Emilie Je Pense À Jean-Jacques

Bénédicte

Kim

Adèle, Manon, Maïlys, Clément, Maria, Maxence, Jody, Matej, Cédric, Emilie Camille Valentin

Corentin

Aurélien

Simon

Ben

Kévin Lorenzo

ORIGINS OF LIFE AND HOMOCHIRALITY PREFACE * 1 st integral 2D GC * Slow injection * Overlapping of Analytes

Keywords: Chiralité, origine de la vie, GC×GC, dérivatisation, acides aminés, sucres Chirality, origin-of-life, GC×GC, derivatization, amino acids, sugars. IX REMERCIEMENTS

L et les sucres D, ainsi que la détection de CPL dans l'ISM. Les moyens d'étudier ces excès sont discutés en combinant un outil analytique puissant tel que la chromatographie en phase gazeuse multidimensionnelle intégrale couplée à un spectromètre de masse à temps de vol (GC×GC-TOFMS) pour analyser des mélanges complexes, et des procédures de dérivatisation appropriées pour améliorer la volatilité, la détection et l'énantioséparation de biomolécules chirales. La dérivatisation des acides aminés a été VI largement étudiée tandis que la dérivatisation des sucres est plus délicate en raison de leur cyclisation, et doit être adaptée à l'objectif de l'analyse. Pour étudier simultanément les acides aminés et les sucres dans des échantillons extraterrestres ou analogues, une méthodologie pour l'échantillonnage, l'extraction, la purification et le fractionnement de ces échantillons a été développée en utilisant la chromatographie par échange d'ions. Les applications de cette méthodologie, ou de certaines de ses parties, sur des échantillons issus de la synthèse d'acides aminés à partir d'hexaméthylènetétramine et de la synthèse de sucres à partir de formaldéhyde dans des conditions simulant les processus d'altération du corps parent des météorites sont mises en évidence.

LES ORIGINES DE L'ASYMETRIE BIOMOLECULAIRE

Etude de la matière extraterrestre par chromatographie bidimensionnelle en phase gazeuse Résumé : La définition de la "vie" n'a pas encore fait l'objet d'un consensus scientifique, mais elle peut être comprise comme des organismes ayant un cycle de vie construit sur des cellules pouvant subir diverses réactions chimiques (métabolisme). Ces organismes sont maintenus dans un état d'homéostasie mais peuvent croître par division cellulaire, se reproduire, évoluer et s'adapter à leur environnement.

Malgré son abondance actuelle sur Terre, il faut garder à l'esprit que la vie est apparue à un moment donné, s'est répandue et a donné lieu à une diversité et une complexité considérable. Au niveau moléculaire, la vie est apparue sur Terre grâce à des blocs de construction chimiques clés capables de polymériser et/ou de s'auto-assembler, générant des macromolécules telles que l'ADN, l'ARN et les protéines. Ces structures ont la caractéristique unique et cruciale d'être constituées de monomères chiraux, dont un seul énantiomère a été sélectionné pour construire la vie. Cette prédominance d'un énantiomère sur l'autre est commune à tous les organismes et est appelée homochiralité. Abiotiquement, cette particularité est difficile à décrire car elle implique la sélection d'un énantiomère par rapport à son image miroir, ce qui n'est généralement pas le cas sans support biologique : des proportions racémiques sont censées être obtenues dans ces conditions. Cet événement de rupture de symétrie, ou la création d'un excès énantiomérique suivi de son amplification est toujours une question en suspens mais est d'une importance fondamentale pour tenter d'expliquer l'apparition de la vie. Parmi les hypothèses existantes présentées ici, un scénario plausible pourrait être l'interaction entre la lumière polarisée circulairement (CPL) et la matière organique, induisant des excès énantiomériques. En effet, une photolyse et/ou une photosynthèse énantiosélective utilisant cette lumière chirale pourrait s'être produite sur des particules de poussière dans le milieu interstellaire (ISM), et des composés enrichis en énantiomères pourraient avoir été incorporés dans des météorites et des astéroïdes qui ont ensuite ensemencé la Terre primitive.

Par conséquent, l'étude des matériaux extraterrestres tels que les météorites, les astéroïdes et les comètes devient essentielle pour obtenir des indices sur l'origine de la vie. La littérature soutient l'existence d'une source extraterrestre de composés enrichis en énantiomères, tels que les acides aminés Figure 6. Sublimation chamber used for interstellar ice analogs studies. Figure 7. Strecker synthesis of an α-amino acid from an aldehyde and Michael addition of ammonia to acrylonitrile leading to a β-amino acid under abiotic conditions. Figure 8. Formose reaction chemical network. Figure 9. Synthesis of hexamethylene tetranime (HMT) from formaldehyde and ammonia reagents. Figure 10. Plausible chemical pathway leading to HMT under abiotic conditions using methanol, ammonia and UV-light. Figure 11. Schematic of two-dimensional gas chromatography (GC×GC). Table 1. Components of the interstellar medium. Table 2. Average petrological characteristics of carbonaceous chondrites. Table 3. Amino acid amounts in Murchison and their calculation in mol/L for our analyses. Table 4. Example of amounts of sugars and sugar-related compounds in Murchison and their calculation for our analyses. Table 5. Evolution of early thermal and valve modulators used in GC×GC. Table 6. Acquisition rate of GC×GC compatible detectors. Table 7. Physical and chemical properties of native cyclodextrins. Table 8. Anomer ratios for a series of aldoses in aqueous solution. Table 9. 20 to 22 proteinogenic amino acids including the 9 essential amino acids. Table 10. Relative counter ion selectivity for AG 50W-X8 resin. Table 11. List of mechanisms involved in ion exchange chromatography. Table 12. Extraction methods of various targeted compounds in meteoritic samples. Table 13. Characterization of N-trifluoroacetyl methyl ester amino acid derivatives by mass spectra, retention time and method detection limit. Table 14. Repeatability tests on 9 samples analyzed once for each amino acid at 5×10 -5 M. Table 15. Regression data of the MeOH/TFAA calibration curve. Table 16. Relative standard deviation of 8 samples analyzed once using 5×10 -8 M of each amino acid. Table 17. Investigation of alanine contamination using 2 mL of dried reagent, hydrolyzed with H2O/AcOCl and derivatized as ECHFBE derivatives. Table 18. Alanine contamination in three different grades of TFAA. Table 19. Recovery tests after 7 days using 5×10 -5 M for each amino acid. Table 20. Characterization of MBA/TFAA sugar derivatives by mass spectra and retention time. Table 21. Comparison of 5% eeL doped amino acids at 10 -5 M with and without ion exchange chromatography. Table 22. Recovery of DL-alanine and DL-ribose by ion exchange chromatography for 3 and 4 bed volumes and with 1 M NH4OH. Table 23. Recoveries of DL-alanine and DL-ribose using ion exchange chromatography for 4 BV and using 2 M NH4OH. Table 24. Ribose recovery depending on the drying procedure before derivatization. Table 25. Amino acids recoveries after water extraction. Table 26. Recoveries of Ala and Iva after mortar-doped serpentine, water hydrolysis and IEC. Table 27. Comparison of the amino acid content reported in the original study and in the present work. Table 28. Comparison of the amino acids content between the work of V. Vinogradoff et al. and the presented study. Table 29. Overview of samples studied in this work. Table 30. Qualitative analysis of some relevant formose reaction products using BSTFA derivatization. Countless scientific and philosophical questions about the origins of life remain unanswered and may never find a meaningful explanation. Yet, a question as simple as "what is life?" remains itself an open debate [START_REF] Tetz | A New Biological Definition of Life[END_REF] . It can, however, be understood as organisms having a life cycle built on cells that can undergo various chemical reactions of metabolism. These organisms are maintained in a state of homeostasis but can grow by cell division, reproduce, evolve, and adapt to their environment. Since ancient times, scientists have believed that life arose spontaneously under favorable conditions from nonliving matter, through what is called spontaneous generation. According to this theory, worms, maggots, and insects for example, could arise from organic matter (heterogenesis) from dead flesh or from inorganic matter (abiogenesis) such as dust particles.

