Innovative separation methods in bioprocessing 181 7. 2. 2 Kinetics and mass transfer The physical behaviour of the system has an effect on the development of novel separation processes. Processes can be divided into equilibrium and rate processes. In equilibrium processes selective separation depends on the attainment of a favourable equilibrium state. This, for example, includes liquid-liquid extraction and ion exchange chromatography. Rate processes, on the other hand, separate different proteins on basis of their response to an imposed field(such as an electric field). Mobility and similar properties determine the selectivity of this type of operation; a successful pro s one in which the proteins have markedly different mobilities( e.g. electrophoresis) In a number of protein n processes the residence time in the reactor is insufficient for equilibrium to be achieved and the kinetics of adsorption play an important role for example in affinity chromatography and in the CArE (continuous dsorption recycle extraction) process. New developments in materials have recently shown dramatic advances in overcoming mass transfer limitations in processes such as perfusion and membrane chromatography and adsorption resulting in extremely fast separations. Some recent examples of novel techniques, which exploit the principles discussed above and provide useful analyses for optimal design of operations, include expanded bed(fluidised bed)adsorption of proteins, which allows direct broth extraction; cross-flow electrofiltration of disrupted microbial cells and for improved ultrafiltration proteins; mathematical modelling of partitioning and phase behaviour in liquid-liquid extraction; mathematical modelling of chromatographic columns; perfusion and membrane chromatography; and advanced reversed phase chromatography using HPLC The potential for scale-up of many of these systems is analysed and discussed 7.3 LIQUID-LIQUID EXTRACTION: INTRODUCTION Liquid-liquid extraction as a technology has been used in the antibiotics industry for several decades and it is now beginning to be recognised as a potentially useful separation step in protein recovery and separation, particularly because it can readily be scaled-up and can, if necessary, be operated on a continuous basis. The physicochemica factors of the protein that determine partitioning are also starting to be understood. It is a easonably high-capacity process ar offer good selectivity for the desired protein product. However, poor solubility of the large protein molecules in typical organic solvents restricts the range of solvents available for use in such a separation process Two classes of solvents that appear to offer advantages for protein recovery for protein separations are aqueous polymer /salt(in some cases also polymer/polymer) systems and reverse micellar solutions. In both cases two phases are formed and the separation exploits the difference in partitioning of the proteins in the feed and extraction phases. In the aqueous polymer/salt separation systems the partitioning of the protein occurs between two immiscible aqueous phases; one rich in a polymer(usually polyethylene glycol, PEG)and the other in a salt(e. g. phosphate or sulphate). These systems show lon-denaturing solvent environment, small interfacial resistance to mass transfer, relatively high protein capacity and high selectivity. On the other hand reverse micelles exploit the solubilising properties of surfactants that can aggregate in organic solvents to form so-called inverted or reverse micelles. These aggregates consist of a polar core ofInnovative separation methods in bioprocessing 18 1 7.2.2 Kinetics and mass transfer The physical behaviour of the system has an effect on the development of novel separation processes. Processes can be divided into equilibrium and rate processes. In equilibrium processes selective separation depends on the attainment of a favourable equilibrium state. This, for example, includes liquid-liquid extraction and ion exchange chromatography, Rate processes, on the other hand, separate different proteins on the basis of their response to an imposed field (such as an electric field). Mobility and other similar properties determine the selectivity of this type of operation; a successful process is one in which the proteins have markedly different mobilities (e.g. electrophoresis). In a number of protein separation processes the residence time in the reactor is insufficient for equilibrium to be achieved and the kinetics of adsorption play an important role for example in affinity chromatography ana in the CARE (continuous adsorption recycle extraction) process. New developments in materials have recently shown dramatic advances in overcoming mass transfer limitations in processes such as perfusion and membrane chromatography and adsorption resulting in extremely fast separations, Some recent examples of novel techniques, which exploit the principles discussed above and provide useful analyses for optimal design of operations, include expanded bed (fluidised bed) adsorption of proteins, which allows direct broth extraction; cross-flow electrofiltration of disrupted microbial cells and for improved ultrafiltration of proteins; mathematical modelling of partitioning and phase behaviour in liquid-liquid extraction; mathematical modelling of chromatographic columns; perfusion and membrane chromatography; and advanced reversed phase chromatography using HPLC. The potential for scale-up of many of these systems is analysed and discussed. 7.3 LIQUID-LIQUID EXTRACTION: INTRODUCTION Liquid-liquid extraction as a technology has been used in the antibiotics industry for several decades and it is now beginning to be recognised as a potentially useful separation step in protein recovery and separation, particularly because it can readily be scaled-up and can, if necessary, be operated on a continuous basis. The physicochemical factors of the protein that determine partitioning are also starting to be understood. It is a reasonably high-capacity process and can offer good selectivity for the desired protein product. However, poor solubility of the large protein molecules in typical organic solvents restricts the range of solvents available for use in such a separation process. Two classes of solvents that appear to offer advantages for protein recovery for protein separations are aqueous polymer/salt (in some cases also polymer/polymer) systems and reverse micellar solutions. In both cases two phases are formed and the separation exploits the difference in partitioning of the proteins in the feed and extraction phases. In the aqueous polymer/salt separation systems the partitioning of the protein occurs between two immiscible aqueous phases; one rich in a polymer (usually polyethylene glycol, PEG) and the other in a salt (e.g. phosphate or sulphate). These systems show a non-denaturing solvent environment, small interfacial resistance to mass transfer, relatively high protein capacity and high selectivity. On the other hand reverse micelles exploit the solubilising properties of surfactants that can aggregate in organic solvents to form so-called inverted or reverse micelles. These aggregates consist of a polar core of