Stephanie Peper

Experimental Methods for Phase Equilibria at High Pressures

Knowledge of high-pressure phase equilibria is crucial in many fields, e.g., for the design and optimization of high-pressure chemical and separation processes, carbon capture and storage, hydrate formation, applications of ionic liquids, and geological processes. This review presents the variety of methods to measure phase equilibria at high pressures and, following a classification, discusses the measurement principles, advantages, challenges, and error sources. Examples of application areas are given. A detailed knowledge and understanding of the different methods is fundamental not only for choosing the most suitable method for a certain task but also for the evaluation of experimental data. The discrepancy between the (sometimes low) true accuracy of published experimental data and the (high) accuracy claimed by authors is addressed. Some essential requirements for the generation of valuable experimental results are summarized.

Separation of ibuprofen enantiomers by supercritical fluid simulated moving bed chromatography

The continuous separation of R(−)-ibuprofen and S(+)-ibuprofen by supercritical fluid simulated moving bed chromatography has been developed. First, experiments were performed at a low concentration level. Therefore, a starting set of operating parameters was calculated using the “triangle theory,” which has been developed in the frame of equilibrium theory. Subsequently, the feed concentration was increased. A dynamic simulation program, based on a plug flow model with axial dispersion and linear mass transfer resistance, was used to predict the effect of operating variables on the process performance. Adsorption isotherms were determined with the elution by a characteristic point method. The experimental results were compared both with the model predictions based on the triangle theory and with the results of simulations.

Enzyme catalysis in organic solvents: influence of water content, solvent composition and temperature on Candida rugosa lipase catalyzed transesterification.

In the present study the influence of water content, solvent composition and reaction temperature on the transesterification of 1-phenylpropan-2-ol catalyzed by Candida rugosa lipase was examined. Reactions were carried out in different mixtures of hexane and tetrahydrofurane. The studies showed that an increasing water content of the organic solvent results in an increasing enzyme activity and a decreasing enantiomeric excess. Furthermore, a significant influence of the solvent hydrophilicity both on the enzyme activity and on the enantiomeric excess was found. An increase in solvent hydrophilicity leads to a decrease of enzyme activity and an increase of the enantiomeric excess. This indicates that the enzyme becomes more selective with decreasing flexibility. Similar effects were found by variation of the reaction temperature. Taken together, the decrease in conversion and the increase in selectivity with increasing solvent hydrophilicity are induced by the different water contents on the enzyme surface and not by the solvent itself.

Simulated moving bed chromatography with supercritical fluids for the resolution of bi-naphthol enantiomers and phytol isomers

The combination of the simulated moving bed (SMB) technique with supercritical fluid chromatography (SFC) leads to a process with unique features. Besides the known advantages of the SMB process, the use of supercritical carbon dioxide as the mobile phase offers the advantages of reduction in organic solvents and an easy eluent/solute separation. Because of the low viscosity and high diffusion coefficients of supercritical fluids, a high efficiency is possible. The steps of process development for SMB SFC are presented using the separations of the bi-naphthol enantiomers and phytol isomers as examples. The development of a packed column SFC method at an analytical scale is shown for the separation of the bi-naphthol enantiomers on a chiral stationary phase and CO(2) with a modifier as the mobile phase. The influence of the modifier, modifier content, and column configuration on productivity of the SMB SFC process was investigated by simulation. The first set of experiments was performed in the SMB separation of phytol isomers at low concentration to test the feasibility of the SMB SFC high purity separation of the binary mixtures. In the second set of experiments, the productivity of the process was increased by increasing the feed concentration up to 54 grams feed per liter stationary phase (SP) and hour (g(feed)/l(SP) h).

