Lab 5 and Lab 6 Macromolecules Detection PDF

Lab 5 and Lab 6 Detection of biological macromolecules Cells, the fundamental units of life, are composed of various combinations of organic macromolecules: carbohydrates, lipids, proteins, and nucleic acids. This lab exercise is designed to show you the qualitative tests com...

Lab 5 and Lab 6 Detection of biological macromolecules Cells, the fundamental units of life, are composed of various combinations of organic macromolecules: carbohydrates, lipids, proteins, and nucleic acids. This lab exercise is designed to show you the qualitative tests commonly used to detect their presence. Objectives Test for the presence of monosaccharides by using the Benedict’s test Test for the presence of starch, a polysaccharide, by using the Lugol’s iodine test Test for the presence of lipids by using the Sudan test Test for the presence of proteins by using the Biuret reagent test Determine the identity of an unknown substance by detecting the presence of organic macromolecules using the above tests Introduction Biological macromolecules are defined as large molecules (polymer) made up of smaller organic molecules (monomer). There are four classes of macromolecules: 1) Carbohydrates The monomer of carbohydrates is monosaccharide, Carbohydrates have the general formula [CH2O] n (where n is the number of repeating units). The main function of carbohydrates is short-term energy storage (such as glucose). A secondary function is intermediate-term energy storage (as in starch for plants and glycogen for animals). In addition, some carbohydrates are involved as structural components in cells, such as cellulose in the cell walls of plants and chitin in the exoskeleton of arthropods. Sugars are structurally the simplest carbohydrates. Types of carbohydrates: A- Monosaccharides: are single (mono) sugars. Important monosaccharides include ribose, glucose, and fructose (an isomer of glucose), galactose. B- Disaccharides: are formed when two monosaccharides are chemically bonded together. Monosaccharides are linked into disaccharides and polysaccharides by a reaction that requires the loss of one water molecule is called dehydration synthesis. Sucrose, a common plant disaccharide, is composed of the monosaccharide’s glucose and fructose. Lactose, which is a sugar found in milk, is a disaccharide composed of glucose and the monosaccharide galactose, maltose disaccharide composed of two glucose C- Polysaccharides: are large molecules composed of individual monosaccharide units. A common plant polysaccharide is starch, which is made up of many glucose subunits. Glycogen is another polysaccharide that accumulates in the vertebrate liver and serves as a storage product. Cellulose is a polysaccharide found in plant cell walls that forms the fibrous and rigid part of the cell wall. In terms of human diets, cellulose is indigestible and thus forms an important part of dietary fiber. To test for the presence of monosaccharides and disaccharides, we will use the Benedict’s test for reducing sugars. Benedict’s reagent is clear blue (from the presence of cupric copper ions, Cu++), but when combined and heated to boiling with a substance containing reducing sugar, the cupric ions are reduced to a cuprous form (Cu+) and then oxidized to form copper oxide (Cu2O). Copper oxide is a brownish-orange substance that is insoluble in water. Therefore, a positive reaction in a Benedict’s test is the change of the clear light blue solution to an opaque orange-brown solution or brick red in a boiling water bath. This color change indicates the presence of reducing sugar (all monosaccharide and all common disaccharide except sucrose) in a given solution. To test for the presence of starch in given solution, Lugol’s (IKI) iodine solution is used, it reacts with starch to produce a purple or black color. 2) Lipids Lipids are primarily involved in long-term energy storage. They are generally insoluble in polar substances such as water. Additionally, lipids serve as structural components (as in the case of phospholipids which are the major building blocks in cell membranes) and as “messengers” (hormones) that play roles in communications within and between cells. Fats and oils function as an energy source. Animals convert excess sugars (beyond their glycogen storage capacities) into fats. Some seeds and fruits have energy stored as oils (for example, corn oil, peanut oil, palm oil, canola oil, and sunflower oil). Fat molecules consist of a glycerol molecule (a 3- carbon alcohol) and three fatty acids (of varying lengths). Another use of fats is as isolator.  Waxes are an important structural component for many organisms, such as the cuticle, a waxy layer covering the leaves and stems of many land plans, as well as protective coverings on skin and fur of animals. Lipids may be detected in a number of ways. The application of a fat-soluble dye known as Sudan III will allow lipids to be detected. The dye soluble in fats and can be seen in a simple test , so positive result in Sudan III test indicated by seeing the solubility. 3) Proteins: Proteins monomer is amino acid which has an amino end (NH2) and a carboxylic acid end (COOH). The R indicates the variable or differing component (also known as the “R- group” of each amino acid. Amino acids linked together by joining the amino end of one molecule to the carboxyl end of another. Removal of a water molecule, which is known as dehydration synthesis, allows the formation of a specialized type of covalent bond known as a peptide bond. Proteins are very important in biological systems as control and structural elements like structural proteins in the cell membrane, muscle tissues, another examples of protein, enzymes and proteinaceous hormones, carry out a number of biological control functions, enzymes are chemicals that act as organic catalysts. Biuret is a reagent that will change color from blue to violet when it detects peptide bonds between amino acids of a protein. Violet is a positive test, while blue is a negative test. Part A: Carbohydrates Detection 1) Reducing sugar detection Materials - Hot plate - Sucrose solution - 400mL beaker - Starch solution - Test tubes - Fructose solution - Test tube holder - Glucose solution as positive control - Test tube rack - Distilled water as negative control - Benedict’s Reagent - marker Procedure 1.Prepare a water bath by filling a beaker slightly over halfway with tap water. 