Magali Ader
email: ader@ipgp.jussieu.fr

Pierre Cartigny

Jean-Paul Boudou

Jae-Ho Oh

Eddy Petit

Marc Javoy

Nitrogen isotopic evolution of carbonaceous matter during metamorphism: Methodology and preliminary results

Keywords: Nitrogen isotopes, Metamorphism, Kerogen, Carbonaceous matter, Graphite

Analysis of nitrogen content and isotopic composition of sedimentary organic matter (atomic N / C ratio higher than 2%) are usually performed on bulk samples using C-H-N elemental analysis or sealed-tube combustion, neglecting the associated fixed ammonium or assuming that its δ 15 N is similar to that of the organic nitrogen. Numerous bulk sample δ 15 N data (taken as estimates for organic matter δ 15 N) have thus been published, improving our understanding of the past and present nitrogen cycles in the oceans or lakes and their relations with trophic structures and/or climate (e.g. Rau et al., 1987;[START_REF] Talbot | A high resolution palaeoclimatic record for the last solution 27,500 years in tropical West Africa from the carbon and nitrogen isotopic composition of lacustrine organic matter[END_REF][START_REF] Altabet | Sedimentary nitrogen isotopic ratio as a recorder for surface ocean nitrate utilization[END_REF][START_REF] Altabet | Climaterelated variations in denitrification in the Arabian Sea from sediment 15 N/ 14 N ratios[END_REF][START_REF] Caplan | Demise of a Devonian-Carboniferous carbonate ramp by eutrophication[END_REF][START_REF] Holmes | Reconstruction of past nutrient utilization in the eastern Angola basin based on sedimentary 15 N/ 14 N ratios[END_REF][START_REF] Schubert | Nitrogen and carbon isotopic composition of marine and terrestrial organic matter in Arctic Ocean sediments: implications for nutrient utilization and organic matter composition[END_REF][START_REF] Altabet | The effect of millennial-scale changes in Arabian Sea denitrification on atmospheric CO2[END_REF][START_REF] Ganeshram | Reduced nitrogen fixation in the glacial ocean inferred from changes in marine nitrogen and phosphorus inventories[END_REF]. However, with kerogen maturation and following carbonaceous matter (CM) metamorphism, a significant proportion of the organic nitrogen is converted into ammonium substituting for potassium in K-bearing minerals, such as clays, micas, K-feldspar, etc. [START_REF] Juster | NH4-bearing illite in very low grade metamorphic rocks associated with coal, northeastern Pennsylvania[END_REF]Daniels andAltaner, 1990, 1993;[START_REF] Sucha | Ammonium illite from anchimetamorphic shales associated with anthracite in the Zemplinicum of the western Carpathians[END_REF]. 8 15 N of this neo-formed mineral nitrogen can be different from its organic source [START_REF] Williams | Nitrogen isotope geochemistry of organic matter and minerals during diagenesis and hydrocarbon migration[END_REF]Ader et al., 1998b), so that organic nitrogen 8 15 N measurements should be performed on isolated carbonaceous matter and mineral ones on mineral separates (e.g. [START_REF] Teagle | The behavior of nitrogen and nitrogen isotopes during metamorphism and mineralization: evidence from the Otago and Alpine Schists, New Zealand[END_REF]. Due to the time-consuming HCl/HF attack necessary for this isolation step, and to the difficult 6 15 N determination for samples with low atomic N / C ratios, only few data on organic nitrogen content and 8 15 N are available in metamorphic rocks (Ader et al., 1998a;[START_REF] Beaumont | Nitrogen isotopic ratios of kerogens in Precambrian cherts: a record of the evolution of atmosphere chemistry?[END_REF]Jia and Kerrich, 2004a,b;[START_REF] Ueno | Carbon isotopes and petrography of kerogens in 3.5-Ga hydrothermal silica dikes in the North Pole area, Western Australia[END_REF][START_REF] Van Zuilen | Nitrogen and argon isotopic signatures in graphite from the 3.8-Ga-old Isua Supracrustal Belt, Southern West Greenland[END_REF] as well as in extraterrestrial material (e.g. [START_REF] Kerridge | Isotopic characterization of kerogen-like material in the Murchison carbonaceous chondrite[END_REF][START_REF] Sephton | Investigating the variations in carbon and nitrogen isotopes in carbonaceous chondrites[END_REF].

These data were obtained using different methods such as: sealed-tube combustion (Ader et al., 1998a;[START_REF] Beaumont | Nitrogen isotopic ratios of kerogens in Precambrian cherts: a record of the evolution of atmosphere chemistry?[END_REF], CHN analyser [START_REF] Ueno | Carbon isotopes and petrography of kerogens in 3.5-Ga hydrothermal silica dikes in the North Pole area, Western Australia[END_REF]Jia and Kerrich, 2004b) or on-line steppedpyrolysis/combustion [START_REF] Van Zuilen | Nitrogen and argon isotopic signatures in graphite from the 3.8-Ga-old Isua Supracrustal Belt, Southern West Greenland[END_REF]. So far, these methods have not been compared to each other for carbonaceous-rich metasediments. Consequently, different datasets cannot be compared either, while their use as a paleoenvironment reconstruction tool for the Archean period [START_REF] Beaumont | Nitrogen isotopic ratios of kerogens in Precambrian cherts: a record of the evolution of atmosphere chemistry?[END_REF]Jia and Kerrich, 2004b;[START_REF] Ueno | Carbon isotopes and petrography of kerogens in 3.5-Ga hydrothermal silica dikes in the North Pole area, Western Australia[END_REF], relies on the assumption that organic nitrogen 8 15 N do not change with increasing maturation. Partial studies performed on homogenous series of sedimentary organic matter from terrestrial plant matter that is lacking in lipids or waxy matter, showed that mean organic nitrogen 8 15 N does not change until the oil window [START_REF] Boudou | Unexpected enrichment of nitrogen during the diagenetic evolution of sedimentary organic matter[END_REF] and during anchi and epi-metamorphism (Ader et al., 1998a), where the mean organic atomic N / C ratio decreases from 2% in anthracite to 0.5% in meta-anthracite [START_REF] Volkova | Properties of high-rank coals and dispersed organic matter of associated rocks in the Donets Basin (U.S.S.R.)[END_REF]Daniels andAltaner, 1990, 1993;Ader et al., 1998a). This organic nitrogen 8 15 N steadiness still needs to be established for higher metamorphic grades (green-schist and amphibolite grades) corresponding to the meta-anthracite and semi-graphite stages, i.e. the ultimate stages of H, O, N, S removal from the carbonaceous matter, which not necessarily lead to long range crystalline ordering and well crystallized graphite (graphite). For such intermediate to high metamorphic grades, ammonium 6 15 N has been proposed as a tracer for the reconstruction of nitrogen cycle in the Archean (Boyd, 2001a,b;Jia and Kerrich, 2004a;[START_REF] Papineau | Nitrogen isotopic composition of ammoniated phyllosilicates: case studies from Precambrian metamorphosed sedimentary rocks[END_REF]. However, because metamorphic thermal devolatilization progressively enriches the residual fixed ammonium in 15 N [START_REF] Haendel | Isotopic variations of the fixed nitrogen in metamorphic rocks[END_REF][START_REF] Bebout | Nitrogen-isotope compositions of metasedimentary rocks in the Catalina Schist, California: implications for metamorphic devolatilization history[END_REF][START_REF] Mingram | Ammonium concentration and nitrogen isotope composition in metasedimentary rocks from different tectonometamorphic units of the European Variscan Belt[END_REF], ammonium 6 15 N in metamorphic rocks can only be considered as a higher estimate of the initial 6 15 N. For all these reasons, measuring the change in carbonaceous matter 815N during increasing metamorphism represents a necessary contribution towards modelling the nitrogen transfer in the crust, as well as deciphering the evolution of the biosphere during the Archean period.

To achieve this goal, the present paper (1) provides a thorough comparison of sealed-tube combustion to one type of on-line combustion method, (2) addresses the problem of nitrogen contamination during carbonaceous matter extraction, and (3) presents preliminary data on nitrogen content and isotopic composition of carbonaceous matter in a meta-anthracite to graphite sequence.

Geological background and material

Terminology

In this paper we use the term of kerogen in its genetic meaning: organic matter insoluble in common organic solvents and in boiling HF/HCl, and able to generate and expel hydrocarbons if its level of thermal maturity was higher. In the case of metamorphism, this simple analytical definition would lead to consider all insoluble allotropic carbon forms as kerogen. It is the reason why, beyond the point of convergence of the kerogen paths, or maceral path in the case of coal, in a H / C versus O / C van Krevelen diagram [START_REF] Durand | Elemental analysis of kerogens (C,H, O,N,S,Fe)[END_REF], one should no more speak of kerogen but of carbonaceous matter (CM). We will use this term to design solid HF/ HCl extractable amorphous carbonaceous material (a solid high in content of the element carbon without long range crystalline order and structurally in a non-graphitic state), semi-graphite or graphite-d1 and graphite. Semigraphite is characterised by a relatively intense 002 peak at about 3.35-3.36 Å, which however, is noticeably broadened and more or less skewed toward lower angles. The 002 peak spacing is more or less large but the reflections other than 002 are absent. Graphite is characterised by 3.35-3.36 Å as well as other considerably weaker reflections at higher angles (e.g., 100, 101, 004, [START_REF] Landis | Graphitization of dispersed carbonaceous material in metamorphic rocks[END_REF]Kwieciñska and Petersen, 2004). In the case of coal (rock containing more than 40% of carbon, whatever the kerogen or maceral path), the metaanthracite rank, within the ASTM D388-05 standard anthracite rank, starts for a volatile matter content equal or less than 2% dry ash free. Up to a value of vitrinite Rmax of 5%, the beginning of graphitization causes a decrease in Rmin. This stage of thermal metamorphism defines the transition from anthracite to meta-anthracite stage (ASTM classification), which ends at R max of 6.5% (Kwieciñska and Petersen, 2004).

Sample set

The sample set (Table 1) is composed of (1) hard solid bitumen, resulting from a primary algal phase (representative of type I or type II kerogen) followed by a generation of bitumen in the course of thermal evolution of Table 1 Samples presentation: maturation degree, provider name, sampling location and details on the carbonaceous matter type, origin, or metamorphic history are given when available [START_REF] Oh | Structural study of graphitization in the Moongyeong coalfield, South Korea[END_REF]Jehlička, 1994;Jehlička et al., 1997;[START_REF] Zheng | Raman spectra of coal-based graphite[END_REF][START_REF] Zheng | Observations of microstructure and reflectivity of coal graphites for two locations in China[END_REF] WM-GP-M8-1 anthracite [START_REF] Hryckowian | Experimental studies of anthracite coals at high pressures and temperatures[END_REF]Levine and Davis,1989) treated at 3.5 GPa, 750 °C, 24 h in gold cell [START_REF] Boudou | Etude expérimentale du comportement sous haute pression de l'azote organique sédimentaire. 1 er Forum de Technologie des Hautes Pressions[END_REF] the sedimentary series, and ending by their high-temperature transformation related to a basalt extrusion, (2) meta-anthracite, semi-graphite and not well crystallized graphite samples derived from vitrinite rich-coals representative of type III kerogen (sedimentary organic matter derived from terrestrial plant matter that is lacking in lipids or waxy matter) (3) HF/HCl purified graphites resulting from regional or contact metamorphic transformation of carbonaceous matter dispersed in a rock matrix.

