Jacques Fages
email: jacques.fages@mines-albi.fr

Elisabeth Rodier

Jean-Jacques Letourneau

Martial Sauceau

Spiro D Alexandratos

Polypropylene Grafting in Supercritical Carbon Dioxide

Polypropylene (PP), one of the most widely low-cost commodity polymers, has a hydrophobic nature which prevents its use from a large variety of applications. Functionalisation, through free radical grafting of polar vinyl monomers offers an effective route towards higher added value applications especially for environmental remediation, wastewater treatment, and hydrometallurgy.

Classic processes for the modification of polypropylene by polar monomers includes reactive extrusion in the molten state and chemical reaction in the solid-state as well as in liquid solvents. Recently, the use of supercritical carbon dioxide (Sc-CO 2 ) has appeared as an innovative and interesting medium because of its environmentally friendly characteristics and its properties of sorption, swelling and foaming of polymers.

In this study, PP was put in an autoclave in different forms: pellets, fibres or powders. Vinylbenzyl chloride (VBC) was added along with benzoyl peroxide as initiator of the polymerisation. The autoclave was filled with CO 2 , pressurised and let for a certain amount of time under controlled pressure and temperature. After opening, the swollen polymer was washed with acetone, analysed by infra-red spectroscopy and the chlorine content was determined. When there is sufficient chlorine the samples were used for further modification with a phosphate ligand in order to perform metal ions complexation from water solutions.

Microphotographs as well as infra-red spectra show clearly that effective grafting with covalent binding is obtained under sc-CO 2 conditions not only at the surface but also into the pellets. It also appears that the reaction is more complete when polypropylene has previously been pulverised. This may come from a larger surface area on one hand and a different crystalline ratio on the other hand.

A two-step process, one aimed at the polymer swelling and the second aimed at the grafting has been successfully compared to the single step process. The influence of several operating parameters was tested: duration of the experiments, pressure and temperature, peroxide content and VBC/PP ratio.

After reflux treatment with triethyl phosphite, the best result achieved was a phosphorus elemental analysis of 1.25 mmol/g. This value shows that a high degree of polymerization has been reached and that the polyVBC is grafted on the PP chains, rather than coated on the surface.

The next study will deal with the capacity of such grafted PP samples to remove heavy metal ions from wastewater.

INTRODUCTION

Water is the one commodity on which life on Earth most depends yet it is one that has been taken for granted and used as a waste repository throughout our history. With the advent of the Industrial Revolution, pollution of our waterways has quickened to the extent that it is difficult to find any body of water that has not been impacted by human activity. This now includes the aquifers, rivers, and lakes on which much of humanity relies upon for its drinking water. Purification of water in the environment is thus one of the most pressing problems facing all countries today. The conventional techniques for removing contaminants from water are insufficiently selective for removing the lethal yet low amounts of contaminants such as lead, mercury, and cadmium metal ions. There is thus a need to develop methods to remove contaminants from water and to do so in a cost-effective manner.

This paper describes a preliminary study aimed at producing a low-cost and accessible process for producing a plastic filter to be used in the removal of toxic metal ions from water in the environment. The filter will be a modified polypropylene (PP) membrane. Polypropylene is one of the most widely low-cost commodity polymer. However, its hydrophobic nature prevents its use from a large variety of applications. To overcome these limitations free radical grafting of polar vinyl monomers offers an effective route towards higher added value applications. This functionalisation, opens a larger compatibility spectrum of the polyolefin with hydrophilic systems. The modification of PP with polar ligands will permit a new level of applicability to environmental remediation, wastewater treatment, and hydrometallurgy. In these applications, polymers are often applied as beads (ionexchange or chelating resins) requiring a flow of the aqueous stream through a column. The modification of PP will now permit the preparation of a wide range of ion-selective membranes for applications in which flow through membranes is preferred over flow through a column of beads.

Classic processes for the modification of polypropylene by polar monomers includes reactive extrusion in the molten state and chemical reaction in the solid-state as well as in liquid solvents. Recently, the use of supercritical carbon dioxide (Sc-CO 2 ) has appeared as an innovative and interesting medium because of its environmentally friendly characteristics and its properties of sorption, swelling and foaming of polymers. This paper describes a method which utilizes supercritical CO 2 in the modification of polypropylene and then further modify this polymer with ion-selective ligands for application to environmental remediation.

MATERIALS AND METHODS

The experiments were carried out in a multifunctional high-pressure pilot plant (Separex Champigneulles, France). A flowsheet of the apparatus is shown on figure 1. Carbon dioxide is cooled and stored in a liquid CO 2 storage tank (2). It is put in motion and pressurized by a membrane pump (Lewa, Germany) (3). In this study, PP was put in a 500 mL autoclave (5) in different forms: pellets, fibres or powders. Vinylbenzyl chloride (VBC) was added along with benzoyl peroxide as initiator of the VBC polymerisation. The autoclave was then filled with CO 2 , which was previously heated by passing through a heat exchanger (4) to become supercritical until the desired pressure was reached. The pressurised autoclave was let for a certain amount of time under controlled pressure and temperature.

