You are on page 1of 14

UDDD1104 Cell Biology UTAR

Title: Macromolecules Objectives:


1. Describe and carry out the Benedicts test that indicates the presence of reducing sugars specifically. 2. Describe and carry out the Iodine Test that indicates the presence of starch specifically. 3. Describe and carry out the hydrolysis process on the non-reducing disaccharide and polysaccharide. 4. Describe and carry out the Biuret test that indicates the presence of proteins. 5. Describe and carry out the paper test that indicates the presence of lipids.

Introduction: Carbohydrates, proteins, and fats are all essential nutrients. We cannot manufacture this macromolecule so we must obtain them from our environment. In this lab, with the use of indicators as chemical detection tools, you will analyze a variety of given solutions for the presence of nutrients. Detection is based upon observing a chemical change that takes place most often a change in color. Carbohydrates are consist of carbon, oxygen and hydrogen they contain are generally in proportion to form water with the general formula C n (H2O)n. It is a major food source and a key form of energy for most organisms. When combined together to form polymers, carbohydrates can function as long term food storage molecules, as protective membranes for organisms and cells, and as the main structural support for
1

UDDD1104 Cell Biology UTAR

plants and constituents of many cells and their contents (Nordqvist, 2009). Carbohydrates have several roles in living organisms, including energy transportation, as well as being structural components of plants and arthropods. Carbohydrate derivates are actively involved in fertilization, immune systems, the development of disease, blood clotting and development. The simplest form of carbohydrates is the monosaccharide. 'Mono' means 'one' and 'saccharide' means 'sugar'. Monosaccharides are either aldoses or ketoses. Aldoses such as glucose consists of a carbon backbone and a carbonyl group (C=O) located at the end of the chain. Ketoses such as fructose consists of a carbon backbone with a carbonyl group located at any other carbon in the chain. The remaining carbon atoms are bound to hydroxyl groups (-OH). Monosaccharides with three carbon atoms are called , those with four are called tetroses, five are called pentoses, six are hexoses, and so on. These two systems of classification are often combined. For example, glucose is an aldohexose (a six-carbon aldehyde), ribose is an aldopentose (a fivecarbon aldehyde), and fructose is a ketohexose (a six-carbon ketone).

Monosaccharide is the smallest possible sugar unit include glucose, galactose or fructose. When two monosaccarides are joined together through a bond, a disaccharide is formed. Maltose is a sugar composed of two glucose molecules. Nelson and David L. (2005) concluded that other common disaccharides include lactose and sucrose. The structure of carbohydrates featuring two or more monosaccharides is held together covalently with a glycosidic bond. Sucrose is found in table sugar, and is often formed as a result of photosynthesis (sunlight absorbed by chlorophyll reacting with other
2

UDDD1104 Cell Biology UTAR

compounds in plants). If one glucose molecule bond with a galactose molecule will get lactose, which is commonly found in milk. Starch is a glucose polymers made up of Amylose and Amylopectin. Amylose molecule chains are linear (long but no branches) while Amylopectin molecules are long and branch out. Some Amylopectin molecules are made of several thousand glucose units. Starches are not water soluble. Humans and animals digest them by hydrolysis and our bodies have amylases which break them down. Rich sources of starches for humans include potatoes, rice and wheat. Proteins are the molecules contain nitrogen, carbon, hydrogen and oxygen. They act as biological catalysts enzymes, form structural parts of organisms, participate in cell signal and recognition factors, and act as molecules of immunity. Proteins can also be a source of fuel (Bailey, 2012). These molecules provide human with pigment. They also enable cells to move, to have a specific shape, and to recognize material around them. The lipid is a group of macromolecules that are insoluble in water which consist of carbon, hydrogen, and oxygen atoms. The major kind s of lipids in biological world include fats, phospholipid, and steroids. They fats or triacylglycerols are made up of glycerol (an alcohol) and fatty acids (a monocarboxylic acid) which are bounded together by ester linkages. It is the main constituents of all membranes in all cells (cell walls) food storage molecules, intermediates in signaling pathways, Vitamins A, D, E and K, cholesterol. (Mandal, 2011)

UDDD1104 Cell Biology UTAR

Investigation 1: Monosaccharides and Disaccharides Materials: Benedicts reagent, 1% solutions of glucose, fructose, lactose, sucrose, and starch, test tube, beaker, hot plate Methods: 1. A boiling water bath was made by filling a beaker about half full of water and heating it on a hot plate. 2. A test tube was placed in 1 ml of a 1% glucose solution and 5 ml of Benedicts solution. 3. A control was prepared. 4. The two tubes were placed in boiling water for 2-3 minutes. 5. The colour of the solution was observed and noted whether a precipitate has formed. 6. The test with 1% solutions of fructose, lactose, sucrose and starch were repeated. 7. All of the results were recorded table 1.1.