Meta-anthracites are here the less mature samples with an atomic N / C ratio inferior to 5 × 10 -3 [START_REF] Volkova | Properties of high-rank coals and dispersed organic matter of associated rocks in the Donets Basin (U.S.S.R.)[END_REF]Ader et al., 1998a). At the top of the maturation scale, graphite can be considered as pure carbon supposedly containing only nitrogen traces. Highly carbonized hard solid bitumens from basaltic rocks, which cross-cut a black shale formation of Upper Proterozoic age in central and western part of the Bohemian Massif, Czech Republic, are characterized by a low atomic H / C ratio of about 0.1. They differ from dispersed kerogens isolated from regionally metamorphosed black shales of the same primary sedimentary basin [START_REF] Jehlicka | Etude structurale de matières organiques soumises a des processus métamorphiques[END_REF][START_REF] Jehlicka | Raman microspectrometry of accumulated non-graphitized solid bitumens[END_REF][START_REF] Jehlicka | Evidence for fullerenes in solid bitumen from pillow lavas of Proterozoic age from Mitov (Bohemian Massif, Czech Republic)[END_REF].

In a first part, sealed-tube and on-line combustion methods for isotopic ratio determination of nitrogen are tested and compared between each other for a set of carbonaceous-rich metasedimentary rocks of variable types and origins.

In a second part, the impact of graphitization on both N-content and 615N of the kerogen extracted from organicrich metasedimentary rocks is investigated using a subset of 14 samples, selected in coal bodies from three different formations and affected to various degrees by regional and contact metamorphism. Carbonaceous matter from vitrinite-rich coals can be considered as representative of the disseminated sedimentary organic of the same type III kerogen [START_REF] Durand | Elemental analysis of kerogens (C,H, O,N,S,Fe)[END_REF]: the most important form of kerogen which gives rise to large carbonaceous matter and graphite deposits in the earth crust. According to reference information already available, kerogen 6 1 5 N of coal of various age and location varies within a relatively narrow range of -3.4%o to +5.5%o [START_REF] Parwel | Natural variations in the relative abundances of the nitrogen isotopes[END_REF][START_REF] Boudou | Unexpected enrichment of nitrogen during the diagenetic evolution of sedimentary organic matter[END_REF][START_REF] Rigby | The isotopic composition of nitrogen in Australian coals and oil shales[END_REF][START_REF] Whiticar | Stable isotopes geochemistry of coals, humic kerogens and related natural gases[END_REF]Ader et al., 1998a). This initial range of 8 15 N is narrower than the range of 6 1 5 N in marine sediments (-2.7%o up to + 12%o) [START_REF] Peters | Correlation of carbon and nitrogen stable isotope ratios in sedimentary organic matter[END_REF]Rau et al., 1987), so that the 615N change along the kerogen maturation paths (from sedimentary deposition to the metamorphism onset) can be more straightforwardly determined with this type of kerogen, though it would be useful to investigate the different kerogen (I and II) maturation paths in this respect.

At first sight, vitrinite-rich coal generated from higher plants that started to evolve in the Phanerozoic, should not be appropriate to study Archean sedimentary organic matter predominantly derived from marine unicellular organisms (more like type I and/or type II kerogen) and it should be stated that coal may not represent the N-systematics of Archean sedimentary kerogen. In fact, the rock samples used in this work have been chosen because they are very rich in carbon and purer carbonaceous matter can be extracted from them as a result. This carbonaceous matter contains more than 97% of carbon. Assuming that it contains some residual heteroelements, its atomic H / C ratio would be lower than 0.37: a value located much below the ordinary type I, II, II-S, III kerogen or maceral paths plotted in a H / C versus O / C van Krevelen's diagram [START_REF] Durand | Elemental analysis of kerogens (C,H, O,N,S,Fe)[END_REF] where one can easily predict the possible average molecular structure of this hydrogen depleted solid as van Krevelen himself did [START_REF] Van Krevelen | Coal[END_REF][START_REF] Van Krevelen | Properties of Polymer: Their Correlation with Chemical Structure; Lymer: Their Estimation and Prediction from Additive Group Contribution[END_REF]. In this post-kerogen region, one can no more speak of kerogen but of carbonaceous matter (including kerogen residue and solid bitumen). Whatever the initial kerogen source, the only imaginable model for a carbonaceous matter resulting from long-term thermal reaction and containing more than 97% of carbon, is a solid made of a combination of basic structural units (BSU), along a well-known structural and textural model developed by Oberlin and co-workers [START_REF] Bonijoly | A possible mechanism for natural graphite formation[END_REF][START_REF] Oberlin | Carbonization and graphitization[END_REF][START_REF] Beyssac | Graphitization in a high pressure, low-temperature metamorphic gradient: a Raman microspectroscopy and HRTEM study[END_REF], where nitrogen is involved in polyaromatic systems (pyridinic and cyclazine or quaternary type nitrogen).

Among the 14 samples specifically selected for studying the impact of graphitization on both carbonaceous N-content and 6 15 N, 6 samples (DS series) belong to the Permian anthracite bodies of the Sadong formation, which underwent several deformations, with thrusting [START_REF] Kim | Structures of the Ojeongsan area, Moongyeong coal field, South Korea[END_REF] and low-grade (chlorite grade) regional metamorphism. Powder XRD analysis showed that, apart from quartz, DS and BM6 samples contain muscovite and illite as major mineral species. DS1 and DS2 contain chlorite, in addition to muscovite and illite. These phyllosilicates, which account for an important part of the total mineral matter are present in inclusions in the carbonaceous matrix and may contain some ammonium. Samples DS4 to DS7 were collected in the Daeseong mine within 1.5 m of a Cretaceous porphyritic dyke (5 m thick; intrusion temperature: about 700 °C; and intrusion pressure: a few kbar). The samples DS1 and DS2 were collected 1 km away from the dyke and were not affected by the contact metamorphism. One sample (BU1 sample) belongs to the Boeun (Bu) anthracite field (South Korea, 30 km away from the Moongyeong coal basin) deposited at the same time as the Sadong formation and affected by a similar tectonic and metamorphic history. This sample is considered as an equivalent of the DS1 and DS2 samples. Three samples (BM series) were collected from a coal body in the Bongmyeong mine (Jurassic Bongmyeongri formation) within 700 m of the border of a granite intrusion. Contact metamorphism was developed between 2 and 5 kbar, up to a distance of several kilometres from the granite intrusion. The maturation degree of DS and BM samples CM was evaluated using the graphitic interlayer spacing d002 calculated from X-ray diffraction, and TEM observation [START_REF] Oh | Structural study of graphitization in the Moongyeong coalfield, South Korea[END_REF]. Based on those data, the samples are sorted by increasing maturity order in Table 1. The remaining four samples (GL series) were collected from the Lutang graphite deposit in Hunan province and the Panshi graphite deposit in Jiling province in China. The Lutang deposit was formed as a result of regional metamorphism of early Paelozoic (Upper Permian) coalbearing formation. They were graphitized in response to the thermal contact metamorphism caused by the intrusion of Indosinian Qitianling granite. The major rock types in the deposit are dark green slate and hornfels. In addition to graphitization, the country rocks of the coal bodies have also been subjected to silicification and andalucitization. The content of andalucite in andalucite hornstone may reach 10% to 35%. Previous studies reported the microstructure, reflectivity [START_REF] Zheng | Observations of microstructure and reflectivity of coal graphites for two locations in China[END_REF], and the Raman spectra [START_REF] Zheng | Raman spectra of coal-based graphite[END_REF] of the graphitized coal bodies from the Upper Permian Leping group but the data are not available for the GL samples studied here. GL reference numbers GL4-3, GL3-3, GL2-3 and GL 1-3 are arranged in order of an increasing distance from the intrusion body.

There is still a lot of debate about the relative importance of pressure-temperature profiles for coal/kerogen graphite formation (e.g. [START_REF] Landis | Graphitization of dispersed carbonaceous material in metamorphic rocks[END_REF]Buseck and Bo-Jun,1985;[START_REF] Beyssac | Graphitization in a high pressure, low-temperature metamorphic gradient: a Raman microspectroscopy and HRTEM study[END_REF]. In the present work, low pressure and high temperature (LP-HT) contact metamorphism series were preferred to high pressure-low temperature (HP-LT) regional metamorphic series, which involve high degree of strain, because contact metamorphism offers more homogeneous series to follow the metamorphic transformation from a single carbonaceous matter source.

Methods

The major analytical problem associated with Ncontent and δ 15 N measurements of carbonaceous-rich metamorphic rocks (characterised by low atomic N / C ratio b5 × 10 -3 ) comes from the large amounts of CO2 produced during combustion and from the possible production of associated traces of carbon monoxide. Moreover, high partial pressure of CO2 prevents an efficient reduction of trace amounts of NOx into N2 (Boyd et al., 1994). This problem was overcome using CaO for both sealed-tube [START_REF] Kendall | Combustion tube method for measurement of nitrogen isotope ratios using calcium oxide for total removal of carbon dioxide and water[END_REF]Ader et al., 1998a) and on-line combustion [START_REF] Boyd | Noncryogenic purification of nanomole quantities of nitrogen gas for isotopic analysis[END_REF]. At 600 °C, CaO reacts with CO2 to form CaCO3, lowering the partial pressure of CO2 and therefore allowing the reduction of NOx. The 8 15 N measurement of low nitrogen amounts requiring low blanks, the apparatus used in the present study involves a static mass-spectrometer coupled to an ultra-high vacuum line (Fig. 1) allowing 6 15 N measurement with a precision of ±0.5%o (2Q) on 1-2 nmol of N2 gas. It has already yielded measurements of both N-contents and 8 15 N of diamonds (on-line combustion; [START_REF] Cartigny | The origin and formation of metamorphic microdiamonds from the Kokchetav massif, Kazakhstan: a nitrogen and carbon isotopic study[END_REF], metamorphic rocks (sealed-tube pyrolysis; Boyd, 1997; sealed-tube combustion; [START_REF] Busigny | Massive recycling of nitrogen and other fluid-mobile elements (K, Rb, Cs, H) in a cold slab environment: evidence from HP to UHP oceanic metasediments of the Schistes Lustrés nappe (western Alps, Europe)[END_REF] and oceanic crustal rocks (sealed-tube combustion; [START_REF] Busigny | Quantification and isotopic analysis of nitrogen in rocks at the ppm level using sealed-tube combustion technique[END_REF]. Although the sealed-tube and on-line combustion methods have already been described for the previous sample types, they had to be adapted for the N content and 615N analyses of organic-rich metasediments.

