Subnanomolar Sensitive Stable Isotopic Determination in CO₂ by Tunable Infrared Laser Absorption Spectroscopy

Abstract

Oxygen and carbon stable isotope ratios (¹⁸O/¹⁶O, ¹³C/¹²C, and ¹⁷O/¹⁶O) of CO₂ have been crucial in helping us understand Earth and planetary systems. These ratios have also been used in medicine for the noninvasive diagnosis of diseases from exhaled breath and for quantifying biochemical or metabolic reactions and in determining the production area of agricultural products. The current method for measuring the stable isotope ratios of CO₂ is primarily gas-source isotope ratio mass spectroscopy (IRMS). Due to the recent demand for isotopic microanalysis of carbonates and organic compounds, the sample size required for isotopic measurements has been reduced to approximately 2 nmol CO₂ (equivalent to 0.2 μg CaCO₃ and 24 ng carbon) by using high-precision IRMS. We report a novel method using tunable mid-infrared laser direct absorption spectroscopy (TILDAS) for sensitive measurements of ¹⁸O/¹⁶O and ¹³C/¹²C in subnanomolar CO₂. This method can accurately measure ¹⁸O/¹⁶O and ¹³C/¹²C in CO₂ with a repeatability of less than 0.03‰ (n = 28) in a range of 0.3 nmol (equivalent to 0.03 μg CaCO₃ and 3.8 ng carbon) to 30 nmol. This is a sample size 1 order of magnitude smaller than currently available sensitive analytical techniques. In addition, the TILDAS system measures ¹⁷O/¹⁶O simultaneously with a repeatability of less than 0.06‰ (n = 28). Our method is a major advance in supersensitive CO₂ stable isotopic analyses for various fields.

Graphical Abstract

INTRODUCTION

Light-element stable isotope ratios have contributed substantially to advancements in a wide range of research fields, such as Earth and planetary sciences, medical sciences, and food traceability. In particular, the stable isotope ratios of oxygen and carbon (δ¹⁸O and δ¹³C) in carbonates have been used to reconstruct detailed climate histories for Earth.¹ The 17O-excess, based on the measurement of δ¹⁷O, in carbonates has recently been identified as having great potential as a new environmental proxy.² The stable isotope ratios of CO₂ in exhaled breath have also been used in medicine for the noninvasive diagnosis of diseases, such as H. pylori infection, and studying metabolic alterations in pathophysiology, as well as in metabolism research as tracers to quantify biochemical or metabolic reactions.^3,4 Also, CO₂ isotopes have been used for tracing the geographical origin of agricultural products, especially rice, beef, and honey, and of food materials to protect the brand names and intellectual property rights of these products.⁵ Gas-source isotope ratio mass spectroscopy (IRMS) has been the dominant method for measuring δ¹⁸O, δ¹³C, and δ¹⁷O in CO₂ for decades. In response to the demand for isotopic microanalysis of carbonates and organic compounds, research has focused on achieving δ¹⁸O and δ¹³C measurements in submilligram samples using the modified continuous-flow IRMS system.^6,7 Consequently, the IRMS detection limit has been reduced to approximately 2 nmol CO₂ (equivalent to 0.2 μg CaCO₃ and 24 ng carbon) with standard deviations of 0.1‰ for δ¹³C and 0.18‰ for δ¹⁸O.⁷ However, the IRMS systems have appeared to reach a limit of the signal/noise balance for high-sensitivity isotopic detection. Furthermore, there are operational difficulties, such as mass overlap of multiple CO₂ isotopologues and contaminants, arduous sample preparation and purification, and the requirement for dedicated laboratory space and a high power requirements.

Laser spectroscopy has increasingly been used as an alternative to conventional IRMS for measuring isotopic ratios in atmospheric CO₂, CO₂ derived from carbonates, and exhaled CO₂.^8–12 Laser systems use the mid-IR spectral region, which allows highly sensitive isotopic measurements owing to the characteristic rotational–vibrational bands of the strong fundamental vibrations in this region. In addition, laser spectroscopy does not suffer from the mass overlap of multiple CO₂ isotopologues because each CO₂ isotopologue has specific absorption peaks. Laser isotope analysis systems are also cheaper, have lower power requirements, have a benchtop-sized footprint, and do not require high-purity gases or high-vacuum systems. Sakai et al. (2017)¹³ demonstrated the potential for high-sensitivity and high-precision detection of ¹⁸O/¹⁶O, ¹³C/¹²C, and ¹⁷O/¹⁶O of CO₂ with sample sizes as low as 20 nmol CO₂ (equivalent to 2 μg CaCO₃ and 240 ng carbon) using a tunable mid-infrared laser direct absorption spectroscopy (TILDAS) system coupled with a high-precision CO₂ preparation system.¹⁴

Herein, we report ¹⁸O/¹⁶O, ¹³C/¹²C, and ¹⁷O/¹⁶O analyses for subnanomolar levels of CO₂ (nanogram quantities of CaCO₃/carbon) by TILDAS coupled with a new high-precision CO₂ gas preparation system, which provides a sensitivity of 1 order of magnitude higher than currently available analytical techniques.^7,13,14 Our method is a major advance in supersensitive CO₂ stable isotopic analyses for various fields.