LIST OF TABLES

Since Miller-Urey's 1952 experiment, which simulated early Earth conditions using simple molecules such as methane, ammonia, dihydrogen, water, and electrical sparks that synthesized amino acids abiotically, a door was left-open to the likelihood of life's building blocks to randomly appear on Earth in a mixture of primitive gases. The experiment prompted serious criticism of the conditions necessary for such reactions to occur. Temperature, pH, concentrations, dihydrogen content and a reducing atmosphere were all pointed out. Nevertheless, it paved the way for further investigations, as this primordial soup concept has since been widely explored. Hydrothermal vents, using volcanic hotspots as energetic source were later proposed as potential location for the origin of life due to their high methane, ammonia, and mineral composition. Wet-dry cycles constitute a later study, suggesting that biosignatures, like amino acids polymerize into oligo and polypeptides via alternating cycles of wet nighttime cooling and hot daytime drying cycles near lakes. In addition, meteorite studies have highlighted the possibility of an extraterrestrial seeding process of early Earth with various prebiotic molecules, raising many questions about the role of asteroids, comets and the interstellar medium in the synthesis and delivery of these compounds.

Ultimately, tracing the origins of life involves a multidisciplinary investigation at the interface between chemistry, biology, physics, astrophysics, and geology. In this context, particular emphasis is placed on meteorites and extraterrestrial samples, as well as on the means to extract and analyze them.

CHAPTER I

Origins of life and homochirality

INTRODUCTION & OBJECTIVES

Living organisms are assembled using specific chemical building blocks, leading to complex structures and self-assembly at the molecular level. Compounds such as lipids, carbohydrates (sugars), nucleobases, phosphates and amino acids are essential for the proper functioning of organisms. The combination of some of these molecules covalently and/or supramolecularly bond together can give rise to structures essential to life, such as DNA (Figure 1), which encodes the genetic information through chains of nucleotides, or proteins, assuring many roles, including enzyme catalysis. Interestingly, it was discovered that the monomers involved in DNA and proteins have a specific configuration, as shown in Figure 2. These monomers are chiral and therefore cannot be superimposed on their mirror image. This chiral property is conferred by asymmetric carbons that are linked to four different atoms or groups and lead to an R or S configuration. As molecules can contain several asymmetric carbons, enantiomers are defined as compounds having all of them of opposite configuration while the others are called diastereomers. In the case of amino acids and sugars, enantiomers are defined as D or L after the historical definition of optically active (+)-glyceraldehyde as D-glyceraldehyde. Thus, DNA is built from D-sugars (along with phosphates and nucleobases), while proteins use exclusively L-amino acids. On a larger scale, atypical structures of DNA and proteins are evidenced as a double helix and an α-helix or β-sheet respectively. These examples show that the genetic material of life depends on enantiomers, which is in some ways difficult to understand given that abiotic chemical reactions tend to produce compounds in racemic proportions. Intuitively, a D or L predominance in biomolecules makes sense, as it allows for better recognition and replication in enzymatic reactions. Interestingly, a complete opposition -i.e., a "mirror world" using Damino acids and L-sugars -should yield identically efficient life [START_REF] Avalos | From Parity to Chirality: Chemical Implications Revisited[END_REF] . Indeed, DNA, for example, would have the same double helix structure, but with L-nucleotides instead of D-nucleotides, which would give an opposite direction to the helix. It should also be noted that although life is primarily built on one specific configuration -D-sugars and L-amino acids -the other enantiomer exists in living organisms [START_REF] Aliashkevich | New Insights Into the Mechanisms and Biological Roles of D-Amino Acids in Complex Eco-Systems[END_REF][START_REF] Pikuta | Bacterial Utilization of L -Sugars and D -Amino Acids[END_REF][START_REF] Fujii | No Title Found[END_REF] .

Furthermore, to obtain such polymers, it is questionable whether homochiral prebiotic building blocks are exclusively required. Theoretical calculations have shown that homochirality and polymers should go hand in hand, and that polymerization kinetics are altered by cross-inhibition of enantiomers [START_REF] Sandars | A Toy Model for the Generation of Homochirality during Polymerization[END_REF] . Nevertheless, having an excess of one enantiomer should only enhance this selectivity-based process.

