Biochemistry Lecture Notes PDF

BIOCHEMISTRY Under supervision of Dr/ Eman abd elmaksoud QUALITATIVE ANALYSIS This term we will study how to detect unknown sample carbohydrates lipid Protein CARBOHYDRATES Classified acc. To 1. Number of sugar unit into 1. Monosaccharide ( glucose,fructose,manno...

BIOCHEMISTRY Under supervision of Dr/ Eman abd elmaksoud QUALITATIVE ANALYSIS This term we will study how to detect unknown sample carbohydrates lipid Protein CARBOHYDRATES Classified acc. To 1. Number of sugar unit into 1. Monosaccharide ( glucose,fructose,mannose,galactose) 2. Disaccharide (lactose,maltose,sucrose) 3. Oligosaccharide (3-10)Carbon 4. Polysaccharide >10 Carbon  MONOSACHARIDE According to the number of carbon atoms and active group The samallest and the simplest sugar is Glyceraldehyde TRIOSE 3C TETROSE 4C PENTOSE 5C HEXOSE 6C HEPOTOSE 7C ALDOSE Glyceraldehyde erythrose Ribose glucose L-glycero-D-manno- heptose KETOSE Dihydroxyacetone erythrulose Ribulose fructose mannoheptulose QUALITATIVE TESTS FOR CARBOHYDRATES 1. Molisch test 2. Fehling test 3. Benedict test 4. Barafoed test 5. Seliwanoffs test 6. Hydrolysis of starch  Unknown Molisch test +ve -ve carbohydrates non carbohydrate glucose, fructose, lactose , starch Benedict test +ve -ve Glucose , fructose, lactose starch Barfoed test +ve -ve glucose, fructose lactose Seliwanoffs test +ve -ve fructose glucose MOLISCH`S TEST Aim : detection of carbohydrate presence Reagents : 1. α-naphthol 2. Sulfuric acid PROCEDURE In a test tube contain 1ml of test solution add on the wall of + + Few drops the test tube of α- add 1ml of naphthol conc. Sulfuric acid slowly mix well RESULT +ve -ve Purple to violet ring No ring Sample contain carbohydrates Sample my be lipid or protein ( glucose,fructose,lactose,starch) PRINCIPLE 1. Addition of concentrated sulfuric acid cause hydrolysis of carbohydrates to simple form 2. Dehydration of monosaccharide and will give 5 carbon atom cyclic compound 1. Furfural for pentose 2. Hydroxy methyl furfural for hexose 3. Then α-naphthol will combine with the product (Furfural, Hydroxy methyl furfural) and give purple ring at the junction between acid and solution AVOID shaking of test tube to avoid burning of sugar Appear as dark brown color This reaction didn’t give positive with trioses and tetroses Why ??? BENEDICT`S TEST Aim : differentiate between reducing and non reducing sugar (which contain free aldehyde or ketone group ) Differentiate mono and disaccharides except sucrose (NON Reducing) from polysaccharide Reagents : 1. Benedict reagent which contain 1. sodium carbonate, provides the alkaline conditions 2. sodium citrate, complexes with the copper (II) ions so that they do not deteriorate to copper(I) ions during storage. 3. copper(II) sulfate pentahydrate TEST 1ml of benedict reagent In boiling water for maximum 5 minutes +  Experiment Observation Inference Substance in water + 3 mL Benedict's solution, then boil Red, green, or yellow Reducing sugar, such as for few minutes and allow to precipitate is obtained glucose, is present cool. Substance in water + 3 mL Benedict's solution, then boil Solution remains clear or is a Reducing sugar is not for few minutes and allow to little blue present cool. PRINCIPLE When Benedict’s solution and simple carbohydrates are heated, the solution changes to orange red/ brick red. This reaction is caused by the reducing property of simple carbohydrates. The copper (II) ions in the Benedict’s solution are reduced to Copper (I) ions, which causes the color change. AVOID heating of solution more than 5 minutes as heat will cause hydrolysis of polysaccharide also sucrose and give false positive result BARFOED`S TEST Aim : 1. To distinguish reducing monosaccharides from disaccharides. Reagents 1. Barfoed reagent 1. copper acetate 2. Diluted acetic acid. TEST 2ml of barfoed reagent In boiling water for maximum 3 minutes Brick red ppt + PRINCIPLE Barfoed’s test is used for distinguishing monosaccharides from reducing disaccharides. Monosaccharides usually react in about 1-2 minute while the reducing disaccharides take much longer time between 7-12 minutes to react with the reagent. Brick red ppt is obtained in this test which is due to formation of cuprous oxide. SELIWANOFF`S TEST Aim : To detect the presence of ketohexoses in a given sample. To distinguish ketoses from aldoses. Raegent 0.05% resorcinol (m-hydroxybenzene) concentrated HCl TEST 2ml of seliwanoff`s reagent In boiling water for maximum 2 minutes Red color +  The acid hydrolysis of polysaccharides and oligosaccharides yields simpler sugars. Ketoses are more rapidly dehydrated than aldoses. Ketoses undergo dehydration in the presence of concentrated acid to yield 5- hydroxymethyl furfural. The dehydrated ketose reacts with two equivalents of resorcinol in a series of condensation reactions to produce a complex (not a precipitate), termed xanthenoid, with deep cherry red color. Aldoses may react slightly to produce a faint pink to cherry red color if the test is PRINCIPLE prolonged. The product and reaction time of the oxidation reaction helps to distinguish between carbohydrates. Other carbohydrates like sucrose and inulin also give a positive result for this test as these are hydrolyzed by acid to give fructose. WHO CAN IMAGINE WHAT TO DO TO PROVE THAT SOLUTION CONTAIN : 1. Starch 2. Sucrose 3. Fructose 4. Lactose 5. Glucose 6. Galactose