Reading instructions to centrifugation, precipitation and filtration •Centrifugation Wilson&Walker (edition 6)
chap. 3 + PP slides
•Precipitation Wilson&Walker (edition 6) p. 366 from ”Prel pur steps” - 373 to ”Charge” + PP slides Filtration Copies from Prot. Biotechnol., Walsh&Headon (Filtration, Conc.by UF, Diafilt.) p. 50 - 54 to Aq two-phase part and p. 61 - 65 to Charge + PP slides
Biochemical analysis and separation techniques HT2007/AV
Separation and analysis in biochemistry
Host cells: Bacteria, Yeast, Animal cells Biochemical analysis and separation techniques HT2007/AV
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Centrifugation Analytical Centrifugation
Preparative Processes
Sedimentation in a gravitational field (G) > g = 9.8m/s2 (g = gravitation field of earth)
Biochemical analysis and separation techniques HT2007/AV
RCF - Relative centrifugal field (g-force) f = frequency of rotation (rps) = ω/2π [1/s]
r = radius Normal acceleration (gravitation field) G = aN = ω2 r [m/s2] ω [radian/s] = angular velocity g [m/s2] = gravitational field of earth rps = revolutions per second rpm = revolutions per minute
RCF = G/g = 1.118 x 10-5 f2 r,
f [rpm], r [cm] Biochemical analysis and separation techniques HT2007/AV
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v - Sedimentation velocity r F0 = v f, (v = sedimentation velocity, f = friction coefficient)
F = aN m = 4/3 πr3p (ρp - ρm) ω2r Friction coefficient for a spherical non-hydrated particle f = 6πηrp (Stokes equation) (η = viscosity of media, rp = particle radius) Equilibrium, F = F0
dr/dt = v = 2r2p (ρp - ρm) ω2r/9η Biochemical analysis and separation techniques HT2007/AV
t - Sedimentation time dr v= = kr dt t
where
1
" 2 2rp2 ( # p $ # m ) k= 9%
rmax
dr rmin r
" dt = k " 0
!
!
rt rmin rav
! 1 r t = ln max k rmin
rav gives RCFav
Particles/molecules can be separated with respect to differences in ρ and/or rp (Mw)
rb rmax
!
Biochemical analysis and separation techniques HT2007/AV
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f/f0 - frictional ratio dr v= = kr dt
!
Non ideal particle/molecule
v=
dr kr = dt f f0
f = friction coefficient for a non ideal particle, i.e. hydrated and/or non spherical
!
f0 = theoretical friction coefficient for an ideal particle with the same relative mol weight and density f/f0 ≈ 1 - 1.4 globular proteins
Particles/Molecules with the same mass can be separated with respect to shape/conformation
Biochemical analysis and separation techniques HT2007/AV
s - sedimentation coefficient sobs (sec) = v/G
(G = ω2 r and v =dr/dt)
s20,w = standard sedimentation coefficient s020,w = s20,w at infinite dilution
s 2 0,w = s o b s !
