Materials Included In Kit

Additional Materials Required

Prelab Preparation

• Bromine water: Prepare 0.5 M sodium bromide and hydrochloric acid solutions. Combine 25 mL of 0.5 M NaBr and 25 mL of 0.5 M HCl in a dark glass (amber) bottle. Add 10 mL of 5% sodium hypochlorite solution (bleach) and gently swirl to mix the reactants. Cap the bottle and store it in a secure location. Carry out this procedure in an operating fume hood. Do not mix the HCl and NaOCl solutions directly in the absence of NaBr. We recommend the purchase of the Bromine Water Kit (Flinn Catalog No. AP4502) for the safe preparation of bromine water in any amount in less than five minutes. Bromine water is very difficult to store because bromine vapor will escape from almost any container and begin to attack nearby metal surfaces. We recommend making only enough bromine water for your immediate needs. Do not store.
• Sodium thiosulfate solution, 50%: Add 125 g of sodium thiosulfate pentahydrate (hypo, Na₂S₂O₃•5H₂O) to 150 mL of distilled or deionized water. Stir to dissolve, then dilute to 250 mL. The solution concentration is 50% by mass–volume.

Safety Precautions

Carry out all procedures in an operating fume hood. Bromine water is toxic by inhalation and ingestion and may cause skin and eye burns. Do not breathe bromine vapor. Sudan III solution is an alcohol-based solution and is a flammable liquid. Hexane is also a flammable liquid and a dangerous fire risk. Keep away from heat, sparks and open flames. Avoid exposure of all chemicals to eyes and skin. Hexane is a volatile liquid and may cause drowsiness or dizziness if inhaled. Avoid breathing vapors, mist or spray. Some students may experience a food allergy to peanuts. Students who are allergic to nuts should not do the peanut oil extraction activity. Food-grade items that have been brought into the lab are considered laboratory chemicals and are for lab use only. Do not taste or ingest any materials in the chemistry lab. Wear chemical splash goggles, chemical-resistant gloves and a lab coat or chemical-resistant apron. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

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. Solid or liquid samples of lipids and proteins, as well as Sudan III solution, may be packaged for landfill disposal or rinsed down the drain with water according to Flinn Suggested Disposal Method #26a or 26b, respectively. Always segregate excess or leftover reactive chemicals, such as bromine water, a strong oxidizer, to avoid potential undesirable side reactions that may release heat or generate gases. Leftover test mixtures containing flammable liquids such as hexane should be transfered in the hood to a properly labeled flammable organic waste container for eventual licensed hazardous waste disposal. Record the amount and identity of chemicals added to the waste container as well as the date. Excess or leftover bromine water may be reduced by reaction with sodium thiosulfate, according to Flinn Suggested Disposal Method #12a.

Lab Hints

• The experimental work for this lab can reasonably be completed in a typical 2-hour lab period. A few minutes of follow-up time are required on a subsequent day for students to mass the dry peanut residue.
• Preparation of bromine water 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 containers in central locations for immediate collection of leftover hazardous chemicals: (1) a flammable liquid container for hexane solubility test mixtures; and (2) a beaker filled with 100 mL of 50% sodium thiosulfate solution to reduce any excess or unreacted bromine. Both containers must be kept in the hood.
• The three parts of the experiment can be performed in any order. Consider staggering the starting points for different student groups—set up separate hoods for each activity 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 solubility observations. Advise students to “call it as they see it.”
• The test for unsaturation may 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 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. Fresh nuts work better than dry roasted nuts.
• The peanut oil extraction procedure can be extended to study the properties of the oil and the residual peanut “meal.” Evaporate the solvent from the hexane/peanut oil extract using a rotary evaporater 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 (Flinn Catalog No. B0050, Biuret Test Solution) and for carbohydrates using Benedict’s test (Flinn Catalog No. 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). See the “Making Soap” experiment in this manual. The reaction is called saponification.
• Encourage students to read the nutritional information labels on a variety of food items to learn about the distribution of saturated and unsaturated fats in different foods.
• The bromine test for unsaturation is similar to a test that is used by the food industry to characterize fats and oils. The iodine number, based on the reaction of ICl with fats and oils, is used as a quantitative measure of unsaturation. The greater the iodine number, the higher the percent unsaturation in a fat or oil. ICl is used in the iodine number test because it is more reactive than I₂.
• Bromine water does not store well. Prepare only as much solution as is needed for this lab.