2. Place the water-filled beaker on the electric hot plate, turn it on high, and bring the water to a boil. 3. label 5 test tubes using the marker. 4. Place 10 drops of Benedict’s reagent in each tube. 5. In the tube labeled 1, place 10 drops of distilled water as negative control. In the tube labeled 2, place 10 drops of glucose solution as positive control. In the tube labeled 3, place 10 drops of fructose solution. In the tube labeled 4, place 10 drops of sucrose solution. In the tube labeled 5, place 10 drops of starch. 6. Record the color of each tube before heating in the space provided in Table 1. 7. Place the five test tubes into the boiling water, being careful not to burn yourself. 8. Allow the test tubes to stay in the boiling water for five minutes. Carefully use the test tube holder to remove each tube and place it in the test tube rack. Record your observations in Table 1. Note: reducing sugars cause a color change in Benedict’s solution from its original color to green, yellow, orange, or red. 9. In the last column of the data table, indicate presence of the reducing sugar with a +, and absence with a -. 2) Starch detection (polysaccharide) Materials -Lugol’s (iodine) - Test tube rack - distilled water as negative control - marker -starch as positive control - glucose solution - Test tubes - mayonnaise brands - potato juice - rice water Procedure 1) put 15 drops of distilled water test tube labeled with the letter W. 2) put 15 drops of starch solution in test tube labeled with the letter S. 3) put 15 drops of glucose solution in test tube labeled with the letter G. 4) put 15 drops of rice water in another test tube and label it with the letter R. 5) Place 15 drops of potato juice in another test tube and label it with the letter P. 6) In each of the test tubes, place 2 drops of Lugol’s iodine. Record the results in Table 2. 7) In the last column of the table, indicate the presence or absence of starch polysaccharide in each tube, using a (+) symbol to indicate the presence, and a (-) symbol to indicate the absence. Part B : Lipid detection : Materials -Sudan III dye - palm oil(P) - Distilled water as negative control (W) - coconut oil (C) - Vegetable oil as positive control (V) - plastic sheet - marker - orange juice(O) - stick -unknown (U) Procedure 1) Mark a small dry piece of plastic sheet with a marker as shown below 2) Place two drops of distilled water in the circle next to the letter W 3) Place two drops of Vegetable oil in the circle next to the letter V. 4) Place two drops of palm oil in the circle next to the letter P. 5) Place two drops of orange juice in the circle next to letter O. 6) Place two drops of unknown in the circle next to letter U. 7) put small amount of the Sudan III dye powder on each spot above. 8) Gently agitate each spot with separate stick.  9) Notice the solubility of each sample with Sudan III dye. 10) Fill table 3 with soluble or insoluble. Part C: protein detection: Materials _Biuret reagent _ Glucose solution _ Test tubes and its rack _ milk _ Egg white solution as positive control _ marker _ Distilled water as negative control _polypeptide solution Procedure 1) Label 5 small test tubes 1- 5, as indicated in Table 4. 2) Place 5 drops of the indicated material in each of the labeled test tubes. 3) Place 5 drops of Biuret Reagent into each of the labeled test tubes. 4) Record the color and other observations in Table 4. 5) In the last column of the data table, indicate the presence of proteins with a (+) and the absence of proteins with a (-).  Lab 4 and lab 5 Report Introduction ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… …………………………………………………………………………………………………….. Results Part A: Carbohydrates Detection 1) Table (1): Reducing sugar detection Test Sample Color before Color after (+) or tube reaction reaction (–) number result 1 Distilled water (- ve) control 2 Glucose solution (+ve) control 3 Sucrose solution 4 Starch solution 5 Fructose solution 2) Table (2): Starch detection. Test Sample Color before Color after (+) or tube reaction reaction (–) number result 1 Distilled water (- ve) control 2 starch solution (+ve) control 3 glucose solution 4 Rice water solution 5 potato juice Part B: Table (3) lipid detection symbol Sample Soluble insoluble W distilled water V Vegetable oil P palm oil O orange juice U unknown Part C: table (4) protein detection Test Sample Color before Color after (+) or (–) tube reaction reaction result number 1 distilled water 2 Egg white as positive control 3 Glucose solution 4 polypeptide solution 5 Milk Discussion: Q1 : complete the table below: Indicator Target Positive Result Color Negative Result Color Macromolecule Benedicts Solution Lugol’s Solution Biuret’s Solution Q2: Does dH2Ocontain any macromolecules? Why did we test dH2O with each of the indicators? ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… Q3: List the positive controls used for each of the tests. What is the purpose of each of these positive controls? ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… Q4: Macromolecule present ……………………………………………………………………… Q5: if a student tested starch by benedict’s reagents and got orange/ brick red color. What are your explanations for this result? ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… Q5: write the chemical equation that represent the reaction between reducing sugar and benedict’s reagent ……………………………………………………………………………………………………… Q7: If you conducted biuret test and the water sample give purple color (note: the water sample is pure), how could you get this result? ……………………………………………………………………………………………………… Q8: suppose your lunch was grilled chicken, fried potato, salad with olive oil and Coca- Cola list what will give a positive result when tested by Benedicts reagent: ………………………………. Biuret solution ……………………………………… Sudan III dye ………………………………………. Iodine solution ……………………………………… The end