12C Carbon Isotope Ratio in Carbonates

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Determination of The 13C/12C Carbon Isotope Ratio in Carbonates and Bicarbonates by 13C NMR Spectroscopy Concetta Pironti, Raffaele Cucciniello, Federica Camin, Agostino Tonon, Oriana Motta, and Antonio Proto Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02473 • Publication Date (Web): 13 Sep 2017 Downloaded from http://pubs.acs.org on September 27, 2017

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Analytical Chemistry

Determination of the 13C/12C Carbon Isotope Ratio in Carbonates and Bicarbonates by 13C NMR Spectroscopy Concetta Pironti,‡ Raffaele Cucciniello, ‡* Federica Camin,§ Agostino Tonon,§ Oriana Motta,ǂ Antonio Proto‡ ‡

Department of Chemistry and Biology, University of Salerno, via Giovanni Paolo II 132, 84084 Fisciano (SA), Italy § Department

of Food Quality and Nutrition, Research and Innovation Centre, Fondazione Edmund Mach (FEM), Via E. Mach 1, 38010 San Michele all' Adige (TN), Italy ǂ

Department of Medicine Surgery and Dentistry “Scuola Medica Salernitana”, University of Salerno, via S. Allende 1, 84081 Baronissi (SA), Italy

ABSTRACT: This paper is the first study focused on the innovative application of 13C NMR (Nuclear Magnetic Resonance) spectroscopy to determine the bulk 13C/12C carbon isotope ratio, at natural abundance, in inorganic carbonates and bicarbonates. In the past, 13C NMR spectroscopy (irm-13C NMR) was mainly used to measure isotope ratio monitoring with the potential of conducting 13

C position-specific isotope analysis of organic molecules with high precision.

The reliability of the newly developed methodology for the determination of stable carbon isotope ratio was evaluated in comparison with the method chosen in the past for these measurements, i.e. Isotope Ratio Mass Spectrometry (IRMS), with very encouraging results. We determined the 13C/12C ratio of carbonates and bicarbonates (≈50-100 mg) with a precision in the order of 1 ‰ in the presence of a relaxation agent, such as Cr(acac)3, and CH313COONa as internal standard. The method was first applied to soluble inorganic carbonates and bicarbonates and then extended to insoluble carbonates by converting them to Na2CO3, following a simple procedure and without observing isotopic fractionation. Here we demonstrate that 13C NMR spectroscopy can also be successfully adopted to characterize the 13C/12C isotope ratio in inorganic carbonates and bicarbonates with applications in different fields, such as cultural heritage and geological studies.

studies, biology, archaeology, forensics and food science. Sta-

1. Introduction The 13C/12C carbon isotope ratio (δ13C) has been recognized as

ble carbon isotope analysis, in fact, provides a powerful tool to

a valuable chemical parameter covering a wide range of scien-

trace the source and fate of CO2 in the environment1-2, it allows

tific domains, such as climatology, ecology, environmental

the characterization of the geographical origin of food3 and has been recognized as a molecular marker in biological studies.4-5

ACS Paragon Plus Environment


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Isotope Ratio Mass Spectrometry (IRMS) represents the pre-

reduced molar fraction (fi/Fi) of 13C is used to determine the site-

ferred method for analyses of the bulk 13C/12C carbon isotope

specific δ13C values for each carbon in a target molecule. These

ratio at natural abundance due to the relative high accuracy

values are further combined with bulk δ13C values obtained by

(0.1‰) and sensitivity (up to 0.01‰).6-7 However, due to the

IRMS. By using an internal standard, such as dimethylsufone,

increasing interest in this parameter, several alternative analyt-

Bayle and co-workers determined δ13C with high accuracy.17

ical methods to IRMS have been designed and applied in the δ13C analysis, such as mid-infrared laser spectroscopy,8 non-

The research of new independent methodologies for δ13C analysis is a powerful approach that allows to validate the accuracy

dispersive infrared spectrometry (NDIRS)9 and Fourier Transobtained by using the reference method such as, in this case, form infrared spectrometry (FT-IR),10-11 which offer the adIRMS. vantage of being less expensive and complex. In recent years, NMR spectroscopy has been deeply investiMoreover, since the 1980’s, SNIF 2H NMR (Site Specific Natgated as an ideal tool to study the sequestration and storage of ural Isotope Fractionation Deuterium Nuclear Magnetic ResoCO2 in geologic formations and has been identified as a promnance spectroscopy) has been used to measure the site-specific ising strategy to reduce the impact of greenhouse gases on isotope ratios of D/H at natural abundance. The technique has global warming.18 In this scenario, Diefenbacher and co-workbeen extended to a wide range of applications, such as metaers19 developed a NMR probe solution to study the reaction of 12

