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AP BIOLOGY NAME______________________ Sept. 28/29/30, 2010 LAB 2: ORGANIC MOLECULES PURPOSE: To learn how to identify several major types of organic molecules found in living systems. OVERVIEW A cell is a living chemistry laboratory in which most functions take the form of interactions between organic (carbon-containing) molecules. Most organic molecules found in living systems can be classified as carbohydrates, fats, proteins, or nucleotides. Each of these classes of molecules has specific properties that can be identified by simple chemical tests. In this lab you will be learning how to identify: • reducing sugars using the Benedict’s test (one major group of monosaccharides and disaccharides) • polysaccharides using Lugol’s iodine • lipids using the Sudan IV test • proteins using the Biuret reagent • amino acids using Ninhydrin EXERCISE 1: TESTING FOR CARBOHYDRATES The basic structural unit of carbohydrates is the monosaccharide (or single sugar). Monosachharides are classified by the number of carbons they contain: for example, trioses have three carbons, pentoses have five carbons, and hexoses have six carbons. Glucose Fructose Figure 1. Glucose, an aldose sugar, and fructose, a ketose sugar. Both are hexoses. Monosaccharides are also characterized by the presence of a carbonyl group, either a terminal aldehyde group or an internal ketone group. Both of these groups contain a double bonded oxygen atom that will react with Benedict’s reagent to form a colored precipitate. When two monosaccharides are joined together (via dehydration synthesis), they form a disaccharide. If the reactive aldehyde or ketone groups are involved in the bond between the monosaccharide units, the disaccharide will not react with Benedict’s reagent. Sucrose is an example of such a disaccharide. If only one carbonyl group is involved in the bond, the other is free to react with the reagent. Maltose is such a disaccharide. Sugars with free aldehyde or ketone groups, whether monosaccharides or disaccharides, are called reducing sugars. These sugars are oxidized (lose electrons) by the Cu 2+ in the Benedict’s reagent and the Benedict’s reagent is reduced (gains the electrons lost by the sugar). Hence the name reducing sugar! Figure 2. Two disaccharides: maltose and sucrose. Monosaccharides may join together via dehydration synthesis to form long chains, known as polysaccharides. These chains may be either straight or branched. Starch is an example of a polysaccharide formed entirely of glucose units. Starch does not show a reaction with Benedict’s reagent because the number of free aldehyde groups (found only at the end of each chain) is small in proportion to the rest of the molecule. Many polysaccharides will react with Lugol’s reagent (iodine/potassium iodide, I2KI). Lugol’s reagent changes from a brownish or yellowish color to blue-black when starch is present, but there is no color change in the presence of monosaccharides or disaccharides In this section the lab, you will be using Benedict’s reagent to test for the presence of reducing sugars and Lugol’s iodine to test for the presence of starch. PROCEDURE: PART A: BENEDICT’S TEST FOR REDUCING SUGARS 1. Set up a row of nine test tubes. Use a marker to number the tubes 1 through 9. 2. Add 2 ml of the solutions listed in Table 1, matching the number of the solution to the number on the tube. 3. Add one dropperful (approximately 2 ml) of Benedict’s reagent to each tube. 4. Mix the reagent and the sample by agitating the solution in each tube from side to side. Record the original color of each tube’s contents in Table 1 in the appropriate column. 5. Heat the test tubes in boiling water for 3 minutes. Record the color of each tube at the end of three minutes in Table 1. PART B: LUGOL’S TEST FOR STARCH 6. Place five microscope slides on top of a white piece of paper. Divide each down the middle with a line drawn with a marker. Number each half, from 1 to 9. 7. Place a drop or two of each of the solutions listed in Table 1 on the slides, matching the number of the solution with the number on the space on the slide. 8. Record the original color of each solution in Table 1 in the appropriate column. 9. Add one or two drops of Lugol’s reagent to sample of solution. Mix using toothpicks. Use a clean toothpick for each numbered sample. 