Carole Reymond

Agnès Le Masle
email: agnes.le-masle@ifpen.fr

Nadège Charon

Cyril Colas

A rational

Keywords: LC-UV/MS, High resolution mass spectrometry, Design of experiments approach, Ionization efficiency, Lignocellulosic biomass

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Liquid chromatography (LC) hyphenated with mass spectrometry (MS) began to be studied much later than gas chromatography/mass spectrometry (GC/MS) because of the technical difficulties imposed by such hyphenation (desolvation in particular). It is only since thirty years that LC/MS has a real rise thanks to the development of ionization sources at atmospheric pressure. Despite the strong interest of the scientific community specially in metabolomics research, LC/MS is not systematically used as a routine technique [START_REF] Forcisi | Liquid chromatography-mass spectrometry in metabolomics research: mass analyzers in ultra high pressure liquid chromatography coupling[END_REF]. Preliminary tests and an important step of optimization must be done when an analysis is set up. The main causes are the incompatibility of some chromatographic methods to mass spectrometry (solvent flow rate, additive, etc.) and the large number of parameters to manage in order to control the ionization efficiency inside the atmospheric pressure ionization source (API). Indeed, the signal obtained depends on several factors such as the flow rates of nebulising and drying gases, the sprayer position, the temperatures of the API and transfer capillary, capillary voltage, etc. Optimizing each of these parameters can be tedious and most of the time it is based on the experience that can have a regular user of a LC/MS system. Moreover, the procedure of varying one variable at a time does not guarantee the achievement of optimal conditions. Conversely, although barely used in the literature, the chemometric approach is based on a rational experimental model, which allows the simultaneous variation of all the experimental parameters. The use of an experimental plan also makes it possible to determine the number of necessary experiments and avoids the redundancies of information. It can also take into account the possible interactions between the different parameters. In our knowledge, only few publications used a design of experiments approach to optimize the ion source-mass spectrometry parameters mainly for pharmaceuticals issues [START_REF] Paíga | Optimization of the Ion Source-Mass Spectrometry Parameters in Non-Steroidal Anti-Inflammatory and Analgesic Pharmaceuticals Analysis by a Design of Experiments Approach[END_REF][START_REF] Grand-Guillaume Perrenoud | Coupling state-of-the-art supercritical fluid chromatography and mass spectrometry: From hyphenation interface optimization to high-sensitivity analysis of pharmaceutical compounds[END_REF][START_REF] Peris-Díaz | Multiobjective optimization of liquid chromatography-triple-quadrupole mass spectrometry analysis of underivatized human urinary amino acids through chemometrics[END_REF]. Main of them focus on improving signal of target analytes for pharmaceuticals issues. Perrenoud et al. compared the sensitivity of a UHPSFC-ESI/MS/MS versus a UHPLC-ESI/MS/MS analysis for pharmaceutical compounds using a face-centered central composite design to optimize capillary voltage, desolvation temperature and drying gas flow rate [START_REF] Grand-Guillaume Perrenoud | Coupling state-of-the-art supercritical fluid chromatography and mass spectrometry: From hyphenation interface optimization to high-sensitivity analysis of pharmaceutical compounds[END_REF].

In addition, LC/MS has become more and more interesting to analyze complex matrices containing for example heavy or thermosensitive products that cannot be analyzed by GC/MS. This is particularly true for oxygenated compounds coming from the conversion of lignocellulosic biomass [START_REF] Jarrell | Characterization of organosolv switchgrass lignin by using high performance liquid chromatography/high resolution tandem mass spectrometry using hydroxide-doped negativeion mode electrospray ionization[END_REF][START_REF] Kiyota | Analysis of soluble lignin in sugarcane by ultrahigh performance liquid chromatography-tandem mass spectrometry with a do-it-yourself oligomer database[END_REF][START_REF] Owen | High-performance liquid chromatography/high-resolution multiple stage tandem mass spectrometry using negative-ion-mode hydroxide-doped electrospray ionization for the characterization of lignin degradation products[END_REF]. In the present challenging energy transition context, biomass seems as a promising source to produce renewable energy, biofuels and chemical intermediates by mean of several transformation pathways [START_REF] Diego | Upgrading of biomass: alternative ways for biomass treatment[END_REF][START_REF] Harmsen | Literature review of physical and chemical pretreatment processes for lignocellulosic biomass[END_REF]. As regards thermochemical pathway, lignocellulosic materials, mainly composed of lignin, cellulose and hemicellulose, are liquefied by fast pyrolysis at 400-450°C to produce bio-oils which might be further upgrated to biofuels [START_REF] Bridgwater | Review of fast pyrolysis of biomass and product upgrading[END_REF][START_REF] Meier | State-of-the-art of fast pyrolysis in IEA bioenergy member countries[END_REF]. The resulting liquid matrices are highly complex since they contain a wide diversity of oxygenated compounds (carboxylic acids, ketones, aldehydes, phenols, furans, carbohydrates, etc.). The large range of polarities and molecular weights but also heat sensitivity behavior of some compounds explain why chemical characterization of such liquids is still a challenge today [START_REF] Charon | Multi-technique characterization of fast pyrolysis oils[END_REF]. Without prior derivatization step, GC and comprehensive twodimensional gas chromatography (GC×GC) could hardness detect compounds with a molecular weight higher than 200 g/mol [START_REF] Marsman | Identification of components in fast pyrolysis oil and upgraded products by comprehensive two-dimensional gas chromatography and flame ionisation detection[END_REF][14][START_REF] Staš | Overview of Analytical Methods Used for Chemical Characterization of Pyrolysis Bio-oil[END_REF]. However liquid chromatography (LC and LC×LC) appears to be a suitable technique thanks to its capacity to elute compounds with high polarity such as carbohydrates and/or high molecular weight [START_REF] Le Masle | Development of on-line comprehensive two-dimensional liquid chromatography method for the separation of biomass compounds[END_REF][START_REF] Tomasini | Complementary analytical liquid chromatography methods for the characterization of aqueous phase from pyrolysis of lignocellulosic biomasses[END_REF]. More recently Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR/MS) has gained an increasing attention [START_REF] Hertzog | Toward Controlled Ionization Conditions for ESI-FT-ICR-MS Analysis of Bio-Oils from Lignocellulosic Material[END_REF][START_REF] Hertzog | Combination of electrospray ionization, atmospheric pressure photoionization and laser desorption ionization Fourier transform ion cyclotronic resonance mass spectrometry for the investigation of complex mixtures -Application to the petroleomic analysis of bio-oils[END_REF][START_REF] Liu | Characterization of Red Pine Pyrolysis Bio-oil by Gas Chromatography-Mass Spectrometry and Negative-Ion Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry[END_REF][START_REF] Jarvis | Characterization of Pine Pellet and Peanut Hull Pyrolysis Bio-oils by Negative-Ion Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry[END_REF][START_REF] Smith | Bio-oil Analysis Using Negative Electrospray Ionization: Comparative Study of High-Resolution Mass Spectrometers and Phenolic versus Sugaric Components[END_REF][START_REF] Abdelnur | Characterization of Bio-oils from Different Pyrolysis Process Steps and Biomass Using High-Resolution Mass Spectrometry[END_REF]. Highresolution mass spectrometry (HRMS) is a powerful technique for the analysis of many kinds of mixtures and provides an important list of chemical molecular formulae allowing a deeper understanding of the composition of bio-oils. Moreover, the use of several atmospheric pressure ionization sources is essential to detect a large range of molecules [START_REF] Staš | Application of orbitrap mass spectrometry for analysis of model bio-oil compounds and fast pyrolysis bio-oils from different biomass sources[END_REF]. Some works have also demonstrated that additives like ammonium chloride or sodium hydroxide could enhance ionization efficiency of hemicellulosic and lignin derivative compounds [START_REF] Hertzog | Toward Controlled Ionization Conditions for ESI-FT-ICR-MS Analysis of Bio-Oils from Lignocellulosic Material[END_REF][START_REF] Boes | Characterization of biofuel refinery byproduct via selective electrospray ionization tandem mass spectrometry[END_REF][START_REF] Boes | Mass Spectrometry Exposes Undocumented Lignin-Carbohydrate Complexes in Biorefinery Pretreatment Stream[END_REF]. However, one issue associated with complex mixture analysis based on mass spectrometry is signal suppression due to competition between analytes during the ionization process, especially with ESI. The hyphenation with liquid chromatography should theoretically reduce this phenomenon but also allow to differentiate isomers. Although a quite high number of papers using HRMS with sample direct introduction have been published on biomass samples [START_REF] Hertzog | Toward Controlled Ionization Conditions for ESI-FT-ICR-MS Analysis of Bio-Oils from Lignocellulosic Material[END_REF][START_REF] Jarvis | Characterization of Pine Pellet and Peanut Hull Pyrolysis Bio-oils by Negative-Ion Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry[END_REF][START_REF] Abdelnur | Characterization of Bio-oils from Different Pyrolysis Process Steps and Biomass Using High-Resolution Mass Spectrometry[END_REF][START_REF] Staš | Application of orbitrap mass spectrometry for analysis of model bio-oil compounds and fast pyrolysis bio-oils from different biomass sources[END_REF][START_REF] Boes | Characterization of biofuel refinery byproduct via selective electrospray ionization tandem mass spectrometry[END_REF], a much smaller number of studies was dedicated to the hyphenation of LC with HRMS [START_REF] Jarrell | Characterization of organosolv switchgrass lignin by using high performance liquid chromatography/high resolution tandem mass spectrometry using hydroxide-doped negativeion mode electrospray ionization[END_REF][START_REF] Owen | High-performance liquid chromatography/high-resolution multiple stage tandem mass spectrometry using negative-ion-mode hydroxide-doped electrospray ionization for the characterization of lignin degradation products[END_REF][START_REF] Prothmann | Ultra-high-performance supercritical fluid chromatography with quadrupole-time-of-flight mass spectrometry (UHPSFC/QTOF-MS) for analysis of lignin-derived monomeric compounds in processed lignin samples[END_REF].