Figure 1 -Experimental set-up for the polymer grafting experiments under supercritical carbon dioxide

After opening, the swollen polymer was washed with acetone, analysed by FTIR spectroscopy and the chlorine content was determined. When there is sufficient chlorine the samples were used for further modification with a phosphate ligand (triethyl phosphite) in order to perform metal ions complexation from water solutions. Scanning Electron Microscopy (SEM) was also used to visualise the processed polymer.

RESULTS

SEM microphotographs as well as FTIR spectra show clearly that effective grafting with covalent binding is obtained under sc-CO 2 conditions not only at the surface of PP pellets but also into the pellets.

A two-step process, one aimed at the PP swelling and the second aimed at the grafting has been successfully compared to the single step process. In a typical experiment using this approach, polypropylene (PP) pellets (2 mm diameter) were added into a reactor with a volume of 500 mL along with vinylbenzyl chloride (VBC, 10 mL per 2 g PP) and benzoyl peroxide (BPO, 1% based on monomer weight), supercritical CO 2 (scCO 2 ) was introduced at an initial pressure of 12 MPa and contacted with the reactants in two steps:

-2 h at 35°C followed by -18 h at 80°C. The reactor was then cooled, the pellets washed extensively with acetone, and dried. The extent of grafting was followed by ATR (the band at 1265 cm -1 is due to C-Cl).

Figure 2 shows the spectrum obtained.

Figure 2. Polypropylene fiber processed with sc-CO2

The influence of several operating parameters was tested: duration of the experiments, pressure and temperature, peroxide content and VBC/PP ratio. This series of experiments with varying conditions proved the feasibility of the modification.

Most importantly, the polyVBC was permanently bound as shown by converting the -CH 2 Cl moieties into phosphonic acid -CH 2 P(O)(OH) 2 by reflux treatment with triethyl phosphite. On figure 3 below, the ATR spectrum shows the broad region around 1103 cm -1 which is representative of the phosphonic acid moiety. The phosphorus capacity of 1.25 mmol/g is indicative of a high level of grafting. This value is quite significant, indicating that the degree of polymerization is high, the polyVBC is grafted on the PP chains, rather than coated on the surface. It also appeared that the reaction is more complete when the PP has previously been pulverised. This may come from a larger surface area on one hand and a different crystallinity ratio on the other hand.

CONCLUSION AND PERSPECTIVES

These first promising results led us to define what could be the next experiments to achieve. Our specific objective is now to determine the conditions that permit a high degree of modification of polypropylene (PP) with vinylbenzyl chloride (VBC) using supercritical CO 2 as the solvent and benzoyl peroxide (BPO) as the initiator. A unique feature of this research will be the preparation of membranes with higher degrees of modification than previously achieved with BPO by focusing on the swelling time with scCO 2 , its initial pressure, and the ratio of VBC to scCO 2 . The most novel aspect of this research is that, once polypropylene is successfully grafted with polyVBC, the polymer chains will be modified with ion-selective ligands using chemistry that we have established from research on polymer beads, and then the membranes utilized for the detoxification of environmentally critical bodies of water such as sources of drinking water.

The general objective of this study is to prepare ion-selective polypropylene membranes in a costeffective manner that can be used for the removal of toxic metal ions from rivers, groundwater, and other sources of drinking water.

Figure 3 .

 3 Figure 3. Polypropylene fiber modified with phosphonic acid ligands: phosphorus capacity of 1.25 mmol/g

ACKNOWLEDGMENT

We gratefully acknowledge the Fulbright Scholar Program administered by the Council for International Exchange of Scholars and the Commission Franco-Américaine for support of one of us (SA) while at l'Ecole des Mines d'Albi.


Maria Dolores Fernandez-Garcia

Romain Volle

Marie-Line Joffret

Serge Alain Sadeuh-Mba

Ionela Gouandjika-Vasilache

Ousmane Kebe

Michael R Wiley

Manasi Majumdar

Etienne Simon-Loriere

Anavaj Sakuntabhai

Marie-Line Joffret

Gustavo Palacios

Javier Martin

Francis Delpeyroux

Kader Ndiaye

Maël Bessaud

M D Fernandez-Garcia

M.-L Joffret

Genetic Characterization of Enterovirus A71 Circulating in Africa

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E nterovirus A71 (EV-A71; species Enterovirus A, genus Enterovirus, family Picornaviridae) is a common etiologic agent of hand, foot and mouth disease in young children. In addition, EV-A71 has been associated with severe and sometimes fatal neurologic diseases, including aseptic meningitis, encephalitis, and poliomyelitis-like acute flaccid paralysis (AFP) [START_REF] Solomon | Virology, epidemiology, pathogenesis, and control of enterovirus 71[END_REF][START_REF] Chang | The current status of the disease caused by enterovirus 71 infections: Epidemiology, pathogenesis, molecular epidemiology, and vaccine development[END_REF].

