Murchison Meteorite Analysis Using Tetramethylammonium Hydroxide (TMAH) Thermochemolysis Under Simulated Sample Analysis at Mars (SAM) Pyrolysis‐Gas Chromatography‐Mass Spectrometry Conditions

The Sample Analysis at Mars (SAM) instrument aboard the Curiosity Rover at Gale crater can characterize organic molecules from scooped and drilled samples via pyrolysis of solid materials. In addition, SAM can conduct wet chemistry experiments which enhance the detection of organic molecules bound in macromolecules and convert polar organic compounds into volatile derivatives amenable to gas chromatography‐mass spectrometry analyses. Specifically, N‐tert‐butyldimethylsilyl‐N‐methyltrifluoroacetamide (MTBSTFA) is a silylation reagent whereas tetramethylammonium hydroxide (TMAH) is a thermochemolysis methylation reagent. Shortly after arriving at Mars, the SAM team discovered that at least one of the MTBSFTA cups was leaking, contributing to a continuous background inside SAM with the potential to interfere with future TMAH reactions. Therefore, here we characterized possible interactions between the two reagents to determine byproducts and implications for the detection of indigenous organics. SAM‐like pyrolysis experiments supplemented with flash pyrolysis were accordingly conducted with fragments of the Murchison meteorite as a reference for exogenous organic matter delivered to Mars. Flash TMAH experiments yielded various aromatic acids, dicarboxylic acids, and amino acids while SAM‐like pyrolysis presented mixtures of methylated and non‐methylated compounds due to decreased reaction efficiency at slower ramp rates. All experiments in the presence of simulated MTBSTFA vapor produced pervasive silylated byproducts which co‐elute and obscure the identification of Murchison‐derived compounds. Despite challenges, a significant diversity of pyrolyzates and TMAH derivatives could still be identified in flash pyrolysis in presence of MTBSTFA. However SAM‐like experiments with TMAH and MTBSTFA are hindered by both decreased methylation yields and additional co‐eluting compounds.

The SAM instrument suite is comprised of a sample manipulation system (SMS), pyrolysis oven, gas chromatograph (GC), tunable laser spectrometer (TLS), and a quadrupole mass spectrometer (QMS) not dissimilar in nature to benchtop gas chromatograph-mass spectrometers (GC-MS) found in laboratories (Mahaffy et al., 2012).When interfaced with a pyrolysis unit like on SAM, py-GC-MS can enable the direct analysis of solid materials via the thermal extraction of a sample's volatile organic fraction directly onto the GC column.SAM can detect organic compounds via the pyrolysis of drilled rock or scooped regolith samples to release volatiles that are either: (a) directed directly toward the QMS for evolved gas analysis (EGA), (b) directed toward the TLS for isotopic measurements, or (c) sent to a hydrocarbon trap which concentrates and later desorbs pyrolyzates onto one or several of the six capillary columns for GC-MS analysis (Mahaffy et al., 2012).Furthermore, SAM is capable of conducting wet chemistry experiments which enhance the thermal decomposition of macromolecules into discrete compounds and convert polar molecules into GC-amenable volatile derivatives via silylation or methylation (del Rio et al., 1996;Metcalffe & Wang, 1981).Specifically: N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) in dimethylformamide (DMF) (4:1, v/v) is a derivatization reagent that adds a tert-butyldimethylsilyl (t-BDMS) group to reactive polar groups (e.g., hydroxyl, carboxyl, amines, and amides) at low temperature (∼75°C) resulting in volatile and thermally stable derivatives (Schummer et al., 2009); Tetramethylammonium hydroxide (TMAH) (25 wt.%) in methanol is a thermochemolysis reagent used to enhance the liberation of macromolecularly bound compounds at high temperature (>500°C) and simultaneously convert reactive polar groups into methyl ester and/or methyl ether derivatives (He et al., 2020a;Williams et al., 2019).
Soon after landing at Gale crater, it was discovered that at least one of the MTBSFTA-containing wet-chemistry cups inside SAM was leaking (Freissinet et al., 2015;Glavin et al., 2013).MTBSTFA vapor consequently became a persistent background element posing unique challenges to the interpretation of pyrolysis experiments conducted by SAM (Freissinet et al., 2015;Glavin et al., 2013).Still, analyses of Martian samples and the deliberate use of MTBTFSA has permitted the detection of indigenous assemblages of organic compounds including aromatic compounds, sulfur-containing compounds, chlorine-containing compounds, and silylated derivatives (Eigenbrode et al., 2018;Freissinet et al., 2015;Millan et al., 2022;Szopa et al., 2020).As the search for preserved organics on Mars continues, SAM conducted the first in situ TMAH thermochemolysis experiment at the Mary Anning drill site in the Glen Torridon region (Eigenbrode et al., 2011(Eigenbrode et al., , 2020;;Williams et al., 2021).This site was selected due to an abundance of clays that can adsorb and concentrate organics (Millan et al., 2022), minimal diagenetic alteration (Williams et al., 2021), and minimal iron oxide phases which are known to influence extraction efficiency (Williams et al., 2019).Fines from the Mary Anning drill sample were delivered into a freshly punctured TMAH wet chemistry cup then heated at a rate of 35°C min −1 from 50 to 860°C.Volatiles were directed toward the QMS for EGA and toward the hydrocarbon trap to be concentrated and desorbed for GC-MS analysis (Eigenbrode et al., 2020;Williams et al., 2021).Preliminary assessments indicate that various methylated, oxygen-, sulfur-, and nitrogen-bearing aromatic organics not previously detected by SAM have been identified (Williams et al., 2021).However, because SAM contains only two TMAH wet chemistry cups, a blank thermochemolysis sample was not conducted to characterize background elements.The identification of some compounds consequently cannot preclude an origin from known SAM internal sources (e.g., hydrocarbon and injection traps).It also remains unknown how persisting MTBSTFA vapor can potentially interact with TMAH and influence the range of possible derivatives that are yet to be identified on Mars (Williams et al., 2019).

