Materials Included In Kit

Additional Materials Required

Prelab Preparation

Bromine Water Kit
The following solutions are provided for bromine water preparation: 0.5 M sodium bromide, 0.5 M hydrochloric acid and 5% sodium hypochlorite.

Carry out this procedure in an operating fume hood. To prepare 60 mL of 0.5% v/v bromine water: (1) Pour the sodium bromide (0.5 M NaBr, 25 mL) and the hydrochloric acid (0.5 M HCl, 25 mL) solutions into the bottle marked “Bromine Water.” (2) Add the sodium hypochlorite solution (5% NaOCl, 10 mL) to the bottle and swirl gently to mix the reactants. (3) Cap the bottle and store it in a secure location. Do NOT mix the HCl and NaOCl solutions directly in the absence of NaBr.

Safety Precautions

Sodium hypochlorite solution reacts with acids to generate toxic chlorine. In this lab sodium hypochlorite is reacted with hydrochloric acid to generate small amounts of very dilute halogen solutions for use by the students. This step should only be performed by the teacher and in the amounts indicated. Follow the directions carefully and work in an operating fume hood. Bromine water is toxic by inhalation and ingestion and is a skin irritant. Work with bromine water in an operating fume hood only. Avoid breathing the vapor. Do not allow bromine water to come in contact with skin or clothing. Sudan III solution is an alcohol-based solution and is a flammable liquid. Hexane is a a flammable liquid and a dangerous fire risk. Do not allow any flames in the laboratory during this activity. Avoid exposure of all chemicals to eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Please review 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. Lipid and protein samples and Sudan III solution may be disposed of according to Flinn Suggested Disposal Method #26a or 26b, as appropriate. Hexane may be allowed to evaporate in a shallow pan placed in a fume hood, according to Flinn Suggested Disposal Method #18a. The organic waste solutions remaining after the hexane solubility test in Part A may be disposed of according to Flinn Suggested Disposal Method #18a or 18b. Excess bromine water is readily neutralized by reaction with sodium thiosulfate, according to Flinn Suggested Disposal Method #12a. Sodium thiosulfate solution has been provided in this kit to allow for immediate and safe disposal of unreacted bromine water samples in Part B.

Teacher Tips

• Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. The experimental work for this lab can reasonably be completed in one 50-minute lab period. A few minutes of follow-up time are required the day after lab for students to mass the dry peanut residue from Part C.
• Preparation of the bromine water solution requires about 15 minutes of prep time prior to class. This may be done 1–2 days before lab if the resulting solution is stored in a properly capped and labeled bottle in a secure location.
• To improve safety in the lab, set up two waste beakers in central locations for immediate collection of laboratory waste: (1) an “Organic Waste” beaker for the hexane solubility test samples in Part A; and (2) a “Bromine Waste” beaker filled with 100 mL of 50% sodium thiosulfate solution for the bromine test samples in Part B. Both waste beakers should be placed in the hood.
• Parts A, B, and C can be performed in any order. Consider staggering the starting points for different student groups—set up separate stations for Parts A, B, and C and have students rotate among the stations to complete the activity.
• Encourage students to label their pipets to avoid contamination and waste.
• Students sometimes become very indecisive about the solubility observations in Part A. Advise students to “call it as they see it.”

Further Extensions

• The test for unsaturation in Part B can be modified to rank different oils with respect to the relative amounts of saturated versus unsaturated fatty acids. Add bromine water dropwise to a given amount of oil until the red color of bromine persists and does not disappear. Consider this procedure only if there is enough time and sufficient hood space to avoid overcrowding.
• Are all nuts the same? Students might enjoy carrying out the extraction procedure in Part C with different types of nuts to compare how much fat they contain. Almonds, for instance, are a lower calorie alternative to peanuts because they contain less fat. This could be a cooperative class exercise to compare the fat content in peanuts, walnuts, almonds, pecans, etc.
• The extraction procedure in Part C can be extended to study the properties of peanut oil and the residual peanut “meal.” Evaporate the solvent from the hexane/peanut oil extract and examine the behavior of peanut oil in the solubility and unsaturation tests. Dissolve the remaining solid peanut residue in water (most of it dissolves) and test it for protein using the Biuret Test (B0050, Biuret Test Solution) and for carbohydrate using Benedict’s Test (B0171, Benedict’s Qualitative Solution).
• One of the oldest chemical reactions known, dating back thousands of years, is the hydrolysis reaction of fats and oils with strong base to make soaps (fatty acid salts). The reaction is called saponification. An excellent kit is available from Flinn Scientific (Catalog No. AP4856, Soap-Making Kit) to allow students to make soap and study its properties.

