Lab 2 Carbohydrate Chemistry Updated PDF

Faculty of Medicine Academic Year: 2024-2025 Year: 1 Semester 1 : Human Body Function (HBF) Module: 102 Lab 2 – carbohydrates chemistry By:Dr. Hanaa B. Atya Lecturer of Biochemistry & Molecula...

Faculty of Medicine Academic Year: 2024-2025 Year: 1 Semester 1 : Human Body Function (HBF) Module: 102 Lab 2 – carbohydrates chemistry By:Dr. Hanaa B. Atya Lecturer of Biochemistry & Molecular Biology Department Biochemistry & Molecular Biology :12/5/2024 2 2  Objectives  Knowledge Acquisition: Understand the structure, function, and classification of carbohydrates, including monosaccharides, disaccharides, and polysaccharides.  Practical Skills Development: Gain hands-on experience in laboratory techniques for carbohydrate analysis, such as Benedict's test, and Barford's test,  Data Interpretation: Develop the ability to analyze and interpret experimental data, drawing conclusions about carbohydrate presence and concentration based on lab results. 12/08/2024 HBF - 102 3 3  Introduction  Carbohydrates, are defined as the polyhydroxy aldehydes or polyhydroxy ketones.  Most , but not all carbohydrate have a formula (CH2O)n.  In human body, the D-glucose is used.  Simple sugars ends with –ose. 12/08/2024 HBF - 102 4 4 Classification of carbohydrates: 12/08/2024 HBF - 102 5 A- Classification based on complexity: 1. Simple sugar (one unit) : Monosaccharides contain one monosaccharide unit. 2. Complex sugar (more than one) :  Disaccharides contain two monosaccharide units.  Oligosaccharides contain 3-9 monosaccharide units.  Polysaccharides can contain more than 9 monosaccharide units.  Complex carbohydrates can be broken down into smaller sugar units through a process known as 12/08/2024 HBF - 102 6 hydrolysis.  B- Classification based on reactivity:  Reducing and non - Reducing sugar :  If the oxygen on the anomeric carbon of a sugar is not attached to any other structure, that sugar can act as a reducing agent and is termed a reducing sugar. attached anomeric carbon FREE anomeric carbon Non-Reducing sugar FREE anomeric carbon Reducing sugar Reducing sugar 12/08/2024 HBF - 102 B- Classification based on reactivity All monosaccharides are reducing sugars; they all have a free reactive carbonyl (C=O) group. Some disaccharides have exposed carbonyl groups and are also reducing sugars like lactose. While other disaccharides such as sucrose are non-reducing sugars and will not react with Benedict's solution. Large polymers of glucose, such as starch, are not reducing sugars. In starch, the glucose units are linked together, and there are no free aldehyde or ketone groups availableHBF 12/08/2024 for reduction reactions. - 102 8  Solubility of sugars [physical property]:  Monosaccharide and disaccharide can be dissolved freely in water because water is a polar.  Polysaccharide cannot be dissolved easily in water, because, it has high molecular weight, which give colloidal solutions in water.(e.g. starch) 12/08/2024 HBF - 102 9 The iodine test for polysaccharides The iodine test is a chemical test used to distinguish mono- or disaccharides from certain polysaccharides like starch, dextrin, and glycogen. Principle of Iodine Test The iodine test is based on that polyiodide ions form colored adsorption complex with glucose residue of starch (blue-black), dextrin (violet), or glycogen (reddish-brown). Monosaccharides, disaccharides, and un-branched polysaccharides like cellulose remain colorless. 12/08/2024 HBF - 102 10  Chemical properties of carbohydrates:  Molisch's Test: To identify the carbohydrate from other macromolecules.  Benedict test: For the presence of reducing sugars.  Barfoed’s Test: To identify the presence of reducing sugars, specifically distinguishing between monosaccharides (which react quickly) and disaccharides (which react more slowly).  Bial’s Test: To distinguish between pentose monosaccharide and hexose monosaccharide (to detect pentoses).  Seliwanoff's Test: To distinguish between aldoses and ketoses (to 12/08/2024 HBF - 102 11 detect ketoses). Experiment 1 : Molisch test  Objective: To identify the carbohydrate from other macromolecules lipids and proteins (this test is specific for all carbohydrates).  Principle: Two solutions are used : (H2SO4 and α-naphthol ) The sulfuric acid (H2SO4) will dehydrate the sugar, The dehydrated sugars will further react with α- naphthol present in the test reagent to produce a 12/08/2024 HBF - 102 12 purple product. Molisch’s Test Procedure 1. 