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
make 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 contain 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. For the students to know how to evaluate the quality of a raw milk if it is bad or good
milk.

2. To test the presence of proteins on the food samples specifically the milk, salt
solution, egg white, and mashed banana.

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
(Fehlings), 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

n/a n/a n/a n/a n/a n/a n/a

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.

After the bromine was added to the solution, from its original color which is orange
the bromine solution becomes colorless.

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 saturated because the milk contains 62% saturated fatty acids,
30% monounsaturated fatty acids, 4% polyunsaturated fatty acids, and 4% trans fatty
acids that includes in the conjugate linoleic acid.

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

Bromine decolorization is a simple qualitative test for unsaturation. Although

bromine is a dark red-brown liquid, alkenes and dibromoalkanes are colorless. When a
dilute solution of bromine in an inert colorless solvent, such as dichloromethane, is
applied to an alkene, it rapidly decolorizes. Most saturated chemicals, on the other
hand, do not decolorize bromine solutions. Oxidizing chemicals react with carbon-
carbon double bonds as well. This reaction can be used to differentiate between alkenes
and alkanes. The reagent in the Baeyer test is alkaline permanganate. The reagent is
reduced to manganese dioxide when the alkene is oxidized. The observed color shift is
from purple (MnO4) to brown (MnO2). Furthermore, given the test circumstances, most
alkanes do not react with permanganate. Alkanes are inert to cold concentrated sulfuric
acid, whereas alkenes react. They either dissolve to generate an alkyl hydrogen sulfate
or make polymers or tars, which are frequently dark in color. In this portion of the
experiment, you will use these three tests to establish that the alkene you created
contains a carbon-carbon double bond. You will also test a structurally similar but
saturated hydrocarbon alkane as a comparative.

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
n/a n/a n/a n/a n/a

True Value – Experimental Value x 100

True Value
What happens when you add the concentrated acetic acid into milk solution?
Protein may be present when concentrated acetic acid is added to milk
solution. The greater the concentration of acetic acid used, the greater the
presence of protein. According to the test, milk becomes violet, indicating the
presence of protein.

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.

Proteins can also be detected using the biuret test. This is because proteins
are made up of polypeptides, which are made up of amino acids linked together
by peptide bonds. The longer the polypeptide chain, the more peptide bonds there
are, and hence the more vivid the violet color when tested with biuret. A few
drops of this reagent will transform the color of an aqueous sample containing
peptide-bonded chemicals from pale to vivid violet. The intensity of the violet
color relies on the amount of peptide bonds in the sample.
 Are your observations consistent with your expectations? Explain briefly.

Consistently, my observations match my predictions since the results show

that the longer the polypeptide chain, the more violet color appears.

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?

Benedict reagent is a difficult blend or reagent of the pentahydrate of sodium

citrate, sodium carbonate and copper sulfate. The test was used to identify plain carbs
that reduce sugars such as monosaccharide and disaccharide in particular. The test takes
place by adding and heating the Benedict reagent in the sample, which means that
sugar is reduced when changed in color.

What is the aim of performing Iodine Test?

The test to detect starch in the sample is the iodine test, or also known as the
starch test. When the sample contains starch, iodine reacts to the sample and forms a
blue-black mix. This test is performed by adding and shaking potassium iodide solution.
The color of the sample changes from yellow brown to blue-black in the case of starch.

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)
evaporating dish evaporating dish milk the evaporating of milk
 with milk evaporating dish with milk
dish
n/a n/a n/a n/a n/a n/a n/a

True Value – Experimental Value x 100

True Value

VI. CONCLUSION

Milk is an aqueous solution made up of several components, including

carbohydrates, lipids, proteins, and phosphate. The concentration of each of these
components will vary depending on the source of the milk as well as the techniques used
to prepare the milk. A large polymeric structure composed of amino acids is called
proteins. An amino acid is a general term for one of twenty distinct compounds.
Carbohydrates are saccharides. Lactose, the primary carbohydrate in milk and is
composed of the monosaccharides glucose and galactose. Milk is an emulsified or colloid
of fat and sugar globules in an aqueous solution.