Experiment 3 5i4h5p

EXPERIMENT 3 GENERAL REACTIONS OF CARBOHYDRATES Carbohydrates are vital sources of energy for both plants and animals. They also serve as skeletal structure for plants and storage for chemical energy for both plants and animals. In of chemical structure, carbohydrates are derivatives of aldehydes and ketones. Their interesting chemical reactions are due to such functional groups. Any material containing carbohydrates yields positive result in the Molisch Test. This test is based on the dehydration of monosaccharides by concentrated sulfuric acid to form furfural derivatives and the subsequent reaction with alpha napthol to form colored complexes. A similar reaction can be observed through Anthrone Test which is also based on the formation of furfural derivatives. Anthrone is a keto form of 9 – hydroxyl – anthracene which, when dissolved in concentrated sulfuric acid mixed with carbohydrates forms a blue or green color. Carbohydrates with free aldehyde or ketone group have reducing properties. They are oxidized by alkaline solution of cupric sulfate (Fehling’s and Benedict’s Reagents) producing a reddish brown precipitate because of the reduction of cuprous ions. Nylander’s Reagent, an alkaline solution of bismuth subnitrate when added to reducing sugars, produces a black precipitate due to the formation of metallic bismuth. Barcode’s reagent, a solution of cupric acetate in weak acetic acid, serves as another test as it is acted upon by reducing monosaccharides but not by reducing disaccharides. The simplest carbohydrates called monosaccharides consist of only one saccharide unit and thus cannot be hydrolyzed. The most abundant in the group is glucose. Other common monosaccharides are fructose, galactose, ribose and deoxyribose. Disaccharides consist of monosaccharide units ed by a glycosidic linkage. Common disaccharides include sucrose, lactose and maltose. Polysaccharides, the most complex of all carbohydrates, composed of many saccharide units, can be hydrolyzed by enzymes or by heating with dilute strong acids to yield monosaccharides. OBJECTIVES 1. To describe the properties and structures of carbohydrates in general 2. To differentiate reducing sugars from non – reducing sugars MATERIALS 1% glucose 1% sucrose 1% glycogen reagent 1% xylose 1% lactose 1% maltose

1% starch 1%silver nitrate 1% Ba(OH )2

conc. Sulfuric acid cupric acetate

glacial acetic acid Benedict’s reagent

6M NaOH

Nylander’s

phonolphthalein NH 4 OH

picric acid

Fehling’s reagent

dil. HCl

Molisch reagent

DATA AND ANSWER SHEET Alano, Danielle Daye Almario, Stephanie Aquino, Florevie – asst. leader Astillero, Jeremy – secretary Bron, Nicola Faye – leader

Remarks/Rating:_____________ Dr. Noemi Madrid Instructor Group no. 4, Lab 1 July 7, 2016

BS BIOLOGY II – A MTh 1:00 – 7:00pm; TF 2:30 – 4:00pm EXPERIMENT 3 GENERAL REACTIONS OF CARBOHYDRATES PROCEDURE: A. Reaction with Acids 1. Molisch Test a. To 1mL of 1% glucose, add 2 drops of Molisch reagent. Mix thoroughly b. Incline the test tube and allow 1mL of concentrated H 2 SO 4 acid to flow down the side of the test tube. Do not shake. Note the color formed at the junction of the two layers. c. Repeat the procedure using 1% dextrose, 1%sucrose, 1%starch, 1%maltose.

Molisch test result for the test compounds – glucose, dextrose, sucrose, starch and maltose. Test Compound Glucose

Dextrose

Observation The glucose test compound exhibited a positive result which is indicated by the appearance of violet or purple colored precipitate. Dextrose is a monosaccharide and Molisch test gives a positive result to all types of carbohydrates that’s why the test

Sucrose Starch Maltose

compound dextrose exhibited a violet colored precipitate. In this test compound – sucrose, also exhibited a positive result just like the first two test compounds. In this test compound – starch, it formed a violet/purple precipitate which indicates a positive result in Molisch test. Maltose is a disaccharide which is eventually hydrolyzed to monosaccharide in the Molisch test which results to the formation of violet/purple precipitate that is a positive result.

