CSB 30103

Downstream processing of
bio-product
(Chapter 4: Sedimentation)
Contact:
Mrs. Rozyanti Mohamad
06-551 2077 / 012-652 0389
rozyanti@unikl.edu.my

Sedimentation
Sedimentation is the tendency for
particles in suspension to settle out of the fluid due to
gravitational or centrifugal acceleration

g = 9.81 ms-2

2R
R: distance of particle
from the center of
rotation
: angular velocity
(rad/s)

Equation of motion
Nearly all analyses of separation by sedimentation begin
with the equation of motion of a particle with:
oRadius,
oDensity,
oMass, m=(4/3)
i : inertial acceleration
b : buoyancy of particles
d : Stokes drag force

Assuming the particles is spherical:

m (dv/dt) = FiR + FbR + FdR
= [(4/3) 2R] + [(4/3) 02R] + [6 v]

(Eq. 4.1)

Equation of motion
Velocity in a centrifugal field at steady state or
centrifugation equation, :
2a 2 ( 0 ) 2 R
=
......( Eq.4.2)
9

If the particles are allowed to sediment only in the

presence of gravity (g = 9.8 m/s2), the equation become:
2a 2 ( 0 ) g
=
......( Eq.4.3)
9
where

: dynamic viscosity
o
: medium density

Equation of motion
If the particle moving out, R is not constant (at t = 0, R =
R0).
The equation become:
R 2a 2 ( 0 ) 2t
ln =
......( Eq.4.4) Where t = time
9
R0

Centrifuge equation useful that relates time to the

distance traveled by the particle.

Creeping flow
o Creeping flow conditions are usually satisfied in
sedimentation, whose Reynolds number is very small
(Re < 1).
Re =

2a

......( Eq.4.5)

Equilibrium sedimentation (isopycnic)

o When particle density and solvent
density is equal, the velocity is zero
and the process is called isopycnic
separation
o Separation based on density and
buoyancy
o Centrifugation suitable to
determine particle or
macromolecule density

Equilibrium sedimentation (isopycnic)

Practically, there are three routes in establishing of isopycnic
sedimentation:

1
Layer the solution of
decreasing density
Starting from the
bottom until the vessel
is filled.
The resulting gradient is
like a staircase.

2
Centrifuge at extremely
high speed
Resulting in isothermal
stratification of densityforming solute.

3
Gradient mixing method
Consist of two
cylindrical container :
one containing
concentrated solution
and another containing
dilute solution
Equipped with stirring
apparatus and link each
other
produce outflow with
linear salt gradient.

Hindered settling
o When concentration of
sedimenting particles
increase, velocity
become decrease.
o In hindered settling,
particles are so close
and they have a major
effect on each other.

Sedimentation coefficient
o When a body force is applied, velocity through a viscous
medium is usually proportional to the accelerating field
o In the case of sedimentation, the resulting constant (a property
of both particle and medium) is sedimentation coefficient
defined as;

S 2
R

Where
= dR/dt
S = the Svedberg (1S = 10-13s)

oFrom Eq. 4.3;

2a 2 ( 0 )
S=
......( Eq.4.6)
9

Equivalent time
o First, we define G as the dimensionless acceleration, the ratio
of centrifugal to gravitational acceleration for a particular
centrifuge:
2R
G
g

o The unit is gs (multiples of earths gravitational acceleration).

Equivalent time
o A rough approximation of the difficulty of a given separation by
centrifugation is the product of the dimensionless acceleration
(G) and time required for separation (t), called as equivalent
time:
2R
Equivalent time Gt =
t ......(Eq .4.7)
g

o Typical values of Gt:

o
o
o
o

Eukaryotic cells
Protein precipitates
Bacteria
Ribosome

= 0.3 x 106 s
= 9 x 106 s
= 18 x 106 s
= 1,100 x 106 s

Sigma analysis
o Commonly used in industry
o Use the operation constant () to characterize a centrifuge into
which feed flows at volumetric flowrate (Q):
Q = {g}{}
Where;
2a 2 ( 0 ) g
vg = sedimentation velocity at 1xg: g =

= the geometry and speed of centrifuge or crosssectional area of centrifuge

Sigma analysis
o The sedimentation velocity at 1xg can directly
determine using:
2a 2 ( 0 ) g
g =
9

o In laboratory, useful equation for determine vg :

g =

R
g ln
R0

2t

Centrifuge:
Basis of separation

Basis of separation

A centrifuge is used to separate particles or even

macromolecules: cells, sub-cellular components,
proteins, nucleic acids, etc

Size
Shape
Density

Centrifuge:
Methods of separation
o Methodology:
o Utilizes density difference between the
particles/macromolecules and the medium in which these
are dispersed
o Dispersed systems are subjected to artificially induced
gravitational fields

o The most common types:

o Tubular bowl, multichamber, disk-nozzle, disk-intermitten
discharge, scroll and basket