In the present study, the aim was to develop an effective LC-UV/MS method to reach optimal signal. We have used a response surface methodology with central composite designs (CCD) in order to increase the ionization efficiency of model compounds related to bio-oils. LC/MS experiments were carried out using a LC/FT-ICR/MS hyphenated system. One experiment design were done for each ionization mode: electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) both in positive and negative modes. Ahead the experiment designs, preliminary tests were made to fix some parameters like additives, split ratio and sprayer position). The objective was to develop a rational optimization of LC/MS method and thus to have a better control of the ionization efficiency. Our four methods were then applied to a lignocellulosic biomass pyrolysis oil.

Experimental

Chemicals

Methanol and formic acid were MS grade, purchased from VWR (Fontenay sous Bois, France). Deionized water was produced by a Milli-Q water purifier (Millipore SAS, Molsheim, France). Ammonium hydroxide, ammonium formate, 1-(4-hydroxy-3-methoxyphenyl)ethan-1-one (acetovanillone), phenylethanone (acetophenone), 1-(4-ethylphenyl)ethanone (4ethylacetophenone), 1,3-cyclopentanedione, 2-phenylbenzo[h]chromen-4-one (α-naphthoflavone), 2,5-hexanedione, 3-methyl-2-cyclohexen-1-one, 3-methyl-2-cyclopenten-1-one, 4methylcyclohexanone, benzene-1-,2-diol (catechol), 2-methoxyphenol (guaiacol), 1,3-dimethoxy-2hydroxybenzene (syringol), (3-phenoxyphenyl)methanol, 4-(hydroxymethyl)-2-methoxyphenol (vanillyl alcohol), butane-1,2,3,4-tetrol (erythritol), but-2-ene-1,4-dioic acid (fumaric acid), octodecanoic acid (stearic acid), benzoic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 4methylbenzoic acid (p-toluic acid), 4-hydroxy-3-methoxybenzoic acid (vanillic acid), 4-hydroxy-3,5dimethoxybenzoic acid (syringic acid), benzaldehyde, 4-hydroxybenzaldehyde and 4-hydroxy-3methoxybenzaldehyde (vanillin) were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France).

The fast pyrolysis bio-oil used in this study was produced from softwood sawdust and provided by IFP Energies nouvelles (Solaize, France).

Sample preparation

Model mixtures used for CCD were prepared in methanol at a concentration of 1 mM for each compound. One mixture contained acetovanillone, acetophenone, 4-ethylacetophenone, 1,3-cyclopentanedione, α-naphthoflavone, catechol, guaiacol, syringol, (3-phenoxyphenyl)methanol, vanillyl alcohol and erythritol. The second was composed of fumaric acid, stearic acid, benzoic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, p-toluic acid, o-toluic acid, vanillic acid, syringic acid, benzaldehyde, 4-hydroxybenzaldehyde and vanillin (Table 1). The fast pyrolysis bio-oil was 10 times diluted in methanol before injection.

Instrumentation

All experiments were performed using an Agilent 1290 UHPLC system (Palo Alto, CA) consisting of a binary pump, an autosampler, a temperature-controlled column compartment and a photo diode array (PDA) detector (volume UV cell 1 µL). The extra-column variance was measured at 9 µL². The chromatographic system was hyphenated to a Thermo Scientific LTQ-FT-ICR (Bremen, Germany) composed of a linear ion trap and an ion cyclotron resonance cell in a 7 Tesla superconducting magnet. Ionization was carried out with the ESI and APCI sources both working in positive or negative modes.

High performance liquid chromatography/high-resolution mass spectrometry

For LC/MS analysis, 1 µL of each sample was injected. Separation was achieved on a Kinetex C18 column (100 × 3 mm, particle size 2.6 µm, Phenomenex, Le Pecq, France) at 30°C. The mobile phase solvents used were a 0.01% (v/v) formic acid in water (A) and a 0.01% (v/v) formic acid in methanol (B) at 600 µL/min. A linear gradient was used as follows: 0.0 -4.0 minutes, 1%B; 4.0 -30.0 minutes, from 1% B to 99% B; 30.0 -35.0 minutes, 99% B; 35.0 -36.0 minutes, from 99% B to 1% B; 36.0-40.0 minutes, 1% B. UV signal was recorded from 210 to 400 nm.

To avoid excessive pressure on the UV cell and to be in optimal conditions for desolvation in ESI, LC flow was splited between UV detector and ESI source (Figure 1). Flow rates of nebulising, drying and sweep gases as well as transfer capillary temperature and voltage were optimized using a design of experiments approach (DoE). The spray voltage was -3.5 kV in negative mode and 4 kV in positive one. For APCI source, flow rates of nebulising and drying gases, as well as vaporizer temperature, transfer capillary temperature and voltage were optimized with a DoE. Discharge current was fixed at 20 µA in positive and negative modes. No sweep gas was used. For both API sources, a delta of 25 V between tube lens voltage and transfer capillary voltage was kept constant. The sprayer position was also optimized.

In order to have at least 10 points for each chromatogram peak, a resolving power of 12500 at m/z 400 was used for the FT-ICR spectrometer. The mass range was set to m/z 92-1000 with 2 µscans averaging. External calibration was accomplished with CalMix+ and CalMix-(Thermo Scientific) in positive and negative mode respectively.

For Flow Injection Analysis (FIA), LC conditions were unchanged. MS conditions with APCI source were as follow: nebulising gas flow rate 40 AU, drying gas flow rate 20 AU, sweep gas flow rate 0 AU, capillary voltage -50V, capillary temperature 250°C, tube lens voltage -75V. MS conditions with ESI source were : nebulising gas flow rate 30 AU, drying gas flow rate 25 AU, sweep gas flow rate 0 AU, capillary voltage -30V, capillary temperature 350°C, tube lens voltage -55V.

Direct introduction (-)ESI/FT-ICR/MS

Sample was diluted in methanol (1:50; v/v) prior to the introduction by infusion (5 μL/min). The number of µscans was set at 8 and 50 scans were accumulated with a resolution of 100000. Flow rate of nebulising gas was set to 5 AU. No drying and sweep gases were used. The spray voltage was -2.8 kV. Transfer capillary temperature, transfer capillary voltage and tube lens voltage were respectively set to 275 °C, -35 V and -110 V.

Software and MS data processing

Extracted ion chromatograms (EICs) were traced and integrated with Xcalibur 2.1 software (Thermo Scientific).

Construction of CCD, quadratic modellings of model molecules and optimization of MS parameters were done on Design Expert 10 software (Stat-Ease).

For molecular formulae calculation, homemade software was used. The following parameters were applied: elemental composition 12 C 1-100 , 1 H 1-100 , 16 O 1-100 and mass accuracy ≤ 2 ppm.

Results and discussion

Methodology for optimizing desolvation and ionization parameters

The objective of this work was to develop a rational strategy to optimize parameters of a LC-UV/MS analysis of complex matrices coming from biomass such as bio-oils. When liquid chromatography is hyphenated to mass spectrometry, desolvation and ionization of molecules are critical steps. Indeed, when LC is used without MS, there is no restriction about additive/salt concentrations neither about the LC flow rate which is directly related to chromatographic column's dimensions. To make LC/MS hyphenation possible, many parameters have to be adjusted: LC flow rate, split ratio between LC and MS system, nebulising, drying and sweep gas flow rates, transfer capillary temperature and voltage, tube lens voltage, vaporizer temperature for APCI, spray voltage or discharge current, API position are the main conditions to be investigated. To increase ionization efficiency, a make-up such as ammonium hydroxide in negative mode could also be added at the outlet of the LC system. In light of the large number of factors, to succeed this hyphenation and reach the real optimum, we propose a DoE methodology using central composite designs. Thereafter, a quadratic model was built, taking into account second order interactions.

This development was carried out on model compounds which are representative of bio-oils according to previous work [START_REF] Crepier | Ultra-high performance supercritical fluid chromatography hyphenated to atmospheric pressure chemical ionization high resolution mass spectrometry for the characterization of fast pyrolysis bio-oils[END_REF]. Two mixtures were used (Table 1):

-one for negative-ion ESI and APCI modes made of carboxylic acids and aldehydes -one for positive-ion ESI and APCI modes made of ketones and alcohols.

The main advantage to use model compounds was to cleverly optimize each ionization mode depending on the ability of compounds to accept or lose proton. Indeed, for example there is no point to improve the ionization of carboxylic acids in positive mode while they are well ionized in negative one thanks to the labile proton on the COOH function.

To build the four CCD, five significant parameters were chosen for each ionization mode in order to limit the number of experiments: flow rates of nebulising, drying and sweep gases, transfer capillary temperature and voltage (see part 3.2.). Tube lens voltage was associated with transfer capillary voltage by keeping a difference of 25 V between them in order to prevent ion source fragmentation. Other parameters which need manual adjustments were not included in the DoE and thus were optimized and fixed in advance: make-up and split of LC mobile phase at the entrance of mass spectrometer as well as the API source position. Impact of additives also called make-ups was also studied (see 3.1.). Chromatographic conditions were chosen to be compatible with mass spectrometry: for example concentration of formic acid was as low as possible but sufficient to keep analytes in their protonated form. Figure 1 illustrates the parameters which were either optimized in advance or with a DoE for ESI mode. To avoid any discharge and/or damage, authors decided to keep spray voltage at -3.5 kV and 4.0 kV for negative and positive ESI modes respectively. Moreover, according to previous publication about effects of several parameters linked to ion generation region on signal intensity using a similar ion source (electrospray ionization MAX2 source, Thermo Scientific); spray voltage was not found as a significant parameters at the opposite of the nebulising gas, capillary voltage and tube lens voltage. [START_REF] Lemonakis | Optimization of parameters affecting signal intensity in an LTQ-orbitrap in negative ion mode: A design of experiments approach[END_REF].

