Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Obtain enough regular margarine (with fat) or corn oil for each student group’s initial test and a small amount (10 mL) of a clear liquid salad dressing (not fat free) or corn oil for the unknown.
2. Add 100 mL of distilled water to the albumin bottle. Cap and shake to mix.
3. The dichloroindophenol solution and the Vitamin C solution should be prepared no more than one day in advance and refrigerated until use. Prepare the 0.025% dichloroindophenol solution by dissolving the contents of the bottle enclosed in the kit in about 500 mL of distilled or deionized water in a one-liter volumetric flask. Fill the volumetric flask up to the one-liter mark with distilled or deionized water. Cap and invert to mix.
   
   Prepare a 100 mg/100 mL (0.1%) vitamin C solution by first crushing the two tablets (1000 mg) with a mortar and pestle. Fill a one-liter volumetric flask with about 500 mL of distilled or deionized water and transfer the crushed tablets to the flask. Fill the volumetric flask up to the one-liter mark with water. Mix thoroughly on a magnetic stirrer. Do not heat. There will be a small amount of undissolved binder material; this is not the ascorbic acid.
4. Prepare a 1% starch solution by wetting 10 grams of soluble starch with a few milliliters of distilled or deionized water. Add the starch slurry to approximately one liter of boiling water. Stir and continue heating until the starch is dissolved. Allow the solution to cool. The shelf life of a starch solution is very poor. Do not store for more than two weeks.
5. Prepare the stomach content solutions of the four murdered individuals following these recipes:
   {13930_Preparation_Table_1}

Safety Precautions

Food items brought into a lab are considered chemicals and, as such, should not be ingested. Benedict’s solution contains cupric sulfate and is moderately toxic and a body tissue irritant. Iodine solutions are irritating to eyes, irritating and mildly corrosive to skin and moderately toxic by ingestion. Biuret solution contains copper sulfate in sodium hydroxide solution. It is corrosive to all body tissue, especially eyes. Sudan III is a biological stain and will stain skin and clothing. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water after completion of laboratory work. Please consult current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Protein test solution should be neutralized using dilute hydrochloric acid solution and rinsed down the drain with excess water according to Flinn Suggested Disposal Method #10. All other test solutions can be disposed of according to Flinn Suggested Disposal Method #26b.

Teacher Tips

• Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. The laboratory activity will likely require two 50-minute class periods to complete.
• It is important to establish the appearance of positive tests for each test (Parts 1–5) before testing the unknowns (Part 6).
• It is important for students to realize that tests like those in this simulation are not “proof” of criminal activity but that it is one more piece of data pointing officials toward guilty or innocent parties.
• Background material is provided for each test procedure. This Background information can be used as appropriate for your course goals and student population.
• In Part 5, step 5, stopper the test tube with a cork or rubber stopper or a piece of Parafilm.

Further Extensions

Part 1. Vitamin C
Try other juices, vitamin supplements or make solutions with vitamin C rich fruits or vegetables (like limes). Test other foods for the presence of vitamin C. Do any foods have vitamin C that you wouldn’t guess have vitamin C?

Part 2. Reducing Sugars
Test for reducing sugars in foods. An example of how to prepare a food sample follows:
Place an apple slice in a mortar along with a few milliliters of distilled water. Crush the apple with a pestle and add water until a mashed apple solution is obtained. Follow the procedures for the Benedict’s test, substituting 8 drops of the apple solution for the dextrose solution.

Part 3. Starches
Test other foodstuffs for starch using the iodine test. Iodine can be placed directly on the food for the test.

Part 4. Proteins
Test other protein-containing foods like gelatin, beans, cheese and milk. This is done by making homogenized solutions and substituting them for the albumin solution in the test procedure.

Part 5. Fats
Test other food stuffs for fat. Sometimes the nature of the food stuff clouds the Sudan III test. Below is an alternative procedure that can also be used to test for fat. It utilizes salad dressing.

