Exercise no.

7
ANALYSIS OF MILK FOR THE LIPIDS, CARBOHYDRATES AND PROTEINS
I. Introduction

Milk is an emulsion or colloid of butterfat globules within an aqueous

fluid. Each fat globule is surrounded by a membrane consisting of
phospholipids and proteins which emulsifiers that keep the individual globules
from joining together into noticeable grains of butterfat.
The largest structures in the fluid potion of the milk are casein protein
micelles (aggregates of several thousand protein molecules) bounded with the
help of nanometer-scale particle of calcium phosphate. The casein protein
makes up around 80% of the protein milk, by weight.
The type of carbohydrates found in the milk structure is lactose (the
combination of glucose and galactose) which gives milk its sweet taste and
contributes about 40% of whole calories of milk. Human milk contains on
average, 1.1% protein, 4.2% fat, 7.0% lactose, and supplies 72 kcal of energy
per 100 grams. Cow milk contains, on average 3.4% protein, 3.6% fat, 4.6%
lactose, 0.7 minerals, and supplies 66 kcal of energy per 100 grams.
II. OBJECTIVES
At the end of the experiment, each student will be able to:

1. determine the primary components of milk,

2. determine the percent composition of milk in terms of
protein, carbohydrates, and lipids.

III. MATERIALS
2-50ml beaker, triple beam balance, 1-100ml beaker, 3-10ml graduated
cylinder, 2 watch glass, test tube (big) with cork, 250ml beaker, Pasteur
pipette, 8-test tubes, test tube brush, test tube rack, test tube holder, Buchner
funnel, filter paper, water bath, electric stove, spatula, stirring rod, evaporating
dish, wire gauze

25ml methylene chloride, 1ml bromine solution, 1ml conc. Acetic acid, 1ml
Biuret test/reagent (0.01M CuSO4), 1 ml 6M NaOH, 6ml Benedict’s solution
(Fehling’s), 1ml iodine solution
IV. PROCEDURE
PART I. Determining the Percent Fat in whole milk
 1. Weigh a dry, clean, empty 100 mL beaker and record the mass.

2. Using a 10 mL graduated cylinder, measure out of 5 mL of milk (raw) and pour

into the beaker and record the mass of the beaker and milk.

3. Determine and record the mass of just milk. Then, add 20 mL of water into the
beaker.

4. Pour all of the milk into a large test tube. In the fume hood add 25 mL methylene
chloride to the milk and cork the tube. Methylene chloride is a nonpolar solvent
which with will not mix the water but will take the fat out of the water since fat is
also nonpolar.

5. Shake the test tube for 30 seconds trying not to get the cork wet: Let the content
of the test tube separate into layers.

6. Using a Pasteur pipet remove the milk layer leaving behind the methylene
chloride/ fat layer in a beaker and set aside the test tube with methylene
chloride/fat layer. Put the milk in the beaker and weight.

7. Weigh the beaker again with the milk and record your data and set aside.

Raw Milk Methylene Chloride % composition

Weight (g) Weight (g) of Weight (g) of Weight (g) Weight (g) of Weight (g) of
of the the beaker the milk of the the beaker with the milk
beaker with milk beaker milk
100g 105g 5g 100g 110g 10g 4.76%

True Value – Experimental Value x 100

True Value

PART II. Determining if the Milk Fat contains Unsaturated Fat

NOTE: Do this part of the experiment in the hood!!

8. To the test tube with the methylene chloride and removed fat, add 3 drops of bromine
solution.
9. Record the color of the solution in the test tube after the bromine has been added.
NOTE: Keep the milk in PART I in step 6 for the next part, pour the organic layer
with the fat, methylene chloride and bromine solution into the liquid waste
container.

Questions:
Is the fat content saturated or unsaturated? Explain.

The fat content is unsaturated. After the bromine solution was added, it was
decolorized.

Is there any other chemical test to determine the unsaturation of the structures? If
any, explain the test briefly.

Yes. Aside from Bromine test, the Alkaline potassium permanganate test, also
known as Baeyer’s test, is another method to determine unsaturation in an organic
compound. The KMnO4 has a deep purple color so when it is added to an
unsaturated compound, the MnO4- gets reduced to MnO2 which forms a brown
precipitate, which indicates a positive test and implies the presence of double
bond. Meanwhile, if KMnO4 is not decolorized, it implies for negative test which
means that double bond is not present.

PART III. Determine the % protein in whole milk

NOTE: Do this part of the experiment in the hood!!

10. Weight the watch glass in grams.

11. To the milk layer from PART I, add 1-3 drops of concentrated acid. DO NOT
smell the fumes of the acid.
12. Swirl the beaker for 30 seconds and the let sit for a few minutes.
13. Record your observation.
14. Using Buchner funnel and filter paper, filter out the liquid from the precipitate.
DO NOT discard the liquid. You will use it later.
15. Once the liquid has been separated from the precipitate, spread the filter paper
with the solid onto a watch the glass and put that on top of a water bath (250 mL
beaker, 2/3 full water on a hot plate). The purpose is to dry the solid so that you
can weigh it.
16. Weigh your solid and record the mass.
Milk from the filter paper % composition
Weight of the Weight (g) Weight (g) of the Weight (g) of
milk (PART 1 of the watch watch glass with the milk
step 6) glass milk
2g 50.489g 52.489g 1.5g 98.55%

True Value – Experimental Value x 100

 True Value

What happens when you add the concentrated acetic acid into milk solution?