Adaptation of the LC downstream flow for MS compatibility

To optimize a LC/MS interface, it is primordial to well understand the process of ionization in order to focus on key parameters and adjust the flow rate and/or the composition of mobile phases to drive them compatible with ion source specificities.

In the case of ESI, the compounds diluted in the mobile phase are introduced by a capillary in an area at atmospheric pressure where an electric field of a few kV is applied. This electric field causes an accumulation of charges on the surface of the liquid causing the formation of a cone at the end of the capillary (Taylor cone). When coulombic repulsions exceed the surface tension of the liquid, charged droplets are then emitted from the tip of the cone. At this stage, several theories concerning the ionization process compete. The best known one is the residual charge theory introduced in 1968 by Dole et al. [START_REF] Dole | Molecular Beams of Macroions[END_REF]. According to them, a succession of fissions of the droplets alternating with the evaporation of the solvent cause the release of ions in the gas phase. Conversely, according to other theories, like the model of ion evaporation (Iribarne and Thomson), the ions would be ejected from the droplets or even ejected directly from the Taylor cone [START_REF] Iribarne | On the evaporation of small ions from charged droplets[END_REF]. In fact, the models may be complementary, and ionic evaporation may occur in the later stages of the residual charge theory [START_REF] Wilm | Principles of electrospray ionization[END_REF][START_REF] Kebarle | On the mechanisms by which the charged droplets produced by electrospray lead to gas phase ions[END_REF]. It should be noted that the formation of ions in the gas phase often ends not in the atmospheric pressure area but at the interface of the latter and the next area at reduced pressure.

Unlike ESI for which ions are pre-formed in solution, APCI requires that the molecules go into the gas phase prior to ionization. For this reason, the ionization source is heated between 300 and 500°C (vaporizer temperature) depending on the flow rate and the composition of the liquid. The formed vapor then passes through a stream of electrons emitted by a corona discharge which creates plasma around the needle.

Flow splitting

LC flow rate was 600 µL/min, which was too high for ESI. According to manufacturer, ESI could manage a flow of 1 mL/min, but optimal operation is obtained between 100 and 400 µL/min. To reduce the flow rate, a T-split was installed after the chromatographic column to prevent UV cell damages (Figure 1). Split ratio was calculated by divided flow rate at the outlet of the UV detector by the LC flow rate. The main drawback to split the mobile phase is solute dispersion, especially when a low split ratio has to be considered. Regarding theoretical considerations, total solute dispersion (σ² total ) can be assessed by peak variance coming from dispersion inside the column (σ² col ) and extracolumn dispersion (σ² ext ) (Eq 1). Extra-column dispersion results from injection, tubing, detector and split ratio (Eq 2) [START_REF] Bernardin | Suitable interface for coupling liquid chromatography to inductively coupled plasma-mass spectrometry for the analysis of organic matrices. 1 Theoretical and experimental considerations on solute dispersion[END_REF].

(1)

² ² ² (2) ² ² ² ² ²
To limit the loss of theoretical plates (N col ), extra-column dispersion must be as low as possible. The ratio between σ² col and σ² ext (β²) represents the percentage of remaining plates. Here, we were interested in the ratio between σ² col and σ² split . For a compound with a retention factor at elution (k e ) of 3, σ² col is equal to 187 µL² according to Eq 3.

(3) ² ² With V 0 the column dead volume and N col estimated by dividing the column length (L col ) by 3 times the pore size (d p ).

To calculate the total dispersion, Dorsey-Foley equation was used (Eq.4).

.

With F the mobile phase flow rate, As the peak asymmetry and W 0.1 the peak width both at 10% of the peak height respectively.

By removing the chromatographic column, σ² total corresponds to σ² ext . In order to measure only the dispersion due to flow splitter, T-split was first removed to calculate the dispersion from injection, tubing and detection. Then this measured dispersion was subtracted to σ² total to obtain only the split dispersion. Several split ratios were tested by changing the LC flow rate and keeping constant the flow coming into the mass spectrometer. By doing this, we could consider that dispersion of mass spectrometer was constant. The variation of tubing's dispersion related to LC flow modification was considered negligible before split dispersion (from 100 to 600 µL/min). Figure 2 shows the percentage of remaining plates according to split ratios. With a ratio of 0.20 (1:5), around 60% of plates were lost only because of the T-split which may lead to mix resolved peaks. To keep at least 70% of theoretical plates, a split ratio of minimum 0.45 (1:2.2) was needed which corresponds to a flow of 270 µL/min directed into the mass spectrometer. Thus, this ratio was used later on.

For APCI, no flow splitter was needed. Thanks to the tubular oven, APCI can handle higher flow rate than ESI. Moreover, APCI is a mass dependent ionization technique, which means a T-split would decrease the MS signal. At the opposite, ESI is concentration dependent, thus with adapted desolvation conditions, a flow splitter does not impact the MS signal [START_REF] Wolrab | Direct coupling of supercritical fluid chromatography with tandem mass spectrometry for the analysis of amino acids and related compounds: Comparing electrospray ionization and atmospheric pressure chemical ionization[END_REF].

Sprayer position

To optimize the source position of APCI and ESI, 4 model molecules were injected in FIA (Flow Injection Analysis) at a concentration of 1 mM. On the API used, four positions were possible from A which is the closest position to the transfer ion capillary, to D the furthest one. Each EIC was integrated and measured areas as a function of the source position are plotted in Figure 3.

For APCI, the source position did not have a real impact on the areas of EIC and thus on the ionization efficiency of model compounds. For ESI, areas of compounds could be 5 times lower for position A compared to position D. So for all other experiments, API was fixed in D position. These observations were consistent with the ionization process of each API source. Indeed, in APCI source, the whole mobile phase is vaporized and the ionization occurs in the gas phase by the use of a corona discharge. At the opposite in ESI source, ionization takes place in liquid phase with the formation of a spray due to high voltage applied between the ESI needle and the ion sweep cone. Thus the position of ESI sprayer have a direct impact on the electric field (E) which becomes more intense by bringing the sprayer closer (Eq.5).

(

5) E • •

With V the applied voltage, A an empirical constant estimated by Smith to be equal to 0.667, r the capillary emitter radius (cm) and d the distance between the ESI sprayer and the ground plane (cm) [START_REF] Thompson | Investigation of electrospray ionization and electrostatic focusing devices using a three-dimensional electrospray current density profiler[END_REF][START_REF] Smith | The Electrohydrodynamic Atomization of Liquids[END_REF].

It is important to note that position source optimum depends on many other parameters such as nebulising gas flow rate or transfer ion capillary voltage. These parameters were optimized later with a DoE.

Influence of additives

It is well known that mobile phase composition plays an important role during desolvation and ionization steps [START_REF] Dams | Influence of the eluent composition on the ionization efficiency for morphine of pneumatically assisted electrospray, atmospheric-pressure chemical ionization and sonic spray[END_REF][START_REF] Huffman | Effect of polar protic and polar aprotic solvents on negative-ion electrospray ionization and chromatographic separation of small acidic molecules[END_REF][START_REF] Kostiainen | Effect of Solvent on Dynamic Range and Sensitivity in Pneumaticallyassisted Electrospray (Ion Spray) Mass Spectrometry[END_REF][START_REF] Kostiainen | Effect of eluent on the ionization process in liquid chromatographymass spectrometry[END_REF][START_REF] Kebarle | From ions in solution to ions in the gas phase -the mechanism of electrospray mass spectrometry[END_REF]. In ESI, the solvent used must have a sufficient dielectric constant to form charged droplets as well as a low surface tension. Moreover, additives should be used with a concentration as low as possible. Indeed, ESI is a competitive ionization process. When several compounds are present in an electrospray droplet, they compete for a limited number of charged sites at the droplet surface, which can lead to ion suppression. In this study, the mobile phase contained 0.01% (v/v) of formic acid in order to keep carboxylic acids under their protonated forms during chromatographic separation. The measured pH of aqueous solvent was 3.2. For ESI positiveion mode, formic acid was a good source of protons; however for negative-ion mode, a pH around 8 would be preferred to help anion formation in the liquid phase. To evaluate the effect on the mass signal of formic acid added in the mobile phase as well as two other additives, 4 model compounds (vanillic acid, syringic acid, vanillin and syringaldehyde) were injected in negative ESI using formic acid, ammonium hydroxide and ammonium formate at concentrations varying from 0.1 µM to 50 mM.

Unexpectedly, formic acid did not deteriorate ionization efficiency at moderate concentrations (Figure 4). Loss of signal started around 1 mM. Between 0.1 µM and 1 mM, ionization of carboxylic acids was not really impacted by formic acid, whereas aldehyde responses were exalted. This phenomenon is called "wrong-way-round ionization" [START_REF] Kostiainen | Effect of eluent on the ionization process in liquid chromatographymass spectrometry[END_REF][START_REF] Wu | Favorable effects of weak acids on negative-ion electrospray ionization mass spectrometry[END_REF]. ESI process is complex and many characteristics of mobile phase composition influence ionization process such as volatility, surface tension, conductivity, ionic strength, pH, gas-phase ion-molecule reactions, etc. Indeed, even if an analyte is ionized in liquid phase, ionization of neutral analytes present in the gas phase could also occur by proton transfer reactions involving charged eluent species from solvents and/or additives. Such exaltation of aldehydes responses may be explained by the fact that ionization took place by gas-phase proton transfer or it may happen at the liquid-gas interface droplet. It is known that redox reactions occurring at ESI sprayer, i.e. reduction for negative-ion mode, create a significant difference of pH between the solution and the droplets [START_REF] Kebarle | From ions in solution to ions in the gas phase -the mechanism of electrospray mass spectrometry[END_REF]. Addition of protons from acids could facilitate chemical reduction and so decrease the pH at the surface of droplets [START_REF] Cech | Practical implications of some recent studies in electrospray ionization fundamentals[END_REF]. Thus even if solutes were not ionized inside the mobile phase, modification of pH droplets could make ionization in liquid phase possible. However, this theory was not in agreement with the fact that carboxylic acids had an almost constant response although aldehydes shown an increasing of their responses, except if carboxylic acids were already well ionized without formic acid because of their low pKa.