Testing salad dressing for fat 

1. Add 12 mL of regular salad dressing and 12 mL of fat-free salad dressing to separate 100-mL beakers.
2. Add 8 mL of hexanes to each beaker and stir the contents of each beaker for one minute with the stirring rods.
3. Carefully decant the hexanes solvent (top layer) using a pipet, from the beakers into separate evaporating dishes. Do not pour any of the salad dressing into the evaporating dishes.
4. Set the evaporating dishes in an operating fume hood or other well-ventilated area so that the hexanes solvent may evaporate quickly and safely. (This should take approximately 20–30 minutes.)
5. Once the hexanes have completely evaporated, use the spatula to sprinkle a few grains of Sudan III onto the residue left on each Petri dish.
6. Streak the Sudan III solid across the dish using a stirring rod or spatula. The Sudan III in the Petri dish from the regular dressing will dissolve in the fatty residue left, and red streaks will appear. The solid’s appearance will not change when streaked across the dish containing the residue from the fat-free dressing.

Sample Data

1. What food type(s) do all the dead individual have in their stomach?

   All of the dead individuals had sugar in their stomach.

2. What Ross Catering employee(s) should be the first to be interrogated by the police? Why?

   Jim Lockpicker should be interrogated since he apparently prepared the candy dishes which were loaded with sugar. Others could also be interrogated like Charles Lightfingers since many fruits also contain sugars.

Discussion

Part 1. Vitamin C
The reaction for the vitamin C test can be summarized as follows:

Using the vitamin C solution as a standard, the amount of vitamin C in fruit juices can be calculated. If it takes 29 drops of vitamin C solution and 77 drops of orange juice to neutralize 10 mL of dichloroindophenol solution, the calculations are as follows:

(Drops standard)(Concentration standard) = (Drops unknown)(Concentration unknown)
(29 drops) (100 mg/100 mL of vitamin C) = (77 drops) (n mg/100 mL of vitamin C)
n = 38 mg

Therefore, the vitamin C concentration of the orange juice is 38 mg/100 mL.

Listed are a few juices and their ranges of vitamin C content. Vitamin C is nutritionally important. Its exact functions in the body are poorly understood. It is known to be necessary for the production of the protein collagen, which is a vital part of various connective tissues such as bone and cartilage. A deficiency in vitamin C can result in a disease known as scurvy, the symptoms of which are bleeding, spongy gums and a tendency to bruise easily. You may have heard of British soldiers historically referred to as “limeys.” The name limey was given to the sailors because during long voyages they would eat limes to prevent scurvy.

Since the body requires vitamin C on a continuing regular basis, it should be part of the daily diet. High concentrations can be found in citrus fruits, tomatoes and cabbage. Potatoes, leafy green vegetables and fresh fruits are also good sources. Nutritionists generally agree that a daily adult intake of 60–70 mg is enough to replenish normal losses and to provide a satisfactory level for cellular needs.

Part 2. Reducing Sugars
Carbohydrates are the most abundant class of organic compounds found in plants. The basic building blocks of all carbohydrates are simple sugars, such as glucose (blood sugar) and fructose (fruit sugar), and are called monosaccharides. Other more complex carbohydrates are the addition products of two, three or even thousands of simple sugars. These compounds are called disaccharides, trisaccharides, and polysaccharides, respectively. Examples of disaccharides include sucrose (table sugar), lactose (milk sugar) and maltose (malt sugar). The most common polysaccharide is starch, which is composed of thousands of glucose units joined together.

Simple carbohydrates can be identified using Benedict’s test. The Benedict’s test reagent consists of copper(II) complex ions in aqueous solution. All monosaccharides and most disaccharides are called reducing sugars because they are capable of reducing the copper(II) ions in Benedict’s solution. (Sucrose is the exception to this rule. Although sucrose is a disaccharide, it is a nonreducing sugar.) Polysaccharides do not react with Benedict’s solution and are also called nonreducing sugars. The presence of reducing sugars in foods is observed by a positive Benedict’s test result—the blue color due to Cu(II) complex ions disappears and a red precipitate consisting of reduced Cu(I) oxide (Cu₂O) appears. Depending on the amount of reducing sugars present in foods, the final color of a positive Benedict’s test result may range from green to yellow to red. A negative Benedict’s test result will show no change—the solution remains bright blue and no precipitate is observed. The overall reaction for a positive Benedict’s test is represented in Equation 1 using glucose as an example. Part 3. Starches
The most common polysaccharides are starch and cellulose. Starch serves as an energy storage molecule in plants, where it typically clumps into visible grains. The most familiar sources of dietary starch are potatoes, beans (legumes) and cereal grains (corn, wheat, barley). Reaction with iodine (the iodine test) is used to identify the presence of starch in foods. A positive iodine test result is observed by the appearance of a dark blue color due to the formation of a starch–iodine absorption complex. Cellulose serves a structural role in plants (cell walls) and is not digested in the human stomach.