10.1029/2023JE007968
3 of 18 In the study presented here, we therefore conduct SAM-like pyrolysis experiments to characterize interactions between the MTBSTFA and TMAH wet chemistry reagents and the range of detectable organics from a Mars-like simulant.Specifically, the Murchison carbonaceous chondrite was utilized as a representative sample for exogenous organic matter that could have been delivered to the surface of Mars by meteorite or interstellar dust particle influx and subsequently detected by SAM (Chyba & Sagan, 1992;Sasselov et al., 2020).The goal of this study was to: (a) determine the range of TMAH thermochemolysis products/derivatives detectable under SAM-like pyrolysis conditions; and (b) identify interactions between MTBSTFA, TMAH, and Murchison-derived organics.Hydrocarbon traps normally used to concentrate volatiles on SAM were intentionally bypassed to exclude possible contributions from degradation products and replaced by an in-line cryogenic trap.Experiments were conducted by pyrolyzing Murchison under a SAM-like pyrolysis ramp (35°C min −1 ) and comparing results to flash pyrolysis (10°C ms −1 ) to corroborate analyte identification.We intend findings from this study will provide insight into the potential range of compounds expected to be detected or that have yet to be identified in the SAM data gathered at the Mary Anning drill site and future use of the remaining TMAH wet-chemistry cup on Mars.

Murchison Meteorite
Experiments were conducted on a subsampled portion from a 90 g fragment of the Murchison meteorite sample with intact fusion crust from Geoscience Australia (GA# 3943).Murchison was selected as an analog sample since it has been a well-studied organically rich primitive meteorite with a wide range of potentially prebiotically important molecules (Glavin et al., 2018 and references therein).

Sample Preparation
Samples for all pyrolysis experiments were prepared by loading ∼0.5-1 mg of Murchison powder, obtained by gently abrading the surface of a subsampled portion with a five-times DCM-cleaned stainless-steel spatula, into combusted (16 hr at 550°C) quartz pyroprobe tubes containing a quartz rod and wool stage (CDS Analytical, Catalogue: 10A1-3015, 10A1-3016L, 1001-0345) (Miller et al., 2015).

TMAH Thermochemolysis
Samples for TMAH thermochemolysis were prepared inside a fume hood by adding 5 μL of TMAH wt.25% in methanol (Sigma-Aldrich, 334901) to Murchison-containing pyrolysis tubes and immediately transferred to the pyroprobe autosampler for pyrolysis.

