Faces in Defoe's Roxana

Anne Rouhette, UCA Roxana's chameleon-like narrator constantly re-invents herself and reveals a relish for and a mastery of disguise, in the literal sense. Yet at the very moment when she parades in her infamous Turkish dress before a masked audience, Roxana is perhaps the least disguised of all in the sense that unlike the others, she offers her naked face for everyone to see and admire: I had no mask, neither did I paint, and yet I had the day of all the ladies that appeared at the ball, I mean of those that appeared with faces on. As for those masked, nothing could be said of them, no doubt there might be many finer than I was… (Roxana 1801 ) This is not the only time that she insists on the purely natural quality of her beauty; she previously referred to her face as "the mere work of nature" (R 72). But this passage encapsulates the double movement which characterizes so much of Roxana and of Roxana, built as it is upon a tension in which Roxana's face arguably plays a crucial role and which takes several forms: between exposure (the face wears no make-up) and concealment (the body is disguised), revelation and hiding behind a mask, naming and anonymity, the natural and the artificial, etc. In this she resembles Defoe's other fictional autobiographers, as Homer O. Brown explains:

Defoe's narrators seem obsessed with concealing themselves, but the impulse leading them towards exposure appears equally strong. Complete concealment is impossible, perhaps not even desirable. On the one hand there is the insistence on building a faceless shelter around the self, but, on the other, a recurring compulsion to move out into the open2 .

But the traditional dichotomy between face and facelessness, face and mask (another formulation of the one which opposes self and persona), is somehow subverted in the very passage I just quoted, which thanks to the phrase "with faces on" also suggests a form of interchangeability or at least of equivalence between "face" and "mask", two terms which at first sight have antithetical meanings. At Roxana's balls, one can almost indifferently put on a mask or a face, bringing onto highly unstable ground the whole question of identity and presenting a very modern view of the self as performative, as a series of roles or masks as several critics have argued3 . But Roxana's repeated definition of herself as a whore also hints at the need for a more stable view of the self, and it is this pull into two opposite directions that I would like to explore here from the perspective of Defoe's complex approach to the human face, more specifically to the female face.

I will begin with the analogy between face and mask in the passage quoted above, an assimilation warranted by the dictionary, in which the phrase "put on a face" is synonymous with "put on a mask or a disguise". A caveat first: given the supposedly highly moral context set up by the preface, we should not endow too quickly the concepts of "face" and "mask"

with the moral values they are often associated with, in the West at least4 , where "face" is equated with truth and receives a positive value while "mask" stands for lying, a negative value which the word "grimace" rather than "mask" carries in the novel5 . They concern very different characters, the foolish husband and the reliable landlord, to very different effect: the Brewer only pretends to be the businessman he most certainly is not while the Landlord will prove to be a true and kind friend to Roxana. Rather than providing a moral commentary or explaining that their appearance, of business or of kindness, corresponds to a hypothetical essence, Roxana's formulations stop at the surface of these characters, suggesting that access to truth is limited to that surface, that it is impossible to go beyond what is offered to view, to reach the interiority of these characters. She makes the same point when discussing her own situation later on in the novel:

I had begun a little, as I have said above, to reflect upon my manner of living, and to think of putting a new face upon it, and nothing moved me to it more than the consideration of my having three children, who were now grown up; and yet that while I was in that station of life I could not converse with them or make myself known to them; and this gave me a great deal of uneasiness […..] "Why, Amy," says I, "is it not possible for me to shift my being from this part of the town and go and live in another part of the city, or another part of the country, and be as entirely concealed as if I had never been known?" "Yes," says Amy, "I believe it might; but then you must put off all your equipages and servants, coaches and horses, change your liveries-nay, your own clothes, and, if it was possible, your very face." (R 207-208) Here again Roxana limits herself to the surface, considering change from a purely external perspective 6 : she does not write about changing her manner of living, but about changing the face, the appearance of her manner of living 7 . This is of course to be linked to her entire career: instead of trying to change herself and truly reform, which would require a minimum of introspection, Roxana merely puts on one disguise after the other, and we'll return to the question of her superficiality below.

However, the move from the figurative to the literal sense of "face" in the second paragraph of the passage quoted above articulates the fundamental problem at the heart of such a conception (of the self, of knowledge, of self-knowledge): "you must... change your liveries-nay, your own clothes, and, if it was possible, your very face" (R 208). Defoe almost juxtaposes the two here to reveal how irreconcilable they are by contrasting the versality of the one (the figurative) with the unescapable fixity of the other (the literal). For as Amy points out, it is literally impossible to have several faces; "I'll be a Quaker today" does not amount to "I"ll have a Quaker's face today". To the figurative or artificial faces I just mentioned, to Roxana's disguises corresponds only one literal or natural face, which acts as a marker of identity and in Roxana's case introduces a form of stability, of singularity, in her shape-shifting plurality. That accomplished actress and mistress of deception, who selfconsciously refers to her career as "the vast Variety of Scenes that I had acted my Part in" (R 200), can be recognized thanks to her face, which metonymically functions as her identity.

The same goes for instance for the thieves who attack and murder the jeweller. They remain shrouded in anonymity and escape punishment largely because their faces remain hidden: "as to their faces, that he could know nothing of, because they had all of them masks on" (R 54).

Seeing Roxana's face is "knowing" her, in the sense of recognizing her, of knowing who she is, whatever is the name that she goes by at the time; but also in a deeper sense of "knowing"

6 As Elizabeth Napier explains, "When Roxana […] speaks of her intended reformation after her dismissal of the lewd lord as "putting a new Face upon it" (207), her use of the metaphor of the mask reveals her tragic inability to conceive of change in any other than a superficial way. Her Element is superficial and sensual… such a focus on outsides, however often she regards herself in the glass or in Amy or Susan, will do little more than postpone the work of self-analysis in which she must engage" (Defoe's Major Fiction, op. cit, 46). 7 Cf. also "I must go back here, after telling openly the wicked things I did, to mention something which, however, had the face of doing good" (R 188): is Roxana implying that this "something"inquiring about her children and trying to help themappears to be good but is not, appears to be good and is good, or is she merely focusing on surface again and implying nothing at all? as we will see later. She holds up twice a fan before her face to avoid being identified, first by the Brewer, then by the Dutch Merchant:

I could not be deceived; I passed so near him that I almost brushed him with my clothes, and looked him full in the face, but having my fan before my face, so that he could not know me. (R 85) "Well, well," says she, "I would have him see thee for all that, as plainly as thou hast seen him. Nonetheless, very often her tears and blushes8 reveal what she tries to conceal but to no avail (or so she says), as in the following instance:

I could never hear him say so, but though secretly it pleased me, yet it so closely touched me another way that I could not refrain sighing, and sometimes tears; and one time in particular it so affected me that I could not conceal it from him; but when he saw tears run down my face, there was no concealing the occasion from him; he was too importunate to be denied in a thing of that moment; so I frankly answered […] (R 80-81, emphasis added).

This represents a degree of transparency surprising in the most opaque of Defoe's narrators 9 , but the legibility of Roxana's face is again limited to the surface, her face lending itself only to a surface reading, not unlike her narrative which is very difficult to see through. It occurs only in specific situations, as she reacts to a particularly overwhelming feeling; the Prince or the Quakeress read one expression on Roxana's face, not her personality, which remains hidden from them (she lies to them both and is not detected). Like Roxana herself when she uses the phrase "put on a face" for the Brewer and the landlord or in other circumstances, as we have seen, they stop at the surface and do not access what a modern reader would call her "inner self", which is a problematic concept in Defoe anyway 10 . At this point I'd like to replace briefly these considerations in a wider context regarding the symbolical value of the face in the early 18 th century, a time when it was not uncommon to try and decipher personality traits on a person's face 11 , to render the human face legible and understandable, a reliable signifier for the character's signified. Physiognomy represented an epistemological system assimilating face and personality; you could know a person, not just their identity but their character and propensities, by analysing their facial features. This perspective would be granted a mock-scientific veneer at the end of century with Lavater's theories, but at the beginning of the century, and especially in fiction, the correspondence between what a person looks like and his or her worth takes on a moral dimension: to a person's outer beauty corresponds, or should correspond, his or her inner beauty (increasingly "her" as the century moves on). Of course Roxana's great beauty conceals a morally despicable personality. But Defoe articulates a reflexion that goes far deeper than the commonplace "beware a pretty face", he does not merely stress the opposition between appearance and essence, he interrogates the epistemological assumptions upon which such a system of reading human beings is based. Furthermore, he explores the impact of Roxana's beauty on her life and on her character, questioning the link between a woman's face and her fate.

