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Ultra-centrifugation and Sedimentation

1.Introduction
The ultra-centrifuge plays an important role in biology and medicine. It is used in both
preparative and analytical applications. The former generally involves isolating a desired
component from a mixture. The latter refers to determining some properties of macromolecules.
The process depends upon Newtonian point mechanics and the sedimentation process. The latter
involves the properties of fluids.
The ultracentrifuge was developed in the early twentieth century by the Swedish physical
chemist Theodore Svedberg at Upsalla University as part of his study of colloids. He recieved
the Nobel Prize in chemistry in 1926.
The basis of the relevant Newtonian mechanics is the influence of force on motion.
Essentially if a force acts on a particle the particle motion is altered. More specifically the
particle velocity changes. The rate at which this change occurs is called the acceleration and is
proportional to the force. The constant of proportionality is the mass. In the simplest of situations
this can be represented mathematically by introducing the symbols, F,m, and a for force to give
F'ma

(1)

As written, this equation implies certain assumptions. It assumes, without more detailed
definition, that only one force acts upon the object. To correct this F must be defined as the sum
of all forces acting on the object. To make this more clear the above relation could be written as
j Fi'ma
n

(2)

i'1

where F has been replaced by the sum over all forces acting on the object.
There is however one more catch. To fully describe quantities associated with motion it
is necessary to provide directional information. For example the statement that Tundra swans are
migrating 100km per day in the spring is incomplete. The complete approximate description
would state they are migrating north west at 100 km per day. These are vector quantities which
provide information about both magnitude and direction. As such they in general require 3
numbers to provide a complete description. Such quantities will be designated by bold type in
the notes and we finally arrive at
j Fi'ma
n

i'1

(3)

A detailed mathematical description of a vector quantity requires the introduction of a coordinate


system. In the standard Cartesian coordinate system of three mutually perpendicular axes usually
designated x-,y-, and z-axes a vector is written
V'Vxx%V
yy%V
zz

(4)

where x,
y and z are unit vectors pointing along the x-,y- and z-axes and Vx,Vy and Vz are the
vector components.

Figure 1 The position vector

An important special case is the vector for which the components are the coordinates of a
point of interest, generally referred to as the position vector and designated r, as illustrated in
Figure 1. A physical quantity that depends upon its position in space, ie is described by a spatial
function can be written f(r). While this can be considered a short form for f(x,y,z) where x,y,z
2

represent general point coordinates, it also can be considered more general and relevant to any
coordinate system.
The magnitude of a vector, corresponding to the length of the geometric representation , is related
to the components through the Pythagoras theorem as:
2

V' Vx %Vy %Vz

(5)

The components can also be related to the magnitude through the polar angle and azimuthal
angle indicated in Figure 1 by:
Vx'Vsincos
Vy'Vsinsin
Vz'Vcos

(6)

2.Gravitational Sedimentation
The phenomenon of sedimentation is the dominant feature of the ultracentrifugation process. In
nature the most common example is the settling to the bottom of a particle of debris blown on the
surface of a pond. This phenomenon plays an important role in geology and in particular in
paleontology where under ideal circumstances the sedimentation rates can be used to estimate the
age of fossils.
Taking the pond to be calm the motion is vertically downward and driven by the gravitational
attraction between the particle mass and the mass of the earth. This is the simplest example of
forced motion of a particle through a fluid. There is only one relevant direction and the motion is
referred to as one-dimensional, since it can be described in a space of one dimension. The
relevant vectors all point either up or down.
Define a coordinate system in which the z-axis points vertically down. Since all relevant vectors
point either up or down they are either parallel, ( = 0) or anti-parallel ( = ). For the parallel
case the general vector of magnitude V will be V'Vz since cos(0)=1, while for the antiparallel
case V'&Vz since cos() = -1. It is common practice in such cases to treat the vectors simply as
algebraic quantities in which the sign designates direction.
There are two types of interactions between the particle and the fluid molecules that play an
important role in determining the nature of the motion.

3.Buoyancy
The first of these is buoyancy which involves the concept of pressure, a special form of stress,
involving in general a force acting in a distributed manner over an area. The pressure is the force
per unit area acting inward on any surface. Consider a plane of water parallel to the surface of the
water with an area of 1 cm2 at a depth z cm. Then the volume of the column of water pressing
down on the surface is z cm3, and the mass is 0z ,where 0 is the fluid density. The pressure, or
force per unit area is then 0zg . Note that since the system is in equilibrium the water under the
column is pushing upward with identical pressure.