EV-A71 is classified into 7 genogroups (A-G). Genogroup A includes the prototype strain BrCr that was isolated in the United States in 1969 [START_REF] Solomon | Virology, epidemiology, pathogenesis, and control of enterovirus 71[END_REF][START_REF] Chang | The current status of the disease caused by enterovirus 71 infections: Epidemiology, pathogenesis, molecular epidemiology, and vaccine development[END_REF]. Most EV-A71 isolates belong to genogroups B or C, which are each further divided into subgenogroups [START_REF] Solomon | Virology, epidemiology, pathogenesis, and control of enterovirus 71[END_REF][START_REF] Chang | The current status of the disease caused by enterovirus 71 infections: Epidemiology, pathogenesis, molecular epidemiology, and vaccine development[END_REF]. Subgenogroups B4, B5, and C4 are mainly restricted to countries in Asia, whereas C1 and C2 circulate primarily in Europe and the Asia-Pacific region [START_REF] Solomon | Virology, epidemiology, pathogenesis, and control of enterovirus 71[END_REF]. Genogroup D and the newly proposed genogroup G appear to be indigenous to India, whereas genogroups E and F were recently discovered in Africa and Madagascar, respectively [START_REF] Bessaud | Molecular comparison and evolutionary analyses of VP1 nucleotide sequences of new African human enterovirus 71 isolates reveal a wide genetic diversity[END_REF].

Although EV-A71 has been reported in many parts of the world, its epidemiology remains largely unexplored in Africa. An EV-A71 outbreak was documented in 2000 in Kenya, where HIV-infected orphans were infected by EV-A71 genogroup C (4). Several AFP cases have been associated with EV-A71 infection during 2000-2013 throughout Africa: in Democratic Republic of the Congo (5) (2000, n = 1); Nigeria ( 6 [START_REF] Faleye | Direct detection and identification of enteroviruses from faeces of healthy Nigerian children using a cell-culture independent RT-seminested PCR assay[END_REF]. Molecular identification of all these isolates was based only on the analysis of sequences of the viral protein (VP) 1 capsid protein region.

Recombination events may be associated with the emergence and global expansion of new groups of EV-A71 that have induced large outbreaks of hand, foot and mouth disease with high rates of illness and death [START_REF] Leitch | The association of recombination events in the founding and emergence of subgenogroup evolutionary lineages of human enterovirus 71[END_REF]. For EV-A71, genetic exchanges have been described both within a given genogroup and with other types of enterovirus A (EV-A), usually in nonstructural genome regions P2 and P3 [START_REF] Solomon | Virology, epidemiology, pathogenesis, and control of enterovirus 71[END_REF][START_REF] Leitch | The association of recombination events in the founding and emergence of subgenogroup evolutionary lineages of human enterovirus 71[END_REF][START_REF] Yoke-Fun | Phylogenetic evidence for inter-typic recombination in the emergence of human enterovirus 71 subgenotypes[END_REF]. However, before 2017, no complete genome sequence of EV-A71 detected in Africa has been reported, diminishing the power of such analysis. We examined the complete genome of most EV-A71 isolates reported to date in Africa to characterize the evolutionary mechanisms of genetic variability.

The Study

We sequenced the full genome of 8 EV-A71 isolates obtained from patients with AFP (Table ): isolates 14-157, 14-250, , and 15-355 from West Africa and isolates 08-041, 08-146, and 03-008 from Central Africa. We isolated and typed these isolates as previously described (7-10) and obtained nearly complete genomic sequences using degenerated primers [START_REF] Yoke-Fun | Phylogenetic evidence for inter-typic recombination in the emergence of human enterovirus 71 subgenotypes[END_REF] and additional primers designed for gene-walking (available on request) or unbiased sequencing methods [START_REF] Kugelman | US Army Medical Research Institute of Infectious Diseases; National Institutes of Health; Integrated Research Facility-Frederick Ebola Response Team 2014-2015[END_REF]. We determined the 5′-terminal sequences by means of a RACE kit (Roche, Munich, Germany). We deposited viral genomes in GenBank (accession numbers in Table ) and submitted sequence alignments under BioProject PRJNA422891. We aligned sequences using ClustalW software (http://www.clustal.org).

