Austin Peay State University Department of Chemistry
CHEM 1021
ISOLATION OF CASEIN FROM MILK
Purpose: In this lab you will be isolating the proteins casein and lactalbumin from a sample of milk. You
will use these values to determine the percent protein in milk compared to the listed value on the box.
Caution: Wear eye protection because of the possible shattering of dropped glassware and because
10% acetic acid is used in this experiment. If any of this acid comes in contact with your skin, wash it off
immediately with plenty of water. Also, avoid breathing vapors of this solution and of course, do not
drink any of the solutions in this experiment.
Introduction: Milk is a complex biological mixture of chemicals that serves as the primary food source
for infant mammals. Milk contains most of the biological molecules necessary to sustain life including
water, a variety of vitamins, minerals, proteins, sugars, and lipids (DNA, although necessary for life, is
made by your body and therefore is not needed on a dietary level). Although most mammals stop
drinking milk upon maturity, many (but not all) human cultures continue drinking milk and consuming
milk products (for example, cheese, butter, and cream) throughout their entire life.
It would be quite challenging to isolate every component of milk because some of the chemicals
are present in very small quantities. You can very easily, however, isolate the most abundant
components from each other. A common example of this
Table 1. Milk components (% composition)
is removing the fat from cow’s milk to make skim milk or
Cow
Human
Goat
removing the sugar from milk to make lactose‐free milk.
Water
87.8
87.4
87.0
Table 1 shows the average composition of milk from
Protein
3.0
1.4
3.3
Lipids
3.9
4.0
4.2
several mammals consumed by humans.
A small
Sugars
4.6
7.0
4.8
discussion of the components of milk is warranted and
Minerals
0.7
0.2
0.7
described below.
Lipids:
If you were asked if fat mixes with water, chances are you would say “no”. However, milk
contains around 4% fat in water. This means that in 100 mL of water, about 4 mL of fats would be
present. This is possible because milk contains, in addition to fats, a number of lipids called
phospholipids. These molecules are similar to the fatty acids you studied in class, but have a negatively‐
charged phosphate group attached to one end (Figure 1).
Figure 1. Example phospholipid
O
O
O
P
O
hydrophobic ( water hating) area
O
hydrophilic ( water loving) area
The negatively charged phosphate group, like other charged, polar groups, is quite soluble in
water. The non‐polar, uncharged hydrocarbon area is completely insoluble in water. The dual
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Austin Peay State University Department of Chemistry
CHEM 1021
ISOLATION OF CASEIN FROM MILK
personality of the phospholipid
(chemists
would
call
it
amphipathic) lets the milk fat be
soluble in the milk water by
aligning a number of its
hydrophobic areas with the
other fats, with the phosphates
facing out toward the water
(Figure 2). This makes a mixture
called an emulsion, meaning a
fat dissolved in water by means
of an amphipathic molecule, called an emulsifying agent (soaps are emulsifying agents that work to
dissolve grease). In this lab, you will be using powdered milk that has already had the fat removed.
Proteins:
There are three main proteins in milk; casein (case‐in), lactalbumin (lact‐al‐bumin), and
lactoglobulin (lact‐o‐glob‐ulin). In this lab you will be isolating the casein, and a mixture of the
lactalbumin and lactogobulin proteins (they isolate under the same conditions and are difficult to
separate from each other).
Chances are you have consumed casein without knowing it. Casein can precipitate from water
when its normal structure is destroyed. We call this process protein denaturation. The curds in cottage
cheese are precipitated casein protein (the enzyme rennin is used in this case to precipitate the protein)
and the Indian cheese paneer is made by adding an acid to milk to precipitate the curds. Many cheeses
are made by precipitating casein and removing it from the left over liquid, the whey. The lactalbumin
proteins are easily precipitated by heating. After casein proteins are removed, heating the whey
solution can provide the solid protein. Although a minor component, the lactoglobulin proteins are the
immune proteins present in milk that protect a baby from illness until it can develop its own immune
system.
Sugars:
The main carbohydrate present in milk is the sugar lactose. Lactose is a disaccharide containing
the monosaccharides galactose and glucose. Mammals produce an enzyme called lactase that breaks
the disaccharide into its monomers during digestion. Many mammals stop producing lactase after
maturity leading them to be lactose intolerant—they lack the ability to digest the sugar in milk.
