Estimation of nitrogen-to-carbon ratios of organics and carbon materials at the submicrometer scale

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Estimation of nitrogen-to-carbon ratios of organics and carbon materials at the submicrometer scale

Julien Alleon, Sylvain Bernard, Laurent Remusat, François Robert

To cite this version:

Julien Alleon, Sylvain Bernard, Laurent Remusat, François Robert. Estimation of nitrogen-to-carbon ratios of organics and carbon materials at the submicrometer scale. Carbon, Elsevier, 2014, 84, pp.290-298. �10.1016/j.carbon.2014.11.044�. �hal-01090504�

Estimation of nitrogen-to-carbon ratios of organics and carbon

materials at the submicrometer scale

Julien Alleo n, Sylvain Bernard^*, Laurent Remusat, François Ro bert

Insti tut de Minéralogie, de Physiqu e des M atériaux et de Cosmochimi e (IMPMC), Sorbonne Universités - CNRS UMR 7590, Muséum National d’Histoire Naturelle, UPMC Univ Paris 06, IRD UMR 206, 61 rue Buffon, 75005 Paris, France.

* correspo nding author : sb ernard@mnhn.fr

Abstract:

Precisely estimating nitrog en-to-ca rbo n (N/C) ratio o f carbonaceous materials at the submicrometer sca le is a challenge in bo th natural a nd material sciences. Following recent a ttempts repo rted in the literature, the present paper discusses t wo metho ds of quantifica tio n o f the N/C ra tio or o rga nics and carbon materia ls at the submicrometer scale using na noscale secondary ion mass spect ro metry (NanoSIMS) a nd X-ray absorptio n spectro scopy (XAS). The present data highlig ht the need to use a t least two standa rds to build a ca libratio n line in order to precisely and a ccurately (±

0 .009 – 95 % co nfidence level) estimate N/C va lues of unknown carbo n samples using NanoSIMS. As shown here using a set of reference compounds, STXM-based XAS a llows directly estimating N/C ratio s of organics without using any standard with errors as low as 0.007.

1 Introduction:

Understand ing the impact of nitrogen incorporation o n carbo n material p roperties at the sub micrometer scale is impo rtant for man y scientific d omains ranging fro m pedolo gy and the stud y o f o rganics in so ils (e.g., [1,2]) to

* Corres pondin g author. Tel : +33 (0)1 40 79 35 32. E -mail: sbernard@mnhn.fr

microp aleonto lo gy and the search for the o ldest traces of life in rocks (e.g., [3,4 ]), through m aterial science and the stud y o f prop erties of N-dop ed carbo n materials (e.g., [5–7]).

Pro cesses occu rring in soils, such as seq uestratio n of organics, may play a crucial role in reducing the atmospher ic CO₂ enrichment and improving b iomass/agrono mic p roductivit y for instance ([8–10 ]). These processes are intimately contro lled b y organic carb on and nitrogen fluxes [11,12]. As soils ma y appear ver y hetero geneo us at all scales of observation [11,1 2], better constraining su ch processes requ ires precise estimatio ns of the N/C ratios of o rganics to be do ne at the su bmicrometer sale.

Precisely estim ating the co ncentrations o f nitrogen at the sub micro meter scale ma y also help identifying organic r emains of life in ancient rocks. For instance, the invo lvement of nitrogen, together with phosphorous and sulphur, in o rganic micro structures found in Gyr-old cherts has b een used to discuss their b iogenicity (e.g., [13–15 ]). In additio n, precisely estim ating N/C ratios of individ ual m icrofossils at the su bmicrometer scale might provid e estimatio ns of the d egree of their preservatio n [16].

Nitrogen doping may profou ndly affect electronic properties of carbo n materials and brings along tremendous technolo gical implications [5–7 ]. As the quantit y of nitro gen that might be inco rporated var ies depending on the technique u sed (e.g., magnetron sputtering, laser ab latio n, p yro lysis or chem ical vapor d epo sitio n) and as carb on nanomaterials may be strongly heterogeneous [17,18], b etter understand ing the properties of carbon materials req uires precisel y quantifying their N/C ratios at the submicrometer scale.

A nu mber of recent stud ies have attem pted to propose new wa ys to p recisely quantif y organic N/C ratio at a small scale. For instance, Hatto n et al.

[1] have used Nanoscale Secondar y Io n Mass Spectrometr y (Nano SIMS) to p ro vide estimatio ns of the N/C ratio of soil organics. Yet, their method ology relies on an inter nal calibratio n requiring a lar ge range of N/C values to be covered b y the investigated materials and is thus no t universally applicab le. Mo re recently, Tho men et al. [19] have p roposed a way to calibrate NanoSIMS data to estimate the N/C ratio of organics. Yet, due to large topo grap hical effects, their calib ration lacks the precision requ ired to obtain meaningful data in the small

range of N/C values t yp ical of terrestrial environments. On the other hand, Cod y et al. [2 0,21 ] and van Dommele et al. [22] have u sed synchrotron-based X-ray absorption spectroscop y (XAS) and X-ray p hotoelectro n spectrosco p y (XPS), resp ectively, to quantif y the N/C ratio of submicrometric organic p articles or carbo n materials. Unfortunately, altho ugh it would have accredited their methodology, these authors d id not repo rt any measurement o n reference compounds.

The present co ntr ibutio n presents carefu l examinatio ns of two methods recently repo rted in the liter ature for p recise qu antificatio n o f the N/C atomic ratio of o rganics at the su bmicrometer scale b ased o n nanoscale secondary io n mass sp ectro metry (NanoSIMS) measurements and X-ray absorption spectroscop y (XAS) data collected u sing scanning transmissio n X-ray microscop y (STXM).

