Centrifugal_Separation_v2c.docx
CentrifugalSeparation
An exploration of the similarities between density and molecular separation techniques using
centrifugation.
1
1.1
OBJECTIVES
EXPERIMENTAL GOAL
Students will use small polyethylene microspheres to observe the differences between gravity and
centrifugal force and to use solutions of different densities to determine the density of a specific sample or
molecule.
1.2
PREREQUISITE SKILLS AND KNOWLEDGE
Students should have some familiarity with using Excel. Prior knowledge from the density and buoyancy
lab should be applied here.
1.3
RESEARCH SKILLS
After this lab, students will have had practice in:
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1.4
following laboratory protocols
using a laboratory notebook
using a mini-centrifuge
making a percent solution
diluting solutions
organizing data
using Excel to streamline calculations
LEARNING OBJECTIVES
After this lab, students will be able to:
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Calculate the density of an object.
Determine what density of solution is needed to make a small object or molecule float or sink.
Determine the centrifuge speed necessary to more quickly separate a objects in a fluid
2
2.1.1
PRE-EXPERIMENT ASSIGNMENT
Density
Density is an intensive property, in that it is intrinsic to the material and does not depend on how much of
the material you have. In this lab, we will be interested in mass density, the proportionality of mass to
volume of a given material.
The symbol for mass density (and for many other densities as well) is the Greek letter  (rho). The mass
density of a given amount of a substance is the ratio of its mass m to its volume V:
 = m/V
Since density is a derived unit, it does not have its own SI unit, but is composed of the SI units kilogram
(kg) and meter (m): kg/m3. While a commonly used unit of density is g/mL, in this lab you will use SI
units.
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2.1.2
The Centrifuge
Operation of the centrifuge is straightforward:
Always make sure the rotor is loaded symmetrically. If you have only a single tube to spin, fill a
second tube with the same weight of material to place directly across from it.
Once the tubes are in place, put the stainless steel top over the tubes, and make sure it is securely
attached. It should snap on.
Close the hinged lid until it clicks into place.
Use the arrows to select the time and speed.
Click on the START/STOP button to start the run.
If you hear anything clattering, push the START/STOP button to abort the run. There is something
loose that needs to be attended to.
When the run is complete the centrifuge will stop and the hinged lid will open automatically.
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Centrifugal Separation 1 |3
2.1.3
Centrifugal* vs. Centripetal Force
Centrifugal force, Fc, is the force that causes an object moving in a circular path to move outward and
away from the center of motion. Centrifugal force should not be confused with centripetal force which is
the force that is necessary to keep an object moving in a curved path and that is directed inward toward
the center of rotation.
As a kid, do you remember riding the Gravitron or
Starship (shown to the right) ride at amusement
parks? This ride uses both centrifugal and centripetal
force for your enjoyment. As you spin, you reach a
speed of 24 rotations per minute, which is the speed
needed to force your body against the side of the
starship. It is the side of the starship that is the
centripetal force to keep your body moving in a
circular motion. It is this combination of body mass
and speed that is needed to create an appealing ride,
dependent on both centrifugal and centripetal force.
The difference between centripetal force, and the
fictitious centrifugal force can be best represented in
the image to the right. As the individual is rotating an
object on a string, the centrifugal force causes the object
at the end of the string to be pushed outward. The string
restricting the outward movement of the object is the
centripetal force acting on the object.
Centrifugal force is the force that acts on an object in a
spinning environment, such as a centrifuge. The object’s
mass m, the rotational speed v, and distance r from the
center of the rotational axis all affect the centrifugal
force, as described in Equation (1).
(1)
The velocity of a centrifuge is not given in meters per
second, however, but in rotations per minute RPM.
How would you compute this conversion (from RPM to meters per second)?
To answer this question it might be helpful to recall that the circumference of a circle is 2πr.
Consider the Mini-Spin centrifuge you have been using in the X-Lab, which has an r = 6.00 cm. Calculate
the velocity in m/s if the rotor is moving at 1000 RPM.
Now calculate the centrifugal force felt by one microsphere from Tube Number 1 in section 3.3.
Compare the equation for the centrifugal force to Newton’s Second Law (refer to the Motion lab).
Compare the acceleration experienced by the tiny microsphere from Tube Number 1 to the acceleration
due to gravity (g = 9.8 m/s2). How many g does the microsphere experience?
