prin bio 1-4?

List of common unit operations

filtration: based on particle size

centrifugation: based on particle density

drying: based on water changes

crystallization: based on phase changes

chromatography: based on spread of particles

mixing: with chemical compositions or w/o

homogenisation: chemical or physical treatment / breaking cells

adsorption: physiochemical treatment, adhesion of gas/solid/liquid materials onto cell surface

solvent extraction: separate compound from mixture by dissolving it in solvent

Products

whole cell as product like yeast

product inside cell like mRNA vaccines or astaxanthin pigments

extracellular products like collagen or human insulin

Steps of downstream processing

separation of cells and culture media inside fermenter

for intracellular products: conc. cells in wet paste get broken. the products inside cells aare released into mixture. filtration/centriguation happens removing cell debris and leaving product suspended and then primary isolation is used

for extracellular products, the culture media is cell free and use primary isolation with products extract but with some impurities

purification either with crystallisation or drying to get single final product with high purity

What is centrifugation?

separation process based on different density of materials by application of a force greater than gravity

used for cell removed, cell recovery, elimination of cell debris, precipitate and crystal collections

Effectiveness of centrifugation depends on

size of cell, shape of cell, viscosity of liquid, centrifugal radius, time for sedimentation, rotational speed, density of liquid and cell

Benchtop centrifuge

for small scale, used in labs

need to balance the samples on the opposite side to be same weight

use centrifuge tubes to avoid spills

liquid = supernatant

sediment = extract

Tubular bowl

used in food and pharmaceutical industries

spins 13k - 16k times gravity force to reach the wall within the residence time of liquid machine

ultracentrifuge: narrow type of tubular bowl for fine precipitates like ribosomes from high density solutions

used when difficult separations with high centrifuge are required

Disc stack bowl

common in bioprocessing with minimum solid-liquid density difference (0.01-0.01 kmm³)

solids must be removed periodically

heavier components goes to outward wall of the ball

spins 5k to 15k times gravity force

RCF

radial/centrifugal force generated by the spinning rotor as expressed relative to earth’s gravitional force

Stokes law involves….

friction F

buoyancy B

centrifugal force C

How to increase particle velocity in given centrifuge

increase RPM/speed

increase particle diameter

increase density difference ps-pb

decrease viscosity of suspending fluid

What happens when scaling up?

more mechanical stress so radius should increase

Define mixing

physical operation that reduces non uniforms in fluid by eliminating gradients of concentration, temperature and other properties

Mixing is used for…

blend/dissolve soluble components into liquid

disperse gases through liquid in small bubbles for cells to access

promote heat transfer to or from liquids

optimize environment for cell culture

deliver nutrient elements to cells throughout the vessels

maintain suspension of cells in liquid media

Parts of mixer

impeller - rotating parts use to increase fliud flow

Baffle - assist mixing and create turbulence in fluid by breaking up circular flow generated by rotation of impeller (1/10 - 1/12 of tank diameter)

Sparger - helps deliver air to vessel

stirrer shaft - supports impeller weight and transmit torque from stirrer motor to the impeller

Torque

tendency of force to cause an object to rotate

Impellers for air dispersion

six flat blade disc turbine

propeller

pitched blade turbine

for industrial fermentation

Impellers not for air dispersion

anchor

gate anchor

helical screw

Factors affecting choice of impeller

liquid viscosity

shear flow requirement

levels of volecity/dispersion

pumping/flow capacity

Rotational flow

circular flow move liquid in streamline aroud stirrer shaft but little top to bottom mixing which produces high mechanical stresses

Radial/horizontal flow

generated by impeller with blades parallel to stirrer shaft

one stream flows to top of tank, the other to bottom of tank

Axial flow

generated by impeller with inclined or pitched blades that make angle of less than 90 degrees

fluid goes along the wall then draw back towards impeller (loop)

useful when strong vertical current required

Bioprocessing mixing types

non aerated - without gase

aerated - with gas

Gassing …..

reduces power requirement because of decrease in density of fluid

Chromatography is…

separation process for resolving mixtures into individual components based on differential migration

Gas chromatography

mobile phase is gas and used for volatile compounds

Solid chromatography

mobile phase is liquid and used for separating solid phases

Adsorption chromatography

adsorbent has power to hold molescules at their surface

holding force comes from van der walls and H bonding

performance deponds on chemical composition or surface and molecule polarity

TLC works base on adsorption chromatography

Partition chromatography

based on unequal distribution of solute between two immiscible solvents

achieved by fixing one solvent to a support

normal phase - stationary phase is more polar than mobile phase

reverse phase - stationary phase is less polar than mobile phase

Ion exchange chromatography

based on electrostatic attration between solute and dense clusters of charged groups

solutes travel by changing pH or ionic strength of liquid phase

cation exchange - molecule of interest is + charge

anion exchange - molecule of interest is - charge

Gel chromatography

molecules in solution are separated using column packed with gel particles of define porosity

speed of components depend on their molecular size

large molecules move quickly and appear first in chromatogram

Affinity chromatography

based on specific binding interaction between immobilised ligand and its binding partner

similar key and lock

biomolecules are removed by applying elution buffer, which disrupts the binding

Differential migration

solution contain solutes A and B with different equilibrium affinities

solute move faster if it is more easily desorbed

velocity depends on equilibrium distributions between stationery and mobile phase

Zone spreading

effectiveness of chromatography also depends on elution bands on individual solutes, which should remain comapct and without overlapping
important when migration rates of solutions are very close that can cause overlap

Axial diffusion

molecular diffusion of solute which occur from regions of high to low concentratios

broaden soluble peak

Eddy diffusion

differntial motion of material due to unpredictable local variations in flow velocity

causes concentration differences in various locations

Local non equilibrium effects

equilibrium cannot be established at any locations with the continuously changing liquid concentration

increasing particle SA which improve mass transfer can reduce these non equilibrium effects

increase liquid flor wate will increase these effects as rate of adsorption fails to keep up