Did all the samples give the same result? YES for the results observed. Molisch test is based from dehydration of carbohydrate with sulfuric acid to form furfural. With alpha naphtol in the reagent molish furfural is colored purple. All carbohydrates monosaccharides, disaccharides, and polysaccharides give a positive reaction, and nucleic acids and glycoproteins also give a positive reaction, as all these compounds are eventually hydrolyzed to monosacharides by strong mineral acids such as H2SO4 which is used in this experiment. This is the reason why the five test compounds namely; glucose, dextrose, sucrose, starch and maltose all exhibited a positive result. B. Reaction with Alkali 1. Moore’s Test

The set-up of boiling hot water bath with test tubes containing 1mL of 1%glucose and 2mL of Ba(OH )2 and 1mL of 1%glucose and 2mL of NaOH, respectively. a. To 1mL of 1% glucose, add 2mL of NaOH. Heat in a boiling hot water bath. What changes in color and odor have you observed?

b. To 1mL of 1% glucose. Add 2mL of 1% Ba(OH )2 . Place in a boiling hot water bath. What changes in color and odor have you observed?

We have observed that in Moore’s test, there was a color change when the 1mL of 1%glucose was added with NaOH which is an alkali. The solution turned into dark yellow and has the liberated the smell of caramel.

We have observed that in Moore’s test, there was no color change when the 1mL of 1%glucose was added with Ba(OH )2 , which is a weak alkali.

How do you for the difference in the results using NaOH and Ba(OH )2 ? When the 1%glucose was heated with an alkali which is NaOH, it turns yellow, and liberated the odor of caramel. This is due to the liberation of aldehyde which subsequent polymerizes to form a resinous substance, caramel. Glucose, allowed to stand in the presence of weak alkali (BaOH) and is converted into a mixture containing glucose with the proportion that reached an equilibrium. C. Reducing Property Preparation of Tollen’s Reagent To 5mL of 1% AgNO 3

solution, add 1 drop of 6M NaOH. Add ammonia

solution dropwise, shaking after each addition until the precipitate initially formed is dissolved. Avoid adding more than what is necessary to dissolve the precipitate. 1. Tollen’s Test

Place 6 drops of 1% glucose into a clean test tube and add 2mL of Tollen’s reagent. Mix well. Allow to stand for 5minutes. If no result is obtained, warm in a water bath for 2-3minutes. What did you observe?

In the picture above, we have observed that there was a silver mirror-like formed in the test 1+¿ tube. This is because the aldehyde was oxidized to a carboxylic acid while the Ag¿ is reduced to silver metal, which deposits as a thin film on the inner surface of the test tube.

2. Fehling’s Test a. Prepare Fehling’s reagent by mixing 1mL of Fehling’s A, 1mL of Fehling’s B and 8mL of distilled water in a big test tube. b. Place 1mL of Fehling’s reagent in a test tube and add 5 drops of 1% glucose solution. Heat in a boiling water bath. Note the color produced. c. Repeat the test on 1% dextrose, 1% sucrose, 1% starch and 1% lactose solutions.

Prepared Fehling’s reagent which is a mixture of 1mL of Fehling’s A, 1mL of Fehling’s B and 8mL of distilled water.

The picture above shows test results for the 5 test compounds – glucose, dextrose, sucrose, starch and lactose. Test Compound 1% glucose

1% dextrose

1% sucrose

Observation We have observed that it changed from bluish color of solution to yellowish/greenish and had a red precipitate at the bottom, which indicates a positive result. It also changed in color from bluish solution to yellowish/greenish solution and formed a red precipitate at the bottom of the test tube which also indicates a positive result. Sucrose also changed in color from bluish to greenish solution but is darker than dextrose and did not produce a red precipitate at the bottom which indicates a negative result.

1% starch

1% lactose

Starch also changed in color from bluish solution to greenish one however, it did not have a red precipitate at the bottom, which indicates a negative result. Lactose, after heating turned from bluish solution to yellow green solution and did have a red precipitate at the bottom, which is a positive result.

Which compounds did not give positive result to Fehling’s test? Explain why. Sucrose and Starch did not give a positive result to Fehling’s test because in this test the presence of aldehydes but not ketones is detected by reduction of the deep blue solution of copper(II) to a red precipitate of insoluble copper oxide. The test is commonly used for reducing sugars but is known to be NOT specific for aldehydes. For example, fructose gives a positive test with Fehling's solution so does acetoin. Two solutions are required: A positive test is indicated by a green suspension and a red precipitate. Fehling's solution contains copper (II) ions complexed with tartrate ions in sodium hydroxide solution. Complexing the copper (II) ions with tartrate ions prevents precipitation of copper (II) hydroxide. Aldehydes reduce the complexed copper (II) ion to copper (I) oxide. Because the solution is alkaline, the aldehyde itself is oxidized to a salt of the corresponding carboxylic acid. 3. Nylander’s Test a. Mix 15 drops of 1% glucose with an equal amount on Nylander’s solution in a test tube. Heat for 5 minutes on a boiling water bath. Note the color produced. b. Repeat the test on 1% dextrose, 1% sucrose, 1% starch and 1% lactose solutions.

