Hydrophobicity, amphilicity, and flexibility: Relation between molecular protein properties and the macroscopic effects of surface activity.

Their surface activity enables proteins to form and stabilize foam, which can be used for in situ product separation or foam fractionation. Thus, it would be highly desirable to predict the surface activity of proteins based on their molecular properties like hydrophobicity, amphilicity, or structure on primary, secondary, and tertiary level. Ionic strength and pH were adjusted to gain maximum surface activity. The surface activity decreased in the order α lactalbumin > ß­lactoglobulin > trypsinogen > papain. For the theoretical analysis, the database was extended by including 2 hydrophobins into the investigation, since they are known to exhibit an outstanding surface activity. No relation to the macroscopic behavior was found considering the hydrophobicity. I.e., the non-hydrophobins did not differ significantly from each other, and from the hydrophobins, one was significantly hydrophobic, and the other was significantly hydrophilic. Also, no relations were found considering the amphilicity of the secondary structure elements. However, taking into account the tertiary protein structure, it was found that for most of the proteins investigated, the presence of non-buried amphiphilic secondary structure elements in combination with a certain amount of flexibility correlates with the surface activity.

Contribution of Secondary Structure Changes to the Surface Activity of Proteins.

A major difference between proteins and surfactants is that proteins are capable of changing their structure during refolding processes in the adsorbed state. It is often reported that these interfacial structure changes increase the surface activity of proteins. In order to investigate this, the surface activity of 5 proteins was determined in foam fractionation experiments, where pH and ionic strength were adjusted separately to gain the maximum surface activity for each protein. Infrared Reflection Absorption Spectroscopy was performed for each protein to analyze the changes in secondary structure after adsorption. In order to investigate quick structural changes, transmission Fourier Transform Infrared spectroscopy was performed to gain information about the secondary structure of the dissolved, non-adsorbed proteins. It was found that most proteins maintain a native-like conformation when adsorbed at the interface. With increasing time, most of the proteins investigated increased the amount of ß-sheets at the interface. This slow process went along with a slow increase in surface pressure. A correlation between structural changes on molecular level and surface activity on macroscopic level could not be found. However, the absolute increase of surface pressure at the very beginning of the adsorption process correlated with the surface activity of the proteins, suggesting that the slow processes on molecular level do not have a considerable impact on macroscopic surface activity.

Influence of thermally induced structure changes in diluted ß-lactoglobulin solutions on their surface activity and behavior in foam fractionation.

Surface activity is an intrinsic protein feature, leading to the capability of aqueous protein solutions to form foam. This feature provides opportunities for downstream processing, such as usage of foam fractionation for purification. In order to investigate the impact of the surface activity on the performance of the foam fractionation process, protein solutions with different surface activity were produced by different thermal denaturation of aqueous ß-lactoglobulin solutions. The effectiveness of the denaturation procedure was verified with circular dichroic spectroscopy, and the impact on surface activity was determined via dynamic surface tension measurement. The increased surface activity resulted in higher foamate flow rates. Furthermore, the effects could be correlated with secondary structure changes and with the dynamic surface pressure. The new result of this study is that the effect of the denaturation of a protein on foam fractionation depends on the protein concentration. At the lower feed concentration, effects became visible, which could not be observed at the higher concentration.

Characterization and correlation of mobile phase dispersion of aqueous-organic solvent systems in centrifugal partition chromatography.

