Sedimentation HowTo

Paul S. Russo 05/02/10

Sedimentation
Introduction
Sedimentation is one of the great classical methods for polymer characterization.
There are two varieties, equilibrium and velocity. They are usually done on the same
instrument, an analytical ultracentrifuge. This device can determine the concentration of
a polymeric solute as a function of position from the center of a rapidly rotating cell.

In equilibrium sedimentation, the particle is not actually sent all the way to the
bottom of the cell, resulting in a “sediment.” Rather, a “low” centrifugal field is used to
create a concentration gradient--more particles near the bottom of the cell than near the
top. The gradient is opposed by diffusion, and dynamic equilibrium is slowly achieved.
Just as chemical potential measurements (e.g., membrane osmometry) yield valuable
information like molecular weight, so do measurements in a combined centrifugal and
chemical potential field. Whereas a membrane osmometer uses a physical barrier to
create a sharp concentration boundary, and whereas light scattering responds to smaller,
spontaneous osmotic potentials, equilibrium analytical ultracentrifugation uses
gravitational potential to create a smooth but large concentration gradient. The point is,
all these methods produce absolute molecular weight information because they take
advantage of the chemical potential. Osmometry is the most limited in molecular weight
range. Equilibrium analytical ultracentrifugation and light scattering can be applied to
practically any macromolecule, and the former technique can even be used for very small
molecules (like sugar). Equilibrium analytical ultracentrifugation is extremely powerful
for aggregating or associating systems. The disadvantage to equilibrium analytical
ultracentrifugation is profound slowness. This problem is ameliorated to some extent in
new machines that measure many samples simultaneously. The method will never have
the throughput of GPC/LS, but thanks to the excellent design of the new instrument,
comparatively little time is spent fiddling around.

Velocity sedimentation is a transport technique, akin to diffusion or viscosity.

The rate at which a particle sinks is measured. And it really does sink--all the way to the
bottom if you let it--because the field is much higher than in the equilibrium method.
Velocity sedimentation does not produce absolute molecular weights, except by
combination with a second transport method. It measures friction and is therefore useful
for shape information. Some of us have research interests in the friction itself and do not
care much about molecular properties (ahem!).

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Method Applications Angular Velocity Time to measure

Equilibrium Absolute M Relatively low Several days
Aggregation

Velocity Sedimentation Relatively high Several hours

Coefficient
M via
Sedimentation &
Diffusion
Mutual friction

Background
These methods, once essential for biochemistry, almost disappeared! Modern
biochemists have a raft of simpler, cheaper and adequate-if-boring methods for the rather
qualitative information they usually require. Most important are gel electrophoresis and
gel filtration. The classic analytical ultracentrifugation instrument was the Beckman
Model E. A triumph of technology and a tribute to the lengths that people will go to for
science, the Model E was nonetheless tedious to operate. Hardly anyone would actually
try to build their own instrument--it requires serious technology. A few devotees
lovingly maintained and even modified their treasured Model E’s. In this way, the old
technique was kept alive while newer methods prospered.

All the new methods fail at a certain, very important task: characterizing
molecular weights and associations at very low concentrations. This is important in the
pharmaceutical industry, and perhaps that explains the resurgence of the method.
Another factor is that computers can remove the tedium of data analysis. Anyway, a new
Beckman analytical ultracentrifuge (AUC) finally appeared, the XLA. There are two
kinds of concentration detectors for XLA’s: UV-Vis absorption and interference. Our
XLA has only the first of these: the absorption (at practically any wavelength you
desire) can be measured as a function of position from the cell, as the instrument is
running. Since concentration is related to absorbance (Beer’s Law) one can essentially
measure c(r) vs. r. This can be done with a precision of about 5 microns, and the
absorption data are of excellent quality. The machine runs at extremely stable speeds,
with good temperature control. Two things our machine cannot do: work in a solvent
that absorbs more strongly than the polymer dispersed in it; and, work with solutes that
do not absorb. Even these cases can be handled on XLA’s equipped with interference
optics.