Nylander’s Test results for the 5 test compounds – sucrose, starch, glucose, lactose and dextrose, respectively. Test Compound 1% glucose

1% dextrose

Observation We have observed that glucose changed in color from clear solution to muddy yellow solution (dark solution) which indicates a positive result. Dextrose also changed in color from clear solution to a dark or

1% sucrose

1% starch 1% lactose

black solution which indicates also a positive solution. Sucrose, unlike the first two – glucose and dextrose, did not change in color. It remained clear afting heating which indicates a negative result. Starch, like sucrose, did not change in color. It also remained clear after heating which is also an indication of negative result. Lactose, also exhibited a positive result which is indicated by the change in color from clear to muddy yellow or dark colored solution.

Which compounds gave positive result? Glucose, Dextrose and Lactose Which compound did not give a positive result? Sucrose and Starch for the black precipitate formed. Nylander's test is a chemical test used for detecting the presence of reducing sugars. Glucose or fructose reduces bismuth oxynitrate to bismuth under alkaline conditions. When Nylander's reagent, which consists of bismuth nitrate, potassium sodium tartrate and potassium hydroxide, is added to a solution with reducing sugars, a black precipitate of metallic bismuth is formed. 4. Benedict’s Test – (NOT PERFORMED because of lack of reagent) a. To 1mL of Benedict’s reagent in a test tube, add 1mL of 1% glucose solution. Heat in a boiling water bath for 2 minutes and allow to stand. Note the time required to show a green or red color. b. Repeat the test on 1% solutions of dextrose, sucrose, lactose, starch and glycogen. What did you observe with the test compounds? Glucose, Dextrose, Sucrose, Starch, Lactose & Glycogen We did not perform this experiment however, the expected results of this test would be glucose, dextrose, lactose and glycogen would give a positive result while sucrose and starch would yield to a negative result. This is because Benedict's test determines whether a monosaccharide or disaccharide is a reducing sugar, and is hence similar in purpose to the Tollen’s and Fehling's test. This makes use of a single solution of copper(II) citrate which does not deteriorate as quickly on standing. Again Benedict’s solution contains copper sulphate. Reducing sugars reduce soluble bluer copper sulphate, containing copper(II) ions to insoluble red-brown copper oxide containing copper(I). The latter is seen as a precipitate. To give a positive test, the carbohydrate must contain a hemiacetal which will hydrolyse in aqueous solution to the aldehyde form. Benedict's reagent is an alkaline solution containing Cu(II) ions, which oxidize the aldehyde to a carboxylic acid. In turn, the cupric ions are reduced to cuprous oxide, which forms a red precipitate. The colour ranges from green to yellow to orange to brick-red depending on the amount of reducing sugar in the sample; with a sample containing 1% glucose, the precipitate is usually brick-red. Benedicts's test will give a color change for any mono- or disaccharide containing a hemiacetal or hemiketal group. Since sucrose or table sugar does not contain these groups, it will not give a positive test.

D. Hydrolysis

1. Prepare the rice solution by placing a spatula of cooked ric in a mortar and pounding using a pestle. Add 10mL of distilled water. Stir. 2. Put 2mL of the rice solution in a test tube, add 1mL of 6M HCl and heat in a water bath for 15 minutes to hydrolyze. 3. Neutralize the resulting mixture with about 10 drops of 6M NaOH. Test by adding a drop of phenolphthalein. A light pink color should be observed. 4. Add 2mL of Fehling’s reagent (1mL of Fehling’s A and 1mL of Fehling’s B). Heat in a water bath for 2 minutes. Was there a change in color? Yes, the solution turned into pinkish green that is slightly like gray in color. Is a positive result obtained? Yes 5. In another test tube, mix 2mL of the rice solution (from step 1) 1mL of Fehling’s A and 1mL of Fehling’s B. heat in water bath for 2 minutes. Was there a change in color? Yes, since before heating, the color was blue, but as it was heated it slowly changed to green. Is a positive result obtained? Yes What does it indicate? The yellowish-green precipitate indicated that the sugars were reduced. What do you think is the purpose of adding HCl to the rice and heating the mixture in a water bath? The addition of HCl to the rice and heating the mixture in a water bath is for the Polysaccharides, which are the most complex of all carbohydrate compounds, composed of many saccharide units is hydrolyzed and yield monosaccharides. REFERENCES: Andrews, Ryan. All about Carbohydrates: How carbs affect your health and performance. Retrieved from http://www.precisionnutrition.com/all-about carbohydrates Baudier KM, Kaschock-Marenda SD, Patel N, Diangelus KL, O'Donnell S, et al. (2014) Erythritol, a Non-Nutritive Sugar Alcohol Sweetener and the Main Component of Truvia®, Is a Palatable Ingested Insecticide. PLoS ONE 9(6): e98949. doi:10.1371/journal.pone.0098949