13

14

bolic analyses , climate studies , environmental studies and carbon dioxide sequestration in a water solution, at elevated food chemistry. It is also the official method applied by the OIV pressure and temperature. Furthermore, an in-situ measurement (Organizzazione Internazionale della Vigna e del Vino - Interof the development of MgCO3 under CO2 sequestration-like national Organization of Vines and Wine) as well as being conditions and in the presence of Mg(OH)2, as unsoluble reacadopted by the European Commission to control the addition of tive compound, was also performed.20 13C NMR spectroscopy sugar to wine.15-16 was also favorably used as quantitative spectroscopic method In the last years, NMR spectroscopy has also been used to conduct

13

C position-specific isotope ratio monitoring (irm-13C

for the determination of [CO2]/[HCO3-] and [HCO3-]/[CO32-] ratios nondestructively.21 Moreover, solid-state

13

C NMR was

NMR) with a precision better than 1‰.4 Bayle and co-workers

also used as an effective tool to quantitatively distinguish and

applied this methodology to vanillin and identified its geo-

characterize magnesium carbonate phases, such as magnesite,

graphic origin on the basis of the 13C isotopic profiles.17 On the

hydromagnesite, dypingite and nesquehonite. Results have

one hand, this approach offers some advantages such as the pos-

demonstrated that NMR spectroscopy represents a valid tool for

sibility to define the isotopomer composition in a target mole-

the distinction of carbonate species with small structural differ-

cule, despite the determination of only bulk δ13C values for

ences among each other.22

IRMS. On the other hand, irm-13C NMR is not directly linked to international standards as much as IRMS and, generally, the


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Thus, considering the high potential of NMR spectroscopy, we

distilled water and dried under vacuum at 300°C for 2 h to con-

attempt in this work to apply this technique to a new methodol-

vert NaHCO3 in Na2CO3, following the well-known Solvay

ogy to determine the bulk δ13C values, at natural abundance, in

method. Na2CO3 was characterized by thermogravimetric anal-

certain inorganic carbonates and bicarbonates, using sodium ac-

ysis by using a Netzsch TG 209 apparatus. The analyses were

etate as internal standard. The use of 13C NMR spectroscopy to

carried out on samples with a mass of about 10 mg placed inside

C/12C carbon isotope ratio is unprece-

an alumina crucible. The sample temperature was then in-

dented, to the best of our knowledge, which could favor its ap-

creased at a heating rate of 10°C min-1 from room temperature

plicability in several research fields. Measurements were car-

up to 800°C under an inert atmosphere of nitrogen.

determine the bulk

13

ried out in comparison with IRMS and NDIRS to assess the re-

2.2 Stable carbon isotope ratio analysis The carbon isotope ratio was expressed in δ‰ relative to V-

liability of the proposed methodology.

PDB (Vienna-Pee Dee Belemnite), according to the following

2. Materials and Methods

IUPAC protocol:

2.1 Materials and samples preparation Sodium carbonate (Na2CO3), potassium carbonate (K2CO3), ce-

δ = (Rsample – Rstandard)/Rstandard

sium carbonate (Cs2CO3), calcium carbonate (CaCO3), ammowhere R is the ratio between the heavier isotope and the lighter nium carbonate ((NH4)2CO3), sodium hydrogen carbonate (Naone. HCO3), potassium hydrogen carbonate (KHCO3), ammonium hydrogen carbonate (NH5CO3), chromium acetylacetonate

2.2.1 Isotopic Analysis of Bulk materials by Elemental Analysis/ Isotope Ratio Mass Spectrometry (EA/IRMS)

(Cr(acac)3) and sodium acetate (CH313COONa) were purchased

A Delta Plus V Isotope Ratio Mass Spectrometer (ThermoFin-

from Sigma Aldrich (Saint Louis, Missouri, USA). Oxalic acid

nigan, Bremen, Germany) equipped with a Flash EA 1112 Ele-

(international standard) was purchased from the International

mental Analyzer (ThermoFinnigan) was used to measure δ13C.