Many questions then arise from this asymmetry present in all living organisms -also called biological homochirality. Several major questions stand out: how and where the building blocks of life were formed as well as how and where the enantiomeric selection could have been occurred and amplified to lead to present homochiral life. Obviously, no scientific consensus has been reached on these two subjects, but several hypotheses have been developed and opened the way to promising leads. Since temperatures were high during the Earth's formation, it is unlikely that hydrocarbon chains were preserved under these harsh conditions. A plausible hypothesis remains that is that extraterrestrial carbonaceous materials fed the Earth after it cooled. Indeed, more than 160 molecular species [START_REF] Herbst | Complex Organic Interstellar Molecules[END_REF][START_REF] Irvine | The Molecular Composition of Dense Interstellar Clouds[END_REF] including carboxylic acids, alcohols, aldehydes, amines, ethers, hydrocarbons and polycyclic aromatic hydrocarbons (PAHs) have been found in the gas or solid phase -at various frequencies -in star-forming regions reinforcing an extraterrestrial carbon source. Thus, meteorites are of great interest because many biosignatures such as amino acids and sugar derivatives have been revealed after their analysis. Assuming such extraterrestrial delivery, the chemical reactions producing these biosignatures must be compatible with interstellar conditions and subsequent survival under early Earth' geochemical conditions. This severely restricts the pathways for obtaining amino acids and sugars, which are generally explained by a

Strecker reaction for amino acids and a formose reaction for sugars.

With respect to homochirality, it is not clear whether it has a deterministic or probabilistic origin. This problem is usually approached as a two-step process: the first part consists of symmetry breaking associated with the creation of small enantiomeric excesses, and the second part consists of the propagation of these small ees through amplification processes. Since small enantiomeric excesses have been found in meteorites, it is likely that the symmetry breaking event occurred in space under interstellar conditions. These excesses may have subsequently seeded and spread on Earth.

Objectives

The questions on the original source of biosignatures and the origin of homochirality are addressed in the first chapter of this manuscript. As any ee found in extraterrestrial material and brought to the early Earth is of special interest to potentially answer the question on the origin of life, the analytical techniques useful to analyze them are detailed in the second chapter. The main objective of this thesis concerns the development of an analytical method to extract, purify, fractionate, derivatize and analyze an extraterrestrial sample and is outlined in the third chapter. Finally, the application of the developed methodologies on relevant extraterrestrial samples are reported in the fourth chapter.

DETERMINATE SYMMETRY BREAKING MECHANISMS & AMPLIFICATION PROCESSES

Apart from the necessity that life chose one molecular handedness of each of its fundamental chiral building blocks because of its efficiency in developing more complex structures during evolution, abiotic theories explaining initial symmetry breaking event can be grouped into random and determinate mechanisms. Random mechanisms are the result of spontaneous symmetry breaking by crystallization, asymmetric adsorption onto minerals, and/or polymerization with minerals [START_REF] Bonner | Origins of Chiral Homogeneity in Nature[END_REF] . Several determined chiral influences have been proposed over time, but only two hypotheses are the most popular and detailed here: parity violation in the weak force and circularly polarized light [START_REF] Macdermott | 8.2 Perspective and Concepts: Biomolecular Significance of Homochirality: The Origin of the Homochiral Signature of Life[END_REF] . The third true chiral influence (true chirality has been defined as parity-odd and time-even by L.D. Barron 11 ) concerns static magnetic fields collinear with unpolarized light [START_REF] Rikken | Enantioselective Magnetochiral Photochemistry[END_REF] . Other hypotheses have been proposed but are not considered because they are not truly chiral.

Parity violation in the weak force

The four fundamental interactions -gravitational, electromagnetic, strong nuclear and weak nuclearwere assumed to possess parity properties. However, the weak nuclear force was shown to violate parity in 1957 by Wu et al. [START_REF] Wu | Experimental Test of Parity Conservation in Beta Decay[END_REF] using [START_REF] Powner | Synthesis of Activated Pyrimidine Ribonucleotides in Prebiotically Plausible Conditions[END_REF] Co. A cooled and magnetically aligned unstable isotope of Co ( 60 Co) was used, as it decays to [START_REF] Powner | Synthesis of Activated Pyrimidine Ribonucleotides in Prebiotically Plausible Conditions[END_REF] Ni by β-decay. An electron, an antineutrino and gamma rays are emitted during this decay. Since electromagnetic waves are known to respect the conservation of parity, the rate of emission of gamma rays was compared to the rate of emission of electrons in two distinct directions at low temperature. It was then shown that the electrons were emitted in a privileged direction -opposite to the gamma rays and the nuclear spin vector -thus violating parity.

The chemical implications of this parity violation concern two types of weak interactions: the charged current interaction and the weak neutral current [START_REF] Hegstrom | Parity Violation and Chiral Symmetry Breaking of a Racemic Mixture[END_REF] . The first is responsible for the emission of left-handed beta electrons during beta decay and radiolyses the enantiomers at different rates via electromagnetic interactions. The second is responsible for the energy differences between the enantiomers (PVED) due to the different interactions between the nuclei and the electrons. The range of this interaction is shorter and like that of the electromagnetic interaction. Theoretical calculations of the difference in parity violation energy (PVED) between the D and L amino acid enantiomers suggest that the L form would be slightly predominant over the D form due to its lower intrinsic energy. The order of magnitude of this energy difference has been estimated [START_REF] Reiner | Evaluation of Enantioselective Gas Chromatography for the Determination of Minute Deviations from Racemic Composition of α-Amino Acids with Emphasis on Tyrosine: Accuracy and Precision of the Method[END_REF][START_REF] Tranter | The Parity Violating Energy Differences between the Enantiomers of α-Amino Acids[END_REF] to be 10 -14 -10 -13 J/mol but cannot be verified experimentally given the extremely small value of the expected difference. Another general skepticism is that the surprisingly found energetic advantage of the biologically just fitting, namely the L-amino acid enantiomer depended on the exact conformation of the compound, which is rather variable in its structure in vacuo, solid state, or dissolved (in water) state [START_REF] Hans-Peter Thiemann | A Short History of the Investigation of the Origin of the Terrestrial Biosphere's Homochirality (Some Personal Remarks, Recalling Historical Milestones)[END_REF] . Moreover, PVED would be insufficient to lead to an amplification in the autocatalytic Soai reaction despite its efficiency in amplifying very small ees [START_REF] Hawbaker | Energy Threshold for Chiral Symmetry Breaking in Molecular Self-Replication[END_REF] . Also, other theoretical calculations tend to say that beta radiolysis creates a larger chiral perturbation than weak neutral currents, up to six orders of magnitude [START_REF] Hegstrom | Weak Neutral Current and β Radiolysis Effects on the Origin of Biomolecular Chirality[END_REF] .

