Experiment 5 Carbohydrates Biochemistry Laboratory 1st Semester PDF

EXPERIMENT 5 Carbohydrates Biochemistry Laboratory 1st Semester A.Y. 2024 - 2025  EXPERIMENT 5: Carbohydrates OBJECTIVES 1. To contrast monosaccharides, disaccharides and polysaccharides based on their properties and reactions to general and specific tests performed for carbohydrates. 2....

EXPERIMENT 5 Carbohydrates Biochemistry Laboratory 1st Semester A.Y. 2024 - 2025  EXPERIMENT 5: Carbohydrates OBJECTIVES 1. To contrast monosaccharides, disaccharides and polysaccharides based on their properties and reactions to general and specific tests performed for carbohydrates. 2. To identify and classify an unknown sample of carbohydrate by comparing the results of the unknown to the various tests with that of the known test substances.  ► Synthesized by plants using sunlight to convert CO2 and H2O to glucose and O2. ► Polymers include starch and cellulose. ► Starch is storage unit for solar energy. Carbohydrates ► Most sugars have formula Cn(H2O)n, “hydrate of carbon.” ► Sakkharon (Greek)- sugar 1 BIOLOGICAL SIGNIFICANCE Carbohydrates Staple of the human diet and its oxidation is the central energy-yielding pathway in most non-photosynthetic cells. (SUGAR AND STARCH) Serves as structural and protective elements in the cell walls of bacteria and plants and in the connective tissues and cell coats of animals. Lubricates skeletal joints and provide adhesion between cells. Complex polymers attached to proteins or lipids, act as signals that determine the intracellular location or metabolic fate of these glycoconjugates. Glycoproteins and Glycolipids The carbohydrate chain enables them to act as receptor molecules ○ This allows them to bind with substances at the cell surface ○ Receptor types include: Signalling receptors which bind to hormones and neurotransmitters Receptors involved in endocytosis Receptors involved in cell adhesion and stabilisation Cell adhesion allows cells to attach to each other to form tissues Some act as cell markers, or antigens, for cell identification ○ E.g. this allows the immune system to determine whether or not a cell belongs in the body, or whether it is a pathogen BIOLOGICAL SIGNIFICANCE Carbohydrates act as fuel in the body. Their oxidation to ATP, carbon dioxide and water is a major source of energy. 2. Certain products of carbohydrate metabolism act as catalyst of oxidation. 3. Carbohydrates can be used as starting materials for the biological synthesis of other types of compounds in the body. CARBOHYDRATES Include both simple sugars (i.e., little ring-shaped molecules made of carbon, hydrogen, and oxygen – either alone or in pairs) and more complex carbohydrates (formed when these rings link up together to make long chains). Carbohydrates SUGAR Sugar refers to a family of molecules called saccharides Monosaccharide (mono means one) - one sugar molecule Disaccharides (di means 2) - two sugar molecules linked together Oligosaccharides (oligo means “a few”) - 3-10 sugar molecules Polysaccharides (poly means “many”) - more than 10 sugar units  < TARGET="display"> glucose + glucose galactose + glucose glucose + fructose GLYCOSIDIC BONDING Monosaccharides link together through glycosidic bonding Glycosidic bonding is when an “OH” group from the carbon of one monosaccharide bonds with an “H” from the carbon of another monosaccharide Together, this forms H2O or a water molecule, which goes away Unlocking Terms Hydroxyl groups - simple structures consisting of an oxygen atom with two lone pairs bonded to a hydrogen atom (-OH) Polyhydroxy – containing more than one hydroxyl group in the molecule Aldehyde - an organic compound in which the carbonyl group (C=O) is attached to a carbon atom at the end of a carbon chain Ketone - the carbonyl group is attached to a carbon atom within the carbon chain Classification of Carbohydrates ► Monosaccharides or simple sugars ► polyhydroxyaldehydes or aldoses ► polyhydroxyketones or ketoses ► Oligosaccharides can be hydrolyzed to a few monosaccharides. ► Polysaccharides hydrolyze to many monosaccharide units. e.g., starch and cellulose have > 1000 glucose units. 