To reach a high separation efficiency using Centrifugal Partition Chromatography (CPC), the fluid dynamical behavior of the liquid-liquid two-phase systems must be clearly understood. The fluid dynamics, namely the dispersion, the coalescence, and the stationary phase retention, have a high impact on a separation. Especially the mobile phase dispersion influences the mass transfer during a separation. In this study, the mobile phase dispersion of different aqueous-organic solvent systems was characterized for ascending and descending mode via video analysis. Thereby the influence of the physical properties of the solvent systems, the operating parameters, and the geometry of the chamber inlet was investigated systematically using dimensional analysis. With the help of the dimensionless numbers Ohnesorge number (OhCPC), Eötvös number (EoCPC), and Weber number (WeCPC) the impact of the solvent system, the plant parameters, and the operating parameters on the mobile phase dispersion could be described. Inside the three dimensional area, spanned by the dimensionless numbers, each state of mobile phase dispersion (undispersed, low dispersed, highly dispersed, and atomized) could be allocated to an individual region for both operating modes. Moreover, differences in mobile phase deflection depending on the operating mode and a possible reason for these were described.

Correlating the phase settling behavior of aqueous-organic solvent systems in a centrifugal partition chromatograph.

The prediction of the performance of a Centrifugal Partition Chromatography (CPC) is a difficult but desirable task. The partitioning of the sample, as well as the fluid dynamical phenomena dispersion, coalescence, and stationary phase retention have to be individually understood. Therefore, the phase settling behavior of different aqueous-organic solvent systems and with this, the dependency of the stationary phase retention in CPC was investigated in this study. On the one hand, batch settling experiments were performed, and the settling velocity of aqueous-organic solvent systems was investigated. With this it was possible to correlate the stationary phase retention in CPC in both operating modes. For descending mode operation a high settling velocity of the lower phase and for ascending mode a high settling velocity of the upper phase is needed for a stable operation and a high stationary phase retention. On the other hand, the dimensionless numbers Capillary number (Ca) and Morton number (Mo) were used to generate a universally applicable correlation for the stationary phase retention in ascending mode. It was shown, that a high stationary phase retention correlates with low values of Ca and Mo, whereas the influence of Mo is neglectable in the parameter space investigated. Within this correlation, the individual influence of each influencing parameter on the stationary phase retention was included. Moreover, this correlation was compared to descriptions for descending mode given in literature. With this it was shown that the minimal stationary phase retention is correlatable to the point of phase inversion.

Correlating physical properties of aqueous-organic solvent systems and stationary phase retention in a centrifugal partition chromatograph in descending mode.

The performance of the Centrifugal Partition Chromatography (CPC) as a liquid-liquid chromatographic technique depends strongly on the two-phase solvent system used. Thereby the individual influence of the retention of the stationary phase, the coalescence, and the dispersion of the mobile phase in the chambers must be understood to select appropriate solvent systems and reach high separation efficiencies. In this study, an optical measurement system was used to investigate the influence of the physical properties of the Arizona solvent systems on the stationary phase retention in descending mode. Therefore, physical properties like density, viscosity, and interfacial tension were measured as well as the stationary phase retention. Using dimensionless numbers, a correlation between the stationary phase retention and the influencing parameters could be determined. The correlation was validated using data from the literature. Additionally, the solvent systems were modified by additives to identify the validity of the correlation. It was proven that the dimensionless numbers Capillary number (Ca) and Morton number (Mo) can be used to predict the stationary phase retention of other liquid-liquid solvent systems as well as for different operating conditions.

Mass Transfer of Proteins in Aqueous Two-Phase Systems.

Aqueous Two-Phase Extraction is known to be a gentle separation technique for biochemical molecules where product partitioning is fast. However, the reason for the high mass transfer rates has not been investigated, yet. Many researchers claim that the low interfacial tension facilitates the formation of very small droplets and with it a large interfacial area causing a fast partitioning. However, an experimental evidence for this hypothesis has not been published yet. In this study, the mass transfer coefficients of two proteins, namely lysozyme and bromelain, were determined by providing a defined interfacial area for partitioning. Compared to low molecular weight solutes the mass transfer coefficient for the proteins investigated was small proving for the first time that the large interfacial area and not fast diffusion seems to be the reason for fast protein partitioning.

Modelling centrifugal partition chromatography separation behavior to characterize influencing hydrodynamic effects on separation efficiency.

