Laurent Leclercq

Xiaoling Leclercq

Alexis Guillard

Laurent Rodriguez

Olivier Braun

Cédrick Favero

Herve Cottet
email: herve.cottet@umontpellier.fr

Hervé Cottet

Characterization of ultrahigh molar mass polyelectrolytes by capillary electrophoresis

Keywords: high molar mass polymer, capillary gel electrophoresis, sieving, molar mass distribution, polyacrylamide derivatives

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

High to ultrahigh molar mass (> 1 MDa) anionic polyacrylamides and their analogues are polyelectrolytes which are widely used as flocculent in wastewater treatment [START_REF] Guezennec | Transfer and degradation of polyacrylamide-based flocculants in hydrosystems: a review[END_REF], as soil conditioner [START_REF] Seybold | Polyacrylamide review: soil conditioning and environmental fate[END_REF], as drag reducer [START_REF] Rodvelt | Use of a salt-tolerant friction reducer improves production in Utica completions[END_REF], and as viscosity enhancer in Enhanced Oil Recovery for flooding of trapped crude oil from the reservoir [START_REF] Thomas | Some key features to consider when studying acrylamide-based polymers for chemical enhanced oil recovery[END_REF]. In the oil field application, however, loss of flooding efficiency often arises from shear sensitive chain scissions in porous media and rheological performance deterioration provoked by salinity and high temperature [START_REF] Sabhapondit | Characterization of acrylamide polymers for enhanced oil recovery[END_REF][START_REF] Tolstikh | Degradation and stabilization of polyacrylamide in polymer flooding conditions[END_REF]. It is therefore desirable to thoroughly characterize these polyelectrolytes including their chemical nature (charge density) and molar mass distributions, and to study the impacts of shear stress, salinity and temperature on stability and rheological behaviors for optimal operations. Nevertheless, the characterization of such ultra-high molar mass polyelectrolytes with broad molar mass dispersion using classical methods such as size exclusion chromatography (SEC) is challenging due to the occurrence of chain breakage in size-exclusion chromatography, possible adsorption leading to abnormal elution [START_REF] Nguyen | Chain scission in transient extensional flow kinetics and molecular weight dependence[END_REF][START_REF] Zaitoun | Shear stability of EOR polymers[END_REF][START_REF] Rodriquez | Analyse dimensionnelle, comportement thermique et mécanique de polymères en solution aqueuse à base de 2-acrylamido-2-méthylpropane sulfonate : Application en récupération assistée d'hydrocarbures[END_REF][START_REF] Rodriguez | Monitoring thermal and mechanical stability of enhanced oil recovery (EOR) acrylamide based polymers (PAM) through intrinsic viscosity (IV): Determination using a new capillary rheology technique[END_REF][START_REF] Jouenne | Method for determining the weight-average molecular weight of a water-soluble high molecular weight polymer[END_REF], and especially the limited range of the molar mass analyzable with currently available SEC columns. In-batch techniques such as multi-angle laser light scattering (MALLS) [START_REF] Wang | Molecular weight and the Mark-Houwink relation for ultra-high molecular weight charged polyacrylamide determined using automatic batch mode multi-angle light scattering[END_REF][START_REF] Buchholz | The use of light scattering for precise characterization of polymers for DNA sequencing by capillary electrophoresis[END_REF], or open-medium separation techniques such as Field-Flow Fractionation (FFF) [START_REF] Woo | Characterization of ultrahigh-molecular weight cationic polyacrylamide using frit-inlet asymmetrical flow field-flow fractionation and multi-angle light scattering[END_REF][START_REF] Dalsania | Characterization of ultrahigh-molecular-weight oilfield polyacrylamides under different pH environments by use of asymmetrical-flow field-flow fractionation and multiangle-light-scattering detector[END_REF][START_REF] Leeman | Asymmetrical flow field-flow fractionation coupled with multi-angle light scattering and refractive index detections for characterization of ultra-high molar mass poly(acrylamide) flocculants[END_REF][START_REF] Hecker | Flow field-flow fractionation of high-molecular-mass polyacrylamide[END_REF] can be used with a certain success to extend the analysis limits.

Capillary Electrophoresis (CE) is also an attractive separation technique without stationary phase, which was originally mainly developed for biomacromolecules such as DNA [START_REF] Viovy | Electrophoresis of DNA and other polyelectrolytes: Physical mechanisms[END_REF], proteins [START_REF]Handbook of Capillary Electrophoresis[END_REF] and polysaccharides [START_REF] Volpi | Capillary Electrophoresis of Carbohydrates: From Monosaccharides to Complex Polysaccharides[END_REF]. More recently, CE has been used for the characterization of synthetic polyelectrolytes [START_REF] Dolnik | Capillary gel electrophoresis[END_REF][START_REF] Cottet | Nonaqueous and aqueous capillary electrophoresis of synthetic polymers[END_REF][START_REF] Cottet | Capillary Electrophoresis: Methods and Protocols[END_REF][START_REF] Chamieh | Quantitative analysis in capillary electrophoresis: transformation of raw electropherograms into continuous distributions[END_REF][START_REF] Thevarajah | Quantifying the heterogeneity of chemical structures in complex charged polymers through the dispersity of their distributions of electrophoretic mobilities or of compositions[END_REF][START_REF] Thevarajah | Determination of the distributions of degrees of acetylation of chitosan[END_REF], owing to its rapidity of analysis, minor sample consumption and orthogonal separation mechanism compared to chromatography.

Free solution capillary electrophoresis allows to characterize the polyelectrolytes according to their charge density since the polyelectrolyte electrophoretic mobility in absence of gel is independent of the molar mass, due to free draining behavior above a typical molar mass of about 20,000 g/mol [START_REF] Cottet | A semi-empirical approach to the modeling of the electrophoretic mobility in free solution: Application to polystyrenesulfonates of various sulfonation rates[END_REF]. It was demonstrated that the distribution in charge density and the associated dispersity indexes can be determined by free solution CE [START_REF] Chamieh | Quantitative analysis in capillary electrophoresis: transformation of raw electropherograms into continuous distributions[END_REF][START_REF] Thevarajah | Quantifying the heterogeneity of chemical structures in complex charged polymers through the dispersity of their distributions of electrophoretic mobilities or of compositions[END_REF]. The characterization of highly charged polyelectrolytes above the Manning condensation threshold [START_REF] Manning | Counterion binding in polyelectrolyte theory[END_REF] is challenging, due to the low dependence of the effective mobility against the polyelectrolyte charge density above this threshold [START_REF] Hoagland | Gel and free solution electrophoresis of variably charged polymers[END_REF][START_REF] Gao | Separation of polyelectrolytes of variable compositions by free-zone capillary electrophoresis[END_REF][START_REF] Cottet | Charge-and size-based separations of polyelectrolytes by heart-cutting twodimensional capillary electrophoresis[END_REF]. Indeed, due to counter-ion condensation, the polyelectrolyte effective charge density levels off above the Manning condensation threshold. Despite the low selectivity, it is however still possible to characterize the charge density distribution of highly charged polyelectrolyte by adjusting the ionic strength of the background electrolyte in counter electroosmotic mode free solution CE [START_REF] Desvignes | Separation and characterization of highly charged polyelectrolytes using free-solution capillary electrophoresis[END_REF].

Increasing the ionic strength lowers the electroosmotic mobility, and consequently the apparent polyelectrolyte electrophoretic mobility, resulting in a better selectivity of the separation according to the charge density.

For polyelectrolytes of constant charge-to-mass ratio, Capillary Gel Electrophoresis (CGE) using entangled or diluted neutral sieving polymer solution, can be employed for molar massbased separation of polyelectrolytes [START_REF] Viovy | Electrophoresis of DNA and other polyelectrolytes: Physical mechanisms[END_REF]. The electrophoretic migration of a polyelectrolyte through a polymer network has been elucidated via different separation mechanisms, depending on shape and size of the solute relative to the pore (or blob) size, and on the applied electric field [START_REF] Heller | Principles of DNA separation with capillary electrophoresis[END_REF][START_REF] Cottet | Electrophoretic behaviour of fully sulfonated polystyrenes in capillaries filled with entangled polymer solutuions[END_REF]. In the Ogston model [START_REF] Ogston | The spaces in a uniform random suspension of fibres[END_REF], a small sized solute is described as a rigid sphere migrating through a porous medium with a relatively large pore size. The reptation model [START_REF] De Gennes | Scaling concept in polymer physics[END_REF][START_REF] Slater | On the reptation theory of gel electrophoresis[END_REF] depicts a snake-like motion of a solute that is too large to pass freely through the pores of the network and must deform / unfold to pass through the matrix. The biased reptation migration (BRM) [START_REF] Dolnik | Capillary gel electrophoresis[END_REF][START_REF] Lumpkin | Theory of gel electrophoresis of DNA[END_REF][START_REF] Grossman | Experimental and theoretical studies of DNA separations by capillary electrophoresis in entangled polymer solutions[END_REF] describes the forced reptation of a polyelectrolyte under electric field, with orientation and stretching of the chain, leading to electric field-and size-dependent effective electrophoretic mobility, µ [START_REF] Dolnik | Capillary gel electrophoresis[END_REF][START_REF] Grossman | Experimental and theoretical studies of DNA separations by capillary electrophoresis in entangled polymer solutions[END_REF]:

  2 1 E b T N      : (1)
where b is a function related to the solute and the network, and N is the number of monomers of the polyelectrolyte. For polymer size comparable to or smaller than the pore size (or blob size) and at low electric field, µ is a linear function of the inversed size, corresponding to pure reptation regime. For large polyelectrolytes, the size-dependence of the mobility disappears, and the electric field term becomes predominant. These regimes were both identified for DNA separation, as well as for synthetic polyelectrolytes such as polystyrene sulfonates (PSS) [START_REF] Heller | Principles of DNA separation with capillary electrophoresis[END_REF][START_REF] Cottet | Electrophoretic behaviour of fully sulfonated polystyrenes in capillaries filled with entangled polymer solutuions[END_REF][START_REF] Cottet | The effect of blob size and network dynamics on the size-based separation of polystyrenesulfonates by capillary electrophoresis in the presence of entangled polymer solutions[END_REF].

