Separation of Soluble Products - Extraction and Precipitation PDF

Primary Isolation of Products Dr. Prangya Ranjan Rout DBT, NIT Jalandhar  Separation of Soluble Products Most microbial products, such as antibiotics, organic acids, solvents, amino acids, and extracellular enzymes, are soluble. Various unit operations have been develo...

Primary Isolation of Products Dr. Prangya Ranjan Rout DBT, NIT Jalandhar  Separation of Soluble Products Most microbial products, such as antibiotics, organic acids, solvents, amino acids, and extracellular enzymes, are soluble. Various unit operations have been developed to recover such soluble products, including extraction, adsorption, ultrafiltration, and chromatography These group of unit operations used for primary isolation of fermentation products relies on mass transfer to achieve separation of components between phases. Equipment for these separations may consist of a series of stages in which the phases make intimate contact so that material can be transferred between them  Extraction Extraction is a process in which two phases come into contact with the objective of transferring a solute from one phase to the other. The phases are most commonly immiscible liquids, and the solute is in soluble form. An organic solvent is often used when the solute to be extracted is stable in the organic solvent (low molecular weight antibiotics). It is not feasible to extract proteins with organic solvents, since proteins are often denatured on contact with the organic solvent. Proteins can be extracted by means of two immiscible liquid phases that consist of solutions of two water-soluble polymers. Extraction is advantageous as it can bring about a significant reduction in volume.  Liquid-Liquid Extraction The separation of a component from a liquid mixture by treatment with a solvent in which the desired component is preferentially soluble is known as liquid–liquid extraction. Separation of biomolecules in liquid–liquid extraction depends on the partitioning of the biomolecules between the liquid phases. Liquid-Liquid Extraction  Liquid-Liquid Extraction The value of K defines the ease of extraction, relatively high K value, good separation of the aqueous and solvent phases, then it may be possible to use a single-stage extraction system. A value of 50 indicates that the extraction should be straightforward whereas a value of 0.1 shows that the extraction will be difficult and that a multistage process will be required. Liquid-Liquid Extraction Liquid-Liquid Extraction Liquid-Liquid Extraction  Aqueous two-phase liquid Extraction Aqueous two-phase extraction is an approach for the extraction of soluble proteins such as enzymes and other sensible products. Aqueous solvents that form two distinct phases facilitate separation of proteins, cell fragments, and organelles. Two-phase aqueous systems are produced when particular polymers or a polymer and salt are dissolved together in water. The liquid containing incompatible polymers, such as polyethylene glycol (PEG) and dextran partitions into two phases, each containing 85 to 99% water.  Aqueous two-phase liquid Extraction Some components used to form aqueous two-phase systems are: Aqueous two-phase liquid Extraction  Aqueous two-phase liquid Extraction Aqueous two-phase extraction is used to recover α-amylase from solution. A polyethylene glycol-dextran mixture is added and the solution separates into two phases. The partition coefficient is 4.2. Calculate the maximum possible enzyme recovery when: (a) The volume ratio of the upper to lower phase is 5.0 (b) The volume ratio of the upper to lower phase is 0.5:  Batch and Staged Extraction In a single-stage extraction process, one feed stream contacts one extraction solvent stream, and the mixture divides into equilibrium extract and raffinate phases. Two immiscible liquids enters with volumetric flow rates Li1 and Li2; these streams contain A at concentrations CAi1 and CAi2, respectively. Two phases exits with volumetric flow rates Lo1 and Lo2; and component A’s concentrations CAo1 and CAo2, respectively.  Batch and Staged Extraction To achieve high bioproduct recovery in extraction, it is frequently necessary to use more than one extraction stage. Extraction processes are usually set up where the extraction solvent and the feed run either co-current or countercurrent to each other. There are n mixer/separator vessels in line and the raffinate goes from vessel 1 to vessel in co-current mechanism. Fresh solvent is added to each stage, the feed and extracting solvent pass through the cascade in the same direction.  