Investigation 2: Starch Materials: Starch solution, iodine reagent, test tubes Methods: 1. A starch solution was prepared by mixing thoroughly 2g of starch with 10 ml of water and then pouring this mixture into 200 ml of boiling water. 2. Two test tubes were prepared and labeled them 1 and 2. 3. A few ml of the starch solution were put in Tube 1. This is the positive control. 4. Tube 2 was prepared and is the negative control.
4

UDDD1104 Cell Biology UTAR

5. A few drops of iodine reagent were put into each tube. 6. The observations were recorded in table 1.2.

Investigation 3: Hydrolysis of Carbohydrates Materials: Starch solution, sucrose solution, 2N HCL solution. benedicts reagent, iodine reagent, test tubes, pipettes, water bath Methods: 1. Eight test tubes were prepared and labeled them 1 through 8. The test tubes were lined up in order in a test tube rack. 2. Two large test tubes were prepared and labeled them starch and sucrose 3. 6 ml starch solution and 3 ml 2N HCl were pipetted into the tube labeled starch. 4. 5 ml sucrose solution and 1 ml 2N HCl were pipetted into the tube labeled sucrose. 5. Each tube was swirled gently to mix the contents. Sampling 6. 1 ml of solution was drawn from the sucrose tube and put it in Tube 1. 7. Using a different pipette, 1 ml of solution from the starch tube was drwan and put it in Tube 3. (Tube 2 was skipped for now.) 8. An additional ml of solution from the starch tube was drawn and put it in Tube 4. 9. The extra-large starch and sucrose tubes were placed in boiling water bath. The time was noted. 10. After 2 or 3 minutes, 1 ml of solution from the sucrose tube was drawn and put it in Tube 2. The sucrose solution was then removed from the water bath.

UDDD1104 Cell Biology UTAR

11. After 5 minutes, 1 ml of solution was drawn from the starch tube and put it in Tube 5. 12. A second ml of starch solution was put in Tube 6. 13. Waited 10 more minutes and then steps 11 and 12 were repeated, putting the solution in Tube 7 and 8. Testing for Starch and Sugar 14. 5 ml of Benedicts reagent were added to Tubes 1, 2, 3, 5, and 7. These tubes were placed in the boiling water bath for 5 minutes. 15. 3 or 4 drops of iodine reagent were added to Tubes 4, 6, and 8. 16. The tubes from the water bath were removed and waited 5 minutes for them to cool. The results were recorded in table 1.3.

Investigation 4: Proteins Materials: 1% egg albumin, concentrated KOH, 0.5% CuSO4, test tubes Methods: 1. Two test tubes were prepared and labeled them 1 and 2. 2. 3 ml of 1 % egg albumin were put into Tube 1. 3. Tube 2 was the control. What 4. An equal volume of concentrated KOH (~ 20%) were added to both tubes. Mixed thoroughly. 5. 1 ml of 0.5% CuSO4 was added slowly and mixed. 6. After 2 minutes, the colour was recored in each tube.

Investigation 5: Lipids
6

UDDD1104 Cell Biology UTAR

Materials: Brown paper, vegetable oil, water Methods: 1. A small square of brown paper was prepared. oil on one half and water on the other were written. 2. A tiny drop of vegetable oil was put on the half of the paper labeled oil. It was rubbed gently with fingertip. 3. As a negative control, a tiny drop of water was puton the half of the paper labeled water. It was rubbed gently with a different fingertip to avoid contamination. 4. The spots were allowed to dry. 5. When the spots are dry, the paper was hold up to the light. 6. Observations were recorded in table 1.5.