Answers to Prelab Questions

1. The C=C double bonds in unsaturated oils react with hydrogen to give “hydrogenated” compounds. What food items can you find on your kitchen or grocery store shelves that have hydrogenated oils listed as a major ingredient? (Hint: The snack food section is a good place to look.) What happens to the consistency of an oil when it is hydrogenated? What are the advantages of this change in consistency in food processing? What are the disadvantages of using hydrogenated oils?

   “Partially hydrogenated vegetable oil” is listed as a main ingredient in cookies, granola bars, crackers, margarine, potato chips, peanut butter, scalloped potato mixes, etc. Hydrogenation converts unsaturated fatty acids to saturated derivatives and thus changes the consistency of an oil to a semi-solid form. The advantage of this in food processing is in improving the texture of food items with a high fat content. The chief disadvantage, of course, it that saturated fats are less healthy than unsaturated ones.

2. The fat content in milk can be estimated by extracting the fat with hexane. A 50.0-g sample of whole milk was mixed with hexane and gently stirred. Two liquid layers were obtained—a lower, “milky” layer and and an upper layer of hexane. The layers were separated and the mass of the lower milk layer was found to be 47.8 g. What is the percentage of fat in whole milk?

   The difference in mass of the milk sample before and after extraction (50.0 – 47.8 g) equals the amount of fat contained in milk. Thus, 50.0 g of milk contains 2.2 g of fat. The percentage of fat in whole milk is calculated as follows:

   (2.2 g fat /50.0 g milk) x 100% = 4.4% fat

Sample Data

Data Table A. Classification and Identification of Lipids

Part B. Test for Unsaturation

1. What is the initial color of bromine water solution? Dark orange-red.
2. What is the initial appearance of samples 1–3 immediately after the addition of bromine water? Each sample consists of two layers, a lower, dark orange layer due to bromine and an upper, pale yellow layer of vegetable oil.
3. Describe the final appearance of test samples 1–3 after three minutes. Sample 1 (coconut oil) did not change in appearance. The bromine color had not changed after three minutes. Samples 2 and 3 (corn oil and olive oil) changed significantly—the bromine color disappeared completely and the lower aqueous layer was left clear and colorless while the top oil layer changed into a cloudy white emulsion.
4. Which seed oils reacted with bromine? Why? Corn oil and olive oil. They contain C=C double bonds.

Data Table C. Extraction of Peanut Oil

Answers to Questions

1. Does the solubility behavior of the test samples in Part A fit the pattern predicted based on the definition of lipids? Explain. Which samples in Part A are lipids?

   All of the lipid samples (coconut oil, corn oil, olive oil and cholesterol) were insoluble in water and soluble in hexane. Thus, lipids dissolve in a nonpolar organic solvent. The only non-lipid sample (albumin) had the opposite pattern—dissolving in water but not in hexane.

2. Compare the effect of Sudan III staining solution on solid and liquid lipids.

   Sudan III stained the liquid oil samples a uniform pink color, while the solid cholesterol sample absorbed the stain to give it a speckled appearance.

3. Sudan III stain can be used to identify fat storage granules in a seed. Discuss how this might be done and what might be observed.

   Cut a cross-section of a seed and add a few drops of Sudan III. Fat granules should appear a darker pink than the other parts of the seed.

4. The following information was obtained from the nutritional labels of various seed oils. Do the results of the bromine test for unsaturation agree with the information provided on the food labels?
   {13379_Answers_Table_6}
   The results of the bromine test agree with the information reported on the nutritional labels. Both corn oil and olive oil contain about 85% unsaturated fat and thus “decolorize” bromine. Coconut oil has less than 10% unsaturation and does not react noticeably with bromine.
5. How could the bromine test be modified to be able to rank different seed oils with respect to the amount of unsaturation in each?