EXPERIMENTAL SECTION

TILDAS.

Spectroscopic measurements were performed with a TILDAS (Aerodyne Research Inc.) system¹³ (Figure 1). The instrument used a quantum cascade laser (QCL) tuned to 2310 cm⁻¹ to measure the absorption spectrum of the four CO₂ isotopologues, ¹²C¹⁶O₂, ¹²C¹⁷C¹⁶O, ¹²C¹⁸O¹⁶O, and ¹³C¹⁶O₂ (Figure 2).

Figure 1.

Schematic of the subnanomolar CO₂ measurement system. (a) The system, consisting of a CO₂ injection unit, CO₂ volume adjustment unit, and TILDAS system. (b) Detail of the leak-tight CO₂ injection unit.

Figure 2.

Example of absorption spectrum for 1.7 nmol CO₂ obtained by the TILDAS system with a 36 m absorption path at a total multipass absorption cell pressure of 15 Torr (2000 Pa). Background noise (designated by the blue range in the figure) had a transmittance of 6.59 × 10⁻⁴ on average and very stable noise spectrum shapes, which allowed for high-sensitivity analyses even when the dimensionality of the absorption spectra is low. The QCL scans the laser frequency range using 800 channels (point number) at a rate of 1.4 kHz, as shown on the horizontal axis.

In our modified instrument, the multipass absorption cell is miniaturized (approximately 200 mL in volume) and vacuum-tight (0.018 Torr/h; 2.4 Pa/h), allowing CO₂ gas to be introduced into the pre-evacuated cell and isolated at a low pressure for highly stable and repeatable measurements. The optical path length in the multipass cell is 36 m long. The detector is a thermoelectrically cooled photovoltaic detector. The optical bench is thermally insulated and the temperatures of the bench and multipass absorption cell are regulated using air–liquid heat exchangers and cooling liquid supplied by a recirculating chiller. The inside of the optical bench housing is purged with N₂ gas to prevent laser light absorption by ambient CO₂ outside of the multipass cell. The careful stabilization of cell temperature and sample pressure allows samples to be measured at high precision and with high repeatability. Laser control, data acquisition, and real-time signal processing are performed by dedicated software. The output for each of the isotopologues is calculated by nonlinear least-squares fitting of its absorption spectrum, yielding a peak area that can be used to calculate the ratio with other peaks.

CO₂ Preparation System for Measuring Stable Isotopes.

Our CO₂ preparation system is divided into a CO₂ injection unit and a CO₂ gas volume adjustment unit, which enables us to deliver quantitative amounts of CO₂ gas precisely to the TILDAS multipass cell (Figure 1). The CO₂ preparation system consists of a stainless-steel vacuum-tight line with electromagnetic valves (Series 99, Parker Hannifin), a capacitance manometer (722B, MKS Instruments, Inc.), metering valves for N₂ purging (SS-2MA, Seagelok), an actuator-driven bellows (14 to 98 mL), all of which are connected to a dry pump (DeoDry 15E, Kashiyama Industries Ltd.). All inner surfaces of the stainless-steel vacuum-tight line are mirror polished to eliminate gas adsorption (arithmetic mean roughness is approximately 0.04 μm). All components are controlled by custom-built LabView-based software.

The CO₂ samples in glass vials are injected with a 5 μL leak-tight plunger-in-needle syringe (5BR-7, SGE Inc.) through the injection port (Figure 1b). The syringe needle is inserted through the top rubber lid of the sealed glass vial filled with 0.25 MPa CO₂ (>99.9995%, Takachiho Chemical Co.) to collect the sample gas. The injection port, through which CO₂ samples are injected into the preparation system, is sealed and isolated from room air via an 8 mm septum (Thermolite Septa, Restek), which is embedded in a stainless threaded closure that can maintain leak-tight conditions (0.003 Torr (0.39 Pa)/h). The amount of injected CO₂ sample gas is measured with a capacitance manometer and expanded into the adjustable volume bellows, which allows the CO₂ gas pressure to be controlled within ±0.001 Torr (0.13 Pa), if needed. The target amount of CO₂ gas can then be expanded into the TILDAS multipass cell for stable isotopic analysis.

Steps in Spectroscopic Gas Measurement.