Figure 2 Buoyancy
Consider now an object of thickness and area A immersed in a fluid, in this case water, at a
depth d. The downward pressure on the upper surface is then 0dg and the upward pressure on the
bottom surface is 0 {d+}g. Multiplying each pressure by the area A gives the force on each
surface. Note that an upward directed force is anti-parallel to the z-axis and hence is negative.
The total force, called the buoyancy force is the algebraic sum of the two forces acting on the
object, giving
F'&0Ag
'&m0g

(7)

The latter result expresses the fact that the product of the fluid density and the volume of the
object is the mass of fluid with the same volume as the object, usually referred to as the fluid
displaced.
Denoting the algebraic sum of the gravitational force acting downward (ie in the positive
direction) and the buoyancy force acting upward as the applied force gives:
Fapplied'Fgravity&Fb
'(m&m0)g

(8)

In this form the object acts as if it had an effective mass. It is more useful to rewrite equation (8)
in an alternative form making use of the fact that m'V and m0'0V where V is the volume of the
object and is the density of the object. Then
Fapplied'm(1&m0/m)g
'm(1&0/)g

(9)

An example of the evolutionary application of this result is the development of an organ in fish
referred to as the swim bladder. The swim bladder is an organ which can be filled with air under
control of the fish. This primarily increases the volume of the fish with a minor increase in its
mass thus lowering its density. Hence the fish can alter its density so that for >0 the applied
force is positive and acts downward and the fish descends to greater depths. For <0 the applied
force is negative, acts in the upward direction and the fish ascends towards the surface. Finally for
'0 the applied force vanishes and the fish can remain at a constant depth.
The form given in Equ (9) is entirely adequate for describing the forces on a solid macroscopic
object such as a grain of sand settling to the bottom of the pond. In modern ultracentrifugation in
many cases the object involved is a molecule. In principle the molecular density is the ratio of the
molecular mass to the molecular volume. However , the molecule often interacts with the fluid
environment. For the grain of sand the quantity v'1/ represents the volume of the sand per unit
mass. It is this quantity, referred to as the specific volume, which is appropriate for all cases
including objects at the molecular level. A more detailed explanation is given in Appendix 1.
Finally then, we arrive at the commonly used form of the applied force given in the following
equation:
Fapplied'(1&0v)mg

(10)

4.Resistive Force and Viscosity


The second interaction between an object and a fluid only exists when the object is in relative
motion with respect to the fluid. It is a frictional type of force which will be referred to here as the
resistive force. It is commonly experienced as the force of the wind. It has the important property
of opposing the motion. At the molecular level this can be appreciated by the fact that in order to
move through the fluid the object must push its way through the fluid molecules. In addition the
magnitude of the resistive force increases with velocity. For example winds are considered to be
gale force when they begin to exceed 61 km/h. In the region of interest for sedimentation the
force magnitude varies linearly with the magnitude of the velocity and can then be written
Fres'&v

(11)

where the minus sign indicates that the resistive force vector is anti-parallel to the velocity
vector. The constant of proportionality, designated depends upon both the properties of the fluid
and the sedimenting object. Anyone who compares stirring honey with stirring water realizes that
honey resists the motion of the stirrer much more than does the water. One aspect of this is the
strength of the intermolecular forces in the fluid. Another aspect which also exhibits the
difference is based upon the fact that a fluid does not retain its shape. If one tips a glass of water
the surface rapidly adjusts so that it is level and no longer makes a right angle to the sides as it did
when the glass was vertical. While the same thing ultimately happens with a thick fluid such as
honey, the re-adjustment takes place slowly with this fluid initially almost behaving as a solid in
retaining its shape. Such fluids as honey are referred to as viscous. The viscous behaviour is
quantified by a parameter referred to as the coefficient of viscosity and commonly designated as
.

surface

bottom
Figure 3. Viscous flow
The formal definition of the coefficient of viscosity is based upon the situation shown in Fig. 3
which represents the flow of a liquid of depth d over a stationary surface, designated bottom in
the figure. The velocity of the liquid in the plane in contact with the bottom is zero. The velocity
then increases linearly reaching a maximum v at the upper surface. The rate of increase with
distance from the bottom is v/d. The force per unit area driving the surface layer can be written
F/A. The two quantities are related through the coefficient of velocity by
F/A'v/d

(12)

The fundamental unit associated with the viscosity is kg-m-1-sec-1. The coefficient of viscosity of
water at 20oC is 10-3 kg-m-1-sec-1.
The resistive force constant for a sphere of diameter d moving through a liquid with coefficient of
viscosity was shown to be
'3d

(13)

by Irish physicist George Stokes at Cambridge in 1851, and as a consequence Eqn(13) is referred
to as Stokes theorem.
7

5.Motion in sedimentation

As we have seen and is illustrated in Fig.4 an object settling in a pond is acted upon by
three forces, gravity, buoyancy and a resistive force. The first two are constant to a good
approximation and can be combined into a single applied force as given in Eq.10. The magnitude
of the resistive force increases with the velocity of the object. The constant of proportionality

Fb

Fres

Fg
Figure 4. Sedimentation forces
depends upon the viscosity of the water and, for a spherical particle on the particle diameter.
More generally for an arbitrary shape the dependance on the particle geometry is more complex
and cant be determined analytically, In the case at hand the resistive force is directed upward as
the motion is downward. The total force acting on the particle is then given by
F'Fapplied&v

(14)

An important aspect of this relationship is the existence of a special velocity, vt'Fapplied/ referred
to as the terminal velocity for which the total force on the particle vanishes. Since the
acceleration, ie the rate of change of velocity, is proportional to the force acting, at the terminal
velocity the rate of change vanishes. In other words at this point steady motion is reached and the
8

particle descends at the terminal velocity. The behaviour of the velocity in the transient period
between starting from rest and reaching the terminal velocity is illustrated in Fig.5.