Sedimentation Velocity

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A mechanical picture of sedimentation suffices to explain most aspects of the

velocity experiment. In the absence of an applied field, a polymer in solution will just
rattle around. If a field (say, a centripetal field caused by spinning the sample at an
angular frequency, ω) is applied, the molecule quickly accelerates and very soon reaches
its terminal velocity. The situation in the sector-shaped analytical ultracentrifuge cell
looks like this:

r = a; meniscus
r = b; bottom
Figure 1
ω
Fb
Fd

Fc

r
Attainment of terminal velocity signifies a balance of forces (a molecule feeling a net
force would accelerate). The balance between centripetal, drag and buoyant forces is
expressed:

Ftotal = Fd + Fb + Fc = 0 Eq. 1

Fc = centripetal force = ω 2rm Eq. 2

where m is the particle mass and -ω r represents the centripetal acceleration.
2

Fd = viscous drag force = -f⋅ v Eq. 3

where f is the translational friction coefficient and v the linear velocity

Fb = buoyancy force = -ω 2rmo Eq. 4

where mo is the mass of solvent displaced by the particle

Thus,

ω 2r(m-mo) - f⋅ v = 0 Eq. 5

What is mo ? We could take it as the volume displaced times the solution density (it’s not
obvious yet why it should be the solution density—that comes from thermodynamics, as
we will see below). The displaced volume could be taken as the product of the mass and
the partial specific volume: mo = mv~2 ρ . The force balance equation becomes:

ω 2rm(1- ρv~2 ) - f⋅ v = 0 Eq. 6

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The particle mass is the molar mass divided by Avogadro’s number, m = M/Na, and we
define the sedimentation coefficient, S, to be the velocity obtained divided by the angular
acceleration applied:
v M (1 − ρv~2 )
S= = Eq. 7
ω 2r Na f

Things to note:
• This development applies to dilute solutions. We can indicate this by writing a
superscript on the S – e.g., So – to indicate extrapolation to infinite dilution.
• If v~2 = 1 / ρ then sedimentation will not occur and no experiments can be done
• If you knew f and v ~ independently, then you could get M from S. This knowledge
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is available from diffusion (e.g., dynamic light scattering). Combining transport
methods is often beneficial, their high precision compensating partially for the
absence of absolute molecular weight information from any single transport method.
• S has units of s/radian2. One doesn’t usually worry about the radian2
• 1 × 10-13 s is called a Svedberg, and given the symbol S
• Many biological components are identified by their sedimentation coefficients.
Example: 4S RNA has S = 4 × 10-13.
• Theodor Svedberg (1884-1971) won the Nobel Prize in
Chemistry in 1926; he deserved the award just for surviving
the dangerous early instruments.
• S measurements are often quoted as if they were made at
20oC in water, even if they were not. The correction
recognizes that f is proportional to viscosity, η , and the
1 − ρv~ term also has to be adjusted.
2

~
o o η T , solvent 1 − ρ 20, waterv 2 20,w ater
S 20, water = S T , solventη ~ Eq. 8
20, water 1 − ρ T , solventv 2 T ,solvent

Measuring S

The first thing to emphasize is that, like any transport measurement, you should
extrapolate S to infinite dilution in order to obtain molecularly meaningful parameters.
The Beckman XLA uses extremely sensitive absorption optics, so in practice it may be
possible to measure at one really low concentration—so close to c=0 that you don’t
worry about the extrapolation. Older machines using Schlieren detection systems did not
afford this luxury. That’s why you see so much emphasis on concentration dependence
in the old literature. Modern reasons for studying the concentration dependence include
better understanding of semidilute behavior, but that’s another story.

An experimental data set (Figure 2) consists of radial scans (Absorbance vs.

radius) acquired at several times during the sedimentation. One hopes that the sample is
not sedimenting much during the relatively short time required to scan the radius.

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2.0 50,000 RPM

o
T=20.2 C
1.5 Times in s, in order: 1409, 2663, 3908, 5197,
6473, 7757, 9104, 10538

1.0
Absorbance

0.5

0.0

-0.5 Meniscus
real data from Igor & Bricker

-1.0

5.5 6.0 6.5 7.0 7.5

r/cm

Figure 2. Typical data set for sedimentation velocity.

The XLA is a dual-beam instrument; incredibly, it can distinguish absorbance from the
reference half of its dual cell and the sample half, even though both are within 1 cm of
each other and spinning at up to 60,000 RPM. The sharp, strong spikes (around 6 cm)
are due to the menisci of reference and sample compartments.

Other noteworthy features

• Although absorbance is plotted, Beer’s law says these curves really represent
concentration profiles. The constants of proportionality between absorbance and
concentration are not important.
• The “boundary” is very broad in this case (a small biopolymer, M ≈ 20,000). This
broadening is caused by diffusion.
• Diffusion is your enemy in sedimentation velocity; it complicates the identification of
the “boundary”. There is nothing you can do about it, though.
• The “boundary” moves towards higher and higher radii with time.
• A “pellet” is forming on the bottom (the sudden upturn at high r).
• The “plateau value” concentration decreases with time (because the cell is fatter at
the bottom than it is at the top, so as to fight convection; this dilution effect is called
radial dilution)