Circularly polarized light

Circularly polarized light (CPL) shown in Figure 3 is a true chiral entity 11 capable of interacting differentially with chiral material through photochemical reactions and revealing the optical activity of an enantiomer.

Three different mechanisms of photochemistry triggered by CPL have been reported so far. Asymmetric photolysis corresponding to the selective decomposition of one enantiomer over the other, asymmetric photosynthesis corresponding to the selective synthesis of one enantiomer and photoisomerization concerning the selective rearrangement of one enantiomer into the other [START_REF] Inoue | Chiral Photochemistry[END_REF] . Thus, the interaction of CPL with chiral molecules can lead to the creation of enantiomeric excesses.

Supernovae were first considered as a potential source of extraterrestrial CPL because of the synchrotron radiation they emit, which could lead to the creation of enantiomeric excesses. However, CPL has not been detected in neutron star regions and this hypothesis has been ruled out [START_REF] Cocke | Upper Limit to Circular Polarization of Optical Pulsar NP 0532[END_REF] . On the other hand, infrared (IR)-CPL has been revealed in several star-forming regions such as the Orion nebulae OMC-1 [START_REF] Bailey | Circular Polarization in Star-Formation Regions: Implications for Biomolecular Homochirality[END_REF] and BN/KL [START_REF] Fukue | Extended High Circular Polarization in the Orion Massive Star Forming Region: Implications for the Origin of Homochirality in the Solar System[END_REF] with polarization rates up to 17% and in NGC6334-V [START_REF] Kwon | CIRCULAR POLARIZATION IMAGES OF NGC 6334-V[END_REF] with polarization rates up to 22%. The evidence of these large-scale CPL regions reinforces the potential role of CPL in triggering ees in extraterrestrial material. Circularly polarized light could be the result of Mie scattering of linearly polarized light due to non-spherical dust grains aligned in a magnetic field [START_REF] Lucas | UV Circular Polarisation in Star Formation Regions: The Origin of Homochirality? Orig[END_REF][START_REF] Yoo | Enhancement of Chiroptical Signals by Circular Differential Mie Scattering of Nanoparticles[END_REF] or other processes of multiple scattering, dichroism, or twisted magnetic field lines [START_REF] Bailey | Astronomical Sources of Circularly Polarized Light and the Origin of Homochirality[END_REF][START_REF] Meinert | Photochemical Models on the Absolute Asymmetric Formation of Amino Acids in Interstellar Space[END_REF] . Calculations have confirmed that such IR-CPL intensities could also extend into the UV range and that absorption by interstellar dust prevents its direct detection [START_REF] Bailey | Circular Polarization in Star-Formation Regions: Implications for Biomolecular Homochirality[END_REF] . Because circularly polarized light is a true chiral entity that can be circularly polarized to the left (l-CPL) or right (r-CPL), circular dichroism (Δε) measurements can be performed on enantiopure compounds.

Circular dichroism reports the difference in the molar extinction coefficient of an enantiopure compound between the two CPL helices at a given wavelength. In other words, it represents the difference in absorption of an enantiomer between l-CPL and r-CPL. Often, the anisotropy factor g -defined as Δε/ε (Equation 1) -which better describes the kinetics using the rate constants k D and k L for D and L enantiomers, and the photoreactions involved using the molar absorption coefficients ε D and ε L for D and L enantiomers, respectively, is used to predict any net effect of the CPL in asymmetric photoreactions (Figure 4). In addition, the induced ee can be related to the anisotropy factor to predict its maximum value [START_REF] Meinert | Anisotropy Spectra of Amino Acids[END_REF][START_REF] Balavoine | Preparation of Chiral Compounds with High Optical Purity by Irradiation with Circularly Polarized Light, a Model Reaction for the Prebiotic Generation of Optical Activity[END_REF] . In Equation 2, the ee also depends on the extent of photolysis ξ. Ultimately, the chiral photoinduced formation of a specific enantiomer and the extent of ee depend on the wavelength and helicity of the CPL. The first experiments using CPL were performed by Kuhn & Braun 31 in the liquid phase using a racemic mixture of ethyl α-bromopropanoic ester which paved the way for enantioselective photolysis experiments of racemic amino acids decades later [START_REF] Flores | Asymmetric Photolysis of (RS)-Leucine with Circularly Polarized Ultraviolet Light[END_REF] . The reported ees for leucine in liquid solution (212.8 nm) were -2.5% and 1.98% using l-CPL and r-CPL, respectively. The first enantioselective laboratory syntheses using CPL were performed in 1971 by H. Kagan et al. [START_REF] Kagan | Photochemistry with Circularly Polarised Light. II) Asymmetric Synthesis of Octa and Nonahelicene[END_REF] using helicenes. The optical rotations reported in these experiments were approximately [α]D = -21 ± 1 ° using r-CPL and [α]D = 21 ± 1 ° using l-CPL. Since then, CD and anisotropy studies of relevant biosignatures such as amino acids [START_REF] Myrgorodska | Light on Chirality: Absolute Asymmetric Formation of Chiral Molecules Relevant in Prebiotic Evolution[END_REF][START_REF] Snyder | Circular Dichroism of the Alkyl Amino Acids in the Vacuum Ultraviolet[END_REF] and sugars [START_REF] Matsuo | Vacuum-Ultraviolet Circular Dichroism Study of Saccharides by Synchrotron Radiation Spectrophotometry[END_REF][START_REF] Nielsen | Synchrotron Radiation Circular Dichroism Spectroscopy of Ribose and Deoxyribose Sugars, Adenosine, AMP and DAMP Nucleotides[END_REF] have been performed under various conditions, followed by corresponding asymmetric photolysis experiments. In the solid phase, racemic films of leucine exhibited an ee of 2.6% with r-CPL and -0.88% with l-CPL at 182 nm [START_REF] Meierhenrich | Asymmetric Vacuum UV Photolysis of the Amino Acid Leucine in the Solid State[END_REF] . After being studied in the liquid and solid phase [START_REF] Meinert | Anisotropy Spectra of Amino Acids[END_REF][START_REF] Meinert | Photonenergy-Controlled Symmetry Breaking with Circularly Polarized Light[END_REF] , amino acids were recently studied in the gas phase to circumvent intermolecular and solvent interactions 40 by synchrotron radiation circular dichroism [START_REF] Meinert | Amino Acid Gas Phase Circular Dichroism and Implications for the Origin of Biomolecular Asymmetry[END_REF] and photoelectron circular dichroism spectroscopy (PECD). PECD experiments on alanine at the astrochemically relevant Lyman-α line (10.2 eV ≈ 121 nm) revealed an ee of about 4% in the photoproducts [START_REF] Tia | VUV Photodynamics and Chiral Asymmetry in the Photoionization of Gas Phase Alanine Enantiomers[END_REF] . It should be noted that the magnitude of ees induced by CPLs is comparable to, but generally lower than the ones found in meteorites [START_REF] Glavin | The Search for Chiral Asymmetry as a Potential Biosignature in Our Solar System[END_REF] , supporting the UV-CPL hypothesis. Thus, given the observable and reproducible laboratory results on ee induction, CPL appears to be a promising candidate in terms of the initial symmetry-breaking event in the biomolecules of life.