A qualitative understanding of the behaviour can be made by considering the transient period to
be divided into a large number of short intervals and approximating the behaviour based upon the
situation at the beginning of each interval. At the beginning of the first interval the particle
velocity is zero as is the resistive force. In this interval, using that approximation the particle will
accelerate at a constant rate determined by the ratio of the applied force to the mass. At the end of
the interval the particle will have reached a velocity the magnitude of which is the product of the
acceleration and the time interval. Thus at the beginning of the second interval the resistive force
is acting which reduces the total force. This in turn reduces the acceleration. At the end of the
second interval the velocity will have again increased but the increase will be less than in the first
interval. In general, for each successive interval the resistive force will be larger and the increase
in velocity will be less than that for the previous interval. Geometrically since the acceleration is
decreasing with time, the slope of the v(t) curve will decrease with time eventually approaching
zero at which point steady motion at the constant terminal velocity is reached. A quantitative
mathematical analysis is given in Appendix 2. A characteristic time essentially characterizing the

Figure 5 Transient behaviour of sedimentation velocity


duration of the transient period is the ratio of the particle mass to the resistive force constant,
9

'm/ . The abscissa in the figure is the ratio of time to characteristic time. That is to say
becomes a convenient time unit. Roughly speaking the transient period is about 6 . The vertical
scale corresponds to the ratio of the velocity at a given time to the terminal velocity, so the steady
motion is when the ratio is one.
6.The sedimentation coefficient
Expressing fully the applied force as given in Eqn (10) leads to a terminal velocity of
vt'(1&0v)mg/

(15)

Since g is an acceleration then the terminal velocity must be equivalent to the product of g and a
time, so Eqn(15) would take the form vt'gs where

s'(1&0v)m/

(16)

is an effective time referred to as the sedimentation coefficient. Notice that it is also the product
of the buoyancy correction factor represented by the bracketed term and the characteristic time
referenced in Fig.5. Eqn(16) is referred to as the Svedberg equation. The sedimentation
coefficient is the fundamental experimental descriptor of an object associated with
ultracentrifugation. While the unit of time in the international system is seconds, here abbreviated
by sec to avoid confusion, the unit used in practice in biology is the Svedberg, S=10-13 sec.
The sedimentation coefficient is often used as an adjective in describing biological organelles and
molecules. For example the E. Coli ribosome is described as consisting of 50S and 30S subunits.
The measured value is usually adjusted to the value for sedimentation in water at 20o C, s20,W (see
appendix 3).
s20,W'

(1&20,Wv)
(1&0v)20,W

@s

(17)

Using vt'gs it is easy to calculate the terminal velocity of the 50S subunit settling under gravity to
be 4.910-11 m/sec. This corresponds to 1.55 mm/year. It is pretty obvious that medical or
biological applications of gravitational settling are rare. There is one however as discussed in the
next section.

10

7.Erythrocyte Sedimentation Rates


The erythrocyte (red blood cell) sedimentation rate (ESR) determination is a simple and
inexpensive laboratory test that is frequently ordered in clinical medicine. The sedimentation rate
is quoted as the observed terminal velocity reached during gravitational sedimentation of the
erythrocytes in plasma. An ESR greater than 100 mm per hour is associated with a low falsepositive rate for a serious underlying disease.
As shown in Fig. 6 a combined determination of the ferritin concentration and sedimentation rate
is represented by a point in the x-y plane formed by the sedimentation rate-ferritin concentration
axes. The plane is divided into three regions providing a recommended diagnosis. For example a
sample with a sedimentation rate of 40 mm/hr and a ferritin concentration of 80 g/l indicates
iron deficiency is highly unlikely. In this application the sedimentation constant is not used as
such, although it could be calculated from the data. For example for a terminal velocity of 100
mm/hr it is straightforward to determine s by dividing by the gravitational constant. If g is used in
the standard units then the terminal velocity must be altered to 2.8 10-5 m/sec. This gives a
sedimentation coefficient corresponding to s = 2.8 10-6 sec or 2.8 107 S. Clearly the situation
in which gravitational settling is at all observable is in the region of sedimentation coefficients for
which the Svedberg is not an appropriate unit. On the other hand, observation of gravitational
settling in the region where the sedimentation coefficient is described appropriately by the
Svedberg is not possible. For this reason it is necessary to introduce an effective force of much
greater magnitude than the earths gravity, which arises in circular motion and is the fundamental
principle of the centrifuge.