Results: Part 1 Investigation 1: Monosaccharides and Disaccharides Tube 1 2 water The solution turned color from blue to orange and form 3 4 5 Fructose orange precipitate. The solution turned color from blue to orange and form Lactose Sucrose orange precipitate. The solution maintained in blue color, negative result
7

Sample Glucose

Observation The solution turned color from blue to orange and form orange precipitate.

Distilled The solution maintained in blue color, negative result

UDDD1104 Cell Biology UTAR

Starch

The solution maintained in blue color, negative result

Table1. 1 Result of Benedicts solution test for small sugars

Investigation 2: Starch Tube 1 2 Sample Starch Distilled water negative result.


Table1. 2 Result of Iodine solution test for starch

Observation The solution form dark blue precipitate, positive result. The solution maintained in orange color (iodines color),

Investigation 3: Hydrolysis of carbohydrates

Time Tube (Min) The solution maintained in 1 Sucrose 0 blue color, negative result The solution turned color 2 3 from blue to orange and form orange precipitate. The solution maintained in 3 0 blue color, negative result The solution form dark blue 4 5 0 5 The solution turned color from blue to orange and form orange precipitate, positive Benedicts reaction
8

Benedicts reagent

Iodine Reagent

Starch

precipitate, positive iodine reaction.

UDDD1104 Cell Biology UTAR

No color changes, negative 6 5 iodine reaction. The solution turned color from blue to orange and form 7 15 orange precipitate, positive Benedicts reaction No color changes, negative 8 15 iodine reaction.
Table1. 3 Result of hydrolysis of carbohydrates with Benedict and iodine test

Investigation 4: Protein Tube 1 2 water


Table1. 4 Result of protein present test

Sample Egg albumin Distilled

Observation Violent color is formed in the solution, positive Biuret test No color changes, negative Biuret test

Investigation 5: Lipid Brown Sample paper A B Water Oil Not translucent (negative test result) Translucent (positive test result) Observation

Table1. 5 Result of lipid present test (Grease spot test)

UDDD1104 Cell Biology UTAR

Discussion: The Benedict's test allows detecting the presence of reducing sugars (sugars with a free aldehyde or ketone group). All monosaccharides (glucose, fructose and galactose) are reducing sugars; they all have a free reactive carbonyl group. Some disaccharides (lactose) have exposed carbonyl groups and are also reducing sugars. Other disaccharides such as sucrose are non-reducing sugars and will not react with Benedict's solution. Starches are also non-reducing sugars. The copper sulfate (CuSO4) present in Benedict's solution reacts with electrons from the aldehyde or ketone group of the reducing sugar to form cuprous oxide (Cu2O), a red-brown precipitate. The iodine test is used to test for the presence of Starch. Iodine solution dissolved in an aqueous solution of potassium iodide and reacts with starch producing a dark blue color. This reaction is the result of the formation of polyiodide chains from the reaction of starch and iodine. The amylose, or straight chain portion of starch, forms helices where iodine molecules assemble, forming a dark blue/black color. The amylopectin, or branched portion of starch, forms much shorter helices and iodine molecules are unable to assemble, leading the color to be of an orange/yellow hue. The blue-black color is not produced as starch is broken down or hydrolyzed into smaller carbohydrate units. Therefore, this test can determine completion of hydrolysis when a color change does not occur. Starch is made up of a long chain of glucose molecules held together by glycosidic bonds. Glucose is basically a bunch of things stuck to a six-member ring. One carbon in the ring is bond to oxygen which is bound to the carbon of another ring.
10