Part 4. Proteins
Proteins are large natural polymers composed of amino acids joined together in chain-like fashion via peptide linkages. The so-called polypeptide chains that make up all proteins can fold up on themselves to form spherically shaped globular proteins such as enzymes or they can aggregate to form protein fibers such as collagen. Examples of food proteins include albumin (egg white) and casein (milk). Gelatin is a mixture of proteins obtained by hydrolysis of collagen in animal skin, ligaments and tendons.

The presence of proteins in foods can be identified using the biuret test. Biuret solution contains copper sulfate dissolved in very strong base. The dissolved copper(II) ions coordinate with nitrogen and oxygen atoms in two or more adjacent peptide linkages in a protein molecule to form purple-colored complex ions. A positive biuret test result is marked by the appearance of a lavender or purple color. The intensity of the purple color depends on the nature of the protein and on how much protein is present. The overall reaction for a positive biuret test is summarized in Equation 2. Part 5. Fats
Fats and oils are members of a biological class of compounds called lipids. Lipids are classified based on a simple physical property—they are insoluble in water. This property of lipids makes them different from carbohydrates and proteins, both of which readily dissolve in water. Fats and oils and other lipids dissolve in nonpolar organic solvents such as hexane, ether, and toluene and are usually obtained from plant and animal tissues by extraction with an organic solvent. The Sudan III test is a classic test for identifying lipids. Sudan III is a special dye that is attracted to and dissolves in nonpolar compounds. It does not dissolve in water or other polar solvents. Sudan III is used as a “fat stain” to identify lipids in foods, seeds and animal tissues.

Introduction

Mr. and Mrs. Henderson are well known and respected in their community. Mr. Henderson is a criminal attorney and has been responsible for the conviction of many unusual individuals over his 31-year career. Last Saturday the Hendersons hosted a large precharity ball party at their home. The party had barely started (only the first few guests had arrived) when police were summoned to the Henderson home. Four people were found dead on the floor near the hors d’oeuvre table. When the crime scene investigator arrived at the Henderson home, the food had already been removed from the premises and the party had been cancelled. The initial suspicion was that the individuals had been poisoned from the hors d’oeuvres. During the ensuing autopsies, stomach content samples were removed from each of the dead individuals and the samples were sent to the crime lab for testing.

The Ross Catering Co. that supplied the food was known to hire former convicts to work in their kitchens. Each person in the hors d’oeuvres department is a specialist and known for specific hors d’oeuvres. As various trays and platters are completed and ready for delivery, the preparer signs off on the job. Thus, for each item on the food table there is a record of who prepared the food. The documents showed that the following individuals signed off on the various hors d’oeuvres. (Coincidentally, all of the individuals were former convictions of Mr. Henderson and all had reason to seek revenge.)

Fruit Platters (Vitamin C)—Charles Lightfingers
Candy Arrangements (Sugar)—Jim Lockpicker
Pasta Platters (Starches)—Sam Cartaker
Fish Platters (Proteins)—Steven Gunit
Chip Dishes (Fats)—Mike Axer

Part 1. Vitamin C Testing
Vitamin C (ascorbic acid) is one of the important substances that contributes to the maintenance of good health. It should be included regularly in a normal diet. Ascorbic acid is water soluble and a reducing agent. In this part of the lab, an indicator solution (dichloroindophenol) will be used to test for vitamin C. The blue indicator solution will become colorless in the presence of vitamin C and remains blue in the absence of vitamin C.

Part 2. Reducing Sugars
Benedict’s qualitative solution is a test reagent that reacts positively with reducing sugars. All monosaccharides and most disaccharides are reducing sugars—that is, they possess a free, or potentially free carbonyl group (C=O) that reacts to reduce Benedict’s solution. A positive Benedict’s test can be seen by the formation of a brownish–red cuprous oxide precipitate when heated. A negative test is shown if the Benedict’s solution remains blue when heated.

Part 3. Starches
Starch is a complex carbohydrate and a primary source of energy in the food we eat. Iodine is a good indicator for starch because it forms an adsorption complex with a characteristic royal blue or purple color when starch is present. The iodine remains a yellow/orange color when starch is not present.