Each measurement is processed as follows. First, the multipass cell and the whole preparation line is flushed with N₂ gas supplied from the metering valve (Figure 1), followed by vacuum pumping the cell and lines to 0.1 Torr using a dry pump. After flushing and vacuuming, the baseline signals are measured for 1 min. Then, the measured quantity of CO₂ is introduced into the multipass cell, and introduced N₂ through the capacitance manometer adjusts the total pressure (15 Torr) in the cell to obtain the pressure broadening condition (broadening of the absorption peaks) that enhances precision and stability of the absorption peaks calculation by increasing the number of laser scanning data points for each peak.¹³ After 3 min delay to allow complete mixing, the CO₂ sample is measured at 1.4 kHz and integrated for 5 min resolution. After the measurement, the multipass absorption cell is again flushed with N₂ from the metering valve and evacuated.

RESULTS AND DISCUSSION

Spectroscopic measurements were performed with a TILDAS system¹³ connected to our high-precision CO₂ preparation system (Figure 1). The instrument used a QCL tuned to 2310 cm⁻¹ to measure the absorption spectrum of the four CO₂ isotopologues, ¹²C¹⁶O₂, ¹²C¹⁷O¹⁶O, ¹²C¹⁸O¹⁶O, and ¹³C¹⁶O₂ (Figure 2). The sensitivity and repeatability (the standard error) of the measurements were evaluated for various amounts of CO₂ gas, from 0.3 to 30 nmol (equivalent to 0.03 to 3 μg of CaCO₃ and 3.8 to 240 ng carbon) over 4 days; no measurements of the standard CO₂ materials for instrumental and analytical calibrations were performed (Figure 3). We obtained the absorption peaks ratios for R¹⁸O (¹²C¹⁸O¹⁶O/¹²C¹⁶O₂), R¹³C (¹³C¹⁶O₂/¹²C¹⁶O₂), and R¹⁷O (¹²C¹⁷O¹⁶O/¹²C¹⁶O₂) that were normalized using the linear relationships between the pressure/temperature changes and the spectral absorption, as previously reported¹³ (Figure 3). All results were highly reproducible for all CO₂ concentrations used in this study (0.3 to 30 nmol CO₂) for the 4-day measurements. The repeatability of the isotopic ratios was 5.8 × 10⁻⁵ for R¹⁸O, 3.1 × 10⁻⁵ for R¹³C, and 1.8 × 10⁻⁴ for R¹⁷O (n = 28). We found slight residues of the linear temperature dependencies of R¹⁸O, R¹³C, and R¹⁷O after calibration¹³ (R² values of the linear regression lines were all 0.998). The calibration of the residual temperature dependency improved the repeatability by about 2-fold (2.8 × 10⁻⁵ for R¹⁸O, 1.7 × 10⁻⁵ for R¹³C, and 5.5 × 10⁻⁵ for R¹⁷O (greater than 3-fold); n = 28). The standard errors of R¹⁸O and R¹³C decreased as the amount of CO₂ decreased (Figure 3b), and were all better than approximately 0.001. In contrast, although the standard errors of R¹⁷O showed the same trend, they were much larger than those of R¹⁸O and R¹³C because the amplitude of the ¹²C¹⁷O¹⁶O absorption peak was much smaller than those of the ¹²C¹⁶O₂, ¹²C¹⁸O¹⁶O, and ¹³C¹⁶O₂ peaks (Figure 2). The small ¹²C¹⁷O¹⁶O peak could still be detected, suggesting that even smaller samples could be analyzed for R¹⁸O and R¹³C isotopologues. Therefore, our TILDAS with the high-precision CO₂ preparation system could be used to determine R¹⁸O, R¹³C, and R¹⁷O for as low as 0.3 nmol CO₂ (equivalent to 0.03 μg CaCO₃ and 3.8 ng carbon) without calibration with standard materials, which is a sample size that is 1 order of magnitude smaller than that in the sensitive IRMS systems. Furthermore, because of this apparent repeatability over different days, it is possible to apply post- or preanalysis calculations that correlate the isotopic results to isotopic standards (VPDB scale), which is an important advance compared with IRMS CO₂ isotope analyses.

Figure 3.

(a) R¹⁸O (¹²C¹⁸O¹⁶O/¹²C¹⁶O₂), R¹³C (¹³C¹⁶O₂/¹²C¹⁶O₂), and R¹⁷O (¹²C¹⁷O¹⁶O/¹²C¹⁶O₂) from the TILDAS system. Note these isotopic mixing ratios are using line strengths of high-resolution transmission molecular absorption database (HITRAN), which are scaled by standard isotopic abundances. The repeatabilities of R¹⁸O, R¹³C, and R¹⁷O between 0.3 and 3 nmol CO₂ (equivalent to 0.03 to 3 μg CaCO₃ and 3.4 to 240 ng carbon) are better than 0.028‰ for R¹⁸O, 0.017‰ for R¹³C, and 0.055‰ for R¹⁷O (n = 28) after normalization (24.5 °C and 10 Torr). All analyses were done in 4 days with no standard gas for scaling the isotopic values. (b) Standard errors of R¹⁸O, R¹³C, and R¹⁷O for a 5 min measurement at a total cell pressure of 15 Torr (2000 Pa) are <0.001 for R¹⁸O and R¹³C and <0.01 for R¹⁷O.