Fro m resu lts o btained on a set of organic reference compounds, the present contrib ution d iscusses the validit y, precisio n, advantages and limitations of these two methods.

2 . Materials & Metho ds

2 .1. Samp le selection

Six homogeneous o rganic co mpound s of vario us chemical structures cover ing a wide range of N/C valu es have b een selected for the present stud y (Table 1). These compounds have been used as reference materials to estimate how p recise and accu rate can be the qu antificatio ns of N/C atomic ratios u sin g NanoSIMS and XAS. Among these reference samples, two natural t ype I and t ype III kero gens, a synthetic epoxide resin, two am ino acids ( L-Phen ylalanine and L- Tr yptophan) and an o rganic compound synthesized b y plasma dischar ge on a N2 – CO gaseous mixtu re (e.g., Nebulotron; [23]). Bu lk N and C concentratio ns have b een measured b y thermal co nd uctivit y o n 1-2 mg aliquots (SGS Multilab) (Table 1 ).

Samp le N/C

B ul k

± 1ı (10^-4)

CN^-/C2-

(mean 3 0 ROI)

± 1ı

(10^-2)

N/C

X AS

graphical error (10^-2)

Kero gen typ e III

0 .016 0 .80 0.141 1 .60 0 .018 0 .70

Kero gen typ e I

0 .025 1 .25 0.238 2 .50 0 .025 1 .00

Resin 0 .053 2 .65 0.491 2 .20 0 .053 2 .00

Phen ylalani ne

0 .111 5 .55 0.961 7 .10 0 .111 1 .00

Tr yptophan 0 .182 9 .10 1.576 2 .90 0 .182 1 .00

Nebulotron 0 .746 37.3 NA NA 0 .700 1 .00

Table 1: Bulk N/C measurements (thermal conductivit y – SGS Multilab ), CN^-/C2-

io nic ratio s measured b y NanoSIMS and N/C ratios estimated b y STXM-based XAS.

2 .2. Samp le Preparation

For NanoSIMS exp eriments, samples have been pressed at 0.5 ton onto clean indiu m fo ils, co ated with 15 nm of gold and d egassed in the Nano SIMS vacuum chamb er for two days b efore analyses. For STXM experiments, the same compounds have been finely crushed, pipetted in ultrapure water and depo sited o n 50 nm thick silicon nitride wind ows.

2 .3. NanoSIMS

The originalit y of the NanoSIMS design is that primar y and seco ndar y io n b eams are sharing the same op tical s ystem. The primar y b eam hits the samp le p erpendicularly to its su rface, which reduces the fo cal length and aberrations compared to regu lar SIMS, thus leading to a probe diameter as small as 50 nm [24 ]. Using the CAMECA NanoSIMS 50 at MNHN (Par is, France), seco ndar y

molecular ions ^{1 2}C¹⁴N^- and ^{1 2}C2-

are collected simu ltaneously. Due to their extr emely high electro n affinit y [25,26] and higher stabilit y, the yield of secondar y CN^- is ver y high comp ared to the o ne o f N^-. Fu rthermo re, the CN^-/C2-

ratio ap pears as a more appropriate proxy for N/C atomic ratio than CN^-/C^- which can be seen as a p ro xy o f nitrogen concentratio n [19].

A 30 μ m entrance slit (ES3) and a 350 μ m ap erture slit (AS1 ) have b een u sed to reduce secondar y ion ener getic and angu lar dispersions, respectively.

Befo re measu rements, Cesium (Cs⁺) has been imp lanted during 7 min using a 200 p A primary cu rrent and a 300 μ m apertu re diaphragm on 13×13 μ m² areas.

Hence, a d ose o f about 3×10^{1 7} Cs.cm^-² has been imp lanted befo re analysis to ensure statio nar y regime for seco ndar y io ns emissio n [19]. Analyses have then b een performed on 10 x10 μ m² areas (i.e., on areas smaller than the imp lanted o nes to avoid edge artifacts) u sing a 0.5 pA primar y current and a 150 μ m aperture diap hragm, i.e., with a b eam size of abo ut 70 nm. Seven c ycles have b een stacked to generate o ne image (256 × 256 pixels fo r 10 × 10 μ m² areas with a counting time of 1 ms/px); three images per sample have b een collected .

Data acquired b y NanoSIMS have been corrected fo r a 44 ns dead time o n each electron multiplier and have been pro cessed u sing Lim age software (developed b y L. Nittler, Car negie Institution, Washingto n DC, USA). Fo r each analyzed area (i.e., for each image), 10 regions of inter est ( ROI) of 0.7 × 0.7 μ m² have b een manually d efined on regio ns of samples with ver y low to pographic var iatio ns (Figure 1; see sectio n 3.2 .2 for details).

Altho ugh there is no isob aric interference with ^{1 2}C^{1 3}C¹H^- ions, ^{1 3}C2-

and

1 2C^{1 4}N^- ions canno t be separated on mass 26 . Yet, this iso baric inter ference is negligib le, even for N-poo r samples. For instance, during one cycle of measurement on the t ype III kerogen, 130000 counts per second (cp s) of ¹²C2-

and 2 0000 cps of ^{1 2}C^{1 4}N^- have been collected on masses 24 and 26 respectively.

Based on natu ral abund ances [27], less than 1 ‰ o f these 2 0000 cp s are accounting from 13C2-

ions (§ 15 cps).