He does so by clearly taking the human face outside of the natural realm. Contrary to the body, which needs to be dressed to become culturally and socially acceptable, and in spite of Roxana's insistence on her face as "the meer work of nature" (R 72), the face in general and Roxana's in particular are not natural givens but cultural constructs, a system of signs, as 9 When Roxana dissimulates, there are no direct, explicit references to her face, see p. 260 for instance. 10 As Elizabeth Napier writes, "inwardness is a concept which remains largely alien to Defoe, at least in its modern psychological sense. Defoe in part still clings to a paradigm of the self that figures it as external… Modern readers, with their gaze directed inward, can only go so far, as a result, in a quest for [D's characters] "true" selves." (Defoe's Major Fiction, op. cit., p. xxi). This is of course a particularly valid point for a character who claims to speak of herself "as if I was speaking of another-body" (R 6). See also Peter New, "Why Roxana can Never Find Herself", MLR, 91: 2 (Apr., 1996), 317-329. 11 See Belting,Faces,op. cit.,104 various philosophers and art historians in particular have shown 12 . This does not come only from the fact that its expressions are construed as signs for others to interpret, as we have seen; more fundamentally, the complete lack of details concerning Roxana's supposedly famous face (eye or hair colour, for instance) and its relative imperviousness to age grant it an abstract quality reminiscent of the artifice of a mask (which does not age either), leading readers to consider it and her in the light of an abstraction that is "beauty" or "woman" rather than a particularity. Faces play a symbolic role, which is borne out in the novel by the only face which stands out, apart from Roxana's: the Jew who harasses Roxana is gratified with "a devil's face" (R 115); in other words, Roxana sees his natural face through the prism of what she imagines of his personality. As for Roxana herself, the symbolic value she attributes to her own face, the prism through which she sees it, is made perfectly clear when she realizes that her Prince doubts the purity of her skin: "I put a Handkerchief into his Hand, and taking his Hand into mine, I made him wipe my Face so hard, that he was unwilling to do it, for fear of hurting me" (R 72). The violence of this gesture and the pain she inflicts upon herself by proxy in the process of cleaning her face are particularly striking, especially since it is a twofold process, first with the handkerchief and then with the" Cup-full of hot Water" she asks for in the next paragraph. It is difficult not to think of the cleansing role of water in an episode perhaps reminiscent of Lady Macbeth's compulsive hand-washing: "will these hands ne'er be clean?" (V, 1) becomes "will this face ne'er be clean?" Roxana's face, not hands, is the instrument of the sin she tries to scrub away, all the more so as she will refer to her "dirty History" two pages later (R 75, emphasis added). Her alluring face becomes the very mark of her whoring, making the saying especially apt for her: her face really is her fortune.

The saying is true in the two meanings of "fortune". First, as regards wealth, I don't need to develop that the pleasure of contemplating her beautiful face and of possessing her desirable body enable her to run a hugely profitable trade; the link of character with coinage and the commodification that it entails 13 , the assimilation of Roxana to a sum of money, are sufficiently evident for me to move to the other sense of fortune, destiny. The moment that seals Roxana's fate, when she chooses to be a prostitute, occurs not with the Landlord (I will not go into the reasons why she sleeps with him here) but with the Prince, at one very specific moment when he invites her to look at her face in a mirror:

12 See for instance "Visagéité" in Mille Plateaux by Gilles Deleuze and Félix Guattari, Paris, Minuit, 1980. 13 Deidre Lynch in particular has argued that Defoe's construction of character links his "persons" with the commodity market in which their search for stability unfold. "We are, in part, drawn again and again to Defoe because of the ontological unpalatability of this premise and the fact that it may be true", Napier comments (Defoe's Major Fiction, op. cit., xxii).

He stood up, and taking me by the hand, led me to a large looking-glass, which made up the pier in the front of the parlour. "Look there, madam," said he; "is it fit that that face" (pointing to my figure in the glass) "should go back to Poictou? No, madam," says he; "stay and make some gentleman of quality happy, that may, in return, make you forget all your sorrows;" and with that he took me in his arms, and kissing me twice, told me he would see me again, but with less ceremony. (R 59-60)

Even though the Prince designates her figure, it is her face that he draws attention to, a face which by metonymy comes to represent Roxana herself and determines her "career" of "making gentlemen happy" -the repetition of the verb "take" and the kisses in this short passage leaves no doubt as to the nature of the happiness to be conferred. Her face is her fate, seeing her is knowing her indeed. Beyond that, to return to that crucial scene, the Prince invites Roxana to look at herself in a mirror, and -since they're both lookingto look at herself being looked at, construing her as turned towards the outside, not merely subjecting herself to the "male gaze" but identifying her perspective with it (note the objectification in "that face"she is looking upon herself as an object to be looked at). She is asked to identify with the surface of a face seen in a mirror, not even her face, a part of her own body, but "that face", which eventually reduces her to a surface clearly external to herself, or, to put it differently, to pure spectacle, a view of herself which she endorses and to which the numerous tableaux in which she describes herself throughout the novel testify 14this point has been addressed in particular by James Maddox, who insists on the role played by her time with the Prince in Roxana's self-formation and definition 15 .

Because of the abstract quality of Roxana's face, such a line of reasoning may be extended to the female face in general, an interpretation suggested by the research conducted by film critics such as Laura Mulvey or art historians like John Berger. Mulvey's famous analysis of how women characters on-screen are coded with "to-be-looked-at-ness 16 " concerns primarily Hollywood film actresses but her claims can be extended to all women in 14 E.g. R 16-17. 15 ".. during [Roxana's] time [with the Prince] her growing consciousness of her beauty comes to reinforce her already well-developed attention to the observing eyes of others. Roxana, acutely sensitive to the possible scorn in others' eyes, longs to see admiration beaming from the eyes of one so prestigious as the prince and so she becomes victim to that other-directedness which is the occupational hazard of the very beautiful… Exactly because she both fears and reveres the eyes of others, Roxana labors to present herself as a polished, twodimensional, depthless surface; she wishes to be looked at, but never seen into. The strategy of this selfpresentation is especially clear in her account of the three years she spent living in absolute seclusion in Paris as the prince's mistress.

[…] the fascinating glimpses that Roxana gives of those years reveal […] an intensely public world, in which the prince is spectator at Roxana's formal self-displays. Each of the lovers, after all, exists for the other as a polished porcelain surface: Roxana is in love with the prince's rank, while the prince is in love with Roxana's great beauty. There is in Roxana's descriptions of these scenes nothing like our idea of a "private self"; instead, their three years are spent in a constant display of their different forms of prestige." James Maddox, "On Defoe's Roxana", ELH, 51: 4 (Winter, 1984), 672-73. 16 In "Visual Pleasure and Narrative Cinema", Screen 16: 3, October 1975, 6-18. the line of art historian John Berger's contention that men and women have a different kind of presence, or more accurately, that their presence has been construed differently over the centuries, that they have learned to see themselves in the world differently. Berger explains that to be a woman in culture is "to be looked at":

Men act and women appear. Men look at women. Women watch themselves being looked at. This determines not only most relations between men and women but also the relation of women to themselves. The surveyor of woman in herself is male: the surveyed female. Thus she turns herself into an object-and most particularly an object of vision: a sight. (Ways of Seeing, London, Penguin, 1972, 47) To conclude, I'd like to raise the question whether the face of such a woman is still a face. It no longer is that most human of all body parts, the part that connects us to other human beings, an interface between interiority and exteriority; on the contrary, its being perceived as purely two-dimensional prevents us partly from recognizing it as human, stopping us at the threshold of the human, at the "pre-face", so to speak. What should be familiar is defamiliarized. The more naked the face, and thus theoretically the more open to scrutiny, the more uncanny (in the Freudian sense) the experience of the viewer; this feeling accounts to some extent for the uneasy experience that is reading Roxana, a novel whose narrative may be dressed up like its heroine's body, but where the surface of the narrator's words alienates readers instead of provoking their sympathy.

