SCHOOL OF PHARMACY BIOCHEMISTRY SGS0033

LAB REPORT 5.1 CARBOHYDRATE DETECTION TEST (MOLISCH TEST) LAB REPORT 5.3 CARBOHYDRATE DETECTION TEST (BARFOEDS TEST)

NAME I.D. NO. LECTURER D.O.S.

: DHIVIYA NAVARASAN : 012009050718 : MS. KALAIVANI BATUMALAIE : 24 AUGUST 2010

INTRODUCTION : A carbohydrate is an organic compound that is made up of carbon, hydrogen and oxygen in the ratio of 1:2:1. The general formula of carbohydrate is Cm(H2O)n. Generally, carbohydrates are referred to as hydrates of carbon, although in most cases they are known as polyhydroxy aldehydes and ketones. Carbohydrates, also known as saccharides, are basically divided into four main groups which are the monosaccharides, disaccharides, oligosaccharides and polysaccharides. Carbohydrates are the most important source of energy for the body. 1g of carbohydrate provides 4.2 kcal of energy. Cells in the body need glucose in order for the production of energy in the form of adenosine triphosphate. The brain cells and the erythrocytes are completely dependant on glucose as an energy source. If there is too much energy in the body, in the form of carbohydrates, it is transformed into fat and stored in the body as glycogen. About 30% of the energy is lost when carbohydrates are stored as fat. Another important function of carbohydrates is to act as energy storage. The body's own glycogen stores act as a readily available energy reserve. 1/3 of the glycogen is stored in the liver and about 2/3 is stored in the muscles. Dietary fibres are also carbohydrates. It used to be believed that fibres could not be used by the human body, as the digestive juices in the body did not contain enzymes capable of breaking down these compounds. Some other important functions of carbohydrates are: Sparing the use of proteins for energy Breakdown of fatty acids and preventing ketosis Biological recognition processes Flavor and Sweeteners As an anticoagulant (heparin) As an antigen (contains oligosaccharides) As hormones As raw material for industries like paper and textile Precursor for organic compounds

There are two types of carbohydrates which are the simple carbohydrate and

complex carbohydrate. All carbohydrates are made up of units of sugar, also called as saccharide units. Carbohydrates that contain only one sugar unit, monosaccharides, and two sugar units, disaccharides, are referred to as simple sugars or simple carbohydrates. Simple sugars are sweet in taste and are broken down quickly in the body to release energy. Three of the most common monosaccharides are glucose, galactose and fructose. Glucose is the primary form of sugar stored in the human body for energy. Fructose is the main sugar found in most fruits whereas galactose is found in milk. These simple sugars cannot be hydrolyzed further into any simpler forms. Disaccharides have two sugar units bonded together. Most common forms of disaccharides are maltose, lactose and sucrose. Maltose which is also known as barley sugar is made up of two units of glucose. It is mostly found in barley, beer and malt extract. On the other hand, lactose is made up of one unit of glucose and one unit of galactose. It is found in milk and dairy products as it is milk sugar. Sucrose, that is produced from the combination of one unit of glucose and one unit of fructose, is known as beet and cane sugar. It can be found in sugar cane, sugar beet, and household sugar too. Simple sugars are directly absorbed into the bloodstream to produce an instant source of energy. Complex carbohydrates are polymers of the simple sugars. In other words, the complex carbohydrates are long chains of simple sugar units bonded together. Polysaccharides are starch, glycogen and also cellulose. Starch is the principal polysaccharide used by plants to store glucose for later use as energy. Plants often store starch in seeds or other specialized organs. For example, common sources of starch include rice, beans, wheat, corn, potatoes, and so on. When humans eat starch, an enzyme that occurs in saliva and in the intestines called amylase breaks the bonds between the repeating glucose units, thus allowing the sugar to be absorbed into the bloodstream. Once absorbed into the bloodstream, the human body distributes glucose to the areas where it is needed for energy or stores it as its own special polymer - glycogen. Glycogen, another polymer of glucose, is the polysaccharide used by animals to store energy. Excess glucose is bonded together to form glycogen molecules, which the animal stores in the liver and muscle tissue as an "instant" source of energy. Both starch and glycogen are polymers of glucose; however, starch is a long, straight chain of glucose units, whereas glycogen is a branched chain of glucose units. Another important polysaccharide is cellulose. Cellulose is yet a third polymer

