Isolation and Characterization of Protein and Sugar from Milk
Milk is an important food for mammalian development. It is the only food ingested by young mammals in the weeks following birth; therefore, it must contain an enormous amount of nutritionally significant vitamins, minerals, proteins, carbohydrates, and lipids. Due to the varying chemical properties of each of these compounds, it is easy to separate many of them using common organic chemistry techniques. In this experiment, you will separate several of the chemical substances found in milk. First, you will isolate two proteins, casein and albumin. The remaining milk mixture will then be used as a source of sugar, α-lactose. After you isolate the milk sugar, you will perform several chemical tests on this material. Fats, which are present in whole milk, are not isolated in this experiment because powdered nonfat milk is used.
After isolation of the various chemical compounds, characterization of the optically active compounds can be performed using polarimetry. The sugar isolated in this experiment, α-lactose, is optically active and slowly interconverts to β-lactose in water:
α-lactose β-lactose [α] = 92.6° [α] = 34.0°
A compound will consistently have the same specific rotation under identical experimental conditions. The specific rotation is related to a compound’s concentration through Biot’s law: α = [α] ℓ c where α is the observed optical rotation in units of degrees, [α] is the specific rotation in units of degrees (the formal unit for specific rotation is degrees dm-1 mL g-1, but scientific literature uses just degrees), ℓ is the length of the cell in units of dm, and c is the sample concentration in units of grams per milliliter.
OBJECTIVES
In this experiment, you will
Isolate several of the chemical compounds found in milk, including casein, α-lactose, and albumin.
Determine your yield for each of these compounds.
Observe the optical rotation of α-lactose using polarimetry.
MATERIALS
LabQuest or computer interface watch glass
LabQuest App or Logger Pro
100 mL and 50 mL beakers
Vernier Polarimeter
95% ethanol
Polarimeter sample cell apparatus for heating and monitoring
250 mL Erlenmeyer flask temperature (e.g., sand bath)
10% acetic acid bench-top centrifuge powdered nonfat milk vacuum filtration apparatus calcium carbonate
PROCEDURE
Part I Isolation and Purification of Casein
1. Obtain and wear goggles. Protect your arms and hands by wearing a long-sleeve lab coat and gloves. Conduct this reaction in a fume hood.
2. Dissolve 4.0 g of powdered nonfat milk in 10 mL of water in a 100 mL beaker. Heat the solution on a sand bath or aluminum block to about 40°C, monitoring the temperature with a thermometer or temperature probe.
3. When the mixture has reached 40°C, add 1 mL of 10% acetic acid dropwise to the warm milk. Maintain the solution temperature at 40°C and, after every 5 drops of acid, stir the solution gently using a small spatula. Using the spatula, push the precipitated casein onto the side of the beaker so that most of the liquid drains from the solid.
4. Transfer the congealed casein to a 50 mL beaker in small portions. If any liquid separates from the casein, use a Pasteur pipet to transfer the liquid back into the original 100 mL beaker.
5. Slowly continue the dropwise addition of the 1 mL 10% acetic acid solution to the 100 mL beaker to complete the casein precipitation. Remove as much casein as possible but avoid adding excess of acetic acid to the milk solution, as this will cause the lactose in the milk to hydrolyze to glucose and galactose.
6. When most of the casein has been removed from the milk solution, add 0.2 g of calcium carbonate to the milk in the 100 mL beaker. Stir this mixture for a few minutes and save it for use in the isolation of lactose (Part II). Caution: this mixture should be used as soon as possible, during the same lab period.
7. Transfer the casein from
