PERALES - CHEM 411 Logbook Experiment 1

Perales, Kent Benedict B.
– 3CHEM1
DATE: September 7, 2020
Experiment no. 1
The separation and hydrolysis of the protein casein from a non-fat milk
OBJECTIVES
 To denature the casein protein from a non-fat milk
 To divide the protein residue into three groups, namely: acid hydrolysis solution, base
hydrolysis solution, and intact protein
 To observe the appearances that happen within the three protein residue groups
MATERIALS AND INSTRUMENTS
A. Materials
 5.0 g of non-fat milk
 50 mL graduated cylinder
 Stirring rod
 Preheated water (55 °C)
 50 mL 10 % Acetic acid
 8N H2SO4
 2.0 g of Ba(OH)2
 Saturated Ba(OH)2
 Watch glass
 Blue micropipettor (1.0mL)

 Small and Large beakers
 Small and Large Erlenmeyer Flasks
 Filter papers
 Funnel
 15 mL distilled water
B. Instrument
 Portable pH meter
PROCEDURE
The first step in the separation and hydrolysis of the protein casein from non-fat milk was
to prepare all materials and instruments needed for the experiment. Once done, the non-fat milk
was being transferred by a beaker that contained a 55°C pre-heated water as shown in Figure
1(a). Right after being transferred, the mixture should have stirred by the stirring rod. The initial
pH of the mixture should be measured using a portable pH meter as shown in Figure 1(b). A
blue micropipettor had been used by continuously dropping a 10% acetic acid into the mixture
until its pH has reached 4.6. Once the pH reached 4.6, the final volume of the 10% acetic acid in
the 50-mL graduated cylinder was recorded as shown in Figure 1(c). The set-up consisted of a
filter paper was placed within a funnel placed on the mouth of a 50 mL Erlenmeyer flask to filter
the mixture. The residue was being pressed in between filter papers to remove its excess water as
shown in Figure 1(d). Afterward, the residue separated by three parts: acid hydrolysis + 4 mL
8N H2SO4, intact protein, and base hydrolysis + 2.0 g Ba(OH)2 in 5.0 mL boiling water.
Observations were made regarding the appearance of the acid hydrolysis solution and base
hydrolysis solution. The mixture all over again observed right after it has been autoclaved for 5
hours at a pressure of 15psi. The acid hydrolysis was neutralized by the addition of saturated
Ba(OH)2. However, base hydrolysis was neutralized by the addition of 8N H 2SO4. Then, a set-up
consisted of a filter paper was placed within a funnel placed on the mouth of a 50 mL
Erlenmeyer flask. The acid and base hydrolysis solutions were filtrated and collected separately.
Then it was dissolved with the intact protein in a 50-mL beaker that contained a 15-mL of
distilled water. Once done, the three solutions would be stored and used for the next experiment.
(a) (b)
(c) (d)
Figure 1. Isolation of casein protein from non-fat milk

RESULTS
In the isolation of casein protein, as shown in Figure 1, non-fat milk was made use of to
separate the fat. Since whole-fat milk will coagulate with casein, it would be hard for it to be
separated. Aside from that, it was heated no more than 55°C because it would breakdown and
destroy the casein protein. Casein protein would be soluble in water since the net charge of the
casein protein at the normal pH of milk is negative. The casein protein managed to have an
isoelectric point of 4.6. In the experiment, it showed that the casein protein precipitated at a pH
of 4.63 with the addition of 10% acetic acid. Its color appeared to change from white to
yellowish-white.
After filtrating and removing its excess water, 2.0 g of Ba(OH) 2 has been prepared and
used for base hydrolysis of the casein protein to determine what would be its effects. The
reagents used for hydrolysis were H2SO4/Ba(OH)2 instead of HCl/NaOH because
H2SO4/Ba(OH)2 can be easily extracted after hydrolysis. The intact protein as shown in Figure 2,
it changed from solid-white to a solution after its acid hydrolysis. On the other hand, the base
hydrolysate as shown in Figure 2, it turned to a yellow solution from solid white after its acid
hydrolysis.
Figure 2. Acid and base mixtures changed colors after applying acid hydrolysis
After acid hydrolysis, autoclaving was the next step in the experiment. This process
would take for at least five hours with pressure at 15 psi. Autoclaving is being used for
sterilization that uses steam to kill all foreign contaminants under high pressure over a long
period. After autoclaving was performed, the acid hydrolyzate turned from a white solution to a
black solution as shown in Figure 3. And for the base hydrolyzate, it turned from a yellow
solution to a yellow-orange solution as shown in Figure 3.
Figure 3. Acid and base mixtures changed colors after the process of autoclaving
Right after autoclaving, the process of neutralization was performed; this is to make sure
the pH of the solution becomes 7.0 or neutral. In the experiment, saturated Ba(OH)2 was added in
the acid hydrolyzate. And for the base hydrolyzate, 8N H2SO4 was added to neutralize the pH.
The intact protein was submerged in a 15-mL distilled water to prevent it from being exposed
and contaminated. After the neutralization, the mixtures were filtrated and collected separately. It
turned from a black solution to a clear light brown solution for the acid hydrolysate as shown in
Figure 4. And for the base hydrolyzate, it turned from a yellow-orange solution to a clear
yellow-orange solution as shown in Figure 4.
Figure 4. Acid and base mixtures changed colors after the process of neutralization
POSTLAB QUESTIONS
1. Non-fat milk was made use to separate fat instead of whole fat milk because whole-fat
milk will coagulate the protein casein and it would be difficult to separate. There's no
simple way to separate the bonding.
2. Milk solution can only be heated no more than 55°C because it breakdown/destroy the
structure of its protein.
3. The principle behind the isolation of casein from milk is that most it shows a minimum
solubility at their isoelectric pH. The experiment was done through the continuous
addition of 10% acetic acid until it reached its isoelectric point of casein that is around
pH 4.6.
4. Autoclaving was used for sterilization to kill all foreign contaminants under high pressure
over a long period that were present in the mixtures/hydrolyzates.

5. H2SO4/Ba(OH)2 used instead of HCl/NaOH in the acid/alkaline hydrolysis of proteins
because they can be easily extracted once the process of neutralization has started.

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Perales, Kent Benedict B.

DATE: September 7, 2020

 To denature the casein protein from a non-fat milk

hydrolysis solution, and intact protein

MATERIALS AND INSTRUMENTS

 5.0 g of non-fat milk

 Preheated water (55 °C)

 Blue micropipettor (1.0mL)

 Small and Large Erlenmeyer Flasks

Figure 1. Isolation of casein protein from non-fat milk

reagents used for hydrolysis were H2SO4/Ba(OH)2 instead of HCl/NaOH because

solution to a yellow-orange solution as shown in Figure 3.

yellow-orange solution as shown in Figure 4.

simple way to separate the bonding.

structure of its protein.

over a long period that were present in the mixtures/hydrolyzates.

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