Influence of reaction conditions on the enantioselectivity of biocatalyzed C–C bond formations under high pressure conditions

Benzoylformate decarboxylase (BFD, EC 4.1.1.7) is a homotetrameric thiamine diphosphate (ThDP)-dependent enzyme which catalyzes the synthesis of chiral 2-hydroxyketones accepting a broad range of aldehydes as substrates. In this study the synthesis of 2-hydroxypropiophenone (2-HPP) from benzaldehyde and acetaldehyde was catalyzed by three BFD variants namely BFD F464I, BFD A460I and BFD A460I-F464I. This paper reports the effect of hydrostatic pressure up to 290 MPa when the reactions were carried out at different benzaldehyde concentrations (5-40 mM) as well as at different pH values (7.0-8.5). Acetaldehyde concentration was fixed at 400 mM in all biotransformations. Reactions performed at high benzaldehyde concentrations and at high hydrostatic pressures showed an increase in (R)-2-HPP formation catalyzed by all BFD variants. For BFD A460I-F464I we observed an increase in the ee of (R)-2-HPP up to 80%, whereas at atmospheric conditions this variant synthesizes (R)-2-HPP with an ee of only 50%. Alkaline conditions (up to pH 8.5) and high hydrostatic pressures resulted in an increase of (R)-2-HPP synthesis, especially in the case of BFD A460I and BFD F464I.

Influence of the Hydrostatic Pressure and pH on the Asymmetric 2-Hydroxyketone Formation Catalyzed by Pseudomonas putida Benzoylformate Decarboxylase and Variants Thereof

Benzoylformate decarboxylase (BFD) from Pseudomonas putida is a thiamine diphosphate‐dependent (ThDP) enzyme that catalyzes the asymmetric CC bond formation to (S)‐2‐hydroxypropiophenone [(S)‐HPP] starting from benzaldehyde and acetaldehyde. The enantioselectivity of BFD was shown to be a function of temperature and substrate concentration. It can additionally be changed by site‐directed mutagenesis on hot spot positions in the active site. In this article, we present the effect of hydrostatic pressure up to 250 MPa on the enantioselectivity for the recombinant wtBFD as well as for the variants BFD F464I, BFD A460I, and BFD A460I‐F464I. A general tendency toward lower amounts of (S)‐HPP could be observed at increasing pressures. For two of these variants an increase in pressure even caused an inversion in the enantioselectivity and thus increasing enantiomeric excesses, respectively. A pressure‐induced increase in enantioselectivity could therefore be observed for the first time in biocatalysis to the best of our knowledge. Furthermore, the pH is shown to be a parameter that also significantly influences the enantioselectivity of the reaction mentioned above. Biotechnol. Bioeng. 2010; 106: 18–26.

High Pressure Phase Equilibria Measurement for Mixtures Comprising Food Substances

In the processing of food materials, separation processes are frequently performed at high pressures, namely in the extraction of aromas, flavours, or oils through supercritical technology, in the fractionation of liquids, in chromatographic separations, etc. The design and optimization of such processes rely on the phase equilibria of the systems in question. A wide variety of methods and techniques is available for high-pressure phase equilibria determinations. A detailed knowledge and understanding of the different methods is essential for the appropriate choice of the most suitable method for a certain determination. In the present chapter, the various methods and techniques available for the study of phase equilibria at high pressures are presented, together with considerations on the main characteristics, advantages, challenges and common error sources for each of the methods. Examples of studies related to food processing are provided, and the trends concerning the most commonly used methods for application to the food industry are analysed.

Comparison of Purification Efficiencies of Different Affinity Separation Techniques

An affinity separation method for the isolation of recombinantly produced benzoylformiate decarboxylase (BFD) is presented based on the interaction of the enzyme to its cofactor. Enzyme/cofactor interactions are well suited for affinity purification processes with regard to its high affinity and selectivity. Problems can be induced due to harsh desorption conditions that probably diminish the activity of the purified enzyme. The implementation of boronic acids to immobilize the cofactor introduces a gentle release by a simple pH shift from 8.5 to 6. The disadvantages of m- aminophenyl boronic acids are the high nonspecific adsorption induced by the introduction of a positive charge and a hydrophobic phenyl ring. It was shown that the disadvantages prevail and boronic acid resins are not suitable for protein purification from complex matrices. In contrast to the affinity separation method, where the cofactor was directly immobilized onto the support, a better separation performance could be achieved, which is reflected by a purification factor of 4.