g = Δε ε = 2 × ε l-CPL -ε r-CPL ε l-CPL + ε r-CPL = 2 × ε D -ε L ε D + ε L = 2 × k D -k L k D + k L Equation 1 𝑒𝑒 ≥ (1 -(1 -𝜉)
Nevertheless, chance and determined mechanisms alone are insufficient to explain biological homochirality given the low ees obtained, which are of the order of a few percent only. An amplification mechanism to increase this imbalance is therefore mandatory. Several amplification models have been proposed that may be responsible for this phenomenon. Chemical models use chemical transformations such as catalysis-based amplification to preferentially synthesize an enantiomer [START_REF] Blackmond | The Origin of Biological Homochirality[END_REF] , while physical models exploit physicochemical properties such as solubility, crystallization rates, or physical behavior of phases to enantioenrich a phase [START_REF] Blackmond | The Origin of Biological Homochirality[END_REF][START_REF] Jacques | Racemates, and Resolutions[END_REF] .

Viedma ripening

Spontaneous deracemization of enantiomers has been used as a separation technique, but only a few compounds, about 15% [START_REF] Viedma | Evolution of Solid Phase Homochirality for a Proteinogenic Amino Acid[END_REF] , show this predisposition. This is because conglomerate crystallization and solution racemization must occur at the same time under given conditions. Originally discovered by the crystallization of sodium chlorate NaClO3 [START_REF] Viedma | Chiral Symmetry Breaking During Crystallization: Complete Chiral Purity Induced by Nonlinear Autocatalysis and Recycling[END_REF] , it has since given way to other techniques. During the Viedma ripening process, the solution must be saturated, stirred and may be racemic. Under these conditions, renucleation cannot take place, only growth dynamics and dissolution are affected. The use of glass beads to shake the flask is interesting because it leads to many smaller crystals with higher solubility and thus to a slight supersaturation of the compound in solution. To compensate for this effect, the accretion rate on the existing NaClO3 crystals is increased. The important characteristic of NaClO3 is its achiral nature in solution which gives it the ability to accrete indifferently on D or L crystals. It rather depends on the size of the existing crystal according to the Ostwald maturation process [START_REF] Ostwald | Studien Über Die Bildung Und Umwandlung Fester Körper: 1. Abhandlung: Übersättigung Und Überkaltung[END_REF] , where large crystals are favored.

Thus, if a chiral crystal predominates randomly, this will eventually lead to the growth of this enantiomorphic solid and the creation of an ee. Since this is an unnatural equilibrium change, this attrition-promoted deracemization of conglomerates has been termed Viedma ripening, in reference to the Ostwald ripening cited above. In addition to NaClO3, a biologically relevant amino acid derivative was reported [START_REF] Noorduin | Emergence of a Single Solid Chiral State from a Nearly Racemic Amino Acid Derivative[END_REF] , providing the first confirmation of the feasibility of this process. This enantioenrichment was also performed on the proteinogenic amino acid aspartic by C. Viedma et al. [START_REF] Viedma | Evolution of Solid Phase Homochirality for a Proteinogenic Amino Acid[END_REF] as it is the only one of the 20 proteinogenic amino acids capable of crystallizing as homochiral D and L crystals [START_REF] Jacques | Racemates, and Resolutions[END_REF][START_REF] Viedma | Evolution of Solid Phase Homochirality for a Proteinogenic Amino Acid[END_REF] .

Catalysis-based amplification

Catalytic amplification mechanisms rely on far-from-equilibrium conditions consistent with prebiotic conditions and were introduced by F.C. Frank 50 using model equations. Several hypotheses have been described over time, such as the asymmetric autocatalytic reaction, the reaction with asymmetric selfamplification, the asymmetric catalytic reaction with a "hyperpositive" nonlinear effect, and the asymmetric catalytic reaction without nonlinear effects, and have been reviewed by K. P. Bryliakov [START_REF] Bryliakov | Chemical Mechanisms of Prebiotic Chirality Amplification[END_REF] . The genesis of these mechanisms lies in a slight enantiomeric imbalance due to random fluctuations or a deterministic symmetry-breaking event described previously. This imbalance can lead to an amplification because one enantiomer catalyzes its own formation or is potentially able to inhibit the synthesis of the other enantiomer.

