UNIT OPERASI BIOPROSES (UOB)

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1 TPE4211 UNIT OPERASI BIOPROSES (UOB) YUSRON SUGIARTO KULIAH 7

2 MATERI KULIAH No Pokok Bahasan Waktu (Jam) 1. Pengantar 2. Satuan dimensi 2 x 50 3 Kesetimbangan Massa 2 x Kesetimbangan Energi 2 x Kesetimbangan Unsteady-State 2 x 50 Massa dan Energi 6. Unit operasi: Filtrasi 2 x Unit operasi: Sentrifugasi 2 x 50

3 CENTRIFUGATION

4 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.

5 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.

6 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.

7 Centrifugation Theory and Practice Routine centrifuge rotors Calculation of g-force Differential centrifugation Density gradient theory

8 Centrifuge rotors Swinging-bucket axis of rotation Spinning g At rest g Fixed-angle

9 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.

10 Geometry of rotors r max r av r min axis of rotation a r min r av r max r min r av r max b c Sedimentation path length

11 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 swingingbucket rotor (a) is routinely described by three parameters, the r min, r av and r max (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 g min, g av and g max.

Geometry of rotors 2 In a fixed angle rotor the value of r min, r av and r max (and the corresponding RCFs) is modulated by the angle of the tube to the vertical; the difference between r min and r

12 Geometry of rotors 2 In a fixed angle rotor the value of r min, r av and r max (and the corresponding RCFs) is modulated by the angle of the tube to the vertical; the difference between r min and r max 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 r max r min. For tubes of equal volume and dimensions, the sedimentation path length is longest for a swinging-bucket rotor and shortest for a narrow-angle fixedangle rotor, although in Training File 2 rotors are described, which have even shorter sedimentation path lengths.

13 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: t 1 k 1 k t 2 2

14 Calculation of RCF and Q RCF Q x r Q 299 RCF r RCF = Relative Centrifugal Force (g-force) Q = rpm; r = radius in cm

15 RCF in swinging-bucket and fixedangle rotors at 40,000 rpm Beckman SW41 swinging-bucket (13 ml) g min = 119,850g; g av = 196,770g; g max = 273,690g Beckman 70.1Ti fixed-angle rotor (13 ml) g min = 72,450g; g av = 109,120g; g max = 146,680g

16 Velocity of sedimentation of a particle v d 2 ( p l ) g 18 v = velocity of sedimentation p = density of particle g = centrifugal force d = diameter of particle l = density of liquid = viscosity of liquid

17 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.

18 Size of major cell organelles Nucleus Plasma membrane sheets Golgi tubules Mitochondria Lysosomes/peroxisomes Microsomal vesicles 4-12 m 3-20 m 1-2 m m m m

19 Differential centrifugation of a tissue homogenate (I) Decant supernatant 1000g/10 min etc. 3000g/10 min

20 Differential centrifugation of a tissue homogenate (II) 1. Homogenate 1000g for 10 min 2. Supernatant from g 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

21 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

22 Differential centrifugation (IV) Poor resolution and recovery because of: Particle size heterogeneity Particles starting out at r min have furthest to travel but initially experience lowest RCF Smaller particles close to r max have only a short distance to travel and experience the highest RCF

23 Differential centrifugation (V) Swinging-bucket rotor: Long sedimentation path length g max >>> g min Fixed-angle rotor: Shorter sedimentation path length g max > g min

24 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.

25 Density gradient centrifugation Density Barrier Discontinuous Continuous

26 How does a gradient separate different particles? Least dense Most dense

27 Predictions from equation (I) v d 2 ( p l ) g 18 When p > l : v is +ve When p = l : v is 0

28 Predictions from equation (II) v d 2 ( p l ) g 18 When p < l : v is - ve

29 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

30 1 2 Buoyant density banding Equilibrium density banding Isopycnic banding 3 4 5

31 3 Formats for separation of particles according to their density When density of particle < density of liquid V is -ve

32 Resolution of density gradients Density Barrier Discontinuous Continuous I II

33 Problems with top loading

34 Separation of particles according to size p >> l : v is +ve for all particles throughout the gradient

35 THANK YOU...

Theory module: 11(MCQ) CENTRIFUGATION

Theory module: 11(MCQ) CENTRIFUGATION Theory module: 11(MCQ) 1. A primary objective of cell fractionation is A. to crack the cell wall so the cytoplasmic contents can be released. B. to identify the enzymes outside the organelles. C. to view