In this work, we report the size-based characterization of high to ultra-high anionic poly(acrylic acid-co-acrylamide) (molar mass ranging between 22 kDa -15 MDa), including standards and industrial samples with charge density comprises between 25 -33% (expressed as a molar content in acrylic acid) by CGE using replaceable polymer solution as sieving matrix. A thorough optimization of the operating conditions including the nature of the separating polymer, the electric field strength, the ionic strength and the sample concentration, was investigated to permit the molar mass characterization of the APAM samples.

Experimental

Chemicals

Tris(hydroxymethyl)aminomethane (Tris), anthraquinone-2-sulfonic acid sodium salt monohydrate 97%, hydroxyethylcellulose (HEC, M w 1.3  10 6 g/mol) and polyethylene oxide (PEO, average M w 2  10 6 g/mol, 5  10 6 g/mol and 8  10 6 g/mol) were purchased from Aldrich (Saint Quentin Fallavier, France), pullulan from Aureobasidium pullulans (M w 575  10 3 g/mol) from Chem Cruz (Heidelberg, Germany), dextran from Leuconostoc mesenteroids (M w 1.5 -2.8  10 6 g/mol) from Sigma (Saint Quentin Fallavier, France), dextran SGO (M w 50  10 6 g/mol) kindly provided by SNF (Andrézieux, France), PEO (M w 1  10 6 g/mol) from Alfa Aesar (Illkirch, France), hydrochloric acid 37% and potassium chloride 99.6% from VWR (Fontenay-sous-Bois, France), sodium chloride 99.5% from Fluka (Illkirch, France), lithium chloride anhydrous 99% and lithium hydroxide 98% from Sigma-Aldrich (Saint Quentin Fallavier, France) and boric acid from Fluka (Illkirch, France).

Samples

All samples of anionic poly(acrylic acid-co-acrylamide) (APAM) in white powder were provided by SNF. Standard APAM samples were synthesized from monomers of sodium acrylate and acrylamide by Controlled Radical Polymerization (CRP) leading to narrow distributed molar mass and anionicity [START_REF] Read | Low temperature RAFT/MADIX gel polymerisation: access to controlled ultra-high molar mass polyacrylamides[END_REF]. Industrial samples of APAM (labelled as IPAM) were obtained by hydrolysis of polyacrylamide in a NaOH medium to reach expected anionicity. The molar mass and PDI were determined by Size Exclusion Chromatography with a Multi Angle Laser Light Scattering detector (SEC-MALS) [START_REF] Jouenne | Method for determining the weight-average molecular weight of a water-soluble high molecular weight polymer[END_REF] for the APAM standards, and by batch Static Light Scattering (SLS) for the industrial samples (IPAM). PDI and weight-average molar masses are gathered in Table 1.

For capillary electrophoresis, all samples were used as received and non-filtered. Stock polyelectrolyte solutions were prepared at 5 g/L as following: 1 g sample was carefully added into the vortex formed by a pitched-blade impeller in 200 mL buffer and agitated at 500 rpm for 6 h. As buffer system 20 mM Tris/Cl buffer containing 35.5 mM LiCl, pH 8.0 was selected, and as comparison, a borate buffer (50 mM boric acid /Li buffer containing 7 mM LiCl, pH 8.0) was also tested. Before injection, the stock solutions were diluted with the background electrolyte (i.e. the sieving medium excluding the sieving polymer) to the desired injected concentration and homogenized on an orbital mixer for 3 min. Stock solutions kept at 5°C were warmed up to room temperature before use.

Free solution and Capillary Gel Electrophoresis

Free solution and CGE were performed on a Beckman P/ACE MDQ apparatus (Sciex, Villebon sur Yvette, France). Capillaries were prepared from a fused silica tubing (Composite Metal Services, Photon Lines, France). Unless otherwise stated, the capillary dimensions were LiCl concentration (n mM) was also used, the total ionic strength being (7 + n) mM. For CGE, the sieving polymers (pullulan, dextran, PEO) were used as received except HEC that was purified by dialysis at a cutoff of 50,000 against deionized water overnight and then lyophilized. The BGE was made up by dissolving the sieving polymer in the buffer of selected ionic strength under gentle magnetic agitation overnight.

Before injection, 1 mL of APAM sample solution was obtained by dilution of the stock 5 g/L APAM solution in the BGE (without sieving polymer) to the desired injected concentration (comprised between 0.5 and 2 g/L), mixed with 25 L of DMF (at 0.2 % in water, electro-osmotic flow marker) and 20 L of anthraquinone-2-sulfonic acid sodium salt (2 % in water, mobility marker) on an orbital mixer. Samples were injected at 0.5 psi for 5 s.

Separation voltage of +2 kV (unless otherwise stated) was applied in normal mode (i.e. positive polarity with counter-electroosmotic mode). UV absorption was monitored at 200 nm. Temperature of the sample chamber and of the capillary cassette was set at 25°C.

Data processing of the electropherograms

The electropherograms were transformed into effective mobility scale. The effective mobility was calculated as:

11 eof lL t t V       (2)
where eof t is the elution time of the neutral marker (DMF), t the migration time of the solute, l the effective capillary length to the detector, L the total capillary length, V the separation electric voltage. In this transformation, the y-axis was transformed to a mobility massweighted distribution function P() using the following equation:

2 ( ) ( ) ( ) ( ) 1 A t A t tt P A t t d t dt                 (3)
with A(t) the UV absorbance of the detector [START_REF] Chamieh | Quantitative analysis in capillary electrophoresis: transformation of raw electropherograms into continuous distributions[END_REF]. The average effective mobility of the polymer calculated on the whole polymer distribution was determined according to:

( ) ( ) ( ) ( ) P dµ A t t dµ µ P dµ A t t dµ        (4a)
The detector signal is recorded in digitalized form of N data data points (A(t i ); t i ) varying from the initial (i = 1) to the end time point (i = N data ) of the polymer signal, eq (4a) can be rewritten in its discretized form (4b):

1 1 1 1 ( ) ( ) ( ) ( ) data data N i i i i i i N i i i i i A t t µ A t t              (4b)
To get the molar mass distribution, it is first required to normalize the effective mobility µ obtained in sieving conditions to the average free-solution effective mobility 0 µ obtained in the same electrophoretic conditions but in the absence of sieving matrix. This normalization is important to take into account to correct from the possible differences in polyelectrolyte charge density from one sample to the other. For that, the effective mobility distributions were transformed in distribution of µ/ 0 µ using the following equation:

  0 0 0 () Pµ P A t t µ µ d µ dµ             (5)
Assuming the charge density of samples was evenly distributed according to the molar mass distribution, the molar mass distribution P(M w ) is obtained using a calibration curve obtained with APAM standards according to equations ( 5) and ( 6) [START_REF] Chamieh | Quantitative analysis in capillary electrophoresis: transformation of raw electropherograms into continuous distributions[END_REF]:

0 w M µ     (6) 1 0 1/ 0 0 0 ( ) ( ) P µ P M A t t µ µ dM d µ                    (7)
The calculation of the weight-and the number-average molar mass leading to the dispersity index can finally be calculated for each sample using equations (8-10):

() () w P M MdM M P M dM    (8) () (
)

n P M dM M PM dM M    (9) w n M PDI M  (10)

Size-Exclusion Chromatography (SEC)

Weight and number average molecular mass, PDI, and radius-of-gyration of APAM standards were collected by a SEC-MALS system. The SEC system used consisted of an on-line degasser, a high-pressure pump (Agilent 1260 Infinity I), an automatic sampler (Agilent 1260 Infinity I) a 8 × 300 mm SEC column packed with polyhydroxymethacrylate-based gel (OHpak columns, Shodex, Japan), a MALS detector (Dawn Heleos II, Wyatt Technologies) and a refractive index detector (Optilab, Wyatt Technologies). Experimental conditions of the analysis have been realized according to Jouenne et al. study [START_REF] Jouenne | Method for determining the weight-average molecular weight of a water-soluble high molecular weight polymer[END_REF]. A 0.5M sodium nitrate solution and 55 mM HEPES sodium buffer (pH 8) was used as the eluent. The eluent was filtered through a 0.1 µm cellulose membrane before use. The flow rate was set at 0.3 mL/min mL/min. Sample concentration of 0.02% (w/V) in the eluent. Before injection, the sample solutions were filtered through a 1.2µm cellulose disposable membrane. The injection volume was 100 µL. The detector cells of MALS and RI were kept at ambient temperature. The value of the refractive index increment (dn/dc) of each sample was measured by using the Optilab detector (Wyatt Technology). Data acquisition and processing were carried out using the ASTRA software (version 6.1, Wyatt Technologies).