*
NOTE: In Newtonian physics, the term “centrifugal force” describes a fictitious force. Refer, for
example, to the following links: https://en.wikipedia.org/wiki/Centrifugal_force and
http://www.physicsclassroom.com/class/circles/Lesson-1/The-Forbidden-F-Word
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2.1.4
Centrifugal Separation of Biomolecules
Centrifuges are used to speed up separation of large
biomolecules in laboratory settings. For example, E. coli
cells can be separated quickly from an LB growth medium
solution via centrifugation. This process can occur without
centrifugation by gravitational force, FG. However,
centrifugal force, FC, is commonly used to speed up this
separation process. Shown in the image to the left are three
forces that affect a cell or biomolecule in solution: buoyant
force, FB, drag force, FD, and gravitational force, FG. Based
on the image to the left, in which direction is the object
moving?1
Recall that an object will move in the direction determined
by the forces of gravity and buoyancy. Combining these
gives a sort of modified force of gravity:
(1)
.
When a centrifuge is used to separate cells, biomolecules,
.
and other particles, fast rotational force multiplies this
Thus the acceleration of the particle is often given in
multiples of the acceleration g due to gravity.
What if the buoyant force is greater than the gravitational force for the object in a particular fluid? In
which direction will the object move if the object in the fluid is placed in a centrifuge?
The time it takes for suspended particles to reach the top or bottom of a centrifuge tube—thus separating
from the solution—is called the separation time. Often, the goal of a centrifugation is to produce a pellet
at the bottom of a centrifuge tube, so that the fluid above (supernatant) can be easily removed either by
decanting or pipetting. What factors do you think have an effect on the separation time?
2.2
PREPARE FOR THIS EXPERIMENT
Read through the entire lab procedure and prepare your lab notebook, including the calculations to make
1 mL of 6%, 10%, and 14% w/v sucrose solutions from a 20% w/v sucrose sample. Include any necessary
formulae. Set up an Excel table to simplify the necessary calculations.
When you feel ready for the lab, test your preparation with the Pre-Experiment Quiz on e-Learning.
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3
3.1
LABORATORY MANUAL
MATERIALS CHECK OFF LIST
Each group of (2 - 3) students will have:
Laptop computer
5 mL of 20%w/v sucrose solution
One microfuge tube with blue microspheres labeled 1
One microfuge tube with blue microspheres labeled 2
4-5 tubes with a small amount of pink microspheres labeled 3
source of DI water
50 mL beaker
Set of automatic micropipettes with associated tips
1 or 2 groups will share:
Mini-Spin centrifuge
Tube
Number
Diameter
-4
Density
Color
3
1
BLPMS
3.55 x 10 m
1075 kg/m
Blue
2
BLPMS
1.25 x 10-4 m
1075 kg/m3
Blue
-
Pink
3
3.2
Product #
Microsphere
UV-PMS-BR
-5
5.3 x 10 m
SAFETY AND WASTE DISPOSAL PROTOCOLS, INCLUDING WASTE LABELS TO BE PREPARED
A lab coat, leg coverings, and closed-toe shoes must be worn. Eating, drinking or applying lotion to the
skin is not allowed in the laboratory.
Keep water away from electronics and electrical outlets. Do not discard the polyethylene microspheres
after lab. Give the microfuge tubes containing the solution and microspheres to your instructor to
save for future use.
3.3
EXPERIMENTAL PROCEDURE
3.3.1
Determine the effect of particle size on separation
Note the density and diameter of each microsphere in the table above.
Q1. Record differences in the visible size of the microspheres in tubes 1 and 2. Which tube contains the
largest microspheres?
Q2. Calculate the mass of one microsphere from Tube Number 1 and one from Tube Number 2.
Q3. The density of water is 998.2 kg/m3. Do you predict these microspheres to float or sink when added
to water? Why?
1. Add 1 mL of distilled water to each of the microfuge tubes labeled 1 and 2, containing blue
polyethylene microspheres.
2. Close the lids and invert the tubes vigorously, or quickly vortex to ensure all the microspheres are
suspended into the water.
Q4. What happens to the microspheres when you hold the tube upright and still for 30 seconds? Do the
microspheres sink or float rapidly, or does it take time for separation to occur?
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Q5. Consider the differences in separation rate. What properties of the microspheres are different?
Discuss the possible effect of each difference on the separation rate.
3. Invert or vortex both tubes a few times so that the polyethylene microspheres have been resuspended and fully mixed within the water.
4. Place both tubes 1 and 2 in the micro-centrifuge and centrifuge at 5,000 RPM for a “short spin”.
Observe and record their separation.
