LABORATORY REPORT

Faculty: Science

Course: Biomedical Science

Unit Code & Title: UDDD 1134 Biochemistry and Metabolism

Semester: Jan

Academic Year: 2018

Title: Centrifugation

Instructor: Ms Sangeetha Arullappan

Declaration:

I certify that this assignment is my own work. Where material has been used
from other sources it has been properly acknowledged.

TAN YEU LUN

Student’s Name : ……………………………………..
16ADB02666
ID Number : ……………………………………..

Signature : ……………………………………..

Date of Submission 30.1.2018

: ........................................................
 UNIVERSITI TUNKU ABDUL RAHMAN
Faculty : FSc Unit Code : UDDD1134
Course : Biomedical Science Unit Title : Biochemistry and Metabolism
Year/Semester : Y1S3 Lecturer : Ms. Sangeetha Arullappan

Experiment 1: Centrifugation (Milk Samples)

Marking Scheme for Experiment 1:

No. Criteria Allocated Marks Assessed Marks

1. Objectives 10
2. Materials and Methods 10
3. Results 10
4. Discussion 10
5. Conclusion 5
6. References & In-text citations 5
Harvard Style
7. Overall Performance: Grammar and 10
Format
Total 60

Assessor’s Feedback:

………………………………………………………………………………………………
………………………………………………………………………………………………
………………………………………………………………………………………………
………………………………………………………………………………………………
Title: Centrifugation

Objectives:

1) To determine the relationship between the speed of centrifugation and the

separation of coagulated milk from acetic acid after centrifugation.
2) To observe the layers of suspension formed after centrifugation of mixture of
milk and acetic acid at various centrifugal speed.
3) To observe the isolation of casein from milk by adding acetic acid.

Materials:

Centrifuge, Centrifuge tubes (15 mL), Microcentrifuge tubes (1.5 mL), Micropipettes
(P1000), Pipettes and pumps, Fresh milk, Acetic acid (50%).

Methods:

1. About 4 mL of fresh milk was drawn into centrifuge tube which contained
50% of acetic acid (10 mL).
2. The centrifuge tube was inverted few times gently to mix the mixture.
3. The milk sample was centrifuged at 1500 rpm for 10 minutes to pellet the
coagulated milk solid.
4. One milliliter of milk was transferred to the centrifuge tube and centrifuged in
5000 rpm for 15 minutes.
5. To the microcentrifuge tube, 1 mL of milk was transferred and centrifuged in
13000 rpm for 15 minutes.
6. The differences between these tubes were observed at the end of the
procedure.

1
Result:

Centrifugation Result

4 mL of fresh milk and 10 mL of acetic

acid (50%) at 1500 rpm for 10 minutes

1 mL of milk sample from the first

sample at 5000 rpm for 15 minutes

1 mL of milk sample from the first

sample at 13000 rpm for 15 minutes

2
Discussion:

Centrifugation is a technique used for the separation of particles from a

solution according to their size, shape, density, viscosity of the medium and rotor
speed (Majekodunmi, 2015). The particles are suspended in a liquid medium and
placed in a centrifuge tube. The tube is then placed in a rotor and spun at a defined
speed. Separation through sedimentation could be done naturally with the earth
gravity, nevertheless, it would take ages. Centrifugation is making that natural process
much faster. The result of the sedimentation due to centrifugation depends on the
density of both sample and solution, temperature, distance of the particles
displacement and the rotation speed (Fisher Scientific, 2017). Rotation of the rotor
about a central axis generates a centrifugal force upon the particles in the suspension.
When the velocity of the rotation and radius of the rotor increase, the force exerted on
a sediment particle increases. Thus, centrifugation with higher centrifugal speed may
produce more complete separation compared to centrifugation with lower centrifugal
speed.

From the experiment, the first step of the experiment is adding the milk into
the acetic acid. After mixing the milk with acetic acid, although they are not obvious,
but there are some coagulated milk floating in the milk sample. This is because the
milk had been stored for a long period, some of the milk was coagulated before
mixing with the acetic acid. Due to long period of exposing to the environment, the
milk sample is affected by several environmental factors and forming the coagulated
milk before starting the experiment. At this stage, no layer is formed. The next step is
to separate the precipitation by centrifuging the mixture at 1500 rpm for 10 minutes.
After the first centrifugation, there is no any obvious layer separation in the milk
sample and it appears as cloudy. At the top of the sample, a thin colorless layer is
formed. The coagulated milk is settled at the bottom of the sample. The 1 mL of the
milk is extracted from the upper layer of the first sample is then transferred to another
centrifuge tube. The centrifuge tube which contained the sample is centrifuged at
5000 rpm for 15 minutes. After the second centrifugation, 2 layers are formed in the
sample. The upper layer which appears in cloudy and the lower layer appear in
colorless. Another 1 mL of the milk is extracted from the upper layer of the first
sample is then transferred to the microcentrifuge tube. The sample in the

3
microcentrifuge tube is centrifuged at 13000 rpm for 15 minutes. After high-speed
centrifugation, the clear separation of 2 layers is observed. The coagulated milk found
on the top layer which is more condensed than the upper layer of the sample which
centrifuged at 5000 rpm.

