unknown Carbohydrate Solution Carbohydrates identification tests, part 2  Remember ! All monosaccharides are reducing sugars; they all have a free reactive carbonyl group. Some disaccharides have exposed carbonyl groups and are also red...

Determination Of An Unknown Carbohydrate Solution Carbohydrates identification tests, part 2  Remember ! All monosaccharides are reducing sugars; they all have a free reactive carbonyl group. Some disaccharides have exposed carbonyl groups and are also reducing sugars. other disaccharides such as sucrose are non-reducing sugars. All polysaccharides are non reducing sugars. Complex carbohydrates can be broken down into smaller sugar units through a process known as hydrolysis. Chemical reactions for carbohydrates identification Molisch’s test: specific for carbohydrates. Iodine Test: to detect presence of starch Fehling’s test: differentiate between reducing and non-reducing sugars. Benedict's test: differentiate between reducing and non-reducing sugars. Barfoed's test: To distinguish between mono- and di- saccharides. Seliwanoff's test: to distinguish between aldoses and ketoses. Acid Hydrolysis: converts di- and poly-saccharides into monosaccharide (simple sugars). Phenyl Hydrazine Reaction (Osazone formation): differentiate between reducing Sugars  Chemical reactions for carbohydrates identification 5 Barfoed’s test Objective distinguish between mono- and di- saccharides Principle: Barfoed’s test uses copper (ii) ions in a slightly acidic medium. (Copper Acetate) - Reducing monosaccharides are oxidized by the copper ion in solution to form a carboxylic acid and a copper (i) oxide (red ppt). giving a quick result before 10 mins (within 2-3 minutes) of boiling. - Reducing disaccharides undergo the same reaction at a slower rate (after boiling for 10 minutes) Chemical reactions for carbohydrates identification  Chemical reactions for carbohydrates identification 5 Barfoed’s test 3 ml of a Barfoed’s reagent is placed in a test tube. Add 1 ml of sugar sample and heat in water bath. A Red precipitate is formed in case of reducing sugars, determination of sugar is time dependent. Test Observation Result Formation of red ppt. In Solution is monosaccharides Less than 10 minutes (after 2-3 minutes) (glucose, fructose) Barfoed’s Test Formation of red ppt in more than 10 Solution is disaccharides minutes  Chemical reactions for carbohydrates identification 6 Seliwanoff’s test Objective: differentiate between sugars that have a ketone group (ketose) and sugars that have an aldehyde group (aldoses). This test is a timed color reaction specific to ketohexoses Principle: - The reagent of this test consists of resorcinol and concentrated HCl. “acid hydrolysis of complex sugars”, the dehydrated ketose reacts with resorcinol producing a complex (not a precipitate), termed xanthenoid, with cherry red color. - Other carbohydrates like sucrose and inulin also give a positive result for this test as these are hydrolyzed by acid to give fructose.  Chemical reactions for carbohydrates identification 6 Seliwanoff’s test Put 1 ml of sample in test tube Add 3 ml of Seliwanoff’s reagent (Resorcinol and HCl sol.). Boil for 5 minutes in waterbath. Look for the development of Orange to red color.  Chemical reactions for carbohydrates identification Note: One of this test limitations is that prolonged boiling can transform glucose to fructose by the catalytic action of acid and form cherry red-complex giving a false-positive result. Test Observation Result Orange to red color forms Solution is Fructose Seliwanoff’s Test No change in color Solution is Glucose Chemical reactions for carbohydrates identification “Confirmatory tests” 7 Acid hydrolysis test Definition: Acid hydrolysis of disaccharides and polysaccharides produces monosaccharides by breaking the glycosidic links between monomer units in the structure of the molecule. Principle: Complex sugars such as starch are hydrolyzed by hot dilute acids to monosaccharides. - On Polysaccharides testing the hydrolysate; where it does not give the Blue color of the starch (polysaccharides) with iodine and shows reducing properties with Benedict’s test. - On Non-reducing polysaccharides Hydrolysate of sucrose shows reducing property which was not shown by sucrose before hydrolysis. Chemical reactions for carbohydrates identification “Confirmatory tests” 7 Acid hydrolysis test The hydrolysis of starch (polysaccharides) requires a longer time than disaccharides. Disaccharides hydrolyze into 2 monosaccharides, starch (polysaccharides) hydrolyze into many monosaccharides Chemical reactions for carbohydrates identification “Confirmatory tests” 8 Phenyl Hydrazine Reaction (Osazone formation) Objective: differentiate between reducing and non reducing depending on the shape of crystals formed. Principle: Sugars with free aldehyde or ketone group react with phenyl- hydrazine to form yellow insoluble crystalline compounds known as Osazone Result interpretation: Based on the shape and structure of the crystals and their time of appearance, different sugars can be identified. Chemical reactions for carbohydrates identification “Confirmatory tests” Test Observation Result Comment Solution is Glucose, Needle-like Crystals Used for the identification of Fructose Phenyl sugars Hydrazine to differentiate between the Sunflower-like Crystals Solution is Lactose different sugars excreted in urine Reaction Test ex. lactosuria Rosette-like Crystals Solution is Maltose Gluco- or Fructosazone Maltosazone Galacto- or Lactosazone Needle-like Rosette-like Rosette-like  Molisch +ve → CHO I2 (Iodine Test) +ve, Blue Starch -ve Reducing\non-red. sugars Confirm by, Acid Reducing tests Hydrolysis (Fehling\Benedict) → followed by I2 test (-ve) Fehling\Benedict (+ve) Red +ve -ve, Reducing Sugar non-reducing Sucrose Barfoed test Confirm by: 1. Seliwanoff 10 mins → Followed by monosaccharide Lactose Fehling\Benedict Osazone test Seliwanoff’s test +ve -ve Fructose Glucose Osazone *Confirmatory* Scheme for carbohydrates identification Molish Test: 1 - 3 ml of a sample + 2 drops Molisch and mixing - 3 ml of H2SO4 (2+1) gradually on the wall gives “Reddish-violet ring” 2 Idodine test: 5 ml solution + 1 drop of iodine, Starch gives Blue color. Fehling’s test: 3 1 ml Fehling’s A +1 ml Fehling’s B (mix) + 2 ml sugar ->Boil 2-3 minutes “Reddish brown precipitate if reducing sugar”. Bendict: 4 1 ml sample + 2.5 ml of benedict‘s (mix) ->Boil 2-3 minutes “Green to red precipitate if reducing sugar”. Barfoeid test: 5 1 ml barfoeid’s +1 ml sugar solution ->heat in water bath if Red ppt in >10 min “Di-saccharide”, in Boil in waterbath 5 minutes “Orange to red precipitate if Keto-sugar”. Thank you