  " "No, but he shan't," says I, "for I am sure he don't know me in this dress, and I'll take care he shan't see my face, if I can help it;" so I held up my fan before my face, and she saw me resolute in that, so she pressed me no farther…[Quakeress] though thy tongue will not confess it [that you know him], thy face does.(R 218) When she playfully pretends to be a Muslim, on account of the Turkish dress she's wearing, her face, her "Christian's face" (R 175), gives her away. Faces can betray, as Roxana well knows: "What my face might do towards betraying me I knew not, because I could not see

myself" (R 284). She cannot "escape her face", as the Quakeress acknowledges: "though thy tongue will not confess it, thy face does". The Quakeress reads on Roxana's face the confession that is not forthcoming in wordsthe irony being that in the general context of the novel, since the reader obviously cannot "see" Roxana's face but only her words on the page, the extent of her "confession" remains extremely limited.

The face may reveal what language does not or cannot express, or will not (as in this instance). On numerous occasions such as this one, Roxana's face transparently conveys emotions which she is reluctant to put into words, although sometimes her facial expressions require interpretation and explanation, preventing any attempt at generalization: he [the Prince] perceiv'd Tears to run down my Cheeks; My Dear, says he, aloud, what mean these Tears? My Lord, said I, after some little Check, for I cou'd not speak presently, I beseech you to believe me, they are not Tears of Sorrow, but Tears of Joy (R 72)

The references are to Oxford's World Classics edition, hereafter abbreviated as "R"; almost all extracts come from the digital version available on www.gutenberg.org, hence the differences in spelling, punctuation and capitalization.

"The Displaced Self in the Novels of Daniel Defoe," ELH

(1971): 562-90, 569.

Amongst other,s Elizabeth Napier in Defoe's Major Fiction, Accounting for the Self, Newark, U of Delaware P, 2016, Part Two: "'Meer Manage': The Performing Self".

On this subject, see Hans Belting, « Le Visage et le masque », Faces. Une Histoire du visage, trad. Nicolas Weill, Paris, Gallimard, 2017.

See for instance "How did my blood flush up into my face when I reflected how sincerely, how affectionately, this good-humoured gentleman embraced the most cursed piece of hypocrisy that ever came into the arms of an honest man! His was all tenderness, all kindness, and the utmost sincerity; mine all grimace and deceit" (R 300).

Roxana blushes at several point, e.g. "We had some very agreeable conversations upon this subject, and once he told me, with a kind of more than ordinary concern upon his thoughts, that he was greatly beholden to me for taking this hazardous and difficult journey, for that I had kept him honest. I looked up in his face, and coloured as red as fire" (R 103-04); "I coloured as red as blood itself could make a face look" (R 173), etc.


Van-Hoan Le
email: van-hoan.le@univ-lorraine.fr

Marie-Camille Caumon

Alexandre Tarantola

Aurélien Randi

Pascal Robert

Josef Mullis

Quantitative measurements of composition, pressure and density of micro-volumes of CO2-N2 gas mixtures by Raman spectroscopy

Quantitative analysis of gases by Raman spectroscopy is based on relative Raman scattering cross-sections (RRSCS) and the evolution of different spectral parameters (peak position, peak area, peak intensity, etc.). However, most of the calibration data were established at low pressure (low density) and without evaluating the effect of the composition. Using these data may lead to considerable errors, especially when applied to gas mixtures at high pressure as found in natural fluid inclusions. The aim of this study is to reevaluate the RRSCS of CO2 and to establish new calibration data based on the variation of CO2 Fermi diad splitting as a function of pressure (density) and composition over a pressure range of 5 to 600 bars at 22 and 32 °C. A high-pressure optical cell system (HPOC) and a heating-cooling stage were used for Raman in-situ analyses at controlled PTX conditions. Our experimental results show that the RRSCS of CO2 varies slightly with pressure but can be considered constant over the studied pressure range. It can be used to measure the proportion of CO2 in gas mixtures with an uncertainty of about ± 0.5 mol%. Different polynomial equations were provided to calculate pressure and density of CO2-N2 gas mixtures with an uncertainty of ± 20 bar or 0.01 g.cm -3 . A comparison of PVTX properties of natural CO2-N2 fluid inclusions hosted in quartz from the Central Alps (Switzerland) obtained by Raman measurement and as derived from phase transition temperatures by microthermometry experiments shows comparable values.

The exploration of the Raman effect by C.V Raman in 1928 provided a new way for non-destructive analyses of materials under different phase states (solid, liquid and gaseous) to get qualitative and quantitative information (after establishing calibration data). The sensitivity of Raman spectroscopy covers a wide concentration range, down to very low concentration, 1,2 even to sub-ppm levels 3,4 . Raman spectroscopy has been widely used for gas analysis in various domains of investigation such as monitoring of polluted air 5 or automobile exhaust gases, 1 fuel gas analysis, [6][7][8] diagnosis and monitoring of disease states by human breath analysis, 3,4,9 controlling and monitoring of fruit ripening, 10 analyzing of gas bubbles appearing as defects inside industrial glasses to optimize production process. 11 Other applications can also be found in the field of environmental gas sensing, e.g. monitoring of geological storage site of CO2, 12 investigation of biological and/or geochemical gas exchange and migration processes within the different compartment (groundwater, subsurface, surface, atmosphere). [13][14][15][16][17] All applications mentioned above relate exclusively to the analysis of immense and/or small volume of gas at relatively low pressure (< few dozen bars). The present study is dedicated to another case of extreme conditions: the analysis of gas mixtures in micro-volumes at relatively high pressure (up to 600 bar). The main application is the study of fluid inclusions naturally trapped in minerals.

Fluid inclusions (FIs) are small cavities in minerals containing a micro-volume of a geological fluid trapped during or after crystal growth. They are the most reliable relicts recording information about the conditions of crystal formation as well as of paleo-fluid circulations. A quantitative knowledge (composition, pressure, and density) of these fluids provides key information to better understand geological processes, to reconstruct the conditions of paleo-fluid circulations and thereafter for further application such as natural resources exploration. 18 CO2 and N2 are among the most common gases present in a large variety of geological fluids. 18,19 The observation of phase transitions during microthermometry experiments is currently the standard method to investigate fluid inclusion properties. However, some limitations appear when FIs are of small size (< 5 µm), of complex composition or of low density without any observable phase transitions. [20][21][22][23][24] Raman spectroscopy is a complementary method to microthermometry as it can offer fast (from a few seconds to a few minutes), high resolution (down to  1 µm 2 ) and simultaneous non-destructive, qualitative and quantitative analyses. [25][26][27] Several applications of Raman spectroscopy dedicated to the investigation of FIs have been carried out by different research teams since the 1970s (cf. reviews by Burke 25 and Frezzotti et al. 26 ). The determination of the composition, pressure, and density of gas mixtures using Raman spectroscopy requires the knowledge of the Raman scattering cross-section (RSCS) and the behavior of Raman spectral features (peak position, peak area/intensity ratio…) as a function of pressure, density, temperature, and composition. 25,26 RSCS is a specific parameter related to the probability of Raman scattering effect for each vibration. It can be used to determine the concentration of molecules. 28 Due to the difficulty of the determination of absolute values of RSCS, only that of N2 was carefully determined by different techniques. [29][30][31][32] Relative RSCS (RRSCS) values of common gaseous species found in fluid inclusions (CO2, CO, CH2, O2, H2S…) were then measured relatively to RSCS of N2 with an accuracy varying from 5 to 20 %. 25,32 However, all published data of RRSCS were determined at room temperature and 1 -5 atm whereas, according to Wopenka et Pasteris 28 and Seitz et al. 33,34 RRSCS may not just vary as a function of wavelength and temperature, 32 but also as a function of pressure and composition due to changes in molecular interaction. 35 Moreover, although many improvements in Raman instruments were made, the RRSCS data were never reevaluated since the 1970s.