Pyrolysis Gas Chromatography-Mass Spectrometry
SAM is the most resource-intensive instrument aboard Curiosity (Grotzinger et al., 2012) Because of this, SAM operations are optimized to divert power between the various systems (e.g., sample manipulartion system, manifold heaters, mass spectrometer) resulting in conservative pyrolysis ramps (Mahaffy et al., 2012).Experiments were conducted on a CDS Analytical 5250T pyroprobe with autosampler using both flash (10°C ms −1 ) and SAM-like (35°C min −1 ) pyrolysis ramps in the presence and absence of derivatization reagents (Figure 1).We then compared the diversity of pyrolyzates and reaction products released under flash and SAM-like pyrolysis conditions to assist in the characterization of volatile species.Neat samples (e.g., without reagents) and experiments with reagents were heated in the pyrolysis oven under a stream of ultra-high purity He (35 mL min −1 ) from 50 to 600°C while the pyroprobe housing and valves were held at 300°C.Volatiles released from the samples were transferred via a heated transfer line (300°C) directly into an Agilent 6890A gas chromatograph (GC) coupled to an Agilent 5975C (MS) Mass Selective Detector (MSD) system, bypassing a hydrocarbon or injection trap typically utilized on SAM (Figure 1) (Mahaffy et al., 2012).This was implemented to circumvent the introduction of potential trap contaminants and to recover all volatile material which may not be adsorbed or desorbed (either partially or totally).The inlet temperature was held at 300°C and operated with a 10:1 split.The GC was fitted with a J&W DB-5MS fused silica capillary column (60 m × 0.25 mm × 0.25 μm), He carrier flow at 1.5 mL min −1 , and MS transfer line set to 280°C.Flash pyrolysis of samples occurred instantaneously and the GC was programmed with the following parameters previously utilized to simulate SAM (Miller et al., 2015): 35°C oven held for 5 min, followed by a 10°C min −1 ramp to 300°C, then a final isothermal hold at 300°C for 8.5 min (40 min total).The MS was operated in electron impact (EI) mode at 70 eV scanning m/z 10-535 (Miller et al., 2015).SAM-like pyrolysis of samples occurred over ∼16 min as the pyrolysis chamber is heated at 35°C min −1 from 50 to 600°C.A portion of the GC column was accordingly submerged in a dry ice slurry containing 40% ethylene glycol in ethanol kept between −55 and −60°C to cryogenically trap volatiles prior to detection (Figure 1).The GC method and MS acquisition were manually initiated immediately after pyrolysis reached completion and the GC column was removed from the slurry.The MS was operated as above.Other than the lack of the traps, this configuration differs from SAM by utilizing a 60 m column and final 300°C GC temperature for better separation and detection of large molecular weight molecules (SAM limit ∼180-280°C).Pyrolysis blanks preceded all samples to control the cleanliness of the analytical set-up and prevent potential cross-contamination.Blank pyrolysis tubes spiked with an internal standard containing C8-C40 n-alkanes (MilliporeSigma, 40147) were pyrolyzed after every four samples to diagnose potential leaks or significant changes in instrument sensitivity.
Results were analyzed using Agilent MassHunter Qualitative B.08.00 and Quantitative Analysis (for GC-MS) B.08.00 software.All reported compounds were identified using a combination of internal standards (e.g., amino acids standards), mass spectral libraries (NIST17), and interpretation of fragmentation patterns and comparison to reference literature (e.g., Gallois et al., 2007).