Figure 6. Effect of Ferritin concentration on erythrocyte sedimentation rate


11

8.Rotational Motion
The motion of the rigid objects in the ultracentrifuge corresponds to steady circular
motion around the machine axis in a plane. In order to analyse this let the motion take place in the
x-y plane forming a circle of radius r. From Eqn (6), the x-y plane corresponds to z=0, and thus
the polar angle = /2. Then
x(t)'rcos(t)
y(t)'rsin(t)

(18)

For steady motion the angle varies linearly with time according to (t) = t, where the constant of
proportionality, , is referred to as the angular velocity.
As shown in Fig.7 the position vector, shown in red, can be written in terms of its components as
r(t)'x(t)x%y(t)

y , where the components are given in Eqn(17) in terms of the radius, angular
velocity and time. The magnitude of the position vector is constant, but it follows the particle as it
rotates around the circle. By definition the velocity vector is the derivative of the position vector,
giving
dr dx dy
' x%

y
dt dt
dt
'[&rsin(t)]x%[rcos(t)]

(19)

This quantity is referred to as the tangential velocity. The particle acceleration vector is the time
derivative and is of fundamental importance in this application. From Eqn(18)
d 2x d 2y
x%

y
dt 2
dt 2
'&2[x(t)x%y(t)

y]

2
'& r(t)

a'

12

(20)

Figure 7 Circular motion quantities


The magnitude of the particle acceleration is thus r2 and points radially inward .In order for such
an acceleration to exist there must be a force of magnitude mr2 also pointing radially inward
acting on the particle. This force is the centrifugal force.
From the perspective of someone in the rotating system, the particle is at rest since r is constant.
On the basis of Newton law the observer would conclude that, since the total force on an object
at rest must be zero, that there must be an outward directed force opposing the centrifugal force.
Mathematically, the origin of this effective force arises from the from the form of the equations of
motion in the plane polar coordinate system.
Since the effective force is proportional to the mass of the object upon which it acts it is in this
way analogous to the gravitational force. To a good approximation the sedimentation of a particle
in a fluid rotating in an ultracentrifuge is analogous to the gravitational settling discussed above
with an effective gravitation acceleration given by geff'r2 .
9.Ultracentrifuge equivalent gravity
The rotational speed of an ultracentrifuge is specified in rotations per minute, rather than the
angular velocity, normally expressed in radians/sec. It must be emphasized that the radian is a
dimensionless quantity since it is the ratio of two quantities each of which has the dimension of
length. The dimension of angular velocity is then inverse time.
Ultracentrifuge rotational speeds of 100,000 rpm are available. The relationship converting the
rotational speed to angular velocity rests upon the fact that a rotation corresponds to an angle of
2, and is

13

'

(2)rpm
60

(21)

For r=5 cm, geff = 5.5106 m/sec2. For a sedimentation of 50S the terminal velocity for this case is
9.9 cm/h. Manufacturers often quote geff in comparison to g, ie dividing by 9.8 m/sec2, in which
case geff is quoted as just under 5.6105 g.
10.The ultracentrifuge equation
The consequence of using the rotational effective gravitational quantity geff in describing the
sedimentation process is to introduce a slight complication. The terminal velocity for a particle
with sedimentation coefficient s now becomes
vt(r)'sr2

(22)

Clearly, unlike settling under gravity, in this case the terminal velocity is not constant. In fact this
approach is not strictly correct, but is an excellent approximation under the circumstances in
which it is used.. Because the velocity is not constant, the determination of the sedimentation
coefficient from experimental observation of the motion is more complicated that in gravitational
settling, where one merely needs to measure the time it takes for the particle to move a specific
distance and calculate the ratio of distance over time..
The terminal velocity in this case refers to the motion down the centrifuge tube, which is radial,
and hence is the rate of change of the distance from the axis of rotation, r. Hence the relation
between the radial coordinate and time is given as:
dr
'sr2
dt

(23)

The mathematical function proportional to its own derivative is the exponential so that the
solution is
2

r(t)'r(0)e s t

(24)

In order to determine the sedimentation coefficient from observation of the radial distance at two
times with interval t, the natural logarithm of each side of Eqn(23) leads to ln[r(t)]'ln[r(0)]%s2t ,
giving:

14

s'

ln[r(t)/r(0)]
2t

(25)

Eqn (24) is referred to as the centrifuge equation and is used to determine the sedimentation
coefficient from the experimental data.
11.Current density
While we have so far concentrated on a single particle, sedimentation is collectively a flow
phenomenon quantitatively described by a current density. This concept is most easily understood
in the simple case of a fluid flowing in a pipe.