"T ,w (1 # $ % 2 0,w ) " ! l ! " 2 0,w "w (1 # $ % T ,l )
ν = specific volume of a solute = 1/ρ solute, non hydrated η = viscosity ρ = density T = experimental temperature w = water l = solvent in experiment
Biochemical analysis and separation techniques HT2007/AV
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Typical size range of sedimentation coefficients - s Substances
s(S)
Soluble prote i n s
2-25
Nucleic acids
3-100
Ribosome s
20-200
Viruses
40-100 0
Membranes
100-100x103
Mitochond r i a
20x103
Cell nucleus
4000x103- 40000x103
1 S = 10-13 seconds where S = Svedberg unit
Biochemical analysis and separation techniques HT2007/AV
Centrifuges - type - application Type Small table centrifuges Micro centrifuges
Volume (ml) - 10 - 1.5
RCF (x g) Application 3 000-7 000 Cells, precipitat e s - 10 00 0
Refrigerated centrifuges
- 4 000
- 6 500
Cells, precipitat e s
Refrigerated high speed Continuous
- 1 500 1500 ml/ m i n
- 60 00 0
Cells, cell debris, Larger organelles Protein precipitates
Preparative ultracentrifuges
- 6 ml x 8 - 40 ml x 8
- 800 000 - 500 000
Preparation cell organelles, macromolecules
Analytical ultracentrifuges
0.4 - 1
- 500 000
Sedimentation data Cell organelles, macromolecules
- 10 00 0
Cell removal, Cell debris removal, precipitate s
Process 20 litres/h– centrifuges/separatores more then Semi-continuous, continuous 1000 litres / h
Biochemical analysis and separation techniques HT2007/AV
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Centrifuge rotor types Rotor types Swing-out rotor Angular rotor Vertical rotor
Rotor construction material Brass, steel (for low speed) Aluminium alloy Titan alloy
(for high speed)
Carbon fibre
Biochemical analysis and separation techniques HT2007/AV
Swing-out rotor From: Wilson and Walker, Principal and Techniques in Practical Biochemistry, 5th ed 2000, Cambridge University Press
Biochemical analysis and separation techniques HT2007/AV
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Fixed-angel rotor From: Wilson and Walker, Principal and Techniques in Practical Biochemistry, 5th ed 2000, Cambridge University Press
Biochemical analysis and separation techniques HT2007/AV
Vertical rotor From: Wilson and Walker, Principal and Techniques in Practical Biochemistry, 5th ed 2000, Cambridge University Press
Biochemical analysis and separation techniques HT2007/AV
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Centrifuge rotor care and maintenance Rotor-/centrifug maintenance •Corrosion - avoid acids, strong alkaline detergents •Keep the rotor clean, use D.I. water, dry place for storage •Balance the sample •Reduce the specified maximum rotational speed, M, when the solvent density, N > 1.2 g/ml Mn = [1.2xM2/N]1/2 Tube material, tube choice •Glass, cellulose ester, polycarbonate, polyethylene, polypropylene, nylon, stainless steel •Sample volume, sample type (ex. organic solvent), rotor type, rpm, temperature Biochemical analysis and separation techniques HT2007/AV
Preparative centrifugation Differential centrifugation • Differences in size, shape and density different sedimentation velocity • Pellet fraction - a mix of components •Supernatant - pure, low yield •Fractionation by stepwise increase of RCF (ex. Fig. 3.5 p 120 in Wilson and Walker ed 6) From: Wilson and Walker, Principal and Techniques in Practical Biochemistry, 5th ed 2000, Cambridge University Press
Biochemical analysis and separation techniques HT2007/AV
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Centrifugation - preparation of density gradients Continuous gradient • Linear • Convex • Concave • Gradient mixer top loading (hydrophilic tube) bottom loading (hydrophobic tube)
Step gradient • Underlay method • Overlay method Biochemical analysis and separation techniques HT2007/AV
Centrifugation - gradient material
Ionic strength, pH, viscosity, Density-max, UV abs, Osmotic effect, inert towards biological material etc.
