Materials Included In Kit

Additional Materials Required

Prelab Preparation

Carbohydrate and protein reference solutions must be freshly prepared for each class period. Add 50 mL of distilled water to 1 g of solid in a 150-mL Erlenmeyer flask.

Safety Precautions

Acetic acid and sodium hydroxide solutions are corrosive liquids. Ethyl alcohol is a flammable organic solvent and a dangerous fire risk. Benedict’s solution contains copper tartrate and is an alkaline solution. Biuret solution is a highly alkaline solution and is corrosive to eyes and body tissue. Do not allow students to ingest any of the milk samples before or after this laboratory. The samples have been stored with non-food-grade laboratory chemicals and are for lab use only. Wear chemical splash goggles, chemical-resistant gloves, and a chemical-resistant apron. Avoid exposure to eyes and skin. 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. All solutions from this activity can be disposed of according to Flinn Suggested Disposal Method #26b.

Teacher Tips

• Enough materials are provided in this Super Value Kit for 5 classes of 30 students each, working in pairs (75 total student groups. About 10 percent extra has been included with the kit to allow the teacher to test the procedure beforehand. The teacher is encouraged to perform the experiment ahead of time in order to guide the students through the procedure.
• The laboratory kit requires two 50-minute laboratory periods to complete. It is recommended that Parts A, B and C be completed during one lab period and Part D as soon as possible thereafter. Part C is optional. If Part C is not done, the experiment can be completed in one lab period. In order to do this, students would test the protein samples before they are completely dry. This will not affect the Biuret or Benedict’s test results.
• Solid protein samples from Parts A and B should be dried overnight in a secure location. In addition, students must also be able to save the filtrate solution from Part C for testing in Part D. The time required to complete Part D will not take up the entire second 50-minute period, and enough time should remain for the class to perform the lab calculations and analysis together.
• It is important that acetic acid be added slowly and with continuous stirring to the milk solution in Part A. If the acid is added too quickly, the casein will precipitate out as a gelatinous solid and will plug the filter funnel. Filtration time will be increased significantly. If this happens, it is recommended that students remove the filtrate in small portions using a Beral-type pipet.
• Extreme caution is urged when the students filter the hot solution in Part B. Students should wear gloves and should use “hot hands” to hold the Erlenmeyer flask. The teacher should demonstrate this technique to the class and should monitor the students closely. If the hot gripping device is not available, teachers can make a pouring “handle” by folding two pieces of paper toweling lengthwise to form a thick, 1-inch wide strip. Wrap the thick strip snugly around the neck of the Erlenmeyer flask and use it as a handle to grab the flask.
• Skim milk is recommended for the quantitative determination of protein because of the absence of fat, which co-precipitates with protein when milk is acidified. The experiment can be extended, however, for the determination of fat in whole milk as well. The fat can be separated first, before acid is added, by extraction with a nonpolar organic solvent such as hexane in a graduated cylinder. If the separation is done very carefully, the mass of milk remaining after extraction equals the mass of whole milk minus the fat content. Alternatively, the precipitate obtained upon addition of mild acid can be washed with several portions of acetone after the aqueous filtrate has been removed. The acetone portions are then combined and evaporated to leave behind the mass of fat residue in whole milk.
• A second consumer chemistry kit dealing with the analysis of the nutritional value of milk has also been developed. Boning Up on Calcium—Student Laboratory Kit provides the chemicals and a detailed procedure to perform a microscale analysis of the amount of calcium in milk.

Answers to Prelab Questions

1. Name the principal proteins present in milk and give their functions.

   The principal proteins in milk are casein, lactalbumin and lactoglobulin. Casein, which constitutes 80% of the total protein content in milk, serves as a nutritional source of amino acids for young mammals. Lactalbumin is involved in the synthesis of lactose, while lactoglobulin is an antibody protein that carries the immunological properties of milk to protect young mammals until their own immune systems develop.

2. Consult nutritional labels for whole milk, low-fat (2%) milk and nonfat skim milk and compare their protein, carbohydrate and fat contents.

   The following information was obtained from representative nutritional labels. Amounts are given per 1-cup (240-mL) serving size of milk. Data will vary depending on the brand of milk.

   {12096_Answers_Table_3}
3. What is the difference between whole milk, 2% milk and skim milk?