21 Monosaccharides ► What is the basis for classification? glucose, a D-aldohexose fructose, a D-ketohexose 22  Monosaccharides ► Classified by: ► aldose or ketose ► number of carbons in chain ► configuration of chiral carbon farthest from the carbonyl group glucose, a D-aldohexose fructose, a D-ketohexose => 23  < TARGET="display"> Classify each of the monosaccharides: Disaccharides ► Consist of 2 monosaccharides bonded together ► Lactose, sucrose, maltose ► Contain glycosidic bond  < TARGET="display">  < TARGET="display">  < TARGET="display">  < TARGET="display"> Polysaccharides ► >10 monosaccharides ► Most are made up of hundreds of monosaccharides bonded together ► Types ► Starch: Storage in plants ► Glycogen: Storage in animals ► Dietary Fiber/cellulose: Not used for energy by humans but key for grazing animals Carbohydrates IODINE TEST  Iodine Test The iodine test is used to detect the presence of polysaccharides. Starch will form a dark-blue complex with iodine. This test has a variation termed starch-iodine test that is performed to indicate the presence of glucose made by plants in the leaves. Objectives of the Iodine Test To detect the presence of polysaccharides, primarily starch.  < TARGET="display"> Iodine Test Iodine Test Iodine Test Iodine Test Iodine Test  PROPERTIES OF CARBOHYDRATES PHYSICAL PROPERTIES OF MONO & DISACCHARIDES ► These substances are crystalline, soluble in water, and moderately soluble in dilute alcohol. practically insoluble in absolute alcohol, ether, and the usual organic solvents. A Little Recap! Oxidation can also be defined as the addition of oxygen to a molecule or the removal of hydrogen. (loss of electrons) Reduction is therefore the addition of hydrogen or the removal of oxygen. (gain of electrons) MOLISCH TEST  CHEMICAL PROPERTIES 1. The Action Of Non-oxidizing Acids With dilute acids disaccharides and polysaccharides are hydrolyzed to monosaccharides. In higher concentration, such acids act on monosaccharides by dehydrating them, forming furfural derivatives. Hexoses form hydroxymethylfurfural; whereas pentoses form furfural. Unlocking Terms Pentose is a five-carbon monosaccharide. Hexose contains six carbon atoms. Hydroxymethylfurfural (HMF) - a chemical that forms when sugars (like in fruits or honey) are heated or broken down under acidic conditions. Furfural is like HMF’s cousin, but it comes from plants, especially things like corncobs or wheat bran.  CHEMICAL PROPERTIES a. Molisch Test ► the sugar solution is mixed with alpha-naphthol and layered with concentrated H2S04, forming a violet ring. ► The acid acts as a dehydrating agent, producing furfural derivatives which combine with alpha-naphthol to form the colored product. ► This is a general test for carbohydrates. Molisch Test The formation of a purple or a purplish-red ring at the point of contact between the H2SO4 and the analyte + Molisch’s reagent mixture confirms the presence of carbohydrates in the analyte. Molisch Test A positive reaction for Molisch’s test is given by almost all carbohydrates (exceptions include tetroses & trioses). It can be noted that even some glycoproteins and nucleic acids give positive results for this test (since they tend to undergo hydrolysis when exposed to strong mineral acids and form monosaccharides).  REMEMBER! Molisch Test - It is important to take note that: 1. Molisch’s test is a chemical test that detects the presence of carbohydrates in an analyte. 1. Molisch’s test principles are based on the dehydration of sulphuric acid into furfural.  REMEMBER! Molisch Test - It is important to take note that: Molisch Test  Molisch Test Test Solutions Observations Glucose + Sucrose + Starch + SELIWANOFF TEST CHEMICAL PROPERTIES b. The Seliwanoff Test ► involves the action of concentrated HCl and resorcinol on the sugar. ► a red color is developed rapidly in the presence of a ketose sugar, such as fructose. Seliwanoff’s Test We can perform Seliwanoff’s Test to distinguish between aldose and ketose. By conducting this test, the color of aldose changes to light pink whereas the color of ketose turns into deep cherry red. REMEMBER! Seliwanoff’s Test Seliwanoff’s Test Test Solutions Observation Glucose - Fructose + (deep red) Galactose - Maltose - Sucrose + (red) Lactose -  GALACTARIC / MUCIC ACID TEST Mucic/Galactaric Acid Test Oxidation of Sugars c. Saccharic acids Both the aldehyde and primary alcohol groups of the aldoses are oxidized to carboxyl groups