UDDD1104 Cell Biology UTAR

The C-O-C bond can be broken through hydrolysis to release the constituent glucoses. Hydrochloric acid (HCl) serves as an acid catalyst in water to hydrolyze carbohydrate polymers. HCl puts H+ in the starch; the lone pairs of the oxygen in the C-O-C bond will attack this H+. The oxygen is bond to three things and it is unstable, because the acid allows this destabilization to occur, it lets hydrolysis of the bond occur. The hydrolysis of sucrose will obtained two monosaccharides: glucose and fructose while the hydrolysis of starch will obtain glucose linkage. In the part 1 of the experiment, hydrolysis of carbohydrates has been conducted. Test tube 1 and 2 were contained sucrose which used to hydrolyze and tested the presence of non-reducing sugar and reducing sugar by Benedicts test. Test tube 1 did not placed in the water bath after added HCl and gave negative test at the end while test tube 2 was placed in the 100C water bath to heat it. The hydrolysis only processed in test tube 2. During the heating process, sucrose has been broken into 2 monosaccharides which are glucose and fructose, therefore, orange precipitate was formed in the test tube 2 after water bath. Furthermore, test tube 3, 4, 5, 6, 7 and 8 are contained starch. Test tube 3, 5 and 7 were used to test the presence of monosaccharides or disaccharides by Benedicts test while test tube 4, 6 and 8 were used to test the presence of starch by iodine test. Test tube 3 and 4 contain starch that did not placed in the water bath after added HCl, the hydrolysis do not take place in this tube, hence, gave the negative Benedicts test in test tube 3 and positive iodine test in test tube 4. In this stage, starch has not being hydrolyze and do not contain constituent glucoses. After 5 minutes, starch has been hydrolyzed in the water bath (100C) and was tested again with Benedicts solution in test tube 5 and iodine solution in test tube 6. Starch has been
11

UDDD1104 Cell Biology UTAR

broken through hydrolysis to release the constituent glucoses; therefore, it gave positive test in Benedicts test in test tube 5 while gave negative test in iodine test in test tube 6. After 15 minutes, the starch was tested again with Benedicts solution in test tube 7 and iodine solution in test tube 8 after the hydrolysis of starch. Finally, all starch has been broken through hydrolysis to release the constituent glucoses; therefore, it gave positive test in Benedicts test in test tube 7 while gave negative test in iodine test in test tube 8. The color of orange precipitate in test tube 7 was more concentrated than test tube 5; this is because test tube 7 has more constituent glucoses than test tube 5 which test tube 7 was hydrolyze with longer time than test tube 5.

The biuret test is based on the ability of Cu (II) ions to form a violet-coloured chelate complex with peptide bonds (-CONH-groups) in alkaline conditions. Biuret reagent contains: hydrated copper sulphate which provides the Cu (II) ions form the chelate complex. Cu (II) ions give the reagent its characteristic violet blue color; second reagent is potassium hydroxide solution which does not participate in the reaction but provides the alkaline medium. The biuret reagent is not named after someone but a substance called biuret (H2NC (O) NHC (O) NH) - the result of the condensation of 2 molecules of urea. The reagent is so named because the peptide bonds in biuret give a positive result for the test. The distilled water added with potassium hydroxide and copper sulphate is a negative control in this test. The working principle of grease spot test is that most lipids have a high boiling point. So, they are non-volatile. In room temperature, the spot of water can absorb enough heat from the air and evaporated. But the spot of lipid is inside the sheet of paper, it diffracts light. So, light can pass from one side of the paper to another side.
12

UDDD1104 Cell Biology UTAR

This gives the phenomenon of translucent. When there is no liquid in the paper, there is no diffraction. So, light cannot pass it through. Conclusion: Monosaccharides and disaccharides give positive test in Benedicts test. Starches give positive test in Iodine test. Protein gives positive test in Biuret test. Instant noodles contained lipid which gives translucent phenomenon on brown paper. Besides, hydrolysis is a way to break long chain of glucose molecules into constituent glucoses. Starch can be hydrolyzed and release constituent glucoses and hence give positive Benedicts reaction.

References: Bailey, Available R., at: 2012. Protein Function. [Online]

http://biology.about.com/od/molecularbiology/a/aa101904a.htm

[Accessed 14 March 2013]. Mandal, Available A., at: 2011. What are Lipids?. [Online]

http://www.news-medical.net/health/What-are-Lipids.aspx

[Accessed 14 March 2013].


Nelson, David L. Lehninger Principles of Biochemistry 4th ed . W.H. Freeman and Company. 2005

Published by HYPERLINK "http://contributor.yahoo.com/user/755354/ac_writer.html" AC Writer


13

UDDD1104 Cell Biology UTAR

Nordqvist, C., 2009. What Are Carbohydrates? What Is Glucose?. [Online] Available at: http://www.medicalnewstoday.com/articles/161547.php

[Accessed 14 March 2013]. University Tunku Abdul Rahman, 2013. Lab manualExperiment

5&6:Macromolecules and macromolecules in food, Kampar, Perak: Universiti Tunke Abdul Rahman.

14