Specific Reactions of Carbohydrates Specific reactions characterize different carbohydrates. Groups of carbohydrates may be differentiated by their particular reactions with the same reagent. Some examples are: Hexoses which are monosaccharides with six carbon atoms and pentoses which have five carbon atoms are differentiated by the Bial’s Orcinol Test. The furfural formed from the dehydration of a pentose with orcinol forms a blue and green color while that from a hexose is muddy brown. Ketoses (carbohydrates with ketone functional group) give cherry red color within two minutes with Seliwanoff’s test while aldoses which are carbihydrates with aldehyde functional group require a longer time. This test involve the reaction of resorcinol and acid on the sugar, forming hydroxyl methyl furfural as a result of dehydration. Reducing sugars forms osazone crystals when heated with an excess phenylhydrazine HCl. This reaction serves to identify the sugars by the structure of the crystals and the time required to form them. Similarly, upon oxidation of nitric acid, hexose produces crystals that are soluble in diluted acid and water. Galactose in particular produces muscle acid, a dicarboxylic acid and an isomer of saccharide acid which is identified by its insolubility in acid and water. Polysaccharides form characteristic colored complexes with iodine. Starch gives a blue color with iodine solution. Dextrin, a product of partial hydrolysis of starch gives red color, and glycogen, a highly branched complex polysaccharide gives a pale red brown. The differences in color of the complexes is due to the structure of these polysaccharides. Starch is made up of linear chains of glucose unit of amylose which undergo helical formation. A helix containing 6glucose unit is enough to accommodate large molecules like iodine. Thus, branched polysaccharides like glycogen, gives a less intense color because of interruption in the helices. Objectives 1. To differentiate the reactions of hexoses from pentoses, aldoses and ketoses. 2. To identify different tests for specific carbohydrates. 3. To identify an unknown carbohydrate sample. Materials 1% fructose solution 1% galactose solution 1% glucose solution 1% lactose solution 1% dextrose solution 1% glycogen solution Bial’s Orcinol Reagent Seliwanoff’s Reagent 95% ethyl alcohol 1% starch solution

1% sucrose solution iodine solution galactose powder glucose powder lactose powder sucrose powder sodium acetate conc nitric acid

DATA AND ANSWER SHEET EXPERIMENT 4 SPECIFIC REACTION OF CARBOHYDRATES PROCEDURE: A. Bial’s Orcinol Test 1. Pipet 0.5mL of 1% glucose solution into a test tube and add 1.0mL of Orcinol’s reagent. Place the test tube in hot water bath. 2. Repeat the test on 1% fructose, 1% amylose and an unknown solution. Which of the test compounds gave a positive result? NONE To which group of carbohydrates group of carbohydrates does it belong? The compound that could give a positive result to Bial’s Orcinol test should belong to the group of pentoses. B. Mucic Acid Test 1. Into 4 separate test tubes, place a pinch of each galactose, glucose, lactose and an unknown. Add 1mL of water and 1mL of concentrated nitric acid. 2. Heat the test tube with their contents in a boiling water bath for an hour. 3. Cool at room temperature. In Juce crystal formation by scratching the tube with a clean stirring rod. Collect some crystal formation from each test tube 4. Examine with a microscope and draw them as seen through the microscope lens. Galactose

The only test compound that yielded to a positive result was galactose. This is because the mucic acid test is used to identify the presence of the sugar galactose in food or in synthetics manufacture. When concentrated nitric acid is heated together with galactose,

Glucose

Lactose

Unknown

No crystals were formed

No crystals were formed

No crystals were formed

a dicarboxylic acid called mucic acid forms as a white precipitate, which counts as a positive result.