   Whole milk, 2% and skim milk differ primarily in the amount of fat present. Whole milk contains 4% butterfat, which is reduced to 2% in 2% milk. In skim milk all of the fat content has been removed.

4. Name and describe the qualitative color tests that will be used to identify the protein and carbohydrate components of milk.

   Protein is identified by means of the Biuret test, reaction with blue copper(II) ions in a highly basic solution. Proteins form a characteristic lavender-colored product with copper(II) ions. Carbohydrates are identified by means of Benedict’s test, a redox reaction with blue copper(II) ions. A positive Benedict’s test is marked by the appearance of an orange-red precipitate of reduced copper(I) oxide. Proteins do not react with the copper ions in Benedict’s solution because the solution is not basic enough.

5. What conditions will be used to precipitate casein, the main protein in milk?

   Casein is precipitated from milk when the solution is made slightly acidic by the addition of 1 M acetic acid. The solution is also gently warmed.

Sample Data

Data Table 1. Protein and Carbohydrate Content of Skim

Data Table 2. Qualitative Tests for Protein and Carbohydrate

Answers to Questions

1. Based on the combined mass of protein isolated from 20 mL of skim milk, calculate the percent composition of total protein in skim milk. What percent of the total protein content is due to the casein fraction? (Assume the density of milk is equal to 1 g/mL.)
   {12096_Answers_Equation_1}
2. Using your experimental value for the percent protein in milk, calculate the mass of protein in grams that would be present in 1 serving size (1 cup, 240 mL) of skim milk. Compare this result with the information provided on the nutritional label for the protein content in 1 serving of skim milk.

   240 mL/1 cup x 0.038 g protein/mL milk = 9.1 g protein per cup
   The nutritional label says there is 8–9 g of protein per 1 cup serving of milk.

3. Based on the amount of lactose isolated from 1 mL of skim milk (filtrate), calculate the percent composition of lactose in skim milk.
   {12096_Answers_Equation_2}
4. Using your experimental value for the percent lactose in milk, calculate the mass of lactose in grams that would be present in 1 serving size (1 cup, 240 mL) of skim milk. Compare this result with the information provided on the nutritional label for the carbohydrate (sugar) content in 1 serving of skim milk.

   240 mL/1 cup x 0.05 g lactose/mL milk = 12 g lactose per cup
   The nutritional label says there are 13 g of carbohydrate per 1 cup serving of milk.

5. Based on the results of the Biuret and Benedict’s tests, comment on the effectiveness of the experimental procedures for the separation and isolation of the protein and carbohydrate fractions of skim milk.

   Both the casein and lactalbumin protein fractions exhibited positive Biuret test results and negative Benedict’s test results. These two results taken together verify that the proteins have been completely separated from the carbohydrate fraction of skim milk in the protein isolation procedure. The filtrate remaining after removal of the two protein fractions exhibited a positive Benedict’s test result and negative Biuret test. These results confirm the identity of lactose as a reducing sugar and also prove that the carbohydrate fraction is not contaminated with any residual protein.

Introduction

From glossy magazine ads to flashy billboards, the message to drink more milk is all around us. Whole milk is a natural, nutritionally complete food source. It contains all of the essential classes of biological molecules—proteins, carbohydrates and fats—that are important in nutrition. The purpose of this lab is to separate the protein and carbohydrate components of skim milk and verify their identity.

Concepts

• Protein vs. carbohydrate vs. fat
• Biuret test
• Benedict’s test

Background

Milk and milk products have been a major food source from earliest recorded history. The principal components of whole milk are proteins (3.5%), carbohydrates (5%) and fat (4%). In addition, milk is also an important source of a variety of essential minerals and vitamins in the diet. In this lab activity, the biochemical nature of proteins and carbohydrates is explored by analyzing the nutritional components of skim milk (milk without the fat component).

Proteins
Proteins are essential constituents of all living cells and are vital for proper cell structure and function. A single cell contains thousands of different proteins, each with a unique structure and function. Although the structures of proteins vary dramatically, all proteins have a similar composition and share a common structural backbone. All proteins are polymers composed of amino acid molecules joined together via peptide linkages to give very long-chain molecules (see Figure 1). Although there are only 20 different varieties of naturally occurring amino acids, the number of ways that these can combine is almost infinite, giving rise to the tremendous number and variety of proteins found in nature.

