UNIT OPERASI BIOPROSES (UOB)

TPE4211

YUSRON SUGIARTO KULIAH 7

MATERI KULIAH No Pokok Bahasan Waktu

(Jam) 1. Pengantar 2. Satuan dimensi 2 x 50 3 Kesetimbangan Massa 2 x 50

4. Kesetimbangan Energi 2 x 50 5. Kesetimbangan Unsteady-State

Massa dan Energi 2 x 50

6. Unit operasi: Filtrasi 2 x 50 7. Unit operasi: Sentrifugasi 2 x 50

CENTRIFUGATION

Centrifugation is used to separate materials of different density when a force greater than gravity is desired.

In bioprocessing, centrifugation is used to remove cells from fermentation broth, to eliminate cell debris, to collect precipitates, and to prepare fermentation media such as in clarification of molasses or production of wort for brewing.

CENTRIFUGATION

Equipment for centrifugation is more expensive than for filtration; however centrifugation is often effective when the particles are very small and difficult to filter. Centrifugation of fermentation broth produces a thick, concentrated cell sludge or cream containing more liquid than filter cake.

CENTRIFUGATION

In centrifugation of biological solids such as cells, the particles are very small, the viscosity of the medium can be relatively high, and the particle density is very similar to the suspending fluid. These disadvantages are easily overcome in the laboratory with small centrifuges operated at high speed.

CENTRIFUGATION

Centrifugation Theory and Practice

• Routine centrifuge rotors • Calculation of g-force

• Differential centrifugation • Density gradient theory

Centrifuge rotors

Fixed-angle

axis of rotation

At rest

Swinging-bucket

g

Spinning g

Centrifuge rotors There are two traditional types of rotor: swinging-bucket and fixed-angle 1. In the swinging-bucket rotor, at rest, the tube and bucket are vertical and the meniscus of the liquid is at 90° to the earth’s vertical centrifugal field. During acceleration of the rotor the bucket, tube and meniscus reorient through 90° in the spinning rotor’s radial centrifugal field. 2.In the fixed-angle rotor only the meniscus is free to reorient – one of the reasons why open-topped tubes in particular must not be filled beyond the recommended level in a fixed-angle rotor, if spillage is to be avoided.

Geometry of rotors

b c

rmax

rav

rmin

rmax

rav

rmin

Sedimentation path length

axis of rotation

a

rmax rav rmin

Geometry of rotors The g-force or relative centrifugal force (RCF) in a rotor tube increases linearly with the radius, so the geometry of the tube with respect to the axis of rotation is important in determining the suitability of a rotor for a particular particle separation. 1 The rotor (or tube) of the swinging-bucket rotor (a) is routinely described by three parameters, the rmin, rav and rmax (the distance from the axis of rotation to the top, midpoint and bottom of the tube) and the RCF at each point is described as gmin, gav and gmax.

Geometry of rotors 2 In a fixed angle rotor the value of rmin, rav and rmax (and the corresponding RCFs) is modulated by the angle of the tube to the vertical; the difference between rmin and rmax being greater in a rotor whose tubes are held at a wide angle to the vertical (b) than in one with a narrow angle (c). 3 The sedimentation path length of the rotor (or tube) is rmax – rmin. For tubes of equal volume and dimensions, the sedimentation path length is longest for a swinging-bucket rotor and shortest for a narrow-angle fixed-angle rotor, although in Training File 2 rotors are described, which have even shorter sedimentation path lengths.

k’-factor of rotors • The k’-factor is a measure of the time taken for

a particle to sediment through a sucrose gradient

• The most efficient rotors which operate at a high RCF and have a low sedimentation path length therefore have the lowest k’-factors

• The centrifugation times (t) and k’-factors for two different rotors (1 and 2) are related by:

2

211

k

tkt

Calculation of RCF and Q 2

100018.11

QrxRCF

r

RCFQ 299

RCF = Relative Centrifugal Force (g-force) Q = rpm; r = radius in cm

RCF in swinging-bucket and fixed-angle rotors at 40,000 rpm

• Beckman SW41 swinging-bucket (13 ml) • gmin = 119,850g; gav = 196,770g; • gmax = 273,690g • Beckman 70.1Ti fixed-angle rotor (13 ml) • gmin = 72,450g; gav = 109,120g; • gmax = 146,680g

gd

vlp

18

)(2

Velocity of sedimentation of a particle

v = velocity of sedimentation d = diameter of particle

p = density of particle l = density of liquid

= viscosity of liquid g = centrifugal force

Differential centrifugation • Density of liquid is uniform • Density of liquid << Density of particles • Viscosity of the liquid is low • Consequence: Rate of particle sedimentation depends

mainly on its size and the applied g-force.

Size of major cell organelles • Nucleus 4-12 m

• Plasma membrane sheets 3-20 m

• Golgi tubules 1-2 m

• Mitochondria 0.4-2.5 m

• Lysosomes/peroxisomes 0.4-0.8 m

• Microsomal vesicles 0.05-0.3m

Differential centrifugation of a tissue homogenate (I)

1000g/10 min

Decant supernatant

3000g/10 min etc.

Differential centrifugation of a tissue homogenate (II)

1. Homogenate – 1000g for 10 min 2. Supernatant from 1 – 3000g for 10 min 3. Supernatant from 2 – 15,000g for 15 min 4. Supernatant from 3 – 100,000g for 45 min • Pellet 1 – nuclear • Pellet 2 – “heavy” mitochondrial • Pellet 3 – “light” mitochondrial • Pellet 4 – microsomal

Differential centrifugation (III) Expected content of pellets

• 1000g pellet: nuclei, plasma membrane sheets

• 3000g pellet: large mitochondria, Golgi tubules

• 15,000g pellet: small mitochondria, lysosomes, peroxisomes

• 100,000g pellet: microsomes

Differential centrifugation (IV)

Poor resolution and recovery because of:

• Particle size heterogeneity

• Particles starting out at rmin have furthest to travel but initially experience lowest RCF

• Smaller particles close to rmax have only a short distance to travel and experience the highest RCF

Differential centrifugation (V)

Fixed-angle rotor: Shorter sedimentation path length gmax > gmin

Swinging-bucket rotor: Long sedimentation path length gmax >>> gmin

Differential centrifugation (VI) • Rate of sedimentation can be modulated by

particle density • Nuclei have an unusually rapid sedimentation

rate because of their size AND high density • Golgi tubules do not sediment at 3000g, in spite

of their size: they have an unusually low sedimentation rate because of their very low density: (p - l) becomes rate limiting.

Density Barrier Discontinuous Continuous

Density gradient centrifugation

How does a gradient separate different particles?

Least dense

Most dense

gd

vlp

18

)(2

When p > l : v is +ve When p = l : v is 0

Predictions from equation (I)

gd

vlp

18

)(2

When p < l : v is -ve

Predictions from equation (II)

Summary of previous slides

• A particle will sediment through a solution if particle density > solution density

• If particle density < solution density, particle will float through solution

• When particle density = solution density the particle stop sedimenting or floating

Buoyant density banding

Equilibrium density banding

Isopycnic banding

1

5

2

3

4

1

2

3

3 Formats for separation of particles according to their density

When density of particle < density of liquid V is -ve

Discontinuous

Resolution of density gradients Continuous Density Barrier

I II

Problems with top loading

p >> l : v is +ve for all particles throughout the gradient

Separation of particles according to size

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