2 .4. STXM-based XAS

STXM is a synchrotron-based transmission sp ectro microscop y technique u sing a mo nochromated X-ray beam produ ced b y s ynchrotron rad iation. This technique allows bo th m icrosco pic o bservations - i.e., imaging at the 15-nm scale - and spectroscop ic measu rements - i.e., recording X-ray absorptio n spectra wit h a sp ectral resolution of 0.1 eV which provid e information on element speciatio n at the same spatial scale. Measurements of the present contr ibution have b ee n done u sing the 10 ID-1 SM beamline [28] located at the Canad ian Light Source (CLS). Beamline 1 0ID-1 uses soft X-rays (130– 2500 eV) gener ated with an elliptically po larized undulator inserted in the 2 .9 GeV s ynchro tro n sto rage rin g (250–150 mA). The microscope chamber is evacu ated to 10 0 mTo rr after samp le insertion and back-filled with He. A 100 nm thick titanium filt er is used t o remove second order light when working at the C and N K-edges. Energ y calibratio n is accomplished u sing the well-resolved 3p Rydber g peak at 294.96 eV of gaseous CO2 for the C K-edge.

Here, X-ray ab sorp tion sp ectra have been ob tained b y collecting image stacks, i.e., b y rastering selected areas o f samples in the x- y d irections at energ y incr ements of 0.25 eV over the 270 – 450 eV energ y r ange u sing the lo w energ y grating o f the 10ID-1 SM b eamline. This energ y range co vers the carbon (270- 340 eV) and the nitrogen (390-450 eV) absorptio n ranges. Stack measu rements have been performed with a dwell time of the o rd er of a millisecond or less p er p ixel to avoid irradiation d amages following the procedu res for X-ray microscop y studies of r adiatio n sensitive samp les recommended b y [29].

Importantly, with these settings, radiatio n damage per unit of analytical information has been sho wn to be typ ically 100 -1000 times lo wer in STXM-based XANES spectroscop y comp ared to TEM-based EELS [30]. Note that a minimu m o f 20 pixels have to be summed to have a go od signal/noise ratio with these settings. Alignment o f images of stacks, selection of p ixels, and extraction of XAS spectra have b een do ne using the aXis2 000 so ftware (ver 2.1n). X-ray absorption spectra are measu rements o f ab so rbance (A) (also defined as transmissivit y or op tical densit y) which corresponds to the opposite of the natural lo gar ithm of the ratio between the intensity of the radiatio n p assin g through the sample ( I) and the intensit y o f the incident r adiatio n ( I₀) measu red o n a samp le-free location o n the silicon nitr ide wind ow (A=-ln(I/I0)). Estimations of

N/C ratios from X-ray absorption sp ectra have been do ne here u sing the R stud io software (http://www.r-project.org/), although any programming langu age ma y be u sed.

3 . Estimatio n of N/C atomic ratios

3 .1. NanoSIMS estimatio n of N/C ratios

The distr ibutio n of the mean measured CN^-/C2-

values of all ROIs plotted against bulk N/C values for reference samp les is well explained b y a linear regressio n mod el (R² > 0.99 ; Figure 2). No te that the regression line do es not cross the origin. This m ight be due to the adso rption of N2 onto the sample surface d espite high vacuum. N monoatoms coming from the b eam- indu ced b reaking o f adsorbed N2 may participate to no no rganic CN^- io ns fo rmatio n [31].

In any case, the present d ata highlight the need to u se at least two stand ards to build such a calib ration line in o rd er to p recisely and accu rately (± 0.009 – 95 % confid ence level) estimate N/C valu es of unkno wn carb on samples.

3 .2. STXM-based XAS estimation o f N/C ratio s

According to the Beer-Lamb ert law [32], the intensit y of the radiatio n p assing throu gh the sample (I) is related to the intensit y o f the incid ent rad iatio n (I0) following the equation I = I0e^{- μ lρ} with μ the mass absorptio n co efficient, l the thickness o f the sample and ρ its vo lumetric mass d ensit y. The ab so rp tion signal (A=-ln (I/I0)), i.e., the absorbance or optical densit y, is thus the product o f the thickness times the vo lumetric mass densit y times the mass absorption co efficient (A=μ lρ). According to Henke et al. [33,34], for a given element, the mass absorption coefficient (μ ) is d irectly proportio nal to the ato mic p hotoabsorptio n cross section (ıa), which is itself d irectly proportional to the f2 co mpo nent (imaginar y p art) of the co mplex atomic scattering facto r. (Note that the f₂ components of mo st elem ents can be found online:

http://henke.lbl.gov/op tical_co nstants/).

The optical prop erties of any material in the pho ton ener gy r ange b elow and above the edge regio ns of its co nstituted elem ents can be d escr ibed b y the atomic scatter ing factors [33 ,34]. Thu s, for a given area o f a sample (i.e., for a given thickness and a given volumetric mass densit y), an atomic ratio can be d irectly estimated b y divid ing the coefficients used to fit the sum of the f₂ components of carbon and nitro gen to the measured absorption signal b elow and above the ed ge regions. The ab so rp tion signal over the carbo n and nitrogen edge regions (280-330 eV and 3 95-430 eV, respectively) is not fitted as this signal is dominated b y peaks co rresp ond ing to electronic transitions as well as b y broad spectral features correspo nding to highly delo calized excited states sometimes referred to as virtual state transitions or the overlapp ing contrib ution of Feshb ach reso nances [35 ,36]. A typ ical resulting fit in the ener gy range cover ing the C and N K-edges, i.e., the sum KC×f2 (c a r b on ) + KN×f2 (ni t roge n), is shown on Figure 3. The ratio KN/KC co nstitu tes an estimatio n of the N/C ratio o f the investigated carbo n material.