Principle of centrifugation:
Stokes Law
The concept of using centrifugal force to efficiently separate
components of differing densities
The fundamental principle that applies to centrifugation is
Stokes Law
Formula for determining the rate of sedimentation
It states that a particle moving through viscous liquid attains a
constant velocity or sedimentation rate
The rate can be very slow for particles whose:
(1) density is close to that of the liquid
(2) for particles whose diameter is small
(3) where the viscosity is high

Principle of centrifugation:
Stokes Law
Stokes Law: =

d 2 2r (Pp Pi )
18

......(Eq .4.8)

Where:
= rate of sedimentation (cms-1)
D = particle diameter (cm)
= angular velocity about the axis(radianss-1)
r = distance of particle from the axis of rotation (cm)
Pp = density of particle (g cm-3)
Pi = density of liquid (g cm-3)
= viscosity of liquid (g cm-1sec-1)

Principle of centrifugation:
Centrifugal acceleration
Replacing gravitational acceleration with the acceleration
generated by a rotating centrifuge results in faster
sedimentation.
Centrifugal acceleration can be thousands of times greater
than that of gravity, so the centrifugal sedimentation rate is
thousands of times greater.
What is RPM..?
What is RCF..?

Principle of centrifugation:
Centrifugal acceleration
RPM
(Revolutions per
minute)

RCF
(Relative centrifugal
force) is measured in
force x gravity or gforce.

Describe how fast the centrifuge is going

The rotor, regardless of its size, is revolving
at that rate
The force applied to the contents varies by
the size of the centrifuge rotor.

The force exerted on the contents of the

rotor, resulting from the revolutions of the
rotor
It is RCF, not RPM that separates aqueous
solutions in the centrifuge.

Principle of centrifugation:
Centrifugal acceleration
Relationship between RCF and RPM:
2

RPM

RCF = 11.2 r
1000

Where;
RCF
RPM
r

= relative centrifugal force

= speed of rotation
= radius of rotation in centimeters

Exercise #1
The sedimentation coefficient for smaller ribosomes from E.coli
was determine at 70S. Estimate how long it would take to
completely clarify a suspension of these ribosomes in a high
speed centrifuge operating at 10,000 rpm with a tube containing
the ribosomes suspension in which the maximum distance of
travel particles radially outward is 1 cm and the initial distance
from the center of rotation to the particle nearest is 4 cm.

Exercise #1 (solution)
Based on the equation for sedimentation coefficient;
dR
dR 1
2
s=
s dt =
2
dt R
R

When we integrate this equation at t=0 and R=R0 (distance from

center of rotation to the particles nearest to center of rotation:
2 st = ln

R
R0

Exercise #1 (solution)
To determine the maximum time required, we evaluate R at the
maximum travel of the cells from the center of rotation (5 cm)
R
1h

ln
(
)
ln
5
4
R
3600 s
t= 20=
= 8.1 h
2
s
rev 2 rad 1 min

13
x
x
10,000
70x10 s
min
rev
60 s

Exercise #2
If bacterial cell debris has Gt = 54 x 106 s, what is the
centrifuge speed needed for a full sedimentation in 2 h
of time using 10 cm diameter centrifuge?

Exercise #2 (solution)
From the equation for equivalent time:
1
2

m
2R
rad
Gtg 54 x 10 s x 9.81 s 2
Gt =
t=
= 1,213
=

g
s
Rt 0.05 m x 2(3600)s
1
2

rad 1 rev 60 s
= 1,213
x
x
s 2 rad min
= 11,590 rpm

Exercise #3 (solution)
1) Calculate the speed of rotation in RPM. Given:
(a) r = 30 mm, RCF = 6,000 x g
13,360 rpm
(b) r = 100 mm, RCF = 8,000 x g 8,452 rpm
2) Solve RCF value for:
(a) RPM = 40,000, r = 105.4 mm
(b) RPM = 80,000, r = 210.8 mm

188,877xg
1,511,014xg

3) Calculate the radius of rotation in mm if:

(a) RCF = 5,000 x g, RPM = 10,000 4.464 cm
(b) RCF = 7,000 x g, RPM = 5,000 25 cm

Production centrifuge
The concept of centrifugal force (a.k.a centripetal force) can be
applied in rotating devices such as centrifuges, when they are
analyzed in a rotating coordinate system.
A centripetal force is a force that makes a body follow a curved
path.

A body experiencing uniform circular

motion requires a centripetal force,
towards the axis as shown, to
maintain its circular path.