The presence of proteins can be detected using a simple color test based on the reaction of protein molecules with copper ions in basic solution (pH >13). When a protein is allowed to react with CuSO₄ in the presence of a strong base, the Cu²⁺ ions bind to the nitrogen atoms and carbonyl groups in the protein to form a distinctive and stable purple coordination complex. This is called the Biuret test.

There are three main proteins in milk: casein, lactalbumin and lactoglobulin(s). The chief nutritional protein in milk is casein, comprising 80% of the total protein content; it basically serves as a reservoir of amino acids for the body to synthesize its needed proteins. Casein is a phosphoprotein—it contains a large number of phosphate groups attached to the amino acid side chains in the polymer structure. The negatively charged phosphate groups are balanced by positive calcium ions and are thus responsible for the high nutritional calcium content in milk. Because casein is almost completely insoluble in water, it can be easily precipitated from milk by the addition of a small amount of acid at 40 °C.

The second group of proteins in milk is made up of smaller proteins that are much more water soluble. There are two main proteins in this group—lactalbumin and lactoglobulin. Lactalbumin is an enzyme that is involved in the synthesis of lactose, the principal carbohydrate component in milk. Lactoglobulin is a generic name for a group of proteins that are responsible for the immunological properties of milk. These proteins have a role far greater than their low quantity suggests, since they constitute the “protective” function of milk. Lactalbumin and lactoglobulin remain in solution when the casein is precipitated from milk upon treatment with acid, but can be forced out of solution by heating milk to higher temperatures (80–90 °C).

Carbohydrates
Carbohydrates constitute the second class of biochemical and nutritional components in milk. Monosaccha­rides such as glucose (“blood sugar,” also called dextrose) and fructose are simple carbohydrates and are the fundamental units that make up all other carbohydrate molecules. When two monosaccharide units are joined together, they form disaccharides. Examples of disaccharides include sucrose (cane or table sugar), maltose (malt sugar) and lactose (milk sugar). Polysaccha­rides are complex carbohydrate polymers composed of many simple sugar molecules joined together. The most familiar polysaccharides are starch and cellulose. The monosaccharide glucose is the principal energy source for metabolism while the polysaccharide starch serves as a way of storing chemical energy in plants.

The principal carbohydrate present in milk is lactose, a disaccharide composed of one unit of glucose combined with one unit of galactose (see Figure 2). Lactose can be separated from milk by crystallization via prolonged heating to remove most of the water content. The filtrate remaining after the protein and fat components have been removed is heated at 90 °C until the liquid has evaporated. Lactose is isolated as a white powder and is identified by means of Benedict’s test, a general test for “reducing” sugars. Reducing sugars include lactose and maltose (disaccharides) as well as all monosaccharides (such as glucose and galactose). Benedict’s test is based on an oxidation–reduction reaction between the sugar molecule and Cu²⁺ ion in Benedict’s reagent. The intense blue color of Benedict’s solution fades during a positive test result, which is marked by the formation of an orange-red precipitate consisting of copper(I) oxide (Cu₂O). Fats
The third class of nutrients in whole milk are fats and oils. Fats and oils are water-insoluble substances found in both plant and animal cells. They can be separated from plant and animal products using nonpolar organic solvents, such as hexane. Fats and oils are members of a diverse class of nonpolar biological molecules called lipids. Other examples of lipids include phospholipids, cholesterol, steroid hormones, and fat-soluble vitamins such as Vitamin A and D. Despite their current political “incorrectness,” fats and oils are essential nutrients that are required for cell membrane structure and function. Fats and oils are also a very efficient way of storing chemical energy and are thus the most important means of long-term energy storage in cells.

Whole milk is an emulsion, in which the fat molecules are dispersed in the form of tiny fat globules. The emulsion is stabilized by the presence of protein molecules that adhere to the surface of the fat and prevent it from coalescing and separating out of solution. The fat can be removed from whole milk by extraction with hexane or a similar organic solvent. In skim milk, the fat content has been removed from whole milk by centrifuging it. Fat is less dense than water. When milk is spun very fast in a centrifuge, the fat portion separates out as an upper creamy layer. The upper fat layer is then “skimmed” off—skim milk is the more dense portion that remains behind in a lower layer.