To investigate the genetic relationship between Africa and global EV-A71 isolates, we constructed subgenomic phylogenetic trees based on the P1, P2, and P3 regions of the genome (Figure 1). We identified viral isolates showing related sequences in 1 of these 3 regions by BLAST search (http://www.ncbi.nlm.nih.gov/BLAST) and included them in the corresponding datasets used for analyses. We completed these datasets with a representative global set of EV-A71 sequences available in GenBank and belonging to the different EV-A71 genogroups (https:///wwwnc.cdc.gov/ EID/article/24/4/17-1783-Techapp1.pdf). As expected, in the structural P1 region, the 8 isolates we studied clustered within their respective genogroups (C1, C2, and E), previ-ously established by VP1-based typing (Figure 1, panel A). In particular, the isolates of genogroup E consistently clustered together (bootstrap value 100%), confirming their belonging to the EV-A71 type and their divergence from the other isolates belonging to the common genogroups A, B, and C. Analysis of the nonstructural P2 and P3 genome regions were in agreement with these data. However, the genetic heterogeneity, <12%, observed among the complete genome of genogroup E sequences highly suggested that they have circulated and diverged for years in a large geographic area in Africa. The unique Africa EV-A71-C1 strain clustered with other C1 strains originating worldwide, regardless of which genome region we analyzed. In contrast, the nonstructural sequences of Africa EV-A71 isolates of subgenogroup C2 did not cluster with their non-Africa C2 counterparts or with any of the existing EV-A71 genogroups. The incongruent phylogenetic relationships of Africa C2 strains in the different regions of the genome suggested that recombination events have occurred during evolution. To examine further recombination events, we analyzed EV-A71-C2 study strains by similarity plot against potential parental genomes (Figure 2). This analysis showed that sequences 14-157, 14-250, and 15-355 had high similarity (>95%). By contrast, 13-365 diverged from the other C2 isolates around nt 5600 in the P3 region, suggesting a recombination breakpoint. The analysis showed high sequence similarity (>97%) between the studied EV-A71-C2 isolates and other subgenogroup C2 strains over the P1 capsid region. Conversely, in the noncapsid region, sequence similarity between Africa EV-A71-C2 isolates and classical subgenogroup C2 isolates (e.g., GenBank accession no. HQ647175) was much lower (66%-77%). This finding confirmed a recombination event of the Africa EV-A71 C2 lineage with an unknown enterovirus, the most likely breakpoint being located between nt 3596 and 3740, within the 2A gene. Sequence identity of EV-A71-C2 study strains with their closest related viruses (coxsackievirus A10 [CV-A10], CV-A5, EV-A120, and EV-A71 genogroup E strains) in the 3′ half of the genome was <87.7%.

Of note, we found much higher sequence identity with the full-genome sequence of CV-A14 isolate in our database, obtained in 2014 from a patient with AFP in Senegal [START_REF] Fernandez-Garcia | Identification and molecular characterization of non-polio enteroviruses from children with acute flaccid paralysis in West Africa, 2013-2014[END_REF]. This strain features a high similarity value (>97%) with the 3′ half of the genomes of EV-A71-C2 West Africa strains (Figure 2), indicating that their P3 regions share a recent common ancestor. Because these strains belong to 2 different types, this finding strongly suggests that genetic exchanges occurred through intertypic recombination. This result cannot be a result of cross-contamination during the sequencing process because the CV-A14 and EV-A71 isolates were sequenced on 2 different platforms. tensively circulating in Africa. We also suggest that the common ancestor of EV-A71-C2 strains in West Africa has undergone recombination with >1 EV-A circulating in Africa. Genogroup E and recombinant C2 appear to be indigenous to Africa; they have not yet been detected elsewhere. Further exploration of environmental or clinical samples using deep sequencing technology would be of interest to determine the extent of EV-A71 circulation in Africa in the absence of AFP cases. Systematic surveillance based on full-genome sequencing could also serve to monitor these viruses for potential recombinations and to study their role in the emergence of new EV-A71 variants in Africa.

Conclusions

  ) (2004, n = 1, genogroup E); Central African Republic (7) (2003, n = 1, genogroup E); Cameroon (8) (2008, n = 2, genogroup E); Niger (9) (2013, n = 1, genogroup E); and Senegal, Mauritania, and Guinea (9) (2013-2014, n = 3, subgenogroup C2). Four additional EV-A71 strains were obtained from captive gorillas in Cameroon during 2006-2008 (n = 2, genogroup E) (10) and from healthy children in Nigeria in 2014 (n = 2, genogroup E)

Figure 1 .