In Fig.8 the ideal case of a non-viscous fluid flowing in a pipe reaches the open end and drops
into a container. The fluid velocity is v and the cross sectional area is A. In a time interval t all the
fluid nearer the end of the pipe than the distance vt will be ejected into the container which will

Figure 8. Current density


collect a total amount with volume Avt. If the number of fluid molecules per unit volume is n then
the number collected is nAvt. The collection, or flow rate is then F = nAv.

15

In a realistic situation it is necessary to take viscosity into account, in which case the velocity
varies from a maximum at the centre of the tube to zero at the edge. In this case it is necessary to
decompose the fluid into an infinitesimal number of sub-fluids each with an infinitesimal area dA.
Each of the sub-fluids flows along the tube at the velocity associated with its distance from the
pipe centre. The contribution of the sub-fluid to the flow rate is then dF = nv dA. The quantity j =
nv is referred to as the current density and corresponds to the flow rate per unit area, with
common units of cm-2-sec-1.
12.Brownian Motion and Diffusion
In 1827 An English Botanist named Robert Brown observed that pollen grains observed under a
microscope were continually in motion. It was shown by Einstein that this motion resulted from
the motion of the molecules in the fluid in which the grains were located. The pollen motion
resulted from collisions with the molecules. The grain motion was random and on average the
grain did not depart from its original location. The motion of the molecules is referred to as
thermal energy. It is a fundamental fact of nature that molecules move randomly and the kinetic
energy at absolute temperature T is (3/2) kT, where k is called the Boltzmann constant.
If one adds, for example, a drop of ink in a container of water, over time the ink will spread out
until eventually it is uniformly distributed over the entire volume. This process is diffusion and
results from the random motion of the ink molecules. The following highly simplified example
illustrates how a non-uniform distribution becomes uniform as a result of random motion.

In the upper container of figure 9 sixteen molecules are confined in the left portion of a container
and eight molecules of the same kind as in the left portion are confined in the right half. Since the

16
Figure 9 Simplified diffusion.

molecules are moving randomly the probability of moving in the direction from left to right
equals the probability of moving from right to left. On average then 8 molecules in the left portion
move toward the right and 4 molecules in the right portion move toward the left. If the divider is
removed so the molecules can intermix, then 12 molecules will result in each box. In this case at
any time an equal number of molecules will be moving from the left region to the right region and
from the right region to the left, so the numbers in each half will remain constant, ie a uniform
distribution has been reached. Note that in the process of reaching a uniform distribution there is a
net flow in the direction from the region of higher to the region of lower concentration.
In general the direction of flow is that in which the decrease in concentration is greatest. This is
similar to the direction of steepest decent for an individual on a hill. Since the flow stops when
the density is uniform, the flow rate and hence current density must be related to the rate of
decrease of the density. Studies have lead to Ficks law which states the magnitude of the
diffusive current is proportional to the rate of change of density. If one chooses the x-axis to point
in the direction of maximum decrease then Ficks law becomes
jD(x)'&D

dn
dx

(26)

where D is the diffusion coefficient. Note that since the x-axis is chosen in the direction of
maximum decrease of the number density, n(x) the derivative is negative. Hence the current
density is positive, ie pointing along the x-axis.
13.Analytical Ultracentrifugation
The analytical ultracentrifuge is equipped with an optical system which records the variation of
the number density of the sedimenting particles with the distance from the axis of rotation,
designated r. The measurement may be based upon the light absorption properties of the
sedimenting molecules or upon their effect on the index of refraction of the solution.
The sedimentation process results in a flow down the tube which corresponds to a sedimentation
current density js(r)'n(r)vt(r) pointing along the radial axis. This flow then results in a depletion
of the particles at the top of the tube. This non-uniform concentration distribution then induces a
diffusive flow opposing the sedimentation flow. The concentration distribution at a given time
then reflects both the effects of sedimentation and diffusion.
Figure 10 shows data taken with the ultra-centrifuge at Leeds University. A simulation to
illustrate the information contained in the data is given in Fig.11.

17

reference
meniscus

cell bottom
(outer radius)

sample
meniscus

lower
plateau
( = loading
concentration
at zero time)

0.8
0.7
0.6

Absorbance

0.5
0.4
0.3
0.2
0.1

upper
plateau

0.0
-0.1
-0.2
5.9

6.0

6.1

6.2

6.3

6.4

6.5
6.6
Radius

6.7

6.8

6.9

7.0

7.1

7.2

Figure 10 Typical sedimentation data. Courtesy of Mr.A.J.Baron, Manager,


Analytical Ultracentrifuge Facility, Astbury Center for Structural Molecular
Biology, School of Biochemistry and Molecular Biology, University of Leeds.