Sample preparation and purification from modern organic contamination

Samples were ground into a fine powder (< 160 µm) to provide homogeneous starting material. For some of them, the carbonaceous matter (CM) was isolated using the conventional HF/HCl kerogen preparation method [START_REF] Durand | Procedures for kerogen isolation[END_REF]. Between 0.5 and 3 mg of bulk-rock and/or isolated CM powder were embedded in 3 × 3 mm platinum foil (0.02 mm thick) previously purified by heating at 1050 °C in air. Taking the example of living bacteria atomic N / C ratio (-100 1 ), a contamination of 500 ppm (µg/g) of modern organic carbon would correspond to a contamination of -50 ppm of nitrogen. Therefore, when analysing trace amounts of nitrogen, any modern organic contamination must be removed. For diamonds, metamorphic rocks, oceanic crustal rocks and graphites, modern organic contamination is removed by off-line precombustion at 600 °C [START_REF] Boyd | Improved techniques for the extraction, purification of nanomoles quantities of nitrogen gas: the nitrogen content of diamond[END_REF], on-line precombustion at 575 or 450 °C [START_REF] Boyd | Determination of the ammonium content ofpotassic rocks and minerals by capacitance manometry: a prelude to the calibration of FTIR microscopes[END_REF][START_REF] Busigny | Quantification and isotopic analysis of nitrogen in rocks at the ppm level using sealed-tube combustion technique[END_REF] and on-line precombustion at 450 °C [START_REF] Van Zuilen | Nitrogen and argon isotopic signatures in graphite from the 3.8-Ga-old Isua Supracrustal Belt, Southern West Greenland[END_REF], respectively. For samples containing CM of lower maturity than graphite, such precombustion temperatures will initiate the combustion of the CM itself [START_REF] Gilmour | Stable carbon isotopic analysis of sedimentary organic matter by stepped combustion[END_REF]. In this study the samples were precombusted off-line in a muffle furnace for 15 min at 400 °C, a procedure commonly used for destroying modern organic matter. Mass loss after the precombustion was lower than 1% and N-content and 615N results obtained on the raw and pre-combusted semigraphitesample GL 3-1 are consistent (Table 2), demonstrating that the sample's nitrogen is not affected by the precombustion step.

Sealed-tube combustion

The sealed-tube combustion procedure is slightly modified from the method detailed recently by [START_REF] Busigny | Quantification and isotopic analysis of nitrogen in rocks at the ppm level using sealed-tube combustion technique[END_REF]. Only the modifications will be presented here. The major difference lies in the amounts of CaO and CuO used in the sealed-tubes: 200 mg of CuO wires and 100 mg of CaO granules, instead of 120 mg and 15 mg respectively used by [START_REF] Busigny | Quantification and isotopic analysis of nitrogen in rocks at the ppm level using sealed-tube combustion technique[END_REF] for carbonaceous-poor rocks (less than 0.1% C). For carbonaceous-rich samples the amount of CuO (oxidant) and CaO have to be sufficient to allow the combustion of the carbon and the fixation of the evolved CO2 respectively. In return, no Cu is needed as it is formed during the combustion step from the reduction of the CuO. Measured nitrogen blanks for this method are of 1.1 ± 0.5 nmol N with a mean 8 15 N value of 4±6%o (1Q). The reagents being the major source of nitrogen contamination, the blanks are higher by a factor of 2 compared to the 0.6 ± 0.3 nmol reported by [START_REF] Busigny | Quantification and isotopic analysis of nitrogen in rocks at the ppm level using sealed-tube combustion technique[END_REF].

The other modification deals with the last purification step of the sealed-tube and sample. Instead of being purified at 450 °C under vacuum first and then under oxygen, the sample is degassed at 400 °C under vacuum only, to avoid the onset of combustion. Because this modification might participate in increasing the blanks, the amount of 40 Ar was monitored and found to be higher for samples than for blanks. Assuming that 40 Ar comes from atmospheric contamination the corresponding N2 contamination (i.e. the nitrogen blank) can be calculated. The nitrogen blank amount calculated by this method for the sealed-tube blank analyses corresponds to the effectively measured 1.1 ± 0.5 nmol. In contrast, for sample analyses, the calculated blanks (3 ± 1.5 nmol, see Tables 3, 4 and5) are higher but remain negligible, except for semi-graphite and graphite samples, which require a blank correction. However, these nitrogen blanks must only be considered as maximum estimates since a Blanks are calculated using the 40 Ar, N contents are corrected from them when available, or otherwise from the blank of analysis (1.1 ±0.5 nmol).

δ 15 N values are not corrected from the blank. a Differences between on-line and sealed-tube combustion results: Δ15N= δ 15N sealed-tube -δ 15N on-line and ΔN / C=(N / Csealed-tube -N / Con-line)/N / Csealed-tube.

b Measurement performed by sealed-tube combustion and dual inlet mass spectrometry reported in Ader et al. (1998a,b).

contribution to the atmospheric 40 Ar from 40Arproduced by radioactive decay of 40 K in the rock matrix or carbonaceous matter cannot be ruled out.

The combusted sealed-tube is introduced in the cracker section of the extraction-line (Fig. 1) and degassed for 48 h until the vacuum reaches the value of 2 × 10 -8 torr. Gases contained in the tube are ready to be released and transferred into the purification section.

Samples for the determination of δ 13 C and carbon content were prepared by sealed-tube combustion in a similar but simpler way than for δ 15 N analyses. After CuO and quartz tube purification at 950 °C in air, between 1 and 5 mg of powder sample (bulk rock or extracted carbonaceous matter) is introduced in the tube on top of the CuO. The tube is then evacuated for one night under vacuum at ambient temperature and sealed when the vacuum is better than 10 -5 torr. Combustion of the sealed-tube is done at 950 °C for 6 h. Purification (using a liquid nitrogen trap) and quantification (using a Toepler manometer) of the produced CO2 were performed on a dedicated vacuum line. δ 13 C measurements were performed on a dual-inlet Delta E Finnigan Mat mass spectrometer. Reproducibility is better than ±5% of the measured value for carbon content and ±0.1‰ for the δ13C.

On-line combustion

The on-line combustion section is made of three distinct subsections (Fig. 1 and [START_REF] Boyd | Improved techniques for the extraction, purification of nanomoles quantities of nitrogen gas: the nitrogen content of diamond[END_REF]: (i) the sample reactor, (ii) a CuO furnace providing O2 to combust the sample and (iii) a Cu-CaO furnace allowing both the sequestration of CO2 as CaCO3 and the reduction of NOx into N2. The sample embedded in platinum foil is degassed (b10 -7 torr) in a side arm of the vacuum line at ambient temperature overnight. The sample is then introduced in the sample reactor at a temperature of 400 °C or higher in the case of graphite analysis. When the pressure reaches the minimum value of 2 × 10 -8 torr,the CaO-Cu furnace (when unused set at 850 °C) is cooled to 100 °C to prevent oxidation of the Cu during the combustion period. The combustion system is isolated from the pumps; the reactor and CuO furnaces are set to the temperature of 1100 and 930 °C respectively for the combustion. A liquid nitrogen trap is placed below the reactor, trapping the evolved CO2. As a consequence the oxygen pressure is not diluted by stagnant CO2 and the rate of combustion is enhanced [START_REF] Javoy | Nitrogen and carbon isotopic composition in the diamonds of Mbuji Mayi (Zaïre)[END_REF], 2 mg of graphite being combusted in less than 15 min. After the combustion, the temperature of the CuO furnace is set down to 450 °C to re-fix oxygen. The CaO-Cu furnace temperature is then raised to 600 °C and the liquid nitrogen trap is removed. The CO2 reacts with the CaO and the NOx react with Cu to produce N2. The CaO-Cu furnace is then cooled to 100 °C in order to reduce the residual pressure of CO2. The residual N2 is ready to be transferred into the purification section. Nitrogen blanks are of 0.28±0.14 nmol N. The CO2 produced during the combustion and fixed as CaCO3 is released by setting the CaO furnace to 850 °C. It is quantified using a calibrated piezo-resistive gauge (precision ± 5%) and analysed for the δ 13 C on a conventional dualinlet Delta E Finnigan Mat mass spectrometer.

Nitrogen purification, quantification and isotopic analysis

The gases produced either by sealed-tube or on-line combustion are transferred into the purification section (Fig. 1) where (i) traces of CO if any are oxidized to CO2 by CuO heated at 450 °C and (ii) all condensable gases are trapped cryogenically. The purified N2 is quantified using a capacitance manometer before being introduced in the triple collector static vacuum mass spectrometer connected to the line. The amount of nitrogen is measured with a precision better than ± 8% (2σ) and the δ15N values is measured with an accuracy of ±0.5‰ (2σ) [START_REF] Busigny | Quantification and isotopic analysis of nitrogen in rocks at the ppm level using sealed-tube combustion technique[END_REF] CO lead to a significant increase of a15N values [START_REF] Javoy | Nitrogen and carbon isotopic composition in the diamonds of Mbuji Mayi (Zaïre)[END_REF][START_REF] Beaumont | Determination of the CO contribution to the 15 N/ 14 N ratio measured by mass spectrometry[END_REF], the purity of the analysed N2 with respect to CO is checked for each sample by monitoring both the mass-to-charge ratio of 12 and half-life of N2 species.

Results and discussion

4.1. Comparison of sealed-tube and on-line combustion methods to determine N-content and 8 15 N in carbonaceous-rich metasediments Nine bulk samples and the carbonaceous matter (extracted by HF/HCl) of one of them (GL 3-1) were analysed by both methods. Nine of the eleven graphite separates were analysed by on-line combustion, the other two by sealed-tube combustion. Results are reported in Table 3 along with the C-content and 6 13 C obtained simultaneously by on-line combustion.

For on-line combustion the N-content reproducibility is better than ±4% (1Q) of the measured value (Table 3). The 615N reproducibility (1Q) calculated for samples analysed several times (GP, PIES-6770, GL 3-1) are comprised between ± 1%o and ±2%o. This is rather poor compared to the expected ±0.5%o (1Q) obtained for the analyses of micro-diamonds using exactly the same equipment and analytical protocol (see Table 1 in [START_REF] Cartigny | The origin and formation of metamorphic microdiamonds from the Kokchetav massif, Kazakhstan: a nitrogen and carbon isotopic study[END_REF]. 4), bulk samples analyses are considered as replicate of the CM ones.

b Organic N / C in ppm=atomic organic N-to-organic C ratio× 106.