Similarly to formic acid behavior, ammonium hydroxide and ammonium formate enhanced aldehyde responses, especially vanillin signal. These additives increased the pH, thus ionization could occur directly inside mobile phase. The main differences between ammonium hydroxide and formate were an enhancement less important for ammonium formate than ammonium hydroxide and an important ion suppression at high concentration of ammonium formate which did not occur for ammonium hydroxide. This last phenomenon could be due to the anionic part of ammonium formate HCOO -which is bigger than OH -for ammonium hydroxide and so had a better affinity for droplet surface leading to a competition with solutes at high concentrations.

During this study, 0.01% (v/v) of formic acid was added to mobile phase, which corresponds to 2.7 mM. These conditions were not unfavorable as predicted; thereby no other additive was used at the outlet of LC system.

Optimization of ESI and APCI desolvation conditions by mean of central composite design

CCD was chosen in this study in order to take into account second order interactions. For each ionization mode, a DoE was established including the 5 key parameters. For ESI, nebulising, drying and sweep gas flow rates, transfer ion capillary temperature and voltage were studied. For APCI, preliminary experiments showed that sweep gas flow rate did not influence ionization, thus it was excluded of the design and vaporizer temperature was added to the study. The investigated ranges for each variable are summarized in Table 2. These ranges had to be restricted to make the later second order modelling possible and in the same time sufficient to consider a relevant signal.

Each CCD was composed of a factorial design with 2 k experiments (k is the number of factors, here 5), each factorial point was repeated 3 times -2×k axial points also repeated 3 times -5 central points.

In this work, points were repeated whatever the conditions were, not only on central point as it usually done in literature. In this way repeatability of runs was estimated and unstable ionization conditions were pointed out, especially spray that may be not stable in the ESI source. In total, 131 injections of our model mixture were done for the four ionization modes. EIC were integrated for the 12 model compounds. Measured areas were used as responses of CCD and were exploited by creating one quadratic model per molecule. Models were constructed with 80% of experiments, the remaining 20% part was used as validating points. Last step was optimization of model compound responses simultaneously with desirability function in order to obtain optimized conditions. Optimization of negative APCI is developed in this paper as example.

To correlate responses with factors, a second order model with interaction was fitted to the experimental data. The general form of second order function is:

(6) ∑ ∑ ∑ ∑
Where y is the response, β 0 the constant, β i the first order coefficients, β ii the quadratic coefficients and β ij the interactive coefficients.

A model involving 5 factors requires calculation of 21 coefficients. First, to simplify the model, only significant quadratic and interactive coefficients with a p-value lower than 0.1 were kept. Analysis of variance (ANOVA) for response surface reduced quadratic model of vanillic acid is shown in Table 3.

The model F-value was equal to 58.42 indicating that the model is significant (P<0.05) and there was only 0.01% chance that the F-value this could occur due to noise. P-value less than 0.05 indicates that model terms are significant (with α = 0.05). In this case A, B, D, E, AB, AC, AE, BC, BD, CD, CE, A², C² and D² were significant model terms. First order coefficient C, corresponding to capillary temperature, was not significant; however quadratic coefficient C² and several interactive coefficients with C were significant. R-squared value was 0.9148 representing a good agreement between experimental results and the model predictions. Moreover R-squared increased every time when an independent variable was added to the model, thus regression model containing more variables than another one is expected to provide a better fit (main risk being model overfitting in this case). On the contrary, adjusted Rsquared increased only if a new term improved the model fit otherwise it decreased. Predicted Rsquared was used to determine how well model makes predictions. To calculate this value, a data point from the dataset was removed, then the regression equation was calculated, the last step was to evaluate how well the model predicts the missing point. This procedure was done for all data points. In this case, predicted R-squared was 0.8786 and it was in good agreement with the adjusted R-squared of 0.8992.

Once the model was built, several model evaluation plots could be traced. Figure 5 shows the experimental data versus predicted value plot for the vanillic acid model. It can be observed that points are around the line, which means model fit to experimental data. Moreover, validation points, notched points on the graph, were quite well predicted.

Interactions between factors could be represented with response surface. As an example, Figure 6 illustrates the response surface of ion transfer capillary temperature and ion transfer capillary voltage for vanillic acid. For low capillary voltage, the optimum capillary temperature was higher than 350°C while for high capillary voltage, a capillary temperature lower than 350°C was preferred.

When all the models for compounds were built, a desirability function was used to maximize at the same time all the responses for predicted optimal conditions (Table 4). Figure 7 illustrates perturbations at optimized conditions for the desirability and for two model compounds (vanillic acid and 4-hydroxybenzaldehyde). Optimal conditions will be applied for complex and unknown samples containing similar analytes than chosen model compounds in this study. Thus a compromise was made in order to obtain a sufficient signal for all model compounds without reaching the maximum. For example, the value of C factor, ion transfer capillary temperature, was optimized at 376°C, but it would be better for vanillic acid to use a lower temperature. As regards 4-hydroxybenzaldehyde, a slightly higher temperature would give a better response. Another remark concerns the stability of optimal conditions. The perturbation of desirability shows a plate, except for B factor (drying gas). This means that for example, if needed, smaller vaporizer temperature (E factor) could be used without having important consequence on signal. Globally, higher optimal nebulising and drying gas flow rates were obtained in positive modes than in negative ones. To facilitate desolvation, high temperatures were preferred in negative modes.

Application to a lignocellulosic biomass pyrolysis oil

Optimized ionization conditions were applied to a lignocellulosic biomass pyrolysis oil. LC-UV/HRMS analysis resulted from five complementary detections: UV, ESI and APCI in positive and negative modes. Moreover, all UV spectra were recorded from 210 to 400 nm. Molecular formulae were identified based only on carbon, hydrogen and oxygen elemental composition with an accuracy of 2 ppm (see part 2.5).

UV chromatogram at 254 nm and base peak chromatogram (BPC) of a lignocellulosic biomass pyrolysis oil are shown in Figure 8 as examples. Optimized ESI negative-ion mode showed a high sensitivity, especially between 10 and 30 minutes.

By integrating all BPC data coming from ESI negative-ion mode, two main zones are clearly distinguished on the mass spectrum (Figure 9). The first one includes m/z ratios varying between 100 and 450 whereas the second area ranges from 450 to 900 m/z ratios. When the pyrolytic oil was infused into the FT-ICR mass spectrometer without any chromatographic separation dimension, the second zone was not as well detected because of ion suppression.

When dealing with complex samples producing a large number of data to cope with, van Krevelen diagram is a good way to represent MS data. The highest number of molecular formulae was achieved with ESI negative-ion mode (ca. 5500 formulae). At the opposite only 500 formulae were identified with APCI negative-ion mode. Since they contain significant contents of sugars and other heat sensitive compounds, lignocellulosic bio-oils are known to be thermally unstable. So during MS analysis, compounds burned and formed a black substrate deposited on the tube oven of the APCI source because of the too high temperature (450°C). Experiments at 350°C did not improved the signal. ESI is a softer ionization source than APCI, thus it is more suitable for this kind of sample. However, APCI do not formed adducts during ionization process which can simplify data interpretation. In comparison to negative ionization mode, only 900 molecular formulae could be calculated with positive ionization mode (APCI and ESI). A van Krevelen diagram including all ionization modes was built (Figure 10). Compounds eluted with less than 20% of MeOH were represented in red, in blue those eluted between 20 and 80% of MeOH and in green with more than 80%. Red points correspond mainly to sugaric compounds coming from the degradation of cellulose and hemicellulose. These carbohydrates are very polar compounds and thus were non-retained on apolar stationary phase. Compounds showing a higher affinity with the stationary phase (blue points) correspond to pyrolytic lignin (phenolic compounds). Finally, green points were eluted with more than 80% of MeOH, they might correspond to lipids (fatty and resin acids).

It has to be noted that only 2500 molecular formulae could be attributed by direct introduction in negative ESI against 5500 with LC/MS due to matrix effects leading to ion suppression. Figure 9 already proved that heavy compounds (m/z > 500) were not well ionized with direct introduction. To target molecule class which suffer from ion suppression in infusion, the heteroatom class distribution for LC/MS and direct introduction, with oxygen families ranging from O 0 to O 25 is presented in Figure 11. It appears that molecules having low number of oxygen atoms (< 4) were not detected with direct introduction. Thus prior separation with liquid chromatography greatly enhances the detection of heavy and moderately polar molecules.

In addition to limit ion suppression, liquid chromatography hyphenated to mass spectrometry allowed to distinguish isomers which is not possible with direct introduction. An example is presented in Figure 12. EIC of m/z 259.0975 ± 0.0005 in negative ESI corresponding to C 15 H 16 O 4 shows four intense peaks and other smaller ones at different retention times indicating the presence of several isomers.

Conclusion

Despite LC-UV/HRMS analysis has shown a great interest for analysis of complex matrices since several years, no rational optimization methodology was proposed in order to obtain a sensitive but also controlled analytical method. In this work, a design of experiment was used to improve the desolvation and ionization steps of a system involving a reversed phase column hyphenated to a FT-ICR high resolution mass spectrometer. The effects of five experimental key parameters were studied using ESI and APCI sources in negative and positive-ion modes. In our study, more than 130 experiments were performed for each ionization mode, using a synthetic solution containing 12 oxygenated model compounds. This chemometric approach allowed to take into account all possible second order interactions, which will not be possible with the classic optimization 'one variable at a time'. Quadratic models were built for each model compound, then their responses were maximized at the same time to conclude about optimal conditions. Moreover, some parameters (split ratio, sprayer position and additives) were investigated prior to carry out the design of experiments. As regards ESI analyses, a T-split was added in order to decrease the mobile phase flow rate at the entrance of the mass spectrometer. However dispersion induced by such system must be considered to keep enough theoretical plates and narrow peaks and achieve a satisfactory chromatographic separation. Signal detected in ESI was highly dependent on sprayer position, contrary to APCI. In our conditions (LC mobile phase containing 0.01% of formic acid), no supplementary additive was needed at the outlet of the chromatographic column.