Batch Static Light Scattering (SLS)

SLS batch analysis was carried out on a Dawn HELEOS II equipment (Wyatt Technology, Toulouse, France). Polymer solutions were prepared in an aqueous mobile phase (0.5 M NaNO 3 ) at 0.5 wt % concentration of polymer and mixed with a mechanical stirrer (at 400 rpm), at room temperature for 2 h, to obtain dissolution. The 0.5 wt % polymer solutions were further diluted to 0.01 wt % and stirred with a stir bar at 200 rpm for 1 h. The diluted solutions were then filtered through 1.2 m syringe filters to remove dust and any other large particle contaminants. The filtered 100 ppm solutions were diluted sequentially to 4-5 different concentrations with a set of two syringe pumps, and then directly injected into a the MALS detector. The MALS cell temperature was set at 30°C. The flow rate was 0.3 mL/min. The scattering data were collected at 17 different angles with an incident laser wavelength of 664 nm. Calibration of the MALS detectors was done using HPLC grade toluene. The data analysis was conducted with Astra 6 software provided by Wyatt Technology (Toulouse, France). In a batch MALS measurement, the angular and concentration dependent light scattering data are fitted with Ornstein-Zernick formalism: where R is the excess Rayleigh ratio, K the optical constant, C the polymer concentration, M w the weight-average molar mass, D f the fractional size, q the scattering factor and R g the radius of gyration.

Results and discussion

Free solution capillary electrophoresis

The electropherograms of the APAM standards in free solution CE obtained in a 20 mM Tris-HCl buffer containing 85.5 mM LiCl, pH 8 (96.5 mM ionic strength) are presented in Fig. 1, both in time (Fig. 1A) and in effective electrophoretic mobility scales (Fig. 1B). LiCl was used instead of NaCl to reduce the conductivity, since Li + has a lower ionic mobility than Na + . The separation is performed under counter-electroosmotic mode on fused silica capillary, using positive polarity. The transformation of the electropherograms in effective electrophoretic mobility scale allows to get rid of the EOF fluctuations, as it can be observed by the perfect alignment of the acrylic acid peak (peak 3) in Fig. 1B. As observed on the distributions, the effective mobilities of the APAM standards vary between -20 TU and -25 TU (where TU stands for Tiselius Unit, 10 -9 m 2 s -1 V -1 ), depending on their polymer charge density distributions. The mobility distributions of the APAM standards largely overlap one each other, with slightly faster average mobility for APAM50 and APAM10, in good agreement with their higher anionicity (see Table 1). The average free solution electrophoretic effective mobility 0 µ were calculated for each APAM sample according to equation (4b) and are gathered in Table 1.

Evaluation of the nature of the sieving polymer

As a sieving media, several neutral linear polysaccharides or synthetic polymers were considered: pullulan (575 KDa), dextran (1.6-2.8 MDa), dextran SGO (50 MDa), hydroxyethyl cellulose (1.3 MDa) and four polyethylene oxides (PEO) of molar mass varying between 1.5-8 MDa. Polymers with relatively high molar masses were used in order to reach large mesh size or blob size  b (i.e. low polymer concentration) in entangled conditions (C > C * ). The entanglement threshold C* depends on the polymer molar mass, and can be derived from the intrinsic viscosity using equation ( 12) [START_REF] Heller | Principles of DNA separation with capillary electrophoresis[END_REF]:

  1.5 C    (12)
The C * values of the polymers used in this work are given in Table 2. The 'blob size'  b is a decreasing function of the polymer concentration, and does not depend on the polymer molar mass (as far as the solution is entangled, otherwise it is not defined) according to equation [START_REF] Buchholz | The use of light scattering for precise characterization of polymers for DNA sequencing by capillary electrophoresis[END_REF] [40]: 2. For HEC, PEO and pullulan, the used concentrations were chosen close to C* to get the largest blob size (164-174 nm for HEC and PEO) and to limit the viscosity of the entangled polymer solution.

The separation of the 6 APAM standards were investigated using pullulan, dextran, dextran SGO or HEC at 46.5 mM ionic strength and at a constant electric voltage of +2 kV (50 V/cm) (except + 8 kV, i.e. 200 V/cm, for pullulan). The best results were obtained with HEC entangled solution, where the 4 first APAM standards with molar mass between 0.22×10 6 and 3.93×10 6 g/mol were partially separated (see Fig. 2). However, the analysis of APAM50

4.18×10 6 g/mol and APAM60 7.34×10 6 g/mol was perturbed by the aggregation phenomenon during the CGE (discussed in the next section), that generates spikes during the electrophoretic separation. Compared to the decreasing mobility with molar mass observed in the presence of HEC, lower (or even zero) selectivity according to the APAM molar mass was recorded in pullulan, dextran or dextran SGO solutions (see Fig. SI1 to SI3), whatever the concentration of the separating polymer tested (see Table 2). The lacking sieving ability of these polymers might be related to a too low molar mass of the polymer for pullulan or by the presence of ramifications for dextrans, which may lead to short mesh lifetime [START_REF] Cottet | The effect of blob size and network dynamics on the size-based separation of polystyrenesulfonates by capillary electrophoresis in the presence of entangled polymer solutions[END_REF]. It can also be explained by differences in chain stiffness (pullulan and dextran, 0.4 nm persistence length, being much more flexible than rigid HEC 26-40 nm [START_REF] Martínez-Richa | Determination of molecular size of O-(2-hydroxyethyl)cellulose (HEC) and its relationship to the mechanism of enzymatic hydrolysis by cellulases[END_REF]). As for PEO, un-entangled 1.5 g/L solutions (PEO 1.5, 2 or 5 MDa) at 96.5 mM ionic strength provided good molar mass selectivity up to about 3-4×10 6 g/mol (i.e. up to APAM30, see Fig. SI4 A andB), however, the detection sensitivity was overall lower than in the HEC media. The least sensitivity of detection was observed in entangled PEO 8 MDa solution, together with lower baseline stability and aggregation of APAM60 (see Fig. SI4 C and D).

Reduction of aggregation phenomenon

The phenomenon of electrohydrodynamically-induced aggregation was already encountered in CE of long-chain DNA (> 30 kbp) [START_REF] Magnúsdóttir | Electrohydrodynamically induced aggregation during constant and pulsed field capillary electrophoresis of DNA[END_REF][START_REF] Venzac | On-chip conductometric detection of short DNA sequences via electro-hydrodynamic aggregation[END_REF] or polysaccharides [START_REF] Stefansson | Electrohydrodynamic instabilities and segregation of polysaccharides in capillary polymer solution electrophoresis[END_REF]. It was explained as the electroneutrality breakdown between the polyanions and the co-and counterions of the BGE under action of electric field, that extended on a scale of a few microns far beyond the Debye layer (typically, 1 to 2 nm) [START_REF] Magnúsdóttir | Electrohydrodynamically induced aggregation during constant and pulsed field capillary electrophoresis of DNA[END_REF]. The resulted non-linear electric force is capable to produce an electrohydrodynamic flow and in turn concentration fluctuation that leads to the aggregation of the polyanions. The electric field threshold E a at which aggregation occurs is a function of the polymer concentration C p and the electrolyte concentration C s according to the following scaling law:

~s a p C E C  ( 14
)
where  is the solution viscosity. Equation ( 14) predicts that aggregation occurs at higher electric field strength by increasing the electrolyte concentration and/or by decreasing the polymer concentration.

Impact of ionic strength and sample concentration on aggregation reduction

In addition to its beneficial effect on aggregation phenomenon, it is known that higher ionic strength decreases the electrophoretic mobility of solutes and electroosmotic mobility [START_REF] Ibrahim | Extracting information from the ionic strength dependence of the electrophoretic mobility by use of the slope-plot[END_REF], and also leads to more compact conformations of polyelectrolytes [START_REF] Cottet | On the use of the activation energy concept to investigate analyte and network deformation in entangled polymer solution capillary electrophoresis of synthetic polyelectrolytes[END_REF]. The impact of ionic strength on aggregation was significant: by increasing ionic strength from 46.5 mM to 96.5 mM at 2 kV (50 V/cm) in the HEC solution, APAM50 and APAM60 aggregated much less, as demonstrated in Fig. 3 with better peak shape for the APAM signals. Further increase of the background electrolyte ionic strength from 96.5 mM to 136.5 mM (or even 196.5 mM) did not lead to improvement of the separation, while the total run time was severely increased due to decreased apparent electrophoretic mobility, both in sieving and free solution conditions. Thus, 96.5 mM ionic strength was considered as a good balance between separation performance and analysis time. As expected [START_REF] Magnúsdóttir | Electrohydrodynamically induced aggregation during constant and pulsed field capillary electrophoresis of DNA[END_REF], lower injected concentration (1.5 g/L vs 2 g/L) also tends to reduce the adverse effect of aggregation. However, due to limited sensitivity of detection of APAM, it was hardly possible to reduce the injected concentration below 0.5 g/L.