 1 Figure 1. Phylogenetic relationships of EV-A71 isolates from patients with acute flaccid paralysis in Africa based on 3 coding regions: A) P1, B) P2, and C) P3. Apart from the studied sequences, subgenomic datasets included their best nucleotide sequence matches identified by NCBI BLAST search (http://www.ncbi.nlm.nih.gov/BLAST) as well as representative sequences of different EV-A71 genogroups and subgenogroups originating worldwide. Trees were constructed from the nucleotide sequence alignment using MEGA 5.0 software (http:// megasoftware.net/) with the neighbor-joining method. Distances were computed using the Kimura 2-parameter model. The robustness of the nodes was tested by 1,000 bootstrap replications. Bootstrap support values >75 are shown in nodes and indicate a strong support for the tree topology. For clarity, CV-A10, CV-A5, and EV-A71 subgenogroups C3, C4, and C5 have been collapsed. Study strains are indicated by laboratory code, country of origin, and year of isolation; previously published strains are indicated by GenBank accession number, isolate code, country of origin, and year of isolation. Black triangles indicate EV-A71 strains from this study; black square indicates the CV-A14 strain from this study. Strains gathered in brackets belong to EV-A71 genogroups or subgenogroups; strains marked in blue color belong to other species of EV-A. Scale bars indicate nucleotide substitutions per site. CV, coxsackievirus; EV, enterovirus.

Figure 2 .

 2 Figure2. Identification of recombinant sequences in the genome of EV-A71 C2 isolates from patients with acute flaccid paralysis in Africa (14-157, 14-250, 13-365, 15-355) by similarity plot against potential parent genomes (CV-A14 strain 14-254; EV-A71 genogroup E strains 13-194, 08-146, and 03-008) and from GenBank (CV-A10, CV-A5, EV-A120). Similarity plot analysis was performed using SimPlot version 3.5.1 (http://sray.med.som.jhmi.edu/SCRoftware/simplot) on the basis of full-length genomes. For the analysis, we used a window of 600 nt moving in 20-nt steps. Approximate nt positions in the enterovirus genome are indicated. The enterovirus genetic map is shown in the top panel. We used the genome of EV-A71 study strain 14-157 as a query sequence. UTR, untranslated region.

Table .

 . Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 24, No. 4, April 2018 755 Description of enterovirus isolates from patients with acute flaccid paralysis in Africa that were sequenced for characterization of enterovirus A71*

			Patient age at			Genogroup or
	Strain (reference)	Country of isolation	diagnosis, y	Year	Virus	subgenogroup Genbank accession no.
	14-157 (9)	Senegal	3	2014	EV-A71	C2	MG672480
	14-250 (9)	Mauritania	1.6	2014	EV-A71	C2	MG672481
	13-365 (9)	Guinea	1.7	2013	EV-A71	C2	MG672479
	15-355 (this study)	Senegal	2.4	2015	EV-A71	C2	MG013988
	13-194 (9)	Niger	1.3	2013	EV-A71	E	MG672478
	03-008 (7)	Central African Republic	1.9	2003	EV-A71	E	LT719068
	08-146 (8)	Cameroon	2.6	2008	EV-A71	E	LT719066
	08-041 (8)	Cameroon	1.7	2008	EV-A71	C1	LT719067
	14-254 (15)	Senegal	3	2014	CV-A14	NA	MG672482

*NA, not available.

Acknowledgments

We thank Karla Prieto and Catherine Pratt, who assisted in obtaining nearly complete genomes of West Africa strains, and Joseph Chitty for analysis of the next-generation sequencing data. The next-generation sequencing equipment at Institut Pasteur of Dakar was provided by the Defense Biological Product Assurance Office under the Targeted Acquisition of Reference Materials Augmenting Capabilities Initiative. This work was supported by the IPD, the Pasteur Institute's Transverse Research Program PTR484, Actions Concertées Inter-Pasteuriennes A22-16, Fondation Total Grant S-CM15010-05B, Roux Howard Cantarini postdoctoral fellowship, and Grant Calmette and Yersin from the International Directorate of the Institut Pasteur.

About the Author

Dr. Fernandez-Garcia is a scientist with a PhD degree in virology and is involved in research and surveillance of enterovirus infections at Institut Pasteur of Dakar, Senegal. Her research interests include infectious disease epidemiology and public health microbiology.


Iman Abdallah

Nicolas Ratel-Ramond

C Magen

Béatrice Pécassou

Robin Cours

Alexandre Arnoult

Marc Respaud

Jean-François F Bobo

Gérard Benassayag

Etienne Snoeck

B Pecassou

N Biziere

Structural and magnetic properties of He+ irradiated Co2MnSi Heusler alloys

Keywords: Heusler Alloys, Structure, X-ray diffraction, HAADF-STEM

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

average magnetization of the alloy, which is due to D0 3 disorder and local defects induced by irradiation.