   Add bromine dropwise to oil until the added bromine does not react. Compare the color of the lower aqueous solution against a “blank” that does not react with bromine. Count the number of drops of bromine added until the color due to unreacted bromine persists and is the same as the blank. Rank oils based on the number of drops of bromine added—the more unsaturated the oil, the more bromine required for complete reaction.

6. Using the mass of peanuts before and after extraction of “peanut oil,” calculate the percentage of fat in peanuts.

   Mass of “peanut oil” removed by extraction = 3.02 – 2.22 g = 0.80 g
   Percent fat in peanuts = (0.80 g/3.02 g) x 100% = 27%

7. The nutritional label for peanuts lists the following information. How does your experimental value for the percentage of fat in peanuts (Question 6) compare with that reported on the nutritional label?
   {13379_Answers_Table_7}

   Amount of fat listed on nutritional label for peanuts = (14 g/29 g) x 100% = 48%
   Only about half of the total fat was removed by this extraction procedure.

8. Describe some ways the extraction procedure in Part C could be improved to obtain better agreement between the experimental and known values of the amount of fat in peanuts.

   Increase the volume of solvent used, increase the length of time of the extraction procedure, carry out the extraction at a higher temperature, perform multiple extractions, stir the ground peanuts with the solvent continuously, grind the peanuts to a very fine powder before carrying out the extraction.

Introduction

Fats and oils, waxes and cholesterol, steroid hormones and Vitamins A and D—all of these natural products belong to the diverse class of biological molecules called lipids. What are the properties of lipids? What role do lipids play in the chemistry of life?

Concepts

• Lipids
• Polar and nonpolar compounds
• Extraction
• Triglycerides
• Saturated vs. unsaturated

Background

Biological substances that are insoluble in water are classified as lipids. This characteristic physical property of lipids makes them quite different from other types of biological molecules—carbohydrates, proteins and nucleic acids—that dissolve readily in water. Lipids do dissolve in nonpolar organic solvents, such as hexane, ether and toluene. Lipids are thus usually obtained from plant and animal tissues by extraction with one of these solvents.

The structures of lipids are extremely varied. Members of the lipid class of biological molecules include:

• Fats and oils, such as butter and corn oil, that are familiar to us due to their role in nutrition
• Phospholipids, also known as membrane lipids, that make up the cell membrane structure in all living organisms
• Steroid hormones, such as estrogen and progesterone, that regulate cell activity.

The biological functions of lipids are equally as diverse as their structures. Fats and oils, for example, are used almost universally as stored forms of energy within cells and organisms. In addition to acting as energy sources, fats are stored in adipose tissue that insulates and protects internal organs. Phospholipid molecules are responsible for the “lipid bilayer” structures that form protective membranes around cells and cell structures. The steroid hormones, which are synthesized in the body from cholesterol, act as chemical messengers, carrying signals from one part of the body to another.

Triglycerides
Fats and oils—referred to collectively as triglycerides—have the same basic structure. Triglycerides consist of a glycerol backbone and three attached fatty acid residues. Triglycerides differ in the types of fatty acids attached to the glycerol backbone. Fatty acids are long-chain carboxylic acids, consisting of a long, straight-chain hydrocarbon “tail” (CH₃–CH₂–CH₂–CH₂–) with a carboxyl group (–COOH) at one end. Fatty acids range in length from 10 to 20 carbon atoms and always contain an even number of carbon atoms. The most common number of carbon atoms is 16–18. The hydrocarbon chains in fatty acids can be saturated or unsaturated. Saturated fatty acids contain only C—C single bonds in the hydrocarbon chain, while unsaturated fatty acids contain at least one C=C double bond. The presence of C=C double bonds reduces the number of hydrogen atoms that are attached to the carbon atoms—these fatty acids are “unsaturated” with respect to the number of hydrogen atoms. Fatty acids that contain several double bonds are called polyunsaturated.