Milk is a mixture of many types of proteins. Milk proteins are classified into
three main groups of proteins which are caseins (80%), whey proteins and minor
proteins. Casein, the main protein in milk. Casein exists in milk as the calcium salt,
calcium caseinate. It is a mixture of at least three similar proteins which differ
primarily in molecular weight and the amount of phosphorus groups they contain (α, β
and κ caseins), they form a micelle, or a solubilized unit (Hurley, 2009). These
micelles are responsible for the white opaque appearance of milk. At some pH value,
the net charge of the protein will be zero. That pH value is known as the isoelectric
point (IEP) of the protein and is generally the pH at which the protein is least soluble.
For casein, the IEP is approximately 4.6. Therefore, it is insoluble in solutions of pH
less than 4.6 and it is the pH value at which acid casein is precipitated. In milk, which
has a pH of about 6.6, the casein micelles have a net negative charge and are quite
stable. During the addition of acid into the milk, the negative charges on the outer
surface of the micelle are neutralized, and the neutral casein precipitates, with the
calcium ions remaining in solution (Minard, 2000).

Acetic acid also known as ethanoic acid, CH3COOH which is the weak acid
play a role in this experiment to precipitate out the casein in the milk by lowering the
pH of the milk to reach the isoelectric point of casein at pH 4.6 (Brennan, 2017). The
coagulated milk which floating in the milk composed of casein formed after
introducing acetic acid into the milk. Although the precipitation is not obvious, still
can be observed.

There is no any obvious layer separation in the milk sample and it appears in
cloudy after the first centrifugation. This is due to the low centrifugal speed of the
first centrifugation. The low centrifugal speed causes the sample did not undergo a
complete centrifugation which means that the separation of the casein precipitate and
the acetic acid did not complete result in cloudy solution. Some of the coagulated
milk is trap at the bottom of the sample. This is due to the coagulated milk formed
before the first centrifugation and even before mixing with acetic acid. A colorless

4
layer formed at the top of the sample may be the fat contained in the milk as fat is less
dense than milk protein (Hurley, 2009). Since the density of milk is less dense than
the acetic acid, the milk will be on the top after the first centrifugation. Therefore, the
sample is drawn from the upper layer for the second centrifugation.

In the second centrifugation, the sample from the first centrifugation

undergoes centrifugation at two different speed separately which are 5000 rpm and
13000 rpm. For the sample undergo centrifugation at 5000 rpm, 2 layers are formed in
the sample. The upper layer which appears in cloudy and the lower layer appear in
colorless. The higher spinning forces result in the much more clearer separation
between the casein precipitate and the acetic acid. The thin upper layer which appears
in cloudy is the coagulated milk which is the casein precipitate while the lower layer
appears in colorless is the mixture of whey milk and the acetic acid. The whey milk is
the milk that its casein protein has been removed. The mixture at the bottom layer
showed that the separation is still not completed.

A clear separation of 2 layers is formed after high-speed centrifugation

(13000 rpm) which are the thicker top white layer and the more colorless bottom layer.
The coagulated milk found in the top layer which is more condensed than the upper
layer of the sample which centrifuged at 5000 rpm. This is due to the higher
centrifugal force allow more complete separation between the milk and the acetic acid.
The colorless layer at the bottom is left only the acetic acid and may be few
carbohydrates. From the observations of the 3 samples, we can say that the higher the
centrifugal speed, the more substances can be separated from the sample.

Last but not least, there are some precautions that need to be considered
when carrying out the experiment. The acetic acid should be treated carefully
although it is a weak acid. While adding the acid, pipette must be used instead of
pouring it out from the bottle. The rotor of the centrifuge must be balanced in the
opposite sides by a tube with similar mass in orders to ensure an equal spin. The lid of
the centrifuge must be closed tightly before starting the centrifuge (CRS LLC, 2016).
The milk sample prepared should be as fresh as possible to ensure that there is no any
coagulated milk formed before the experiment. This is to ensure that the result
obtained is more accurate. The centrifuge tube should be carried vertically and
carefully to prevent the supernatant from mixing with the pellet.

5
Conclusion:

The higher the centrifugal speed of the centrifugation which will produce a greater
centrifugal force. The greater the centrifugal force, the more complete separation
between the coagulated milk and the acetic acid.

References:

1. Brennan, J., 2017. What Is Sodium Caseinate? [online] Available at:

<https://www.livestrong.com/article/495564-what-is-sodium-caseinate>
[Accessed 26 January 2018].

2. CRS LLC, 2016. CR Scientific: Catalog: Laboratory Centrifuges: Centrifuge

Use and Safety Tips. [online] Available at:
<http://www.crscientific.com/centrifuge-safety.html> [Accessed 25 January
2018].

3. Fisher Scientific, 2017. Centrifugation Theory. [online] Available at:

<https://www.fishersci.se/se/en/scientific-products/centrifuge-
guide/centrifugation-theory.html> [Accessed 29 January 2018].

4. Hurley, W.L., 2009. Milk Composition. [online] Available at:

<http://ansci.illinois.edu/static/ansc438/Milkcompsynth/milkcompsynthresour
ces.html> [Accessed 26 January 2018].

5. Majekodunmi, S.O., 2015. A Review on Centrifugation in the Pharmaceutical

Industry. American Journal of Biomedical Engineering, 5(2), pp. 67-68.

6. Minard, R., 2000. Isolation of Casein, Lactose, and Albumin from Milk.
[online] Available at:
<http://courses.chem.psu.edu/chem36/Web%20Syn06/Exp112Syn06.pdf>
[Accessed 26 January 2018].