Optimized conditions were applied to a lignocellulosic biomass fast pyrolysis oil to illustrate the efficiency of such a methodology. LC-FT-ICR/MS analysis in ESI negative-ion mode provides a detailed characterization of our complex bio-oil, delivering more than 5500 molecular formulae. Hyphenation coupling a chromatographic separation to high-resolution mass spectrometry was demonstrated as on one hand, LC first dimension enables to alleviate ion matrix effects and differentiate isomers having the same molecular formulae but different retention times, and on the other hand, precise and reliable molecular formulae are proposed for a large number of oxygenated compounds.

Table 1 :Figure 1 :Figure 2 :Figure 3 :Figure 4 : 2 : 3 :Figure 5 :Figure 6 :

 112342356 Model mixtures for negative-ion (left) and positive-ion (right) modes Key parameters for ESI detection optimized with DoE (red boxed text) and before DoE (purple boxed text) Percentage of remaining plates according to the split ratio Areas of EIC for 4 model compounds according to the ESI and APCI position (negative-ion mode). [LC and MS conditions : see Experimental part] Effect of formic acid, ammonium hydroxide and ammonium formate on negative-ion ESI mode responses of 4 model compounds. The vertical axis represents the percentage ratio of peak area in the presence of additive to peak area in the absence of additiveTable Central composite designs for ESI and APCI both in positive and negative modes Table ANOVA for response surface reduced quadratic model of vanillic acid in negative APCI Experimental data versus predicted value plot for the vanillic acid model Response surface plot of ion transfer capillary temperature and ion transfer capillary voltage. Nebulising gas flow rate, drying gas flow rate and vaporizer temperature were fixed at respectively 30 AU, 20 AU and 350°C in this representation.

Figure 7 : 4 :Figure 8 : 9 :Figure 10 :

 748910 Figure 10: Van Krevelen diagram of a lignocellulosic biomass pyrolysis oil.Figure 11: Comparison of heteroatom class distributions between LC-(-)ESI/FT-ICR/MS and (-)ESI/FT-ICR/MS Figure 12: Extraction ion chromatogram of a lignocellulosic biomass pyrolysis oil for of m/z 259.0975 +/-0.0005

Figure 11 :Figure 12 :

 1112 Figure 10: Van Krevelen diagram of a lignocellulosic biomass pyrolysis oil.Figure 11: Comparison of heteroatom class distributions between LC-(-)ESI/FT-ICR/MS and (-)ESI/FT-ICR/MS Figure 12: Extraction ion chromatogram of a lignocellulosic biomass pyrolysis oil for of m/z 259.0975 +/-0.0005

Acknowledgments

The authors thank Abdelaziz Faraj from IFP Energies nouvelles for his help with design of experiments.


Xavier Mangenot

Jean-François Deconinck

Magali Bonifacie

Virgile Rouchon

Pierre-Yves Collin

Marta Didier Quesne

Jean-Pierre Gasparrini

Sizun

Jean-Francois Deconinck

Yves Collin

Didier Quesne

Marta Gasparrini

Jean-Pierre Sizun

C E Bennett

FORTEY D Calmels

Munnecke A & Vandenbroucke

Thermal and exhumation histories of the northern subalpine

Keywords: subalpine massifs, ∆ ∆ ∆ ∆ 47 clumped isotope thermometry, Thermicity, Clay 45 diagenesis, Kinetic models. 46

published or not. The documents may come

and chlorite at the expense of smectite in the more internal domain of the northern subalpine 140 chains. They interpreted the mineralogical evolution as the expression of the tectonic overload 141 (prealpine nappes) in the eastern part of the subalpine basin. Consistently, more recent data on 142 vitrinite reflectance revealed an increase of organic matter thermal maturation in the 143 northeastern part of the subalpine chains (Gorin & Monteil, 1990, Butler, 1991;Moss, 1992).

144 Such a combination of independent thermal indicators indicates that the northeastern part of 145 the chain suffered higher temperatures, due to the greater tectonic loads exerted onto the 146 subalpine chains. Thermal data and modeling results suggest that the eastern Bornes and 147 Bauges massifs have experienced a burial depth up to 5 km greater than their western areas 148 (Moss, 1992). Butler (1991) estimated maximal temperatures induced by this tectonic load 149 around 180°C in the eastern domain and 60-70°C in the western domain. This study focuses on a Late Cretaceous calcareous unit called the "sublithographic 155 limestones" ranging in age from the Turonian to the Campanian (Villard, 1991). The major 156 rock-forming constituents are micrite particles, calcareous ooze, and allochems fragments, 157 including planktonic and benthonic foraminifera and calcispheres (coccolithes fragments).

158

These limestones were chosen for the two following reasons: (1) the "sublithographic 159 limestones" consist of a fine grained and homogeneous micritic formation containing small 160 amounts of clay minerals (<5%), allowing for clay diagenesis and ∆ 47 composition to be 161 measured conjointly on the same samples, and (2) it is a well exposed unit across the fold and 162 thrust belt this allowing for collecting samples through a large range of low-grade 163 metamorphism domains. The "sublithographic limestones" are generally well-exposed to the 164 heart of the synclines of the Bauges and Bornes massifs. They superimpose condensed 165 horizons of glauconite and phosphate bearing green sandstones of Aptian/Albian age 166 (Delamette et al., 1997) and are unconformably overlain by Cenozoic deposits (Fig. 2). A 167 total of 110 samples were collected from 18 sites (Fig. 1) for XRD analyses, from which 8 168

Scanning Electron Microscopy (SEM) and petrophysical properties 190 191

To ensure the homogeneity of the petrographic characteristics of all the samples studied, we 192 made SEM characterization of micrite microtexture on fresh surfaces, without acid attack, and 193 after gold plating, and we measured the petrophysical properties (porosity and permeability) 194 from plugs obtained from the same samples. Water porosimetry was performed to quantify 195 total porosity (connected porosity) using Archimedes method (Richard et al., 2007, Cavailhes 196 et al., 2013). Plugs were first dried in an oven at 60°C until they reached a stable mass (m d ) 197 and then put upright in an airtight enclosure for 24 hours. at room temperature under vacuum 198 at 2.6 Pa. They were progressively imbibed from their base with degassed deionized water, 199 under a dynamic vacuum. The total porosity φ (%) is given by: φ = [(m 1 -m d ) / (m 1 -m 2 )] × 200 100, where m 1 is the mass of the sample saturated with water and m 2 is the mass of the 201 sample saturated with water weighed under water (hydrostatic underwater weighing).

202

Permeability measurements (K) were performed on the same plugs with a gas permeameter 203 equipped with a Hassler cell using nitrogen and the steady state flow method. They were 204 made under an effective pressure of 0.1 MPa and then corrected for the Klinkenberg effect 205 using a graphical method (Zinsner & Pellerin, 2007;Cavailhes et al., 2013) The convergence criteria between measured data versus model predictions were 262 iteratively defined to minimize: (1) the discrepancy between the whole dataset obtained by the 263 mean of three independent paleothermometers (%illite in I-S, Ro% and T∆ 47 ), and (2) the gap 264 between the measured data for each paleothermometer and the values computed 265 mathematically for each proxy (%illite in I-S, Ro% and T∆ 47 ), given the different time-266 temperature paths to evaluate. A good match between data and models was observed by 267 considering typical errors of ±0.1% for Ro%, ±10-15°C for T∆ 47 and ±10% for %illite in I-S. The semi-quantitative XRD bulk rock analyses showed that the studied limestone unit 272 is mostly composed by calcite (>95%) with a minor proportion of clay minerals (<5%) 273 including glauconite in some samples. XRD analyses on HCl-insoluble fraction revealed trace 274 concentration of quartz, opal CT and K-feldspars. Table 1 shows the results of the increases, and these I-S now mostly correspond to R1 ordering scheme. The last type

295

(Sections C14 to C18) shows higher proportions of chlorite and illite, a lower amount of I-S 296 with more than 90% illite layers, now corresponding to R3 ordering scheme (Fig. 3). The stable isotopes data (δ 18 O carb /δ 13 C carb /∆ 47 ) acquired on eight samples collected in 316 the three different geographical domains (A, B and C) are reported in Table 1 and illustrated 317 with different colors in the cross plots of Figure 5. Despite the fact that the samples were 318 collected in different localities tens of kilometers apart, they all exhibit small ranges of 319 variations in δ 18 O carb (between -3.15‰ and -3.89‰; n=8 samples) and δ 13 C carb (from 1.6‰ to 320 2.90 ‰; n=7 -sample A1 is offset with a δ 13 C carb = 0.04‰). This small range of variation 321 likely reflects the fact that all samples precipitated (or recrystallized) in comparable 322 environmental conditions (i.e., temperature, oxygen isotope composition of the parent fluid,

323

water to rock ratios).

325

The ∆ 47 values of the samples investigated here are varying between 0.634‰ and 326 0.400‰. The external reproducibility out of 2 to 4 replicate analyses of the same micrite 327 powder ranges between ±0.010‰ and ±0.049‰ (± 1 S.D., with one offset for sample B12 328 ±0.076‰; Table 1). This is larger than the external reproducibility obtained in this study on 329 homogeneous standards (± 0.018‰, n=49) and likely reflects the relative isotopic 330 heterogeneity of the micrite samples investigated here (compared to homogeneous standards).

