Experiment 2: Centrifugation Title: Centrifugation of fresh milk and Acetic Acid Introduction: Centrifugation is a process which materials suspended

in a liquid medium undergo separation or concentrate due to the effect of gravity. Particles with different density and masses are settled at different rates in a tube which are response to gravity. Centrifuge is an instrument which can spin carrier vessels at high rotation speed and very high centrifugal force to allow centrifugation. The centrifugal force generated is proportional to the rotation rate of the rotor and the distance between the rotor center and the centrifuge tube. There are three general classes of centrifuges: low speed, about 5000 rpm, high speed machine can up to about 25000 rpm and ultracentrifuges turn at up to 1000000 rpm. Microcentrifuge are common in laboratories which generate between 10000 rpm and 13000 rpm and 0.5 or 1.5 mL of disposable plastic tubes are used. Microcentrifuge can only setting for speed (rpm) but not relative centrifugal force (rcf): relative centrifugal force, g=(1.118 10-5)rs2 (r=radius of the rotor in cm, s=speed of centrifuge in rpm) Objective(s): 1. To separate coagulated milk from mixture of fresh milk and Acetic Acid

by Centrifugation. 2. To determine the relationship of speed of centrifugation with the

coagulation of milk which separate from mixture 3. To study the process of centrifugation

Materials: Centrifuge, Falcon tube (50mL), Centrifuge tubes (15mL), Microcentrifuge tubes (1.5mL), Micropipettes, blue tips (P-1000), Pipettes (10mL & 5mL), Plastic Pipette Pump, Fresh Milk, 50% Acetic Acid Procedure: 1. 10mL of 50% of acetic acid was drawn by pipettes with plastic pipette

pump and released into 50mL Falcon tube. 5mL of fresh milk was then drawn and released into falcon tube where 10mL of acetic acid contained. 2. 3. Falcon tube was inverted gently few times and both solution mixed. Milk sample was centrifuged at 1300 rpm for 10 minutes, the

coagulated milk solid was pelleted. 4. 1mL of milk was transferred from falcon tube to centrifuge tube. The

centrifuge tube was centrifuged in 5000 rpm for 15 minutes. 5. 1mL of milk was transferred from centrifuge tube to microcentrifuge

tube. The microcentrifuge tube was centrifuged in 13000 rpm for 15 minutes. 6. Differences between these tubes was observed and drawn in result at

the end of the procedure.

Results: Centrifugation First Centrifugation Centrifuge tube

Second Centrifugation

Third Centrifugation

Discussion: Centrifugation is a type of separation techniques which separation of particles within different masses suspended in solution are based on the centrifugal field and assume that density, relative molecular mass and shape are under investigation and their separation affected by gravitational field and centrifugal force. Centrifuge is instrument that spin samples in high speed which can allow solution in centrifuge tube undergo centrifugation. The rotor speed indicates the centrifugal separation of particles. The rotor is accelerated to a certain speed, and particles move at a rate which determine by size, shape and by the centrifugal force. Acetic acid is an organic compound with the chemical formula CH3CO2H and it is a type of weak acid. 50% of Acetic acid was added in this experiment is to clot and precipitate out casein from milk by adjust the pH and isoelectric point of casein. After mixing 50% of Acetic acid with milk, the precipitate of casein cannot be seen clearly, this is the reason why we place the centrifuge tube that contain mixture into centrifuge. The first centrifugation centrifuge sample at 1,300 rpm for 10 minutes, but the sample in centrifuge tube after 10 minutes does not have obvious layer separation, the sample appear as cloudy solution. This is due to the separation of casein precipitate with acetic acid does not complete in lowspeed centrifugation. Since the separation do not complete, the experiment continue with second centrifugation which 1mL of the bottom layer of the sample that centrifuged was transferred in to another centrifuge tube. Second

centrifugation occurs in 5,000 rpm for 15 minutes result in 2 layers of sample in the centrifuge tube. The upper layer is the cloudy coagulated milk (casein) and the lower layer is the colourless acetic acid. When the rotor spin in high speed, particles which are initially mixed together will separate due to its size and density towards gravitational force. Coagulated milk locates at the upper layer due to its lower density. Although the layer separation can be seen after second centrifugation, the separation do not consider complete yet. Upper layer of the sample that undergone second centrifugation was transferred about 1mL to microcentrifuge tube and third centrifugation centrifuge in 13,000 rpm for 15 minutes. This result in separation of 2 layers, the upper layer is the milky coagulated milk layer which is more condense than the upper layer of second centrifugation and the lower layer is the colourless layer of acetic acid. This is because it had been gone through two times centrifugation, the separation of coagulated milk from acetic acid is more to complete in this third centrifugation and the higher speed of spinning causing the coagulated milk separate more completely due to greater centrifugal force. There are some precaution must be follow to avoid any accident happen in the laboratory and spoiling of any devices. Short-term exposure to acetic acid would cause irritation of eyes, lungs, throat and nose. Avoid acetic acid in contact directly to eyes, skin and mouth is the best way of prevention. Libs of the centrifuge must be closed before operating. Content to be centrifuge must be balance before turning on the centrifuge. Samples must be arrange oppositely with same size and same volume in the centrifuge. Make sure the rotor is stop before opening the centrifuge lids. Specified tube for

certain centrifuge must be used. Tube must not contain volume more that its capacity so that sample in tubes would not spill during rotor spinning. These steps must be follow when using centrifuge to prevent spoiling.

Conclusion: The increase of rotor spinning speed leads to greater centrifugal force and the separation of coagulated milk from acetic acid more completely.

References 1. Wilson.K and Walker.J.M, 2000. Principles and Techniques of Practical

Biochemistry, 5th ed. United Kingdom: The Press Syndicate of the University of Cambridge, p 263. 2. Babara H.E et al, 2000. Basic medical Laboratory Techniques, 4th ed.

Delmar Cengage Learning, p 69. 3. Spurlock. D, 1999, Isolation and Identification of Casein From Milk Notes [online]. Available at:

Course

http://homepages.ius.edu/dspurloc/c122/casein.htm [Accessed : 25 January 2013]