by nitric acid producing saccharic acids. reaction is used for the detection of galactose, because it forms insoluble saccharic acid (mucic acid). Mucic/Galactaric Acid Test Mucic acid test is a test that is highly specific and is used for the detection of the presence of galactose and lactose. It is also termed galactaric acid that is named after the product of the reaction. Objectives of Mucic acid test To detect the presence of galactose and lactose in a given sample. To distinguish between the galactose containing saccharides and other sugars. Mucic/Galactaric Acid Test Mucic/Galactaric Acid Test Mucic/Galactaric Acid Test Mucic/Galactaric Acid Test  Galactaric Acid Test Test Solutions Observation Glucose No insoluble crystal Galactose With insoluble crystal Lactose With insoluble crystal  Tests on the Ability of Carbohydrates/Sugars to REDUCE METALS: REDUCING SUGARS Reducing Sugar A reducing sugar is one that reduces another compound and is itself oxidized; that is, the carbonyl carbon of the sugar is oxidized to a carboxyl group. BENEDICT’S TEST Benedict’s Test Benedict's test is used to detect the presence of reducing sugars. Any monosaccharide or disaccharide that can form a free aldehyde group in solution will react with Benedict’s reagent to form a brick-red, brown, green or occasionally yellow precipitate. A colored solution with no precipitate is a negative result.  < TARGET="display">  < TARGET="display">  Benedict’s Test simple carbohydrates containing a free ketone or aldehyde functional group can be identified with this test. The test is based on Benedict’s reagent (also known as Benedict’s solution), which is a complex mixture of sodium citrate, sodium carbonate, and the pentahydrate of copper(II) sulfate.  Benedict’s Test Principle When a reducing sugar is subjected to heat in the presence of an alkali, it gets converted into an enediol (which is a relatively powerful reducing agent). Therefore, when reducing sugars are present in the analyte, the cupric ions (Cu2+) in Benedict’s reagent are reduced to cuprous ions (Cu+). These cuprous ions form copper(I) oxide with the reaction mixture and precipitate out as a brick-red coloured compound. Benedict’s Test Benedict’s Test Benedict’s Test BARFOED’S TEST  Barfoed’s Test c. Barfoed's Solution made up of cupric acetate to which have been added a few drops of acetic acid. used to distinguish between mono- and disaccharides. Monosaccharides reduce Barfoed's solution fairly rapidly forming Cu2O. Disaccharides which reduce Fehling's solution rapidly reduce Barfoed's solution very slowly. Barfoed’s Test Barfoed’s Test Barfoed’s Test NYLANDER’S TEST  Nylander’s Test d. Nylander's Solution contains bismuth subnitrate, potassium hydroxide, and Rochelle salt. On boiling with a solution of a reducing sugar, black metallic bismuth precipitates. Nylander’s Test Nylander’s Test is used to detect and confirm the presence of sugars with reducing properties. The formation of the dark bismuth precipitate serves as a definitive indicator, emphasizing the test’s reliability and precision. Tests Depending on the Ability of Carbohydrate to Reduce Metals Samples Test Solutions Benedict’s Barfoed Nylander’s + (intense Glucose + + blue) + (intense Fructose + + blue) Galactose + + (intense blue) + Maltose + + (light blue) + Sucrose - - - Lactose + + (light blue) + IDENTIFICATION OF SUGARS BY OSAZONE FORMATION  Osazone Formation Test 6. Reactions of Sugars with Phenylhydrazine If a solution of reducing sugar is heated with phenylhydrazine, a yellow precipitate is finally obtained (osazone). The importance is that they are easily formed from reducing sugars and may be used to identify sugars. differentiated by their microscopic appearance and also by their melting points. Osazone Formation Test Osazone test is a chemical test used to detect reducing sugars. This test even allows the differentiation of different reducing sugars on the basis of the time of appearance of the complex. This test is also termed Phenylhydrazine test based on the reagent used for this test. Reducing sugars, characterized by a free carbonyl group (either aldehyde or ketone), are the primary focus of the Osazone test. Osazone Formation Test Objectives of Osazone Test To detect reducing sugars. To differentiate reducing sugars from non-reducing sugars. To distinguish different reducing sugars between each other. Reactions of Sugars with Phenylhydrazine  Osazone Formation Both C1 and C2 react with phenylhydrazine. => Chapter 23 99 Osazone Test Osazone Test Osazone Test Osazone Test Osazone Test Osazone Test  Osazone Test False Positives for Non-Reducing Sugars The Osazone test can yield a false-positive result for sucrose if it is boiled for 30 minutes or longer. Sucrose, a non-reducing sugar, may form osazone-like crystals under prolonged heating. This limitation can lead to misidentification of non-reducing sugars as reducing sugars.  