331

In contrast with the regional-scale homogeneity on δ 18 O carb and δ 13 C carb compositions of the The "sublithographic limestones" do not contain organic matter. However, thermal and Bornes) did not experience burial temperature above 70-80°C (Fig. 7). Although the investigated lithology consists of a micritic limestone with a CaCO 3 431 content of up to 95%, and potassium deficiency, the illitization processes described in this 432 study are comparable to those described in argillaceous rocks or ashes layers, confirming that 433 temperature is the dominant controlling factor at regional scale (Środoń & Clauer, 2001). , 1983, , Ferry et al., 1983;;Deconinck & Debrabant, 1985;Levert & Ferry, 1988). 5).

471

This suggests that all the micrites from this study likely recrystallized at higher temperatures 472 than the original one at depositional site. Such hypothesis finds support in two observations.

473

First, the limestone units, initially composed of calcareous ooze with planktonic foraminifera The three proxies (i.e. clay diagenesis, organic matter maturity and carbonate clumped

250

  Furthermore, in order to consider mid-temperature scenarios, we also integrated five 251 Page 6 of 30 Basin Research additional time-temperature paths by adjusting the maximal burial temperature from 80 to 180°C. We computed the expected changes in vitrinite composition, ∆ 47 temperatures and % 253 illite in I-S with time and temperature, for comparison with the measured values. Three 254 empirical kinetic models were used with sequential time-temperature steps of 1Ma: (1) 255 Vitrinite maturation was predicted by the Easy% Ro kinetic model of Sweeney & Burnham, 256 1990; (2) The extent of solid-state C-O bond reordering over time was simulated using the ∆ 47 257 kinetic model of Henkes et al. (2013) (see Shenton et al. 2015 for an application); and (3) the 258 extent of smectite to illite conversion in I-S was calculated following the kinetic model of 259 Huang et al., 1993.260 261

  275mineralogical, petrographic, petrophysical and geochemical analyses of the clay and 276 carbonate components from the investigated limestone unit.

  Clay mineralogyl results are presented in figure2, where the relative proportions of 281 the different minerals identified in the samples from the eighteen sampled sections are 282 reported. From top to base are presented the results from sections located respectively in the 283 external and internal domains (A, B, C) of the Bauges and Bornes massifs. In the studied area, 284 clay minerals include illite, illite/smectite mixed-layers (I-S), chlorite, chlorite/smectite 285 mixed-layers (C-S) and some kaolinite. Small quantities of quartz occur in the clay fraction of 286 all samples. Three types of clay mineral assemblages are distinguished, depending on the 287 geographical location of the samples (c.f. A, B and C domains). The first type (Sections A1 288 to A8) refers to clay mineral assemblages largely dominated by illite/smectite mixed-layers 289 expanding to 17 Å after glycolation and therefore corresponding to I-S R0 ordering scheme 290 (Fig. 3). They are associated with illite and occasionally with small amounts of C-S and 291 kaolinite. The second type of clay mineral assemblages (Sections B9 to B13) is characterized 292 by higher percentages of illite, a decreasing proportion of I-S, and the systematic occurrence 293 of chlorite and C-S. In samples from the B domain, the proportion of illitic sheets in I-S Page 7 of 30 Basin Research

  part (C domain) of the studied subalpine massifs, the clay mineral 406 assemblages are dominated by chlorite, illite and I-S R3 containing a small proportion 407 (mostly below 10%) of smectite sheets. This type of assemblage results from the illitization of 408 I-S R0 through smectite dissolution/precipitation processes (Nadeau et al., 1985) and from the 409 transformation of these smectitic minerals into chlorite. The C-S present in the eastern 410 sections are likely the intermediates of smectite to chlorite transformations. The difference in 411 the composition of clay assemblages between the western and the eastern part of the subalpine 412 massifs was already noticed and interpreted as due to tectonic overload triggered by the 413 prealpine nappes (Deconinck & Debrabant, 1985). New data provided in this study suggest 414 that samples from the C domain likely experienced maximum temperatures up to 140°C, 415 confirming that thermal diagenesis was responsible for the illitization and chloritization of I-S 416 R0 (Fig. 7), the increase of vitrinite reflectance (up to 1.3%) and of Rock-Eval T max 417 (T max >460°C) in these localities. The relationship observed in the studied area between the 418 Rock-Eval T max and the illite content in illite-smectite (I-S) mixed layers agrees with the one 419 observed by Dellisanti et al. (2010) in similar syn-orogenic successions from the Northern 420 Apennines (Italy). These results are also concordant with the maximum burial temperature of 421 Page 10 of 30 Basin Research 180°C estimated in the studied area by Butler, (1991). minerals and organic maturity data indicate that the B domain experienced a moderate 426 thermal influence compared to the external and internal domains (A and C, respectively).

434

  However, in the B and C domains, chloritization processes are progressively increasing from 435 West to East where and the chlorite proportion reaches 50% of the clay fraction. Such chlorite 436 proportions cannot be explained by depositional heterogeneities linked to detrital input. 437 Indeed, the hot and hydrolyzing climates dominating the Late Cretaceous time did not allow 438 chlorite preservation. Furthermore, in the absence of diagenetic transformations, the chlorite 439 proportions are always very low in Upper Cretaceous sediments (Jeans et al., 2001; 440 Deconinck et al., 2005). The chloritization processes in the studied carbonate succession were 441 probably favored by the magnesium release from the transformation of High-Magnesium 442 Calcite (HMC) into Low-Magnesium Calcite (LMC) during diagenesis. This process is 443 particularly evident in marl-limestone alternations of the subalpine ranges where I-S R0 444 evolved into illites within the marls and into chlorites within the limestones (e.g. Deconinck 445 & Chamley

  In the studied sublithographic limestones, the micrite samples record contrasting ∆ 47 450 temperatures clustered in two distinct groups, depending on their geographic locations. The 451 micrites from the A and B domains have T∆ 47 clustered at 51±8°C (n=4 samples) which 452 sharply contrast with the micrites from the more internal C domain, displaying T∆ 47 clustered 453 at 183±17°C (n=4 samples). Given the fact that all the investigated micritic calcites from all 454 domains show very similar micro-texture, petrophysical properties, as well as δ 13 C and 455 δ 18 O carb isotopic compositions (Fig.4, 5 and Table1), it is unlikely that this sharp difference in 456 T∆ 47 values could result from different recrystallization events. We suggest that the high 457 temperatures recorded in samples of the eastern C domain result from 13 C-18 O bonds re-458 equilibration via solid-state diffusion at temperatures higher than the blocking temperature. 459 Such hypothesis also finds support in the fact that: (1) the calculated δ 18 O water of about 19‰ 460 for micrites of the C domain are too high to be reconciled with the most common δ 18 O water of 461 diagenetic fluids in sedimentary basins (Karhaka et al. 2003); (2) clay mineral and vitrinite 462 reflectance data indicate a maximal burial temperature up to 160-180°C for this area.

474

  and coccoliths fragments, now exhibit numerous petrographic hints of recrystallization (i.e.475particle shapes, type of contact between grains and crystallometry parameters). Second, the 476 measured T∆ 47 show higher temperatures than coeval shallow seawater(i.e., 20-26°C 477 followingJenkyns et al. 1994). Noticeably, the T∆ 47 of micrite samples from the external 478 domains (A and B), not affected by ∆ 47 resetting, point toward a recrystallization event 479 occurring during shallow burial, at temperatures close to 50°C and by fluids with δ 18 O water 480 values of +1.7±1.2‰. Such recrystallization event may correspond to the transformation of 481 the metastable High-Magnesium Calcite (HMC) into Low-Magnesium Calcite (LMC) of the 482 micritic sediment (Schlager & James, 1978). 483 Overall, the micrite samples investigated here exhibit apparent T∆ 47 values that are 484 expected to be an integration of: (1) temperature, timing, and extent of the shallow burial 485 recrystallization occurring for all geographic domains (A, B and C) and (2) an additional 486 overprint by solid-state C-O bond reordering only in samples from the C domain, which was 487 function of the thermal history and of specific C-O bond reordering kinetics of ∆ 47 paleothermometric results acquired on three samples representative of 493 the three geographic domains investigated in this study, we employed a forward modeling 494 approach to find the best thermal scenario to reconcile all the obtained ∆ 47 , vitrinite 495 reflectance and clay mineral data. This approach is devoted to refine the burial and 496 exhumation history of the sampled areas. The model results are shown in Figure 7 where 497 seven different time-temperature scenarios, with varying maximal burial temperature (MBT) 498 are considered. These scenarios are: (1) pre-thrusting burial (88-65Ma); (2) Eocene inversion 499 (65Ma-45Ma); (3) main emplacement of thrust units and associated heat increase (40Ma to 500 8Ma); and (4) erosion (8Ma-present).501 502 First of all, the results of the forward models for each paleothermometric proxy, 503 demonstrate that model predictions and data may converge for a set of thermal histories (TH) 504 Page 12 of 30 Basin Research with maximal burial temperature (MBT) ranging from 60°C to 180°C. More specifically, 505 given the measured Ro%, ∆ 47 and % illite in I-S mixed layer for the three selected samples 506 (c.f. A1, B9 and C16 samples), the forward modeling results predict that: 507 508 TH1-TH2 (MBT between 60°C and 80°C) are the most likely scenarios suggested for 509 A1 sample (A domain) because these time-temperature paths are the only ones prone to 510 generate a substantial agreement between all the available paleothermometric data. As 511 discussed previously, sample A1 exhibits a T∆ 47 of 61±15°C suggesting a recrystallization 512 event under shallow burial temperatures, without any record of the successive solid-state 513 diffusion overprint. In this case, ∆ 47 thermometry only preserves a screenshot of punctual 514 recrystallization temperature and does not provide important information on the sample 515 thermal history. The weak maturity recorded for the A domain by vitrinite reflectance data 516 (Ro%: 0.45±0.05) correlated with a low %illite (:25±4%) in I-S mixed-layers, and is also in 517 good agreement with TH1-TH2 model prediction, suggesting that A1 sample likely 518 experienced maximum burial temperature in the range 60-80°C. 519 520 Following the same model predictions, it comes out that mid-scenarios TH3-TH4, 521 with MBT between 100 and 120°C, are the most susceptible scenarios to conjointly increase 522 the illite content in I-S (between 51 to 93 %illite) and vitrinite reflectance (between 0.55 to 523 0.67%), without providing sufficient activation energy to initiate ∆ 47 kinetic C-O bond 524 reordering. The paleothermometric data available for sample B9 (c.f., Ro%: 0.7±0.1; I% in I-525 S:48±9% and T∆ 47 :54±9°C) from the B domain agree perfectly with model predictions for 526 TH3-TH4. Therefore, sample B9 likely experienced maximal burial temperatures ranging 527 from 100 to 120°C. 528 529 Finally, sample C16 from the C domain exhibits T∆ 47 in excess of 120°C compared to 530 samples A1 and B9, a clear increase of vitrinite reflectance values (Ro% = 1.3±0.2) and a 531 near total illitization of I-S mixed layer (up to 90% illite). These data clearly indicate a 532 significantly higher level of thermal maturity in the C domain. First, ∆ 47 data in sample C16 533 have been previously interpreted as reflecting the attainment of ∆ 47 blocking temperatures 534 reached during the cooling stage (i.e. from 8Ma to present-day), that followed a complete re-535 equilibration of ∆ 47 compositions at maximal burial temperatures (see Fig.7.B for more 536 detail). ∆ 47 kinetic modeling results showed that only TH7 (maximal burial temperature 537 >180°C), or hotter scenarios, may provide sufficient activation energy to generate the 538 observed complete re-equilibration of ∆ 47 in C16 samples. This finding is a strong argument 539 in favor of maximum burial temperatures higher than 180°C in this area. Then, the available 540 Ro% values and the near complete illitization of I-S mixed layer observed in C16 sample, 541 also showing a substantial agreement with TH7 modeling predictions, conclusively point 542 toward a temperature regime with maximum temperature close to 180°C.