Ionic media (CsCl, NaBr, NaI):
Nucleic acids, Proteins, Virus
Sucrose:
Organelles, Virus
Non-ionic aromatic compounds : Broad use
Biochemical analysis and separation techniques HT2007/AV
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Preparative centrifugation Density gradient centrifugation 1. Rate zonal • Maximum density of gradient < highest density of macromolecules/particles to be separated • Separation of component with similar density but different size/shape (eg. proteins) • Time dependent From: Wilson and Walker, Principal and Techniques in Practical Biochemistry, 5th ed 2000, Cambridge University Press
2a. Isopycnic (same density) • Maximum density of gradient > highest density of any particles/macromolecules to be separated • Separation of components with different density, similar size (eg. subcelullar organelles, mitochondria, peroxisomes) • Size dependent, time independent • Pre-prepared density gradient Biochemical analysis and separation techniques HT2007/AV
Preparative centrifugation Density gradient centrifugation 2b. Equilibrium isodensity method The density gradient is formed during the run, run time > 40 h Analytical use eg. for DNA and for separation of different DNA (eg. CsCl - gradient) From: Wilson and Walker, Principal and Techniques in Practical Biochemistry, 5th ed 2000, Cambridge University Press
Biochemical analysis and separation techniques HT2007/AV
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Centrifugation - purpose of density gradient Rate zonal centrifugation • Reduce convection • Reduce effects of vibrations and thermal movement Isopycnic centrifugation • Collect separated zones at equilibrium density
Biochemical analysis and separation techniques HT2007/AV
Centrifugation - sample application on density gradient Rate zonal centrifugation • Aim for small loading zone
Sample volume and concentration critical
• Top loading
Isopycnic centrifugation • Top loading • Bottom loading
Sample volume + distribution non-critical
• Mix with gradient
Biochemical analysis and separation techniques HT2007/AV
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Centrifugation - sample application on gradient • Optimal sample concentration + volume are important for the best resolution • Tube diameter (cm)
sample volume (ml)
1.0
- 0.2
1.6
- 0.5
2.5
-1
• Sample conc. (nucleic acids, proteins)
1µg/ml - 1mg/ml
Biochemical analysis and separation techniques HT2007/AV
Centrifugation - sample recovery from density gradient tube Upward displacement
UV Fraction collector
small sized protein
Chemical analysis Enzymatic analysis
medium sized protein large sized protein
Pump Biochemical analysis and separation techniques HT2007/AV
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Centrifugation - application of preparative rotors
Rotor type Swing-out Angular rotor Vertical rot o r
Centrifugation techniqu e Differential Rate zonal Isopycn i c ++ + +++ ++ + +++
+++: very good, ++:good, +: acceptable, -: bad, 0: not possible
Biochemical analysis and separation techniques HT2007/AV
Help for rotor choice: k - factor k = 2.53"1011
(ln(rmax ) # ln(rmin )) (rpm) 2max
k-factor: the relative pelleting efficiency of a rotor and given by the manufacturer (dimension + speed of rotor) Calculated at max. rotor speed and at density + viscosity ≈ H2O
! max rpm: krevised below
k revised = k "
(rpm) 2max (rpm) 2selected
! k -factor helps to estimate the time for an required pelleting:
t [h] = k/s20,w Biochemical analysis and separation techniques HT2007/AV
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Centrifugation - application of analytical ultracentrifugation •Determination of relative molecular mass •Estimation of purity of macromolecules •Detection of conformational changes in macromolecules
Biochemical analysis and separation techniques HT2007/AV
Analytical ultracentrifugation – detection system UV- Light absorption Refractive index - Schlieren system - Rayleigh system From: Wilson and Walker, Principal and Techniques in Practical Biochemistry, 5th ed 2000, Cambridge University Press
Biochemical analysis and separation techniques HT2007/AV
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Precipitation Soluble macromolecule Change of physicochemical conditions
Precipitation of macromolecule • Salt addition • Temperature • pH • Organic solvent addition • Water soluble polymer addition • Metal ions addition Fractionation by repeated addition of increased salt concentrations [(NH4)2SO4] with separation steps in between (Ex. Tab. 8.3 p. 367, Wilson & Walker) Biochemical analysis and separation techniques HT2007/AV
Precipitation - salting out • NH4SO4 • Na2SO4 • Protein stabilising conditions • Salting out effect (hydrophobic interaction) and salting in (electrostatic repulsion) on going at the same time