Atomic Energy Agency (IAEA) as follows: IAEA-C8, oxalic

The δ13C isotopic values were calculated using 2 homogenized

acid, δ13C=-18.3±0.2.

in-house protein standards, which were themselves calibrated

CaCO3 was converted in water soluble Na2CO3 for 13C NMR

against international reference materials: L-glutamic acid

analysis according to the following procedure: a portion of 2 g

USGS 40 (IAEA International Atomic Energy Agency, Vienna,

of CaCO3 was introduced into a 10 mL glass flask, that was

Austria), fuel oil NBS-22 (IAEA) and sugar IAEA-CH-6 for

evacuated, and 5 mL of orthophosphoric acid was syringed to

13C/12C. The measurement uncertainty, computed using the

produce carbon dioxide. The obtained CO2 gas was collected in

NORDTEST23-24 which combines the internal reproducibility

an impringer, filled with 20 mL of a saturated solution of so-

with the performances results achieved in proficiency test FIT-

dium chloride and with 50 mL of NH3 solution at 30-33% w/w;

PTS, was 0.3‰. The δ13C values were reported relative to Vi-

then it was quantitatively converted in NaHCO3 and Na2CO3.

enna-Pee Dee Belemnite on a scale that was normalized by as-

NaHCO3 and Na2CO3 were removed by filtration, washed with

signing a value of 46.6‰ to LSVEC lithium carbonate (IAEA). 2.2.2 NDIRS



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The samples were reduced to carbon dioxide for NDIRS (Non

Typical values of the experimental parameters were as follows:

Dispersive Infrared Spectroscopy) analysis. A portion of 100

spectrum width (SW) 3000 MHz, pulse width (PW, for a pulse

mg of carbonate was introduced into a 10 mL glass flask, that

angle of 90°) 12μs, memory size (SI) 32K, delay time 150 s,

was evacuated, and 2.5 mL of orthophosphoric acid was sy-

temperature (T) 300 K, zero filling (Z) 32K, number of transi-

ringed to produce carbon dioxide. The CO2 gas produced was

ents (NS) 70, number of experiments per sample (NE) 4. Anal-

collected in a specific aluminized bag. NDIRS spectroscopy

yses time change in the range 1-2 h, based on the chemical dif-

was conducted by means of a Heli-FANplus analyzer (Medimar

ference of the compounds investigated.

s.r.l, Milan, Italy) equipped with a single beam non-dispersive infrared industrial photometer. The aluminized bags were directly connected with the inlet ports of the NDIR spectrometer for sequential measurements. The NDIRS device was interfaced to a computer system that enabled the software-guided measurement and calculation of results.

3. Results and Discussion 3.1 Method optimization for 13C NMR analysis The first part of our work was dedicated to optimizing the spectral parameters for 13C NMR analysis, which was necessary to enhance the precision and accuracy in determining the bulk δ13C

All the chemicals were purchased from Sigma Aldrich (Saint Louis, Missouri, USA).

values. Indeed, we analyzed the influence of the relaxation time (T1), the presence of the relaxation agent, the pulse intervals

NDIRS calibration was performed by using the international standard purchased from the International Atomic Energy Agency (IAEA) (marble, δ C= +2.5 ± 0.1‰). The measure-

(D1) and the use of an internal standard to determine the δ13C in several carbonates and bicarbonates, comparing the results with

13

ment uncertainty was 0.6 ‰. 2.2.3

13C

those previously obtained by means of IRMS. Carbon isotopic

23-24

ratio analyses were carried out on 0.1 g of sample (sodium car-

NMR Spectroscopy

bonate, potassium carbonate, sodium hydrogen carbonate and

The quantitative NMR spectra were recorded using a Bruker 600, with a probe accepting 10 mm o.d. tubes. The sample

ammonium hydrogen carbonate) dissolved in 1 mL of D2O. The longitudinal relaxation times of carbonates and bicarbonates

13

(0.1000 g), CH3 COONa used as internal standard (0.0100 g) and the accurately weighed relaxation reagent Cr(acac)3 (0.0050 g), were added to 0.5 mL of deuterated solvent (D2O) in order to lock the field to the frequency of the spectrometer. Gated decoupling techniques were applied in order to obtain quantitative results. The pulse angle was set at 90° and the pulse

were obtained by the inversion-recovery sequence, a simple two-pulse sequence that creates the initial population disturbance by inverting the spin populations through the application of a 180° pulse. As shown in Table 1, we observed that the relaxation times ranged from 16.050 s (in NaHCO3) to 47.944 s (in K2CO3).

intervals, D, were selected (D> 3T1 max) on the basis of the longitudinal relaxation times, T1, first determined by the inversion recovery method.