Batch and Staged Extraction In a counter-current system, the extracted raffinate passes from vessel 1 to vessel n while the product-enriched solvent is flowing from vessel n to vessel 1. The feed and extracting solvent pass through the cascade in opposite directions. The most efficient system for solvent utilization is countercurrent operation, showing a considerable advantage over batch and co-current systems. Batch- Analytical Methods Analytical Methods  Analytical Methods In a batch steroid extraction process, water containing 6.8 mg/L of a steroid is extracted with initially pure methylene dichloride. The equilibrium constant for the steroid is 170 and the ratio of water to solvent is 82. What is the concentration in the organic after the extraction? What fraction of the steroid has been removed?  Continuous- Analytical Methods 1st 2nd N-1 N Continuous- Analytical Methods 1st 2nd N-1 N Continuous- Analytical Methods 1st 2nd N-1 N  Continuous- Analytical Methods A clarified fermentation beer H containing 260 mg/L of actinomycin D antibiotic is to be extracted in a staged extractor using butyl acetate L. Because the beer’s pH is 3.5, the equilibrium constant K is 57. You plan to let H equal to 450 L/h and L equal 37 L/h. You hope to recover 99% of the antibiotic in the feed. How many stages will you need to accomplish this separation?  Precipitation The first step in the purification of intracellular proteins after cell disruption is usually precipitation, well-established method for recovering products from culture broths. Addition of precipitants and changes in experimental condition are used to reduce the solubility of the product, causing it to precipitate in the form of insoluble particles. Precipitation is applied commonly during the early stages of downstream processing because large reductions in liquid volume can be achieved. The tendency of a protein to precipitate depends on the properties of the solvent, such as pH, ionic strength, and dielectric constant, and the size, charge, and hydrophobicity of the protein.  Precipitation Both attractive and repulsive forces exist between neighboring protein molecules in solution. The balance of these forces, determines whether or not the protein will precipitate. In aqueous solution, water-soluble proteins are folded so that most of their polar, hydrophilic amino acid side chains are presented on the protein surface, while most of the hydrophobic residues are shielded within. When two protein molecules are brought together, they are attracted to each other due to interactions between opposite surface charges and any exposed hydrophobic regions  Precipitation The isoelectric point of a protein is the pH at which the protein carries zero net charge Isoelectric points of many proteins range between 4 and 6. Surface chemistry of globular proteins. (a) Hydrophobic zones and nonuniform charge distribution on the surface of a protein. (b) Formation of the Stern layer of counter-ions and the electrical double layer around two neighbouring soluble proteins  Precipitation When suspended in near-neutral pH, the surfaces of these proteins have a net negative charge. As a consequence, the protein will attract positive ions from the solution to form a close layer of counter-ions over the molecule surface. This is called the Stern layer. When two similarly charged protein molecules are brought together, although they may be attracted by ionic and hydrophobic interactions, they are repulsed by their electrical double layers Another source of repulsion between proteins in aqueous solution is the hydration layers that form around protein molecules. Salting Out Salting Out  Salting Out The hydrophobic zones of the protein surface thus become exposed, providing sites of attraction between neighbouring protein molecules. The increase in surface tension of water by salt follows the well-known Hofmeister series  Salting Out It is important that the salt used be inexpensive. The salt must also be highly soluble in aqueous solutions to achieve the concentrations required. Salt should not have a large heat of solution to avoid temperature increases that might denature the product or affect its solubility. Precipitates formed by salting-out contain large quantities of salt as well as protein; therefore, must be desalted to remove the residual salt. This is usually achieved using dialysis, diafiltration, or gel chromatography  Other methods of Precipitation Precipitation of protein can also be achieved by adding organic solvent addition at low temperatures (T < -5°C), which reduces dielectric constant of the solution. A reduction in the dielectric constant of a solution results in stronger electrostatic forces between the protein molecules and facilitates protein precipitation. Isoelectric precipitation is the precipitation of proteins at their isoelectric point. The use of Ionic polyelectrolytes changes the ionic strength of the media and causes protein precipitation The use of nonionic polymers reduces the amount of water available to interact with proteins and results in precipitation. Thank You