How microwaves can help to study membrane ageing.

This paper studies original protocols of rapid PES/PVP membrane NaOCl degradation allowing at reaching ageing states that are representative of industrial ageing. The long term objective is to propose basis for further fundamental studies aiming at the improvement of the impact of membrane ageing on behaviour in UF (fouling and cleaning mastering). The key of several protocols is the use of ageing acceleration thanks to microwave irradiation, either continuous or pulsed ones, that can be further associated (or not) with short ageing time in UF conditions. To evaluate the representativeness of obtained aged membranes, comparisons are achieved between pristine, voluntary laboratory aged membranes and an industrial membrane at the end of its service-life. Several physico-chemical analyses were used (ATR-FTIR, SEM-EDX, contact angle, SEC-HPLC). Evaluation of UF performances were made in UF of a model protein (Lysozyme, 14,300 g.mol-1). Finally, the proof of concept is done that conditions using MW exist to reach ageing state representative of industrial ageing.

On the impact of ethanol on the rejection and transfer mechanism during ultrafiltration of a charged macromolecule in water/ethanol.

Ultrafiltration (UF) is a sustainable membrane separation technique. It could be useful for the concentration/purification of bio-sourced molecules that are extracted either by pure ethanol or by water/ethanol mixtures. Nevertheless, the process optimization requires an in-depth understanding of the transfer mechanisms of solute through membranes, especially for charged solutes, that are nowadays not sufficiently documented. Previous studies achieved in aqueous media have shown that the rejection of charged solutes by an UF membrane involves at least three mechanisms: convection, diffusion and electrostatic interactions. The present study aims at a systematic analysis of the transfer mechanisms of a model protein (lysozyme) in water/ethanol mixtures (100/0-70/30 v/v) during UF by a zirconia inorganic membrane. The influence of the pH varying in the 4-9 range and of the ionic strength (I) is also discussed. The ionic strength I can be adjusted by addition of an indifferent electrolyte (NaCl) only aiming at the screening of the electrostatic interactions or by addition of a selectively adsorbed electrolyte(KH2PO4) that is able to change the isoelectric pH of the protein and thus to modulate the electrostatic interactions in a different way when compared to NaCl. Of course, both salts have an impact on the protein rejection in UF. The results are analysed using the CDE model previously developed in our group to explain the behaviour of a single protein during UF in water and accounting for convection, diffusion and electrophoretic migration. The applicability of the CDE model in water/ethanol mixtures up to 70/30 v/v is finally shown.

A methodology for monitoring globular milk protein changes induced by ultrafiltration: a dual structural and functional approach.

Understanding filtration mechanisms at a molecular level is important for predicting structural and functional properties of globular milk proteins after membrane operations. This stage is thus highly decisive for the further development of membrane separations as an efficient alternative to chromatographic processes for the fractionation of milk proteins. In this study, we proposed an original and complete analytical package for the examination of the putative effect of filtration at both macroscopic and molecular levels. We then investigated the pertinence of this analytical package during ultrafiltration (UF) of globular milk proteins in both dead-end and crossflow modes. Reverse-phase HPLC combined with statistical computing was shown to be relevant for the assessment of even slight physically induced modifications. Adaptations of circular dichroism and solubility measurements, regarding their respective dependence on temperature and pH, were also useful for an accurate evaluation of functional modifications. At last, immunochemistry was proven to be a pertinent tool for the specific detection of modifications affecting a targeted protein, even in mixed solutions. Moreover, results obtained by such methods were shown to be coherent with data obtained from classical techniques such as fluorescence. For beta-lactoglobulin, some physically induced modifications were noticed in the permeate because of shear stress inside membrane pores. In the case of alpha-lactalbumin dead-end UF, permeation was shown to affect protein characteristics because of an increase in the relative calcium content responsible for a conformational transition from the apo-form to the holo-form of the protein. Finally, during crossflow UF with limited transmission of BSA, observations were coherent with a partial aggregation because of the circulation of proteins in the filtration pilot. Such a hypothesis corroborates results previously mentioned in the literature.

Role of electrophoretic mobility of protein on its retention by an ultrafiltration membrane. Comparison to chromatography mechanisms.