  Guided by the data-model comparisons presented on three representative samples of 545 the studied area, we provide quantitative information on paleotemperatures and, indirectly, on 546 sample burial and exhumation histories. In light of these results, it is clear that samples 547 Page 13 of 30 Basin Research collected from the same stratigraphic interval (Upper Cretaceous "sublithographic 548 limestones"), and from closed geographic areas, may have experienced very contrasting 549 thermal histories, with maximum burial temperatures varying from 60-80°C in the western 550 external domains (A and B), up to 180°C in the internal domain (C) of the studied area. These 551 heterogeneities in the paleothermal record may reflect significant changes in overburden, 552 induced by the emplacement of thrust-sheet units with variable thickness. Indeed, by 553 considering a regional geothermal gradient of 25±5 °C/km for the time encompassing tectonic 554 loading and exhumation (Schegg, 1992b), the paleothermometric data suggest that the 555 prealpine nappes, presently eroded, were 4-7 km and 1.3-2km thick in the eastern and western 556 domains, respectively. These results are consistent with previous estimation of Deville & 557 Sassi (2006) who calculated that more than 4-6 km of prealpine nappes was thrust in the 558 North-West of the studied area. However, we cannot rule out that the East to West increase in 559 paleotemperatures, recorded in this study, may also reflect an heat flow increase without 560 significant additional burial. The definition of the precise process causing this temperature 561 gradient is beyond the scope of this study and would require in-depth thermokinematic 562 modeling to be addressed.

567

  isotope composition) used to reconstruct the thermal history of an Upper Cretaceous 568 limestone unit from the northern subalpine domain (Bauges and Bornes massifs, northern 569 French Alps) provided a coherent dataset showing the existence of a prograde diagenetic 570 gradient from West to East, notably due to the tectonic emplacement of the prealpine nappes. 571 Although the studied sediments correspond to micritic limestones, clay mineral diagenesis is 572 expressed, similarly to what is expected for argillaceous rocks, by the progressive illitization 573 of smectites. The degree of illitization makes it possible to identify three mineralogical zones 574 within the studied massifs: a western zone where sedimentary I-S R0 are preserved, 575 suggesting that the burial temperatures never exceeded 70°C; an intermediate zone marked by 576 the presence of I-S R1, indicating burial temperatures in the range 100-120 °C; and an eastern 577 zone marked by the presence of I-S R3, indicating temperatures in the order of 160 to 180°C. 578 Organic matter maturity data (from T max and Ro%) are consistent with the clay mineral 579 dataset, pointing at an immature organic matter in the western part of the subalpine massif and 580 a prograde increase of T max and Ro% values toward the East. The low ∆ 47 temperatures 581 recorded in micrite samples from the western area of the subalpine massifs are in excess of 582 ~20°C of plausible depositional temperatures, suggesting that micrite recrystallized at such 583 temperatures. However, in the eastern part of the massifs, four samples collected 30km apart, 584 exhibit ∆ 47 temperatures of ~180°C that is a clear evidence of a regional-scale solid-state re-585 ordering process, allowing to apply the newly developed Thermal History Reordering Model 586 (THRM; Henkes et al. 2014). Altogether, the paleothermometric data presented in this study, 587 combined with the results of forward thermal modeling, allow to propose thermal scenarios 588 suggesting that the thickness of the prealpine nappes was between 4 and 7 km and between 589 1.3 to 2 km in the inner and outer parts of the subalpine massifs, respectively.590 a range of geologically relevant temperatures. Geochim. Cosmochim. Acta, 208, 335-738 353.

Figure 3 . 897 Figure 4 .

 38974 Figure 3. Example of X-ray diffraction pattern of I-S (002/003) peak, for the <2µm fraction

  907

Figure 5 . 923 Figure 6 . 934 Figure 7 .

 592369347 Figure 5. Micrite stable isotope data. A. Cross-plot δ 18 O carb versus δ 13 C carb composition of

of the kinetic models

  . They are given Eight samples have been analyzed for their stable isotope composition. These samples 211 have been chosen as representative of each of the three studied areas (domain A, B and C of 212 figure 1), based on petrographic observations and clay mineralogy data that were acquired 213 prior to stable isotope measurements. The required amount of carbonate for replicate ∆ 47 214 measurements of the same sample (between 2 and 4 replicate analyses here; i.e., ~ 20 mg)

		Basin Research
	215
	216	was collected with a dental drill on the same plugs used for petrophysical measurements and
	217	SEM characterization. Measurements of ∆ 47 composition of carbonate samples were
	218	performed at Institut de Physique du Globe de Paris (IPGP, Stable Isotopes Team) using a
	219	Thermo Scientific MAT 253 gas-source mass spectrometer. Carbon, oxygen and clumped
	220	isotopes data were simultaneously acquired on gaseous CO 2 released after reaction of ~ 5 mg
	221	of carbonate powder in 104% phosphoric acid at 90°C (20 min in a common acid bath). The
	222	methods used for carbonate digestion, CO 2 purification, mass spectrometric measurements
	223	and data correction and standardization follow the procedure detailed by Bonifacie et al.
		(2017) and Katz et al. (2017), and includes two kinds of standards : i) gaseous CO 2
	242
	243	These relationships can be simulated through a mathematical (forward) model representing
	244	the time-temperature dependence of the employed paleothermometric technique over
	206 245	geological time. This approach gains a particular interest when different paleothermometers
	207 246	here in millidarcy (mD). are simultaneously employed, because each method acts with its own kinetic response to the
	208	inferred thermal history.
		δ δ δ δ 13 C, δ δ δ δ 18 O, ∆ ∆ ∆ ∆ 47 measurements

224

equilibrated at 25 and 1000°C and ii) homogenous carbonate powders (with a 1:1 ratio for 225 unknown samples/standards). 226 δ 18 O and δ 13 C of the carbonate samples are expressed in per mil with respect to the 227 VPDB standard. To guarantee the accuracy of the ∆ 47 data, we routinely analyzed two 228 carbonate reference materials (IPGP-Carrara marble and 102-GCAZ01b, also reported by 229 Dennis et al., 2011 and many other studies). The ∆ 47 values obtained for these carbonate 230 standards over the course of this study (July 2014-May 2016) are: ∆ 47 = 0.412 ± 0.016‰ 231 (1SD, n = 28) for IPGP-Carrara marble; ∆ 47 = 0.721 ± 0.020‰ (1SD, n = 21) for 102-GC-232 AZ01b. Finally, the corrected ∆ 47 values were converted into temperatures using the 233 composite ∆ 47 -T calibration determined for all carbonate minerals for the 0-300°C 234 temperature range (i.e., Eq. (3) from Bonifacie et al., 2017 that compiles 103 mean ∆ 47 data 235 from seven different laboratories and with proper error propagation, which is ∆ 47 = 0.0422 * 236 10 6 / T + 0.2182 in the 25 °C acid digestion frame). 237 Parameterization 238 Extracting thermal history information from kinetic paleothermometers is best treated 239 with a forward approach, meaning that from quantitative observations in a given sample, such 240 as a clumped isotope temperature or a vitrinite reflectance, we select preferred time-241 temperature history that would best reproduce all the measured paleothermometric data.

247 248

In this study, two distinct prograde thermal history paths, previously proposed by 249 Butler, (1991) were used as an initial scenario to drive the different kinetic models.