The variations of the peak positions of Raman bands of N2 and CO2 as a function of pressure (or density) were also separately investigated, 20,21,24,[36][START_REF] Garrabos | Marsault-Herail[END_REF][38][39][40][41] showing the applicability for pressure and density monitoring. Indeed, the Fermi diad splitting (distance between the two main peaks) of CO2 was used to develop densimeters for pure CO2 or CO2-rich fluid inclusions. 20,21,24,[START_REF] Garrabos | Marsault-Herail[END_REF]39,40,42,43 Nevertheless, the variation of peak positions as well as of CO2 Fermi diad splitting are subject to changes as a function of pressure, density, temperature 39,40 but also composition. 33,34,40 The inappropriate use of calibration data obtained from pure gas for analyzing gas mixtures may, therefore, lead to an over/underestimation of density 40 or pressure. 41 Thus, the effect of composition, pressure, density, and temperature on Raman spectral features should be simultaneously taken into account upon any quantitative analysis. The experimental data of Seitz et al. 33,34 revealed the variation trends of different Raman spectral features of CO2 and N2 as a function of composition (when mixed with CH4) but the results were quite scattered due to the use of low spectral resolution ( 5 cm - 1 ). Consequently, no robust calibration with uncertainty analysis was given.

The present work aims (1) to reevaluate the dependence of RRSCS of CO2 on pressure and composition by using nowadays performance instruments and (2) to establish a new Raman calibration methodology for composition, pressure, and density measurement of any CO2-N2 gas mixtures. Data acquisition was done thanks to the combination of an improved HPOC system [START_REF] Chou | Advances in High-Pressure Technology for Geophysical Applications[END_REF][START_REF] Chou | [END_REF][46] and a heating-cooling stage to control the PT conditions during experiments and to collect numerous data points for statistical purposes. CO2-N2 gas mixtures of different compositions were analyzed by Raman spectroscopy at 22 °C (room temperature) and 32 °C (just above the critical point of pure CO2 in order to avoid any V-L phase transition for any CO2-N2 mixture composition) over the pressure range 5 -600 bar. The relationships between the variation of Raman spectral parameters with pressure (or density) and composition were evaluated to determine the most reliable quantification parameters. Finally, the composition, density, and pressure of natural FIs hosted in quartz from the Central Alps (Switzerland) 47 were obtained with these calibration data, and subsequently compared with microthermometry data.

MATERIALS AND METHODS

Gas mixture preparation. CO2-N2 binary mixtures of different compositions were prepared from high-purity commercial N2 and CO2 gases (99.99 % purity, Air Liquide TM ) at low pressure (< 10 bar) by a gas mixer (GasMix AlyTech TM ). They were subsequently compressed up to 120-150 bars using a home-made compressor system and stored in a stainless-steel reservoir. The composition of the prepared gas mixtures was controlled by gas chromatography (GC) to ensure there was no significant modification during the compression step. The GC was calibrated by measuring several times a commercial CO2-N2 gas mixture (Air Liquide TM ), yielding a standard deviation of about 0.4 mol%. The final composition of the prepared gas mixtures was the average of three GC measurements with a standard uncertainty  0.4 √3  0.3 mol% (1). The compositions of the CO2-N2 gas mixtures used in this study were 10.5, 30.1, 50.3, 60.9, 70.5, 80.1, and 88.6 mol% CO2.

Pressurization system. The reservoir containing the gas mixture was connected to an improved HPOC system, which consists of several valves, stainless steel microtubes and a pump (Figure 1). [START_REF] Chou | Advances in High-Pressure Technology for Geophysical Applications[END_REF][START_REF] Chou | [END_REF][46] One end of the HPOC system was equipped with a manual screw pressure generator. The other end was coupled with a fused silica capillary (FSC) of 200 µm of internal diameter sealed at one end by a hydrogen flame. 46,48 Two pressure transducers were set on the fixed part and on the movable part of the HPOC system to monitor pressure (± 1 bar) inside the whole system. The FSC was set on a customized heating-cooling stage (Linkam CAP500) previously calibrated by measuring the triple point of distilled water (0.0 °C) and of a pure CO2 standard sample (-56.6 °C) to maintain the temperature at 22.0 and 32.0 ± 0.1 °C. The system was evacuated for a minimum of 30 minutes to remove any other gases before loading the investigated gas mixture. Thereby, the gas mixtures were analyzed by Raman spectroscopy through the microcapillary at controlled PT conditions.

Figure 1: Sketch of the HPOC system coupled with a transparent fused silica capillary (FSC) set on a Linkam CAP500 heating-cooling stage. The system consists of a fixed part composed of a manual pressure generator, a pressure transducer (PT1), valves, microtubes, and a pump to purge the system. The movable part connects the system with the heating-cooling stage. It is also equipped with valves and another pressure transducer (PT2).

Raman spectroscopy. Raman measurements were performed using a LabRAM HR spectrometer (Horiba Jobin-Yvon®) equipped with an 1800 groovemm -1 grating with an aperture of confocal hole and slit set at 1000 µm and 200 µm, respectively, giving a spectral resolution of about 1.67 cm -1 (fitted FWHM of Neon peak at 2348 cm -1 ). The excitation radiation was provided by an Ar + laser (Stabilite 2017, Spectra-Physics) at 514.53 nm with a power of 200 mW, focused on the FSC by a 20 X objective (Olympus, NA = 0.4). Each measurement was repeated six times successively at the same PTX conditions for statistical purposes. A spectrum was recorded before loading any gas mixture into the microcapillary to measure the contribution of atmospheric N2 for peak area correction. 35 The same configuration (excitation wavelength, hole, slit, grating) was used for Raman analyses of natural FIs, except the use of a 50 X objective (Olympus, NA = 0.5). To minimize the error due to the subtraction of the N2 peak area, the intensity of the N2 band within FI should be 3 or 4 times higher than that of ambient N2. Thus, the acquisition time ranged from 5 to 30 seconds per accumulation (with 10 accumulations per measurement) depending on the density, size, shape, and depth of FIs. The Raman spectrum of N2 characterized by a peak at  2331 cm -1 was simultaneously recorded with Ne peak at  2348 cm -1 (Figure 2a) for wavelength calibration. [START_REF] Mccreery | Spectroscopy for Chemical Analysis[END_REF] As the N2 band is not symmetrical (due to the asymmetric distribution of J = 0 transition in the Q-branch [START_REF] Bendtsen | [END_REF] ), the spectra of N2 were fitted by an asymmetrical Gaussian-Lorentzian function using the peak fitting tool of LabSpecs 6 software (Horiba) after baseline subtraction. The peak position of N2 was then corrected by that of Ne using Equation 1, where  𝑁 2 and  𝑁𝑒 are the fitted peak positions of N2 and Ne, respectively, and 2348.4318 cm -1 the reference peak position of Ne cited from NIST webbook. 51