5. Invert or vortex both tubes a few times so that the polyethylene microspheres have been resuspended and fully mixed within the water.
6. Centrifuge both tubes at 5,000 rpm for 60 seconds. Observe and record their separation.
Q6. Is the separation of these microspheres dependent on time?
Q7. Describe in words the relationship between particle density, centrifugal force, and time of rotation.
7. Invert or vortex both tubes a few times so that the polyethylene microspheres have been resuspended and fully mixed within the water.
8. Place both tubes 1 and 2 in the micro-centrifuge and centrifuge at 2,000 rpm for a 30 seconds.
Observe and record their separation.
9. Invert or vortex both tubes a few times so that the polyethylene microspheres have been resuspended and fully mixed within the water.
10. Centrifuge both tubes at 10,000 rpm for 30 seconds. Observe and record their separation.
Q8. Is the separation of these microspheres dependent on rotational velocity? If so, in what way?
Q9. Describe in words the relationship between particle density, centrifugal force, and rotational
velocity.
3.3.2
Determine the density of a particle
Calculate the volume of 20% w/v sucrose solution and water needed to make 1 mL of each solution in the
following table of % w/v sucrose dilutions.
1. On the top of one of the tubes labeled 3, write “10% w/v”. Find this w/v percentage in the table
below.
Density of Sucrose Solutions
w/v % Sucrose
0
2
4
6
8
10
12
14
16
18
20
Density (kg/m3)
998.2
1006.0
1013.9
1021.9
1029.9
1038.1
1046.5
1054.9
1063.5
1072.1
1081.0
Q10. Record your observations of the polyethylene microspheres in tube 3. Describe color and size. Do
they look like individual spheres or just powder?
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Q11. From what you learned in section 3.3.1, based on your observation of these microspheres, do you
think they will separate quickly just by standing in the earth’s gravitational force or will they need
centrifugal force to separate quickly?
2. Add the calculated amounts of 20% w/v sucrose solution and DI water to this tube to produce 1
mL of 10% w/v solution.
3. Vortex the tube so the sucrose solution is completely mixed and the polyethylene microspheres
are fully mixed in the solution.
4. Centrifuge the sample with a matching tube for balance for 60 seconds at 10,000 rpm, or until the
microspheres have formed a distinct floating or sunken layer.
Q12. Did the polyethylene microspheres float or sink in the 10% w/v sucrose solution? Does this mean
the microspheres have a higher or lower density than 1038.1 kg/m3?
5. Create a table in Excel with the following columns: % w/v sucrose, density (kg/m3) of sucrose
solution, and float/sunk. After each centrifugation, record your observations of the polyethylene
microspheres in this table. State whether they floated on top or sank to the bottom for the given %
w/v sucrose solution.
6. For your second vial labeled 3, calculate and add the % w/v sucrose solution that lies between
10% w/v sucrose solution and the 0% or 20% w/v sucrose solution that would cause your
microspheres to do the opposite of what they did prior. For example: If your microspheres sank
in 10% w/v sucrose solution, calculate and make a sucrose solution at the midpoint of the
concentrations that would cause your microspheres to float instead.
7. Repeat steps 3-6 until you have found the two sucrose solutions within 2% w/v of each other in
which your microspheres either float or sink.
Q13. Based on the two % w/v sucrose solutions in which the microspheres either floated or sank, state
the range of values for the density of these microspheres.
Q14. Predict what would happen if you were to suspend the microspheres into a sucrose solution with the
same density as the microspheres.
If you have microspheres and time left, test your prediction.
3.4
POST-LAB ASSIGNMENT
Q15. Describe in detail how you would create a table of %w/v sucrose solution densities. What
laboratory glassware and materials would you use? If density is equal to mass/volume, specifically
describe what mass you are measuring and what is the volume.
Q16. If you had a microfuge tube containing a mixture of three polyethylene microspheres of varying
densities 1.000g/cm3, 1.025g/cm3, and 1.07g/cm3 how could you separate them using what you
know about density? Which sucrose solutions would you need? Would you need to transfer some of
the microspheres to a new microfuge tube? Describe this process.
Q17. Try to think of a few ways that centrifugal force might be used outside of the laboratory. Is this
force used in your home? What are some ways that centrifugal force might be helpful in areas
outside of the laboratory? Could you use a centrifuge in your kitchen?
Q18. In the field of physics, centrifugal force is described as a “fictitious” or “false” force. Search google
for information that will help you understand what a physicist means by fictitious or false force and
describe the argument. Based on your research, rewrite section 2.1.3.
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