Figure 4 shows the X-ra y absorptio n sp ectra of the six o rganic reference compounds used for the p resent contr ibu tion and the resp ective resulting fits of the sum KC× f2 (c a rb o n) + KN×f2 ( ni t roge n). As shown o n Figure 4G, the regressio n curve between the ratios KN/KC and the N/C bu lk measurements is ver y close to a straight line with a slope of 1 passing through the origin. Note that the XAS- b ased estim ations of N/C ratios perfectly match the exact values measured o n bulk po wd ers for the purest organic compou nds used in the present co ntr ibutio n (i.e., the s ynthetic epo xide resin and the two pure amino acids). The slight d eviation of the linear regr ession line slope likely resu lts from the chemical hetero geneo us nature of the two kero gens and the Neb ulotron samples at the sub micro meter scale. Imp ortantly, the fact that the regression line p asses through the o rigin suggests that nitrogen adsorbed o nto the sample surface d oes not contrib ute to the absorptio n signal.

4 . Discussion

4 .1. NanoSIMS measu rements

4 .1.1. Linear Resp onse, Rep eatabilit y, Predictio n Inter val, Lim its of Detectio n and Quantificatio n

The p rediction inter val (PI) corresponds to the uncertaint y o n the pred icted N/C valu e inverted from a CN^-/C₂^- m easu rement (Figure 2 ). It differs from the confid ence inter val (CI), which represents the u ncertaint y of the mean values, b y taking into account the fluctuation of CN^-/C2-

measurements aro und the mean value (Figu re 2). The lim it of detection (LOD – 0.009) corresponds to the horizo ntal inter section o f the y- intercep t of the PI with the calibratio n line (Figure 2 ). A measu red CN^-/C2-

ratio lower than the LOD canno t be d istinguished from zero [37]. The limit of qu antificatio n (LOQ – 0.018) correspo nds to the horizo ntal intersectio n of the upp er y- intercept of the PI with the lower b ound of the CI (Figu re 2). Below the LOQ, N/C values cannot be qu antified [37]. With the settings used for the present contr ibu tion, a measured CN^-/C2-

valu e of 0.5 for example will correspo nd to a ‘true’ N/C value o f 0.056 ± 0.009 (2ı). The level of p recision that can be reached u sing Nano SIMS in the present configuratio n is thus twice as better as the one repo rted in the literatu re [19].

We attr ibu te su ch a goo d precisio n level to the low dispersio n arou nd the calib ration cu rve of the data reported in the p resent co ntr ibutio n compared to d ata repo rted b y Thomen et al. [19]. Such impro vement, ind icating a better repeatab ilit y o f Nano SIMS measu rements (Figu re 2 ), mostly r elies on sample p reparation. In co ntrast to Tho men et al. [19] who pressed their samp les at 50 k g o r less, the reference samples measured in the p resent contr ibution have b een p ressed at 0.5 ton, in ord er to limit sample topo grap hy. This is o f primar y importance as the sp uttering yield increases with r ising angle o f incidence of the p rimar y b eam ( fro m the zero - no rmal incidence) due to the ejectio n of primar y recoil atoms [38]. This is well illustrated b y the CN^- intensities measured on the fractured zones of the tr yp tophan reference sample (Figure 1A). In additio n, selecting the ROIs with care, o n flat and smooth areas, enhances bo th linear it y and measurement r ep eatabilit y. Fo llowing these precau tio ns, the final relative standard deviations o f N/C estimatio ns fo r the reference samples u sed in the p resent co ntribution are 1 1 % for the t yp e III kerogen, 10 % for the t ype I kerogen, 4 % for the epoxide resin, 7 % for the p hen ylalanine and 2 % fo r the

tr yptophan. The Neb ulotron reference sample co uld ho wever not be measured b y NanoSIMS (see sectio n 4.1.2).

4 .1.2. Sensitive carbon materials and primary beam d amage

Stead y-state emissio n of secondar y ions requ ires a su fficient implantatio n o f Cs⁺ b efore analysis. Yet, the primary beam ma y severely d amage o rganic surfaces [3 9]. Figures 1C and 1 D show SEM images of the tr yptop han and the Nebulotron reference samples after Nano SIMS measurements, respectively.

While the su rface of the tr yptophan reference samp le seems to have b een u nmodified , the Nebulo tro n reference sample has exper ienced severe structu ral d amage preventing any reliable NanoSIMS measurements. The origin of this structural modification m ight b e related to the aliphatic natu re of the Nebulo tro n reference samp le, which makes it very sensitive to scissio n chain and crosslinking processes. Indeed , thermochemical processes indu ced b y the NanoSIMS io n beam may induce cyclizatio n, aromatizatio n and cond ensatio n of o rganic mater ials [40]. In p articu lar, the io n beam may trigger the productio n of reactive free radicals from the cleavage o f chemical bonds. These radicals ma y chemically react with the surrou nding matr ix and fo rm co njugated doub le bo nds o r two- or three- dimensio nal cross-linked structures [41], which wou ld eventually lead to netwo rk-like structu res similar to those o bserved for the Nebulotron reference samp le (Figure 1D). NanoSIMS may thus not be u sed with sensitive o rganics and carbon mater ials.

4 .2. STXM Measurements

4 .2.1. Limits and Precisio n

Uncertainties on XAS-b ased N/C estimations may ar ise fro m poo r signal- to -noise ratios as well as from lo cal samp le topography which may be responsible for small-angle beam scattering. Yet, these two effects appear negligib le compared to the error o n estimations that might result from poor fit ap preciatio n.