I. Introduction

In the last ten years, full Heusler compounds with general formula X 2 YZ have become a major topic in spintronics, especially for spin torque devices requiring low damping and high spin polarization. Among them, Co 2 MnSi (CMS) is a very promising candidate. It is predicted to be half metallic, with a Curie temperature well above the room temperature [1][2][3][4] and a very low Gilbert damping coefficient as compared to other ferromagnetic metals [5,6].

In all Heusler alloys, the magnetic properties and the atomic order are intimately related. For example, half metallicity has been predicted in CMS for the L2 1 or B2 structure only [7]. The L2 1 structure is the most ordered phase and corresponds to 8 body centered cubic (bcc) sub-lattices having the Co atoms at the corners of the bcc cells and the center sites occupied either by the Mn or Si atoms.

A random distribution between Mn and Si or between Mn and Co atoms corresponds to the B2 or D0 3 order respectively while a random distribution of the Co, Mn and Si atoms between the different atomic sites leads to the disordered A2 phase.

Ion irradiation with light ions is an efficient technique to improve the local chemical order in different magnetic alloys such as FePt [8] or more recently in Heusler alloy [9]. Indeed, Gaier et al.

demonstrated that He + ion irradiation at 30 keV increases the long range order parameter in CMS grown in the B2 phase. Therefore, ion irradiation appears as a very interesting complementary or alternative technique to high temperature annealing, incompatible with microelectronic processes.

Before this, further studies about the structural modification induced by irradiation are needed. In this work, we offer to study the structural modification in both the L2 1 and B2 order in Co 2 MnSi irradiated with 150 keV He + ions.

Several techniques can be used to characterize the crystal structure such as neutron diffraction, nuclear magnetic resonance, X-ray absorption and circular magnetic dichroism (XAS/XMCD), photoemission spectroscopy (HAXPES) or HAADF-STEM techniques [10][11][12][13]. However, X-ray diffraction is an accessible and commonly used technique for macroscopic characterization of the crystal order [14][15][16]. Quantitative information on the presence of the different phases can be extracted from the measurements of the intensity of different diffraction peaks. For example, superlattice (h,k,l) diffraction peaks for which with h, k and l are odd numbers (e.g. 111) only appear when the L2 1 and/or D0 3 phases are present while diffraction peaks for which h+k+l = 4n+2 (e.g. 002)

appear for L2 1 , D0 3 and B2 phases. Finally, the fundamental peaks whose h+k+l = 4n (e.g. 022)

appear for all crystal phases.

One of the major issues encountered in X-ray diffraction experiments to discriminate between the various ordered phases in CMS is that the Co and Mn scattering factors are very close at the Cu Kα edge, making it almost impossible to distinguish between D0 3 and L2 1 phases. This issue can however be overpassed using a Co-Kα source because, due to anomalous diffraction of the Co at the K-edge, the Co and Mn scattering factors becomes very different and Co and Mn atoms can be differentiated.

Thus, the different disorder parameters in CMS can be obtained combining diffraction measurements using Cu and Co Kα sources. The method, based on a model proposed by Niculescu et al. [17], has recently been applied by Takamura et al. [18] for the structural characterization of Co 2 FeSi. In this model α, β and γ are three disorder parameters. α corresponds to the number of Mn atoms located on Si sites and then represents the Mn/Si substitution per CMS unit. Similarly, β and γ corresponds to the number of Co atoms on Si and Mn sites respectively. Then, the structure factors for the different peak of interest are expressed as:

F 111 ∝ (1 -2α -β)(f Mn -f Si ) + (γ -β)(f Co -f Mn ) (1)
F 002 ∝ (1 -2β)(f Co -f Si ) + (1 -2γ)(f Co -f Mn ) (2)
F 022 ∝ 2f Co + f Mn + f Si (3)
where f Co , f Mn and f Si are the scattering factors calculated from Ref [START_REF]International Tables for X-ray Crystallography[END_REF]. We can see from eq. 1 and 2 that if f Co and f Mn are close, as for Cu Kα source, the intensity of the diffraction peaks I 111 (∞ │F 111 │ 2 ) and I 002 (∞│F 002 │ 2 ) are not sensitive to D0 3 order while it is for Co-Kα source in anomalous conditions.

I. Experimental details.

In this study CMS have been grown by magnetron sputtering on MgO (001) single crystals in a Plassys MPU 600 S ultrahigh vacuum (UHV) chamber. Details about the deposition conditions are presented in Ref [START_REF] Ortiz | [END_REF]. The thickness of the CMS layer is 42 +/-1 nm and a 10 nm MgO capping layer is deposited to avoid oxidation. This reference sample is then cut into four pieces, one as a reference and three for He + irradiation at 150 keV performed with a 200A2 Varian ion implanter. The irradiation is performed at room temperature and the fluences for the three samples are 1x10 15 , 5x10 15 and 1x10 16 ions per cm² respectively. The high kinetic energy of the ions prevents from implantation of He + in the CMS film as they stop several hundreds of nm deep in the substrate.