The first and most famous experiment highlighting an asymmetric autocatalytic reaction scenario was reported by K. Soai et al. [START_REF] Soai | Asymmetric Autocatalysis and Amplification of Enantiomeric Excess of a Chiral Molecule[END_REF][START_REF] Soai | Asymmetric Self-Catalytic Reaction. Self-Production of Chiral 1-(3-Pyridyl)Alkanols as Chiral Self-Catalysts in the Enantioselective Addition of Dialkylzinc Reagents to Pyridine-3-Carbaldehyde[END_REF] using alkylzincs. The so-called "Soai reaction" is very sensitive to very small enantiomeric imbalances. In their follow-up work [START_REF] Soai | Asymmetric Autocatalysis and Amplification of Enantiomeric Excess of a Chiral Molecule[END_REF] , the ee reported after several autocatalytic runs showed a huge increase from 2 to 5% ee to 90% using a pyrimidine-like molecule containing a secondary alcohol.

Other catalysis-based amplifications cited take advantage of a chiral catalyst leading to the achiral oxidized form of the initial product and only one of the two enantiomers [START_REF] Talsi | Asymmetric Autoamplification in the Oxidative Kinetic Resolution of Secondary Benzylic Alcohols Catalyzed by Manganese Complexes[END_REF][START_REF] Talsi | Autoamplification-Enhanced Oxidative Kinetic Resolution of Sec -Alcohols and Alkyl Mandelates, and Its Kinetic Model[END_REF] , a chiral auxiliary whose ee (≈ 5%) leads to a higher product ee (≈ 50%) [START_REF] Geiger | Hyperpositive Nonlinear Effects in Asymmetric Catalysis[END_REF] , and a catalyst that allows for a racemoselective, reversible catalytic reaction combined with precipitation-induced chiral amplification [START_REF] Steendam | Emergence of Single-Molecular Chirality from Achiral Reactants[END_REF] . Using the latter, S. Miyagawa et al. [START_REF] Miyagawa | Absolute Asymmetric Strecker Synthesis in a Mixed Aqueous Medium: Reliable Access to Enantioenriched α-Aminonitrile[END_REF] recently showed that spontaneous absolute asymmetric Strecker synthesis could be achieved in a prebiotic environment as enantioenriched chiral amino acid intermediates were obtained from achiral conditions. More importantly, R. Breslow et al. [START_REF] Breslow | L-Amino Acids Catalyze the Formation of an Excess of D-Glyceraldehyde, and Thus of Other D Sugars, under Credible Prebiotic Conditions[END_REF] showed a synergistic process between L-amino acids and D-sugars in a formose reaction, consistent with the current trend of life's homochirality. Finally, the RNA synthesis of Powner/Sutherland 60 combined with the work of Hein et al. [START_REF] Hein | A Route to Enantiopure RNA Precursors from Nearly Racemic Starting Materials[END_REF] offers an enantioselective prebiotic synthesis of activated RNA nucleotide monomers from glyceraldehyde. Therefore, catalysisbased mechanisms are promising candidates as ee amplification processes.

Other asymmetric amplification models

Various other models have been proposed, and can be classified as chemical, physical, or even a combination of both. The eutectic model or the eve crystal are physical models, the former being based on the differences in amino acid solubility between homochiral and heterochiral crystals [START_REF] Morowitz | A Mechanism for the Amplification of Fluctuations in Racemic Mixtures[END_REF] . Heterochiral crystals are less soluble and give one enantiomer the opportunity to predominate in solution. At the eutectic point, the ee of the aqueous solution is generally high, 46% for valine and 99% for serine [START_REF] Klussmann | Thermodynamic Control of Asymmetric Amplification in Amino Acid Catalysis[END_REF] .

However, chiral amplification is impossible for all amino acids because some of them form conglomerates such as threonine and arginine. Other research has also demonstrated the role of small molecules in modifying the composition of ee eutectic via cocrystallization [START_REF] Klussmann | Rationalization and Prediction of Solution Enantiomeric Excess in Ternary Phase Systems[END_REF] . Using oxalic acid in water, the ee of threonine at the eutectic point was found to be 50% (instead of 0%) while using fumaric acid in water, the ee increased from 47% to 99% for valine [START_REF] Klussmann | Emergence of Solution-Phase Homochirality via Crystal Engineering of Amino Acids[END_REF] . The latter uses similar conditions to Viedma ripening but also includes the control of the temperature. As the solution is cooled, only one nucleus can be generated due to the low nucleation kinetics. Further nucleation will be favored because the first homochiral crystal is mechanically disintegrated into smaller crystals [START_REF] Kondepudi | Chiral Symmetry Breaking in Sodium Chlorate Crystallization[END_REF] .

In terms of chemical amplification, we can mention incomplete reaction and polymerization. The former is based on kinetic differences between enantiomers during a stereoselective reaction leading to ees if the reaction is not complete. Tyrosine methyl ester was enriched in the L-enantiomer from 27.4% to 30.8% via its thermal dimerization after 40% completion [START_REF] Langenbeck | Theorie Der Erhaltung Und Entstehung Optischer Aktivität in Der Natur[END_REF] . Polymerization of leucine at approximately 50% completion led to increased ee values in the final product [START_REF] Blair | Experiments of the Amplification of Optical Activity[END_REF] . Starting with a 27% L-ee solution, polyleucine was found to have an L-ee of 39.5% and left the remaining monomers enriched in D. The combination of the two has been treated extensively by M. Klussman et al. [START_REF] Klussmann | Rationalization and Prediction of Solution Enantiomeric Excess in Ternary Phase Systems[END_REF] and Hein et al. [START_REF] Hein | A Route to Enantiopure RNA Precursors from Nearly Racemic Starting Materials[END_REF] using physical enrichment of the enantiomers through catalytic amplification followed by selective crystallization.

BIOSIGNATURES IN INTERSTELLAR ICE ANALOGS

As the possible scenario of seeding the Earth with interstellar matter is addressed in this thesis, the composition and chemical reactivity in interstellar environments as well as means of replicating these chemistries in the laboratory are briefly considered in the following.