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Table 1. Longitudinal relaxation times of carbonates and bicarbonates obtained by the inversion-recovery sequence at 600 MHz. Sample

Cs2CO3 NaHCO3 K2CO3 NH5CO3 CH313CO2Na

Chemical shift (ppm) 168.15 161.20 168.20 160.94 181.50

T1 13C (s) 42.855 16.050 47.944 23.845 36.160

We evaluated the influence of the paramagnetic relaxing complex, such as Cr(acac)3, that allows to reduce the pulse interval values from 10*T1 to 3*T1 and to shorten the analysis time from

Figure 1.13C NMR spectra of sodium carbonate, Na2CO3 ( ) in the presence of CH313CO2Na ( ) , with different recovery delay: a) delay was set equal to T1max (13C); b) delay was set to >3*T1max (13C); c) delay was set to >5*T1max (13C).

Once the spectral parameters were optimized, we evaluated the

2-4 h, that are generally necessary, to 1-2 h.25 The spectra obtained for sodium carbonate, using different values of pulse intervals (D1), are reported in Figure 1. Previously published reports adopted D1 = 10*T1 for 13C-NMR data acquisition4,25,26; however, in our experimental conditions, we found

possibility of using an internal standard, such as sodium acetate. As has been recently reported in the literature, the δ13C determination of vanillin with high accuracy was obtained by using dimethylsufone as standard.13

that D1= 3* T1 was the best compromise for this quantitative

Generally, the site-specific δ13C determination by NMR spec-

analysis. As a matter of fact, as Figure 1 clearly suggests, the

troscopy was obtained by combining the bulk δ13C values ob-

recovery delay influenced the quantitative analysis. We set a

tained by IRMS with the molar fraction (fi/Fi) of 13C; however,

delay of >3*T1max (13C) (spectrum a) and equal to T1max (13C)

determining the bulk δ13C values using NMR with high accu-

(spectrum b) and clearly demonstrated that this value affected

racy, becomes interesting when using an internal standard, such

the determination of 13C/12C for sodium carbonate. Referring to

as sodium acetate. To the best of our knowledge, this work is

δ13C of Na2CO3 obtained by IRMS, we optimized the integral

the first example of the application of NMR spectroscopy to de-

value (D1>3*T1max) until we obtained a comparable value.

termine the bulk isotope carbon composition, at natural abundance. Sodium acetate, CH313CO2Na, was found to be a suitable internal standard due to the associated relaxation time (36.16 s) and chemical shifts (181.5 ppm) that were in the same range of the carbonates and bicarbonates investigated in this work. Furthermore, by using CH313CO2Na only the enriched carbonyl carbon was detected. The sample was prepared by dissolving CH313CO2Na, the sample and Cr(acac)3 in D2O. Isotope carbon


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composition of the bulk sample was evaluated following the

0,0112

equations step by step and using linear calibration. First of all,

0,0111

it was necessary to integrate the spectra: the intensity of 13C was

0,0110

normalized on carbonyl carbon of sodium acetate (internal

0,0109

standard) such as 1 and the signal intensities of the sample were

0,0108

13 C/12C (13C NMR)


0,0107

compared to the standard signal. 13CR is the molar carbon of the

0,0106

internal standard,

13

Csample is the molar 13C of the sample, ob0,0105

tained as the product of the molar carbon of the internal stand-

-50

-40

-30

-20

-10

0

δ13C (‰)

ard and the signal intensities of the sample (Isample), as described Figure 2. Linear calibration plot between the 13C/12C ratio ob-

in the following equations:

13C

R=

13C

tained by 13C-NMR and the δ13C (‰) obtained by IRMS.

gsodium acetate molar masssodium acetate sample =

13C

R

For major clarity, results are also reported in Table 2. The 13

C/12C ratio obtained by the 13C-NMR experiments was deter-

* Isample

mined independently for each carbonate and bicarbonate as the 13C 12C

=

13C

sample

mean value out of four measurements for each sample. The val-

Ctot -13Csample

ues of δ 13C (‰) of standards ranged from -3.32 ‰ (in NaHCO3)