Impact of the electric field strength on aggregation reduction

Since aggregation was induced by action of the electric field, decreasing the electric field strength was an effective counteraction. In the HEC solution at the optimized ionic strength of 96.5 mM, decreasing the applied separation voltage from +2 kV (50 V/cm) to +1 kV (25 V/cm) effectively suppressed the aggregation of high polyelectrolytes (see Fig. SI5), but the peak broadening due to late migration times generated low signal to noise ratio. Therefore, the separation voltage was retained at + 2 kV in the following sections.

Effect of the nature of the buffering species on the size-based separation

Surprisingly, changing the Tris/Li/Cl buffer by a Li/borate/Cl buffer, and keeping constant the other conditions (ionic strength at ~100 mM, pH 8, and 0.8 g/L HEC), led to much lower size-based selectivity of the APMA standards (see Fig. SI6), indicating Tris/Li/Cl buffer was better suited for HEC sizing of APAMs. One possible explanation of this effect could be that the selectivity obtained in HEC/Tris sieving medium is partly due to a transient interaction between the APAM polyelectrolyte and the neutral separating HEC chain (as already observed for DNA separation [START_REF] Barron | Capillary electrophoresis of DNA in uncrosslinked polymer solutions: Evidence for a new mechanism of DNA separation[END_REF]), and not only due to a sieving mechanism. In the context of such prevailing separation mechanism, the nature of the buffering ions could change the strength of the APAM / HEC interaction, which would be higher in the case of Tris/Li/Cl buffer compared to Li/borate/Cl buffer.

Reduced electrophoretic mobility distributions

The effective mobility distributions presented in Fig. 3D were transformed in distribution of µ/ 0 µ according to equation ( 5), as presented in Fig. 4A. This transformation aimed at a correction of the impact of slightly higher charge density values for samples APAM 50 and APAM 10, leading to a smoother 'non-biased' variation of the reduced effective mobility according to the molar mass.

Under the optimized conditions established with APAM standards, CGE was performed on 4 industrial samples (IPAM) of molar mass up to 15 MDa (as estimated by SLS, see Table 1)

and an anionicity between 27 and 33%. The results were presented in Fig. 4B.

Electropherograms obtained in free solution for the IPAM samples under similar electrophoretic conditions can be found in Fig. SI7.

Electrophoretic mobilitymolar mass calibration curve

The size-based selectivity obtained for the APAM (standards) in 0.8 g/L HEC at +2 kV and 96.5 mM ionic strength is quantitatively represented on a Ferguson-like plot (see the red dots in Fig. 5), where the log of the average reduced electrophoretic mobility ( µ / 0 µ ) is M w CGE values of IPAM1 industrial sample (4.3 MDa) is in better agreement with the SEC value (4.3 MDa), but lower than the SLS one (6.5 MDa).

As for the polydispersity, and as expected, APAM standards have relatively low polydispersity indexes ranging between 1.2 and 1.5 in CGE; which are in reasonably good agreement with those obtained by SEC for most of the APAM samples. Significantly smaller PDI were obtained by CGE for APAM 10 and APAM 50 samples compared to SEC, which may be related to higher peak broadening in SEC compared to CGE, as observed in previous studies [START_REF] Thevarajah | Quantifying the heterogeneity of chemical structures in complex charged polymers through the dispersity of their distributions of electrophoretic mobilities or of compositions[END_REF][START_REF] Vander Heyden | Contribution of the polymer standards' polydispersity to the observed band broadening in size-exclusion chromatography[END_REF][START_REF] Popovici | Band broadening in size-exclusion chromatography of polydisperse samples[END_REF]. As for IPAM industrial samples, polydispersity indexes are obviously much higher than for APAM samples, and are comprised between 2.4 and 2.9 for IPAM 1 and IPAM 2 samples. It is worth noting that IPAM samples cannot be analyzed by SEC because of shear degradation occurring through the columns, adsorption phenomena and lack of separation due to actual SEC column technology. The main limitation is due to the exclusion limit of the gel matrix used in SEC [START_REF] Jouenne | Method for determining the weight-average molecular weight of a water-soluble high molecular weight polymer[END_REF]. IPAM 3 and IPAM 4 polydispersities could not be accurately determined by CGE due to the presence of spikes above 10 MDa (aggregation phenomenon).

On the whole, one can conclude that the M w values obtained by CGE are quite consistent with those obtained from other methods within the experimental confidence intervals. CGE can be used to determine average molar mass, molar mass distribution and polydispersity index of APAM/IPAM samples up to about 10 MDa.

Conclusion

The molar mass-based characterization of anionic polyacrylamides by CGE using entangled HEC solutions as sieving media was successfully achieved up to about 10 MDa by unifying two prerequisites: analysis of high polymers was made possible by aggregation reduction and HEC was capable to size the whole range of the molar masses studied. The sample aggregation was reduced by optimization of the operating parameters: low electric field strength (50 V/cm), relatively high ionic strength of 96.5 mM, low sample concentration (~1 g/L). The buffer nature (TRIS) was also found crucial to get appropriate selectivity that is both depending on sieving mechanism and possible polyelectrolyte / HEC transient interaction. Aggregation was also promoted by the presence of separating polymer, while no detectable aggregation was observed in free solution under the same conditions.

Based on the log( µ / 0 µ ) -logM w relationship established with the APAM standards, the molar mass distribution and the polydispersity index of the APAM and IPAM samples were Experimental conditions as in Fig. 3.

  40 cm (30 cm to the detector)  50 m ID. The capillary was first activated by successive flushing (at 20 psi) with 1 M NaOH for 30 min, 0.1 M NaOH 10 min, deionized water 10 min, and finally with the background electrolyte for 10 min. Between runs, capillaries were successively flushed at 20 psi with 1 M NaOH for 15 min, 0.1 M NaOH for 5 min, deionized water for 5 min and BGE for 10 min. The BGE for free solution CE consisted in a 20 mM Tris/Cl buffer solution of pH 8.0 containing varying LiCl concentration (n mM) providing a total ionic strength of (n + 11) mM. A 100 mM borate/Li buffer pH 8.0 containing varying

  where a and K are the Mark-Houwink coefficients, N a the Avogadro constant and  the modified Flory viscosity constant. The characteristics of the polymers including the molar mass M w , the entangled threshold C*, the [] -M w relationship, the used concentrations C, and the blob sizes  b at the used concentrations are gathered in Table

Figure 1 .

 1 Figure 1. Free solution electropherograms of standards (APAM) obtained at 96.5 mM ionic strength in time scale (A) and in effective electrophoretic mobility scale (B). Experimental conditions: Fused silica capillary 40 cm (30 cm to the detector)  50 m ID. BGE: 20 mM Tris/Cl buffer containing 85.5 mM LiCl, pH 8. Separation voltage: + 2 kV. Injection: 0.5 psi, 3 s. Sample concentration: 1 g/L APAM + 0.05% DMF in BGE. UV detection at 200 nm. Temperature: 25°C. Identification: DMF (1), APAM (2) and acylic acid (3). Acrylic acid was originally present in the polymer samples.

Figure 2 .

 2 Figure 2. Electropherograms obtained for the size-based separation of APAM standards in HEC solution at 46.5mM ionic strength and +2 kV in time scale (A) and effective mobility scale (B). Experimental conditions: Fused silica capillary 40 cm (30 cm to the detector)  50 m ID. BGE: HEC: 0.8 g/L (blob size: 164 nm) in 20 mM Tris/Cl buffer containing 35.5 mM LiCl, pH 8. Electric voltage: +2 kV. Injection: 0.5 psi, 3 s. Sample concentration: 1 g/L APAM + 0.05 % DMF. UV detection at 200 nm. Temperature: 25°C. Identification: DMF (1), APAM (2), acrylic acid (3).

Figure 3 .

 3 Figure 3. Electropherograms obtained for the size-based separation of APAM standards in HEC solution at 96.5 mM ionic strength in time scale (A, C) and effective mobility scale (B, D) at different injected concentrations (A, B: 2 g/L; and C, D: 1.5 g/L). Experimental conditions: Fused silica capillary 40 cm (30 cm to the detector)  50 m ID. BGE: HEC: 0.8 g/L (blob size 164 nm) in 20 mM Tris/Cl buffer containing 85.5 mM LiCl, pH 8. Electric voltage: + 2 kV (50 V/cm). Injection: 0.5 psi, 3 s. Sample concentration: 1.5 g/L (C, D) or 2 g/L (A, B) APAM + 0.05% DMF. UV detection at 200 nm. Temperature: 25°C. Identification: DMF (1), APAM (2), mobility marker: anthraquinone sulfonic acid (3).

Figure 4 .Fig. 3 .

 43 Figure 4. Reduced electrophoretic mobility distributions P(µ/ 0 µ ) of APAM satandards (A) and IPAM industrial samples (B) in 0.8 g/L HEC Tris/Cl electrolyte at 96 mM ionic strength. Experimental conditions as in Fig. 3. Injected concentrations at 1.5 g/L (A) and 1 g/L (B).

Figure 5 .

 5 Figure 5. log( µ / 0 µ ) vs log M w plot of APAM standard (•) in 0.8 g/L HEC solution and for IPAM industrial samples with M w from SLS (▼). The calibration curve obtained on the APAM samples reads: log( µ / 0 µ ) = - (0.1129±0.0069)logMw -(0.109±0.0040) (M w in g/mol), R² = 0.9864. Expected molar masses for IPAM industrial samples (•) obtained by CGE by integration of the polymer peak of Fig. 6B using equation (8).