Experiment Overview

This lab activity involves the separation, identification and quantitative analysis of the protein and carbohydrate fractions of skim milk. Skim milk is used rather than whole milk since it lacks fat and thus makes it easier to obtain pure protein and carbohydrate samples. The experimental results will be compared against the information provided on the nutritional label for the amount of protein and carbohydrate in milk.

The principal milk protein casein is removed from skim milk by precipitation with mild acid. Dilute acetic acid is added and the solution is heated gently. Casein is a protein that normally contains many negatively charged phosphate groups surrounded by calcium ions in solution. It is this property that keeps the protein “dissolved” or suspended in milk. As the pH of milk is reduced below 5.0, the phosphate groups lose their negative charge and the calcium ions are replaced by bound hydrogen ions from the added acid. The stable protein suspension is broken and casein precipitates from solution. After drying overnight the protein is isolated in the form of a white solid.

After removal of the main insoluble protein fraction, the filtrate is heated to a higher temperature (80–90 °C) and the second class of milk proteins (lactalbumin and lactoglobulin) precipitates out of solution. The combined mass of the protein fractions can be used to calculate the percent protein composition in milk. The identity of the protein fractions of milk is confirmed by means of the Biuret test and compared against a reference protein solution (egg albumin).

The milk “whey” filtrate remaining after the proteins have been removed is tested with Benedict’s solution to identify the carbohydrates in milk. In an optional exercise, a small portion of the filtrate can be concentrated to dryness by heating for 20–30 minutes. Lactose is isolated as a white solid, and its mass can be used to calculate the percent carbohydrate (sugars) in milk.

The steps involved in characterizing the nutritional components of milk are illustrated by means of a flow chart diagram in Figure 3.

Flow Chart Diagram

Materials

Prelab Questions

Read the Background information and answer the following questions on a separate sheet of paper.

1. Name the principal proteins present in milk and give their functions.
2. Consult nutritional labels for whole milk, low-fat (2%) milk and nonfat skim milk. Compare their protein, carbohydrate and fat contents.
3. What is the difference between whole milk, 2% milk and skim milk?
4. Name and describe the qualitative color tests that will be used to identify the protein and carbohydrate components of milk.
5. What conditions will be used to precipitate casein, the main protein in milk?

Safety Precautions

Acetic acid and sodium hydroxide solutions are corrosive liquids. Ethyl alcohol is a flammable organic solvent and a dangerous fire risk. Benedict’s solution contains copper tartrate and is an alkaline solution. Biuret solution is a highly alkaline solution and is corrosive to eyes and body tissue. Avoid exposure of all chemicals to eyes and skin. 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 A. Isolation of Casein

1. Use a graduated cylinder to add 20 mL of skim milk to a 125-mL Erlenmeyer flask. Record the volume to the nearest tenth or 0.1 mL in Data Table 1.
2. Heat the milk sample to 40 °C on a hot plate at the lowest setting.
3. Using a graduated Beral-type pipet, add 2 mL of 1 M acetic acid solution dropwise with constant stirring.
4. Continue heating the mixture for 5 minutes until a white precipitate appears and the liquid portion is a very pale yellow, but clear, solution.
5. Prepare a gravity filtration setup using a filter funnel fitted with a piece of folded filter paper that has been wet with 1 mL of distilled water. Use a clean 125-mL Erlenmeyer flask as the receiving vessel.
6. Filter the warm solution through the filter funnel.
7. Rinse the contents of the reaction flask with 1 mL of distilled water and add this to the filter funnel.
8. After the filtration is complete, remove the flask containing the filtrate (which should be a clear, cream-colored solution) and place a medium-size test tube under the funnel.
9. Save the filtrate in the Erlenmeyer flask for step 16 in Part B, Isolation of Lactalbumin and Lactoglobulin.
10. Rinse the precipitate in the filter funnel with 1–2 mL of 95% ethyl alcohol and continue the gravity filtration until all of the liquid has been removed.
11. Weigh a labeled watch glass and record the mass in Data Table 1.
12. Remove the filter paper from the funnel and gently scrape the wet solid onto the preweighed watch glass.
13. Use a spatula tip to break the wet solid mass into smaller clumps. Blot the solid gently with paper towels to remove visible liquid and to hasten drying. Do not press the solid down hard onto the watch glass (or it will form a “milk glue”).
14. Set the watch glass and solid in a secure location and allow the solid to air dry overnight.
15. Measure the combined mass of the watch glass and dry casein sample and record the result in Data Table 1. Save the casein for Part D.