For sealed-tube combustion, N-rich samples (>_1000 ppm N; meta-anthracite, semi graphite and solid bitumen) N-content and 6 15 N reproducibility is better than 4% (1Q) and ±0.7%o (1Q) respectively, i.e. better than for on-line combustion analyses although not as good as can be expected from [START_REF] Busigny | Quantification and isotopic analysis of nitrogen in rocks at the ppm level using sealed-tube combustion technique[END_REF]. The results obtained for the sample PIES-6770 are within error of a previous determination using sealed-tube combustion and dual inlet mass spectrometry (N= 0.45 ± 0.03%, 8 15 N=3.45±0.15%0; Ader et al., 1998a). For a low N-content sample (not well crystallized graphite GL 3-4; around 60 ppm N) the results are not reproducible (6 15 N=2.3 ± 1.6% 0 ), reflecting a higher proportion of blank (between 15% and 35%) in the analysed N2. For very low N-content samples (graphites containing less than 3.5 ppm N), the high proportion (more than 88%) of blank in the analysed N2 did not allow any reliable 615N measurement.

The results obtained by both methods are reproducible enough to see that they are frequently different from each other. For example, the PIES-6770 sample 615N measured by on-line combustion is of -2.6±1.3%o when this value measured by sealed-tube combustion is of +3.5 ± 0.7%o. The corresponding atomic N / C ratio is of 4007±200 ppm by on-line combustion and of 4655± 220 ppm by sealed-tube combustion. In Fig. 2, the results acquired on the same starting material are compared by plotting O 1 5 N = a 1 5 Nsealed-tube-a 1 5 Non-line versus A N / C=(N / Csealed-tube-N / C on-line) /N / Csealed-tube. If the methods gave the same results, all the points should fall around zero on both scales. Instead, they define a trend of increasing A 15N with increasing AN / C indicating that on-line combustion yields less nitrogen with lower 615N values (down to -6.2%o) than sealed-tube combustion.

The difference in N content between the two methods cannot be due to a source of extra nitrogen brought by the sealed-tube method -namely atmospheric and/or modern organic contamination -since the blanks are very low and the potential modern organic contaminations have been removed (see Section 3.2 and Busigny et al., 2005). The difference in N content between the two methods can only be due to poor nitrogen recovery by on-line combustion, showing that the on-line combustion method described in this paper is not suitable for the N-content and δ 15 N analysis of carbonaceous-rich metamorphic rocks. In contrast, the sealed-tube combustion technique is a reproducible and accurate method for the determination of nitrogen content and δ 15 N in organic rich metamorphic rocks -provided that the nitrogen concentration is high enough with respect to the nitrogen blank (3 ±1.5 nmol N) -a conclusion in agreement with that of [START_REF] Bräuer | Methodological aspects of the 15 Nanalysis of Precambrian and Palaeozoic sediments rich in organic matter[END_REF] who recently demonstrated that for carbonaceous-rich samples the sealedtube combustion method yields N content and δ15N results identical to digestion techniques. Among the possible reasons for the inadequacy of the on-line combustion for the N content and δ 15 N analysis of carbonaceous-rich rocks, a general dysfunction of the on-line combustion section can be ruled out as a microdiamonds powder analysed during the same period gave fully consistent results (δ 15 N and N content reproducibility are ±0.5‰ (1σ) and ±2.5% (1σ) respectively, n=4). Incomplete combustion of organic nitrogen can also be ruled out as combustions of pure graphite samples yielded 100% of carbon recovery and further combustion of the samples produced only blank amounts of CO2. Incomplete release of fixed-ammonium -as suggested from similar poor N recovery with lower δ 15 N by elemental analysis coupled to mass spectrometry [START_REF] Bräuer | Methodological aspects of the 15 Nanalysis of Precambrian and Palaeozoic sediments rich in organic matter[END_REF]) -can also be ruled out since it was shown that vacuum pyrolysis at 1100 °C using the same apparatus releases most of the fixed nitrogen from metamorphic rocks [START_REF] Boyd | The measurement of 615N in crustal rocks by static vacuum mass spectrometry: application to the origin of the ammonium in the Cornubian batholith, southwest England[END_REF][START_REF] Boyd | Determination of the ammonium content ofpotassic rocks and minerals by capacitance manometry: a prelude to the calibration of FTIR microscopes[END_REF] and the presence of O2 during the combustion step at 1100 °C is expected to enhance the release of fixed ammonium. The fact that the nitrogen content and δ 15 N differences are relatively reproducible for each sample, but very different from one sample to another, points out to a matrix effect. If all the nitrogen is released from the sample during on-line combustion, its incomplete recovery could be explained by secondary gas phase reactions with impurities (such as sulphur and metals, absent from diamonds but frequently present in carbonaceous-rich metamorphic rocks) occurring at some stage of the combustion process. The speciation of the nitrogen (N2, NOx) involved in these reactions and the nature of the condensed phase are unknown but the following observations seem to validate this hypothesis: after several online combustions yellow deposits have been observed on the unheated glass walls adjacent to the Cu/CaO furnace, and blank increase after a combustion (up to 1.4 after instead of 0.3 nmol before, while the 40 Ar remainedconstant) suggested that some nitrogen was stored in the on-line combustion section during the sample combustion and partly remobilised during the following blank. The fact that these reactions do not occur during sealed-tube combustion suggests that they are due to temperature and/or oxygen fugacity contrasts: during combustion, the whole sealed-tube (sample and reactants) is heated at homogeneous temperature, as opposed to the on-line combustion with different subsections characterised by contrasting temperature and oxygen fugacity.

The present results indicate the need to use the sealedtube technique as a reference method as it circumvents all kinds of matrix-related problems.

Consequences of carbonaceous matter extraction: nitrogen in CM versus nitrogen in bulk rock

The impact of the HCl/HF-attack on the δ 15 N of the carbonaceous matter is investigated here with a special interest in potential nitrogen contamination of samples containing less than 100 ppm N, an often-neglected issue. Bulk powder and isolated CM of 10 samples were analysed by sealed-tube combustion. For most of these samples it is assumed that carbon is mostly only present in the carbonaceous matter and is therefore totally preserved during the CM extraction. DS and BM samples did not show carbonate by XRD and all the samples (even the GL samples for which δ 13 C N-20‰ suggest isotopic equilibration with carbonate) show similar δ13C for the bulk sample and the carbonaceous matter, indicating the absence of carbonate.

For the purpose of the discussion, samples are divided in three groups (Table 4).

• The first group is characterised by bulk samples (3 samples) presenting a higher N / C ratio than their CM. These samples illustrate the presence of fixedammonium. Significant differences between δ 15 N in the bulk and in the CM (up to 3.4‰ for DS6) demonstrate that carbonaceous matter extraction is needed. • The second group of samples (4 samples) shows similar atomic N / C ratios in bulk samples and their CM. These samples illustrate the absence of fixedammonium. In this case the similarity between δ15N in bulk samples and their CM demonstrate that the procedure of CM extraction did not affect its δ15N. • In the third group of samples (3 samples) nitrogen associated with the HF/HCl isolated carbonaceous matter displays a higher atomic N / C ratio and a lower δ 15 N (down to -10‰) than in the bulk sample. This illustrates a nitrogen contamination, which may have been introduced through fluorite precipitation during the HCl/HF treatment (see also [START_REF] Van Zuilen | Nitrogen and argon isotopic signatures in graphite from the 3.8-Ga-old Isua Supracrustal Belt, Southern West Greenland[END_REF]. As tested with the BM6 sample, this contamination can be partly removed by a pyrolysis step at 1100 °C. In the present work (Tables 3 and5) we found that atomic N / C ratios (× 10 6 ) are comprised between: (1) 2500 and 4050 in meta-anthracites, (2) 990 and 1600 in semi-graphites, (3) 45 and 110 in not well crystallized graphites, (4) and finally that they are lower than 3 in graphites. Table 5 and Fig. 3 also show that nitrogen is efficiently removed from the carbonaceous matter during graphitization (decreasing interplanar spacing d002) in both DS and BM series. In the absence of a conventional maturation index, the CM atomic N / C ratio could therefore be used as such. The 615N results obtained onthe DS series for the metagenesis stage (i.e. meta-anath-racite to semi-graphite stages) are presented in Fig. 4 versus their CM atomic N / C ratios used as a maturation index. CM atomic N / C ratios (× 10 6 ) are comprised between 990 and 4050. They are not correlated with organic 8 15 N values, which range between -0.7%o ± 0.6%o and +2.7 ± 0.5%o (n = 7). The average 1%o increase between the meta-anthracite samples and the semigraphite samples cannot be considered as significant compared to the 8 15 N internal variability of both the meta-anthracite and semi-graphite samples (respectively 3No and 2No). These results complete the available picture of the trends of organic nitrogen content and 615N during diagenesis and metamorphism. Previous studies did not find any clear type III kerogen 8 15 N change neither during diagenesis, where organic nitrogen is largely preserved (Fig. 4; [START_REF] Boudou | Unexpected enrichment of nitrogen during the diagenetic evolution of sedimentary organic matter[END_REF][START_REF] Rigby | The isotopic composition of nitrogen in Australian coals and oil shales[END_REF][START_REF] Whiticar | Stable isotopes geochemistry of coals, humic kerogens and related natural gases[END_REF], nor during the early stages of metagenesis -anthracite to meta-anthracite stages -(Fig. 4; [START_REF] Rigby | The isotopic composition of nitrogen in Australian coals and oil shales[END_REF]Ader et al., 1998a), in spite of a significant mineralization of organic nitrogen -organic atomic N / C ratio decreasing down to 0.5% (see also [START_REF] Volkova | Properties of high-rank coals and dispersed organic matter of associated rocks in the Donets Basin (U.S.S.R.)[END_REF]Daniels andAltaner, 1990, 1993). The present work brings the first N / C and 6 15 N data on cm nitrogen mineralization process during the next stage of metagenesis -meta-anthracite to semigraphite stages -and suggests that 8 15 N would remain about the same during metamorphism, which implies that, organic nitrogen (whatever the initial kerogen path) would be mineralized without any isotopic fractionation. Fig. 4. Carbonaceous matter 615N for the DS semi-graphitisation series are reported as a function of atomic N / C ratio. Previous data are also reported for comparison: Pennsylvania and Bramsche Massif metaanthracitisation series (Ader et al., 1998a); Mahakam burial diagenesis series [START_REF] Boudou | Unexpected enrichment of nitrogen during the diagenetic evolution of sedimentary organic matter[END_REF].

During the last metagenesis stages -semi-graphite to graphite -mean nitrogen content further decreases down to 10 to 60 ppm N in relatively well crystallized graphite (see also [START_REF] Van Zuilen | Nitrogen and argon isotopic signatures in graphite from the 3.8-Ga-old Isua Supracrustal Belt, Southern West Greenland[END_REF], and down to less than 3 ppm in well crystallized graphite, but its δ 15 N evolution remains to be constrained.

