Ab initio Emulsion Polymerization by RAFT (Reversible Addition–Fragmentation Chain Transfer) through the Addition of Cyclodextrins

A novel process to produce homo‐ and copolymers by RAFT polymerization in emulsion is presented. It is known that RAFT‐controlled radical polymerization can be conducted in emulsion polymerization without disturbing the radical segregation characteristic of this process, thus leading to polymerization rates identical to those encountered in the corresponding nonliving systems. However, RAFT agents are often characterized by very low water solubility and, therefore, they diffuse very slowly from the monomer droplets, where they are initially solubilized, to the reaction loci, i.e., the polymer particles. Accordingly, when used in emulsion polymerization, they are practically excluded from the reaction. In this work, we show that cyclodextrins, well‐known for their ability to form water‐soluble complexes with hydrophobic molecules, facilitate the transport across the H₂O phase of the RAFT agent to the polymer particles. Accordingly, chains grow through the entire process in a controlled way. This leads to the production of low‐polydispersity polymers with well‐defined structure and end functionalities as well as to the possibility of synthesizing block copolymers by a radical mechanism.     

Further insights into the universality of colloidal aggregation

Dynamic light scattering (DLS) performed at various scattering wave vectors provides detailed information about the aggregation kinetics and the cluster mass distribution (CMD) in colloidal dispersions. Detailed modeling of the aggregation kinetics with population balance equations requires a quantitative connection between the CMD and measurable quantities such as the angle dependent hydrodynamic radii obtained by DLS. For this purpose we evaluate and compare various models for the structure factor of fractal aggregates. Additionally, we introduce a simple scattering model that accounts for the contribution of internal cluster dynamics of fractal clusters to the first cumulant of the dynamic structure factor. We show that this contribution allows to quantitatively describe previously measured experimental data on the scattering wave vector dependence of the hydrodynamic radius in diffusion limited cluster-cluster aggregation (DLCA), which was shown to exhibit some kind of universality behavior (master curve). Using the same scattering model, we analyze a similar set of experimental data but in reaction limited cluster-cluster aggregation (RLCA). We find that in this case the crossover from RLCA to DLCA and gravitational settling both have a significant influence on the CMD and consequently on the scattering wave vector dependent DLS data. Only when accounting for both these effects they temporarily compensate each other and a satisfactory representation of the aggregation master curve is possible for the RLCA data at longer times. Indeed, we find that either crossover from RLCA to DLCA or gravitational settling, when present individually, causes the loss of a master curve for aggregation.     

Experimental implementation of automatic 'cycle to cycle' control of a chiral simulated moving bed separation

In the absence of a suitable controller, currently simulated moving beds (SMBs) are operated suboptimally to cope with system uncertainties and to guarantee robustness of operation. Recently, we have developed a 'cycle to cycle' optimizing controller that not only makes use of minimal system information, i.e. only the Henry constants and average bed voidage, but also optimizes the process performance and taps the full economic potential of the SMB technology. The experimental implementation of the 'cycle to cycle' optimizing controller had been carried out for achiral separation. For chiral separation however, application of any online controller has not been possible because an appropriate online monitoring system has not been available. This work reports and discusses the first experimental implementation of the 'cycle to cycle' optimizing control for chiral separations. A mixture of guaifenesin enantiomers is separated on Chiralcel OD columns with ethanol as mobile phase in a eight-column four sections laboratory SMB unit. The results show that the controller, although using minimal information about the retention of the two enantiomers, is able to meet product and process specifications, can optimize the process performance, and is capable of rejecting disturbances that may occur during the operation of the SMB plant.     

Chromatographic behavior of a polyclonal antibody mixture on a strong cation exchanger column. Part I: Adsorption characterization

In this work, the adsorption of a polyclonal antibody (IgG) on a preparative strong cation exchanger is described in detail. In a first stage, the polyclonal mixture is characterized using both chromatographic and non-chromatography methods. This analysis suggested that the mixture can be simplified by lumping the different components of the IgG into two "macro-components", referred to as pseudo-variants in the following. An analytical method for the determination of the concentrations of the two pseudo-variants is developed. Based on this, the mass transport and the adsorption isotherm parameters are determined experimentally using only well known sort-cut methods. This analysis is evidencing the complexity of the characterization when operating in the presence of slow diffusing mixtures of components. Even in the presence of strong competition, mass transport plays a major role in determining the peak shape. In frontal analysis, competition is still operative after full saturation of the column.     