Production centrifuges

Production centrifuges:
Tubular bowl centrifuge
Liquid enters the
bowl through an
opening in the center
of the lower bowl
head.

Particles are
centrifuged towards
the wall of the
cylinder

Clarified liquid will

overflows a ring weir
in the upper bowl
head.

The liquid is pushed

by centrifugal force
toward the periphery
of the rotating bowl.

Production centrifuges:
Tubular bowl centrifuge
The equation of motion is used to determine
the flowrate (Q).
dR 2a 2 ( 0 ) 2 R
=
Radial direction:
9
dt

Axial direction, due to pumped flow:

dz Q
Q
= =
dt A ( R02 R12 )

These equation is combined to give the

trajectory equation: dR
dt = dR
dz dz
dt

Production centrifuges:
Tubular bowl centrifuge
Integrating dR (between R0 and R1) and integrating dz
(between 0 and L) and solve for Q:

2a 2 ( 0 ) L( R02 R12 ) 2

Q=

R
9

ln 0

R1

In term of sigma analysis, Q = {vg}{} where,

L( R 2 R 2 ) 2
0
1

=
R

g ln 0

R1

Production centrifuges:
Disc bowl centrifuge
Feed suspension
enters on the axis
of rotation

Feed is forced to
the bottom of
rotating bowl

Pressure forces
the suspension
upward

Heavier fluid is
forced through
holes at the end
of each disc

Lighter fluid flows

up the disc and
out of the
centrifuge

Dense particles
are ejected
through a nozzle

Flocculation and sedimentation

After cells have been lysis, it is often useful to hasten the
subsequent sedimentation or filtration step by reversibly
increasing the size of the particles to be separated.

Addition of flocculants :

Forming interparticle
molecular bridges
between particles

Reducing the
repulsive forces
between cells.

Flocculation and sedimentation

Sedimentation velocity of flocs:
2 a 2 (1 )( 0 ) 2 R
=
9 ( , a )

Void volume fraction, (reduction of density)

Drag reduction factor,
Floc radius, a

Sedimentation at low accelerations

At low g forces, sedimentation rate slows down and sometimes
be similar with the rate of transport by diffusion
Diffusion, Brownian motion
consequence of a random walk by particles due to thermal
energy kT (k = Boltzmans constant), which represented by:
x 2 = 2 Dt
Where;
<x> = mean square distance
D = diffusion coefficient
t = time

Sedimentation at low accelerations

For spherical particles of radius () undergoing Brownian motion
in a fluid viscosity (), relationship of diffusion coefficient to
thermal energy kT:

D =

kT
6 a

In a concentration gradient, the unidirectional flux of particles is

proportional to D and the gradient, dc/dx, of the particle
concentration c.
Diffusion is not affected by gravity.

Sedimentation at low accelerations

In order to compare sedimentation and diffusion rates:
1. Isothermal
settling
2. Inclined
sedimentation
3. Field-flow
fractionation

Sedimentation at low accelerations:

Isothermal settling
If temperature (T) does not change over the height (h) of an
ensemble of particles, then the mean kinetic energy, which is
proportional to kT, of all particles is the same at all heights.
The potential energy of a particle of mass (m) is usually expressed
as mgh.
If particle are subject to buoyant force, potential energy becomes
V(-o)gh.

Sedimentation at low accelerations:

Isothermal settling
From Boltzmann distribution rule, the concentration of
particles at h at equilibrium is:
V ( 0 ) gh
c ( h ) = c ( 0 ) exp

kT

The Boltzmann distribution rule, explain that:

o concentration (c) is an exponential function of height under isothermal
condition.
o For large, dense particle with potential energy greater than kT
(mammalian cells) will be concentrated at h=0.
o Small particles will have c(h)constant

Sedimentation at low accelerations:

Isothermal settling
Convective motion and Pclet analysis
o Pe number is the ratio of the sedimentation velocity to the
characteristic rate of diffusive transport over distance (L):
Pe =

v
D/L

o If Pe < 0.1: diffusion is dominant and c(h) is distributed

o If Pe > 10: sedimentation dominates

Sedimentation at low accelerations:

Inclined sedimentation
Rapid removal of high density solids can be achieved at 1g by
using inclined sedimentation.
Feed containing
suspended particles is
pumped into the settler
at its lower end

Particle-free overflow
exits the upper end

Particle-rich suspension
leaves in the underflow

Sedimentation at low accelerations:

Inclined sedimentation
Inclined settlers are designed so that the path to the
sedimentation completion of a particle is extremely short (only
few mm) before the sedimented particles begin to be convected
toward the underflow.
If particulate fraction desired, it can be batch concentrated by
recycle of underflow back to the tank while the overflow bleeds
off the supernatant.