10. Record the color of each sample after mixing in Table 1. Table 1. Results for Benedict’s test for reducing sugars and Lugol’s test for starch Tube # and Benedict’s Test Lugol’s Test Contents Original color Final color after Original color Final color after before boiling boiling before adding adding I2KI I2KI 1. Water 2. Starch 3. Glucose 4. Maltose 5. Sucrose 6. Onion Juice 7. Potato Slice 8. Milk 9. Corn Syrup EXERCISE 2: TESTING FOR LIPIDS The word lipid refers to any member of a rather heterogeneous group of organic molecules that are soluble in nonpolar solvents such as chloroform (CHCl3) but insoluble in water. Although lipids include fats, steroids, and phospholipids, this lab will focus primarily on fats. Triglycerides, a popular topic in the discussion of diet and nutrition, are the most common form of fat. They consist of three fatty acid molecules attached to a molecule of glycerol. Triglycerides are found predominantly in adipose tissue and store more energy per gram than any other type of compound found in living tissue. Triglycerides store approximately 9 calories per gram, carbohydrates and proteins both store approximately 4 calories per gram. At room temperature, some lipids are solid (generally those that are found in animals) and are referred to as fats, or more properly as saturated fats. Other triglycerides are liquid at room temperature (generally those found in plants) and are referred to as oils, or more properly as unsaturated fats. Vegetable oil, a liquid fat, is a mixture of triglycerides. Since both liquid and solid fats are nonpolar, you will test for their presence by using Sudan IV, a nonpolar dye that dissolves in nonpolar substances such as fats and oils, but not in polar substances such as water. Figure 3. A tryglyceride, an example of a lipid. Procedure 1. The familiar “grease spot” is the basis of a very simple test for fats. On a piece of unglazed paper, such as brown paper lunch or grocery store bags, place one drop of oil and one drop of water. Allow the drops to dry and observe the difference between the spots where the drops were applied. 2. Label six test tubes in sequence, 1 through 6. Add 1 ml of each substance listed in table 2 to the appropriate tube. 3. Add 3 drops of Sudan IV to each tube and mix. 4. Finally add 2 ml of water to eachg tube. 5. If fats or oils are present in the sample, these will appear as floating red droplets or a floating red layer colored by the Sudan IV. Record your observations in Table 2. Table 2. Results for Sudan IV test for lipids. Tube number and Sudan IV Solubility Reaction Substance 1. Distilled Water 2. Vegetable Oil 3. Onion Juice 4. Hamburger Juice 5. Heavy Cream 6. 1% Milk EXERCISE 3: TESTING FOR PROTEINS AND AMINO ACIDS Proteins are made up of one or more polypeptides, which are linear polymers of smaller molecules called amino acids. Amino acids derive their name from the amino group and the carboxyl group (acidic) that each possesses. Polypeptides are formed when amino acids are joined together by peptide bonds between the amino group of one amino acid and the carboxyl group of another amino acid. Figure 4. The amino acids leucine and proline Figure 5. A peptide bond between 2 amino acids The Biuret reagent reacts with the peptide bonds and therefore reacts with proteins, such as egg albumin, but not with free amino acids, such as glycine and alanine. The Biuret reagent is light blue, but in the presence of polypeptides and proteins it turns violet. Other types of molecules may cause other color changes, but only the violet color indicates the presence of polypeptides. On the other hand, the reagent ninhydrin reacts with the amino group of free amino acids, but not with polypeptides. Ninhydrin turns purple or violet in the presence of the free amino groups in amino acids. In the presence of the amino acid proline, however, it turns yellow. Proline reacts differently because its amino group is not free but is, instead, part of the ring structure of the molecule. PROCEDURE PART A: TESTING FOR PROTEINS WITH THE BIURET REAGENT 1. Obtain seven clean test tubes and number them 1 through 7. 2. Add 2 ml of the solutions listed in Table 3 to the test tubes, matching the number of the substance to the number on the tube. 3. Add 2 ml (one dropperful) of Biuret reagent to each tube. 4. After an incubation period of 2 minutes, record your results in Table 3 and determine which of the samples contain protein. Base you conclusions only on the presence or absence of the violet color. Table 3. Results for Biuret test for the presence of proteins Tube Number and Color with Biuret reagent after 2 minutes Protein Present (+) Contents or Absent (- ) 1. Distilled Water 2. Egg Albumin 3. Potato Starch 4. Glucose 5. Amino Acid 6. Hamburger Juice 7. Chicken Broth PART B: TESTING FOR AMINO ACIDS WITH NINHYDRIN 1. Write the numbers 1 through 6 around the perimeter of a circular piece of filter paper. Put your name and your lab partner’s name on the filter paper. 2. Apply one drop of each of the substances listed in Table 4 in the appropriate spot on the filter paper. 