2ml of Sample is taken in a test tube. 2. 2 drops of Molisch’s reagent (naphthol in 95% ethanol) are added to the sample and mix it. 3. add slowly concentrated sulphuric acid in sloping the test tube by its sides without mixing vigorously so that it will get time to react and will form a layer. Observation A purple ring appears at the interface between the acid and test layers which confirms the presence of carbohydrates. 12/08/2024 HBF - 102 13  Experiment 2 : Benedict's test  Objective: To distinguish between the reducing and non-reducing sugars (to detect the presence )-( Sucrose )+( Glucose )+( Lactose of reducing sugar).  Principle: The copper sulphate (CuSO4) present in Benedict's solution reacts with the aldehyde or ketone group of the reducing sugar in alkaline medium. Reducing sugars are oxidized by the copper ion in solution 12/08/2024 to form a carboxylic acid and a reddish HBF - 102 14  Experiment 3 : Barfoed’s test  Objective: To identify the presence of reducing sugars, specifically distinguishing between monosaccharides (which react quickly) and disaccharidesReducing  Principle: (which react more slowly). saccharides are oxidized by the cupric ion in a slightly acidic medium to form a carboxylic acid and a reddish precipitate of cuprous oxide. Different types of reducing sugars react at different rates No Reduction Reduction  Reducing monosaccharides react quickly  but 12/08/2024 HBF - 102 reducing disaccharides react very slowly. 15 Experiment 4 : Bial’s test  Objective: To distinguish between pentose monosaccharide and hexose monosaccharide (to detect pentoses). Pentoses  Principle: Bial's reagent (orcinol, concentrated HCl and ferric chloride). The test reagent dehydrates pentoses to form furfural, furfural further reacts with orcinol, Hexose and the iron ion present in the test reagent to s produce a bluish or green product. 12/08/2024 HBF - 102 16  Experiment 5 : Seliwanoff's test  Objective: To distinguish between aldoses and ketoses (to detect ketoses).  Principle: Seliwanoff’s reagent (HCl + resorcinol). Fructose Glucose The test reagent dehydrates ketohexoses which further condenses with resorcinol present in the test reagent to produce a cherry red product within two minutes. Aldohexoses react to form the same product. But do so more slowly giving yellow to faint pink colour. 12/08/2024 HBF - 102 17 Glucose test strips (DIP test)  Glucose test strips measure the amount of glucose, or sugar, in a person’s urine.  They are used to monitor glucose levels in people with diabetes.  As high glucose levels in the urine can indicate high blood sugar. 12/08/2024 HBF - 102 18 Practical work Sample (A) Sample (B) You are provided with two biological samples ( A & B ). Determine the presence of glucose in each sample Benedict's 12/08/2024 HBF - 102 test 19 Practical work You are provided with two biological samples ( A & B). Determine the presence of glucose in each sample. Samples Observation Comment ……………………… ……………………… Sample (A) ……………………… ……………………….. ………… ……… ……………………… ……………………… Sample (B) 12/08/2024 …………………….. HBF - 102 …………. 20  Interactive Question What does Molisch's test detect? A) Reducing sugars B) Starch C) All carbohydrates D) Non-reducing sugars In Benedict's test, what color change indicates the presence of reducing sugars? A) Blue to green B) Blue to red 12/08/2024 HBF - 102 21 21  Interactive Question Barfoed’s test differentiates between which types of carbohydrates? A) Monosaccharides and polysaccharides B) Aldoses and ketoses C) Monosaccharides and disaccharides D) Reducing and non-reducing sugars In Seliwanoff’s test, a red color indicates the presence of: A) Aldoses B) Ketoses C) Starch 12/08/2024 HBF - 102 22  Summary  Carbohydrates, are defined as the polyhydroxy aldehydes or polyhydroxy ketones.  Carbohydrates classified as simple sugar (one unit) & Complex sugar (more than one).  Molisch's Test used to identify the carbohydrate from other macromolecules.  Benedict test used for the presence of reducing sugars.  Barfoed’s Test used to identify the presence of reducing sugars, specifically distinguishing between monosaccharides and disaccharides. 12/08/2024 HBF - 102 23 23  References "Fundamentals of Biochemistry: Life at the Molecular Level" by Donald Voet, Judith G. Voet, and Charlotte W. Pratt. This book covers the biochemical principles of carbohydrates, including various tests used to identify them. Chapter 2. pages from 222- 245. 12/08/2024 HBF - 102 24 24 12/08/2024 HBF - 102 25