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![ Solvent based precipitation method
reduced the dielctric constant of
medium in which they are present.
ln {S/Sw}={[A/RT ][1/ew]- [1/e]}
S= solubility of protein, Sw =
solubility of protein in water, A=
constant, e = dielectric constant of
medium, ew = dielectric constant of
water.
Protein have lower solubiltiy in
medium[ethanol] than in water.
lower concentration of organic
solvent are used in precipitation
processes at low temperature
Ex; human plasma protein
purification by cohn fractionation.
Reducing protein solubility by
increasing salt concentration results
in increase in protein protein
hydrophobic interaction.
ln(S)=B – Ks Cs i.e., Cohnequation
B= Constant, Ks= salting out
constant, Cs= salt concentration
At low temp. 4degree celsius, &
constant depend on salt, pH and
protein solution.
More protein precipiated out &
stability increases at low temp. by
synergetic rxn.
Salt used = Ammonium salts, NaCl
Can be dirct addition or saturated
Ex; 30% -50% Ammoniumsalt cut.
A. B.](https://image.slidesharecdn.com/pptbiosep-210221113937/75/BIOSEPARTION-ENGINEERING-19-2048.jpg)
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BIOSEPARTION ENGINEERING
- 1.
- 2. BIOLOGICAL PRODUCTS NATURE OF BIO SEPARATION CELL DISRUPTION & ITS TYPES PRECIPITATION METHODS MEMBRANE BASED BIO SEPARATION
- 3.
- 4. Types ofBiologically derived products based on chemical nature 1. Solvents 2. Organics acids 3. Vitamins 4. Amino acids 5. Antibiotics 6. Sugars and carbohydrates 7. Lipids 8. Nucleic acids 9. Semi-purified proteins 10. Purified proteins 11. Cells 12. Crude cellular extracts 13. Hydrolysates Ethanol, butamol. Acetone Citric acid, lactic acid, butyric acid Ascorbic acid, vitamin B12 Lysine, phenylalanine, glycine Penicillins, rifanpicin, framycetin, Glucose, fructose, starch, dextran, xanthan, gellan Glycerol, fatty acids, corticosteroids, prostaglandins Plasmids, therapeutic DNA, Industrial enzymes, egg proteins, milk proteins, Therapeutic enzymes, cytokines, hormones, Bakers yeast, brewers yeast, freeze dried lactobacillus Yeast extract, soy extracts, animal tissue extract Soy hydrolysates, whey hydrolysates, animal tissue
- 5. Types ofbiological products based on Applications 1. Industrial chemicals 2. Agrochemicals 3. Biopharmaceuticals 4. Food and food additives 5. Nutraceuticals 6. Diagnostic products 7. Commodity chemicals 8. Laboratory reagents 9. Cosmetic products Solvents, organic acids, industrial enzymes Biofertilizers, biopesticides Antibiotics, hormones, monoclonal antibodies, proteins, vaccines, hormones, cytokines, Whey proteins, milk proteins, egg proteins, soy proteins, protein hydrolysates Vitamins, enzymes, coenzymes, cofactors, amino Glucose oxidase, peroxidase, HCG Detergent enzymes, insecticides Bovine serum albumin, ovalbumin, lysozyme plant extracts, animal tissue extracts
- 6. 1. Separation onchemical processes. 2. Low molecular weight compound such as amino acid , vitamins are purified by conventional processes. These are: liquid-liquid extraction, packed bed adsorption, evaporation drying with practically no necessary modificationss. NATURE OF BIO SEPARATION
- 7. Bases of separtionwould be., Density, size, porosity, shape , polarity, solubility, diffusivity, volatality, charges, molecular weight, partition coeeficent,l ight absorption DIFFERENCES BETWEEN BIOLOGICAL SEPARATION AND SYNTHETIC SEPARTTION I. Large volume of dilute streams are needed even for low concentration compound separation.,ex : For monoclonal antibodies in 0.1mg/ml in mammalian cell culture supernatants. II. Bioseparation should be Selective in nature due to similiarty between impurities and product of interest. III. Potential to avoid extreme physiochemical conditions., pH, temperature etc due to degradtion of biological products. IV. Sometimes sub – ambient temperature required for heat labile. V. Solutions are prepared inside bioprocess for inectables therapeutics and endotoxin free compound
- 8. CELL DISRUPTION Theplasma membrane of different oragnism can be destroyed or disrupted by using solvents,detergents,osmoticshock etc.It can be physical or chemical. Disrupted cells can be gram negative, gram positive, mould cells,yeast cells,mammalian cells,cultured plant cells and ground cells etc. The main barrier is the cell wall which is composed of peptidoglycan, teichoic acid and polysaccharides and is about 0.02 to 0.04 microns thick. • Gram negative have multilayered envelops and thinner peptodoglycan. • Yeasts which are unicellular have thick cell walls, typically 0.1 to 0.2 microns in thickness (such as glucans, mannans and chitins.) They are composed of phospholipids arranged in the form of a bi-layer with the hydrophilic groups of the phospholipids molecules facing outside . The hydrophobic residues remain inside the cell membrane where they are shielded from the aqueous environment present both within and outside the cell.