18
Figure 11 Analysis of ultra-centrifuge data

The data consists of the radial concentration profile at a specific time n(r,t) . The profile which
would result in the absence of diffusion is indicated by the sharp edges from zero concentration to
the relative value two which at times t1 and t2 occur at 3 and 6 cm respectively. This movement
down the tube can be used to determine the sedimentation coefficient, s, using Eqn(24).
The actual rather smeared out edges result from the diffusion process as molecules move back
up the tube from the region of high concentration into the depleted region. The detailed shape
provides experimental information regarding the diffusion coefficient D. While in practice the
analysis is carried out on all the data in Fig.10 using a special relationship called the Lamm
equation, Fig. 11 illustrates the principle. The derivatives of the concentration profiles with
respect to r result in the peaked functions represented by the dotted lines that are often referred to
as Gaussians. The Gaussian function describes the frequency of fluctuations of a statistical
quantity around the mean. The root mean square fluctuation is the standard deviation which is a
measure of the uncertainty in a measurement. It is related to the width of the Gaussian as
indicated by Wi;i'1,2 in the figure. The rate of growth of the width determines the diffusion
coefficient D.
Qualitatively, the rate at which the edge of the concentration profile travels down the tube
determines the sedimentation coefficient and the rate at which thesmearing of the edge
increases determines the diffusion coefficient.
Knowledge of these two quantities can be used to determine the molecular weight of a
sedimenting molecule as a result of an important relationship between the diffusion coefficient
and the resistive force constant. In order for particles to undergo any motion in a fluid, they must
overcome the fluid resistance. Hence it is reasonable that the larger the resistive force constant the
smaller the diffusion constant. Since the diffusion process arises from thermal motion it is also
reasonable that the constant will increase with temperature. The exact relationship is given by
D'kT/ where k is the Boltzmann constant which equals the ratio of the ideal gas constant R
divided by Avogadros number NA . From the fundamental relationship between the
sedimentation coefficient and the particle-fluid properties given in Eqn(16)
s (1&0v)m (1&0v)M
'
'
D
RT
D

(27)

where M 'NAm is the molecular weight.


The analytic ultracentrifuge plays an important role in determining the molecular weights of
macromolecules and hence in molecular biology. While the sedimentation coefficient is itself an
19

important parameter, the molecular weight is a far more universal quantity.

14.Equilibrium
A common phenomenon during summer in large urban areas is smog. The smog effect is
produced from extremely small particles referred to as aerosols produced by human activity that
remain suspended in the air. An analogous phenomenon in which the fluid is a liquid rather than a
gas is the formation of a colloid as a result of a chemical reaction.
Since the particles are acted upon by gravity the obvious question is why arent they settling on
solid surfaces as is usual in the sedimentation process. Gravity is in fact the only fundamental
force acting, since the two other effects, buoyancy and the resistive forces are effective forces.
The fact that sedimentation is not occurring means that the concentration profile does not vary
with time, ie is static. Such a situation is referred to as a state of equilibrium. For such a state to
occur there must be no net flow.. This leads to the condition js%jD'0 or jD'&js .Taking the z-axis
to point upward, the sedimentation current density can be written js'&nvtz'&ngs

z . From the
expression in Eqn (25) for the diffusion current density we have
&D

dn
'ngs
dz

(28)

The dependance of the concentration on z, ie altitude in the case of smog on a calm day, is then
n(z)'n(0)e &(gs/D)z

(29)

The concentration decreases exponentially with altitude. The larger the sedimentation coefficient
the more rapid the decrease. In the case of air, the buoyance effect is negligible and the buoyancy
correction factor is one to an excellent approximation. Then s/D = m/( kbT) . The average altitude
is h'kBT/mg .
It should be emphasized that this analysis is based not only on a calm situation, but one in which
the temperature, and hence diffusion coefficient, variation with z is negligible. The equilibrium
situation also takes time to reach from a beginning non-equilibrium concentration distribution.
It is also not obvious as to what distinguishes the equilibrium situation from the non-equilibrium
situation. First of all, the initial concentration profile will not necessarily be that given in Equ(28),
and hence there is an adjustment period until equilibrium is achieved. Secondly, for particles of
20

large mass such that kTmgh , the average altitude becomes extremely small, such that it may
actually be less than the size of the particle, in which case the adjustment is equivalent to the
sedimentation process. In this case, the solid surface associated with the bottom of the pond, or
the centrifuge tube alters the situation.
Molecules with sufficiently small sedimentation coefficients cannot form a sediment in
ultracentrifuges even at the highest rotational velocity. In addition, the gravitational effect can be
varied so that in principle by lowering the speed of operation the formation of a sediment can be
prevented. In either case, eventually the equilibrium state will be reached.
In the centrifuge the sedimentation flow is down the tube corresponding to the positive radial
direction. The diffusive flow is always down the concentration gradient. Substituting geff'r2 , the
terminal velocity is sr2 and the equilibrium condition becomes
D

dn
'n(sr2)
dr

It is customary to describe the concentration distribution in logarithmic terms giving


ln[n(r)/n(0)]'sr 22/2D
mr 22
'(1&0v)
2kBT

(31)