Based on the crystals formed, what is the unknown sample? There were no crystals formed. C. Seliwanoff’s Test 1. Place 2mL of Seliwanoff’s Reagent in a test tube and add 2 drops of the 1% glucose solution. Warm in a water bath. Observe the color formed and its time of formation. 2. Repeat the test using 1% fructose, 1% sucrose and 1% starch solution. To which groups of carbohydrates do they belong?

The picture above shows the observed test results of Seliwanoff’s Test. Carbohydrate 1. Glucose

Group Monosaccharide – Aldose

Observed Color

Light yellow

Time 3 minutes

2. Fructose

Disaccharide – Ketose

3. Sucrose

Disaccharide – Ketose

4. Starch

Monosaccharide

5 minutes

Yellow orange 5 minutes

Orange-red 8 minutes

Light yellow Which of the samples reacted most readily to the test? The glucose readily reacted with the Seliwanoff’s test because it took 3 minutes for it to change in color, however, in the 4 test compounds, the Fructose and Sucrose yielded a positive result because Seliwanoff’s test is a chemical test which distinguishes between aldose and ketose sugars. Ketoses are distinguished from aldoses via their ketone/aldehyde functionality. If the sugar contains a ketone group, it is a ketose. If a sugar contains an aldehyde group, it is an aldose. This test relies on the principle that, when heated, ketoses are more rapidly dehydrated than aldoses.

D. Osazone Formation – (NOT PERFORMED because of lack of reagent) 1. Place into separate test tube 1mL of each of glucose, fructose, galactose, lactose, sucrose, xylose and an unknown carbohydrate sample. 2. In a separate container, mix 6g sodium acetate, 2mL of phenolhydrazine-HCl and 20mL of distilled water. Heat the solution while stirring until it becomes clear. 3. Add 2mL of the hot to each of the test samples in their separate test tubes.

4. Provide each test tube with cotton stoppers, mix well and heat on a boiling water bath for half an hour. Cool at room temperature. Examine the crystals under the microscope. 5. Draw the crystals as observed, if any were formed. Fructose

Glucose

Sucrose

Xylose

Galactose

Lactose

Unknown

We were not able to perform the experiment because of lack of reagent, however, the expected result for Osazone test is the formation of beautiful yellow crystals of osazone needle shaped crystals, Hedgehog crystals and Sunflower shaped crystals because reducing sugars forms ozazone on treating with phenylhydrazine. E. Iodine Test In a spot plate or watch glass, separately place 5 drops of each of the 1% solutions of starch, glycogen and glucose. To each spot, add a drop of iodine solution.

The picture above shows the Iodine test results on the 3 test compounds – starch, glucose and glycogen. What color was observed with: Starch: Starch formed an intense dark blue color.

Glucose: There was no change in color in glucose, it remained light yellow in color. Glycogen: Glycogen formed a pale brown-red color.

SUPPLEMENTARY QUESTIONS: 1. Write the structural formula of glucose and fructose.

2. Write the stages in hydrolysis of Starch and their color reaction with iodine solution. 1. GELATINISATION, involving the dissolution of the nanogram-sized starch granules to form a viscous suspension; their color reaction with iodine solution is intense blue/purple color; 2. LIQUEFACTION, involving the partial hydrolysis of the starch, with concomitant loss in viscosity; their color reaction with iodine solution is pale red brown; 3. SACCHARIFICATION, involving the production of glucose and maltose by further hydrolysis; their color reaction with iodine solution is the same as iodine. 3. for/explain the formation of: a. Osazone crystals: Osazones are formed when the sugars react with a compound known as phenylhydrazine at boiling point. The technique was developed by Emil Fischer, a German chemist, to identify different sugars. Fischer was able to differentiate the types of sugar by studying the crystals that formed from his procedure. b. Mucic acid: When concentrated nitric acid is heated together with galactose, a dicarboxylic acid called mucic acid forms as a white precipitate, which counts as a positive result. REFERENCES: Swan, Ryan. May 26, 2016. Everything You Need to Know about Carbohydrates! (blogpost). Retrieved from http://www.bodybuilding.com/fun/teen-ryanswan16.htm

The Merk Index, Ninth Edition, 1976. Arienti G. “Le basi molecolari della nutrizione”. Seconda edizione. Piccin, 2003 Belitz .H.-D., Grosch W., Schieberle P. “Food Chemistry” 4th ed. Springer, 2009