bioseparation

8/7/2019 Bioseparation

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Bio-separation engineering:Recovery, isolation, purification and polishing of synthesized

by biotechnological processes

Extended definition: Final polishing steps of processes such as biotechnology

based effluent treatment and water purification

Properties of the biological products:Size , Molecular weight , Diffusivity, Sedimentation Coefficient, OsmoticPressure , Electrostatic Charge, Solubility, Partition Coefficient, Light

absorption and Flourescence.

An ideal bioseparation process should combine high

throughput with high selectivity, and should ensure stability

of product.


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Transport Phenomena in

Separation process

Heat Transfer Fluid Flow

Mass Transfer

Mass Transfer Migration of a component in a mixture in the same

phase or from phase to phase because of a difference

in concentration

Rate of a transfer process = driving force

resistance


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Examples of mass transfer

Evaporation of water.

Dissolving ink in water.

Dissolving of Oxygen in the solution to the microorganism in the

fermentation process Reaction occurs when reactants diffuse from the surrounding medium

to the catalyst surface

The driving force for mass transfer

1.Concentration different

2.Pressure different3.Electrical gradient.

Mass transfer takes place from solid to liquid, solid to vapor or gas, liquid

to liquid, liquid to vapour or gas.

Its a complex phenomenon taking place in many of unit operation like

Humidification $ dehumidification, Extraction, Drying, Evaporation,Distilation.


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Mass Transfer

Molecular Diffusion

Gases Liquid Solid

Convective MassTransfer


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Transfer of individual molecules through a fluid by random

movement/ walk proces.

From a region of high concentration to low concentration

E.g. A drop of black liquid dye is added to a cup of water the

dye molecules will diffuse slowly by molecular diffusion to all

parts of the water.

-the liquid can be mechanically agitated by a spoon to increase

the rate of diffusion resulting in

Diffusion of molecules(A-sugar) when the bulk fluid(B-water)

is stationary is given by Ficks law

JA = - DAB. dcA/dx

JA = Flux of A in B( kg-moles/m.s)

c - conc. of A (kgmoles/ m3)

x length along direction of diffusion(m).

DAB- molecular diffusivity of A in B (m2/s)


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:

Amino Acid(A) in water(B)

Flux of A from point 1 to 2 in the medium

Flux of B from point 2 to 1 in the medium

The diffusion constant being same

DAB = DBA

Hence JA = - JB

JA= -DAB dcA= - DAB.cA2 - cA1dx x2 x1

JB = -DBAdcB = - DBA.cB1 cB2dx x2 x1

Equimolar Counter Diffusion i.e. molar flux of A in a certain direction is

matched by the flux of B in the opposite direction.


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Diffusion Cell

DAB = V ln (c1- c2/ c1- c2)

2at

= tortuosity of the membrane

= Thickness of the membrane (m)

= porosity of the membrane

V= Volume of the chamber(m3)

a = area of the membrane

t = time(s)

c1 = solute concentration in chamber 1( kg-moles/m3)

c2 = solute concentration in chamber 2( kg-moles/m3)


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:

Diffusivity of a solute in a liquid medium at a particular temperature by .

Three most widely used correlations :

Stokes- Einstein correlation:DAB = 9.96 x 10

-16 T

VA1/3

Where,

T = absolute temperature (K)

= viscosity of the liquid medium(kg/ms)

VA = solute molar volume at its normal boiling point(m3/kg-mole)

Wilke-Chang correlation: DAB =1.173x10-16(MB)

1/2T

VA0.6

DAB = 9.40 x 10-15T

MA1/3

Polson correlation:

Where ,

= association parameter(2.6 for water)

MB = Molecular weight of the liquid medium(kg/kg-mole)

Where ,

MA = Molecular weight of the solute(kg/kg-mole)

For electrolytes : Nernst-Haskell correlation:

DAB = 8.928 x 10-10T(1/n+ + 1/n_)

(1/ + +1/ _)

Where ,

n+ = valency of the cation n_ = valency of the anion

+= ionic conductance of the cation _ = ionic conductance of the anion


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Dense Solids: by dissolving in it eg: ions through dense membranes

Porous Solids:

Hindered diffusion

JA= Deff(cA1 - cA2)

(x2 x1)

Where,

D = diffusivity of the solute in the liquid within the pores(m2/s)

Deff = effective hindered diffusivity(m2/s)

= tortuosity of the medium

= porosity of the medium

Unhindered diffusion

JA= D(cA1 - cA2)

(x2 x1)


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Using mechanical force or action to increase the rate of molecular diffusion . It takes place in

flowing fluids , particularly when the flow is turbulent in nature i.e. when there are eddies.