Figure 6 .

 6 Figure 6. Area-normalized molar mass distribution (P(logM) ~ P(M) M) of APAM standards (A) and IPAM industrial samples (B) in HEC solution. Experimental conditions as in Fig. 3.

Acknowledgments

This work was partly funded by SNF, under a Cooperative Research and Development Agreement with the University of Montpellier and the CNRS.

plotted against logM w . The weight-average molar mass M w of the APAM standards was obtained by SEC-MALS and varied between 0.22×10 6 and 7.34×10 6 g/mol (chromatograms are presented in Figure SI8). The average effective mobility of the polyelectrolyte migrating in the presence of HEC solution µ , was calculated according to equation (4) to the average effective mobility in free solution ( 0 µ ). The latter was separately determined in the absence of the sieving solution under the same experimental conditions using equation (4b). A linear loglog correlation calculated on the APAM standards (red dots) was obtained leading to the following scaling law by least-square regression on the APAM standards: log( µ / 0 µ ) = (-0.113±0.007)logM w -(0.109±0.004) (M w in 10 6 g/mol), R² = 0.9864 or µ / 0 µ = (0.778±0.007)M w (-0.113±0.007)

. As expected, the slope was far from -1 value predicted for a pure reptation regime, suggesting that the migration was strongly electric field-dependent (biased reptation with orientation), and possibly mixed with a transient polyelectrolyte / HEC interaction, as discussed in section 3.4.

Calculation of molar mass distribution and polydispersity of industrial samples

The molar mass distributions P(M) of the standard and industrial samples were calculated from the electropherogram in the reduced electrophoretic mobility scale according to equation ( 6) and [START_REF] Nguyen | Chain scission in transient extensional flow kinetics and molecular weight dependence[END_REF], where the calibration equation is derived from Fig. 5 as µ / 0 µ = (0.778±0.007)M w (-0.113±0.007)

. Fig. 6 displays the area-normalized molar mass distributions P(log M) ~ P(M) M as a function of logM for the standard and industrial samples. In these optimized conditions, the molar mass distribution can be obtained up to 10 MDa without significant effect of the aggregation. Above 10 MDa, the molar mass distribution can be impacted by the aggregation effect as visualized by the multiple spikes for the IPAM 3 and 4 samples (see Fig. 6B). A comparison on the Ferguson-like plot using M w from SLS is displayed in Fig. 5 for the IPAM samples, showing a reasonable extrapolation of the log ( µ / 0 µ ) vs log M w correlation for molar mass higher than the largest APAM standard (i.e. M w > 7.34 MDa). The average molar mass M w (CGE), of the industrial samples calculated by integration of the molar mass distributions according to equation [START_REF] Zaitoun | Shear stability of EOR polymers[END_REF], as well as the polydispersity of all samples using equation ( 10) are gathered in Table 1. The M w values obtained by CGE for most the IPAM industrial samples: 6.5 MDa (IPAM2), 10.1 MDa (IPAM3) and 14.9 MDa (IPAM4), are similar to those obtained by SLS (8, 10.5 and 15 MDa, respectively) within the confidence interval (calculated as ± one SD on n=3 repetitions). The obtained as far as the molar mass did not exceed 10 MDa. The molar mass values were similar to those determined by SLS, proving the potential of CGE in molar mass based characterization of high ultra-high molar mass polyelectrolytes up to about 10 MDa. Further or future developments may focus on the limitation of the aggregation above this threshold.


Ferenc Martinovic

Quang Nguyen

Fabio Alessandro Deorsola

Samir Bensaid

Regina Palkovits

Werner Paulus

Barbara Bonelli

Francesco Di Renzo

Raffaele Pirone

SO 2 deactivation mechanism of NO oxidation and regeneration of the LaCoO 3 perovskite

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Perovskite structured materials are an interesting type of catalysts due to their tunable catalytic activity, thermal stability and peculiar redox properties. 1 In the automotive exhaust treatment, they have been suggested as a potential low-cost substitute for the platinum group metals Pt-Pd (PGM) widely used in three way catalysts, diesel oxidation catalysts (DOC), catalysed diesel particulate filters (cDPF) and lean NOx traps (LNT). [1][2][3][4][5][6][7] The most promising perovskites are lanthanides with LaBO3 structure, where B is usually Fe, Mn and Co in the +3 oxidation state. Out of these three, LaCoO3 has shown the highest activity in the automotive application, partly due to its interaction and high oxidation ability of NOx. [1][2][3][4][5][6] Despite advances made in hydrodesulphurization, deep removal to produce low sulphur concentration in fuel (<10 ppm) remains a challenge. Hence, sulphur-based compounds are invariably present in the fuel and produce SO2 during combustion. In different degrees, SO2 represents a poison to almost all catalysts used in aftertreatment systems. The most sensitive to sulphur are catalysts that are characterized by an alkaline surface (e.g. barium in LNT), which can form stable sulphates and deactivate the catalyst active site. While there are many detailed studies of SO2 poisoning and deactivation on Pt-Ba based LNT systems, [8][9][10] only few and conflicting reports are available on the deactivation of the perovskite-based catalysts. [11][12][13][14][15][16][17][18][19] The reason is that, in the deactivation studies, the applied reaction conditions are drastically different, depending on the application and/or the aims of the study. For example, in studies concerning the hydrocarbon catalytic oxidation at high temperature (>500 °C) the main results showed that sulphur diffuses in the bulk of the perovskite structure,13,15,20 whereas studies made at lower temperature concluded that the sulphur insertion was limited and the deactivation was mainly a surface phenomenon. 11,14,17 The point at which all agree is the competition for the same catalytic cycle and competition of SO3 and NO2 for the same active site characterized by alkaline character. The first goal of this work is to unravel the deactivation mechanism of NO oxidation by SO2 under relevant reaction conditions that typically occur in diesel exhaust aftertreatment system. The second goal of the work focuses on alternative methods of SO2 poisoning management. The typical methods suggested for the regeneration of sulphur poisoned LNT catalysts involve the injection of large amounts of reductant (such as H2, C3H6, NH3) and high regeneration temperatures (>600 °C), which result in high fuel penalty and catalyst damage. [9][10][11] Based on the deactivation mechanism and different chemical nature of the perovskite-based catalysts compared to the PGM based LNT, several novel methods are suggested: 1. Use of inexpensive Ca(OH)2 as SO2 adsorbent and bed guard. 2. Use of soot for enhancing the release of SO2. Soot is a pollutant abundant on the cDPF, which is one of the main catalytic potential applications of LaCoO3. 3. Facile regeneration at room temperature, by washing with distilled water. In this work all three alternative regeneration methods are investigated and discussed.

Experimental procedures

Catalyst preparation. The LaCoO3 catalyst was prepared by following the citrate sol-gel method. Proper equimolar amounts of La(NO3)3•6H2O and Co(NO3)2•6H2O (each 5 mmol) were dissolved in 60 mL of deionized water. 20 mmol citric acid monohydrate was added under stirring to the mixture and the pH adjusted to 7 by adding ammonia solution (10% v/v). The solution was heated to 80 °C and evaporated under stirring. As the liquid volume decreased, the chelated metallic ions were crosslinked by citric acid and the viscosity increased. The as-obtained gel was dried at 110 °C for 24 hours after which it was crushed and pre-calcined at 350 °C for 1 hour to decompose the citric acid. The obtained powder was ground in a mortar and calcined at 700 °C for 6 hours (5 °C/min heating rate). Regeneration of the poisoned samples was made by placing the sulphated LaCoO3 (LaCoO3-S) in 20 mL of distilled water and by adjusting the pH to 7 by adding ammonia solution. The samples could be also regenerated by using distilled water without adjusting the pH; the ammonia solution was added only to avoid metal leaching and potential sample loss. The slurry was stirred for 30 min at room temperature, after which it was separated by centrifugation. The regenerated sample was dried at 200 °C for 12 h and tested again. Several successive deactivation-regeneration cycles were conducted, and the so obtained samples were referred to as LaCoO3-R followed by a number indicating the number of regeneration cycles. A total of 5 poisoning-reactivation cycles was reached. Catalyst characterization. X-ray diffraction were recorded on a Philips PW 3040 X'Pert instrument with Cu anode for the Kα generation at 40 kV operating voltage. A pixel array detector enabled continuous data acquisition in the 2θ range 20-80°, with a step of 0.013°. The cell parameters and the crystallite sizes were evaluated by Rietveld refinement. The specific surface area was determined by N2 physisorption at -196 °C on Micrometrics Tristar 3020 instrument.

