Hillsborough Community College - Ybor City Campus 1025C Laboratory Exercise 2: Indicator Tests for Important Nutrients Introduction

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1 What are Indicators? Hillsborough Community College - Ybor City Campus 1025C Laboratory Exercise 2: Indicator Tests for Important Nutrients Introduction Indicators are chemicals that help identify the composition of an unknown material. In food analysis, indicators are used to detect the presence of specific organic or inorganic compounds such as sugars, proteins, starch, lipids, or salts. Indicators can be used to perform qualitative analyses which are designed to determine the type (quality) of an unknown substance being tested. Indicators can also be used, in conjunction with appropriate techniques, to perform quantitative analyses which are designed to determine the amount (quantity) of unknown substance being tested. In this exercise we will do only qualitative tests. Qualitative Analyses Benedict s test for simple sugars: Benedict's reagent contains blue copper (II) sulfate (CuSO4) which is reduced to red copper (I) oxide (Cu2O). The copper oxide is insoluble in water and precipitates out of solution. The color of the final solution may appear green to brick red depending on how many of the copper (II) ions are present. Lugol s Iodine (IKI) test for the presence of starch: Iodine dissolved in an aqueous solution of potassium iodide - reacts with starch producing a deep blue-black 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, causes the dark blue/black color. The amylopectin, or branched portion of starch, causes the formation of an orange/yellow hue. When starch is broken down or hydrolyzed into smaller carbohydrate units, the blue-black color is not produced. The iodine solution will also react with glycogen and cellulose, although the color produced is more brown and much less intense. The Sudan IV test for the presence of lipids: Sudan IV (C24H20N4O) is a red, fat-soluble dye used for staining lipids, triglycerides and lipoproteins. Staining is an important chemical technique, offering the ability to visually qualify the presence of the fatty compound of interest without isolating it. The Biuret test for the presence of proteins: Biuret reagent, made of sodium hydroxide and copper (II) sulfate, is used for determining the presence of protein in a sample. The test relies on the reaction between copper ions and peptide bonds in an alkaline solution. A violet color indicates the presence of proteins. Proteins give a strong Biuret reaction because they contain a large number of peptide bonds. The blue reagent changes to pink when combined with short-chain polypeptides which contain smaller numbers of peptide bonds. The silver nitrate test for salt (sodium chloride NaCl): Silver nitrate is a chemical compound with chemical formula AgNO3. It is one of the light-sensitive ingredients in photographic film and is a corrosive compound. Soluble silver salts tend to be very toxic to bacteria and other life forms. To detect the presence of chloride (Cl ) in water (from NaCl or any other source), silver nitrate is added. White silver chloride crystals will form if chloride is present: Ag+(aq) + Cl (aq) AgCl(s) Silver chloride crystals become visible because they are insoluble in water. 12

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3 Name: Hillsborough Community College - Ybor City Campus 1025C Laboratory Exercise 2: Indicator Tests for Important Nutrients In analytical chemistry, indicators are chemicals (reagents) that help identify the composition of an unknown material. In food analysis, indicators are used to detect the presence of specific organic or inorganic compounds such as sugars, proteins, starches, lipids, or salts. Part one of this experiment is designed to familiarize you with the names, procedures and characteristic responses produced by important indicator solutions used in laboratory analysis of unknown substances in foods. These tests are designed to be qualitative not quantitative measures. In other words, they will reveal the types of substances that are present in a material, but not the quantity of the substance present. Part two of this experiment will allow you to apply information acquired from part one to test for the presence of specific types of molecules in an unknown mixture of nutrients. Part I Procedure Work in teams of two. ATTENTION: The chemicals used in Test #1 (Benedict s solution) may harm your skin, clothing, or the environment. In addition to your GOGGLES, please wear GLOVES to do this procedure, and place waste chemicals into the designated DISPOSAL BEAKERS in the fume hood. Put your gloves into the trash can when you have completed the test. TEST #1: The Benedict s test for the presence of simple sugars, such as glucose: Place 4 ml of water into tube #1 and add 2 ml of Benedict s solution. Place 4 ml of glucose solution in tube #2 and add 2 ml of Benedict s solution. Rinse the graduated cylinder well before measuring solutions for the next test. Heat both test tubes in a hot water bath for several minutes. Observe the characteristic change in appearance of the test solution containing glucose and record your observations in the space provided in Table 1. The glucose solution provides an example of the positive response to the Benedict s test. TEST #2: The Lugol s Iodine (IKI) test for the presence of starch: Place 2 ml of water into tube #1 and add 10 drops of IKI. Place 2 ml of starch solution into tube #2 and add 10 drops of IKI. Rinse the graduated cylinder well before measuring solutions for the next test. Observe the characteristic change in appearance of the test solution and record your observations in the space provided in Table 1. The starch solution provides an example of the positive response to the Lugol s Iodine test. TEST #3: The Sudan IV test for the presence of lipids: Obtain a small piece of filter paper and a Petri dish or watch glass. Place a drop of water on the piece of filter paper and allow it to dry. Place a drop of oil (lipid) at a distance from the water spot on the filter paper and allow it to dry. Submerge the filter paper for one minute in a Petri dish that contains just enough Sudan IV solution to cover the paper. Hold the paper above the Petri dish with forceps and rinse away the excess Sudan IV with water. Observe the characteristic appearance of the lipid response and record your observations in the space provided in Table 1. 14