 𝑁 2 (𝑐𝑜𝑟) =  𝑁 2 + (2348.4318 - 𝑁𝑒 )
Eq.1

CO2 has four vibrational modes: symmetrical stretching (1), asymmetrical stretching (3) and a doubly degenerated bending mode (2a and 2b). Only the 1 mode ( 1333 cm -1 ) is Ramanactive. However, the experimental spectrum of CO2 presents two strong bands because of Fermi resonance 52 taking place between the excited vibrational states 1 and the first overtone of 2 (2𝜈 2 = 2 × 667 = 1334 cm -1 ). As these two excited states have nearly the same energy level, they perturb each other and cause a division into two peaks at higher (1388 cm -1 ) and lower (1285 cm -1 ) wavenumbers. This phenomenon is known as the Fermi diad splitting, resulting in two bands denoted  + and  -, respectively. Moreover, the spectrum of CO2 has also two lowintensity bands at 1409 cm -1 and 1265 cm -1 , known as hot bands. Another weak band at 1370 cm -1 corresponds to the signal of 13 CO2 (Figure 2b). Extended interpretation of spectral features of CO2 can be found in literature. [START_REF] Placzek | Rayleigh-Streuung und Raman-Effekt[END_REF] Raman spectra of CO2 were fitted by symmetric Gaussian-Lorentzian function using LabSpec 6 (Horiba) after baseline subtraction. Final values of spectral parameters were the mean of 6 measurements, yielding an uncertainty of about  0.4 % (1) for peak area values, about  0.01 cm -1 (1) for peak position and  0.015 cm -1 for CO2 Fermi diad splitting (1) (see Supporting Information for detailed uncertainty calculations).

The Raman spectra of CO2 and N2 were recorded in two different spectral ranges (1100 to 1580 cm -1 and 2100 to 2525 cm -1 , respectively) with different instrumental efficiency of the spectrometer. 27 All the Raman spectra were thus corrected using an ICS function (Intensity Correction System) integrated into LabSpec 6 software to normalized instrument response with wavelength. The calibration was done using a white lamp of known emission (Raman Calibration Accessory, Kaiser Optical Systems, Inc.). 27 As a result, the instrumental efficiencies at the wavelength of CO2 and N2 peaks ( 𝐶𝑂 2 and  𝑁 2 ) can be considered identical.

Microthermometry. Microthermometry is a standard method to determine PVTX proprieties of natural FIs. It is based on the determination of phase transition temperatures of geological fluid trapped within inclusions. In the present work, microthermometry measurements of natural FIs were made using a THMSG600 heating-cooling stage coupled with an Olympus BX50 microscope. The stage was calibrated at ± 0.1 °C using standard inclusions against the melting point of pure CO2 (-56.6 °C) and of pure H2O (0.0 °C). Herein, the melting temperature Tm(car) and the homogenization temperature Th(car) of the volatile carbonic phase containing the CO2-N2 mixtures trapped within FIs were measured. These phase transition temperatures were then used to determine the composition and density of FIs using the VX diagram of Thiéry et al. 54 GERG-2004 equation of state (EoS) is used to calculate (i) the pressure within natural FIs at a given temperature from density-composition properties derived from microthermometry results and (ii) the density of gas mixtures during Raman measurements (for a given composition, pressure and temperature). The GERG-2004 EoS is known as the most accurate available EoS at the P and T conditions of interest. Concerning the CO2-N2 gas mixtures, the EoS is fitted from 823 experimental data points, covers the entire composition range (from 1 to 98 mol% in CO2) and large pressure and temperature ranges (1 to 2740 bars and -63 to 400 °C). The uncertainties in density are shown to be less than 0.1 % when pressure < 350 bars or less than 0.5 % when pressure < 700 bars in the vapor region, about 0.1 -0.5 % in the liquid region and less than 3 % in the two-phase region. This model is integrated into REFPROP software. [START_REF] Lemmon | Standard Reference Data Program[END_REF] Natural fluid inclusions. A prismatic quartz crystal (Mu 147.2) found in late Alpine tension gashes from the Central Alps (Switzerland) 47 was used for its CO2-N2 natural fluid inclusions. At room temperature, the 2 to 30 µm large FIs are either monophasic (liquid CO2+N2) or biphasic (liquid H2O + vapor CO2-N2) (Figure 3). 15 FIs from 4 different zones were selected to be analyzed by Raman spectroscopy and microthermometry for comparison.

RESULTS AND DISCUSSION

Mixture composition: Evaluation of the RRSCS of CO2. The RRSCS of the two main bands of CO2 ( 𝐶𝑂 2 ) were calculated at different composition and pressure conditions with the use of Equation 2, 28 where 𝐴 𝐶𝑂 2 is the peak area of  + or  -band, 𝐴 𝑁 2 is the peak area of the N2 band, 𝐶 𝐶𝑂 2 and 𝐶 𝑁 2 are the concentration (mol%) of CO2 and N2, respectively.

Figure 4a shows the variation of the RRSCS of the upper band (  + ) and the lower band (  -) as a function of pressure and composition. Both RRSCS are somewhat perturbed at low pressure (<  80 bars), probably due to a significant change of molecular interaction effect. 33,34 Above  80 bars,   + increases slightly whereas   -decreases slightly with increasing pressure. Indeed, from 5 to 600 bars, the RRSCS value only increases by 0.05 (  + ) or decrease by 0.1 (  -), resulting in a difference of only  0.2 mol% CO2. Figure 4b shows the evolution of the sum of the two RRSCS of CO2 (  + +   -) as a function of pressure and composition. A slight perturbation was also observed at a low pressure-range. Above 80 bars, it remains nearly constant up to 600 bars for every composition.

σ CO 2 = 𝐴 𝐶𝑂 2  𝐶 𝑁 2 𝐴 𝑁 2  𝐶 𝐶𝑂2
Eq. 2 A small shift with composition is observed but without a clear correlation (Figure 4a and4b). The shifts are quite small, which is probably due to the small error on the measured composition of gas mixtures and to the sensitivity of the Raman spectrometer. Indeed, each gas mixture was analyzed on a different day, and there is always a fluctuation in the instrumental efficiency from day to day (known as instrumental and random errors).

Table 1: RRSCS of the two bands of CO2 ( + and  -) and their sum. Values in literature were obtained at low pressure (1-5 atm) and room temperature. Our data are obtained in the pressure range 5 -600 bar at 32 °C.

This study

Ref. 29 Ref. 30 Ref. Dubessy et al. 35 stated that the sum of two RRSCS of CO2 should always be preferred for the determination of gas mixture composition because it is constant with pressure. However, Seitz et al. 34 showed in figure 5 that it was advantageous to use   + only rather than the sum of the two for determining the composition of gas mixtures. According to our statistical analyses, the small variations of   + and   -with pressure are negligible. We can, therefore, conclude that   + ,   -and their sum are all almost constant for every gas mixture concentration over the studied pressure range and can be therefore be used to determine the composition of the gas mixture. However, the small variations of   + and   -may become significant at very high pressure. The sum of the two RRSCS of CO2 should, therefore, be used in preference to avoid any effect of pressure. The averaged values calculated from the experimental data (a population of 160 data points) are 1.40 ± 0.03, 0.89 ± 0.02 and 2.29 ± 0.04, for  + ,  -and the sum  + +  -, respectively (uncertainties at 1σ). The values are in agreement with the data from literature but are given with better accuracy (Table 1).