Therefore, once the ratio 13C/12C was evaluated, the δ13C of the sample was calculated on the basis of the linear calibration ob-

to -43.25 ‰ (in NH5CO3), in line with data reported in the literature for these samples.27

tained by using two carbonates (Cs2CO3 and K2CO3) and two bicarbonates (NaHCO3 and NH5CO3). Figure 2 shows the cali-

Table 2. 13C NMR chemical shifts and isotope ratio obtained

bration line obtained by plotting the average absorption intenSample

sity ratios against the reference δ13C values obtained by IRMS. As can be observed, the linear correlation was rather good (correlation coefficient r2=0.9987), leading to a standard deviation of 1.0 ‰ in terms of δ C units. 13

Chemical shift (ppm)

T1 13C (s)

δ13C (‰)

13

C/12C* (NMR)

NaHCO3

161.20

16.050

-3.32

0.01113

Cs2CO3 K2CO3

168.15 168.20

42.855 47.944

NH5CO3

160.94

23.845

-5.20 -27.7 -43.25

0.01103 0.01076 0.01058

by δ13C analysis*.

*13C-NMR analyses are carried out in the presence of 0.005 g of Cr(acac)3.

3.2 Comparison among techniques (13C NMR, IRMS and NDIRS)


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In line with the aim of this study, four samples of carbonates

1 2 and bicarbonates were analyzed to extend the choice of the de3 veloped methodology among the techniques applicable for the 4 5 determination of carbon isotopic composition. Results are sum6 7 marized in Table 3 and the reliability of the innovative method8 9 ology was compared to IRMS and NDIRS. 10 11 12 δ13C SEM* δ13C SEM* δ13C SEM* 13 Sample** (IRMS) (NMR) (NDIRS) 14 15 - 2.0 0.1 1.0 -2.4 0.2 -2.6 16 Na2CO3 -28.9 0.1 30.0 1.0 -29.7 0.2 KHCO 3 17 18 CsHCO3 -33.0 0.1 - 32.0 1.0 -32.0 0.3 19 (NH4)2CO3 -45.7 0.1 - 46.0 1.0 -45.5 0.1 20 21 Table 3. Comparison among techniques (IRMS, NMR and 22 NDIRS) for δ13C analysis of carbonates and bicarbonates. 23 24 25 26 *SEM: Standard error of mean. 27 28 * *13C-NMR analyses are carried out in the presence of the relaxation reagent (Cr(acac)3). 29 30 31 32 33 As highlighted in Table 3, δ13C values obtained by IRMS, 13C 34 35 NMR and NDIRS were very close to each other, bringing to a 36 37 very good agreement among techniques. Precision value of 1‰ 38 39 was obtained with 13C NMR, in line with data obtained by 13C 40 position-specific isotope ratio monitoring4. These results make 41 42 us confident of the fact that the 13C NMR methodology could 43 44 be a valuable alternative to IRMS and NDIRS for the determi45 46 nation of carbon isotopic composition in bulk samples, at natu47 48 ral abundance. 49 50 We thus extended NMR methodology to the analysis of isotopic 51 52 composition of insoluble calcium carbonates, which are of con53 siderable interest due to their importance in several applica54 55 tions, such as environmental studies and in the field of cultural 56 57 heritage.27,28,29 58 59 60

Water insoluble carbonates were converted into soluble carbonates following the procedure reported in the experimental section. The synthesized carbonates were characterized by means of thermogravimetric analysis to ensure the quantitative conversion of NaHCO3 in Na2CO3. In addition, δ13C values of the initial unsoluble carbonates as well as those of the obtained soluble carbonates were determined by IRMS and the results excluded any isotopic fractionation. The obtained carbonates were then analyzed by IRMS, NDIRS and13C-NMR and the results are reported in Table 4 as mean value of three measurements for each sample. The results showed that NMR spectroscopy represents a valid tool to determine the carbon isotope composition of carbonate species in solution; more specifically, calcium carbonate showed a δ13C of -9.8 ± 1.0‰ obtained by NMR, which was very close to the data obtained by IRMS (δ13C= -9.0 ± 0.1‰). Positive results were also obtained for marble samples where δ13C= 1.0 ± 1.0‰ and 1.0 ± 0.1‰ by NMR and by IRMS, respectively. Table 4. Comparison among techniques (IRMS, NMR and NDIRS) for δ13C analysis of carbonates.