Inferences on the process of organic nitrogen mineralization during metagenesis

Our study suggests that, in spite of a large nitrogen mineralization, metamorphism would not produce any significant change in organic δ 15 N. The observed organic nitrogen δ 15 N steadiness provides constraints on both the nitrogen mineralization process and the general natural process of low to high-pressure structural transformations of the carbonaceous matter, which remains to be explored.

After sedimentary deposition and early diagenesis, the organic nitrogen-containing structures appear to be mostly pyrrolic and pyridinic [START_REF] Burchill | Variation of nitrogen content and functionality with rank for some UK bituminous coals[END_REF][START_REF] Wojtowicz | The fate of nitrogen functionalities in coal during pyrolysis and combustion[END_REF][START_REF] Knicker | A solid solid-state 15N NMR spectroscopic investigation of the origin of nitrogen structures in coal[END_REF][START_REF] Kelemen | Thermal chemistry of nitrogen in kerogen and low rank coal[END_REF][START_REF] Kelemen | XPS and 15 N NMR study of nitrogen forms in carbonaceous solids[END_REF][START_REF] Bennett | Hydropyrolysis of algae, bacteria, archaea and lake sediments; insights into the origin of nitrogen compounds in petroleum[END_REF]. The observed δ15N constancy until the onset of oil/gas formation along the type III kerogen maturation path [START_REF] Boudou | Unexpected enrichment of nitrogen during the diagenetic evolution of sedimentary organic matter[END_REF][START_REF] Rigby | The isotopic composition of nitrogen in Australian coals and oil shales[END_REF]Whiticar,1996) can be explained by internal structural rearrangements with only little organic nitrogen loss [START_REF] Boudou | Unexpected enrichment of nitrogen during the diagenetic evolution of sedimentary organic matter[END_REF][START_REF] Boudou | Molecular nitrogen from coal pyrolysis[END_REF]. At higher maturation grade, whatever the initial kerogen path, nitrogen appears to substitute to carbon in condensed polyaromatic systems. In this position, it could not be removed by pure thermal reactions, as shown with model compounds [START_REF] Pan | Experimental study of the effects of temperature and ultra-high pressure on the coalification of bituminous coal[END_REF][START_REF] Stanczyk | Elimination of nitrogen from coal in pyrolysis and hydropyrolysis -a study of coal and model chars[END_REF][START_REF] Kelemen | Thermal chemistry of nitrogen in kerogen and low rank coal[END_REF][START_REF] Zolotov | Stability of condensed hydrocarbons in the solar nebula[END_REF][START_REF] Xiao | Reactions of nitrogen and oxygen surface groups in nanoporous carbons under inert and reducing atmospheres[END_REF], while it could be more easily mineralized by pressure reaction in water media, under sub-or supercritical conditions, with formation of ammonia and organic denitrogenated by products [START_REF] Katritzky | Aqueous high-temperature chemistry. 22. Nitrogen-containing heterocycles in supercritical water at 460 °C[END_REF][START_REF] Nakano | Influence of water agent on high-pressure/high-temperature transformation of graphitic BC2N[END_REF][START_REF] Akimoto | Hydrothermal dechlorination and denitrogenation of municipal-waste-plastics-derived fuel oil under sub-and supercritical conditions[END_REF][START_REF] Akimoto | Hydrothermal denitrogenation of fuel oil derived from municipal waste plastics in a continuous packed-bed reactor[END_REF]. Previous gold cell experimental simulation of the co-evolution of carbonaceous matter and associated silicates during early metamorphism stages using model carbazole-kaolinite or anthracite-kaolinite mixtures showed that metamorphic pressure reaction, in presence of water resulting from the transformation of kaolinite into illite, produces a strong nitrogen mineralization without any mean carbonaceous matter δ 15 N change (Ader et al., 1998a,b). For the more advanced metamorphic grades, experimental hydrothermal process, when applied to coal or amorphous carbon, can leave behind graphitic carbon (u.a. [START_REF] Tagiri | Hydrothermal syntheses of graphite from bituminous coal at 0.5-5 kbar water vapour pressure and 300-600[END_REF][START_REF] Jakobsson | The system C-O in equilibrium with graphite at high pressure and temperature: an experimental study[END_REF][START_REF] Kraft | Carbon formed by hydrothermal treatment of a-SiC crystals[END_REF][START_REF] Libera | Hydrothermal synthesis of graphite tubes using Ni catalyst[END_REF][START_REF] Calderon-Moreno | Non-catalytic growth of multiwall carbon nanotubes in hydrothermal fluids[END_REF], and it is worthy to note that graphite particles have been reported in natural circulating hydrothermal fluids [START_REF] Jedwab | Graphite crystals in hydrothermal vents[END_REF]. The role of water as reactant in kerogen/CM denitrogenation has also been underlined in hydrocarbon and fluid formation by kerogen cracking [START_REF] Everlien | Hydrous pyrolysis of high-maturity Palaeozoic coals and Black Shales from Central Europe and adjacent areasthermodynamic considerations[END_REF][START_REF] Lewan | Experiments on the role of water in petroleum formation[END_REF][START_REF] Price | Evidence and characteristics of hydrolytic disproportionation of organic matter during metasomatic processes[END_REF][START_REF] Schimmelmann | Experimental controls on D/H and 13C/12C ratios of kerogen, bitumen and oil during hydrous pyrolysis[END_REF][START_REF] Mastalerz | Isotopically exchangeable organic hydrogen in coal relates to thermal maturity and maceral composition[END_REF][START_REF] Schwab | Chemical and carbon isotopic evolution of hydrocarbons during prograde metamorphism from 100 °C to 550 °C: case study in the Liassic black shale formation of Central Swiss Alps[END_REF].

It is reasonable to assume that, whatever the initial type of kerogen, water would not penetrate quickly, through a microporous network, into the matrix of the resulting natural carbonaceous matter undergoing metamorphism, but would only slowly penetrate by a thermal activated solid phase diffusion forming an external front around the carbonaceous solid particle, where nitrogen mineralization, by oxy and hydroxyl radical attack, would progress more quickly to completion than the diffusion itself. Observations of the texture of metamorphic carbonaceous matter support this fluid transfer scheme. Anthracites have some mesopores and a closed micropore network [START_REF] Fryer | The micropore structure of disordered carbons determined by high resolution electron microscopy[END_REF]Mahajan,1984;[START_REF] Daulan | Influence of anthracite pretreatment in the preparation of activated carbons[END_REF][START_REF] Radlinski | Application of SAXS and SANS in evaluation of porosity, pore size distribution and surface area of coal[END_REF], which result from early cross-linking processes. Micropores correspond to the global misorientation of small nanometric BSU (usually stacking of two or three aromatic layers of 2-3 nm in diameter, as in most chars and cokes). Locally, 5 to 10 nm BSU domains tend to form pore walls of larger mesopores. Such domains are revealed in the HRTEM 002 dark field images [START_REF] Rouzaud | The characterization of coals and cokes by transmission electron microscopy[END_REF]. The porous texture of carbonaceous matter derived from other kerogen types can be inferred from experimental slow carbonization of graphitizable precursors, such as algal ones, which produces a coalescence of the carbon matrix, foregoing oil/gas formation and leaving behind very little micropores, but large basic structural units which easily form planar graphite by high pressure breaking -along the graphitization Oberlin's model.

Along an incremental denitrogenation model, complete nitrogen loss of the residual carbon would occur at the external interface, before progressing towards the interior part of the solid, instead of occur r ing in the whole bulk solid, as it would be the case in a purely thermal denitrogenation process. Therefore carbonaceous matter denitrogenation with ammonia genesis, by pressure reaction in presence of water, would just concern the external grain interface without affecting the residual bulk nitrogen (very thermostable) where δ 15 N would remain constant -as any possible thermal-reactions of ammonia contained in formation waters with carbonaceous matter would just concern the fully denitrogenated external interface. The water necessary for this reaction does not necessarily need to be introduced from an external source but could simply be produced in situ, by an illitization process [START_REF] Ahn | Interstratification of carbonaceous material within illite[END_REF]. In the case of HP-LT metamorphic series, another type of oriented nitrogen attack supporting the carbonaceous matter δ15N steadiness can be deduced from HRTEM study of the natural graphitisation [START_REF] Oberlin | Graphitization studies of anthracites by high resolution electron microscopy[END_REF][START_REF] Bonijoly | A possible mechanism for natural graphite formation[END_REF][START_REF] Beyssac | Graphitization in a high pressure, low-temperature metamorphic gradient: a Raman microspectroscopy and HRTEM study[END_REF]. High-pressure metamorphism would produce graphitic lamellae by breaking the carbon microtextures from previous lowpressure kerogen metamorphism (e.g. anthracite). From the mechanistic graphitization scheme, one can hypothesize that the rupture of micropore walls and onion like structures would locally produce edge lamellae nitrogen species prone to mineralization by interfacial incremental pressure reaction in water media leaving behind unchanged internal quaternary nitrogen. HP/HT experimental simulations conducted in the absence of water, explain the natural graphitization by tectonic stress, which -in addition to frictional heating and pressure ordering [START_REF] Bustin | Mechanisms of graphite formation from kerogen: experimental evidence[END_REF][START_REF] Nover | Promotion of graphite formation by tectonic stress -a laboratory experiment[END_REF] -is also known to facilitate the hydrothermal fluid circulation. This was explicitly quoted by [START_REF] Suchy | Vitrinite reflectance and shearinduced graphitization in erogenic belts: a case study from the Kandersteg area, Helvetic Alps, Switzerland[END_REF], who suggest that the high temperatures responsible for the formation of 'transitional matter' and 'optical graphite' may be explained by two possible mechanisms: flow of hot brines through micro-shear zones or frictional heating during micro-shearing. In fact, the flow of hydrothermal fluids through organic-rich strata has been proposed as a general cause of coal metamorphism [START_REF] Hower | Mechanisms of coal metamorphism: case studies from Paleozoic coalfields[END_REF].

Implications for the use of metasedimentary carbonaceous matter δ 15 N as a paleoenvironmental indicator

Assuming that the carbonaceous matter, whatever its initial kerogen source, behaves similarly through the metamorphism, an important implication of the δ15N steadiness is that it could be used as an estimate of CM δ 15 N prior to metamorphism. Because of internal variations of the δ 15 N values of the DS series, the accuracy of the estimate is only of ± 1‰ as far as the semi-graphitisation stage (green-schist facies). However, compared to the known range of sedimentary kerogen δ 15 N, such an accuracy is quite good, and is particularly interesting, on one side to trace deep fluid formation (e.g. [START_REF] Gerling | New aspects on the origin of nitrogen in natural gas in northern Germany[END_REF][START_REF] Zhu | The isotopic compositions of molecular nitrogen: implications on their origins in natural gas accumulations[END_REF], on the other side for the study of the Archean biosphere, as all Archean rocks of sedimentary origin have been affected at least by low grade metamorphism (e.g. Jia and Kerrich, 2004a,b). For the moment, the use of CM δ 15 N as an indicator of the initial kerogen δ 15 N is suggested only until the semigraphitestage (i.e. low-grade metamorphic). If the contamination problems of N-poor CM were solved and the CM δ15N steadiness through the graphitisation steps predicted by this study demonstrated, the use of CM δ 15 N as an indicator of initial kerogen δ 15 N could be extended to the graphitisation stage.