Improvement in industrial re-chromatography (recycling) procedure in solvent gradient bio-separation processes

Re-chromatography or recycling impure products obtained from the batch runs of solvent gradient chromatography is commonly practiced in industry to improve product yield. However, as the re-chromatography steps are carried out at the expense of running fresh batches, any improvement in the yield comes as a trade-off with the production time, and hence productivity. In recent studies, on the other hand, it has been suggested that with a properly designed recycling process one can not only improve the yield, but the productivity as well. That study, however, considered a steady-state recycling process, a technology yet to be implemented with bio-chromatographic systems. In the present paper we are reporting a study made on non-steady-state recycling or re-chromatography, as it is typically done in industrial practice. The results point out an amendment to the standard way of designing solvent gradients, which is necessary to improve both the yield and the productivity of an industrial run with recycle. Although the test case used here was the separation of an industrial peptide, Calcitonin, in a reversed-phase column, the general methodology of gradient manipulation, needless to say, is also valid for other solvent gradient processes like ion-exchange, HIC, etc.     

Dependence of aggregate strength, structure, and light scattering properties on primary particle size under turbulent conditions in stirred tank

The steady-state size and structure of aggregates produced under turbulent conditions in stirred tank, for primary particle diameter, d(p), equal to 420 nm and 120 nm, were studied experimentally for various values of the volume average shear rate, G, and solid volume fraction, phi, and compared with data for d(p) = 810 nm. To exclusively investigate the effect of dp, polystyrene latexes with same type and similar density of surface charge groups (sulfate) were used. The mass fractal dimension, d(f), obtained by image analysis, was found to be invariant of d(p) and G, with a value equal to 2.64 +/- 0.18. Small-angle static light scattering was used to characterize the cluster mass distributions by means of the root-mean-square radius of gyration, R(g), and the zero-angle intensity of scattered light, I(0), whose steady-state values proved to be fully reversible with respect to G. The absolute values of R(g) obtained for similar phi and G proved to be independent of d(p), and for all studied conditions, R(g) was proportional to G-1/2. At very low phi, a critical aggregate size for breakage was obtained and used to evaluate the aggregate cohesive force, as a characteristic for the aggregate strength. The aggregate cohesive force was found to be independent of aggregate size, with similar values for the investigated dp. Due to large d(p) and high d(f), the effect of multiple light scattering within the aggregates was found to be present, and by relating the scaling of R(g) with I(0) to d(f), the corresponding correction factors were evaluated. By combination of the independently measured aggregate size and structure, it is possible to experimentally determine the relation between the maximum stable aggregate mass and the hydrodynamic stresses independent of the multiple light scattering present for large d(p) and compact aggregates.     

Electrostatic model for protein adsorption in ion-exchange chromatography and application to monoclonal antibodies,lysozyme and chymotrypsinogen A

A model for the adsorption equilibrium of proteins in ion-exchange chromatography explicitly accounting for the effect of pH and salt concentration in the limit of highly diluted systems was developed. It is based on the use of DLVO theory to estimate the electrostatic interactions between the charged surface of the ion-exchanger and the proteins. The corresponding charge distributions were evaluated as a function of pH and salt concentration using a molecular approach. The model was verified for the adsorption equilibrium of lysozyme, chymotrypsinogen A and four industrial monoclonal antibodies on two strong cation-exchangers. The adsorption equilibrium constants of these proteins were determined experimentally at various pH values and salt concentrations and the model was fitted with a good agreement using three adjustable parameters for each protein in the whole range of experimental conditions. Despite the simplifications of the model regarding the geometry of the protein–ion-exchanger system, the physical meaning of the parameters was retained.     

Role of the ligand density in cation exchange materials for the purification of proteins

The performance of functionalized materials, such as cation exchange resins, is dependent not only on the ligand type and ligand density, but also on the pore accessibility of the target molecule. In the case of large molecules such as antibodies this latter parameter becomes crucial, because the size of such molecules falls somewhere inside the pore size distribution of the resin. The influence of the ligand density and accessibility on the overall performance of the material is explored systematically. Five different materials, having the same chemistry as the strong cation exchange resin Fractogel EMD SO₃⁻ (M) , have been analyzed. These materials only differ in the ligand density. It is shown that the ligand density directly influences the porosity of the materials as well as the pore diffusivity and the dynamic binding capacity. For a given purification problem an optimal ligand density can be found. Based on the above results a new material is proposed, showing superior properties in terms of dynamic binding capacity. This is achieved by an optimization of the ligand density and by a decrease of the particle size of the stationary phase. The material properties are modeled with a general rate model. Further simulations were conducted to evaluate the performance of the new material in comparison with a conventional resin.     

Peptide pore accessibility in reversed-phase chromatography

The effect of salt or peptide concentration on peptide porosity (i.e. the porosity accessible to a given peptide) is investigated on six different reversed-phase stationary phases. The peptide porosity is found to increase with the local concentration of negative charges following a saturation-type function within the same porosity boundaries for both cases. This can induce the formation of anti-Langmuirian peaks in non-adsorbing conditions since the local increase of the ionic strength due to the peptide concentration increases the porosity accessible to the peptide. This behavior can be well reproduced by the ideal model of chromatography assuming non-constant porosity. The acetonitrile adsorption isotherm was also measured on all the considered reversed-phase stationary phases. A comparison between the stationary phases shows a correlation between the amount of acetonitrile accumulated in the pores and the reduced pore accessibility for the peptide.