In addition to the selection or adjustment of phase systems to gain a suitable partition coefficient for the target molecule, the separation efficiency in centrifugal partition chromatography (CPC) is strongly influenced by the hydrodynamic interactions of the mobile and the stationary phase in the chambers. Thus, the hydrodynamic interactions must be investigated and understood in order to enhance a CPC separation run. Different hydrodynamic effects like mass transfer, back mixing and the non-ideal behavior of stationary phase, which cannot be determined directly, are known, but quantifying these effects and their influence on separation performance is barely achieved. In order to understand their influence, a physically detailed mathematical model of a CPC chamber was developed. The model includes a parameter representing the hydrodynamic effects mentioned above and is able to determine the parameters significance by fitting them to separation experiment data. The acquired knowledge is used to correlate the effects of the hydrodynamic influences on the separation performance and can be used to forecast hydrodynamic and separation behavior in a CPC device.

Enzymatic hydrolysis in an aqueous organic two-phase system using centrifugal partition chromatography.

Multi-phase reaction systems, mostly aqueous organic systems, are used in enzyme catalysis to convert hydrophobic substrates which are almost insoluble in aqueous media. In this study, a Centrifugal Partition Chromatograph is used as a compact device for enzymatic multi-phase reaction that combines efficient substrate supply to the aqueous phase and separation of both phases in one apparatus. A process design procedure to systematically select the aqueous and organic phase to achieve stable and efficient reaction rates and operation conditions in Centrifugal Partition Chromatography for efficient mixing and separation of the phases is presented. The procedure is applied to the hydrolysis of 4-nitrophenyl palmitate with a lipase derived from Candida rugosa. It was found that the hydrolysis rate of 4-nitrophenyl palmitate was two times higher in Centrifugal Partition Chromatography than in comparable stirred tank reactor experiments.

Investigation, comparison and design of chambers used in centrifugal partition chromatography on the basis of flow pattern and separation experiments.

In centrifugal partition chromatography (CPC) the separation efficiency is mainly influenced by the hydrodynamic of mobile and stationary phase in the chambers. Thus, the hydrodynamic has to be investigated and understood in order to enhance a CPC separation run. Different chamber geometries have been developed in the past and the influence of several phase systems and CPC operating conditions were investigated for these chambers. However, a direct comparison between the different chamber types has not been performed yet. In order to investigate the direct influence of the chamber design on the hydrodynamic, several chamber designs - partially similar in geometry to commercial available designs - are investigated under standardized conditions in the present study. The results show the influence of geometrical aspects of the chamber design on the hydrodynamic and therewith, on the separation efficiency. As a conclusion of the present study, some ideas for an optimal chamber design for laboratory and industrial purpose are proposed.

Tunable aqueous polymer-phase impregnated resins-technology-a novel approach to aqueous two-phase extraction.

Aqueous Two-Phase Extraction (ATPE) represents a promising unit operation for downstream processing of biotechnological products. The technique provides several advantages such as a biocompatible environment for the extraction of sensitive and biologically active compounds. However, the tendency of some aqueous two-phase systems to form intensive and stable emulsions can lead to long phase separation times causing an increased footprint for the required mixer-settler devices or the need for additional equipment such as centrifuges. In this work, a novel approach to improve ATPE for downstream processing applications called 'Tunable Aqueous Polymer-Phase Impregnated Resins' (TAPPIR(®))-Technology is presented. The technology is based on the immobilization of one aqueous phase inside the pores of a solid support. The second aqueous phase forms the bulk liquid around the impregnated solids. Due to the immobilization of one phase, phase emulsification and phase separation of ATPE are realized in a single step. In this study, a biodegradable and sustainable aqueous two-phase system consisting of aqueous polyethylene glycol/sodiumcitrate solutions was chosen. The impregnation of different macroporous glass and ceramic solids was investigated and could be proven to be stable. Additionally, the separation of the dye Patent blue V was successfully performed with the TAPPIR(®)-Technology. Thus, the "proof of principle" of this technology is presented.

