Title: Centrifugation

Objective:
1) To perform the proper technique of centrifugation.
2) To separate the molecules according sizes of the molecules.
3) To determine the relationship between the speed of centrifugation and the coagulation
of milk of the mixture.
Introduction
Centrifugation is a separation process which uses the action of centrifugal force to promote
accelerated settling of particles in a solid-liquid mixture. Centrifugation used to separate or
concentrate materials in liquid medium based on the effect of gravity on particles. Different
masses settle in a tubes at different rates in response to gravity. The centrifugal force increase
the settling rate by centrifuge.
Centrifuges are devices which spin carrier vessels at high rotation speeds and high
centrifugal force. Centrifugal force generated is proportional to the rotor rotation rate and the
distance between rotor centre and centrifuge tube. Multiple rotor sizes gives flexibility in
choosing centrifugation conditions. Centrifuges comes in different variety with different
speed in centrifugation.
Rotors for a centrifuge are either fixed angles or swinging buckets. Fixed angles work faster,
precipitation faster and increase relative centrifugal force for a given rotor speed and radius.
In the fixed angles rotor, the substitute are forced against the side of centrifuge tube, and slide
down the wall of the tube. Abrasion of the particles along the wall of the centrifuge tube
occurs. Conversely, a swinging bucket, the materials must travel down entire length of the
centrifuge tube through the media within the tube. Swinging bucket appears to have a lower
relative centrifugal force due to the viscosity of the media substance and takes longer to
precipitate. The swinging bucket usually used to separate molecules or organelle on the basis
of their movements through a viscous field.
Materials
Centrifuge (1,500rpm, 5,000rpm, 13,000rpm) Centrifuge tubes (15mL), Micro centrifuge
tubes (1.5mL), Micropipettes blue tip(P-100), Pipettes (5mL,10mL),Pipette pump, Fresh
Milk, 50% Acetic acid, Test tube holder, Micro centrifuge holder, Beaker, Conical flask
Methods
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1. Approximately 4 mL of fresh milk was draw into a centrifuge tube which contains d
50% of acetic acid (10 mL).
2. The mixture was gently inverted several times.
3. The milk sample was centrifuge at 1,500rpm for 10 minutes to pellet the coagulated
milk solid.
4. Approximately 1 mL of milk was transfer to the centrifuge tube and centrifuged in
5,000rpm for 15 minutes.
5. About 1 mL of milk was transfer to the micro centrifuge tube and centrifuged in
13,000rpm for 15 minutes.
6. The differences between these tubes at the end of the experiment were recorded in
result.
Results

First centrifugation
(1,500rpm , 10 minutes)

Second centrifugation
(5000rpm,15 minutes)

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Second centrifugation
(13,000 rpm, 15 minutes)

First centrifugation

Observation
Cloudy mixture

(1,500rpm , 10 minutes)

Sediment of pellet at the bottom of centrifuge tube

Second centrifugation

Two layer formed clearly

(5,000rpm,15 minutes)

Thin layer of precipitate at the upper layer

Semi cloudy supernatant at the lower layer

Third centrifugation

Two layer formed clearly

(13,000rpm, 15 minutes)

Thin layer of precipitate at the upper layer

Clear solution at the lower layer

Discussion
In the experiment, the fresh milk was added with 50 % of acetic acid. Acetic is an organic
compound which is a weak acid. The purpose acetic acid added to the fresh milk is to
coagulate and precipitate casein which is present in milk by alter the pH and isoelectric point
of the casein. However, the precipitate of the casein cannot seen clearly in the experiment.
Thus, centrifugation was carried out to separate the mixture.
At the first centrifugation, which carried out in the centrifuge at low speed 1,500 rpm for 10
minutes. The separation did not complete, the mixture still remain cloudy, the large size pallet

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sediment at the bottom of the centrifuge tube. Thus, the casein precipitate did not separated
completely at low speed.
In the second centrifugation, the mixture was transfer into the centrifuge tube and place in
the centrifuge at 5000 rpm for 15 minutes. After 15 minutes, two layers formed in both. The
upper layer was the thin layer of solid coagulated milk (casein) whereas the lower layer was
the semi cloudy acetic acid solution. When rotor spin, the particles of the mixture will
separate according to the sizes and density towards gravitational force. Coagulated milk
located in the upper part due to its lower density.
The second centrifugation also carried out where 1 mL of the mixture is transfer to the micro
centrifuge tube and centrifugation at 13,000 rpm at 15 minutes. This result in formation of
two layers, the upper layer is the coagulated milk which is more condensed than the second
centrifugation. Conversely, the lower layer is the clear acetic acid solution. The separation is
more obvious than the second centrifugation in centrifuge tube due to the increased of speed
and centrifugal force.
Several precaution should be noticed in the experiment which is two different pipette is used
to pipette the acetic acid and fresh milk to prevent contamination. By mixing the acetic acid
and fresh milk, the mixture must not shake too vigorously to prevent bubbles that would
affect the spinning in the centrifuge. The libs of the centrifuge must closed before operating.
Moreover, the centrifuge must be balance which samples must be arranged oppositely with
same size and same volume in the centrifuge. When transfer the mixture in to the centrifuge
tube and micro centrifuge tube, the volume of the sample should be lower than the maximum
scale of the centrifuge tube to prevent spilling of the sample that may spoil the centrifuge.
Conclusion
The increase of the spinning speed produce higher centrifugal force and the separation of the
coagulated milk more efficiently.
References
CENTRIFUGATION
In-text: (Public.asu.edu, 2016)

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Public.asu.edu. (2016). Centrifugation. [online] Available at:

http://www.public.asu.edu/~laserweb/woodbury/classes/chm467/bioanalytical/centrifugation
%20notes.html [Accessed 20 Jun. 2016]
CENTRIFUGATION LENNTECH
In-text: (Lenntech.com, 2016)
Lenntech.com. (2016). Centrifugation Lenntech. [online] Available at:
http://www.lenntech.com/library/clarification/clarification/centrifugation.htm [Accessed 20
Jun. 2016].
DIFFERENTIAL CENTRIFUGATION
In-text: (Ruf.rice.edu, 2016)
Ruf.rice.edu. (2016). differential centrifugation. [online] Available at:
http://www.ruf.rice.edu/~bioslabs/methods/fractionation/centrifugation.html [Accessed 20
Jun. 2016].
Introduction to the Centrifuge: the Pellet and the Supernatant | Protocol
In-text: (Gregg et al., 2016)
: Gregg, C., Kyryakov, P., Titorenko, V., Staggs, S., See, M., Dubey, J., Villegas, E., Dunford,
E., Neufeld, J., Huntley, J., Robertson, G., Norgard, M., Hoffmann, C., Finsel, I. and Hilbi, H.
(2016). Introduction to the Centrifuge: the Pellet and the Supernatant | Protocol. [online]
Jove.com. Available at: http://www.jove.com/science-education/5019/an-introduction-to-thecentrifuge [Accessed 20 Jun. 2016].
UTAR, 2016. EXP: 2 Centrifugation. Kampar.Faculty of Science, UTAR. Pp 6-7

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