Part 4. Proteins
Biuret solution can be used to determine the presence of protein. In this experiment, biuret is used to test for protein in albumin. Biuret solution will turn to a pinkish or purplish color if protein is present.

Part 5. Fats
Sudan III can be used to test various foods for the presence of fat. In the presence of fat, Sudan III will stain fats and oils pink from their original yellow/white color. Sudan III will not dissolve or disperse in the absence of a fat.

Concepts

• Chemical food tests
• Test standards
• Testing unknowns

Experiment Overview

This forensic simulation consists of two basic parts: (1) Standard tests for various food types are conducted to learn the nature of positive and negative tests for sugars, fats, proteins, vitamin C and starches; (2) The stomach contents of the four dead individuals are tested for the presence of the food types and the test results are added to the police evidence files.

Materials

Safety Precautions

Although vitamin C and 2,6-dichloroindophenol are not considered hazardous, wash hands thoroughly after handling. Food items, once brought into a lab, are considered chemicals and, as such, should not be ingested. Benedict’s solution contains cupric sulfate and is moderately toxic and a body tissue irritant. Use insulated gloves or test tube clamps when handling the heated test tubes during the Benedict’s test procedure. Iodine solutions are irritating to eyes, irritating and mildly corrosive to skin and moderately toxic by ingestion. Biuret solution contains copper sulfate in a sodium hydroxide solution. It is corrosive to all body tissue, especially eyes. It is also moderately toxic by ingestion. Sudan III is a biological stain and will stain skin and clothing. Any food items brought into a laboratory setting are considered chemicals and should not be ingested thereafter. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Part 1. Vitamin C Testing

1. Using a 10-mL graduated cylinder, measure out 10 mL of 0.025% dichloroindophenol solution. Transfer to one of the test tubes.
2. Using a Beral-type pipet, add the vitamin C solution drop by drop to the test tube, counting each drop added until the color changes from blue to the colorless/very light amber endpoint. Be sure to stir or swirl the solution after each drop is added. Record the number of drops required to turn the indicator nearly colorless. Repeat the procedure again to obtain an average value.
3. Consult your instructor for appropriate disposal procedures.

Part 2. Reducing Sugars

1. Add 5 mL of Benedict’s solution and 8 drops of the dextrose solution to a test tube.
2. Heat a beaker of water to boiling or near boiling.
3. Place the test tube in the boiling water bath. Note any changes after three or four minutes.
4. A positive Benedict’s test can be seen by the formation of a brownish-red cuprous oxide precipitate.
5. Consult your instructor for appropriate disposal procedures.

Part 3. Starches

1. Place 2–3 mL of starch solution into a test tube.
2. Test the starch solution sample provided by your teacher by adding one or two drops of a dilute iodine solution. Note the color change.
3. Consult your instructor for appropriate disposal procedures.

Part 4. Proteins

1. Using the graduated cylinder, measure out 5 mL of the albumin solution into a test tube. Add 2.5 mL of the biuret solution.
2. Wait 10–15 minutes for color (pink to purplish) to develop.
3. Consult your instructor for appropriate disposal procedures.

Part 5. Fats

1. Add a small dab of regular margarine to an evaporating dish. Carefully melt the margarine sample on a hot plate at a very low temperature setting. If corn oil is used, go to step 3.
2. While the margarine is melting, prepare a test tube by adding 5 mL of distilled water and a few grains or particles of Sudan III to a test tube.
3. Using a Beral pipet, add 6–7 drops of the melted margarine or corn oil. The melted margarine or corn oil will form a layer on top of the water in the test tube.
4. Stopper the test tube and shake the tube vigorously, holding the stopper with your thumb.
5. The material in the test tube containing the margarine or corn oil should turn a distinct pink color indicating the presence of fat in those areas.
6. Consult your instructor for appropriate disposal procedures.

Part 6 

1. Test the stomach content samples from the four dead victims as directed by your instructor. Test the sample(s) for all five food types: Vitamin C, Starch, Sugar, Fat and Protein.
2. Record results on the Crime Lab Report Worksheet.
3. Collect or share results for all four dead individuals and answer the questions on the Crime Lab Report Worksheet.

Student Worksheet PDF

13930_Student1.pdf