Answers to Prelab Questions

1. The C═C double bonds in unsaturated oils react with hydrogen to give “hydrogenated” compounds. Conduct a scavenger hunt in your kitchen shelves to identify food items that list hydrogenated oils as a major ingredient.

   “Partially hydrogenated vegetable oil” is listed as a main ingredient in many snack foods (e.g., cookies, granola bars, crackers, margarine, potato chips, peanut butter, scalloped potato mixes).

2. Predict the change in consistency that should be observed when an oil is hydrogenated. What are the advantages of this change in consistency in food processing? What are the disadvantages?

   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, is that saturated fats are less healthy than unsaturated ones.

3. The fat content in milk can be determined by extraction 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 milky layer was found to be 47.8 g. Calculate 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:

   {14050_PreLabAnswers_Equation_1}
4. Fatty acids are joined to the glycerol backbone in a triglyceride via ester linkages. Circle and label the ester functional groups in the structure of the following lipid.
   {14050_PreLabAnswers_Figure_3}
5. The C═C double bonds in triglycerides occur naturally in the cis-configuration. Heating and/or treating a tripolyunsaturate glyceride with a metal catalyst, such as in a hydrogenation process, converts it to a more stable trans-configuration. Circle the polyunsaturated fatty acid residue in the lipid shown in Question 4. What would this residue look like in a so-called trans fat? Trans fat:
   {14050_PreLabAnswers_Figure_4}

Answers to Questions

Laboratory Report

Solubility of Lipids

Test for Unsaturation

1. What is the initial color of bromine water? 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 samples 1–3 after two 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? Corn oil and olive oil.

Extraction of Peanut Oil Post-Lab Questions

1. Compare and contrast the solubility results of the tested samples. Explain the observed pattern in terms of the definition of lipids. Which samples 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 nonlipid sample (albumin) had the opposite pattern. It dissolved in water but not in hexane.

2. Compare the effect of Sudan III 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?
   {14050_Answers_Table_5}
   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 “decolorized” bromine. Coconut oil has less than 10% unsaturation and did not react with bromine.
5. How could the bromine test be modified 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 the experimental value for the percentage of fat in peanuts (Question 6) compare with that reported on the nutritional label?
   {14050_Answers_Table_6}
   Amount of fat listed on nutritional label for peanuts = (14 g/28.4 g) x 100% = 49%
   Only about half of the total fat was removed by the extraction procedure.
8. Describe some ways the extraction procedure could be improved to obtain better agreement between the experimental and known values for 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 or 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 compounds called lipids. What are the properties of lipids? What role do lipids play in the chemistry of life?

Concepts

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

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 compounds—carbohydrates, proteins and nucleic acids—that readily dissolve in water. Lipids typically 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 structures and biological functions of lipids are very diverse. Examples of lipids and their biological roles include:

• Fats and oils, such as butter and corn oil, which are used to store energy within cells and organisms. In addition, fats accumulate in adipose tissue that insulates and protects internal organs.
• Phospholipids, also known as membrane lipids, which are responsible for the “lipid bilayer” structures that form protective membranes around cells in all living organisms.
• Steroid hormones, such as estrogen and progesterone, which are synthesized in the body from cholesterol, and act as chemical messengers, carrying signals from one part of the body to another to regulate cell activity.

Triglycerides
Fats and oils have the same basic structure and are referred to as triglycerides or triacyclglycerols. As shown in Figure 1, triglycerides consist of a glycerol backbone with three attached fatty acid residues. Fatty acids are long-chain carboxylic acids consisting of a long hydrocarbon “tail” 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 two most common carbon chain lengths are 16 and 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 in the hydrocarbon tail—these fatty acids are “unsaturated” with respect to the number of hydrogen atoms. Fatty acids that contain more than one double bond 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—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 fats 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. Unsaturated fats may 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 also depends 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 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 obtaining the so-called seed oils (e.g., corn oil, olive 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 extracts are then heated to remove the solvent and the oils are subjected to further heat processing to improve their shelf life and stability.

Experiment Overview

The purpose of this experiment is to identify and classify lipids and examine their properties. There are three parts to the experiment.