By multiplying numerator and denominator by Avogadros number and re-arranging, the


molecular weight can be determined from
M'

2RTln[n(r)/n(0)]
(1&0v)r 22

(32)

This relation can be used to determine the molecular weight of molecules with small
sedimentation coefficients.
15.Preparative Methods
Preparative applications are directed toward separating components in a mixture. There are three
methods, differential, rate zonal and isopycnic centrifugation. The first two separate the
components on the basis of their sedimentation coefficients. Indeed the first is closely related to
the famous historical method of panning for gold. The third is a clever method used to separate
components based upon their density. Combining the latter with one of the first two then has the
21

potential to refine the separation capability since two parameters are defined in the dual process.
Differential centrifugation is similar to the usual chemical technique of forcing more rapid
sedimentation of a precipitate in a standard centrifuge. The liquid phase after the process is
completed is referred to as the supernatant. A highly simplified diagram illustrating the separation
principle is shown in Fig. 12. The left container represents a mixture of two components with
quite different sedimentation coefficients initially uniformly distributed in the liquid.
The container on the right illustrates the distribution after centrifugation. The cluster of

Figure 12 The differential process


components pressed against the bottom of the tube is called a pellet. The component with the
large sedimentation coefficient is entirely pelletized during the time of the run, while only a
portion of the smaller sedimentation coefficient component is. The supernatant is relatively pure
in this component. If the pellet is re-dissolved the new solution is reduced in the undesirable
component. As an example, suppose 20% of this component has been pellatized. If the operation
is repeated the pellet will now contain 20% of the 20%, or 4% of the undesired component.
Repeating the process can further increase the purity.
The rate zonal method involves carefully introducing a layer of the mixture on the surface of the
liquid. The centrifuge is now run for a time less than that required for the desired component or
components to achieve sedimentation. As illustrated in Fig.13, the component with the larger
sedimentation coefficient will form a band further down the tube than the other component. Each
component can then be recovered by careful extraction of the layer with a pipette. It must be
remembered that the edges of the layer are not sharply defined because of diffusion.
Because the terminal velocity increases with radial distance, it is difficult to control the process,
22

since the radial distance increases exponentially with the time. Since the terminal velocity is the
product of the sedimentation coefficient and the effective gravitational field which increases with
r, the development of a system in which s decreases with r has been developed as a method to
prevent the velocity from growing uncontrollably. This is done by creating what is referred to as a
density gradient in the liquid so the density 0(r) increases with r. The sedimentation coefficient
now becomes s(r)'(1&0(r)
v)m/ . The density gradient then produces a buoyancy correction
factor which decreases with the distance down the tube, opposing the increasing effect of the
effective gravitational term r2 on the terminal velocity.
The density gradient can be created by dissolving a solute in the liquid and creating an

Figure 13
equilibrium distribution discussed in the previous section.
The development of this technique lead to the discovery of a preparative method that did not
depend on the actual value of the sedimentation coefficient at all. The inverse of the specific
volume is the density of the component which in particles such as organelles is well-defined. In
this case the buoyancy factor can be written 1&0(r)/j where j is the density of component j. The
buoyancy correction factor then vanishes when 0(r)'j . If the value of r which satisfies this
condition is designated rj , then sj(rj)'0 and the terminal velocity vanishes. Note that for r>rj the
sedimentation coefficient for the component becomes negative. This means that particles of
component j will travel up the tube towards the position at which the sedimentation coefficient
vanishes. For r<rj the particles travel down the tube. These particles will eventually all tend to
congregate at position rj .

23

Figure 14
The process is illustrated in Fig. 15. In this case the fluid density increases from about one g/cm3
to 1.9 g/cm3 at 10 cm. Components one, two and three with densities of 1.18, 1.36, and 1.61
g/cm3 will cluster at 5.1, 6.8 and 8.6 cm respectively.

Figure 15 Isopycnic separation

Appendix 1. Specific Volume


24

The magnitude of the buoyant force is the product of the fluid density, the volume of fluid
displaced by the intruding particle, and the gravitational force constant Fb'0Vdg . For a particle of
mass m the applied force is then
Fapplied'(m&0Vd)g
'm(1&0Vd/m)g