Interstellar medium & grain model

The interstellar medium (ISM) is defined as the matter and radiation occurring between the stars in a galaxy and can itself be divided into several components according to temperature, density, and state of matter. These components are briefly detailed in Table 1 and show a great diversity of physicochemical conditions in different regions. These huge differences are thought to arise from energy injection by supernovas via shock wave compressions and radiative processes resulting from turbulent behavior of the ISM [START_REF] Wolfire | Neutral Atomic Phases of the Interstellar Medium in the Galaxy[END_REF][START_REF] Ehrenfreund | Cosmic Carbon Chemistry: From the Interstellar Medium to the Early Earth[END_REF] . The major components of the ISM are H2 gas (75%) and He (24%) [START_REF] Meinert | Photochemical Models on the Absolute Asymmetric Formation of Amino Acids in Interstellar Space[END_REF] but account for only a few percent of the Galactic mass. The remaining percentage is attributed to silicates and carbon-based particles forming dust suitable for gas accretion and chemical reactions. Among the carbon-containing species, CO is the most abundant with a H2/CO ratio of about 10 4 while the abundance of other compounds varies with the ISM region [START_REF] Wooden | Composition and Evolution of Interstellar Clouds: Comets II[END_REF] .

Given the numerous complex compounds that can be found in the ISM, laboratory simulations attempt to partially reproduce the composition, temperature, pressure, and/or radiation to gain access to primitive extraterrestrial-like samples. A model of interstellar dust grains (Figure 5) has been developed

and aims to reproduce the surface chemistry of such extraterrestrial samples. Dust grains only represent about 1% of the total ISM mass but are fundamental as third bodies to host chemical reactions [START_REF] Burke | Ice in Space: Surface Science Investigations of the Thermal Desorption of Model Interstellar Ices on Dust Grain Analogue Surfaces[END_REF] . A fivestep interaction mechanism has also been proposed to calculate the formation of simple molecules [START_REF] D'hendecourt | Time Dependent Chemistry in Dense Molecular Clouds. I. Grain Surface Reactions[END_REF] . The products of these experiments are referred to as interstellar ice analogs and will be detailed in the next section. Many processes such as UV radiation [START_REF] Chiar | The Nature and Evolution of Interstellar Ices[END_REF] (desorption and/or destruction of small molecules), cosmic rays (creation of secondary electrons capable of triggering chemical reactions), low-energy protons (formation of new radicals and molecular compounds) can be involved in ice chemistry. Finally, these dust particles may undergo heating as they move between regions of the ISM such as hot cores, increasing their temperature from 10 K to about 200 K. This heating effect has been shown to be essential for heavy radicals to be mobilized and participate in the formation of complex organic molecules [START_REF] Garrod | Complex Chemistry in Star-Forming Regions: An Expanded Gas-Grain Warm-up Chemical Model[END_REF][START_REF] Millar | Molecules in High-Mass Star-Forming Regions -Theory and Observation[END_REF] .

Interstellar ice analogs

Depending on their composition, which is mainly H2O, CH3OH, CO, CO2, CH4, NH3, H2CO and HCOOH 78,[START_REF] Ehrenfreund | Laboratory Studies of Thermally Processed H2O-CH3OH-CO2 Ice Mixtures and Their Astrophysical Implications[END_REF] , they can be classified into polar ice [START_REF] D'hendecourt | Time Dependent Chemistry in Dense Molecular Clouds. I. Grain Surface Reactions[END_REF][START_REF] Tielens | Model Calculations of the Molecular Composition of Interstellar Grain Mantles[END_REF] having high H2O content with low amounts of CH3OH, CH4, and NH3, as well as apolar ice [START_REF] Ehrenfreund | Infrared Spectroscopy of Interstellar Apolar Ice Analogs[END_REF] with high CO, CO2, N2, and O2 content. In addition, the influence of water ice morphology is still an issue, as water can be amorphous or crystalline depending on the exact temperature conditions [START_REF] Smoluchowski | Solar System Ice: Amorphous or Crystalline?[END_REF] . Below 150 K, water ice resulting from the direct condensation of steam is amorphous and two categories are distinguished. Below 10 K, amorphous water is dense, while above 10 K, it is lighter and more common. Naturally, this is also a major concern for extraterrestrial samples, as they may have undergone aqueous alteration over time. Amino acid syntheses via this type of set-up were performed by Bernstein et al. [START_REF] Bernstein | Racemic Amino Acids from the Ultraviolet Photolysis of Interstellar Ice Analogues[END_REF] and led to the detection of glycine, alanine and serine using H2O, CH3OH, NH3 and HCN under UV light on a nickel substrate.

Contaminations were ruled out by [START_REF] Wu | Experimental Test of Parity Conservation in Beta Decay[END_REF] C labeling and racemic proportions were obtained for alanine and serine. Another implication of these results is the non-required liquid conditions on the parent bodies.

Indeed, molecular clouds could thus be the seat of amino acid syntheses via photochemical reactions.

Besides, Muñoz Caro et al. [START_REF] Caro | Amino Acids from Ultraviolet Irradiation of Interstellar Ice Analogues[END_REF] identified 16 amino acids using H2O, CH3OH, NH3, CO and CO2 under UV light on an aluminum substrate. Of these amino acids, 6 were found to be proteinogenic and some of them have been reported in previous analyses of meteorites. In addition, diamine compounds were found in these simulated interstellar ices and the authors suggested that they might also be present in meteorites.

Another striking finding was the high proportion of hexamethylenetetramine, which could be a source of prebiotic compounds, as discussed in 1.5 Extraterrestrial synthesis of biosignatures. In these experiments, enantioselective analysis was generally performed to justify the non-contamination of the samples because no ees were expected. However, Bernstein et al. [START_REF] Bernstein | Racemic Amino Acids from the Ultraviolet Photolysis of Interstellar Ice Analogues[END_REF] anticipated at the time the use of UV-CPL in these samples as a potential means to induce ees. Years later, initial investigations into the possibility of creating enantiomeric excesses using UV-CPL illuminating a simple gas mixture of H2O, CH3OH, and NH3 under interstellar conditions showed that L-ees of up to 1.34% ± 0.40% can be obtained [START_REF] De Marcellus | AMINO ACID PRODUCTION BY ULTRAVIOLET IRRADIATION OF ACHIRAL INTERSTELLAR ICE[END_REF] .