Fats are solids, obtained primarily from animal tissue, that contain a large proportion of saturated fatty acids. Oils are liquids, obtained primarily from plants, that contain a greater proportion of unsaturated fatty acids. This key structural difference has important consequences in nutrition, since replacing saturated fats in the diet with polyunsaturated oils may help prevent heart disease. A close look at the nutritional label attached to any food item reveals not only the amount of “fat” in the food, but also the amount of saturated, monounsaturated, and polyunsaturated fats. The role of saturated versus unsaturated fatty acids in nutrition is related to their structures. Unsaturated fatty acids have bends in their structures at the location of the C=C double bonds, and these bends make oils more fluid and less rigid than fats. This structure is also thought to prevent the buildup of solid residues in arteries and veins. Figure 1 shows the structure of a triglyceride containing both saturated and unsaturated fatty acids. Whether a triglyceride is a solid or liquid at room temperature depends not only on the amount of unsaturation in its fatty acid chains, but also on the average number of carbon atoms. Coconut oil, for example, is considered one of the “unhealthy” oils—it contains more than 90% saturated fatty acids. However, because it has a high proportion of relatively short-chain fatty acids (the average chain length is about 12), coconut oil is a liquid at room temperature.

There are two principal methods of separating the so-called seed oils (e.g., corn oil, olive oil, sunflower oil, canola oil) from seeds. Unrefined oils are obtained by “squeezing” seeds under high pressure at elevated temperatures. Refined oils are obtained by extraction—the ground seeds are stirred with hexane or other petroleum solvents, which dissolve the oils. The resulting liquid extracts are then heated to remove the solvent and the oils are subjected to further heat processing to give a clear and uniform product with improved shelf life and stability.

Experiment Overview

Part A—Classification and Identification of Lipids. The basic definition of lipids will be illustrated by examining their solubility in both water and hexane, a nonpolar hydrocarbon solvent. The solubility of lipids will be compared to that of albumin, the main nutritional protein in eggs. The Sudan III test—a classic test for identifying lipids—will also be run. Sudan III is a special dye that is attracted to nonpolar compounds and is thus readily absorbed by lipid droplets. It is used as a “fat stain” in botany and medicine to identify lipids in seeds and tissue samples, respectively.

Part B—Test for Unsaturation. The presence of unsaturation in oils will be detected by mixing the oils with bromine water. Bromine (Br₂) combines with C=C double bonds to form “dibromides,” in which the bromine atoms have added to the two carbon atoms in the double bond. Saturated compounds do not react with bromine under these conditions. Disappearance of the orange-red color due to bromine serves as a positive test for unsaturation. A related chemical reaction is used in the food industry to measure the amount of unsaturated fatty acids in food products.

Part C—Extraction of Peanut Oil. The amount of “peanut oil” in peanuts will be determined by extracting ground peanuts with hexane. By measuring the mass of peanuts before and after solvent extraction, the amount of lipids can be determined and compared with the information provided on the nutritional label for peanuts.

Materials

Prelab Questions

1. The C=C double bonds in unsaturated oils react with hydrogen to give “hydrogenated” compounds. What food items can you find on your kitchen or grocery store shelves that have hydrogenated oils listed as a major ingredient? (Hint: The snack food section is a good place to look.) What happens to the consistency of an oil when it is hydrogenated? What are the advantages of this change in consistency in food processing? What are the disadvantages of using hydrogenated oils?
2. The fat content in milk can be measured by extracting the fat with hexane. A 50.0-g sample of whole milk was mixed with hexane and gently stirred. Two liquid layers were obtained—a lower, “milky” layer and an upper layer of hexane. The layers were separated and the mass of the lower aqueous layer was found to be 47.8 g. What is the percentage of fat in whole milk?