• The solubility of lipids will be studied and compared with that of albumin, a protein isolated from 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. It is used as a “fat stain” in botany and medicine to identify lipids in seeds and tissue samples, respectively.
• 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. Disappearance of the orange-red color due to bromine serves as a positive test for unsaturation.
• The amount of “peanut oil” in peanuts will be determined by extracting 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. Conduct a scavenger hunt in your kitchen shelves to identify food items that list hydrogenated oils as a major ingredient.
2. Predict the change in consistency that should be observed when an oil is hydrogenated. What are the advantages of this change in consistency in food processing? What are the disadvantages?
3. The fat content in milk can be determined by extraction 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 milky layer was found to be 47.8 g. Calculate the percentage of fat in whole milk.
4. Fatty acids are joined to the glycerol backbone in a triglyceride via ester linkages. Circle and label the ester functional groups in the structure of the following lipid.
   {14050_PreLab_Figure_2}
5. The C═C double bonds in triglycerides occur naturally in the cis-configuration. Heating and/or treating a triglyceride with a metal catalyst, such as in a hydrogenation process, converts it to a more stable trans-configuration. Circle the polyunsaturated fatty acid residue in the lipid shown in Question 4. What would this residue look like in a so-called trans fat?

Safety Precautions

Carry out all procedures in an operating fume hood. Bromine water is toxic by inhalation and ingestion and may cause skin and eye burns. Do not breathe bromine vapor. Sudan III solution is an alcohol-based solution and a flammable liquid. Hexane is also a flammable liquid and a dangerous fire risk. Keep away from heat, sparks and open flames. Hexane is a volatile liquid and may cause drowsiness or dizziness if inhaled. Avoid breathing vapors, mist or spray. Avoid exposure of all chemicals to eyes and skin. Some students may experience a food allergy to peanuts. Students who are allergic to nuts should not do the peanut oil extraction activity. Food-grade items that have been brought into the lab are considered laboratory chemicals and are for lab use only. Do not taste or ingest any materials in the chemistry lab. Do not remove any remaining food items from the lab after use. Wear chemical splash goggles, chemical-resistant gloves and a lab coat or chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Solubility 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:
   {14050_Procedure_Table_1}
3. Use a graduated Beral-type pipet to add 2 mL of water to each test tube. Swirl the test tubes to mix the contents.
4. After 2 minutes, observe each mixture carefully. Which samples dissolved in water?
5. Record the results: soluble (S), if the substance dissolves completely in water; slightly soluble (SS), if it partially dissolves; 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 sample mixture.
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. Repeat step 2 to prepare a set of test tubes for analysis.
10. Using a graduated Beral-type pipet, 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 and record the results: S, SS or IS.
12. Pour the contents of the test tubes into a flammable organic waste container.
13. Wash the test tubes and rinse them with distilled or deionized water.

Test for Unsaturation

14. Relabel three clean 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:
    {14050_Procedure_Table_2}
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 2 minutes, observe the appearance of each sample mixture and answer the questions in the Laboratory Report.
19. Carefully pour the contents of the test tubes into a container for leftover or excess bromine in the hood. Rinse the test tubes once with distilled or deionized water and add the rinse water to the waste container.
20. Wash and rinse the test tubes.

Extraction of Peanut Oil

21. Obtain about 3–4 raw peanuts. Break the peanut shells and remove the thin skin covering the peanuts.
22. Transfer the shelled peanuts to a clean and dry mortar. Mash the peanuts with a pestle for 3–5 minutes to obtain finely divided, ground peanuts.
23. 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.
24. Pour approximately 20 mL of hexane into the Erlenmeyer flask and stopper the flask.
25. Swirl the flask periodically for 15 minutes to mix the contents.
26. 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.
27. After 15 minutes, swirl and pour the contents of the Erlenmeyer flask into the funnel. Use a spatula to transfer any remaining solid from the Erlenmeyer flask to the funnel.
28. When the filtration is complete, carefully remove the filter paper containing the peanut residue from the funnel and place it on a labeled watch glass or Petri dish in a secure location. Allow the residue to dry overnight.
29. Collect the hexane filtrate in a flammable organic waste container.
30. Mass the dry peanut residue and filter paper. Describe the appearance of the residue and record its mass.

Student Worksheet PDF

12532_Student1.pdf