Before the analysis, the samples were evacuated at 200 °C for 2 hours. The reported specific surface area was calculated according to the BET method. X-ray photoelectron spectroscopy (XPS) was performed in a PHI Versaprobe apparatus, using Al Kα radiation with band-pass energy of 187.85 eV and 23.5 eV for the high-resolution scan, a 45° take off angle and a 100.0 μm diameter X-ray spot size. The lines of La, Co, O and S were detected. To observe the depth profile and the deactivation mechanism, the deactivated LaCoO3-S was sputtered with Ar+ gun to remove the top layer and the XPS analysis was repeated afterwards. The hydrogen temperature programmed reduction (H2-TPR) was performed on a ThermoScientific TPRDO 1100 instrument equipped with a thermal conductivity detector (TCD) detector. In a typical experiment, the catalyst was pre-treated in-situ under inert flow of Ar at 500 °C for 1 hour to remove any adsorbed species. Afterwards, the sample was cooled down and 20 mL/min of a gas mixture of 5% H2 in Ar was flown over 60 mg of the sample with a 5°C/min rate of temperature increase until 900 °C. The H2-TPR profile for the poisoned LaCoO3-S sample was also repeated in the experimental setup described below, to detect by means of mass spectrometer the various potential species formed during reduction. The soot-TPR, H2-TPR and temperature programmed desorption (TPD) of the poisoned sample was performed in a slightly different setup, with the outlet connected to a mass spectrometer to follow the evolution of all the potential species. Typically, 60 mg of sample (LaCoO3 and LaCoO3-S) was used under 60 mL/min Ar flow and 5 °C/min heating rate. For the soot-TPR the fresh and poisoned catalyst was mixed with soot in a planetary ball mill in 10: 1 catalyst: soot mass ratio. Morphology and elemental composition were determined by field emission scanning electron microscopy-Energy-dispersive X-ray spectroscopy (FESEM-EDS) under high vacuum, using Zeiss MERLIN Gemini II equipped with EDS at 3 keV accelerating voltage and different magnifications. Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS) analysis was performed on Vertex-70 IR spectrometer equipped with Praying Mantis (Harrick) heated reaction cell. The samples LaCoO3 or LaCoO3-S were mixed to KBr in 100:2 mass ratio. The so-prepared samples were placed into an isolated cell and kept under inert N2 flow during the test. LaCoO3-S was heated to 400 °C, 500 °C, 550 °C and 600 °C and held at constant temperature for 30 min after which they were quenched at 200 °C/min cooling rate. All the IR spectra were collected in absorbance mode at 30 °C (2 cm -1 resolution; 64 scans; wavenumber range 1800-800 cm-1). Catalytic tests. The catalytic tests were conducted in a glass tube reactor with i.d. 10 mm. The catalyst was placed on a glass membrane inside the reactor and heated in an isolated vertical furnace with a thermocouple in the catalytic bed for programmable heating. The outlet was connected to ABB Limas and Uras nondispersive infrared and ultraviolet (NDIR and UV) analysers for continuous measurement of CO, CO2, NO, NO2. The deactivation was performed by flowing 600 mL/min of 60 ppm SO2, 500 ppm NO, 4% O2 in N2 over 200 mg of catalyst. To prepare the LaCoO3-S sample, the deactivation was performed at constant temperature of 300 °C as it is a typical temperature found in diesel exhaust. Deactivation was considered complete when the NO oxidation rate dropped to 0, i.e. the NO2/NOx ratio after the catalytic bed was the same as at the inlet. The poisoning time typically lasted 90 minutes. To demonstrate the effectiveness of an adsorbent towards the SO2 management, 200 mg of LaCoO3 was mixed with 100 mg of Ca(OH)2 before performing the deactivation, under the same conditions described before, at 300 °C and also at 500 °C. Different adsorbent catalyst configurations were investigated and compared: 1) LaCoO3 and Ca(OH)2 gently mixed with spatula to obtain "loose contact";

2) LaCoO3 and Ca(OH)2 mixed in planetary ball mill for 15 minutes to obtain "tight contact"; 3) Zoned dual-layer configuration whereby the LaCoO3 bed was located downstream of the Ca(OH)2 bed. To show competitive adsorption of NOx and SO2, the catalyst was first saturated with NOx at 200 °C. When the NOx concentration was stable, NOx flow was switched off and the catalyst purged with N2 to remove weakly adsorbed NOx. Once the NOx concentration in the gas phase decreased to 0, 60 ppm of SO2 in 4% O2 and N2 was introduced to the reactor and the desorbed NOx species were measured. During the whole test, the temperature was kept constant at 200 °C. NO oxidation tests were made under the aforementioned conditions, without SO2 in the feed stream and at constant heating rate of 5 °C/min in the temperature range 150-600 °C. The NO oxidation performance of the fresh catalyst was compared to that of the regenerated LaCoO3-R.

Results and discussion Characterization results.

The XR-diffractograms of the samples LaCoO3, LaCoO3-S and LaCoO3-R5 are presented in Figure 1. In all the samples, only the LaCoO3 perovskite phase is detected without any Co3O4 or La2O3 phases, indicating good crystallization of the sample and the perovskite formation. The cell parameters of the rhombohedral perovskite structure (a = 5.441( 5) and c = 13.10( 5)) are not significantly affected by the treatments. The degree of crystallinity is the same with all the samples, as even after 5 regeneration cycles the normalized peak intensities are the same. The value of crystallite size as calculated by the Williamson-Hall method is 41 nm for the parent LaCoO3 sample, decreases to 32 nm for LaCoO3-S and stabilizes to 28 nm for regenerated samples from LaCoO3-R1 to LaCoO3-R5. The main diffraction peaks of La2(SO4)3, Co2O3, orthorhombic La2O3 and cubic La2O3 (expected at the 2θ values 28.33°, 36.56°, 29.92° and 33.19° respectively) were not detected by XRD (25-30° magnified in Figure 1).

Fig. 1 Powder XRD diffractograms of the LaCoO3, LaCoO3-S and LaCoO3-R5 catalysts.

Conversely several reports, 15,16,20 show the presence of peaks of La2(SO4)3 as a result of SO2 poisoning. The major difference is that for those, the deactivation by SO2 was made at much higher temperature, well above 500 °C, and under different reaction conditions. At high temperature, changes in the perovskite structure can occur more readily and sulphates can diffuse into the bulk by forming a mixed phase. Here the deactivation proceeded at 300 °C (lower temperature) through a different mechanism and, as will be discussed later on, sulphates formed only at the surface, without affecting the crystalline structure of the perovskite. In fact, no changes in both the symmetry and lattice parameters were observed for LaCoO3, LaCoO3-S and LaCoO3-R5. As the total quantity of Sulphur was low and dispersed uniformly on the surface in a nm thin layer (vide infra), the sulphate containing phases could not be detected by XRD. The decrease of crystallite size, as observed by XRD linewidth, is not contradictory with the stability of grain size as observed by FE-SEM. As the decrease in crystallite size is mainly observed after the first catalytic use of the material, it seems likely that the largest grains have been fractured by thermal stress without physical separation of the formed sub-grains, hence with no changes of grain morphology. The particles had a globular shape and were interlinked, forming regular intergranular macropores. The FE-SEM analysis (Figure S1) showed powders with a morphology typical of perovskites synthesized by the citrate complexation method. Both the LaCoO3-S and the LaCoO3-R5 catalysts presented no significant difference in the morphology as compared to the fresh catalyst (Figure S1). No significant difference in the BET specific surface area was observed as well, the corresponding values being 12.6, 12.5, 9.1 m2/g for the LaCoO3, LaCoO3-S and LaCoO3-R5 catalysts, respectively. The slight decrease of specific surface area could be attributed to the filling of the some intergranular spaces by smaller particles (most likely La(OH)3) that formed during the dissolution process. Conversely, significant decrease of the specific surface area was reported after deactivation by SO2 albeit in different reaction conditions, 15,16,20 i.e. when the deactivation was made at high temperature (500-600 °C) for prolonged periods with catalysts calcined at lower temperature than in this study. In those cases, the decrease in the specific surface area could be attributable, at least partially, to thermal sintering during sulphation. In this work, instead, the calcination temperature was 700 °C and the sulphation temperature 300 °C and thus, the specific surface area was stabilized during the calcination and no significant decrease in the surface area was observed due to thermal effect, nor due to sulphation. The total consumption of H2 during H2-TPR was 6.2 mmol/g for both the parent LaCoO3 and the regenerated LaCoO3-R5 (Figure 2). This amount corresponds to the stochiometric reduction of Co3+ to metallic Co via the reduction steps: Co3+ → Co2+ → Co0.21 It is generally accepted that the reduction of LaCoO3 perovskites occurs along the reactions R1-2. This can explain the occurrence of two clearly separated reduction steps, at variance with the reduction of Co3O4 that has only a single peak in the low temperature region. 22 The reduction peak between 200-450 °C corresponds to the reduction of Co3+ to Co2+ (reaction R1). 21 This first reduction step can be separated into several components, showing two distinct reduction temperatures at 325 and 400 °C. This is most likely due to the inhomogeneity and the presence of different types of Co3+ and O2-species in the sample. The second peak between 550-650 °C is due to the reduction of Co2+ to metallic Co0 with the consequent loss of the perovskite crystalline phase and the formation of Co/La2O3 system. Indeed, as expected, the H2 consumption corresponding to the second peak of LaCoO3 and LaCoO3-R5 TPR profiles is twice the H2 consumption of first peak. In contrast, the LaCoO3-S sample did not show any H2 consumption at ow temperature, in that the first peak was absent and reduction started only at 400°C meaning that the Co3+ to Co2+ reduction is deactivated.

Fig. 2 a) H2 consumption during H2-TPR, b) H2S and H2O evolution during the reduction of LaCoO3-S.

