Title: Centrifugation Objective: To investigate the process of centrifugation on mixture of fresh milk and acetic acid.

Introduction: In order to separate or concentrate material that is suspended in a liquid medium, a process called centrifugation can be carrying out. This technique is based on the effect of gravity on particles in suspension. In a tube, two particles of different masses will settle at different rate in response to gravity. The settling rate of an instrument called centrifuge can increase by using centrifugal force. In a variety of applications, centrifuge is used to spin carrier vessels that are centrifuge tubes at high rotation speeds and very high centrifugal force. The centrifugal force generated is proportional to the rotation rate of rotor and the distance between the rotor center and the centrifuge tube Therefore, multiple rotor sizes is used to give flexibility in choosing centrifugation conditions. Each centrifuge has a special graph, which is nomograph or a table which relates rotation rate to centrifugal force for each size of rotor it accepts. Centrifuge have three general classes, that is low speed, which is up to about 5000 rpm, high speed machines which is up to about 25,000 rpm and ultracentrifuges which will turn at up to 100,000 rpm. There are another form of centrifuge that is microcentrafuge. These are simple machines used with 0.5 or 1.5 mL disposable plastic tubes. Most of these machines generate between 10,000 and 13,000 rpm. Many microcentrifuges only have settings for speed (rpm), but not relative centrifugal force. Consequently, a formula for conversion is required to ensure that the appropriate setting is used in an experiment. The relationship between rpm and rcf is as follows: g= (1.118x10 ) rs . Where g is the relative centrifugal force, r is the radius of the rotor in centimeters, and s is the speed of the centrifuge in rpm. Rotors for a centrifuge are either fixed angles or swinging buckets. Fixed angles generally work faster, in which substances precipitate faster and they have an increased relative centrifugal force for a given rotor speed and radius. In a fixed angle motor, the materials are forced against the side of the centrifuge tube, and then slide down the wall of the tube. This action will lead to abrasion of the particles along the wall of the centrifuge tube. For a swinging bucket, the materials must travel down the entire length of the centrifuge tube through the media within the tube. Since the media is usually viscous substance, the swinging bucket appears to have a lower relative centrifugal force, and it takes longer to precipitate anything contained within.

Material: 1. 2. 3. 4. 5. 6. Centrifuge Centrifuge tubes (15 mL) Microcentrifuge tubes (1.5 mL) Micropipettes Fresh Milk Acetic acid

Results: Speed of centrifuge (rpm) Time taken for centrifuge to (minutes) 10 15 the Observation spin Milky solution remain unchanged. Two layers of solution was formed but not that obvious. Two layers was formed obviously in the solution. A milky white pellet was formed at the upper layer with the pale yellowish white layer formed at the lower layer.

1,300 5,000

13,000

15

Firstly, 5 mL of fresh milk was drawn into a centrifuge tube which contain 10 mL of 50% of acetic acid and was inverted few times to mix. Next, the milk sample is centrifuge at 1,300 rpm for 10 minutes to pellet the coagulated milk solid. After 10 minutes the milk sample has been centrifuged, the milky white solution is maintained unchange. After that, 1 mL of the milk was transferred to the centrifuge tube and centrifuge in 5,000 rpm for 15 minutes while another 1 mL of milk was transferred to the microcentrifuge tube and centrifuge in 13,000 rpm for 15 minutes. For the milk sample that undergo centrifugation process in 5,000 rpm, two layers of solution was formed but hardly to see the two layers formed very clearly. For the milk sample that undergo centrifugation process in 13,000 rpm, two layers was formed in the solution. A milky white pellet was formed at the upper layer with the pale yellowish white layer formed at the lower layer.

Discussion: Milk is an emulsion of fat, water, lactose, and a mixture of proteins. The proteins are mostly of a kind called caseins, which have two discernable ends, one likes to be surrounded by water and hates to be surrounded by fats, and the other end is the opposite. In milk these proteins form little spheres called micelles, with the hydrophobic sides all facing in and the hydrophilic sides facing out, that is facing the water. For each protein, their end sticking into the water grabs electrons and winds up with a negative electric charge. All the little spheres are then negatively charged and repel each other, which stops them from clumping together. Therefore, milk stays as milk. An acid is any chemical which loves to give positively charged protons away. The stronger the acid, the stronger the tendency to force protons onto other chemicals. When the acetic acids is added to the milk, the positive hydrogen atoms get attracted to the negative micelles, making them neutral. Now there is no force to push the micelles apart, and eventually they hit each other and stick together, making a tangled mess of protein called curds. For further explanation, in normal conditions milk has a pH of about 6.5-6.7 and at this pH value the casein is without protons, it has a negative charge and therefore the casein micelles are relatively soluble, because they repel each others. In an acid ambient milk coagulates, because casein has its isoelectric point at 4.6 pH, that is at this pH value it has a quantity of positive charges equal to the quantity of negative charges and the positive part of each "micelle" is attracted by the negative part of the others, causing the formation of ionic bonds among the "micelle" working against the dipole-dipole bonds with water, so that the protein precipitates in the form of demineralized casein. From the experiment that we conducted, the milky white materials was formed at the upper layer of the solution in centrifugation process of 13,000 rpm is casein, which is one of the protein that exist in the composition of milk. Conclusion: Through experiment of centrifugation on mixture of fresh milk and acetic acid that we carried out, the milky white materials was formed at the upper layer of the solution in centrifugation process of 13,000 rpm is casein,

References: does milk curdle when acid is added? Retrieved from http://www.boston.com/business/articles/2009/10/26/acid_is_the_key_to_making_ milk_curdle/ on 25.1.2011 2. Acid is the key to making milk curdle. Retrieved from http://www.itisacqui.it/sitob/formagette/curdling.htm on 25.1.2011
3. The 1. Why

Curdling of Milk. Retrieved http://wiki.answers.com/Q/Why_does_milk_curdle_when_acid_is_added on 25.1.2011

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