Although most people can digest lactose upon maturity, there are many who can’t. These people can
buy lactose‐free products, or buy the enzyme supplement lactase to aid in digestion of the sugar.
On a practical note, when bacteria get into milk, they digest the lactose and form the acid lactic
acid. This causes a precipitation of the protein casein. This is what happens when old milk “sours”.
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Austin Peay State University Department of Chemistry
CHEM 1021
ISOLATION OF CASEIN FROM MILK
Equipment:
10% acetic acid solution
10‐mL Graduated cylinder
Powdered milk
spatula
disposable pipettes
nylon hose
filter flask
filter paper
Buchner funnel
Procedure:
Casein isolation
1. Place about 4 g of powdered nonfat milk and 42 mL of water into a beaker and record the mass.
Heat the milk to 40 oC.
2. When the milk has reached 40 oC, start adding the acetic acid solution 5 drops at a time. After
every 5 drops, stir the solution with a spatula. You will see a white solid forming. This is the
casein. Push the casein to the side of the beaker above the solution and push the liquid out and
to compress the protein. Transfer to a watch glass as you go. Stop after 20 drops of the acetic
acid (1 mL). If you see any liquid on the watch glass, pipette it back into the beaker.
3. Add 0.2 g of calcium carbonate to the milk solution in the beaker to neutralize the pH.
4. Transfer the casein to a section of nylon hose. Wrap the protein and gently squeeze over the
top of the beaker to remove as much of the milk solution as possible.
5. Weigh a plastic boat and record. Add the casein and record the mass on the data sheet.
Lactalbumin isolation
6. Heat the mixture to 75 oC for 5 minutes. This precipitates the lactalbumin protein (and any of
the lactoglobulin protein in the milk).
7. Use a filter funnel, flask, and filter paper to separate the solid precipitate from the hot solution.
8. Transfer the lactalbumin to a weigh boat and record mass on data sheet.
Waste Disposal and Cleanup: Properly dispose of all solutions and solids according to your lab
instructor. Thoroughly wash and rinse all glassware and return to their original places.
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CHEM 1021
ISOLATION OF CASEIN FROM MILK
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Austin Peay State University Department of Chemistry
CHEM 1021
ISOLATION OF CASEIN FROM MILK
DATA SHEET
Name: ______________________________________
Partner: _____________________________________
1. Exact mass of dried milk
_________________g
2. Mass of isolated casein
_________________g
3. Mass of lactalbumin
_________________g
Using the box of milk to find numbers we can work with to check quality control:
4. Mass of protein listed per serving on milk box
_________________g/serving
5. Volume of reconstituted milk per serving (in mL)
_________________mL/serving
6. Convert the volume of milk (in #5) to the mass of reconstituted milk.
Assume the density of milk is ~1 g/mL.
7. Percent protein in a reconstituted milk sample
_________________g
#
#
100
_________________%
Compare your data to the listed nutritional information you determined above:
8. Total mass of protein in your sample (#2 + #3)
_________________g
9. Find the percent of total protein in your sample
_________________%
100
10. Find the percent of casein in your sample
_________________%
11. Find the percent of lactalbumin in your sample
_________________%
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Austin Peay State University Department of Chemistry
CHEM 1021
ISOLATION OF CASEIN FROM MILK
POST LAB QUESTIONS
Name:__________________________________
Partner: ________________________________
1. Percent error: Determine the error in this experiment by using the following equation:
100%
_________________%
2. Assuming your experiment had some level of error (in other words, you have a non‐zero
answer for #1), what do you think could be a reason for your error?
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
3. What does the acetic acid do the structure of casein that causes it to precipitate?
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
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Austin Peay State University Department of Chemistry
CHEM 1021
ISOLATION OF CASEIN FROM MILK
PRE‐LAB ASSIGNMENT
Name:_________________________________
1. What is casein?
__________________________________________________________________________________
__________________________________________________________________________________
2. What is the purpose of lactoglobulin?
__________________________________________________________________________________
__________________________________________________________________________________
3. What is an emulsion?
__________________________________________________________________________________
__________________________________________________________________________________
4. What is an example of an emulsifying agent, other than phospholipids? ___________________
5. If you are lactose intolerant, what enzyme are you lacking?
__________________________________________________________________________________
__________________________________________________________________________________
6. What are the monomers that make up lactose? _______________________________________
7. Why does milk spoil? Be as specific as possible.
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
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