ß
Empirical equation: log S/S0 = ß + Kc S0 = protein solubility at zero salt concentration
log S/S0
S = protein solubility ß = f(protein, temp, pH) K = f(protein, salt) slope = -K Salt concentration, c Biochemical analysis and separation techniques HT2007/AV
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Precipitation - organic solvent • Low temp. (0°C) - minimise ”unfolding” - denaturing • Flammable • Sometimes expensive • Methanol, ethanol, isopropanol, acetone, trichloro acetic acid • Reduced water availability for protein due to hydration of organic molecules • Increased electrostatic attractive interaction between proteins due to change of dielectrical properties by organic molecules Electrostatic force, ∆E, between two charged particles with the charges ZA and ZB, respectively, separated by the distance rAB ∆E [J] = ZA ZB ε2 /D rAB D ε = 1 electron charge [As] D = dielectrical constant [As/Vm]
H2O: 80 Methanol: 33 Ethanol: 24 Isopropanol: 20
Biochemical analysis and separation techniques HT2007/AV
Precipitation - water soluble polymers Poly ethylene glycol Mw 6000 - 12000 •HO-[CH2-CH2-O]n-H
n = 130-270
•Typical concentration range
6-12%
•Reduced water availability for protein due to exclusion effect •Mostly protein stabilising conditions Poly ethylenimine •Cationic polymer •Nucleic acid precipitation Protamin sulphate •Mixture of small strongly basic proteins •Nucleic acid precipitation Biochemical analysis and separation techniques HT2007/AV
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Precipitation - metal ions •Irreversible, denaturing effect on protein •Precipitation of DNA eg. Mn2+
Biochemical analysis and separation techniques HT2007/AV
Filtration Application Sterile filtration Buffer exchange- desalting Cell concentration - cell removal Fractionation and concentration of macromolecules
Type of filtration Dead-end filtration Tangential flow filtration (TFF)/ cross flow filtration (CFF)
Tangential flow filtration
Biochemical analysis and separation techniques HT2007/AV
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Filter types Depth filters •Glass fibre, cellulose •irregular network of random oriented fibres Membrane filters •Cellulose nitrate, cellulose acetate, poly sulfone, poly vinyl chloride, poly carbonate •Membrane like thin layer with small pores sitting at a coarser support structure •Absolute filter, elimination of particles larger than a given size is guarantied •Nominal filter, elimination of all particles larger than a given size is not guarantied
Biochemical analysis and separation techniques HT2007/AV
Membrane filtration modes Mode
Pore size
TMP
Typical flux
Application
(trans membrane pressure)
(µm)
(bar)
(l/m2h)
RO
non porous diffusion
30-100
0.5
desalting
Ultrafiltration (UF)
10-3-10-2 (1-1000 kDa)
2-10
10-60
macromolecules (particulate matter)
2-10
10-100
particulate matter (macromolecules)
Microfiltration (MF) 10-1-10
Flux = flow through the filter membrane [volume/filter area and time]
Biochemical analysis and separation techniques HT2007/AV
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Tangential flow filtration (TFF) Pin
Pret
Feed Membrane Pperm Permeate (Filtrate) Driving force: Trans membrane pressure (TMP)
TMP =
p in + p ret " pperm 2
! Biochemical analysis and separation techniques HT2007/AV
Membrane filtration – rejection characteristics
Biochemical analysis and separation techniques HT2007/AV
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Stirred cell membrane filtration unit and flux-TMP relation Stirred cell, 2 – 100 ml Pressurised e.g. with air
Filter membrane Filtrate out Biochemical analysis and separation techniques HT2007/AV
Filtration unit – cassette system
membrane retentate channel
0.1 m2
filtrate channel Out filtrate
in
0.5 m2 Out retentate Biochemical analysis and separation techniques HT2007/AV
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Diafiltration Vw
P
σ = rejection coefficient
Vp
Vw = volume of added solvent (eg. buffer)
V0
C
V0 = initial volume Vp = volume of permeate (filtrate) C0 = initial concentration of a solute molecule CF = final concentration of a solute molecule
v
c =ce
If σ = 0
c
F
=
ce 0
-
F
vw (1"# ) v0
0
c =c
If σ = 1
F
- vw0
0
!
Biochemical analysis and separation techniques HT2007/AV
!
!
Concentration P
σ = rejection coefficient V0 = initial volume
Vp
Vp = volume of permeate (filtrate)
V0
C0 = initial concentration of a solute molecule
C
VF
CF = final concentration of a solute molecule
ConcFact =
V0 VF
σ CF = C0 x ConcFact
If σ = 1 If σ = 0
CF = C0 x ConcFact CF = C0
Biochemical analysis and separation techniques HT2007/AV
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