The investigation of nitrogen in graphite could also help to discriminate biogenic from non-biogenic fluiddeposited graphite. Much attention has been devoted to the process of metamorphic kerogen graphitization to highly crystalline graphite [START_REF] Landis | Graphitization of dispersed carbonaceous material in metamorphic rocks[END_REF][START_REF] Grew | Carbonaceous material in some metamorphic rocks of New England and other areas[END_REF][START_REF] Wopenka | Structural characterization of kerogens to granulite-facies graphite: applicability of Raman microprobe spectroscopy[END_REF][START_REF] Beyssac | Graphitization in a high pressure, low-temperature metamorphic gradient: a Raman microspectroscopy and HRTEM study[END_REF]. Valuable information regarding the degree of metamorphism of host rocks and the nature of the carbon phases, temperatures of equilibration over a wide range of rock types among others, have been obtained based on the study of graphite [START_REF] Ohmoto | Devolatilisation equilibria in graphitic systems[END_REF][START_REF] Cesare | Graphite precipitation in C-O-H fluid inclusions: closed system compositional and density changes, and thermobarometric implications[END_REF][START_REF] Barrenechea | Veintype graphite in the Jurassic volcanic rocks of the external zone of the Betic Cordillera (southern Spain)[END_REF][START_REF] Pasteris | Fluid-deposited graphitic inclusions in quartz: comparison between KTB (German Continental Deep-Drilling) core samples and artificially reequilibrated natural inclusions[END_REF]. However, in some cases, it is not clear if graphite from in situ metamorphism of CM and graphite from precipitation from saturated C-O-H fluids (epigenetic or vein-type graphite) operate separately or, otherwise, if there was some kind of connection between them [START_REF] Luque | Natural fluid-deposited graphite: mineralogical characteristics and mechanisms of formation[END_REF]. Once identified, by combining nitrogen content and δ 15 N with more conventional characterization parameters (crystallinity, mineralogy, δ13 C, etc.), biogenic/metamorphic graphite could be used as a geomarker for tracing Precambrian organic-rich sedimentary basins [START_REF] Sreeraj | Graphite as geomarker and fluid index in east Gondwana terrains[END_REF][START_REF] Dissanayake | Graphite as a geomarker -application to continental reconstruction of pan-African Gondwana terrains[END_REF].

Conclusions

The present work aims to establish a methodology for undertaking the study of nitrogen isotopic evolution of carbonaceous matter during metamorphism. It was shown that, in carbonaceous-rich metasediments, nitrogen content and δ 15 N measurements by on-line combustion are not reliable (probably because of a matrix effect inducing reactions of condensation involving nitrogen). Nitrogen content and δ 15 N measurements were therefore performed by sealed-tube combustion. The required analytical conditions to follow the change of nitrogen content and of δ 15 N were fulfilled in the case of anthracite to semi-graphite series. In the case of more mature samples, it was pointed out that HF/HCl extraction of CM is a possible source of nitrogen contamination, so that, at the semi-graphite and graphite transition, CM nitrogen contents are higher estimates only and δ 15 N analyses are meaningless. In other words, although the δ 15 N analyses of samples with extremely high C / N ratios (N10,000) is feasible, more work concerning the chemical separation of carbonaceous matter from metasediments is still required before reliable δ 15 N analyses of can be obtained.

The overall preliminary results showed that, in the case of selected homogeneous contact-metamorphic sequences, there is no significant evolution of the mean CM δ 15 N during the meta-anthracite and semi-graphite stages. The CM δ 15 N steadiness, which co-occurs with a strong nitrogen loss, can be explained assuming that the external front reaction progresses until nitrogen mineralization is complete, before progressing towards the interior part of the solid. If further field and experimental investigations prove that this model is valid, and assuming that each kerogen path brings its own isotopic signature at the metamorphism onset, where kerogen types converge, carbonaceous-rich rocks could be viewed as isotopic fossils, highly practical to investigate nitrogen from sedimentary kerogen, using metasediments. The present work brings some facts which enable us to think that the metamorphic path of the post-kerogen nitrogen would keep the memory of the initial kerogen: an interesting tool, not only to investigate the Archean biosphere, but also the nitrogen transfer from the atmosphere to the mantle through the photosynthesis sedimentation-metamorphism-subduction sequence, and to extent these studies to a cosmogeochemical approach.

Fig. 1 .

 1 Fig. 1. Schematic representation of the ultra high-vacuum line.

Fig. 2 .

 2 Fig. 2. Results acquired by on-line and sealed-tube combustion methods are compared by plotting A 15 N = 6 15 Nsealed-tube-6 15 Non-line versus AN / C=(N / Csealed-tube-N / Con-line)/N / C sealed-tube. All the points should fall close to zero on both scales if both methods were giving the same results.

Fig. 3 .

 3 Fig. 3. Carbonaceous matter atomic N / C ratio of the DS and BM graphitisation series versus the graphitic interlayer spacing d002 obtained by XRD -used here to evaluate the structural organisation of the carbonaceous matter.

  If up to 100 ppm N contamination can occur during CM extraction of samples initially containing more than 50% of carbon, one can question the quality of CM extraction for most metasedimentary rocks usually containing less 2% carbonaceous matter of low N / C. Controlling the CM purity, and rejecting suspicious ones, as done by Van Zuilen et al. (2005), is then necessary. In our case, the absence of control on the presence and/or total removal of the contamination on data obtained for CM containing very low N content (<200 ppm; BM and GL series) lead us to consider their N content as maximum estimates and 615N as irrelevant. 4.3. Trends of organic nitrogen content and 815N during metamorphism 4.3.1. Results

Table 3 N

 3 content and 615N results obtained by on-line and sealed-tube combustion methods

	CM type			On-line				Sealed-tube			Difference
				combustion			combustion			methoda
	Sample	% C	6 13 C	Blk	Blk % ppm N	61 5 N	Blk	Blk % ppm N	61 5 N	A61 5 N	AN / N
	references	±2%	±0.1%o	nmol		±4% rel	±0.5%o	nmol		±4% rel	±0.5%o	±2%o	±7%
	Solid bitumens
	SB-11		-29.7			1560	-9.4			2095	-4.2	+5.2	25.5
	SB-7		-30.4			1600	-6.7			1830	-4.0	+2.7	12.6
	Meta-anthracites
	PIES-6770		-24.1			3798	-1.1			4600	+3.0
	PIES-6770					3555	-3.5			4200	+4.3
	PIES-6770					3922	-3.1			4300	+3.1
	Average					3758±187 -2.57±1.29			4367±208 +3.47±0.72	+6.0	13.9
	PIES-6770 b									4500±300 +3.45±0.15
	SB-16		-33.4			1543	-4.1			1875	+1.0	+5.1	17.7
	SB-21		-22.0			2240	-4.0			2210	-3.5
	SB-21									2300	-2.9
	Average									2255±64	-3.2±0.42	+0.8	0.7
	Semi-graphites
	GP HT HP		-23.7			2670	-3.9			3281	+3.3
	GP HT HP		-24.0			2580	-1.3			3200	+3.8
	Average					2625±64	-2.60±1.84			3241±57	+3.55±0.35	+6.2	19.0
	SG-K		-21.0			2835	+4.7			2875	+3.5
	SG-K					2770	+2.0			3150	+4.4
	SG-K					2620	+4.4			2600	+3.9
	SG-K					2830	+1.5			2800	n.d.
	Average					2764±100 +3.15±1.63			2856±228 +3.93±0.45	+0.8	3.2
	GL 1-3		-19.3			1186	+3.8	3.4 4	1150	+2.6
	GL 1-3		-19.4			1141	+2.3	3.2 8	1170	+2.4
	GL 1-3							3.8 6	1270	+2.7
	Average					1164±32	+3.05±1.06			1197±64	+2.55±0.15	-0.5	2.8
	GL 1-3 CM		-19.4			1453	+1.1	1.7 7	1490	+2.4
	GL 1-3 CM		-19.4			1383	+1.0
	Average					1418±49	+1.05±0.07					+1.4	4.8
	Not well cryst.
	graphites
	GL 4-3		-14.7	0.72	7.6 42	+3.3	3.5 30	44	+4.0
	GL 4-3		-14.6	0.66 10.6 27	+8.9	2.0 33	67	+1.0
	GL 4-3							1.6 17	67	+2.0
	GL 4-3-degassed		-14.8	0.16	6.2 21	+3.9
	at 700 °C
	GL 4-3-degassed		-14.7	0.34 10.9 24	+4.9
	at 900 °C
	GL 4-3-degassed		-14.6	0.21	7.4 27	+3.5
	at 1100 °C
	GL 4-3-degassed		-14.5	0.29	4.4 25	+7.7
	at 1100 °C
	Average		-14.6			28±7.4	+5.4±2.37			59±13	+2.3±1.6	-3.1	52.6
		±2	±0.11
	Graphites
	FGR 218-1		-23.8	0.40 88	0.5	+24.0
	FGR 240-2		-23.5	0.34 62	1.9	+5.3
	FGR 132-2	n.d. n.d.	0.26 62	1.6	+8.6
	FGR 227-2		-25.9	0.31 50	3.3	+23.5
	FRG 227-1		-25.2	0.24 68	1.3	+16.0
	FGR 2984		-12.6	0.24 68	1.5	+17.0
	FGR 169-8		-15.3	0.36 92	0.3	+10.2
	FGR 241-I		-7.0	0.31 63	1.7	+11.5
	FRG 279-2							2.7 98	0.4	+4.7

Table 4

 4 Atomic N / C ratio and 615N obtained by sealed-tube combustion for bulk samples and their carbonaceous matter (CM)

	. Because isobaric interferences of

a N/ C in ppm: atomic N/ C ratio × 106.

