Centrifugation

Question 1

Uses of centrifugation

Answer 1

Preparative - to separate
Analytical

Question 2

Analytical applications of centrifugation

Answer 2

o Mr of proteins
o Density
o Shape
o Association/Disassociation

Question 3

Movement of particles depends on… (5)

Answer 3

• Applied force
• Density difference with solvent
• Size of particles
• Shape of particles
• Viscosity

Question 4

The rate at which particles sediment in a centrifuge depends on … (4)

Answer 4

o Centrifugal force
o Buoyancy
o Resultant force
o Velocity produced

Question 5

Centripetal force

Answer 5

Force keeping an object moving in a circle.
Acts towards the centre

Question 6

Centrifugal force

Answer 6

Centripetal force → tube & liquid in circle

Question 7

Centrifugation means that over time particles move _

Answer 7

down the tube

Question 8

What is the Archimedes principle?

Answer 8

Upthrust force = weight of solvent displaced
V0 (solvent volume displaced) = Vp (particle volume)

Question 9

What is the resultant force

Answer 9

Resultant force = Centrifugal – Upthrust

Question 10

What is the diffusion coefficient?

Answer 10

How long a particle takes to diffuse across a volume

Question 11

What is the sedimentation velocity?

Answer 11

Speed at which particle moves away from rotation axis

Question 12

What is the sedimentation coefficient s?

Answer 12

Speed of sedimentation / speed of centrifuge

Question 13

If you know size of particle (M) and speed of sedimentation required, you can determine _

Answer 13

w
Preparative centrifugation

Question 14

What do you do in analytical centrifugation?

Answer 14

Set w, determine speed of sedimentation (dx/dt) and D => M
Can calculate mass of proteins accurately

Question 15

Fractionation

Answer 15

Precipitation with increasing salt ie: (NH4)2SO4
To find approximately where precipitation occurs

Question 16

Stages of preparative centrifugation (2)

Answer 16

Precipitation
Cell fractionation

Question 17

What is the sedimentation pattern in analytical centrifugation

Answer 17

o Where sediment is and when
o End with clear solvent with some back diffusion

Question 18

Fitting absorbance curves to Svedburg equation gives s and D and hence _

Answer 18

M

Question 19

Summary of analytical centrifugation

Answer 19

• Method uses sedimentation velocity.
• Absolute method for native Mr
• Sedimentation pattern shows some back diffusion

Question 20

What is the sedimentation equilibrium?

Answer 20

the point at which there is an equilibrium between sediment and solution.
Can use the concentration to give Mr

Question 21

Sedimentation velocity vs sedimentation equilibrium

Answer 21

Sedimentation is fast and good for multiple non-interacting species
Sedimentation equilibrium is slower and gives M of structure, good for single or interacting species

Question 22

True or false:
Both sedimentation velocity and equilibrium give absolute values

Answer 22

True

Question 23

True or false:
Both sedimentation velocity and equilibrium have constant solvent density

Answer 23

True

Question 24

What is density gradient centrifugation?

Answer 24

Sedimentation (Velocity or Equilibrium) in uniform solvent
Separation by size (M) with some shape (D)
Separation by density

Question 25

Two types of Density Gradient Centrifugation

Answer 25

1. Pre-formed gradient: rate zonal centrifugation 2. Gradient formed during experiment: equilibrium (isopycnic)

Question 26

So particle will sediment until the _ of the protein reaches the _ of the solvent

Answer 26

Density

Question 27

Rate zonal centrifugation

Answer 27

Equivalent to sedimentation velocity Material layered on top Sediment on basis of size Slows as rp approaches "ρ0" Fractionate at end of run to remove

Question 28

Equilibrium or isopycnic gradient centrifugation

Answer 28

Solvent usually salt solution e.g. CsCl Gradient forms on centrifugation Simpler experimental setup Molecules move up or down until point where it has the same density as the solvent. Opposed by diffusion, therefore finite band width

Question 29

Rate zonal vs isopycnic

Answer 29

Rate zonal: shallow gradient, time dependent and size separation Isopycnic: steep gradient, sufficient time and density separation

Question 30

The centrifuge apparatus needs to be _

Answer 30

Balanced

Question 31

Swing out rotor

Answer 31

Low speeds, low rmax, used in challenging separations

Question 32

Fixed angle centrifuge

Answer 32

High speed, also micro centrifuges rmax short so more effective pelleting

Question 33

Vertical centrifuge

Answer 33

Isopycnic, high/ultra speeds No pellets formed, no harvesting

Question 34

Relative centrifugal force (RCF)

Answer 34

Multiple of acceleration due to gravity (g-value) Depends on speed, n (revs per minute) And radius of rotation r, (mm) There isn’t a constant RCF throughout the tube, so we use an average.