To evaluate whether a consistent isotope ratio offset is measurable between different CO₂ gases with different isotope ratios, we measured an international CaCO₃ standard (LSVEC) and two CaCO₃ calibrated by the international scale (JNOC-86 and Bio-Std). CaCO₃ was converted to CO₂ gas by the cryogen-free CO₂ preparation system as described in Sakai et al. (2017), then the CO₂ gas was introduced from the add port (Figure 1) of our CO₂ preparation system to TILDAS. All CO₂ gases were derived from less than 3 μg of CaCO₃ at 15.0 Torr total cell pressure measurement condition. The results showed a highly linear relationship between the accepted δ¹³C_PDB and δ¹⁸O_PDB values of the standards and R¹³C and R¹⁸O values measured by TILDAS (Figure 4 and Table 1). Regarding R¹⁷O, there are no international scale that can be used for the comparison but we observed a repeatability of ~0.05 (Table 1).

Figure 4.

Accepted δ¹⁸O and δ¹³C by IRMS values plotted against R¹⁸O and R¹³C by TILDAS.

Table 1.

R¹⁸O (¹²C¹⁸O¹⁶O/¹²C¹⁶O₂), R¹³C (¹³C¹⁶O₂/¹²C¹⁶O₂), and R¹⁷O (¹²C¹⁷O¹⁶O/¹²C¹⁶O₂) from the TILDAS System^a

standard	n	CaCO3 (μg)	R18O	R18O ± 1S.E	R13C	R13C ± 1S.E	R17O	R17O ± 1S.E	cell pressure (Torr)	cell temp (K)	accepted δ18OVPDB	accepted δ13CVPDB
JNOC-86	2	2.1	0.9848	0.0015	1.0061	0.0016	1.1604	0.0507	15.11	297.614	0.538	2.595
Bio-Std	2	3.3	0.9794	0.0010	0.9972	0.0004	1.1441	0.0140	15.09	297.622	−5.56	−2.91
LSVEC	2	2.3	0.9596	0.0019	0.9651	0.0033	1.0536	0.0205	15.08	297.661	−26.7	−46.6

^aCell pressure (Torr), cell temperature (K), and calculated isotopic values obtained from the TILDAS system are also shown. Accepted d values are reported by IRMS. R¹⁸O, R¹³C, and R¹⁷O are normalized at 15.0 Torr in total pressure and 297.65 K (24.5 °C) conditions.

Our method removes the analytical limitation for various high-resolution and high-sensitivity isotopic microanalyses. Coupled with microsampling using a high-resolution micro-milling system, which can achieve a spatial milling resolution of 1 μm,¹⁵ this method allowed us to perform micrometer-order special isotopic analyses of samples such as marine organisms, speleothems, meteorites, and crystal structures of rocks, which currently can only be performed by secondary ion mass spectrometry with a beam spot diameter of ~10 μm.¹⁶ Moreover, ¹⁷O/¹⁶O analysis is difficult by IRMS because the ¹²C¹⁷O¹⁶O isotopologue (~760 ppm) is masked by the much more abundant ¹³C¹⁶O₂ isotopologue (~11000 ppm), which has the same nominal mass. However, conversion of CO₂ to O₂ for measuring O₂, which has no natural interfering isobars, requires a complicated preparation process.¹⁷ Therefore, the direct measurement of ¹⁷O/¹⁶O ratios in nanomolar CO₂ samples by our TILDAS system could be used as a new analytical strategy for samples such as meteorites that have limited quantities of material and large anomalies in ¹⁷O content compared with terrestrial materials.¹⁸ Furthermore, our method for subnanomolar detection of CO₂ allows us to handle smaller sample sizes easily, offering an high-sensitivity alternative to CO₂ isotopic analyses widely used for organic compounds, such as natural materials from Earth or other planets, agricultural products, and human cells, which are commonly measured by gas chromatography/IRMS and elemental analyzer/IRMS.^5,19–21

CONCLUSIONS

In conclusion, there is a value in examining the weak absorption signals for the isotope detection based on precise sample gas handling. TILDAS, with a high-precision CO₂ preparation system, provides a major advance in supersensitive CO₂ stable isotope measurements in a number of fields.