Eg. Stirring of water to dissolve coffee during coffee making

Solid-liquid system Liquid-liquid system

Flux for convective mass transfer:

NA= - (D+E). dcA/dx

Where, E= eddy diffusivity(m2/s)

The above equation can be written as:

NA= kAcA Where, kA = mass transfer coefficient(m/s)


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The average flux across the mass transfer zone can be calculated

from

NA= m/ at

Where ,

m = amount of solute transferred(kg-moles)

a = mass transfer area(m2)

t = transfer time(s)

kA = m/ at cA


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Inter phase Mass Transfer:

C1

C2

Ci2

Ci1Liquid 1

Liquid 2

x1 x2

J = D1 (C1 - Ci1) = D2(Ci2 -C2 )

x1

x2


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bacteria

moulds

Yeast cell Plant cell

Animal cell

Ground tissue


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Gram Negative Bacteria

Sub-micron to 1 micron in size

Cell capsule present

Peptidoglycan layer is thin

Periplasmic space present

Mechanically less robust than gm+

bacteria

Chemically more resistant than gm+bacteria

Gram Positive Bacteria

Sub-micron to 2 microns in size

Have thick cell walls, 0.02-0.04

microns, peptidoglycan +

polysaccharide+ teichoic acid

Phospholipid cell membrane present


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Yeast: 2-20 microns in size, spherical or ellipsoid

Moulds: Bigger and filamentous

Yeasts are unicellular while moulds are multicellular

Very thick cell walls are present in both

Cell wall is mainly composed of polysaccharides such

as glucans, mannans and chitins

Plasma membranes are mainly made up of

phospholipids


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Animal cells do not have cell walls

Animal cells are very fragile

Cultured animal cells are several microns in size

Spherical or ellipsoid

Plant cells can be bigger

Plant cells have thick and robust cell walls mainly

composed of cellulose

Plant cells are difficult to disrupt

Cultured plant cells are less robust than real plant cells


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Types of

CellDisruption

PhysicalMethods

Bead Mill

Rotor-StatorMill

French Press

Ultrasonic

vibrations

Chemical andphysiochemical

methods

detergents

Enzymes

Solvents

OsmoticShock


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Bead mill

Cascading

beads

Cells beingdisrupted

Rolling

beads

Disruption takes place due to the grinding action of the

rolling beads and the impact resulting from the

cascading ones

Bead milling can generate enormous amounts of heat

Cryogenic bead milling : Liquid nitrogen or glycol

cooled unit

Application: Yeast, animal and plant tissue

Small scale: Few kilograms of yeast cells per

hour

Large scale: Hundreds of kilograms per hour.


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Rotor-Stator mill

Cellsuspension

Rotor

Stator

Disrupted

cells

Typical rotation speeds: 10,000 to 50,000 rpm

Mechanism of cell disruption: High shear andturbulence

Application: Tissue based material

Single or multi-pass operation


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Plunger

CylinderCellsuspension

Impactplate

Jet

Orifice

Application: Small-scale recovery of intracellular proteins and DNA

from bacterial and plant cells

Primary mechanism: High shear rates within the orifice

Secondary mechanism: Impingement

Operating pressure: 10,000 to 50,000 psig

French press


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Cell suspension

Ultrasound tip

Ultrasoundgenerator

Application: Bacterial and fungal cells

Mechanism: Cavitation followed by shock waves (

long or short)

Frequency: 25 kHz

Time: Bacterial cells 30 to 60 seconds, yeast cells 2

to 10 minutes

Used in conjunction with chemical methods


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Solubilize the phospholipids of the cell membrane.

Three typesCatonic Anionic Non-ionic

eg: Triton, Tween series


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lysozymeslysozymes,, zymolasezymolase, proteases, proteases

Acetone, SDSAcetone, SDS


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ADVERSE FACTORS DURING DISRUPTION

The following are some of the several adverse factors to be considered while

selecting a disruption method.

* Heat generation* Release of Proteases

* Nucleic acid contamination

* High Foaming

* Heavy metal Toxicity


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bioseparation

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