The ratio of volume displaced to mass, Vd/m , is referred to as the specific volume and designated
v and the buoyancy correction factor becomes 1&0v
Determination of the specific volume can be determined as follows. A solution of volume V
consists of a solvent of mass m0 and density 0 in which is dissolved a solute of mass m. The
measured density of the solution is then
s'(m0%m)/V'm0/V%m/V
The volume of the solution is the sum of the original volume of the solvent and the volume
displaced by the solute. The solvent volume can then be written V&Vd . The ratio of solute mass to
solution volume, m/V is the concentration c and usually given in mg/ml. The solvent mass is
m0'0(V&Vd) ,
giving
s'0(1&Vd/V)%c
Re-arranging gives
Vd'(1&

s&c
0

)V

Then dividing both sides by m


v'(1&

s&c
0

This can be re-arranged into the form usually given

25

)1/c

1 &c
v' & s
c 0 c

' 0 & s %1/0


0 c s c
&
1
' (1& s 0 )
0
c
The specific volume can then be determined from the measurable quantities, s,0 and c.
Appendix 2 Transient Behaviour
The total force acting on a particle in a fluid can be written
F'Fapplied&v

where all forces are aligned with a common axis. The applied force is assumed to be constant. The
direction of the resistive force is antiparallel to the direction of the applied force causing the
motion. Newtons law states that this force equals the product of the mass and acceleration so the
above can be written
ma'F'Fapplied&v

This equation involves two unknowns, the acceleration and the velocity. In order to reduce the
equation to one unknown use is made of the definition of acceleration as the rate of change of
velocity. The above equation can then be written as
m

dv
'F
&v
dtN applied

Since the derivative of a constant is zero it is apparent that vt'Fapplied/ satisfies the equation.
However this cant be the complete solution since a particle can start from rest and ultimately reach
the terminal velocity. The complete form of the unknown velocity must take the form
v(tN)'vt%u(tN)

26

where u(0)'&vt is required to be satisfied if the initial velocity is zero. By substituting Fapplied'vt
The right side of Newtons equation can be written &u(tN) . Again using the fact that the derivative
of a constant is zero the equation reduces to
&m

du
'u(tN)
dtN

This can be integrated according to


u(t)

t )'t

u(0)

t '0

du
'ln[u(t)]&ln[u(0)]'&/m dt )'&t/m
m u
m
)

Then taking anti-logs gives


u(t)'u(0)e &t/m'&vte &t/m

Finally,
v(t)'vt(1&e &t/m)

Appendix 3 Transforming s to s20,w


The fundamental relationship, often referred to as the Svedberg equation , gives the sedimentation
coefficient as
s'(1&0v)m/

This can be taken further for a spherical particle. Let the diameter be d and the density be . Then
the mass, or product of the density and volume is
m'(d 3/6)

27

Using Stokes law, '3d , the sedimentation coefficient in the fluid used in the experiment could
be written
s'(1&0v)

d2
18

For non-spherical particles the above relation can be corrected by multiplication by a geometrical
factor g. The main point is that the relation consists of the particle characteristic parameters,
density, diameter and geometric factor, , d, and g, as well as the fluid parameters, the fluid density
and coefficient of viscosity, 0 and . It is the latter two parameters that are altered to 20,W and
20,W to obtain s20,W , while the particle parameters remain the same. Then
s20,W/s'[
'

(1&20,Wv)d 2
1820,W
(1&20,Wv)

(1&0v)20,W

which leads directly to Eqn(17).

28

][

18
]
(1&0v)d 2

Problems
1. An erythrocyte has a mass of 10.13 10-14 kg and a volume of 910-11 cm3. It sediments in water
with a terminal velocity of 66 mm/hr. Find the resistive force constant in kg-sec-1.
Ans.(6.1610-9 kg-sec-1)
2. A particle with density 4 times the fluid in which it is moving settles under gravity with a
terminal velocity of 0.098 nm/s.(a) Find the sedimentation coefficient in Svedbergs. (b) Find the
characteristic time =m/. (100S, 13.3 psec)
3. The Beckman XL90 centrifuge operates at a frequency of 80,000 rpm with a nominal 8.4 cm
radius..
(a) Find the nominal acceleration in units of g. (6.02105)
(b)Find the terminal velocity of the 40S ribosomal subunit in water at the nominal radius.
(2.3610-3 cm/sec)
4. A liposome sedimenting in an ultracentrifuge operating at a frequency of 100000 rpm is
observed at a radius of 4.0 cm. Following an interval of 1000 sec it has migrated to a radius of 5.6
cm. Find the sedimentation coefficient in Svedbergs. (30.7 S)
5. A ribosome has a diameter of 30 nm and a s20,W =80S. Calculate the ribosomal density. (1.16
g/cm3)
6. The sedimentation coefficient for a spherical particle with density 2.3 g/cm3 sedimenting in a
fluid with density 1.2 g/cm3 and viscosity .026 g cm-1 s-1 is 25S. Find s20,W . (76.8S)
7. As stated, the data in Fig. 2.5 were taken using a centrifuge rotating at 55000 rpm for 5 h.
Estimate the observed sedimentation coefficient from the 50% concentration points of the first
and last scan. Note this is not for water at 20oC. (1.6 S)
8. A fluid is constructed with a density gradient so that the density varies as o=1.1+0.08r g/cm3
where r is the distance down the tube in cm. Find the distance where particles of specific volume
1/1.5 cm3/g will collect in an isopycnic separation.

29