297 298 Micro-textural and petrophysical properties of the carbonate fraction

	299
	300
	301	SEM characterization of micrites microtexture revealed very comparable and
	302	homogeneous properties among all samples (Fig. 5). The micritic particles are essentially
	303	anhedral, compact, with fully coalescent, though distinct, contacts (Deville de Periere et al.,
	304	2011, Régnet et al 2014). A relevant crystallography parameter, defined as the median of the
	305	maximum 2D particle lengths measured on SEM photomicrographs, varies between 1.30 and
	306	1.45µm, and corresponds to fine micrites granulometry (0.5-2µm). Porosity and permeability
	307	measurements out of 58 samples exhibit very homogeneous values; i.e. porosity between
	308	0.40% and 1.36% and permeability lower than 0.0001mD. No significant geographic
		variation was detected, both in term of petrographic texture and petrophysical properties
	314
	315

309

(Table

1

). These results suggest that the carbonate fraction from the investigated sections is 310 not composed by differently sourced particles mixed together and did not experience 311 distinctive recrystallization events.

312 313

Stable isotope composition of micrites (δ δ δ δ 18 O carb /δ δ δ δ 13 C carb /∆ ∆ ∆ ∆ 47 )

  On the other hand, the ∆ 47 values found for domain A and B are compositions are averaged in the following discussion. The ∆ 47 values are distributed in two Table 1 also presents the oxygen isotopic compositions of the water (δ 18 O water ) in 347 equilibrium with the carbonate that can be calculated using both the T∆ 47 and the δ 18 O carb 348 values measured as well as the oxygen isotope fractionation between calcite and water from 349 Watkins et al. (2013). For samples of the A and B domains, the calculated δ 18 O water are

	345
	346
	360
	361
	362	Organic matter maturity
	363

332

micrites from all investigated geographical domains, ∆ 47 values from carbonate samples from 333 the C domain are significantly different from the two other domains. The ∆ 47 values of the C 334 domain are averaging at 0.421±0.015‰ (n=4 samples and 11 analyses in total), which is 335 within the uncertainties on the ∆ 47 values found for calcite marbles (Bonifacie et al., 2013; see 336 for example Carrara marble analyzed as a standard in this study with ∆ 47 = 0.412±0.016‰, 337 1SD, n = 28 analyses). 343 clusters with corresponding temperatures averaging at 183±17°C for the C domain and 344 51±8°C for the A and B domains (

Table 1). 350 averaging at 1.7±1.2‰ (1 S.D.; n=4), i.e., enriched in 18 O compared to expected Late 351 Cretaceous seawater (δ 18 O water ~-1‰ ; Jenkyns et al. 1994). Contrastingly, for the C domain, 352 the calculated oxygen isotope composition of the fluid in equilibrium with the micrite samples 353 are way more enriched in 18 O, with δ 18 O water averaging at 17±1‰ (1 S.D.; n=4) -that is hard 354 to reconcile with known δ 18 O water of crustal fluids in carbonate settings (e.g., Karhaka et al.

355 2003). [It is noteworthy that Dassié et al. (2018) recently showed that δ 18 O water values directly 356 measured in fluid inclusions from diagenetic cements and the δ 18 O water independently 357 calculated from the ∆ 47 and δ 18 O carb data, within the same cements, agree within 1‰. These 358 findings demonstrate the viability of coupling ∆ 47 and δ 18 O carb for determining the δ 18 O water of 359 the mineralizing fluid, at least in absence of solid-state diffusion].

  It is noticeable that the observed δ 18 O carb composition of micrites are slightly depleted in 18 O

	Page 11 of 30	Basin Research
		Following the kinetic model of Henkes et al. 2013, such temperature conditions can lead to a
		fully-re-equilibration of ∆ 47 compositions.
	466
	467	compared to carbonates precipitated in equilibrium with Late Cretaceous shallow seawater
	468	(see light grey box in Figure 5; Veizer et al. 1999). However, the micrite oxygen and carbon
	469	isotopic compositions are comparable to those observed in analogue deep-chalk sediments
	470	with similar facies (e.g. δ 18 O carb between -1.5 and -3‰ for Upper Cretaceous chalk deposits
		from different areas; Jenkyns et al. 1994 and Schiinfeld et al. 1991; black box in Figure

465

  KELE, S., BREITENBACH, S.F.M., CAPEZZUOLI, E., MECKLER, A.N., ZIEGLER, M., 740 MILLAN, I.M., KLUGE, T., DEAK, J., HANSELMANN, K., JOHN, C.M., YAN, H., 741 LIU, Z. & BERNASCONI, S.M. (2015) Temperature dependence of oxygen-and 742 clumped isotope fractionation in carbonates: A study of travertines and tufas in the 6-743 95°C temperature range. Geochim. Cosmochim. Acta 168, 172-192. Taveyanne greywacke in the Helvetic zone of the Swiss Alps.

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	745	KELSON, J.R., HUNTINGTON, K.W., SCHAUER, A.J., SAENGER, C. & LECHLER, A.R.
	746	(2017) Toward a universal carbonate clumped isotope calibration: Diverse synthesis and
	747	preparatory methods suggest a single temperature relationship. Geochim. Cosmochim.
	748	Acta, 197, 104-131.
	749	KISCH, H, J. (1980) Illite crystallinity and coal rank associated with lowest-grade
	metamorphism of the 750
	751	Eclogae Geol. Helv., 73, 753-777.
	752	KHARAKA, Y. K., & HANOR, J. S. (2007). Deep fluids in the continents: 1. Sedimentary
	753	basins. Surface and Ground Water, Weathering and Soils. Book, Treatise on
	754	Geochemistry 5.
	755	KÜBLER, B. & JABOYEDOFF, M. (2000) Illite Cristallinity. C. R. Acad. Sci. Paris, 331,
	756	75-89.
	757	KUBLER, B., MARTINI, J. & VUAGNAT, M. (1974) Very low grade meatmorphism inthe
	758	Western Alps. Sclnreizerische Mineralogische und Petrographische Mitteilungen, 54,
	759	461-469.
	760	LAWSON, M., SHENTON, B.J., STOLPER, D.A., EILER, J.M., RASBURY, E.T.,
	761	BECKER, T.P., PHILLIPS-LANDER, C.M., BUONO, A.S., BECKER, S.P.,
	762	POTTORF, R., GRAY, G.G., YUREWICZ, D. & GOURNAY, J. (2017) Deciphering
	763	the diagenetic history of the El Abra Formation of eastern Mexico using reordered
	764	clumped isotope temperatures and U-Pb dating. Geological Society of America bulletin,
	765	130, 1-13.
	766	LEVERT J., & FERRY S. (1988) Diagenèse argileuse complexe dans le mésozoïque subalpin
	767	révélée par cartographie des proportions relatives d'argiles selon des niveaux
	768	isochrones. Bull. Soc. Géol. Fr., 4, 1029-1038.
	769	LOYD, S.J., CORSETTI, F. A., EILER, J.M. & TRIPATI, A. K. (2012) Determining the
	770	Diagenetic Conditions of Concretion Formation: Assessing Temperatures and Pore
	771	Waters Using Clumped Isotopes. J. Sediment. Res., 82, 1006-1016.
	772	LOYD, S.J., DICKSON, J. A. D., SCHOLLE, P. A. & TRIPATI, A. K. (2013) Extensive,
	773	uplift-related and non-fault-controlled spar precipitation in the Permian Capitan
	774	Formation. Sediment. Geol., 298, 17-27.
	775	MANGENOT, X., BONIFACIE, M., GASPARRINI, M., GOETZ, A., CHADUTEAU, C.,
	776	ADER, M. & ROUCHON, V. (2017) Coupling ∆47 and fluid inclusion thermometry on
	777	carbonate cements to precisely reconstruct the temperature, salinity and δ 18 O of paleo-
	778	groundwater in sedimentary basins. Chem. Geol., 472, 44-57.
	779	MANGENOT, X., GASPARRINI, M., ROUCHON, V. & BONIFACIE, M. (2018) Basin-
	780	scale thermal and fluid flow histories revealed by carbonate clumped isotopes (∆ 47 ) -
	781	Middle Jurassic carbonates of the Paris Basin depocentre. Sedimentology, 65, 123-150
	782	MOLDOVANYI, E.V. & LOHMANN, K.C. (1984) Isotopic and petrographic record of
	783	phreatic diagenesis: lower Cretaceous Sligo and Cupido formations. Journal of
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	786	analysis of clay minerals. Oxford University Press, 378pp
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reflectance (~0.7%) and T max (~437°C) pointing at moderate maturity . Altogether, clay

indistinguishable from each other (respectively of 0.615±0.025‰ and 0.623±0.015‰, n=2 339 samples for each domain, 11 analyses in total). Given the fact that the four samples collected massifs. The estimations of illite content in I-S mixed layers are highlighted with black dots when using the methods of Środoń (1980; 1984) and with red dots when using the method of 889 Inoue et al. (1989). Note how samples from a single stratigraphic interval, within in a small 890 geographic area, can be significantly differentiated based on their clay mineral assemblages 891 and illite content in the I-S mixed layers.

Micrite crystallometry ( μm)

Frequency Frequency

Particle shape : Anhedral-subhedral compact Contact : Coalescent Crystallometry : 1,45μm Ø : 0.56% ; K <0.001MDa

Particle shape : Anhedral-subhedral compact Contact : Coalescent Crystallometry : 1.31μm Ø : 1,07%., K <0.001MDa

Particle shape : Anhedral-subhedral compact Contact : Coalescent Crystallometry : 1.43μm Ø : 0.51% ; K <0.001MDa (5) Oxygen isotope compositions of the fluid in equilibrium with the carbonate calculated using T ∆47 and the fractionation factor of oxygen isotopes between calcite and water of Watkins et al. (2013).

(6) Vitrinite reflectance and Tmax values were measured by Gorin and Monteuil, 1990 in the overlying Cretaceous Wang Formation, and in the same or very close localities.

DOMAIN B DOMAIN C DOMAIN A

Organic matter-bearing Wang Formation (6) Stable isotope data Micrite particles (2) Petrophysical Illite content in I-S (1) Clay fraction (<5%) Carbonated fraction (>95%)

Page 27 of 30 Basin Research