centrifugate.docx

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

1605608 SIAU SIEW CHING

Methods

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)


(13,000 rpm, 15 minutes)

Observation

First centrifugation

(1,500rpm , 10 minutes)

Cloudy mixture

Sediment of pellet at the bottom of centrifuge tube


(5,000rpm,15 minutes)

Two layer formed clearly

Thin layer of precipitate at the upper layer

Semi cloudy supernatant at the lower layer

Third centrifugation

(13,000rpm, 15 minutes)

Two layer formed clearly

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 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)

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-the-centrifuge [Accessed 20 Jun. 2016].

UTAR, 2016. EXP: 2 Centrifugation. Kampar.Faculty of Science, UTAR. Pp 6-7


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centrifugate.docx