Effect of composition, pressure, and density on Raman spectral features of N2 and CO2

a. Variation of the N2 peak position at 32 °C The downshift of the N2 band as a function of pressure and composition is shown in Figure 2a and Figure 5. The uncertainty of the corrected peak position of N2 (± 0.01 cm -1 ) is too small to be shown in Figure 5. At low pressure, the N2 peak position seems to converge to the same value ( 2330 -2330.5 cm -1 ) for all gas compositions that is in agreement with Lamadrid et al. 43 A drastic downshift is reported from 5 to 200 bars, especially for the gas mixtures dominated by CO2. Above 200 bars, it becomes less sensitive to pressure, even becoming nearly constant for gas mixtures dominated by CO2. This stepwise behavior can be explained by the variation of the density of the gas mixtures. For example, the density of the mixture at 11.4 mol% N2 increases drastically from 5 to 200 bar then reaches a plateau until 600 bar (cf. Figure S1). Figure 5 shows that the peak positions of N2 also vary with the composition of gas mixtures. In general, the presence of CO2 causes a greater downshift of the N2 peak position than that of pure N2 at the same pressure. For instance, a downshift of about 2 cm -1 is observed for pure N2 at 600 bars, whereas it is about 3.4 cm -1 for a gas mixture of 11.4 mol% N2. Besides, some curves relative to samples at different N2 concentrations are superimposed or overlap each other (100 and 89.5 mol% N2 or 29.5, 39.9 and 69.9 mol% N2), indicating that the variation of the peak position of N2 as a function of composition is not significant enough to be distinguished for some composition-pressure ranges. Moreover, for unknown reasons, the 89.5% N2 curve shows an abnormal behavior whereas the corresponding associated curve of the Fermi diad splitting of CO2 (the curve of 10.5 mol% CO2 in Figure 6) evolves as expected. The modest reproducibility of the N2 peak position despite wavelength calibration by Ne may be linked to small dayto-day variation in the shape and the position of the neon band because of variations in the positioning of the neon lamp in the optical path of the Raman spectrometer. Thus, a higher-accurate method to wavelength correction is required to use the N2 peak position as a reliable quantitative parameter. After all, we can only conclude here the global variation trend of the N2 peak position with a significant effect of the composition and pressure.

b. Variation of the CO2 Fermi diad splitting at 32 °C Figure 2b shows typical Raman spectra of CO2 and the downshift of CO2 peaks with increasing pressure. The two bands of CO2 were investigated by measuring the distance between them (Fermi diad splitting), so there was no need for absolute wavelength calibration of the spectrometer. Uncertainties of Fermi diad splitting (± 0.015 cm -1 at 1) and of pressure (± 1 bar) are smaller than the data dot size.

Figure 6 represents the variation of the Fermi diad splitting of CO2 at 32 °C as a function of pressure and composition of the CO2-N2 gas mixture. At low pressure, the Fermi diad splitting value is nearly identical ( 102.762 cm -1 ) for any composition. At higher pressure, an effect of the gas mixture composition is clearly observed. In general, the presence of N2 reduces the magnitude of the variation of the Fermi diad splitting. For example, at 600 bars, the Fermi diad splitting shifts down from 105.348 cm -1 (for pure CO2) to 103.093 cm -1 (for the gas mixture at 10.5 mol% CO2). This trend is relatively similar to that observed for CO2 mixed with CH4. 34 The repeatability and the reproducibility of the relationship between Fermi diad splitting, pressure, and composition of the gas mixture are much better than the peak position of N2 (Figure 5). The Fermi diad splitting of CO2 can thus be used as an accurate parameter to determine the pressure of CO2-N2 gas mixtures.

The Fermi diad splitting of CO2 can also be used for the determination of the density of CO2-N2 gas mixtures. For this, the density of every gas mixtures at given PT conditions presented in Figure 6 was calculated by the GERG-2004 EoS. The resulting relationship between Fermi diad splitting of CO2, density, and composition of gas mixtures (at 32 °C) is presented in Figure 7. The Fermi diad splitting increases with the density of the gas mixture and the content of CO2. Note that the shape of the curve with 99.8% CO2 is relatively irregular between 103.7 and 104.3 cm -1 (0.3 -0.7 gcm -3 ). This is likely due to the proximity with the critical temperature of CO2 (31.05 °C) where a small temperature fluctuation may result in a significant variation of density.

Figure 7: Evolution of the Fermi diad splitting of CO2 as a function of composition and density of CO2-N2 gas mixtures at 32 °C. The density was calculated at given temperature, pressure, and composition by the GERG-2004 EoS. Uncertainty on density is smaller than data dot size.

Overall, the data of the present study are in good agreement with recently published densimeters of pure CO2. 39,40 It also agrees well with the previous investigations regarding the Fermi resonance of CO2: (i) with increasing pressure, the Fermi resonance interaction reduces through a decrease of the anharmonic coupling constant (k122), resulting in an increase of the separation between the unperturbed levels (1 and 22) as well as the Fermi diad splitting, [START_REF] Hacura | [END_REF][57][58] and (ii) while increasing the content of N2, the reduction of the Fermi resonance becomes smaller (so the Fermi diad splitting still increases but with a smaller magnitude) in comparison with that of pure CO2 in the same conditions. 57 c. Effect of temperature on the Fermi diad splitting of CO2 The effect of temperature on the variation of CO2 Fermi diad splitting of CO2-N2 gas mixtures was also analyzed conducting experiments at 22 °C. At this temperature, a V-L phase transition is observed for any gas mixture containing >  92 mol% CO2 (Figure 8a). The results are compared with those obtained at 32 °C in Figure 8b and Figure 8c. In general, the magnitude of the variation of the CO2 Fermi diad splitting at 32 °C is smaller than that at 22 °C at the same pressure (Figure 8b) but identical at the same density (Figure 8c). A large gap appears at 60 bars on the curve of pure CO2 at 22°C (< Tc = 31.05 °C) due to the vapor-liquid phase transition. Indeed, at this condition (22 °C and 60 bars), CO2 is in the two-phase LV domain (Figure 8a), and thus both liquid and vapor phases coexist in the microcapillary. Pressure remains constant at 60 bars until the vapor phase is completely converted to liquid. As a result, no data point can be recorded within the density range  0.2 gcm -3 (vapor state) to  0.7 gcm -3 (liquid state). At 32 °C, there is no phase transition (supercritical state) for any CO2-N2 gas mixture (Figure 8a) and data points can be collected over the entire density range. Therefore, only the data acquired at 32 °C were fitted to provide calibration equations. These equations must be used at 32 °C only for pressure determination but can be used (at least) in the range 22 -32 °C for density determination of any gas composition above the critical temperature (Figure 8b and Figure 8c). Fermi diad splitting / cm -1 50% 70% 100% d. Calibration equations to determine the pressure and density of CO2-N2 gas mixtures According to the whole data set shown in Figure 5, Figure 6 and Figure 7, the Fermi diad splitting of CO2 appears as the most reliable quantitative parameter for determining pressure and density of CO2-N2 gas mixtures. Data obtained at 32 °C were fitted to provide calibration equations. In order to minimize uncertainties on the calculated pressure and density, the calibration data were fitted separately for five smaller pressurecomposition (PX) domains (Figure 9). Regions Third-order polynomial equations linking pressure or density to the CO2 Fermi diad splitting and the composition of gas mixtures were computed for each PX domain. The general form of the calibration polynomial is given in Equation 3, where 𝐶 𝐶𝑂2 and  are defined by Equations 4 and 5, respectively. Fitting by a higher-order polynomial does not lead to substantial improvements in the qualitative of measurement. The coefficients (𝑝 𝑖𝑗 , 𝑎, 𝑏, 𝑠𝑡𝑑_𝑎, 𝑠𝑡𝑑_𝑏, with (𝑖 + 𝑗) ≤ 3) of each calibration equations are listed in Table S2 (pressure) and Table S3 (density) in Supporting Information. Two main error sources contribute to the final uncertainty of the calculated pressure and density. The first source is directly associated with the uncertainty of the Fermi diad splitting (± 0.015 cm -1 ) and of the measured composition of the gas mixture (calculated from RRSCS and peak areas with Equation 2). As the regression calibration equations are not linear, the uncertainty of the first error source is not constant but varies with the composition of the gas mixture and the CO2 Fermi diad splitting. The second error source is related to how well the bestfitted calibration equations reproduce the pressure and the density from a given CO2 Fermi diad splitting and gas mixture composition. The uncertainty of each calibration equation was derived from its prediction bounds (at 1) and reported in the last row of Tables S2 andS3. The ultimate uncertainty on measured pressure or density will be the sum of these two error sources, as reported by Fall et al. 39 and Wang et al. 40 for pure CO2.