Sedimentation Equilibrium

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There were some problems in the above treatment. How do we really identify rb?
Why should we use solution density, instead of solvent? These problems stem from our
single particle, mechanical viewpoint. In fact, there are many particles in a
thermodynamically large system. The solution is to use a theory that connects flow of
the molecules (including sedimentation and diffusion from the outset) with a gradient of
an appropriate potential that includes both the effects of concentration and the effects of
the centrifugal field. Define this total potential as:
~ = µ +U
µ 2 c Eq. 12

where µ 2 has its usual meaning (chemical potential of solute) and Uc is the
centripetal potential. The flux of particles through a unit area defined in the sample is
~
developed by multiplying the gradient in the total potential, ∂ µ ( ∂r ) , by some generalized
mobility, L. In computing the total potential gradient, the chemical potential is treated as
a function of T, p and c. The pressure term matters, since pressure varies with position in
the rotor; the actual solution density should be used in computing it (aha!). Skipping a
few, not-too-difficult manipulations, one obtains:

 RT dc 
J = L ω 2 rM (1 − ρv~2 ) −  Eq. 13
 c dr 

The mobility coefficient is c/Naf and so we can write:

~ )
M (1 − ρv RT dc dc
J = ω2 rc 2 − = ω2 rcS − D Eq. 14
Na f N a f dr dr

where S and D are properly defined through the second half of the expression in terms of
flux. Eq. 14 is just provided for comparison with the mechanical picture. Eq. 13 really
makes a better start for the equilibrium experiment. At equilibrium, J = 0 and Eq. 13
becomes:

1 dc ω 2 rM ~ )
= (1 − ρv 2
Eq. 15
c dr RT

This has the solution:

ω 2 M (1 − ρv~2 )( r 2 − a 2 )
c( r ) = c( a) exp( ) Eq. 16
2 RT

Where a is an integration constant, usually taken as the meniscus position or similar.

The equation basically indicates exponential growth in concentration as you move down
the cell. Figure 4 illustrates the growth.

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Igor-Bricker sample
o
T=20.0 C
Absorbance 24,000 RPM v2=0.73 mL/g

40 45 50
2 2
r /cm

Figure 4. Equilibrium analytical ultracentrifugation data.

Since ω, ρ, Τ, and v~ are all known, one can easily determine M. The modern way
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to do this is with a nonlinear least squares fit. One could first linearize the expression
instead:

ω 2 M (1 − ρv~2 )( r 2 − a 2 )
ln[ c (r )] = ln[ c (a )] + Eq. 17
2 RT

If multiple species are present, the concentration profile is a sum of exponentials. Of

great importance to biological chemistry is the case where the multiple species represent
some definite kind of aggregation: e.g., 4 proteins associate into a tetramer. In such
cases, analytical ultracentrifugation can be used to obtain the binding constants and the
stoichiometry of the association. It may have no equal for this.
Analytical ultracentrifuge
In an analytical ultracentrifuge, a sample being spun can be monitored in real time
through an optical detection system, using ultraviolet light absorption and/or interference
optical refractive index sensitive system. This allows the operator to observe the
evolution of the sample concentration versus the axis of rotation profile as a result of the
applied centrifugal field. With modern instrumentation, these observations are
electronically digitized and stored for further mathematical analysis. Two kinds of
experiments are commonly performed on these instruments: sedimentation velocity
experiments and sedimentation equilibrium experiments.

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Sedimentation velocity experiments aim to interpret the entire time-course of

sedimentation, and report on the shape and molar mass of the dissolved macromolecules,
as well as their size-distribution[1]. The size resolution of this method scales
approximately with the square of the particle radii, and by adjusting the rotor speed of
the experiment size-ranges from 100 Da to 10 GDa can be covered. Sedimentation
velocity experiments can also be used to study reversible chemical equilibria between
macromolecular species, by either monitoring the number and molar mass of
macromolecular complexes, by gaining information about the complex composition from
multi-signal analysis exploiting differences in each components spectroscopic signal, or
by following the composition dependence of the sedimentation rates of the
macromolecular system, as described in Gilbert-Jenkins theory.
Sedimentation equilibrium experiments are concerned only with the final steady-state of
the experiment, where sedimentation is balanced by diffusion opposing the concentration
gradients, resulting in a time-independent concentration profile. Sedimentation
equilibrium distributions in the centrifugal field are characterized by Boltzmann
distributions. This experiment is insensitive to the shape of the macromolecule, and
directly reports on the molar mass of the macromolecules and, for chemically reacting
mixtures, on chemical equilibrium constants.
The kinds of information that can be obtained from an analytical ultracentrifuge include
the gross shape of macromolecules, the conformational changes in macromolecules, and
size distributions of macromolecular samples. For macromolecules, such as proteins, that
exist in chemical equilibrium with different non-covalent complexes, the number and
subunit stoichiometry of the complexes and equilibrium constant constants can be
studied.
Preparative ultracentrifuge
Preparative ultracentrifuges are available with a wide variety of rotors suitable for a great
range of experiments. Most rotors are designed to hold tubes that contain the samples.
Swinging bucket rotors allow the tubes to hang on hinges so the tubes reorient to the
horizontal as the rotor initially accelerates. Fixed angle rotors are made of a single block
of metal and hold the tubes in cavities bored at a predetermined angle. Zonal rotors are
designed to contain a large volume of sample in a single central cavity rather than in
tubes. Some zonal rotors are capable of dynamic loading and unloading of samples while
the rotor is spinning at high speed.
Preparative rotors are used in biology for pelleting of fine particulate fractions, such as
cellular organelles (mitochondria, microsomes, ribosomes) and viruses. They can also be
used for gradient separations, in which the tubes are filled from top to bottom with an
increasing concentration of a dense substance in solution. Sucrose gradients are typically
used for separation of cellular organelles. Gradients of caesium salts are used for
separation of nucleic acids. After the sample has spun at high speed for sufficient time to
produce the separation, the rotor is allowed to come to a smooth stop and the gradient is
gently pumped out of each tube to isolate the separated components.