of the monosaccharide glucose. Cellulose differs from starch and glycogen because the glucose units form a two-dimensional structure, with hydrogen bonds holding together nearby polymers, thus giving the molecule added stability. Cellulose, also known as plant fiber, cannot be digested by human beings, therefore cellulose passes through the digestive tract without being absorbed into the body. Some animals, such as cows and termites, contain bacteria in their digestive tract that help them to digest cellulose. Cellulose is a relatively stiff material, and in plants it is used as a structural molecule to add support to the leaves, stem, and other plant parts. Despite the fact that it cannot be used as an energy source in most animals, cellulose fiber is essential in the diet because it helps exercise the digestive track and keep it clean and healthy. Oligosaccharides are carbohydrates that are made up of two to ten units of monosaccharides. They can be liberated on hydrolysis. As they attach to proteins and lipids, these complex carbohydrates then form glycoprotein and glycolipid, which plays a major role in the plasma membrane. There are a few tests to detect the presence of carbohydrates as well as different types of them. One of the test is Molisch Test. Molisch test is a sensitive chemical test that is used to detect the presence of carbohydrates in sample. It is based on the principle of dehydration of the carbohydrate by sulphuric acid in to produce an aldehyde. This aldehyde eventually condenses with two molecules of phenol, usually -naphtol, resulting in a red or purple coloured compound. A positive test is indicated by appearance of a purple ring at the interface between the acid and test layers. This is the result between the interaction of the sulphuric acid with -naphtol. Barfoeds test, on the other hand, is another test that plays a msjor role in detecting carbohydrates. However, this test is mainly to detect the presence of monosaccharides. It is based on the reduction of copper(II) acetate to copper(I) oxide. This results in the formation of a brick red precipitate. Although disaccharides may react, their reaction is much slower. The aldehyde group of the monosaccharides which normally forms a cyclic hemiacetal is oxidised to the carboxylate.

TITLE : Lab 5.1 : Carbohydrates Detection Test (Molisch Test) OBJECTIVE : To detect the presence of carbohydrates qualitatively using Molisch reagent MATERIALS : Molisch reagent Sugar solution Concentrated sulphuric acid

APPARATUS : Dropper Beaker Glass rod Pipette Test tube Test tube rack

PROCEDURES : 1. Two drops of Molisch reagent was added to 2ml of the sugar solution and mixed thoroughly. 2. The test tube was inclined. 5ml of concentrated sulphuric acid, H2SO4 was poured down the side of the test tube gently. 3. A purple colour at the interface of the sugar and acid indicates a positive test. The test was disregarded if green colour appears.

RESULT : TEST SOLUTION Glucose Fructose Lactose Maltose OBSERVATION Purple ring formed Purple ring formed Purple ring formed Purple ring formed POSITIVE/NEGATIVE Positive Positive Positive Positive

DISCUSSION : The reaction between Molisch reagent with glucose, fructose, lactose as well as maltose shows a positive test. The positive reaction is indicated by the formation of purple ring near the interace where the acid meets the carbohydrate (sugar). All carbohydrates show a positive reaction with Molisch reagent because all sugars undergo dehydration reaction in the presence of sulphuric acid. A formation of hydroxymethylfurfural or also known as furfural occurs. In the second step, the furfural reacts with -naphtol in order to form the coloured product which is the purple ring.

Sugars are dehydrated in the presence of concentrated sulphuric acid. This results in the formation of furfuryl derivative which is the furfural or hydroxymethyl furfural. Furfural is derived from the dehydration of pentoses and pentosans. On the other hand, 5-hydroxymethyl furfural is produced from hexoses and hexosans.

1-naphtol reacts with hydroxymethylfurfural. The hydroxyl group of 1-naphtol has a storng electron donating resonance effect that contributes in increasing the electron density in the benzene ring. 1-naphtol acts as a nucleophile and is added to the positive polarized carbon of the

aldehyde group of 5-hydroxymethylfurfural. Water is then eliminated after an intramolecular proton migration. A carbon=carbon (C=C) double bond is also formed.

Now a further nucleophilic addition of 1-naphthol molecule takes place. After an intramolecular proton migration two protons are eliminated leading to a purple furfuryl dipheylmethane dye.

Although this test will detect compounds other than carbohydrates such as glycoproteins, a negative result indicates the absence of carbohydrates. precautions should also be taken during the carrying out of the procedures of this experiment. This is because concentrated sulphuric acid is a highly toxic material that can cause severe burns. It can be fatal if swallowed and is also carcinogenic. It is very destructive of mucous membrane. Besides, 1-naphtol is an eye, skin and respiratory irritant. It can also cause liver damage. Due to the dangerous side effects of these materials used in this experiment, thus safety precautions should be taken. CONCLUSION : The Molisch test is used to detect the presence of sugar or carbohydrates. All carbohydrates gives a positive reaction towards Molisch reagent which is identified by the formation of purple ring. REFERENCE : http://en.wikipedia.org/wiki/Molisch's_test http://www.uniregensburg.de/Fakultaeten/nat_Fak_IV/Organische_Chemie/Didaktik/Ke usch/p30_Z_mol-e.htm http://answers.yahoo.com/question/index?qid=20071004013938AAZkobt http://www.answers.com/topic/alpha-naphthol-test-molisch-s-test-molisch-test-molischreaction http://www.iloveindia.com/nutrition/carbohydrates/functions-of-carbohydrates.html http://www.novafeel.com/nutrition/carbohydrates/carbohydrates.htm http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Carbohydrates.html