X-ray diffraction experiments were performed on a Bruker D8-Discover (Da-Vinci)

diffractometer equipped with a Cu Kα 1 source (λ=0.154 nm) to measure the (002) and (004) peaks while a Panalytical Empyrean diffractometer equipped with a Co Kα 1 source (λ=0.179 nm) has been used to measure the (111), ( 002) and (022) diffraction peaks. Note that the (004) and (022) peaks show similar structure factor and are both 'fundamental' peaks. Figure 1 presents examples of different diffraction peaks obtained either with Co or Cu Kα 1 sources.

II.

Results and discussion. The results for the deduced α, β, γ are presented in Figure 1.d. For all the samples, we observe that the β parameter, i.e. Co/Si disorder, remains almost constant (0.04 +/-0.02) whatever the ion fluence. Additionally α increases from 0.14 +/-0.01 to 0.22 +/-0.01 for the reference sample and the 10 16 irradiated one respectively. While we already observe a small increase of α for 1x10 15 ions/cm², there is a clear step at 5x10 15 . Similarly the γ parameter, i.e. Co/Mn disorder, increases significantly for fluences above 5x10 15 . For lower fluences, the Co-Mn substitution (γ) is not detectable within the uncertainty of the measurements, meaning it is less than 0.02.

Using these disorder parameters one can calculate the probability of presence of the different atoms on their original site considering the L2 1 phase as starting structure (see details in Ref 18). We found values of 98, 86 and 83 % for the Co, Mn and Si respectively for the reference sample. These probabilities fall down to 93, 71 and 75% at 10 16 ions/cm². Considering that α = 0.5 for full B2 order, we can estimate that our sample grows as mixture of L2 1 and B2 phases, with ratios of about 25% of B2 and 75% of L2 1 order (neglecting the small amount of Co/Si disorder).

We also observe structural modifications of the alloy for fluences above 5.10 15 /cm². The 002 and 004 diffraction peaks of the irradiated sample in Figure 1 are slight shifted toward low angle compared to the peak position of the reference sample, indicating an increase of the out-of-plane lattice parameter from 5.67 to 5.69 Ǻ. In contrary the (111) reflection remains at the same Bragg angle. As the (111) peak is related to the L2 1 and/or D0 3 phases only, we may assume that only regions of the thin film presenting the B2 order have their out-of-plane lattice parameter increasing with the irradiation.

While X-ray diffraction gives macroscopic information about the structural order, it does not explain the way the different atomic exchanges get organized in the material. For example, it is difficult to know if the B2 and D0 3 disorders are diluted in the initial matrix or if grains of a particular ordered phase grow from the L2 1 starting structure. To answer this question we performed HAADF-STEM experiments which provide information on the local ordering at the atomic scale.

Measurements have been performed at 300 kV on an FEI Titan 60-300 microscope, equipped with a spherical aberration corrector for the probe. Two lamellas have been prepared by FIB, one extracted from the reference sample and the other one prepared from the sample irradiated at 10 16 ions/cm².

HAADF-STEM studies were realized in the [-110] zone axis. In this orientation and for L2 1 order, the intensity of each atomic column, which increases with Z, corresponds to only one type of atoms. order. We also note that the difference of intensity between Co and Mn columns is weak as expected from the Co and Mn atomic numbers (Z Co = 27). Similarly to classical X-ray diffraction using a Cu Kα source, HAADF-STEM is not very sensitive to D0 3 disorder. In order to demonstrate that the L2 1 order is the main phase of the sample we performed a statistical analysis of maximum peak intensities of the HAADF-STEM images for the reference sample (Figure 2) and for the sample irradiated at 10 16 ions/cm² (Figure 3). For the first one, three distinct intensity distributions corresponding to the Co, Mn and Si atomic columns are observed (Figure 2.c). The values are normalized by the value of the Co intensity at the center of the Co distribution. The appearance of three different intensities in the HAADF-STEM image is in good agreement with the L2 1 order even if some spreading of the intensity distribution is observed. One source of spreading comes from the slight change of thickness across the lamella prepared for STEM experiment. This effect prevents any statistics over very large areas.

In some particular regions of the film other intensity distribution are observed, as the one reported in Figure 2.d which corresponds to statistical analysis performed in the black box in Figure 2.a. The inset in Figure 2.d shows that STEM intensities corresponding to the Mn and Si columns are very similar. Even if the statistical analysis is performed on a small number of atomic columns, it clearly shows that the STEM intensities corresponding to Mn and Si atomic columns converge to a single value, which could be associated to the appearance of the B2 order. This result indicates that very small grains with B2 order are distributed in the L2 1 matrix.