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Sedimentation coefficient
The sedimentation coefficient s of a particle is used to characterize its behaviour in
sedimentation processes, notably centrifugation. It is defined as the ratio of a particle's
sedimentation velocity to the acceleration that is applied to it (causing the sedimentation).

The sedimentation speed vt (in ms−1) is also known as the terminal velocity. It is constant
because the force applied to a particle by gravity or by a centrifuge (measuring typically
in multiples of hundreds of thousands of gravities in an ultracentrifuge) is cancelled by
the viscous resistance of the medium (normally water) through which the particle is
moving. The applied acceleration a (in ms−2) can be either the gravitational acceleration
g, or more commonly the centrifugal acceleration ω2r. In the latter case, ω is the angular
velocity of the rotor and r is the distance of a particle to the rotor axis (radius).
The sedimentation coefficient has the dimensions of a unit of time and is expressed in
svedbergs. One svedberg is defined as exactly 10−13 s. Essentially the sedimentation
coefficient serves to normalize the sedimentation rate of a particle by the acceleration
applied to it. The resulting value is no longer dependent on the acceleration, but depends
only on the properties of the particle and the medium in which it is suspended.
Sedimentation coefficients quoted in literature usually pertain to sedimentation in water
at 20°C.
Bigger particles tend to sediment faster and thus have higher svedberg values.
Sedimentation coefficients are, however, not additive. Sedimentation rate does not
depend only on the mass or volume of a particle, and when two particles bind together
there is inevitably a loss of surface area. Thus when measured separately they will have
svedberg values that may not add up to that of the bound particle.
This is notably the case with the ribosome. Ribosomes are most often identified by their
sedimentation coefficient. For instance, the 70 S ribosome that comes from bacteria
actually has a sedimentation coefficient of 70 svedberg. It is composed of a 50 S subunit
and a 30 S subunit.

Density gradients are used in many different operations:

• To separate particles of different densities (isopycnography,
which is short for "equilibrium density gradient centrifuation)
• To separate particles of different sizes (sedimentation
centrifugation)
• Column elutions that must smoothly go from one concentration
to another
• Isolation of diamond dust (isopycnography)
• Isolation of bovine X-sperm from Y-sperm (dairy industry)
(sedimentation without centrifugation)

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There are several ways for making density gradients including those
that use syringes, twin linked containers, and other devices. Here are
two very simple additional ways to make linear density gradients:
1. This might be simple but it is one that takes many hours: fill a
plastic centrifuge tube with - say - a 10% sucrose solution. Put it
in the freezer. The first part that freezes will be almost pure
water at the top, with only a little sucrose trapped in it, but as
the overlying ice layer gets thicker, more and more sucrose is
trapped within it. Thus when it is completely frozen, the top has
little sucrose and the bottom has much. Upon subsequent
thawing, the bottom melts first, and the melting proceeds
upwards reinforcing the preparation of the gradient.
2. Here is one that takes about 60 seconds of time from start to
finish!

 Isopycnography (Equilibrium Density Gradient Centrifugation),

which is used for separating things on the basis of differing intrinsic
densities. This is a "static" system as the experimental objects come
to rest at points in the tube at which they are in density equilibrium
with the surrounding solvent at that point.

 Sedimentation Centrifugation, which is used to separate things

on the basis of their differing "effective" sizes, or, roughly, on the
basis of molecular weights. This is a "dynamic" situation because the

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experimental objects have started at the top and are settling through
the gradient at rates roughly proportionate to the square roots of their
molecular weights.

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