Sedimentation at low accelerations:

Filed-flow fractionation
Is designed to separate particles of different sizes on the basis of
hydrodynamics of a very thin layer, flat, horizontal channel
Sample suspension is pumped and subjected to the laminar flow
velocity gradient.
Driving force at lower channel is gravity or centrifugal
sedimentation.
Steep velocity gradient occur at the upper and lower channel:
o Distribution of larger particles towards the center
o Distribution of smaller particles towards the lower wall

As particle transport proceed, the particles bunch up according to

their velocity, thus the lower wall is called accumulation wall.

Centrifugal elutriation
Similar to inclined sedimentation and field-flow fractionate
(sedimentation in presence of fluid flow).
In a centrifugal elutriation (a.k.a counterstreaming
centrifugation), fluid is continuously pumped in the opposite
direction to that sedimentation.

Industrial application
Industrial centrifuges can be classified into two main types:
sedimentation and filtering centrifuges.
1. Sedimentation centrifuges
Use centrifugal force to separate solids from liquids, as well as
two liquids with different specific gravities (decanter, disk-stack,
solid-bowl basket and tubular bowl centrifuges)
2. Filtering centrifuges
Use centrifugal force to pass a liquid through a filtration media,
such as a screen or cloth while solids are captured by the filtering
media. Filtering centrifuges primarily deal with perforate basket,
pusher and peeler centrifuges

Industrial application
Wastewater processing
Deals with separation of municipal, farm, DAF (dissolved air
flotation), trap grease, drilling mud, and environmental
wastewater sludges.
Chemical processing
Which produces raw products such as acids, salts, oil refinery
by-products, polymers, oil-water-solids, and so on.
Pharmaceutical and Biotechnology industries
That manufacture drugs, vaccines, medicines, penicillin,
mycelia, E-coli bacteria, algae, enzymatic waste, etc.

Industrial application
Fuel and Biofuel industry
Including synthetic fuels, biodiesel, ethanol, cellulosic ethanol,
algae biomass dewatering; fuel and lube oil purification, etc.
Food Processing
Which deals with refining of vegetable oils, dairy (milk, cheese,
etc.); poultry, swine and beef rendering; yellow, white, and brown
grease separation; fruit and vegetable juice; beer, wine and liquor
clarification, etc.
Mining and mineral processing
Including coal, tar sands, copper, precious metals, calcium
carbonate, kaolin clay, and many more.

Industrial application
Sedimentation
principles
Equation of
motion
Gravitational
acceleration
Centrifugal
acceleration
Sensitivities
Reynolds
No.

Equilibrium
sedimentation

Production
centrifuge

Methods
Layer the
solution of
decreasing
density
Centrifuge
at high
speed
Gradient
mixing
method
Coefficients
Sedimentatio
n coefficient
Equivalent
time
Sigma
analysis

Tubular
centrifuge
Disk
centrifuge

Flocculation
and
sedimentation

Sedimentation
at low
acceleration
Diffusion,
Brownian
motion
Isothermal
settling
Convective
motion and
Peclet No.
Inclined
sedimentation
Field flow
fractionatio
n

Centrifugal
elutriation

Exercise #4
It is desired to achieve complete recovery of bacterial cells from
fermentation broth with a pilot plant scale tubular centrifuge. It
has been already determined that the cells a approximately
spherical with radius of 0.5 m and have density of 1.10 g/cm.
The speed of the centrifuge is 5000 rpm, the bowl diameter is 10
cm, the bowl length is 100 cm, and the outlet opening of the
bowl has a diameter of 4 cm. Estimate the maximum flowrate of
the fermentation broth that can be attained (viscosity of water is
0.01 g/cm.s).

Exercise #4 (solution)
The flow rate can be estimated by:
Q = {g}{}
where

6
3
g
m
10
cm
2 0.5x106 m x (1.10 1.00) 3 x 9.81 2 x
2
3
2 ( 0 )g
cm
s
m
g =
=
g
9

9 0.01

cm s

cm
= 5.45x106
s
2

Exercise #4 (solution)
For complete recovery of tubular bowl centrifuge;

L( R 2 R 2 ) 2
0
1

=
R

g ln 0

R1

rev 2 rad

(100 cm )x 52 2 2 cm 2 x 5000
x

min
rev

=
2
m
5 100 cm 60 s
9.81 2 x ln x
x

s
2
m
min

= 2.01 x 106 cm 2

Exercise #4 (solution)
Therefore,
Q = {g}{}

cm
L
60 s

6
2
= 5.45x106
x
2.01x10
cm
x
x

s
103 cm3 min

= 0.66

L
min