Osazone Test Precipitation Sugar Tested Initial Time Illustration Time Needle-shaped Glucose crystal Needle-shaped Fructose crystal Needle-shaped Galactose crystal Lactose Sunflower-shap ed crystal Hedgehog or Maltose ball-shaped crystal Sucrose No crystal  TESTS/ MOLISCH’S BENEDICT’S BARFOED’S NYLANDER’S SELIWANOFF’S GALACTARIC IODINE OSAZONE SAMPLES (MUCIC) DESCRIPTION -General test for -test for reducing - detect reducing -detect reducing -distinguish -Oxidation of most -The use of Lugol's -If a solution of carbohydrates sugars monosaccharides sugars between monosaccharides iodine reagent (IKI) reducing sugar -+- violet ring -aldoses & in the presence of -black precipitate aldohexoses and by nitric acid is useful to is heated with -H2SO4- ketoses disaccharides. to indicate positive ketohexoses yields soluble distinguish starch phenylhydrazine dehydrating agent -brick red, brown, - uses copper reaction -no. of times dicarboxylic and glycogen from , a yellow green or yellow ions to detect -contains bismuth required for the red acids. other precipitate is ppt. reducing sugars subnitrate, color to appear. -oxidation of polysaccharides. finally obtained in an acidic potassium - positive test for galactose yields - Lugol's iodine (osazone). solution. hydroxide, and ketoses. an insoluble mucic yields a blue-black -The importance -same color Rochelle salt. -Aldohexoses react acid. color in the presence is that they are changes as in -On boiling with a to form the same -Lactose will also of starch. Glycogen easily formed Benedict's test. solution of a product, but do so yield a mucic acid, reacts with Lugol's from reducing -Formation of a reducing sugar, more slowly. due to hydrolysis reagent to give a sugars and may green, red, or black metallic of the glycosidic brown-blue color. be used to yellow precipitate. bismuth linkage between -It is thought that identify sugars. precipitates. its glucose and starch and glycogen -differentiated galactose form helical coils. by their subunits. Iodine atoms can microscopic then fit into the appearance and helices to form a also by their starch-iodine or melting points. glycogen-iodine complex. 1. glucose + + + (intense blue) + - No insoluble No color change + crystal (yellow-brown Needle-shaped solution) crystals 2. galactose + + +(intense blue) + - with insoluble No color change + crystal Needle-shaped crystals 3. fructose + + +(intense blue) + + No insoluble No color change + crystal Needle-shaped crystals 4. sucrose + - - - + (red) No insoluble No color change - crystal 5. maltose + + + (light blue) + - No insoluble No color change + crystal sunflower-shape d crystals  TESTS/ MOLISCH’S BENEDICT’S BARFOED’S NYLANDER’S SELIWANOFF’S GALACTARIC IODINE OSAZONE SAMPLES (MUCIC) DESCRIPTION -General test for -test for reducing - detect reducing -detect reducing -distinguish -Oxidation of most -The use of Lugol's -If a solution of carbohydrates sugars monosaccharides sugars between monosaccharides iodine reagent (IKI) reducing sugar -+- violet ring -aldoses & in the presence of -black precipitate aldohexoses and by nitric acid is useful to is heated with -H2SO4- ketoses disaccharides. to indicate positive ketohexoses yields soluble distinguish starch phenylhydrazine dehydrating agent -brick red, brown, - uses copper reaction -no. of times dicarboxylic and glycogen from , a yellow green or yellow ions to detect -contains bismuth required for the red acids. other precipitate is ppt. reducing sugars subnitrate, color to appear. -oxidation of polysaccharides. finally obtained in an acidic potassium - positive test for galactose yields - Lugol's iodine (osazone). solution. hydroxide, and ketoses. an