While the B2 order in the reference sample is only observed in small regions of similar size as the black box in Figure 2.a, it can be observed in more extended areas for a fluence of 10 16 ions/cm². This is shown in Figure 3.a. The B2 order can be observed either from the intensity profile of the different We also observe for the irradiated sample that the relative STEM intensity of the different atomic columns spread over slightly larger values as compared to the reference sample. The most probable explanation relies on the difference between the two prepared samples, especially the thicknesses which modify the absolute STEM intensity. Another possibility relies on local defects such as vacancies induced by the irradiation.

As already explained, it is very challenging to state about any Co/Mn exchange from HAADF-STEM experiments. However one can argue that if Co/Mn substitutions arise in the B2 phase, one should observe a spreading of the STEM intensity associated to the Mn columns that range between Si and Co ones. This is however not what is observed. Moreover, the relative intensity of the Mn columns compared to the Co ones in the L2 1 phase shows similar values (as for the reference sample).

Therefore we assume that the D0 3 order most probably arises in the L2 1 matrix.

Finally, we measured the magnetization of the four samples (Figure 4) with a QUANTUM design PPMS VSM magnetometer at 300 K. We clearly observe a decrease of the magnetization versus the ion fluence. As demonstrated in Ref [2] the B2 and L2 1 phase have the same magnetic moment (µ 0 Ms ≈ 1.25 T) while it decreases by 10% in the D0 3 phase. Therefore we argue the decrease of the magnetization can be accounted for the local Co-Mn exchange in the L2 1 matrix and vacancies induced by the irradiation.

III. Conclusions.

In conclusion we demonstrated that He + ion irradiation at 150 keV induces Mn/Si exchange and then favors the B2 structure at the cost of the L2 1 phase. However, for fluences above 5x10 15 ions/cm² , the out of plane lattice parameter of the B2 phase increase and might have an impact upon half metallicity. In addition, for this threshold value, Co/Mn substitution occurs in the L2 1 structure leading to a decreased magnetization and so half metallicity. Below this threshold, no significant improvement of L2 1 order has been observed. Therefore our study demonstrates that ion irradiation is an interesting alternative to annealing only for the B2 order as long as fluence is kept under a threshold value.

  According to eq 2 and 3, comparing the experimental I 002 /I 004 ratio to the calculated one allows the extraction of the β values. Then α and γ are deduced from the measurements of the I 002 /I 022 and I 111 /I 022 ratios.

Figure 1 .

 1 Figure 1. (color online) (002) (a) and (111) (b) diffraction peak at Co Kα 1 edge for reference (black) and 10 16 ions/cm² irradiated (red) samples. (c) (002) and (004) diffraction peaks at Cu Kα 1 edge. (d) Disorder parameters obtained from X-ray diffraction experiments.

Figure 2 .

 2 Figure 2.a shows an example of a HAADF-STEM image obtained on the reference sample. The insert shows a zoom on few atomic columns. Figure 2.b shows the intensity profiles of STEM image taken along different lines reported in Figure2.a. The profiles clearly shows the alternation of high and low peak intensity corresponding to Mn (Z = 25) and Si (Z = 14) columns, demonstrating the L2 1

Figure 2 .

 2 Figure 2. (color online) (a) HAADF-STEM image of CMS. The insert at top right is a zoom over 6x6 atomic columns. (b) Intensity profile of the lines denoted by the colored arrows in (a). (c) Statistical analysis of intensity profile obtained from (a) on a region of 11 x 23 atomic columns. (d) statistical analysis of the area denoted by the black box in (a). In insert the intensity profile of the three central lines in the black box.

lines (Figure 3

 3 .b) or statistically over the selected area (Figure3.c). Similar values for the STEM intensity of the Mn and Si columns are measured while the intensity corresponding to the Co columns remains higher. On other part of the sample we still observe the L2 1 order (Figure3.d). We then suggest that ion irradiation induces a L2 1 to B2 transformation, which occurs around the initial B2 grains.

Figure 3 .

 3 Figure 3. (color online) (a) HAADF-STEM image of CMS irradiated at 10 16 ions/cm². The black boxes correspond to a zone with B2 or L2 1 character. (b) Intensity profile of the lines denoted by red and black lines in the B2 or L2 1 boxes in (a). Statistical analysis of the B2 (c) and L2 1 (d) regions in (a).

Figure 4 .

 4 Figure 4. (color online) Evolution of the CMS average magnetization at 300 K as a function of the He + fluence.

Acknowledgments

The authors thank LPCNO for X-ray diffraction facilities. This work has been supported by the French Agence Nationale pour la Recherche (ANR NASSICS 12-JS10-008 01) and the "French RENATECH network".