Investigation of CO2-N2 natural fluid inclusions. The calibrations described above were applied to 15 natural CO2-N2 fluid inclusions trapped within a quartz sample from the Central Alps (Switzerland). Composition, pressure, and density were compared with those derived from microthermometry.

Each FI was analyzed three times by microthermometry to determine the melting temperature Tm(car) and the homogenization temperature of the volatile phase Th(car) (Table S1). These phase transition temperatures were subsequently reported in the VX diagram of Thiéry et al. 54 to calculate the composition and molar volume (density) of the fluid inclusion. As the uncertainties arising from this VX diagram are unknown, only the uncertainty of ± 0.1 °C of the heating-cooling stage to Tm(car) and Th(car) is considered. This uncertainty of ± 0.1 °C can cause either significant or insignificant error depending on the region in the VX diagram. For example, the slopes of the Th(car) lines are less steep in the vapor field than in the liquid field (Figure 8b in 54 ). In this domain, an uncertainty of ± 0.1 °C in Tm(car) can result in a variation of up to 5 mol% in composition and up to 0.09 gcm -3 in density. The second source of error may be an error in graphical reading. Detailed microthermometry results of each FI were reported in Supporting Information (Table S1).

The selected FIs were also analyzed three times by Raman spectroscopy. The averaged values of the peak position and peak area of CO2 and N2 bands were used to calculate composition, pressure, and density through Equations 2 to 5. The composition of the FIs was calculated by the RRSCS of the two CO2 bands and their sum for comparison. The difference between these three values is always less than 1 mol% CO2. Detailed measurements of each FI are presented in Table S4 of Supporting Information. Figure 10 presents a comparison between Raman and microthermometry results. The uncertainty of Raman measurements on composition (< 0.5 mol% CO2) and density (< 0.01 gcm -3 ) is slightly better than that derived from microthermometry measurements (ranging from 1 to 2.2 mol% for composition, and from 0.01 to 0.04 gcm -3 for density). Uncertainty on measured pressure at 32 °C from microthermometry (varying from 2 to 11 bars, Figure 10c) is equivalent to that from Raman ( 15 bars when pressure > 150 bars, and  3 bars when pressure < 150 bars).

Overall, the results derived from the two methods are close and comparable with a relative difference varying from 0.1 -7 % coming from the error sources mentioned above, but also from the unknown error of the thermodynamic models of Soave-Redlich-Kong (1972) and Lee-Kesler (1975) used for the construction of the VX diagram of Thiéry et al. (1994). 54 According to Mullis et al. 47 , the trapping temperature of the geological fluid where Mu 147.2 sample was collected was about 400 °C. Thereby, the trapping pressure (the pressure at trapping temperature) of the geological fluid determined using GERG-2004 EoS is about 1610 ± 20 bar (calculated from Raman results) or 1770 ± 20 bars (calculated from microthermometry results). The difference of density leads to a difference of only 160 bars (9 %) which is of no consequence on geological interpretation.

Raman spectroscopy may be more efficient than microthermometry in some cases. For example, FI4.8 is too small (< 2 µm, Figure 3a) to observe any phase transition with good accuracy. Concerning FI3.5, Th(car) could not be determined precisely because the vapor bubble was located in the dark part of the inclusion at a temperature close to homogenization (Figure 3b). Similarly, FI2.2 could not be analyzed by microthermometry because of bad optical conditions (color, contrast, etc.). These three FIs could, however, be analyzed by Raman spectroscopy. Another disadvantage of microthermometry method (using VX diagram of Ref. 54 ) appears when clathrate is formed and remains above Th(car), meaning that a part of CO2 is still trapped inside the clathrate structure, and thus could lead to an underestimation of the CO2 quantity while using only Th(car). 59,60 The latter problem is not encountered by Raman spectroscopy at 22 and 32 °C.

CONCLUSION

The use of an improved HPOC system consisting of an FSC coupled with a heating-cooling stage and a Raman spectrometer makes it possible to investigate the behavior of CO2-N2 mixtures at controlled pressure and temperature conditions. The experiments can be easily repeated several times for statistical purposes as well as repeatability and reproducibility test. A complete calibration of the Raman signals of CO2-N2 mixtures was thus performed for the first time. The Fermi diad splitting of CO2 was linked to pressure or density for any CO2-N2 gas mixture in the range 22 -32 °C. It was also demonstrated that the RRSCS of CO2 does not depend on composition but slightly on pressure or density. However, this effect is negligible in the studied pressure range (< 600 bar). Thus accurate RRSCS values of the two vibration modes of CO2 and their sum can be either used to determine the composition of CO2-N2 gas mixtures with an uncertainty of about  0.5 mol%. The pressure and the density of CO2-N2 binary gas mixtures can be henceforth calculated by using regression calibration equations that were validated by successful application to natural fluid inclusions from the Central Alps, Switzerland. A detailed comparison was made indicating that Raman spectroscopy is a powerful alternative tool to the microthermometry, providing not only PVX information with comparable accuracy (even better in some cases) but also handling cases for which microthermometry cannot be applied. This study shows the applicability of Raman spectroscopy for gas analysis purposes at extreme conditions (very small object at high pressure) and can be easily extended to any gas mixture.

Figure 2 :

 2 Figure 2: Evolution of (a) N2 and (b) CO2 Raman spectra with pressure. Both gases show a downshift with increasing pressure. The signal of neon (Ne) was simultaneously recorded with N2 spectra for wavenumber calibration.

Figure 3 :

 3 Figure 3: Examples of selected FIs of sample Mu 147.2 (transmitted plane-polarized light at room temperature): (a), (b) and (c) monophasic FIs from zones 4, 3 and 2 containing a CO2-N2 liquid phase; (d) two-phase FI containing H2O (liquid) and a bubble of CO2-N2 vapor.

Figure 4 :

 4 Figure 4: Variation as a function of pressure and composition of (a) the RRSCS of the two bands of CO2 (+ at 1388 cm -1 and at 1285 cm -1 ) and (b) their sum (+ + -).

Figure 5 :

 5 Figure 5: Variation of the fitted peak position of N2 (corrected from Ne peak position) at 32 °C as a function of pressure and composition (mol% N2) of gas mixtures.

Figure 6 :

 6 Figure 6: Evolution of the Fermi diad splitting as a function of composition and pressure of different CO2-N2 gas mixtures at 32 °C. Uncertainties of Fermi diad splitting (± 0.015 cm -1 at 1) and of pressure (± 1 bar) are smaller than the data dot size.

Figure 8 :

 8 Figure 8: (a) Phase diagrams of CO2-N2 gas mixtures exported from data calculated by REFPROP. L: liquid-phase domain; V: vaporphase domain and LV: biphasic liquid-vapor domain. Only critical isochores are drawn for each mixture. (b) and (c) Comparison between the evolution of Fermi diad splitting of pure CO2 and CO2-N2 mixtures (50 and 70 mol% CO2) as a function of pressure and density at 22 and 32 °C.

  (a) and (b) cover the pressure range 5 to 600 bars and the composition range 50 -100 mol% CO2 and 10 -50 mol% CO2, respectively. Regions (c), (d) and (e) cover only the low-pressure range (5 -150 bars) and the composition range 90 -100 mol% CO2, 30 -90 mol% CO2 and 10 -30 mol% CO2, respectively.

Figure 9 :

 9 Figure 9: Pressure-composition (PX) domains for application of polynomial equations a, b, c, d, and e. Experimental data were fitted within each PX domain to provide the best-fitting polynomial equation to minimize uncertainties on the calculated pressure and density.