The appearance of precipitate was observed which indicates the presence of

protein, which is called casein.

PART IV. Test for the presence of protein

NOTE: Still, DO NOT discard the liquid.

17. Put 1ml of milk (PART 1 step 6) as test tube 1 and 1 gram of milk (PART III step
16) as test tube 2.
18. Add about 1 ml of Biuret Reagent (0.01M CuSO4) to both test tubes, #1 and #2.
19. Add 3 drops of 6M NaOH to each test tube and mix.
20. Record your observation.

Question:
Why Biuret test is used to determine the absence or presence of proteins? Explain
by giving your reasons and reactions if necessary.

It is because Biuret test is the simplest method and a rapid way to determine and
detect the peptide bonds in a protein sample and provides stable positive color –
violet. The biuret reagent contains CuSO4 which gives the blue color, the NaOH
that provides the alkaline medium even though it doesn’t participate in the
reaction, and potassium sodium tartrate which stabilizes the chelate complex.
Therefore, the biuret test is based on the ability of Cu (II) ions to coordinate with
lone electron pairs from 4 nitrogen atoms in peptide bond to form the chelate
complex. Since the chelate complex can absorb light at 540 nm, so the color
changes from blue to violet which indicates the presence of proteins.

Are your observations consistent with your expectations? Explain briefly.

Yes. Based on the experiment, it can be observed that after adding CuSO 4 and
NaOH, the color changed to light violet, which is an indication of presence of
protein in milk.

PART V. Determining the percent water and percent Carbohydrate:

21. Label 4 test tube as #1, #2, #3 and #4. Add 2mL of Benedict’s (Fehling’s)
Solution to test tubes #1 and #2. Place them in a water bath for about 4 minutes.
22. Put about 10 drops of the liquid you filtered (PART III step 14) in a test tube #1
and #3 and a few flakes of the solid in the other test tube #2 and #4. Leave tubes
#1 and #2 in the water bath but remove from the heat. Record your observation.
 23. To test #3 and #4, add 10 drops of iodine solution and record your observation.

Questions:
What is the aim of performing Benedict’s test?

The main purpose or aim of the Benedict’s test is to determine the absence or
presence of reducing sugar in simple carbohydrates. A positive test indicates a
color change from blue to brick red.

What is the aim of performing Iodine Test?

The aim of performing Iodine test is to determine the presence or absence of

starch in a given sample. A positive test which indicates the presence of starch is
the appearance of blue-black color.

24. Weigh a clean dry evaporating dish and record the mass.
25. Add 5 mL of fresh milk to the evaporating dish and weigh again and record the
mass.
26. Set the evaporating dish with fresh milk on the top of the water bath and heat. Stir
the milk continuously to prevent burning.
27. Stop heating when the water is gone from the dish. You will no longer see steam
coming from the top of the dish. The dried milk from the stirring rod on the edge
of the evaporating dish.
28. Remove the evaporating dish and dry its bottom before you weigh it.
29. Weigh the dish with the dried milk and record.

HEATING %
COMPOSITION
Weight (g) of the Weight (g) of Weight (g) of Weight (g) of Weight (g) of Weight (g) of
evaporating dish evaporating dish milk the evaporating milk
with milk evaporating dish with milk
dish

39.25g 44.25 5g 39.25g 43.50g 5g 11.5%

True Value – Experimental Value x 100

True Value
VI. CONCLUSION

Based on the tests performed in each experiment, we were able to know and
understand the major concepts and ways in determining the percent fat in whole milk and
if it contains unsaturated fat by using Bromine solution. We were also able to determine
the percent of protein in whole milk as well as using Biuret test to detect the presence of
protein. Lastly, we were able to discover the purpose of using Benedict’s test and Iodine
test in determining the percent water and percent carbohydrate in milk sample.
Therefore, milk provides essential nutrients and is a valuable source of vitamins,
minerals, energy, and protein in human diet. Lactose is the major carbohydrate fraction in
milk. Also, about 98% of milk fat is a mixture of triacylglycerols, with much smaller
amounts of free fatty acids, mono-and diacylglycerols, phospholipids, sterols, and
hydrocarbons. In addition, milk fat also acts as a solvent for the fat-soluble vitamins A,
D, E and K and also supplies essential fatty acids (linoleic, linolenic and arachidonic).
Furthermore, milk proteins represent one of the greatest contributions of milk to human
nutrition, containing 3.5 percent protein on average. Caseins (80%) and whey proteins
(20%, such as lactalbumin and lactoglobulin) make up the protein fraction.