Table 5

 5 Carbonaceous matter atomic N / C (corrected from the blank) ratio and 615N for samples of coal origin

	Sample references	XRD d0 0 2 Å	% C±2%	61 3 C±0.1%o	n a	Blank (%)	Organic N / C ppm b	61 5 N (%o)
	DS2 CM	3.58	89	-23.6	1	3	3464±220	-0.7±0.6
	DS1 CM	3.52	92	-24.2	1	4	4051±240	+1.8±0.6
	BU1 CM	3.52	90	-23.1	1	9	2744±123	+1.9±0.8
	DS5 CM	3.37-3.38	92	-24.5	1		1540±75	+2.7±0.5
	DS6 CM	3.37-3.38	96	-24.1	1	8	1159±80	+0.9±0.8
	DS7 CM	3.37-3.38	96	-24.6	1	5	991±60	+2.0±0.6
	DS4 CM	3.37-3.38	93	-24.3	1	4	1626±110	+1.9±0.6
	GL 1-3 CM		92	-19.4	4		1360±64	+2.4±0.7
	GL 2-3 CM		95	-18.0	2		<110±14
	GL 3-3 CM		95	-21.5	3		:546±15
	GL 4-3 CM		94	-14.7	4		:570±22
	BM6CM	3.364	99	-22.3	3		<112±11
	BM21 CM	3.365	94	-21.6	1		<113±6
	BM44 CM	3.360	100	-21.3	1		:550±8
	a n: number of analyses. For samples having bulk N / C equal to CM N / C (see Table

Acknowledgments

This research was partially funded by a grant from Chevron Petroleum Technology Company, P.O. Box 446, La Habra, CA, U.S.A. We thank all the people who provided the samples without which this study would not have taken place: Eric Daniels (Chevron Petroleum Technology Company) and Jeffrey Levine (Consulting geologist, Tuscaloora, Alabama) for providing Pennsylvanian anthracites and for helpful discussion cites discussion, Peter Gerling (Bundesanstalt für Geowissenschaften und Rohstoffe Geozentrum Hannover) for coal samples from Northern Germany and for constructive comments. FGR graphite separates were provided by Francis Fischer (TIMCAL -Graphite and Technologies); GL samples by Zhe Zheng (Peking University, Department of Geology, Beijing, China); SB samples by Jan Jehlicka (Department of Geochemistry, Charles University, Prague, Czech Republic); PIES 6770 sample and several other coal samples from Bramsche Massif by Angelika Vieth-Redemannn (Geologishes Landesamt NRW, Krefeld Deutschland) and SG SG-K semigraphite sample by Jin Kuili (China University of Mining and Technology, Beijing, China). We also sincerelywish to thank Mark van Zuilen (Scripps Institution of Oceanography, La Jolla) and an anonymous reviewer for their comments and suggestions, which greatly helped improving the quality of the paper. Contribution to IPGP No. 2105. [PD]


T Matrab

M M Chehimi
email: chehimi@paris7.jussieu.fr

J P Boudou

F Benedic

J Wang

N N Naguib

J A Carlisle

Surface functionalization of ultrananocrystalline diamond using atom transfer radical polymerization (ATRP) initiated by electro-grafted aryldiazonium salts

Keywords: ATRP, Diazonium salts, Polymer brushes, Ultrananocrystalline diamond (UNCD), Protein adsorption

The integration of organic adlayers with diamond surfaces imparts many new properties to diamond materials, including lubrication, optical response, chemical sensing, and biocompatibility. The development of new immobilization chemistries is required in which organic layers are covalently attached to the diamond surface. Poly(methyl methacrylate) and polystyrene brushes (PMMA, PS, respectively) were grown from the surfaces of ultrananocrystalline diamond (UNCD) thin films that were modified by the electrochemical reduction of the diazonium salt BF 4 ~,+N2-C6H4-CH(CH3)-Br. The grafted -C6H4-CH(CH3)-Br species were effective in promoting surface initiated atom transfer radical polymerization (SI-ATRP) of the vinyl monomers. Attempts to perform SI-ATRP from undoped diamond films produced by microwave plasma assisted chemical vapour deposition (MPACVD) process was rather unsuccesful due to a very low extent of electrochemical modification by the brominated aryl diazonium salts. The formation of the polymer brushes was confirmed by XPS. The polymer brushes were further evaluated as adsorbents for human serum albumin (HSA) used as a model protein. Protein adsorption decreased in the order PS>PMMA, that is the decreasing trend of hydrophobic character.

Introduction

Atom transfer radical polymerization (ATRP) is one of the most investigated methods of controlled radical polymerization as it can be performed with a variety of functional monomers, at room temperature and in aqueous or organic solvents [1]. In addition, ATRP can be initiated from surfaces for several purposes [2]. The resulting polymer brushes can facilitate the preparation of hybrid materials [3] or biocompatible surfaces [4]. ATRP can be surface-initiated by using, e.g., initiatorterminated silanes [1] and thiols [5], photo-brominated surfaces [6], or macroinitiators [7].

Recently, we have shown that ATRP of vinyl monomers can be surface-initiated by electro-grafted brominated aryl species based on diazonium salts [8]. The use of aryl diazonium salts is indeed an elegant way to modify conducting or semi-conducting substrates such as metals, carbon and hydrogenated silicon [9]. The grafted species can bear a variety of functional groups (e.g., alkyl, and halides) depending on the target applications such as polymer coatings, molecular junctions in electronic circuits, and the detection of molecules of biomedical interest.

The motivation for using aryl diazonium salts to surface initiate ATRP (SI-ATRP) is to design, for example, a salt which is an analogue of 1-phenylethyl bromide (1-PEBr), a frequently used bulk solution ATRP initiator [1]. The corresponding BF44, + N2-C6H4-CH(CH3)-Br salt could readily be electrochemically reduced on glassy carbon (GC) and iron [8]. The resulting -C6H4-CH(CH3)-Br group (1) grafted onto iron permitted the growth of strongly adhering polystyrene, poly(methyl methacrylate) and poly(n-butylacrylate) brushes (PS, PMMA and PBA, respectively) [8].

Performed successfully on metals and carbon, it is expected that the ''ATRP/diazonium salt'' protocol can be extended to ultrananocrystalline diamond (UNCD). UNCD thin films have a variety of interesting bulk and surface properties and potential applications such as low friction wear resistance coatings [10], coatings for biomedical implants [11,12], selective biosensors [13,14], biointerfaces [15], electrodes for the study of the direct electron transfer to proteins [16], and biocompatible surfaces [17]. UNCD films also have the superior properties of diamond such as extreme hardness and mechanical strength, favorable tribological properties, high room-temperature n-type conductivity (when doped with nitrogen) [18], extreme microstructural and morphological stability, and electrochemical properties including high overpotentials for both oxygen and hydrogen evolution [19,20]. The enhanced conductivity of UNCD is due to nitrogen doping at the grain boundaries (GB). In UNCD, the grains are small (less than 15 nm in an amount of no less than 10 10 atoms/cm 3 ) and the number of GBs, where the effective nitrogen doping occurs, is large. The potential for using n-type UNCD and other forms of conducting diamond films as electrochemical biointerfaces with soft materials motivates much of the work reported here [21][22][23]. However, several UNCD applications require control of its surface properties such as surface chemistry, wettability, optical properties, etc. The surface modification of synthetic diamond materials has been investigated by several groups. Two general approaches have been studied: direct chemical functionalization of hydrogen-terminated surface [24,25] or application of preteatment method that renders the surface hydrophobillic (by generation of oxygen-containing surface functionalities) [26,27]. In the first approach, the hydrophobic hydrogenterminated surfaces were functionalized, either photochemically by UV excitation of halogen gases [24], or alkenes [25] thereby introducing chloro-, fluoro-, or aminoalkyl groups, or chemically by thermal decomposition of benzoyl peroxide [28]. In the second approach, oxygen-containing functional groups were introduced onto polycrystalline diamond surfaces by oxygen plasma treatment, anodic polarization, or by strong oxiding acids. The hydrophilic surface can then be modified using standard silane coupling agents [29].

Recently, Carlisle's group has demonstrated the surface modification of UNCD thin films via electrochemical reduction of aryl diazonium salts [30]. It was shown that grafting of 3,5-dichlorophenyl groups renders the UNCD surface hydrophobic, whereas the attachment of 4-aminophenyl groups (obtained by a two-step approach) makes the surface relatively hydrophilic.

In this paper we explore the possibility to use electro-grafted aryl species on undoped and N-doped ultrananocrystalline diamond thin films for the surface-initiated ATRP of vinyl monomers, namely methyl methacrylate and styrene, in the presence of the Cu(I) catalyst and the ligands pentamethylenediethylenetriamine (PMDETA) or bi-pyridine. PMMA and PS brushes grown from electrochemically modified UNCD were characterized by XPS. The polymer-modified UNCD plates were further evaluated as adsorbents for human serum albumin (HSA) at pH 5 in relation to the hydrophobic character of the grafted polymer brushes.

Experimental

The preparation of N-containing conductive UNCD (hereafter called d-UNCD, where d stands for doped) thin film on Si wafers has been described in [31]. d-UNCD thin films were deposited on highly conducting n-type Si(111) substrates (Wafernet Inc., San Jose, CA) using microwave plasma assisted chemical vapor deposition (MPACVD) [CYRANNUS I reactor from IPLAS (Innovative Plasma Systems, Inc., Triosdorf, Germany)]. A mixture of CH 4 , Ar and N2 gases was used for the microwave discharge. The d-UNCD films used in this work were deposited with 5% volume ratio of nitrogen in the plasma, and a flow rate of 1 sccm was maintained for CH 4 . The substrate temperature was about 800 °C, and the input microwave power was about 1.3 kW.

Undoped nanocrystalline diamond films (u-UNCD) were produced by MPACVD process in a home-made bell jar reactor extensively described in previous papers [32]. (100)-oriented silicon wafers of approximately 7 cm 2 in size were used as substrates. Prior to the diamond deposition, the Si substrates were ultrasonically abraded for 1.0 h in a suspension of diamond powder with a grain size of around 40 µm in ethanol [33]. The growth was achieved using a 96% Ar/3% H2/1% CH4 feed gas, under 600 W microwave power, 200 hPa pressure and 250 sccm total gas flow rate, while maintaining the substrate temperature around 1120 K. The deposition time was set at 3.0 h which was suitable to obtain continuous films of approximately 2 µm in thickness. The deposition conditions mentioned above are appropriate for synthesising nanocrystalline diamond layers with a grain size ranging from 10 to 15 nm and a surface roughness (Rms parameter) between 20 and 25 nm, keeping an acceptable diamond purity [34]. After the deposition process, the diamond/Si wafers were cut in order to obtain identical samples of 1 x 2 cm 2 area.

The synthesis and electrochemical reduction of D1 on GC is detailed in Ref. [8]. It is to note that the surface conductivity of u-UNCD [18,35] was responsible for the weak extent of electrochemical reduction of D1 at the surface of u-UNCD.

The growth of brushes on D1-modified ultrananocrystalline diamond was performed as follows : a 100 mL Schlenk flask containing a magnetic stir bar and sealed with a rubber septum was deoxygenated by vacuum followed by back-filling with nitrogen three times. The CuBr powder (1.36 x 10 -3 mol, Aldrich) and the (UNCD +1) plates were introduced into the flask under a nitrogen flow. A mixture containing distilled styrene (106 x 10 -3 mol, Aldrich), 1-PEBr (1.36 x 10 -3 mol, Aldrich) and bipyridine (Bpy, 4.8 x 10 -3 mol, Aldrich), previously degassed, was added to the polymerization flask using a double-tipped needle under a nitrogen flow. The flask w a s p l a c e d i n a n o i l b a t h a t 1 1 0 °C f o r 5 h . T h e polymerization was stopped by cooling and opening the flask to expose the catalyst to air. PMMA brushes were prepared as in Ref. [8].