The adsorbed sulphates had to be removed first by reducing them to H2S (Figure 2b), before both the lattice and surface oxygen of perovskite could be accessible for the subsequent reduction with H2. Interestingly, not all the sulphates had to be removed before the reduction could start, as the H2S and the H2 consumption initiated concurrently. The availability of redox sites for H2 reduction was dependent on the surface coverage and as more of the sulphates were removed, the H2 consumption increased and full reduction was achieved sooner, as shown in Figure 2b. At 600 °C only 50% of the H2S was released, while most of the catalyst was reduced. This provides evidence that the redox sites are blocked by strongly bound sulphates and the reactive oxygen species active for NO oxidation are inactivated. Only after the surface sulphate removal the reduction proceeds. Figure 3 reports the XP-spectra concerning LaCoO3, LaCoO3-S, and LaCoO3-R5 samples. The O 1s XP spectra shows a marked increase in the poisoned sample of the peak at 532 eV which corresponds to the characteristic electronic state of oxygen in sulphate form, originating from the SO4 2-oxygen. 15,[START_REF] Briggs | Handbook of X-ray Photoelectron Spectroscopy[END_REF] Further evidence of surface sulphates comes from the S2p peak as the band centred at 169 eV is a clear indicator for S in the SO4 2-(i.e. S 6+ ) form. [START_REF] Briggs | Handbook of X-ray Photoelectron Spectroscopy[END_REF] The XPS analysis in the Co 2p binding energy region indicates that cobalt is present as Co 3+ as its characteristic peaks are identified at 780 eV and 795 eV. [START_REF] Briggs | Handbook of X-ray Photoelectron Spectroscopy[END_REF] The lack of satellite peaks at 785 eV and at 803 eV allows excluding the occurrence of Co 2+ species in the samples, in agreement with the H 2-TPR results. The XPS analysis of the poisoned sample does not indicate any change in the Co XP spectra and the characteristic peak assignable to cobalt sulphate, expected at 784 eV, is missing. [START_REF] Briggs | Handbook of X-ray Photoelectron Spectroscopy[END_REF] The La 3d XP spectra, in contrast, showed a clear transformation in the poisoned sample since La in the sulphate form was also detected besides the characteristic spectra of the oxide form found in the perovskite. The multiplet splitting in the La 3d3/2 and 3d5/2 regions of the LaCoO3 presented a ΔE of 4.1 and 5.1 eV which is characteristic of La in the oxide form.21,23 After poisoning, the resulting spectra was a combination of the oxide and sulphate form which features a lower ΔE of 3.5 eV. The combination of these two forms were difficult to deconvolute unambiguously as the characteristic positions are only slightly shifted (difference of 0.15 eV) and significantly overlapping. Sputtering with an Ar+ ion gun was used to remove ca. 30 nm surface layer from the LaCoO3-S sample. The XP spectra taken after sputtering showed the same features of the fresh sample (not shown) and the absence of S, confirming that SO2 poisoning mainly affected the surface rather than the bulk after the deactivation was performed at 300 °C. The La/Co and S/La atomic ratios of the samples in the bulk (determined by EDS) and at the surface (XPS) are compared in Table 1. The surface S/La ratio increases to 0.4 and, correspondingly, the La/Co ratio also increases, confirming the formation of lanthanum sulphate. This likely leads to the modification of the perovskite structure due to the reaction of La with S. The reported chemical analysis allows estimating ca. 1 wt% of sulphur in the bulk. However, the surface S/La ratio equals 0.4, i.e. a much lower than the expected 1.5 ratio expected for the complete transformation into La2(SO4)3.

Table 1 XPS and EDS analysis of the surface and bulk characteristics of the presented samples.

The difference between the bulk and surface sulphur contents point out that the sulphate formation is likely restricted to few surface monolayers (~5 nm) at the surface when poisoning occurs at 300 °C. The poisoning is also accompanied by the enrichment of the surface by La relative to Co to La/Co 1.25 from 1.1. This provides further confirmation of the previous results that the sulphates are restricted to the surface and they are bound to the A and not the B site in the ABO3 perovskite. It should be noted that the high La/Co ratio (2.15) after sputtering is most likely the consequence of the preferential ejection of the lighter Co (A = 58.93) with respect to the La (A = 138.91) during the ion sputtering, rather than a change in the chemical composition of the perovskite. Figure 4 reports the DRIFT spectra of the LaCoO3 and LaCoO3-S samples collected at room temperature after heating at 300, 400, 500, 550 and 600 °C to induce sulphate migration. The LaCoO3-S at 300 °C presents two sulphate peaks at 990 and 1383 cm-1 and a broad band centred around 1130 cm-1, likely a combination of a multitude of different vibration modes of amorphous sulphates differently bound to the surface, most commonly ascribed to bidentate sulphates.11,24 Fig. 4 DRIFT spectra of the LaCoO3 and LaCoO3-S and the thermal evolution of the surface and bulk sulfates. LaCoO3-S is heated to 400, 500, 550 and 600 °C with a hold time of 30 minutes each.

Detailed IR studies during sequential adsorption of NO followed by SO2 showed that the replacement of the nitrites/nitrates was highly dependent of the adsorption mode.11 Namely, at first the nitrates bound to multiple sites (meaning in bridging and bidentate configuration) were replaced while the monodentate nitrate form was the last to be substituted by SO2. This implies that the formation and dissociation of the NO2 is strongly hindered by the competitive adsorption. While a single free catalytic site is theoretically enough for the NO oxidation reaction to proceed, it is reasonable to expect that catalytic cycle is strongly inhibited when the fraction of the free sites (occupied by SO4 2-) decreases below a critical value. 25 Between 400 and 600 °C, the characteristic peaks associated with bulk sulphates at the wavenumbers 1069, 1097 and 1204 cm -1 are progressively distinguished. 11,20,[START_REF] Rosso | [END_REF] As the temperature increases, the sulphates become more mobile and migrate from the surface into the bulk of the perovskite and occupy energy states that are more favourable. This migration was also confirmed via XPS (see supplementary material Figure S2) and a uniform low concentration of S was observed through the whole depth profile on a LaCoO3-S that was calcined at 600 °C. As will be discussed later in more detail, this increase sulphate mobility is also evident during the soot-TPR (Figure 6). As the sulphates spillover, the soot consumes one of the oxygen in the sulphate and CO2 and SO2 were simultaneously observed in the gas phase in the corresponding temperature range of 500-600 °C.

Catalytic activity and regeneration

The NO oxidation in the presence of the LaCoO3 catalyst (Figure 5) initiated as low as 200 °C and rapidly increased, reaching very high NO2/NOx ratio (=0.8) and the thermodynamic equilibrium already at 300 °C, even at the high flowrates used in the study. This highlights the potential of LaCoO3 as a low-cost alternative to PGM-based DOC and cDPF. The catalyst was however very sensitive to SO2 (vide infra in Figure 7) and fully deactivated within 90 min. The deactivation was irreversible, and no NO oxidation activity could be recovered after turning off the SO2 flow. The SO2 concentration used in this study was higher than that usually found in the low-sulphur diesel exhausts (5 ppm vs 60 ppm). However, considering that the other parameters such as temperature, space velocity, etc. are similar, this procedure can be reasonably considered an accelerated deactivation giving rise to a calculated SO2 uptake corresponding to ca. 15.000 km of vehicle usage. By considering that a total amount of 6 mmol SO2/gcat passed during 90 minutes of test and we assumed that a vehicle has a fuel consumption of 6 kg/100 km with 10 ppm of S in the fuel (Euro 5 fuel upper limit, EN 590:2009) and a total catalyst load of 200 g is present in the aftertreatment system, the amount of SO2 equivalent to the laboratory test would be reached after ca. 15,000 km. This would imply that during its lifetime the DOC, cDPF etc. would have to be regenerated many times to recover its initial performance. Here, the first regeneration method was washing the catalyst with distilled water. The sulphates, present in small amount and only at the surface, can be easily dissolved and the catalyst activity is fully recovered. This method has been suggested before for the treatment of sulphur poisoned catalysts, including industrial SCR systems. [START_REF] Rosso | [END_REF][26][27][28] Even after 5 regeneration cycles, no significant decrease in activity was observed. It should be noted that the same method could be not appropriate for the PGM-based catalysts, as the active component is expensive, and present only at very low (< 1%) amount and could be easily washed away.

Fig. 5 NO oxidation activity of the fresh and regenerated LaCoO3 samples. Reaction conditions: 200 mg of catalyst, 600 mL min-1 flow rate of 500 ppm NO, 4% O2 in N2, 5 °C min -1 heating rate.