- 10. PHYSICAL METHOD Disruptionin bead mill Disruption using a rotor-stator mill Disruption using French press Disruption using ultrasonic vibrations Disruption using detergents Disruption using enzymes e.g. lysozyme Disruption using solvents Disruption using osmotic shock CHEMICAL METHOD
- 11. ULTRASOUND DISRUPTION HAEMOLYSIS BYOSMOTICSHOCK DISRUPTION Diagrams showing cell disruption method:
- 12. Detergent disruption Frenchpress disruption
- 13. BEAD MILL METHOD This device consists of a stationary block with a tapered cavity stator and a truncated cone shaped rotating object called the rotor. Typical rotation speeds are in the 10,000 to 50,000 rpm range. The cell suspension is fed into the tiny gap between the rotating rotor and the fixed stator. High rate shear generated in the space between the rotor and the stator as well as the turbulence thus generated are responsible for cell disruption. ROTOR-STATOR MILL
- 14. FRENCH PRESS METHOD It consist of cylinder fitted with plunger connencted to hydraulic press. Cells placed inside cylinder and pressurized (10k to 50k psi) using plunger , through an orfice suspension cells emerges at high velocity due to primary shear stress cell starts disrupting. Ultrasonic vibrations of frequency greater than 18kHz are used to disrupt cells. Vibrations create cavitation i.e., formation of bubbles and they reach resonance size where they collapse releasing mechanical energy in form of shock, and disrupts the cell membrane. ULTRASONIC VIBRATION
- 15. DETERGENT DISRUPTION Theydisrupts the cell membranes and phosholipids of mammalian cells mainly. Bacterial celle needs conjunction with lysozyme to disrupts by weaken its walls. Non ionic for bioprocessing havinf least damage effect such as tween series detergents. Need for removal. Enzymes are used to destroy the cell membrane. Such as lysozymes, pectinase used for disruption and breaking the bonds between the membranes. But they are costly. Need for removal after action. ENZYMES DISRUPTION
- 16. OSMOTIC SHOCK Semipremeable cell membrane are transferred into hypotonic solution from isotonic that results into rapid expansion of cell & ruptures called osmotic shock. Used to lyse mammalian cells,remove periplasmic substances by expelation. Acetone act on cell membrane solubilizing its phosholipids and denature the protein. Toluene for fungalcells. Important to remove by volatility. ORGANIC SOLVENT
- 17. Precipitation basedbioseparation essentially involves selcective conversion of a specific dissolved component of a complex mixture to an insoluble form using appropriate physical or physicochemical means. The insoluble form which is obtained as a precipitate is sepatated from the dissolved components by appropriate solid-liquid separation techniques such as centrifugation. Biological macromolecules can be precipitated by: 1. Cooling 2. pH adjustment 3. Addition of solvents such as acetone and ethanol 4. Addition of anti-chaotropic salts such as ammonium sulphate and sodium sulfate 5. Addition of chaotropic salts such as urea and guanidine hydrochloride 6. Addition of biospecific reagents as in immunoprecipitation PRECIPITATION
- 18. USING ORGANIC SOLVENTS • Byreducing dielectric constant USING ANTI-CHAOTOPIC SALTS • By decreasing solubility of proteins PRECIPITATION METHODS A. B .