For PS brushes, the target Mn and Mw/Mn were 71,000 g/mol and 1.22 [36], respectively (M n : number average molecular weight and M w is the weight average molecular weight). UNCD+ 1 +brushes were incubated overnight in 0.5 mg/ ml aqueous solutions of HSA (Sigma) buffered at pH 5 using 0.1 M acetic acid/acetate. The plates were then thoroughly rinsed by 5% volume ration aqueous solution of Tween 20 (Aldrich) and then with distilled water.

XPS spectra were recorded as mentioned in [8] using a Thermo VG Scientific ESCALAB 250 system fitted with a monochromatic Al Ka X-ray source (1486.6 eV). The spot size was 650 Am for standard analysis. The samples were mounted onto conductive double-sided adhesive tapes which enabled us to perform the analyses without the use of the flood-gun, usually employed for insulating materials, even in the case of the u-UNCD samples. Moreover, the raw XPS data obtained with u-UNCD gave C1s spectra centred at 285 eV without any noticeable shift due to static charging effect observed with insulating materials. This is in line with a surface conductivity mentioned in details elsewhere [35].

The lateral uniformity of the D1 electrografting and the growth of polymer brushes along the main axes of the plates were checked using a 400 Am X-ray spot. This gives a macroscopic estimation of the uniformity, at a ¨400 Am scale, of the grafted species.

Results and discussion

The modification of the UNCD substrates by the electrochemical reduction of brominated aryl diazonium salts and the subsequent SI-ATRP of a vinylic monomer is illustrated in Scheme 1. This method was performed in the presence of the sacrificial initiator 1-PEBr added in solution in order to increase the concentration of the Cu II complex and thus to better control the polymerization. Fig. 1 shows XPS survey scans of the uncoated and polymer coated d-UNCD+ 1 plates. The main peaks C1s and O1s are centered at 285 and 530 eV, respectively. The corresponding high resolution C1s regions are shown in the insert. One can also detect Br3d at 71 eV from the grafted aryl species 1 at the surface of d-UNCD+ 1 therefore underlying the successful modification of UNCD by the electrochemical reduction of D1.

The lateral uniformity of the diazonium electrografting and PMMA brush growth were investigated on the UNCD substrates. Fig. 2 shows the Br3d-Br3p region (Fig. 2a) from d -U N C D + 1 a n d t h e C 1 s f i n e s t r u c t u r e f r o m d -UNCD+1+PMMA (Fig. 2b). Both spectra were taken over a few millimeter lines. The spectra have from one spot to another the same structure and intensity which emphasizes a uniform Binding energy (eV) electrografting of the brominated aryl diazonium and a subsequent uniform lateral growth of PMMA chains from the electrochemically modified UNCD. Table 1 reports the surface chemical composition of materials in at.%. The first important observation is that the bromine content of d-UNCD+ 1 is 17 times higher than for u-UNCD plates. It follows that doping is an important procedure for the modification of diamond by aryl diazonium salts. In contrast to undoped UNCD, nitrogen-containing UNCD was observed experimentally to have conductivities approaching that of graphite and yet still be essentially phase-pure diamond [37].

It is clear that for d-UNCD, the growth of PMMA brushes results in a high oxygen content compared to PS brushes as there are two oxygen atoms per MMA repeat unit. Bromine is detected at the surfaces of PS and PMMA brushes grown from d-UNCD+ 1. This indicates that the polymer chains are terminated by bromine, in line with the principles of ATRP [1] and our recent study of polymer brushes grown from the surface of iron.

The contribution of the ester component and shake-up satellite to the C1s peaks of PMMA and PS at d-UNCD are 2.5% and 1%, instead of 20% and 10%, respectively. This indicates that the underlying d-UNCD massively contributes to the total C1s peak intensity, implying very thin polymer coatings (<10 nm, the XPS sampling depth).

In the case of u-UNCD+1, the carbon and oxyen contents before and after growth of PMMA brushes are practically the same.

The detection of Br3d at 71 eV reflects the presence of -CH(CH3)-Br moiety in the initiator and C-Br bonds in polymer chain ends. Cl2p peak was detected, for polymer brushes, 200.0 eV which corresponds to C-Cl bonds. Thus, the polymeric chains could have a chlorine atom at the terminus. N1s and F1s centered at 401 and 685 eV, respectively, are due to traces of supporting electrolytes, which have not been completely removed despite thorough rinsing. Whenever copper is detected, its surface proportion is negligibly small.

It is to note that copper (catalyst) is trapped in the polymer brushes in spite of thorough solvent washing.

As far as the C1s regions are concerned, the d-UNCD+1 exhibits a sharp peak centred at 285 eV with a small component around 286.5 eV due the C-Br bonds and a slight degree of the surface oxidation and contamination. Interestingly, this C1s region has a 7r -> 7r* shake-up satellite due to the electrografted brominated aryl groups. For d-UNCD +1+ PS, the shake-up satellite, characteristic of PS, is also observed.

PMMA brushes impart the most significant changes to the C 1s structure of d-UNCD + 1. Indeed, (i) the characteristic O-C=O component appears at 289 eV together with a distinct component at 286.5 eV due to O-CH3 bonds; (ii) the shake-up satellite from the electrografted aryl type initiator is completely screened by the polymer overlayer.

It is important to note that the characteristic C1s features from PMMA above-mentioned are quasi absent from the u-UNCD + 1 +PMMA surface, thus confirming the quantitative results reported in Table 1 on the quasi absence of polymer brushes at the surface of undoped diamond thin films. Before attachment of HSA, PS brushes have a C1s structure comparable to that of a pure PS with a main feature at 285 eV and a shake-up satellite at 291.5 eV. After protein adsorption, two prominent shoulders were observed at 286. respectively. However, there is evidence for a small component at 288 eV due to the peptidic linkages (N-C=O).

A t t a c h m e n t o f H S

A simple way to monitor protein adsorption onto d-UNCD+ 1 +PS and d-UNCD+ 1 +PMMA is to consider the surface (O+N)/C ratios before and after adsorption. Indeed, the contribution of the protein to the nitrogen and oxygen content is expected to be significant since each amino acid is linked to another via an amide group. Moreover, several aminoacids contain nitrogen and/or oxygen in their residues. Fig. 4 shows the variation of (O+N)/C for the PS and PMMA brushes due to protein adsorption. For d-UNCD+1+ PS, the (O+N)/C ratio increases by a factor of 3 higher than the factor of 1.7 observed for the modification of the d-UNCD + 1 + PMMA surface by the adsorbed HSA. The trend of protein adsorption parallels that of the hydrophobic character of the polymer brushes (PS is more hydrophobic than PMMA [8]) and clearly hydrophobic interactions operate at the proteinbrush-water interface.

Conclusions

ATRP of methylmethacrylate, styrene was initiated from Ndoped ultrananocrystalline diamond (UNCD) surfaces modified by the electrochemical reduction of a brominated aryl diazonium salt, BF44, + N2-C6H4-CH(CH3)-Br (D1). The grafted -C6H4-CH(CH3)-Br acted as ATRP initiator. The PMMA and PS brushes were characterized by XPS and contact angle measurements. The lateral growth of the brushes was found to be homogeneous. Attempts to initiate the ATRP of MMA and styrene from the undoped UNCD was unsuccesful and this is perhaps due to the very negligibly small amount of attached brominated aryl type initiator. Protein adsorption onto the brushes decreased in the order PS>PMMA as judged from XPS measurements, a result that agrees with the superior hydrophobicity of polystyrene.

This paper shows that the ''ATRP/diazonium salt'' protocol is a valuable approach for the controlled surface modification of materials of technological importance by organic coatings. As biointerfaces are concerned, it is also clear that this novel method can be tuned in order to favour or in contrast to limit protein adsorption. Our very recent studies of the minimization of protein adsorption using hydrophilic copolymers of styrene and polyglycidol grown from glassy carbon plates [38] are indeed encouraging in this sense and shall soon be extended to diamond surfaces. This is of fundamental and technological importance given the development of e.g., diamond-coated implants and biosensors with optimized resitance to non specific adsorption.

Scheme 1 .

 1 Scheme 1. Growth of polymer brushes from UNCD surfaces modified by electrografted ATRP initiators based on aryl diazonium salts.

Fig. 1 .

 1 Fig. 1. Survey scans of N-doped diamond (d-UNCD+1) before and after growth of polymer brushes. The corresponding high resolution C1s regions are shown in insert.

Fig. 2 .

 2 Fig. 2. Study of the lateral uniformity of (a) brominated aryl electrografting, and (b) PMMA chains growth by SI-ATRP on UNCD thin films.

  A t o d -U N C D + 1 + P S a n d d -UNCD + 1 +PMMA was monitored using the change in the carbon, nitrogen and oxygen contents while considering the C 1s fine structure. Fig. 3 displays the survey scans of d-UNCD+ 1 +PS and d-UNCD + 1 +PMMA after adsorption of HSA. Both surfaces have a massive increase in the oxygen contents due to the adsorbed protein. Though less clear, the N1s peaks are slightly better detected than in Fig. 1.

  5 and 288 eV corresponding to C-N/C-O bonds and amide groups, respectively. For PMMA, the changes are less significant since PMMA has already oxidized carbon types (C-O and O-C=O) the components of which are centered at 286.5 and 289 eV, Materials d-UNCD +1 d-UNCD+ 1 +PS d-UNCD+ 1 +PMMA u-UNCD+1 u-UNCD+ 1 +PMMA

Fig. 3 .

 3 Fig. 3. Survey scans of d-UNCD+1+PS and d-UNCD+1+PMMA after protein attachment. The corresponding high resolution C 1s regions are shown in insert.

Fig. 4 .

 4 Fig. 4. (O+N)/C atomic ratios determined by XPS for uncoated and HSAcoated d-UNCD+1+PS and d-UNCD+1+PMMA.

Table 1

 1

	Surface chemical compositions of diamond plates as determined by XPS
	C	O	N	Br Cl	Cu	F
	92.1	4.41 1.81 0.85 -	-	0.29
	91.6	5.01 0.98 0.46 0.40	0.06 1.32
	86.2 10.3	1.37 0.59 0.17	0.04 1.28
	93.7	2.12 1.21 0.05 -	-	2.32
	96.9	2.19 0.33 0.09 -	0.12 0.24

Acknowledgements

The authors are grateful to the Conseil re´gional d'Ile de France for financial support through the SESAME 2000 scheme. TM wishes to thank the French Ministry of Higher Education for financial support through a PhD studentship. The work at Argonne National Laboratory was supported by the US Department of Energy, Office of Science, Basic Energy Sciences, under Contact No. W-31-109-ENG-38.