Lysozyme and lactoferrin, two globular proteins, were first studied separately in order to elaborate a strategy for the improvement of their separation by ultrafiltration (UF) with zirconia-based membranes of different charge sign and pore radius. The electrophoretic mobility (mu) at fixed pH and variable ionic strength was used for the characterisation of both proteins and zirconia particles, similar to the active layer of the membrane during the UF run. Specific adsorption of phosphate ions was shown for both proteins resulting in new isoelectric points. The occurrence of electrostatic exclusion mechanism in addition to the molecular sieving in UF of charged solutes was shown for: * Low molecular weight solute: multivalent citrate at pH 6 was specifically adsorbed on zirconia and its transmission through the membrane (defined as the ratio of the concentration in the permeate to that of the feed solution) was reduced in the range 0.001-0.01 mol l(-1) of citrate concentration * Proteins: their transmissions increase when the ionic strength increases (ion-exchange is not the relevant mechanism because transmission is irrespective of the initial charge of the membrane compared with the protein charge). A model based on convection, diffusion and electrophoretic migration mechanisms (CDE model) was proposed to take into account this behaviour. The CDE model predicts the possible existence of a depleted sub-layer of the charged protein in the concentration polarisation layer, located in the close vicinity of the membrane surface. A strategy for the separation of two proteins in mixed solution was proposed by varying both the physico-chemical environment in the feed solution (pH, ionic strength, chemical nature of the electrolyte) and the membrane pore radius. Maximum selectivity was obtained when the target protein (to be transmitted in the permeate side) is close to being uncharged due to specific adsorption of electrolyte ions. Ultrafiltration selectivity is enhanced with membrane of large pore radius, which provides high transmission of the target protein and efficient electrostatic exclusion of the solute to be retained in the retentate side. This UF approach corresponds roughly to the separation of one uncharged and one charged protein from a mixed solution by size exclusion chromatography of the uncharged protein combined with electrostatic exclusion of the charged protein due to packing of similar charge.

Specific adsorption of phosphate ions on proteins evidenced by capillary electrophoresis and reversed-phase high-performance liquid chromatography.

Specific adsorption of phosphate ions at pH=7.0 was studied on different proteins, either counter-ions of phosphate (lysozyme, lactoferrin) or co-ion of phosphate (alpha-lactalbumin). The theoretical electrophoretic mobility of globular proteins lysozyme and alpha-lactalbumin (apo and holo (+1 calcium per molecule) forms) was compared with those measured by capillary electrophoresis in phosphate at pH 7.0, versus the ionic strength (I) in the range 0-0.775 mol L(-1). The specific adsorption of phosphate ions was evidenced by difference. From the experimental charge number (Z(eff)) of protein in phosphate medium, a phosphate content per protein molecule was determined at pH=7.0. * For lactoferrin (pI=8-9), the electrophoretic mobility (mu) was constant and negative, highlighting a charge reversal due to phosphate adsorption. * For alpha-lactalbumin (holo form) experimental mu was roughly constant and more negative than predicted. Z(eff) increased continuously from -4 to -11 in the ionic strength range from 0.005 to 0.775 mol l(-1), respectively. Accordingly, one to six phosphates were bound per molecule, respectively. * For lysozyme, experimental electrophoretic mobility was positive but lower than predicted. Z(eff) was only discrete values +5 for I in the range 0.001-0.020 mol l(-1) and about +3 in the range 0.050-0.500 mol l(-1), whereas the theoretical Z value was +7 at pH = 7.0. Lysozyme bounds one phosphate at low ionic strength and about two-three at higher ionic strength. Reversed-phase HPLC confirms that adsorption of phosphate is different for the three proteins.

Physico-chemical characterization of proteins by capillary electrophoresis.

The electrophoretic mobility of proteins was successfully determined by means of capillary electrophoresis (CE) with various background electrolytes (BGEs). The objective was focused on the variation in BGE physico-chemical composition and the consequential impact on the observed protein charge. Experimental and calculated mobilities, according to Henry's equation, versus ionic strength have been compared. For positively-charged lysozyme, a good agreement between observed and calculated mobilities was observed using triethanolamine chloride at pH 7.0 as the BGE. Mobility close to zero was shown using borate (pH 8.0) and phosphate (pH 7.0) at a low ionic strength of about 20 mmol l(-1), and as a consequence, specific adsorption of oxyanions was evidenced. Lysozyme retention in the case of reversed-phase high-performance liquid chromatography (RP-HPLC) was decreased by the presence of phosphate ions. CE and HPLC are complementary tools for characterizing the behaviour of lysozyme. On the other hand, the mobility of the negatively-charged alpha-lactalbumin remained constant as regards phosphate at pH 7.0 in the 20-200 mmol l(-1) range, contrary to the decrease that had been expected with the increasing ionic strength. beta-Lactoglobulin exhibited increasingly lower mobilities than those expected of boric acid/borate at pH 7.0 and 8.0 (I=20 mmol l(-1)).