A way to quantify su ch an error is illu strated o n Figu re 3. Comp ared to the best

fit (blue cu rve – Figures 3 & 4), poo r fits either slightly overestim ate the carbo n quantit y and underestim ate the nitrogen quantit y or vice ver sa. We d efine the stage at which these poor fits may not be considered as acceptable fits an ymo re u sing the Į criterion such as Įൌ^C୰_C୰^మ_మ^୮୭୭୰_ୠୣୱ୲ ൌ ʹǤͷ, where ⎯r²p o or and ⎯r²be st are, in the energ y range covering the C and N post-ed ge regions, the mean valu e of the squ ared resid uals of a poor fit and of the best fit, resp ectively. A value o f ~2 .5 corresponds to the Į value o f the 95 % confidence inter val of a linear r egressio n model fitting the C po st-edge regio n of the Neb ulotron XAS spectrum.

We thus define here the two ‘extrem e fits’ (i.e. the poorest fits that ma y remain acceptable) as the poo r fits exhibiting Į values of ~2 .5. The difference in N/C ratio s estimated fro m these ‘extreme fits’ ( green and red dashed cu rves sho wn on Figures 3 & 4 ) may be seen as a q uasi-certain interval for the N/C ratio. No te that, excep t for the Resin refer ence sample that exhib its oscillations above the C- and N- ed ges that induce high incertitu de on the fit (± ͲǤͲʹ), this erro r o n N/C ratio estimations from XAS data remains ver y low (” 0.01), even for the t yp e III kero gen reference sample that exhibits a N/C ratio lower tha n 0 .02. Unfortunately, estimating the exact limit o f nitro gen detectio n in organics and carbon materials using STXM-based XAS may not b e done fro m the data reported in the present contr ibution and would require additio nal work o n nitro gen- free o rganics.

4 .2.2. Cau tion for accu rately estimate N/C ratios fro m STXM-based XAS exper iments

In additio n to uncertainties on XAS-based N/C estimations that may arise from small-angle scatter ing caused b y local samp le to pography, sp ecial care is needed when dealing with highly ordered carbon materials such as gr ap hite for instance. The atomic scattering factors can be used to mod el the absorptio n signal if the X-ray scatter ing b y individ ual atoms within a s ystem is essentially u naffected b y the co ndensed state of the s ystem [33,3 4]. This is no t exactly true for samples exhib iting a higher d egree of atomic organizatio n as the y m ay exhibit EXAFS structures (i.e., oscillations above the K-edges) over an ener gy range of abou t 500 eV [42]. In ad dition, nitrogen overestimatio n ma y o ccur when the

investigated organics are clo sely associated with cla y m inerals that may host nitro gen as ammonia within inter layer spaces [4 3,44 ]. Acquiring X-ra y absorption sp ectra over the entire C + N ener gy range thus ap pears important in o rd er to identif y these cla ys minerals based on their po tassiu m (K L-edge regio n - 295-305 eV) and calcium (Ca L-edge region - 340-360 eV) contents.

5 . Co ncluding remar ks: Benefits and Limitatio ns of NanoSIMS and STXM-based XAS for N/C estimatio ns

Fo llowing recent attempts repo rted in the literature, the p resent pap er d iscusses two metho ds o f q uantification of the N/C ratio or organics and carbo n materials using NanoSIMS and STXM-based XAS. As illu strated here, these two techniques allo w accurately estimating N/C ratios at the submicrometer scale with a high level of precision. Carefully preparing the samp les fo r Nano SIMS exper iments has led to a confid ence level fo r N/C estimations of ± 0.009 (9 5 %), which is twice as b etter as previou sly reported in the literature b y Hatton et al.

[1] or Thomen et al. [19]. Similar p recision can b e reached using STXM-based XAS as shown b y the exper iments reported in the p resent co ntr ibution which hence valid ates the metho dology recently adopted b y Cod y et al. [20,21].

Altho ugh these two techniq ues offer the same level of precision and ver y lo w limits of detectio n and quantification, the y do not present the same lim itatio ns. STXM experim ents requ ire samp les to be transp arent to X-rays and hence necessitate to either prepare ultrathin sectio ns o f samples or finely crush the investigated samples to obtain ver y fine-scale po wd ered samp les. In co ntrast, NanoSIMS experim ents may o nly b e done o n p erfectly flat surfaces and thus require either pressing or finely po lishing the investigated samples. Dependin g o n the natu re of the investigated mater ials, such complex sample prep aratio n p ro cedures may constitu te a severe limitatio n. In additio n, Nano SIMS exper iments may not be p erformed on sensitive samp les as the y ma y encompass critical structural mod ificatio ns due to primar y b eam damage.

Bo th techniq ues allow estimating N/C ratios over a wide range of values.

As shown here, STXM-based XAS allo ws directly estim ating N/C ratios of o rganics without using any stand ard. In co ntrast, NanoSIMS-based N/C

estimatio ns require at least two standards to be measured using the sam e analytical settings in order to build the calibration line necessar y to properly inverse CN^-/C2- ratios. Even though it may not be mandato ry, the most rigorous way to precisely estimate the N/C ratio of an u nknown sample wo uld be to use two standards exhibiting N/C ratios that will bracket the unkno wn value.

Obviously, this m ight b e d ifficult to achieve for truly u nknown samples.

The benefits and lim itations of Nano SIMS and STXM-b ased XAS also d iffer for organics closely associated with inorganic p hases or embedded within inorganic matr ices. As stated abo ve, special care is needed when using STXM- b ased XAS to estimate N/C ratio s of o rganics clo sely asso ciated with N-rich inorganic p hases. In co ntrast, such N-rich inorganic phases will not significantl y contrib ute to the cyanide ion (CN^-) co unt r ate and will thu s no t impact, as such, the estimatio ns of N/C ratio s b y NanoSIMS. Still, the p resence of ino rganic p hases may influ ence the ionization rate of organics and carbon mater ials through the so -called matrix effect.