*SEM: Standard error of mean. Sample**

CaCO3 Marble

δ13C (NMR)

SEM *

0.1

- 9.8

1.0

-8.9

0.2

0.1

1.0

1.0

-1.0

0.3

δ13C (IRMS)

SEM*

-9.0 1.0

δ13C

SEM*

(NDIRS)

* *13C-NMR analyses are carried out in the presence of the relaxation reagent (Cr(acac)3).

Because of the great interest in determining the position-specific carbon isotope ratios, at natural abundance, by NMR spectroscopy to identify the origin, authenticity and traceability of several organic molecules in samples (some representative examples were glucose, glycerol, malic acid and vanillin) 30-31, we



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tried to apply the developed methodology also to small organic

The δ13C of oxalic acid was calculated on the basis of the linear

molecules, such as oxalic acid. The sample was prepared by dis-

calibration depicted in Figure 2 and the value obtained (δ13C =-

solving 0.0045 g CH313CO2Na, 0.120 g of oxalic acid and 5.0

19.0±1.0‰) was in line with that measured by IRMS analysis

mg of Cr(acac)3 in 0.5 mL of D2O. The 13C spectrum of oxalic

(δ13C = -18.3 ± 0.5‰).

acid is reported in Figure 3.

4. Conclusions The application of 13C NMR spectroscopy to determine the bulk 13

C/12C carbon isotope ratio, at natural abundance, in inorganic

carbonates and bicarbonates with high accuracy (1.0 ‰) is unprecedented, to the best of our knowledge. Measurement parameters, such as relaxation time (T1), the presence of a relaxation agent, the pulse intervals (D1) and the use of an internal standard were optimized. Thanks to the optimization of the experimental parameters, analysis time was reduced in the range of 1-2 h, based on the chemical difference of the Figure 3. 13C NMR spectrum of oxalic acid ( ) IAEA-C8 (International reference material for stable isotopes) in D2O with CH313CO2Na (●) and relaxation agent Cr(acac)3 .

compounds investigated. The δ13C values obtained by 13C NMR were in line with those

Taking into account that all molar carbon of sodium acetate is 13

C on carbonyl carbon with an intensity such as 1, the signal

intensities of oxalic acid correspond to 0.5282. Based on this assumption,

13

CR represents the molar carbon of the internal

standard, while 13Coxalic acid is the molar 13C of oxalic acid obtained by multiplying the molar carbon of the internal standard with the signal intensities of the acid, as the following equations

obtained by other techniques (IRMS and NDIRS), both for carbonates/bicarbonates and oxalic acid. In sum, this work shows that

13

C NMR methodology can be

used as a valuable alternative to IRMS and NDIRS for δ13C analysis of carbonate and bicarbonate matrices, extending the choice of techniques applicable for the determination of carbon isotopic composition.

suggest:

AUTHOR INFORMATION 13C

R=

13C

0.0045 g = 5.485519329 ∙ 10−5 mol 𝑔𝑔 82.0343 �𝑚𝑚𝑚𝑚𝑚𝑚

oxalic acid = 13C 12C

13C

R * IOA

oxalic acid =

= 2.897446556 ∙ 10−5 mol

13C

oxalic acid

Ctot -13Coxalic acid

= 0.01091

Corresponding Author

*Dr.

Raffaele Cucciniello, tel +39 89969366, e-mail: rcuccini-

[email protected]

ACKNOWLEDGMENTS This work was financially supported by Fondi di Ateneo per la Ricerca di Base (FARB 2016), University of Salerno (Grant no. ORSA 167988),Cle.Pr.In. srl and SNECS Databenc project (CUP


Page 9 of 12


E68C14000050005). We are grateful to Dr. Patrizia Oliva for tech-

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nical assistance.

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Figure 1.13C NMR spectra of sodium carbonate, Na2CO3 ( ) in the presence of CH313CO2Na ( ), with different recovery delay: a) delay was set equal to T1max (13C); b) delay was set to >3*T1max (13C); c) delay was set to >5*T1max (13C).


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0.0112

0.0111

0.0108

13

0.0109

C/12C ( C NMR)

0.0110

13

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0.0107

0.0106

0.0105

-50

-40

-30

-20

-10

0

13C (‰)

Figure 2. Linear calibration plot between the 13C/12C ratio obtained by 13C-NMR and the δ13C (‰) obtained by IRMS.



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Graphical abstract 254x190mm (96 x 96 DPI)


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12C Carbon Isotope Ratio in Carbonates

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