insoluble mucic yields a blue-black -The importance -same color Rochelle salt. -Aldohexoses react acid. color in the presence is that they are changes as in -On boiling with a to form the same -Lactose will also of starch. Glycogen easily formed Benedict's test. solution of a product, but do so yield a mucic acid, reacts with Lugol's from reducing -Formation of a reducing sugar, more slowly. due to hydrolysis reagent to give a sugars and may green, red, or black metallic of the glycosidic brown-blue color. be used to yellow precipitate. bismuth linkage between -It is thought that identify sugars. precipitates. its glucose and starch and glycogen -differentiated galactose form helical coils. by their subunits. Iodine atoms can microscopic then fit into the appearance and helices to form a also by their starch-iodine or melting points. glycogen-iodine complex. 5. maltose + + + (light blue) + - No insoluble No color change + crystal sunflower-sha ped crystals 6. lactose + + + (light blue) + - with insoluble No color change + crystal hedgehog-sha ped crystals 7. starch + Colored solution - - - No insoluble Formed blue-black but no ppt. crystal solution All samples are -mono & -intense -positive to all -fructose is a + for lactose and + for carbohydrates disaccharides blue-reducing reducing ketose sugar while galactose polysaccharides (except for monosaccharaid sugars-see sucrose in hot HCl like starch and sucrose) are e results for is hydrolysed to glycogen reducing while -light Barfoed’s and fructose and complex carbo blue=reducing Benedict’s Tests glucose and the like starch is disaccharides presence of the non-reducing fructose makes sucrose positive for this test.  TESTS/ MOLISCH’S BENEDICT’S BARFOED’S NYLANDER’S SELIWANOFF’S GALACTARIC IODINE OSAZONE SAMPLES (MUCIC) DESCRIPTION -General test for -test for reducing - detect reducing -detect reducing -distinguish -Oxidation of most -The use of Lugol's -If a solution of carbohydrates sugars monosaccharides sugars between monosaccharides iodine reagent (IKI) reducing sugar -+- violet ring -aldoses & in the presence of -black precipitate aldohexoses and by nitric acid is useful to is heated with -H2SO4- ketoses disaccharides. to indicate positive ketohexoses yields soluble distinguish starch phenylhydrazine dehydrating agent -brick red, brown, - uses copper reaction -no. of times dicarboxylic and glycogen from , a yellow green or yellow ions to detect -contains bismuth required for the red acids. other precipitate is ppt. reducing sugars subnitrate, color to appear. -oxidation of polysaccharides. finally obtained in an acidic potassium - positive test for galactose yields - Lugol's iodine (osazone). solution. hydroxide, and ketoses. an insoluble mucic yields a blue-black -The importance -same color Rochelle salt. -Aldohexoses react acid. color in the presence is that they are changes as in -On boiling with a to form the same -Lactose will also of starch. Glycogen easily formed Benedict's test. solution of a product, but do so yield a mucic acid, reacts with Lugol's from reducing -Formation of a reducing sugar, more slowly. due to hydrolysis reagent to give a sugars and may green, red, or black metallic of the glycosidic brown-blue color. be used to yellow precipitate. bismuth linkage between -It is thought that identify sugars. precipitates. its glucose and starch and glycogen -differentiated galactose form helical coils. by their subunits. Iodine atoms can microscopic then fit into the appearance and helices to form a also by their starch-iodine or melting points. glycogen-iodine complex. All samples are -mono & -intense -positive to all -fructose is a + for lactose and + for carbohydrates disaccharides blue-reducing reducing ketose sugar while galactose polysaccharides (except for monosaccharaid sugars-see sucrose in hot HCl like starch and sucrose) are e results for is hydrolysed to glycogen reducing while -light Barfoed’s and fructose and complex carbo blue=reducing Benedict’s Tests glucose and the like starch is disaccharides presence of the non-reducing fructose makes sucrose positive for this test. Reference Guaves, Grace. “ACTIVITY 6 CARBOHYDRATES.” PowerPoint presentation. De La Salle Lipa. Lipa City, Batangas. 2017 Modified by Matibag, Divine Kae Ann T. & Cometa, Zalene Angela. 2024.