  𝑃𝑟𝑒𝑠𝑠𝑢𝑟𝑒 (𝑜𝑟 𝑑𝑒𝑛𝑠𝑖𝑡𝑦) = ∑ ∑ 𝑝 𝑖𝑗 𝐶 𝐶𝑂 2

Figure 10 :

 10 Figure 10: Comparison after analysis of the volatile phase of selected FIs by Raman and microthermometry of the (a) composition, (b) density, and (c) pressure at 32 °C.

ACKNOWLEDGMENTS

This work is a part of the thesis of Van-Hoan Le (Université de Lorraine) who acknowledges the French Ministry of Education and Research and the ICEEL Institut Carnot. The work benefited financial support from CNRS-INSU CESSUR program. The authors are sincerely thankful to Catherine Lorgeoux and Héloïse Verron for their instruction during the Gas Chromatography measurement part, to Silvia Lasala and Romain Privat for a fruitful discussion about the thermodynamic properties of the CO2-N2 system. Two anonymous reviewers are acknowledged for their thorough review and their insightful comments and suggestions.

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website.

Detailed uncertainty calculations and the coefficients of regression polynomial equations 3, 4 and 5 (PDF)

AUTHOR INFORMATION

Corresponding Author * E-mail: van-hoan.le@univ-lorraine.fr

Author Contributions

All authors have approved the final version of the manuscript.


The yin and the yang of p27 Kip1 as a target for cancer therapy B.Eymin and E. Brambilla Schematically, human tumors arise because tumor cells escape from extracellular signals that physiologically monitor cellular proliferation as well as cell death and acquire the property to grow continuously. During tumorigenesis, many proteins that normally control the proper timing of cell growth such as the cyclins or prevent the occurrence of unappropriate apoptosis are abnormally expressed or activated and act as typical oncogenes leading to hyperproliferation and/or protecting tumor cells from death. In contrast, proteins that normally prevent for example damaged cells to progress into the next phase of the cell cycle or act as apoptosis inducers if the damage can not be repaired are frequently inactivated in tumor cells and act as tumor suppressor genes. Therefore, loss of tumor suppressor genes associated with abnormal hyperactivity of oncogenes classically contribute to human carcinogenesis. However, affecting a clear-cut function of tumor suppressor or oncogene remains a subject of debate for certain proteins. p27 Kip1 is a member of the cyclin dependent kinase inhibitory proteins which also include p21 WAF1 , the target gene of p53, p16 INK4a , a member of the retinoblastoma (Rb) pathway and p57 Kip2 . With p16 INK4a , it plays a major role in counteracting the activity of cyclins D 1 and E on Rb phosphorylation and subsequent G 1 -S transition. Thus, it coordinates the activation of cyclin E-cdk2 with accumulation of cyclin D-cdk4 and initiates the timely exit of cells from the cell cycle in response to antimitogenic signals. Beside its classical anti-proliferative function, p27 Kip1 can also promote or inhibit apoptosis depending on the context (1,2). Based on these data, p27 Kip1 presents most of the classical features of a tumor suppressive protein that might be loss in proliferative tumor cells. Unlike most of the tumor suppressor genes which loss of function responds to the classical "two-hit hypothesis" of tumor suppression (Knudson law), p27 Kip1 gene is not deleted (neither loss of heterozygozity nor homozygous deletion) not mutant, and does not suffer aberrant methylation of its 5'-end in tumors. Rather, in mouse models, p27 Kip1 was shown to be a dosage dependent tumor suppressor since loss of a single allele of p27 Kip1 increases the frequency and decreases the latency of tumors to a level intermediate to that seen in wild-type and null counterparts (3). In human tumors, a direct correlation between p27 Kip1 protein levels and survival chances was first noted in colon cancer and later in cancers of the breast, prostate, bladder, ovary, lung and other tissues (for review, see 4). The evidence that reduced p27 Kip1 may contribute to tumor development by either increasing the proliferation of cells or decreasing their apoptosis might explain why the loss of p27 Kip1 is a common marker among many different tumor types and suggest that p27 Kip1 might be a valuable target for both stratifying patient risk and, perhaps, selecting treatment. However, observation that some human tumors exhibit high levels of p27 Kip1 makes this scheme more complex. For example, in aggressive small cell lung cancer, p27 Kip1 overexpression has been suggested to protect cells from apoptosis in unfavorable microenvironments [START_REF] Masuda | Protective function of p27(KIP1) against apoptosis in small cell lung cancer cells in unfavorable microenvironments[END_REF]. In addition, p27 Kip1 might affect the efficacy of chemotherapies as these treatments works better in proliferating rather than in growth-arrested cells. Therefore, we need to keep in mind that modulating p27 Kip1 levels can have unforeseen consequences on therapy depending on the cell type and the contextual clues. In this issue, Ishii and colleagues (6) have studied the consequences of p27 Kip1 overexpression or downregulation in human lung adenocarcinoma. They first show that overexpression of p27 Kip1 inhibits cell growth and rescues cells from death instead of inducing apoptosis. More interestingly, using siRNA technology, they demonstrate that neutralization of endogenous p27 Kip1 induces cell death of several non small cell lung cancer cell lines without affecting their cell cycle status. Finally, after analysis of p27 Kip1 subcellular localization and phosphorylation, they describe the predominant cytoplasmic accumulation of endogenous p27 Kip1 in these tumor cells which might preclude it from regulating the G 1 /S transition. p27 Kip1 activity appears to be directly targeted by its mislocalization to the cytoplasm in colon or ovarian tumors [START_REF] Ciaprrone | Localization and expression of p27Kip1 in multistage colorectal carcinogenesis[END_REF][START_REF] Hurteau | Expression and subcellular localization of the cyclin-dependent kinase inhibitor p27(KIP1) in epithelial ovarian cancer[END_REF] and cytoplasmic p27 Kip1 seems to correlate with poor-long term survival and tumor grades in breast carcinoma [START_REF] Viglietto | Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27Kip1 by PKB/Akt-mediated phosphorylation in breast cancer[END_REF]. Mislocalization effectively inactivates p27 Kip1 inhibitory activity, as cytoplasmic p27 Kip1 is partitioned from its nuclear cyclin-cdk targets. However, as suggested by Ishii and colleagues, such cytoplasmic localization of p27 Kip1 could also contribute to its anti-apoptotic functions. Therefore, before considering therapies, we need to predict how cells in a particular tumor will respond to accumulation or neutralization of p27 Kip1 . In this context, the study of Ishii and co-workers suggests that targeting p27 Kip1 in non small cell lung cancer could inhibit their growth by reducing cell viability. In contrast, in other tumor types, it will be the restoration of p27 Kip1 that will prevent tumor growth, either by inhibiting cellular proliferation or inducing apoptosis. So, depending on the cancer type, the goal of therapy targeting p27 Kip1 will tend to either neutralize or reintroduce it. It is the reason why comprehensive understanding of p27 Kip1 biology would facilitate development of such therapeutic responses in human cancer. So, it would be too simplistic to ascribe p27 Kip1 only as a tumor suppressive protein since its anti-apoptotic function might also predict an "oncogenic" role. As a typical example of this "gain of function" are the highly agressive small cell lung cancer which carry elevated levels of nuclear p27 Kip1 . Obviously, more need to be done to decipher the real contribution of p27 Kip1 in human lung tumor growth. In conclusion, it is now well established that the biological effects of numerous proteins might be quite different, sometimes even opposite, depending on the cellular model, the context as well as their subcellular localization. Therefore, before designing general therapies against cancer, we have to study very carefully the specific proteins involved in each carcinogenetic process as well as to investigate the molecular mechanisms by which the loss or hyperactivity of such proteins contribute to the carcinogenesis. Finally, we have to think about the fact that modulating the level of proteins that controls essential processes such as cellular proliferation and apoptosis might interfere with the response of tumor cells to chemotherapeutic treatments as well as to have unpredicted consequences.