TMAH Thermochemolysis of Murchison Meteorite
Flash pyrolysis of Murchison in the presence of TMAH revealed a distinct suite of methylated compounds (e.g., methyl ethers and methyl esters) previously undetectable by unassisted pyrolysis experiments (Figure 3, Table 2).The pyroprobe with autosampler is operated inside a portable fume hood to prevent the possible release of reagent fumes into the laboratory space and the transfer line is interfaced with the Mass Selective Detector (MSD) GC-MS injector inlet.During cryogenic trapping, the GC oven remains open, and a portion of the column is submerged in a dry ice slurry of 40% ethylene glycol in ethanol.The temperature is kept between −55 and −60°C and monitored with a thermocouple.Once trapping is complete, the column is removed from the slurry, the oven door is closed, and the acquisition method is manually started using the GC control panel.
These include aromatic acids and dicarboxylic acids which are understood to be derived from cleaved ester and diester aliphatic linkages joining the insoluble organic matter (IOM) macromolecular framework (Remusat et al., 2005).The complete suite of compounds includes benzoic acid methyl esters, toluic acid methyl esters, methoxybenzoic acid methyl esters, naphthalene carboxylic acid methyl esters, methylnaphthalene carboxylic acid methyl esters, methoxynaphthalene carboxylic acid methyl esters, short-chain α, ω-dicarboxylic acid dimethyl esters, and amino acid methyl ester and dimethylamines (Figure 3, Table 2).Furthermore, phenol, some organosulfur compounds (e.g., thiophenes), and some alkylbenzenes along with alkylnapthalenes are no longer detected (Table 1).This is because phenol is converted to anisole (i.e., methoxybenzene) while aromatic acids (e.g., benzoic acid, napthalenecarboxylic acid) that might decarboxylate into alkylbenzenes or alkylnapthalenes have now been methylated into volatile, thermally stable derivatives (Hatcher & Clifford, 1994;Martín et al., 1995;Saiz-Jimenez, 1995).Moreover, thiophenes, which may partly have originated from the cyclization of aliphatic sulfides, have now been detected as methyl sulfides (e.g., dimethyl sulfide) (Table 2) (Remusat et al., 2005).Similar to unassisted py-GC-MS experiments (e.g., absence of TMAH), results from TMAH experiments remain consistent with prior thermochemolysis work on the Murchison meteorite (Remusat et al., 2005;Watson et al., 2010).SAM-like pyrolysis with TMAH differed from flash experiments due to the absence of several methylated derivatives which were previously detected (Table 2).Derivatives which have been detected additionally display relative abundances that are approximately two orders of magnitude lower compared to flash conditions (Figure 3).Because of this, chromatograms from SAM-like TMAH thermochemolysis experiments are characterized by a mixture of peaks representing compounds detected in pyrolysis experiments with and without TMAH (Figure 3, Tables 1 and 2).Both phenol and anisole, alkylbenzenes and benzoic acids, alkylnaphthalenes and naphthalenecarboxylic acids, and thiophenes and dimethyl sulfide (among others) were detected within the same sample  (Tables 1 and 2).The co-occurrence of both methylated and non-methylated species suggests partial methylation of compounds from decreased TMAH thermochemolysis reaction rates at slow pyrolysis ramps (Lievens et al., 2013;Onay & Kockar, 2003).Specifically, OH − formation resulting in the release of methyl radicals and trimethylamine from TMAH is pressure dependent (Onel et al., 2013).During TMAH flash thermochemolysis,   a He et al. (2020b).Table 2 Continued samples experienced an immediate and significant pressure increase which enhances the yield of methylated products (He et al., 2020b).During SAM-like thermochemolysis, a constant He flow may sweep away volatiles (including TMAH) and replace warming helium gas within the pyrolysis chamber throughout the temperature ramp, precluding comparable pressures and limiting TMAH exposure (He et al., 2020b).Pyrolyzate methylation is consequently lower under SAM-like conditions resulting in the observed decrease in response and co-occurrence of methylated and non-methylated end members (Figure 3, Tables 1 and 2).

Results From the First TMAH Experiment on Mars
Preliminary analyses of the SAM TMAH thermochemolysis experiment have indicated TMAH and MTBSTFA reaction byproducts including trimethylamine, MSW, BSW, and the 1-fluoronaphthalene internal standard in the TMAH cup have been detected.However, the C9 straight-chain saturated fatty acid standard expected to be derivatized by TMAH into a fatty acid methyl ester, and the pyrene standard, have yet to be identified (Williams et al., 2021).Still, the detection of bands of high molecular weight mass-to-charge ratios (m/z) of 190-485 representing a complex suite of compounds which tentatively include methylated oxygen-, sulfur-, and nitrogen-bearing aromatic organics indicate a successful first TMAH experiment on Mars (Williams et al., 2021).Combined analysis of EGA and GC-MS data corroborate the detection of benzene, toluene, C3-C4 alkylbenzenes, naphthalene, and C1 alkylnaphthalenes.In addition, a C5 alkylbenzene, benzoic acid methyl ester, C2-C4 alkylbenzenamines, dihydronaphthalene, 2-butyl-thiophene, and benzothiophene were independently detected from GC-MS data (Williams et al., 2021).Preliminary identifications are consistent with result from laboratory pyrolysis experiments presented here (Tables 1-3), indicating compounds detected on Mars may represent liberated molecules from a macromolecular source characteristic of meteoritic input (e.g., Derenne & Robert, 2010;Remusat et al., 2005;Sephton, 2002Sephton, , 2012)).Nevertheless, compounds identified from EGA and GC-MS data may additionally represent know internal SAM organics sources including MTBSTFA reaction with the hydrocarbon trap known to produce, amongst others, benzene, toluene, C3 alkylbenzenes, benzoic acid, naphthalene, and diphenylmethane (Buch et al., 2019;Eigenbrode et al., 2018;Freissinet et al., 2015;Glavin et al., 2013;He et al., 2021;Leshin et al., 2013;Miller et al., 2015Miller et al., , 2016)).

