Experiment_732_Isolation_of_a_Protein_from_Milk_1_128129

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Chemistry 07 Experiment 732 V 1.1 Page 1 of 4 Student Name Laboratory Date: Date Report Submitted: __________________________ Student ID Experiment Number and Title Experiment 732: Isolation of a Protein from Milk Experiment 732: Isolation of a Protein from Milk Section 1: Purpose and Summary of experiment Milk is the most nutritionally complete food found in nature. All kinds of milk, human or animal, contain vitamins (principally thiamine, riboflavin, pantothenic acid, and vitamins A, B12, and D), minerals (calcium, potassium, sodium, phosphorus, and trace metals), proteins (mostly casein), carbohydrates (principally lactose), and lipids (fats). The amounts of these nutrients present in different types of milk differ greatly, however. Cows' milk and goats' milk are almost identical in every respect. Human milk contains less than half of the proteins and minerals of cows' or goats' milk, but almost 1.5 times as much sugar. Horses' milk is quite low in proteins and fats compared with the others, whereas reindeer milk is very high in proteins, fats, and minerals, but quite low in carbohydrates. The average composition of whole cows' milk is 87.1% water, 3.4% protein, 3.9% fats, 4.9% carbohydrates, and 0.7% minerals. The only important nutrients lacking in milk are iron and vitamin C. Casein contains all of the common amino acids and is particularly rich in the essential amino acids. Cheese making is based on the precipitation of casein from milk to form solid curds. The liquid whey is drained from the casein curds. The casein curds are concentrated during cooking, with pressure and salting. The flavors are developed with aging. Casein exists in milk as the calcium salt, calcium caseinate. Calcium caseinate has an isoelectric point of pH 4.6. Therefore, it is insoluble in solutions of pH less than 4.6. The pH of milk is about 6.6; therefore, casein has a negative charge at this pH and is solubilized as a salt. If acid is added to milk, the negative charges on the outer surface are neutralized (by protonation) and the neutral protein precipitates, with the calcium ions remaining in solution: Ca-caseinate + 2H +  casein + Ca 2+ In this experiment, you will isolate casein from skim milk by curding the milk with acetic acid. You will then use the xanthoproteic test to test for aromatic amino acids like tryptophan and tyrosine. Xanthoproteic Test The aromatic rings on tyrosine and tryptophan react with nitric acid. In this reaction, the aromatic rings become nitrated. When nitric acid is added to a sample and the mixture is heated, a yellow solution will result if the sample contains tyrosine or tryptophan. When this yellow solution is treated with a strong base (such as NaOH), it turns orange. Since most proteins contain one or both amino acids, most proteins will show a positive reaction in this test.

Chemistry 07 Experiment 732 V 1.1 Page 2 of 4 Section 2: Safety Precautions and Waste Disposal Safety Precautions: Wear your safety goggles. Nitric acid is very corrosive – avoid skin contact with it. If skin contact occurs, flood the area with plenty of running water for 10 minutes. Waste Disposal: At the end of the experiment, all liquid wastes go into the inorganic waste container. Solid wastes go into the solid waste container. Section 3: Procedure Part 1: Isolation of Casein 1. Weigh out 2 grams of powdered non-fat dry milk and dissolve it in 20 mL of warm water in a 100 mL beaker. Record the mass of the powdered non-fat dry milk. (a) Mass of m ilk : ______________ grams 2. Bring the temperature of the solution to 40°C (do not exceed) on a hot plate. Monitor the temperature of the solution with a thermometer. 3. Remove the thermometer, and then add aqueous 10% acetic acid dropwise while stirring with a stirring rod. Do not add the acid too rapidly. Continue adding the acetic acid until no more precipitate forms (slightly less than 2 mL will be required), 4. Stir the precipitated casein until it forms a large amophous mass. 5. Decant most of the liquid whey and transfer the wet precipitate to a vacuum filtration apparatus. 6. With the vacuum running, press the precipitate with a clean cork or spatula to squeeze out as much liquid as possible. 7. Transfer the casein in a 100 mL beaker and add 5 mL 95% ethanol. (CAUTION: HIGHLY FLAMMABLE - NO FLAMES 8. Stir the casein in the ethanol for a few minutes, decant the ethanol, and repeat the process with a second 5 mL portion of ethanol. The ethanol washings remove any small quantities of fat that may have precipitated with the casein. 9. After the second washing with ethanol, collect the casein by vacuum filtration. Wash it on the filter with two small portions of acetone. (b) Mass of filter paper: ______________ grams 10. Place the casein between several layers of paper towels to help dry the product, and let it stand in the air for 10-15 minutes. 11. Weigh the dry casein. c) Mass of casein & filter paper: ______________ grams 1.9980 0.9270

Chemistry 07 Experiment 732 V 1.1 Page 3 of 4 Part 2: Xanthoproteic Test 1. Transfer a small piece of casein to a test tube. 2. Under the hood, add a drop of concentrated nitric acid to test tube and record your observations. Caution: nitric acid must be kept under the hood at all times. Avoid all skin contact with nitric acid. Mix the test tube carefully and place the tubes in a boiling water bath for two minutes. Observe and record the color of the solution. 3. Remove from the water bath and let them cool. When cool, add 10% NaOH to the test tube dropwise until the solution is basic. (You can do this by adding one drop of NaOH, mixing well with a stirring rod, and touching the stirring rod to a piece of red litmus paper. If the litmus paper does not turn blue, add another drop of NaOH to the solution, mix with the stirring rod, and again touch it to a piece of red litmus paper. Repeat this sequence until the litmus paper turns blue – then your solution is basic. You can use the same piece of litmus paper, if you touch the stirring rod to a different dry section each time.) Observe and record any color changes. Xanthoproteic Test Observations Initial Color Color after acid is added Color after base is added Section 4: Calculations 1. Mass of p owdered milk (g) 2. Mass of f ilter paper and casein (g) 3. Mass of filter paper (g) 4. Calculate the mass of casein (g) 5. Calculate the mass percent of casein in dry milk.

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Chemistry 07 Experiment 732 V 1.1 Page 4 of 4 Post-Lab Questions: 1) Based on your observations, what component of milk is responsible for its white color? 2) Interpret the results of the xanthoprotein test. 3) How does your calculated mass percent of casein compare to the average composition of whole cows' milk?