Selection of operating parameters on the basis of hydrodynamics in centrifugal partition chromatography for the purification of nybomycin derivatives.

The selection of solvent systems in centrifugal partition chromatography (CPC) is the most critical point in setting up a separation. Therefore, lots of research was done on the topic in the last decades. But the selection of suitable operating parameters (mobile phase flow rate, rotational speed and mode of operation) with respect to hydrodynamics and pressure drop limit in CPC is still mainly driven by experience of the chromatographer. In this work we used hydrodynamic analysis for the prediction of most suitable operating parameters. After selection of different solvent systems with respect to partition coefficients for the target compound the hydrodynamics were visualized. Based on flow pattern and retention the operating parameters were selected for the purification runs of nybomycin derivatives that were carried out with a 200 ml FCPC(®) rotor. The results have proven that the selection of optimized operating parameters by analysis of hydrodynamics only is possible. As the hydrodynamics are predictable by the physical properties of the solvent system the optimized operating parameters can be estimated, too. Additionally, we found that dispersion and especially retention are improved if the less viscous phase is mobile.

Influence of physical properties and operating parameters on hydrodynamics in Centrifugal Partition Chromatography.

Besides the selection of a suitable biphasic solvent system the separation efficiency in Centrifugal Partition Chromatography (CPC) is mainly influenced by the hydrodynamics in the chambers. The flow pattern, the stationary phase retention and the interfacial area for mass transfer strongly depend on physical properties of the solvent system and operating parameters. In order to measure these parameters we visualized the hydrodynamics in a FCPC-chamber for five different solvent systems with an optical measurement system and calculated the stationary phase retention, interfacial area and the distribution of mobile phase thickness in the chamber. Although inclined chambers were used we found that the Coriolis force always deflected the mobile phase towards the chamber wall reducing the interfacial area. This effect increased for systems with low density difference. We also have shown that the stability of phase systems (stationary phase retention) and its tendency to disperse increased for smaller values of the ratio of interfacial tension and density difference. But also the viscosity ratio and the flow pattern itself had a significant effect on retention and dispersion of the mobile phase. As a result operating parameters should be chosen carefully with respect to physical properties for a CPC system. In order to reduce the effect of the Coriolis force CPC devices with greater rotor radius are desirable.

Multiphase flow modeling in centrifugal partition chromatography.

The separation efficiency in Centrifugal Partition Chromatography (CPC) depends on selection of a suitable biphasic solvent system (distribution ratio, selectivity factor, sample solubility) and is influenced by hydrodynamics in the chambers. Especially the stationary phase retention, the interfacial area for mass transfer and the flow pattern (backmixing) are important parameters. Their relationship with physical properties, operating parameters and chamber geometry is not completely understood and predictions are hardly possible. Experimental flow visualization is expensive and two-dimensional only. Therefore we simulated the flow pattern using a volume-of-fluid (VOF) method, which was implemented in OpenFOAM®. For the three-dimensional simulation of a rotating FCPC®-chamber, gravitational centrifugal and Coriolis forces were added to the conservation equation. For experimental validation the flow pattern of different solvent systems was visualized with an optical measurement system. The amount of mobile phase in a chamber was calculated from gray scale values of videos recorded by an image processing routine in ImageJ®. To visualize the flow of the stationary phase polyethylene particles were used to perform a qualitative particle image velocimetry (PIV) analysis. We found a good agreement between flow patterns and velocity profiles of experiments and simulations. By using the model we found that increasing the chamber depth leads to higher specific interfacial area. Additionally a circular flow in the stationary phase was identified that lowers the interfacial area because it pushes the jet of mobile phase to the chamber wall. The Coriolis force alone gives the impulse for this behavior. As a result the model is easier to handle than experiments and allows 3D prediction of hydrodynamics in the chamber. Additionally it can be used for optimizing geometry and operating parameters for given physical properties of solvent systems.