3. Let the filter paper dry. You may speed up the drying by using a hairdryer. 4. Give your filter paper to your instructor, who will spray the paper with ninhydrin under the fume hood. 5. Leave overnight in the fume hood to dry. 6. When you come to class tomorrow, observe your filter paper and record your observations in Table 4. If the substance reacts with ninhydrin, a pink or purple spot will be seen on the filter paper. Table 4. Results for Ninhydrin test for amino acids. Substance Reaction with ninhydrin (+ or – and color) 1. Distilled Water 2. Egg Albumin 3. Potato Starch 4. Glucose 5. Alanine 6. Glycine EXERCISE 4: TESTING UNKNOWN FOOD SUBSTANCES In this exercise you will be determining the presence of carbohydrates, lipids and proteins in in several unidentified food substance. Once you have determined which types of organic molecules are present in each you will hypothesize as to the true identity of the substance! PROCEDURE: 1. Obtain a sample of each of the unknown substances. 2. Perform the following tests on each sample: Benedict’s, Lugol’s, Sudan IV, and Biuret. Use the procedures given earlier in this handout for each of the tests. 3. Record the results of the tests in Table 5. 4. Based on your results, hypothesize as to the true identity of each food substance. Write your analysis in the space provided under Table 5. Table 5. Analysis of several unknown food substances. Substance # 1 Substance # 2 Substance # 3 Test Results Results Results Benedict’s Lugol’s Sudan IV Biuret Unknown # 1: Contains the following types of organic molecules: ______________________________________________________ Possible identity of Unknown #1: ______________________________________________________ Unknown # 2: Contains the following types of organic molecules: ______________________________________________________ Possible identity of Unknown #1: ______________________________________________________ Unknown # 3: Contains the following types of organic molecules: ______________________________________________________ Possible identity of Unknown #1: DISCUSSION QUESTIONS 1. Explain the limitations of the Benedict’s test in determining whether or not sugar is present in a certain food product. Why do all monosaccharides, but only some disaccharides, react with Benedict’s reagent? 2. What did you learn about the specificity of the Biuret reagent? When would you use ninhydrin instead of the biuret reagent? Give an example 3. The leaves of many plants are coated with a waxy substance that causes them to shed water. Would you expect this substance to “react” with Sudan IV? Explain. 4. Ninhydrin reacts with a mixture of amino acids and turns purple. Could proline be one of the amino acids? Describe a way that you could conclusively determine if proline was present in the mixture? 5. A sample of food product X has a positive test for both Benedict’s and Lugol’s reagents. For what group of nutrients would this food be a good source? Why is this specific group of nutrients needed by living organisms? 6. Nutritionists recommend that some fats be present in a person’s daily diet. Why? What nutrients (biological molecules) does a person obtain from eating fish? Why are these nutrients an important part of a healthy daily diet? 7. Why are vitamins a necessary part of a healthy diet? Give the names of two vitamins and describe what their function is in living systems. 8. Vitamins fall into two general categories: water soluble and fat soluble. Which one of these groups should not be taken in excess? Give an example of one of these vitamins and the dangers associated with taking it in excess. 9. Why are some amino acids called essential amino acids? Give the names of these amino acids. 10. Fatty acids with more than one double bond are considered essential fatty acids. Are these fatty acids considered saturated or unsaturated? Explain why. What are the sources of essential fatty acids? 11. In winter, plants exchange the saturated lipids in their cell membranes for unsaturated lipids. Unsaturated lipids are bent and keep the membrane more fluid because they cannot be stacked closely together. What advantage would this exchange have for herbaceous plants that live through the winter? (Sort of a hint: what happens to bacon grease or the layer of fat on top of soup when you put it in the refrigerator?) 12. Do some online research into what nutritionists recommend for daily calorie and food group intake for a person your age. Create a one-day menu for three meals (breakfast, lunch and dinner) that would meet these requirements. You may also use the food pyramid article given to you in class.
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