The sulphur poisoned catalyst can be regenerated in-situ either by thermal desorption, to which temperatures above 900 °C are required, or at lower temperatures, however above 600 °C, if reducing atmosphere is used (Figure 2 and6). The extremely high temperatures required for thermal regeneration can irreversibly damage the catalyst, which makes reducing treatment preferable and it is used for the Pt-Ba based LNT applications. Typical reductants for LNT are H2, NH3 and C3H6, with a corresponding increase of the regeneration temperature. Such a procedure not only requires large amount of fuel to be injected during the desulphurization, but also irreversibly damages the catalyst which is exposed to high temperature and consequent structural changes. 4,9,[START_REF] Mccarthy | SAE Technical Papers[END_REF][START_REF] Lott | [END_REF] To avoid the high fuel penalties associated with the regeneration by the injection of external reductant, soot was demonstrated here, for the first time, to be an efficient SO2 regeneration medium. From the practical point of view, soot is an interesting alternative as it is a pollutant in the diesel exhaust and abundantly available on the cDPF. From the mechanistic point of view, soot is an interesting reacting species as, unlike most heterogeneous reactions which involve solid-gas interface, the soot oxidation involves solid-solid contact. For this reason, the soot-TPR is a good indicator for the differentiation of the highly reactive surface and lattice oxygen and the oxygen spillover rate. In Figure 6, the evolution of the different products during soot-TPR is shown. In contrast to the H2-TPR, where bound sulphur is reduced to H2S before being released in the gas phase, in the case of soot-TPR the bond between SO4 2-and LaCoO3-S is broken by releasing the more reactive oxygen in the soot oxidation. The reaction products are indeed SO2 and CO2, whereas almost no CO could be detected, despite oxygen free atmosphere, indicating oxygen spillover (reactions R5-9), in the temperature range between 500-600 °C .The increased sulphate mobility and spillover is also clearly observed during the DRIFTS analysis as the sulphate species rapidly change from surface to bulk sulphate at this temperature (figure 4). The same mechanism occurs during the thermal decomposition as, without soot to accept the active oxygen species, the latter are released first by increasing the temperature before the desorption of SO2, i.e. it is necessary that strongly bound sulphates decompose to sulphites before the SO2 desorption. However, during thermal desorption there is no nearby oxygen acceptor and sulphates decomposition requires more energy. As with H2-TPR, the lattice oxygen species are not available until the sulphur species are removed from the surface. This is evident since with the LaCoO3 catalyst soot oxidation was started at lower temperatures, whereas on LaCoO3-S significant soot oxidation is observed right after SO2 is released (Figure 6). As almost all the bound sulphur is released below 600 °C during soot-TPR, the required regeneration temperature is lower than that of H2 regeneration (see figure 2) as well as of thermal decomposition (above 900 °C). At the same time, the complete reduction and the consequent destruction of the perovskite structure is avoided. The reduction of LaCoO3 and LaCoO3-S by soot (R5-R7) and the thermal decomposition and desorption of sulphates (R8-R9) occur according to the following reactions: The temperature required for the soot-mediated sulphate removal is higher (> 500 °C from R5) than that typically encountered in the diesel engine exhaust during normal operation of the diesel engine. However, both the soot buildup and SO2 poisoning have a complementary tendency over the cDPF. In fact, during normal operation the produced NO2 oxidizes the soot continuously thus enabling passive regeneration of the filter. As the catalyst is poisoned by SO2, the NO2 production falls down and thereby the soot builds up on the catalysed filter, that starts to behave as a noncatalysed filter. Once the backpressure is high, fuel injection is required to achieve high temperature and to burn off the accumulated soot. The soot-assisted regeneration is not expected to operate passively, but it could enable lower regeneration temperature with lower amount of additional fuel required. The last phenomenon explored here is not regeneration, but deactivation prevention (Figure 7). The LaCoO3 sample was physically mixed with small amounts of Ca(OH)2, an alkaline compound apt to SO2 capture. Increasing the basicity of the perovskite by doping during synthesis with Sr was shown to increase its resistance to SO2 poisoning, by capturing the sulphates during the poisoning. The approach used here was slightly different, in the sense that the basic compound was added post synthesis. Three configurations were compared, whereby the contact between the catalyst and adsorbent was varied. The LaCoO3 mixed with Ca(OH)2 both in tight contact and in dual-layer enhanced the resistance to poisoning, however the best performance was obtained by the loose contact configuration, confirming that Ca(OH)2 was more efficient in capturing SO3 than SO2. The poisoning was also repeated at 500 °C and the same trends were observed. The adsorbent was also effective at a higher temperature, the only difference being that at 500 °C a longer time was required to reach complete deactivation of the catalyst with respect to poisoning at 300 °C (Fig. S3 †).

When SO2 was in contact with the (LaCoO3 + Ca(OH)2) physical mixture a temporary evolution of NO2 was observed, as SO3 substituted the previously adsorbed NO2 according to reactions (R9)-(R11). The initial deactivation rate was fast since Ca(OH)2 mainly adsorbed the oxidized form of Sox ((R7)-(R9)). After the initial deactivation stage, the catalyst maintained a stable ca. 50% initial activity for a prolonged time. Even after 6 hours of poisoning, a further decrease in the activity was negligible. This method could offer an inexpensive and simple method for the delay and mitigation of deactivation. The two-phase competitive deactivation was demonstrated more clearly (Fig. 7b) on pure LaCoO3, which was saturated with NOx before switching off the NOx flow and turning on SO2. A clear evolution of NO2 was observed upon SO2 introduction, as the previously adsorbed NO2 was being replaced by the SO3 species having higher affinity during the competitive adsorption for the same catalytic active sites. The timespan of this substitution, ca. 20 minutes, corresponded to the timespan of the initial fast deactivation phase.

Conclusions

The deactivation of LaCoO3 by SO2 was investigated under reaction conditions relevant to diesel aftertreatment systems. Although LaCoO3 had excellent NO oxidation activity, it rapidly deactivated in the presence of SO2. Investigation of the deactivated sample by XPS showed that sulphur existed as sulphate species only at the surface, bound to lanthanum rather than to cobalt. As confirmed by DRIFTS and soot-TPR, sulphate mobility and diffusion into the bulk of the perovskite or spillover to soot started only above 500 °C and completed at 600 °C. The bound sulphates blocked gradually the active sites required for NO oxidation. After the initial fast deactivation, which can be ascribed to active site blocking, a second (slower) stage followed, during which multilayered lanthanum sulphates formed at the surface of the catalyst following the deactivation scheme proposed in Fig. 8. The reported activity tests and characterization of the catalyst suggest a two-stage deactivation mechanism: a fast (initial) stage where deactivation occurs by competitive adsorption and chemical blocking of the active sites by sulphates, followed by a second (slower) stage where multilayer lanthanum sulphate forms on the catalyst surface physically blocking the sites. 18,[START_REF] Lott | [END_REF] Reactivity studies showed that the basic and redox sites on the catalyst were blocked by SO4 2-, and for them to be reactivated, the strongly bound sulphur species had to be removed. Washing with water effectively removed the surface sulphates and the reactivated catalyst regained nearly the initial activity, even after 5 poisoning/regeneration cycles. Mixing small amounts of Ca(OH)2 as an adsorbent delayed the complete deactivation and the catalyst retained nearly half of its initial activity for extended period. This method proved, however, ineffective in completely preventing the initial "fast" stage of the deactivation. Finally, soot was explored as a potential regenerating agent, since one of the main potential uses of LaCoO3 would be in catalysed diesel particulate filters. The soot acted as an oxygen acceptor from the surface sulphate and effectively released sulphur as SO2 below 600 °C, a temperature lower than that required for H2 mediated desulphurization.

(R1) 2

 2 LaCoO3 + H2 → 2 LaCoO2.5 + H2O R1 (200-420 °C) (R2) 2 LaCoO2.5 + 2 H2 → 2Co + La2O3 + 2 H2O R2 (500-650 °C) (R3) LaCoO3-xSO3 + 4xH2 → LaCoO3 + H2O + xH2S R3 (500-650 °C)

Fig. 3

 3 Fig. 3 XPS spectra of a) O 1s, b) S 2p, c) Co 2p and d) La 3d band of the fresh, poisoned and regenerated by washing samples.

Fig. 6

 6 Soot-TPR and TPD of the LaCoO3 and LaCoO3-S samples. Reaction conditions: 60 mg of catalyst, 60 mL min-1 flow rate of Ar, 5 °C min-1 heating rate. Note that the different product species are not normalized.

(

  R4) LaCoO3-SO3 + C → LaCoO3 + SO2 + CO2 R5 (500-600 °C) (R5) 4 LaCoO3 + C → 4 LaCoO2.5 + CO2 R6 (550-700 °C) (R6) 2 LaCoO2.5 + C → Co/La2O3 + CO2 R7 (600-800 °C) (R7) LaCoO3-SO3 → LaCoO3-SO2 + O2 R8 (700-800 °C) (R8) LaCoO3-SO2 → LaCoO3 + SO2 R9 (850-1000 °C)

Fig. 7 a

 7 ) Deactivation of LaCoO3 as pure and mixed with Ca)OH)2. Reaction conditions: 200 mg catalyst, 100 mg CaIJOH)2, 600 mL min -1 500 ppm NO, 4% O2, in N2 and 60 ppm SO2 when added, T = 300 °C. b) The competitive adsorption of SO2 on LaCoO3 pre-saturated with NOx, reaction conditions same as before, T = 200 °C.

(

  R9) Ca(OH)2 + SO3 → CaSO4 + H2O (R10) Ca(OH)2 + 2NO2 → Ca(NO3)2 + H2O (R11) 2Ca(NO3)2 + 2SO3 → 2CaSO4 + 5NO2

Acknowledgements

This work was funded through a SINCHEM Grant. SINCHEM is a Joint Doctorate programme selected under the Erasmus Mundus Action 1 Programme (FPA 2013-0037).