- 19. Solvent basedprecipitation method reduced the dielctric constant of medium in which they are present. ln {S/Sw}={[A/RT ][1/ew]- [1/e]} S= solubility of protein, Sw = solubility of protein in water, A= constant, e = dielectric constant of medium, ew = dielectric constant of water. Protein have lower solubiltiy in medium[ethanol] than in water. lower concentration of organic solvent are used in precipitation processes at low temperature Ex; human plasma protein purification by cohn fractionation. Reducing protein solubility by increasing salt concentration results in increase in protein protein hydrophobic interaction. ln(S)=B – Ks Cs i.e., Cohnequation B= Constant, Ks= salting out constant, Cs= salt concentration At low temp. 4degree celsius, & constant depend on salt, pH and protein solution. More protein precipiated out & stability increases at low temp. by synergetic rxn. Salt used = Ammonium salts, NaCl Can be dirct addition or saturated Ex; 30% -50% Ammoniumsalt cut. A. B.
- 20. STAGES OF PRECIPITATION: 1.Mixing 2. Nucleation 3. Diffusion limited growth 4. Convection limited growth • Formation of homogenous mixture and mix it .Time needed for mixiing can be determined by: Tm= (l)(l)/4D ; l= avg eddy length, D= diffusivity. • At supernaturation minute particles form called nucleation . • Diffusion limited growth increases collision between particel hence increses rate of fomation of bigger particles in microns. • Mixing directly proportional to frequency of collision. • Aging process takes place i.e., time given to precipitate & supernatant formation( 6h-12hrs at 4 degree celsius) Mechanism
- 21. Particle-liquid separation Particle-solute separation Solute-solventseparation Solute-solute separation MEMBRANE BASED SEPARATION
- 22. Membranes are dividedinto: 1. Symmetric- similar compostition and morphology 2. 2.Asymmetric- non-identical composition Basically membranes are: 1. Flat sheet membrane 2. Tubular membrane 3. Hollow fibre membrane
- 23. 1. MICROFILTRATION This methodis used for separation of fine particles from solutions. The transmembrane pressure ranges from 1 to 50 psig. Most microfiltration membranes capture particles by surface filtration, i.e. on the surface of the membrane. In some cases depth filtration is also used. Uses: for clarification, sterilization and slurry concentration. 2. ULTRAFILTRATION Membranes retain macromolecules such as proteins while allowing smaller molecules to pass through. (a) separate large molecules from solvents, (b) separate large molecules from smaller molecules, (c) separate large molecules from one another. The primary separation mechanism is size exclusion, but physicochemical interactions between the solutes and the membrane, and operating conditions can influence the process quite significantly. Pressure ranges from 10 to 100 psig. Most UF membranes are asymmetric.
- 24. 3. NANOFILTRATION Allowsalts and other small molecules To pass through but retain larger molecules such as peptides, hormones and sugars. Pressure in NF ranges from 40 to 200 psig. MostNF membranes are composite i.e. asymmetric. 4.Liquid membrane processes TRansport of solutes across a thin layer of a third liquid interposed between two miscible liquids. liquid membranes: 1. Emulsion liquid membranes (ELM) 2. Supported liquid membranes (SLM)
- 25. 5. MEMBRANE CHROMATOGRAPHY Adsorption and chromatographic separations are traditionally carried out using packed beds. Separation mechanisms These include; 1. Affinity binding 2. Ion-exchange interaction 3.Hydrophoboic
- 26. Diagram showingdialysis Solute separation occurs primarily because smaller solutes partition into the membrane better than bigger solutes because the degree to which the membrane restricts the entry of solutes into it increases with solute size. Smaller solutes also diffuse more rapidly than larger ones. 6.DIALYSIS
- 27. REFERENCES: Research gate NCBI WIKIPEDIA BOOK- PRINCIPLES OF BIO- SEPARATION ENGINEERING BY RAJA GHOSH BIOCHEMISTRY TECHNIQUES BOOK BY STRYER THANKYOU