Part B. Isolation of Lactalbumin and Lactoglobulin

16. Place the Erlenmeyer flask containing the clear filtrate from step 9 on the hot plate and heat to 80–90 °C for 10 min. The filtrate will gradually turn cloudy (milky) again, and a granular white solid will precipitate out of solution.
17. Weigh a piece of filter paper and record the mass in Data Table 1.
18. Prepare a gravity filtration setup using a funnel fitted with the piece of preweighed, folded filter paper that has been wet with 1 mL of distilled water. Use a clean 125-mL Erlenmeyer flask as the receiving vessel.
19. Using “hot hands” to hold the Erlenmeyer flask, carefully pour the hot solution through the gravity filtration apparatus.
20. After the filtration is complete, remove the flask containing the filtrate and save the filtrate for Parts C and D.
21. Remove the filter paper from the funnel and place it on a labeled watch glass. Allow the solid to air dry overnight in a safe and secure location.
22. After drying, measure the combined mass of the filter paper and dry protein sample and record the result in Data Table 1. Note: Prior to Part D, set up a hot water bath by placing a 600-mL beaker half full with water on a hot plate at a medium setting.

Part C. Isolation of Lactose (Optional)

23. Pour the filtrate remaining from step 20, Part B into a clean 25- or 50-mL graduated cylinder. Using a Beral-type pipet, add distilled water, as needed, to dilute the filtrate solution to 20 mL (the original milk volume).
24. Label an empty watch glass and measure its mass.
25. Record the mass in Data Table 1.
26. Using a clean, graduated, Beral-type pipet, remove 1 mL of the diluted filtrate solution and add it to the watch glass.
27. Save the remaining filtrate in the graduated cylinder for steps 34 and 39 in Part D.
28. Place the watch glass containing the filtrate on the hot plate and heat it at a medium-high setting until the liquid has evaporated and only a white solid remains. Do not overheat—the solid will begin to turn brown.
29. Using crucible tongs, remove the watch glass from the hot plate and allow it to cool.
30. Measure the combined mass of the watch glass and its contents and record the result in Data Table 1.

Part D. Qualitative Tests for Protein and Carbohydrate

31. Prepare casein solution: dissolve a small amount (<0.1 g) of dry casein from Part A in 5 mL of 1 M sodium hydroxide solution in a test tube.
32. Prepare lactalbumin solution: dissolve a small amount (<0.1 g) of dry lactalbumin from Part B in 5 mL of distilled water in a test tube.
33. Obtain six medium-size test tubes and label them 1–6.
34. Place 1 mL of distilled water (test tube 1), skim milk (test tube 2), casein solution (test tube 3), lactalbumin solution (test tube 4), the filtrate (lactose solution) from step 27 in Part C (test tube 5) and protein reference solution (test tube 6) in separate, labeled test tubes.
35. Add 2 mL of biuret test solution to each test tube.
36. After 3 minutes, record the color of each test sample in Data Table 2. Note whether each result is a positive or negative test result for dissolved protein.
37. Pour the contents of the test tubes into a beaker for later disposal and wash and rinse the test tubes.
38. Relabel the six test tubes, 1–6, if necessary.
39. Place 1 mL of distilled water (test tube 1), skim milk (test tube 2), casein solution (test tube 3), lactalbumin solution (test tube 4), the filtrate (lactose solution) from step 27 in Part C (test tube 5) and carbohydrate reference solution (test tube 6) in separate, labeled test tubes.
40. Add 2 mL of Benedict’s solution to each test tube and place the test tubes in the hot water bath.
41. After 3–5 minutes, record the color and appearance of each sample in Data Table 2. Note whether each result is a positive or negative test result for the presence of a reducing sugar.
42. Using a test tube holder, remove the hot test tubes from the hot water bath and cool them.
43. Consult your instructor for disposal of all laboratory solutions.

Student Worksheet PDF

12096_Student1.pdf