TITLE : Lab 5.3 : Carbohydrates Detection Test (Barfoeds Test) OBJECTIVE : To detect the difference of carbohydrate between monosaccharide and disaccharide. MATERIALS : Barfoeds reagent Sugar solution Concentrated sulphuric acid

APPARATUS : Dropper Test tube Test tube rack Pipette Bunsen burner Beaker Tripod stand Wire gauze Water bath

PROCEDURES : 1. 15 drops of the following carbohydrates were placed in the separate labeled test tubes : glucose, fructose, sucrose, lactose, maltose 2. 1ml of Barfoeds reagent was added to each test tube. The test tubes were heated in a boiling water bath for ten minutes. 3. The test tubes were removed and noted. 4. The results were recorded.

RESULTS : TEST SOLUTION Glucose Fructose Sucrose Lactose Maltose OBSERVATION Brick red precipitate No changes No changes Brick red precipitate No changes POSITIVE/NEGATIVE Positive Negative Negative Positive Negative

DISCUSSION : In this experiment, both monosaccharides as well as disaccharides are used. Glucose and lactose are both monosaccharides whereas disaccharides are undoubtedly fructose, sucrose and maltose. Barfoeds test shows a rapid positive result for monosaccharides. In contrast with that, disaccharides reacts with Barfoeds reagent at a slower rate although it may produce the same result as the monosaccharides in a longer duration of time. Reducing monosaccharides are oxidized by the copper ion in solution to form a carboxylic acid and a reddish precipitate of copper (I) oxide is formed within three minutes. However, disaccharides take about seven to twelve minutes in order to produce the same results. This test is based on the reducing nature of the carbohydrates due to their having aldehyde or ketone groups, that reduce the cupric hydroxide formed in acidic medium to red coloured cuprous oxide. As disaccharides are weak reducing agents, they do not reduce cupric ions in Barfoeds solution under acidic condition. This differs in monosaccharides which are strong reducing agents that are able to reduce cupric ions even in acidic conditions. However, in prolonged heating, acetic acid present in the reagent, hydrolizes disacchraides to monosaccharides which responds to this test. The reagent used in Barfoeds test contains copper acetate and glacial acetic acid.

CONCLUSION : Barfoeds Test is used to differentiate between monosaccharides and disaccharides. Although both sugars may show positive result, but it is the time duration that determines the result of their reaction, thus denoting the difference between them. REFERENCE : http://en.wikipedia.org/wiki/Barfoed's_test http://www.harpercollege.edu/tm-ps/chm/100/dgodambe/thedisk/carbo/barf/barfoed.htm http://www.chemistry.mcmaster.ca/~chem2o6/labmanual/expt11/2o6exp11.html http://en.wikipedia.org/wiki/Chemical_test http://www.harpercollege.edu/tm-ps/chm/100/dgodambe/thedisk/carbo/barf/barfoed.htm

QUESTIONS : 1. Give other test that can be used to detect the presence of carbohydrates. Lugols test (iodine solution) is used to test complex carbohydrates. Positive results are indicated by the change of the iodine solution from yellowish brown to dark purple or black colour. Benedicts test is used to test for reducing sugars or aldehydes. Positive results are indicated by the formation of brick red precipitate. Fehlings solution is used to test for reducing sugars or aldehydes. Positive result is indicated by formation of red precipitate. Tollens test is used test for the presence of aldehydes. Positive result is indicated by a formation of silver mirror.

2. In Molisch Test, why the concentrated sulphuric acid need to be poured slowly down the side of the test tube? The test solution is combined with a small amount of concentrated sulfuric acid and is slowly added down the sides of the sloping test tube, without mixing. If the acid is poured too quickly, it results in a mixing of the contents and, can cause the test results to

be inaccurate. All carbohydrates and even, glycoproteins will give a positive reaction. A positive reaction is indicated by a purple ring forming at the interface between the acid and test layers. The sulfuric acid needs to be slowly poured so that it can have time to get into the test layers. It enhances the depth perception of the reaction. 3. What is the similarity and difference between Benedicts Test and Barfoeds Test? Barfoed's test is used for detecting the presence of monosaccharides. It is based on the reduction of copper(II) (as acetate) to copper(I) oxide (Cu2O), which forms a brick-red precipitate. Disaccharides may also react, but the reaction is much slower. The aldehyde group of the monosaccharide which normally forms a cyclic hemiacetal is oxidized to the carboxylate. A number of other substances, including sodium chloride may interfere. The test is similar to the reaction of Fehling's solution to aldehydes or Benedict's test. There is no difference for a solution to be tested by Benedict`s or by Barfoed`s test, as both detect the presence of reducing sugars. Barfoed`s test is the least known of these three. Both the test detect the presence of reducing sugars and the only difference between the two tests is that further confirmation of a monosaccharides or disaccharides are done in Barfoeds test.