Conclusions
We present results from pyrolysis and thermochemolysis experiments on samples of Murchison meteorite as an analog for organics that might be detectable by the SAM instrument suite on Mars.selected because its organic composition has been well-characterized (e.g., Derenne & Robert, 2010;Komiya & Shimoyama, 1996;Levy et al., 1973;Remusat et al., 2005;Sephton, 2002), it contains compounds previously detected on Mars (Eigenbrode et al., 2018), and because carbonaceous matter from chondrites and interplanetary dust may represent a significant source of organics present in Mars regolith (Chyba & Sagan, 1992;Flynn, 1996;Frantseva et al., 2018).In the presence of TMAH, comparable distributions of aromatic acids, dicarboxylic acids, and amino acids are observed under flash and SAM-like pyrolysis ramps.However, TMAH thermochemolysis experiments demonstrate lower methylation efficiency under SAM-like conditions resulting in the co-occurrence of methylated derivatives at low relative abundances along with non-methylated compounds.This decrease in yield is inferred to result from thermodynamically controlled reactions at slow pyrolysis ramps (Lievens et al., 2013;Onay & Kockar, 2003).Specifically, OH − formation resulting in the release of methyl radicals and trimethylamine (TMA) from TMAH is pressure dependent (Onel et al., 2013).Flash pyrolysis exposes samples to an immediate and significant pressure increase and enhanced production of methylation products while pyrolysis at low ramp rates diminishes the potential for comparable yields (He et al., 2020b).Experiments simulating the co-occurrence of MTBTFA vapor produce various silylated products which obscure the identification of analytes detectable with TMAH alone.These are primarily silylated methanol, MSW, and BSW.Despite challenges posed by co-eluting compounds, most methylated-derivatives could still be identified in flash pyrolysis by comparing retention times and mass spectral fragmentation patterns to reference experiments.In contrast, SAM-like experiments with TMAH and simulated MTBSTFA "vapor" are hindered by both decreased methylation yields and TMAH/MTBSTFA byproducts.Our results indicate that a significant diversity of pyrolyzates and derivatives may be potentially identified from SAM GC-MS data.However, without the availability of analytical blanks, the outcome of TMAH pyrolysis experiments on Mars remains uncertain.

Figure 1 .
Figure1.Pyrolysis-GC-MS setup.The pyroprobe with autosampler is operated inside a portable fume hood to prevent the possible release of reagent fumes into the laboratory space and the transfer line is interfaced with the Mass Selective Detector (MSD) GC-MS injector inlet.During cryogenic trapping, the GC oven remains open, and a portion of the column is submerged in a dry ice slurry of 40% ethylene glycol in ethanol.The temperature is kept between −55 and −60°C and monitored with a thermocouple.Once trapping is complete, the column is removed from the slurry, the oven door is closed, and the acquisition method is manually started using the GC control panel.

Figure 3 .
Figure 3. TMAH thermochemolysis of Murchison meteorite.Total ion chromatogram of pyrolyzed Murchison with 5 μL of TMAH reveals a new suite of methylated compounds (e.g., methyl esters and ethers) previously undetectable.Derivatized compounds included aromatic acids, carboxylic acids, dicarboxylic acids, and amino acids.(a) Flash and (b) SAM-like pyrolysis ramps with a cryogenic trap produced a diversity of methylated derivatives listed in Table2.

Figure 4 .
Figure 4. TMAH thermochemolysis of Murchison meteorite with simulated MTBSTFA background.Total ion chromatogram of pyrolyzed Murchison with 5 μL of TMAH and 0.2 μL of MTBSTFA produces a massive detection of t-BMDS derivatives listed in Table 3.(a) Flash pyrolysis yielded an identical diversity of derivatives as TMAH without MTBSTFA, however, analyte co-elution with silylated compounds posed challenges toward compound identification.(b) SAM-like pyrolysis ramp primarily yielded silylated products which greatly obscured the identification of analytes.

a
He et al. (2021).b Identical mass spectrum to J.

Table 1
Flash and SAM Pyrolysis Products in the Absence and Presence of Derivatization Reagents

Table 2
). Prior results from unassisted and TMAH experiments (without MTBSTFA) (Figures2 and 3) were critical for guiding identifications.Our experiments with simulated vapor therefore suggest that while producing suboptimal results, persisting MTBSTFA vapor should not have considerably altered results from the first TMAH experiment on Mars.However, coelutions from Murchison meteorite was Letters indicate MTBSTFA/DMF and MTBSTFA + TMAH pyrolysis byproducts.

Table 3
Silylated Products From Flash and SAM Pyrolysis With MTBSTFA