Finally, d espite these different limitations, it sho uld be kep t in m ind that NanoSIMS and STXM-based XAS co nstitute highly comp lementar y character ization techniques. As a matter o f fact, co ncomitantly with estimations o f N/C ratios of organics and carb on mater ials, Nano SIMS may provide estimatio ns of their isotopic signatures (į^{1 3}C and į^{1 5}N, for instance) while STXM-based XAS may provide info rmatio n on their mo lecular signatures (carbo n and nitrogen sp eciation, for instance), b oth at the su bmicrometer scale.

Combining these two techniques thus appears particular ly valuable for the fine- scale characterization o f organics and carbon mater ials.

Acknowledg ements

We gratefully acknowled ge su pport from the ERC (project PaleoNano Life - PI: F.

Rob ert) as well as Maïa Ku ga fo r p rovid ing the Nebu lotron reference samp le. The SEM facilit y o f the IMPMC was su pported b y Regio n Ile de France grant SESAME 2006 I-07-593/R, INSU-CNRS, INP-CNRS, Univer sit y Pierre et Marie Curie Par is 6 . The Natio nal NanoSIMS Facilit y at the MNHN was supported b y MNHN, CNRS, Regio n Ile d e France, Ministère de l’Enseignement su périeur et d e la Recherche. STXM-based XAS data were acq uired at beamline 10ID-1 at the CLS, which is sup ported b y the NSERC, the CIHR, the NRC, and the Universit y o f Saskatchewan. Special thanks go to Chithra Karu nakaran and Jian Wang for their expert support of the STXM at the CLS.

Reference

[1] Hatton P-J, Remusat L, Zeller B, Derrien D. A multi- scale approach to d etermine accurate elemental and isoto pic ratios b y nano -scale seco ndar y io n mass sp ectro metry imaging. Rapid Commun Mass Spectrom 2012 ; 26: 1363–71 . [2] Remusat L, Hatton P-J, Nico PS, Zeller B, Kleber M, Derrien D. Nano SIMS stud y of organic m atter associated with soil aggregates: advantages, limitations, and combination with STXM. Environ Sci Technol 2 012; 46: 3943–9.

[3] De Grego rio BT, Sharp TG, Flynn GJ, Wir ick S, Hervig RL. Biogenic o rigin for Earth's oldest putative micro fossils. Geolo gy 2 009; 37: 631–4.

[4] Wacey D, Kilburn MR, Saunders M, Cliff J, Brasier MD. Microfo ssils o f sulp hur-metab olizing cells in 3.4-billio n- year-old rocks o f Wester n Au stralia.

Natu re Geo sci 2011; 4: 698–702.

[5] Wang X, Li X, Zhang L, Yo on Y, Weber PK, Wang H, et al. N-Dop ing of graphene thro ugh electrothermal reactio ns with ammonia. Science 2009 ; 324:

768– 71.

[6] Ayala P, Arenal R, Rümmeli M, Ru bio A, Pichler T. The d oping of carbo n nanotubes with nitrogen and their potential applicatio ns. Carbon 2010; 48: 575–

86.

[7] Yamad a Y, Kim J, Matsuo S, Sato S. Nitrogen-containing gr ap hene analyzed b y X-ra y photoelectron spectrosco p y. Carbon 2014; 70: 59–74.

[8] Lal R. Soil carbon sequestration imp acts on global clim ate change and food securit y. Science 2004; 304: 1623–7.

[9] Schulze ED, Freibauer A. Carbo n u nlocked from soils. Natu re 2 005 ; 437:

205– 206.

[10 ] Young IM, Crawford JW. Interactions and self-organizatio n in the soil- microb e complex. Science 2004; 304: 1634–7.

[11 ] Lehmann J, Solomon D, Kinyanghi J, Dathe L, Wrick S, Jacobsen C. Spatial complexit y of soil o rganic matter forms at nanometre scales. Nature Geo sci 200 8;

1 : 238–42 .

[12 ] Lehmann MF, Ber nasconi SM, Barbieri A, McKenzie JA. Preser vatio n of o rganic matter and alteratio n o f its carb on and nitrogen isotope co mpo sitio n

during simu lated and in situ ear ly sed imentar y d iagenesis. Geochim Cosmo chim Acta 2002; 66: 3573–84.

[13 ] Oehler DZ, Robert F, Walter MR, Sugitani K, Allwo od A, Meib om A, et al.

NanoSIMS: insights to biogenicit y and syngeneit y of Archaean carb onaceous structures. Precamb Res 2009; 1 73: 7 0–8.

[14 ] Van Zuilen MA, Chau ssido n M, Rollion-Bard C, Marty B. Carb onaceous cherts of the Barberton Greenstone Belt, Sou th Africa: isotop ic, chemical and structural characteristics of ind ividu al m icrostructu res. Geochim Cosmo chim Acta 2007; 71: 655–69.

[15] Wacey D, McLoughlin N, Kilburn MR, Saunders M, Cliff JB, Kong C, et al. Nanoscale analysis of pyritized microfossils reveals differential heterotrophic consumption in the ׽1.9- Ga Gunflint chert. Proceed ings of the Natio nal Academy o f Sciences 2013;

100(20 ):8020–4.

[16 ] Schimmelmann A, Lis GP. Nitrogen isotopic exchange during maturatio n of o rganic matter. Org Geo chem 2010 ; 41: 63–70.

[17 ] Rob ertson J, Davis CA. Nitrogen doping of tetrahed ral amorphous carbo n.

Diamond and Related Materials 1995; 4: 4 41–4.

[18 ] Ewels CP, Glerup M. Nitro gen doping in carbon nanotubes. J Nanosci Nanotechnol 2005; 5 : 1345–63.