4 ATTENTION: The chemicals used in Test #4 and Test #5 (Biuret reagent and silver nitrate solution) may harm your skin, clothing, or the environment. In addition to your GOGGLES, please wear GLOVES to do this procedure, and place waste chemicals into the designated DISPOSAL BEAKERS in the fume hood. Put your gloves into the trash can when you have completed the tests. TEST #4: The Biuret test for the presence of proteins: Place 4 ml of water into tube #1 and add 10 drops of Biuret solution. Place 4 ml of protein solution (gelatin) into tube #2 and add 10 drops of Biuret solution. Rinse the graduated cylinder well before measuring solutions for the next test. Observe the characteristic change in appearance of the test solution and record your observations in the space provided in Table 1. The gelatin solution provides an example of the positive response to the Biuret test. TEST #5: The silver nitrate test for salt (sodium chloride NaCl): Place 4 ml of water into tube #1 and add 10 drops of silver nitrate solution. Place 4 ml of salt solution into tube #2 and add 10 drops of silver nitrate. Observe the characteristic change in appearance of the test solution and record your observations in Table 1. The salt solution provides an example of the positive response to the silver nitrate test. BENEDICTS NAME OF TEST TABLE 1: PRACTICE TESTS WITH KNOWN SUBSTANCES OBSERVATIONS OF REAGENT + WATER (- response) OBSERVATIONS OF REAGENT + KNOWN (+ response) LUGOL S IODINE (IKI) SUDAN IV BIURET SILVER NITRATE PART II Select an unknown solution and test its contents using each of the tests above. It is NOT necessary to use a control tube of water for these tests as you did in Part I. Record your results in Table 2. Be sure to record the ID code of your unknown mixture. TABLE 2: OBSERVATIONS OF TESTING AN UNKNOWN FOOD MATERIAL BENEDICT S LUGOL S SUDAN IV BIURET SILVER NITRATE UNKNOWN I.D. CODE (LETTER OR NUMBER ON BOTTLE) Based on my results in Table 2, substances present in my unknown may include: Hillsborough Community College - Ybor City Campus 15

5 1025C Laboratory Exercise 2: Indicator Tests for Important Nutrients Review Questions 1. When would a qualitative test for a food substance be a useful tool? When would a quantitative test be useful? 2. Benedict's test for glucose uses a change in copper from a blue form (Cu II) to a red form (Cu I) to indicate the presence of glucose (or other reducing sugars). What is the purpose of heating this test solution? 3. Lugol's iodine reagent (IKI) is useful to distinguish starch and glycogen from other polysaccharides. Lugol's iodine yields a blue-black color in the presence of starch. Glycogen reacts with Lugol's reagent to give a brown-blue color. Other polysaccharides and monosaccharides yield no color change; the test solution remains the characteristic brown-yellow of the iodine reagent. Why is it important to use a control (the tube with water) in these tests? 16

6 4. Biuret's reagent uses copper like Benedict's reagent for reducing sugars. Biuret's reagent, however reacts with peptide bonds of proteins and polypeptides to change the characteristic blue (Cu II) color to a purple to violet color depending on the number of peptide bonds available. Would you expect this test to develop a purple or a violet color in the presence of proteins? Why? 5. The silver nitrate test for table salt (NaCl) is actually a test for the chloride ion (Cl-) in solution. A positive result is demonstrated by the formation of a white silver chloride precipitate. Under what circumstances might this test be misleading as a qualitative test for NaCl? 17

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