Safety Precautions

Bromine water is toxic by inhalation and ingestion and is a skin irritant. Work with bromine water in an operating fume hood only. Avoid breathing bromine vapor. Sudan III solution is an alcohol-based solution and is a flammable liquid. Hexane is a flammable liquid and a dangerous fire risk. Do not allow any flames in the laboratory during this activity. Avoid exposure of all chemicals to eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Please inform your teacher of any peanut or other allergies to any of the foods used in this laboratory. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Part A. Classification and Identification of Lipids

1. Label a set of five test tubes 1–5 and place them in a test tube rack.
2. Use a Beral-type pipet or a spatula (in the case of solids) to add 10 drops of liquid or a small pinch of solid, respectively, to the appropriate test tube, as follows:
   {13379_Procedure_Table_1}
3. Use a graduated Beral-type pipet to add 2 mL of water to each test tube. Shake or swirl the test tubes to mix the contents.
4. After 2 minutes, observe each mixture carefully. Has the sample dissolved in the water or are two separate layers evident in the tube?
5. Record the solubility results in Data Table A. Describe each sample as soluble S, if it dissolves completely; slightly soluble SS, if if dissolves partially; or insoluble IS if it does not appear to dissolve at all.
6. Add 5 drops of Sudan III staining solution to each test tube. Record the color and appearance of each test solution in Data Table A.
7. Rinse the contents of the test tubes down the drain with excess water. Wash the test tubes and rinse them with distilled or deionized water.
8. Relabel the tubes 1–5, if necessary.
9. Use a Beral-type pipet or a spatula to add 10 drops of liquid or a small pinch of solid, respectively, to the appropriate test tube, as follows:
   {13379_Procedure_Table_2}
10. Use a graduated Beral-type pipet to add 2 mL of hexane to each test tube. Swirl the test tubes to mix the contents.
11. After 2 minutes, observe each mixture carefully. Record the solubility results in Data Table A. Describe each sample as soluble S, if it dissolves completely; slightly soluble SS, if it dissolves partially; or insoluble IS if it does not appear to dissolve at all.
12. Pour the contents of the test tubes into a beaker marked “Organic Waste.”
13. Wash the test tubes and rinse them with distilled or deionized water.

Part B. Test for Unsaturation. (Note: Carry out these experiments in an operating FUME HOOD only!)

14. Relabel three test tubes 1–3, if necessary, and place them in a test-tube rack.
15. Use a Beral-type pipet to add 1 mL of the appropriate liquid to each test tube, as follows:
    {13379_Procedure_Table_3}
16. Add 1 mL of bromine water to each sample. (Caution: Handle with care! Do not breathe bromine vapor. Do not allow bromine to come in contact with skin or clothing.)
17. Gently swirl each test tube once and replace it in the test tube rack.
18. After 3 minutes, observe the appearance of each bromine test mixture. Answer the questions in Part B of the Properties of Lipids Data Sheet.
19. Working in the HOOD, carefully pour the contents of the test tubes into a beaker marked “Bromine Waste.” Rinse them once with distilled or deionized water and add the rinse water to the bromine waste beaker.

Part C. Extraction of Peanut Oil

20. Obtain about 3–4 peanuts.
21. Transfer the shelled peanuts to a clean and dry mortar. Mash the peanuts with a pestle for at least 3–5 minutes to obtain finely divided, ground peanuts.
22. Tare (zero) a 125-mL Erlenmeyer flask and transfer about 3 g of ground peanuts to the flask. Record the exact mass of peanuts used in Data Table C.
23. Pour approximately 20 mL of hexane into the Erlenmeyer flask and stopper the flask.
24. Swirl the flask periodically over the next 15 minutes to mix the contents.
25. While the solvent extraction is proceeding, set up a filter funnel for gravity filtration. Measure and record the mass of a piece of filter paper and place it in a funnel set up over a second Erlenmeyer flask.
26. After 15 minutes, swirl the contents of the extraction flask and pour the contents into the funnel. Use a spatula to transfer any remaining solid from the Erlenmeyer flask to the funnel.
27. When the filtration is complete, carefully remove the filter paper containing the peanut residue from the funnel and place it in a secure location. Write your name on the paper and allow the residue to dry overnight.
28. Mass the peanut residue and filter paper. Describe the appearance of the residue and report its mass in Data Table C.
29. Answer Post-Lab Questions.

Student Worksheet PDF

13379_Student1.pdf