[19 ] Thomen A, Ro bert F, Remusat L. Determinatio n of the nitro gen abu ndance in organic materials b y NanoSIMS qu antitative imaging. J Anal At Spectrom 2014 ; 29: 512– 9.

[20 ] Cod y GD, Ade H, Alexander CMO’D, Araki T, Bu tterwo rth A, Fleckenstein H, et al. Quantitative organic and light-element analysis o f comet 81 P/Wild 2 p articles using C-, N-, and O-ȝ-XANES. Meteo rit Planet Sci 200 8; 43 : 353 –65.

[21 ] Cod y GD, Gupta NS, Briggs DEG, Kilco yne ALD, Summo ns RE, Kenig F, et al. Molecular signature o f chitin-protein comp lex in Paleo zoic arthropods.

Geology 2011; 39 : 255–8.

[22 ] Van Dommele S, Ro mero -Izquirdo A, Br yd so n R, de Jong KP, Bitter JH.

Tu ning nitro gen functio nalities in catalytically gro wn nitrogen-co ntaining carbo n nanotubes. Carbon 2008; 46 : 138–48.

[23 ] Kuga M, Carrasco N, Marty B, Marrocchi Y, Bernard S, Rigaudier T, et al.

Nitrogen isotop ic fractionation during ab io tic s ynthesis of organic solid particles.

Earth Planet Sci Lett 2014 ; 393 : 2–13.

[24 ] Hoppe P. Nano SIMS: A new too l in cosmochemistr y. Ap pl Surf Sci 200 6;

252: 7102 –6.

[25 ] Berkowitz J, Chup ka WA, Walter TA. Photoio nization o f HCN: the electro n affinit y and heat of formation o f CN. J Chem Phys 1 969 ; 50: 1497 –500.

[26 ] Bradforth SE, Kim EH, Arno ld DW, Neu mark DM. Photoelectro n spectroscop y of CN^-, NCO^-, and NCS^-. J Chem Phys 1 993; 98: 800–10.

[27 ] Clode PL, Stern RA, Marshall AT. Subcellular imaging of isotop ically labeled carbo n co mpounds in a bio logical samp le b y ion micro probe (NanoSIMS). Microsc Res Tech 2 007 ; 70: 220–9.

[28 ] Kaznatcheev KV, Karu nakaran Ch, Lanke UD, Urq uhart SG, Obst M, Hitchco ck AP. So ft X-ray spectromicroscop y beamline at the CLS:

commissio ning resu lts. Nu cl Instrum Metho ds Phys Res Sect A 2007; 582: 96–9 . [29 ] Wang J, Mo rin C, Li L, Hitchco ck A, Scholl A, Do ran A. Radiatio n d amage in soft X-ray microscop y. J Electro n Spectrosc Relat Phenom 2009 ; 170(1-3):

25–3 6.

[30 ] Hitchcock AP, D ynes JJ, Johansson G, Wang J, Botton G. Comp arison of NEXAFS microscop y and TEM-EELS for stu dies o f softmatter. Micron 2008;

39(3): 311–9.

[31 ] McMahon G, François Saint-C yr H, Lechene C, Unkefer CJ. CN^- Seco ndar y io ns fo rm b y reco mbinatio n as demo nstrated using multi- iso tope mass spectrometr y of ^{1 3}C- and ^{1 5}N-lab eled p olyglycine. J Am Soc Mass Spectrom 2006 ; 17: 1181 –7.

[32 ] Stöhr J, NEXAFS spectroscop y. Springer 1992; 25 .

[33 ] Henke BL, Lee P, Tanaka TJ, Shimab uku ro RL, Fujikawa BK. Low-energ y X-ray interactio n coefficients: pho toab so rp tion, scatter ing, and reflectio n: E = 100– 2000 eV Z = 1–94 . At Data Nucl Data Tables 1982; 27: 1–144 .

[34 ] Henke BL, Gullikson EM, Davis JC. X-ray interactions: P hotoabsorption, scattering, transmission and reflection at E = 50–30 ,000 eV, Z = 1–92. At Data Nucl Data Tables 1 993 ; 54: 181–342.

[35 ] Bernard S, Beyssac O, Benzerara K, Findling N, Brown Jr GE. XANES, Raman and XRD signatu res of anthracene-based cokes and saccharo se-based chars submitted to high temperature p yrolysis. Carbo n 2 010; 48: 2 506–16.

[36 ] Le Guillou C, Remu sat L, Ber nard S, Brearle y AJ, Leroux H. Amorphizatio n and D/H fractio natio n of kerogens during exper imental electron irradiatio n:

comparison with cho ndritic organic matter. Icaru s 2 013; 226(1): 1 01–10.

[37 ] Mermet J-M. Limit of quantitatio n in ato mic sp ectro metry: an unambigu ous concep t? Sp ectro chim Acta Part B 20 08; 6 3: 166–82.

[38 ] Bay HL, Bohdansk y J, Hofer WO, Roth J. Angu lar distr ibutio n and d ifferential sputtering yields for lo w-ener gy light-io n irrad iation of polycr ystalline nickel and tungsten. App Phys 1980; 21 : 327–33 .

[39 ] Clou gh RL. High-energ y radiatio n and polymers: A review o f commercial p ro cesses and emerging app lications. Nucl Instrum Meth B 2001; 185: 8–33.

[40 ] Svir ido v DV. Chemical aspects of implantation of high-ener gy io ns into polym eric mater ials. Russ Chem Rev 2 002; 71: 315–2 7.

[41 ] Prošková K, Švorþik V, R yb ka V, Hnatowicz V. Selected degradatio n reactions in polyethylene irradiated with Ar+ and Xe+ ions. Rad iat Physi Chem 2000 ; 58: 153– 6.

[42 ] Co melli G, Stöhr J. SEXAFS studies of C=C bond lengths in co nd ensed and chemisorbed mo lecules. Su rf Sci 1988; 200: 35–44.

[43 ] Daniels EJ, Aro nson JL, Altaner SP, Clau er N. Late Perm ian age o f NH4 - b earing illite in anthracite fro m easter n Pennsylvania: tempo ral limits o n coalificatio n in the central Appalachians. Geol So c Am Bull 1994 ; 106 : 760–6.

[44 ] Krooss BM, Jurisch A, Plessen B. Investigatio n o f the fate of nitrogen in Palaeozoic shales o f the Central European Basin. J Geochem Explor 2006 ; 89:

191– 4.

Table Caption

Table 1: Bulk N/C measurements (thermal conductivit y – SGS Multilab ), CN^-/C2-

io nic ratio s measured b y NanoSIMS and N/C ratios estimated b y STXM-based XAS.

Fig ure Ca ption

Fig ure 1: A. NanoSIMS image of Tryptophan (the colo r scale sh ows the secon dary CN^- intensities per pixel). The black squa re co rresponds to on e of the 30 selected ROIs for Tryptophan . B. NanoSIMS imag e of Nebu lotron (the color scale sho ws th e second ary CN^- in tensities per pixel). C. SEM image of the Tryptophan sa mp le a fter NanoSIMS experimen ts. D. SEM image of the Nebulo tron sample a fter Nan oSIMS exp eriments. Darker zones on S EM images correspond to cesium implanted zon es. Red squares on SEM images C. and D.

indica te the measured 10 × 10 μ m² areas sho wn in A. and B., respectively. S cale bar is 2 μ m.

Fig ure 2: Plot sho win g the CN^-/C2-

values extracted from NanoSIMS exp eriments fo r a ll the in vestiga ted referen ce samples but the Nebu lotron sample as a function of their bu lk ato mic N/C ratios. Each d iamond represents a ROI of 0.5 μ m². “LOD” is th e limit of detection, “LOQ” is the limit of quantifica tion and

“PI” and “CI” a re th e p rediction and confidence in terva ls, respectively (see d iscussio n for details).

Fig ure 3: X-ra y ab so rp tion spectrum o f the Nebulotron referen ce sample (black) in the energ y rang e covering the C and N K-edges. Also sho wn in blu e is the b est fit (KC×f2 (c a r b o n) + KN×f2 (ni t r og e n)), the KN/KC ratio correspo nding to th e N/C a tomic ratio. A-B. Details of the carbon and nitrogen po st-edge region s showin g

‘extreme fits’ (in red and green) which are defined as th e poor fits exhibiting Į values of §2.5 (see text fo r details). The green dashed curve o verestimates the carbon quan tity and underestimates the nitrogen quantity while the red dashed curve underestimates the carbon quantity and overestima tes th e nitrogen quan tity. The d ifference in N/C estimations between the two ‘extreme fits’ (the g reen and red da sh ed curves) is defined as the error on th e estimation.

Fig ure 4: A-F. X-ray absorption spectra o f the six investigated reference samples (bla ck), b est fits (blue) and ‘extreme fits’ (red and g reen ). G. Plot showing the N/C values estimated fro m STXM-based XAS experiments (i.e., KN/KC ra tio s) for a ll the investiga ted reference samples as a fun ction of their bulk atomic N/C ratio s. Note that th e regression curve between the ratio s KN/KC and the N/C bulk measu rements is very clo se to a straight line with a slope o f 1 passin g through th e origin. Fo r ea ch reference sample, the error b ar correspond s to the d ifference between estima tio ns fro m th e ‘extreme fits’.

Figure 1

Figure 1

0.00 0.05 0.10 0.15 0.20

0.00.51.01.52.0 0.00.51.01.52.0

linear model R² = 0.99463 PI (95 %) CI (95 %) Kerogen III

Kerogen I Resin

Phenylalanine Tryptophan

CN- /C

- 2

N/C _Bulk

LOD LOQ

Figure 2

Figure 2

270 290 310 330 350 370 390 410 430 450

N/C = 0.700 N/C = 0.710 N/C = 0.690

Figure 3

Energy (eV)

X-Ray Absorption (a.u.)

A

A

B B

Figure 3

N/C _Bulk N/C XAS

Energy (eV)

Energy (eV)

Kerogen Type I Kerogen Type III

Resin

Energy (eV) Energy (eV)

Energy (eV)

X-Ray Absorption (a.u.)

Nebulotron

A B

C D

F E

0.0 0.2 0.4 0.6 0.8

0.00.20.40.60.8 0.00.20.40.60.8

linear model R² = 0.99979

G

Nebulotron Kerogen I Kerogen III

Phenylalanine Resin

Tryptophan

?_}

X-Ray Absorption (a.u.)X-Ray Absorption (a.u.)

Energy (eV)

Figure 4

270 290 310 330 350 370 390 410 430 450

N/C = 0.700 N/C = 0.710 N/C = 0.690 Tryptophan

270 290 310 330 350 370 390 410 430 450

Phenylalanine

N/C = 0.111 N/C = 0.121 N/C = 0.101

270 290 310 330 350 370 390 410 430 450

270 290 310 330 350 370 390 410 430 450

N/C = 0.025 N/C = 0.035 N/C = 0.015

270 290 310 330 350 370 390 410 430 450

N/C = 0.018 N/C = 0.025 N/C = 0.011

270 290 310 330 350 370 390 410 430 450

N/C = 0.053 N/C = 0.073 N/C = 0.033

N/C = 0.182 N/C = 0.192 N/C = 0.172

Figure 4