The authors related the results to previous work on the Murchison meteorite [START_REF] Cronin | Amino Acid Enantiomer Excesses in Meteorites: Origin and Significance[END_REF] where alanine was found in 1.2% excess making this scenario plausible. However, the difficulty of explaining the huge ee values of isovaline, up to 18.5% [START_REF] Pizzarello | Nonracemic Isovaline in the Murchison Meteorite: Chiral Distribution and Mineral Association[END_REF] , with the use of UV-CPL alone was pointed out. Speculations on the nature of the amplification mechanism, via aqueous alteration, has also been made [START_REF] Glavin | Enrichment of the Amino Acid L-Isovaline by Aqueous Alteration on CI and CM Meteorite Parent Bodies[END_REF] .

Phosphine, PH3, is an another abiotically relevant molecule because it has been detected in the circumstellar envelope of a carbon star 90 . It is also thought that it may find an origin on interstellar grains using atomic phosphorus and hydrogen, in a water-like scenario with atomic oxygen and hydrogen.

Phosphorus, especially in its oxidized form of phosphate, plays a crucial role in biology. However, its origin on the early Earth is unlikely due to the limited solubility of inorganic phosphorus under early Earth conditions. Thus, alternative extraterrestrial pathways have been proposed as a source of this element with different degrees of oxidation. From phosphine, carbon dioxide, and water under ionizing interstellar conditions, A.M. Turner et al. [START_REF] Turner | An Interstellar Synthesis of Phosphorus Oxoacids[END_REF] successfully synthesized phosphorus oxoacids such as phosphinic acid H3PO2 P(I), phosphonic acid H3PO3 P(III), phosphoric acid H3PO4 P(V), and pyrophosphoric acid H4P2O7 P(V).

Radical recombination and/or the insertion of electronically excited atomic oxygen obtained by the decomposition of water from carbon dioxide are reported as initiating processes that could have increased the complexity of the phosphinic acid H3PO2 formed first. The authors also addressed that the absence of triphosphoric acid H5P3O10 would be an indicator that the increasing complexity ends with H4P2O7 in the molecular clouds. Triphosphoric acid is considered very important because it is a precursor of the phosphorus backbone of adenosine triphosphate (ATP), providing energy to living cells.

Furthermore, the presence of phosphoric acid and its eventual delivery to Earth would support the findings reported by I. Nam et al. [START_REF] Nam | Abiotic Production of Sugar Phosphates and Uridine Ribonucleoside in Aqueous Microdroplets[END_REF] on sugar phosphates and ribonucleosides. Although thermodynamically and kinetically unfavored in bulk solution, phosphorylation was found to be more efficient using microdroplets and yielded over 6% D-ribose-1-phosphate. It also revealed the presence of uridine, leading the authors to consider microdroplets as a favorable environment for prebiotic ribonucleoside synthesis. Using phosphine, methane and water, Turner et al. [START_REF] Turner | Origin of Alkylphosphonic Acids in the Interstellar Medium[END_REF] detected alkylphosphonic acids such as methylphosphonic acid [CH3P(O)(OH)2], ethylphosphonic acid and propylphosphonic acid.

This validates the detection of alkylphosphonic acids in the Murchison meteorite [START_REF] Cooper | Alkyl Phosphonic Acids and Sulfonic Acids in the Murchison Meteorite[END_REF] and could be the primitive source of bioavailable phosphorus for early organisms.

As previously described, ribose and 2-deoxyribose are fundamental constituents of RNA and DNA as well as other sugars for various nucleic acids. Using H2O, CH3OH, and NH3 subjected to UV light under interstellar conditions, Meinert et al. [START_REF] Meinert | Ribose and Related Sugars from Ultraviolet Irradiation of Interstellar Ice Analogs[END_REF] identified a variety of sugar alcohols (arabitol, xylitol, threitol), sugar acids (ribonic acid, arabinoic acid, xylonic acid), and sugars including arabinose, lyxose, xylose and ribose. Ribose has been later detected for the first time in the Murchison meteorite [START_REF] Furukawa | Extraterrestrial Ribose and Other Sugars in Primitive Meteorites[END_REF] . As the authors point out, such a diversity of compounds can only be achieved by a formose-type reaction detailed in 1.5

Extraterrestrial synthesis of biosignatures. The particularity lies in a non-required divalent catalyst such as calcium, lead or titanium ions [START_REF] Breslow | On the Mechanism of the Formose Reaction[END_REF] for this reaction to occur but a photochemical-driven mechanism.

Nucleobases are essential constituents of DNA and were synthesized abiotically by Y. Oba et al. [START_REF] Oba | Nucleobase Synthesis in Interstellar Ices[END_REF] using H2O, CO, NH3, and CH3OH under UV light and thermal processes. Specifically, pyrimidine (cytosine, uracil and thymine) and purine (adenine, xhantine and hypoxhantine) nucleobases were detected. Although previous studies exist on their synthesis under abiotic conditions using formamide [START_REF] Saladino | Meteorite-Catalyzed Syntheses of Nucleosides and of Other Prebiotic Compounds from Formamide under Proton Irradiation[END_REF] or urea 100 , these compounds and the conditions under which the nucleobases were obtained are less relevant to astrophysical conditions. The reported amounts were found to correlate well with the Murchison meteorite because a factor of 100 was observed between amino acids and nucleobases.

Ultimately, these simulated interstellar ice samples highlight possible formation pathways of relevant organic molecules including chiral biomolecules in extraterrestrial samples. However, such interstellar ice analogs only focus on specific conditions of temperature, pressure, and composition. Minerals, which are known to play a catalytic role and are suspected of triggering ees in amino acids, have so far been excluded from these studies. Analyses of real samples such as meteorites, asteroids, and comets provide a more difficult but more accurate picture of the compounds likely to be delivered to Earth and should be correlated with these experiments. Thus, the composition of extraterrestrial materials will be highlighted in the following section. fraction, about 4%, of meteorite falls and finds [START_REF] Meinert | Photochemical Models on the Absolute Asymmetric Formation of Amino Acids in Interstellar Space[END_REF]103,104 but are not representative of their true proportion because they undergo rapid weathering. Another classification parameter is the aqueous and thermal alteration of the parent body that impacts the mineralogy and texture of the chondrite. This alteration is classified on a scale of 1 to 6, 1 representing the most aqueous alteration and 6 the least. Particular emphasis has been put on the analysis of carbonaceous chondrites (CC) because of their high and diverse composition of organic compounds. Among these CC meteorites, the Murchison meteorite is emblematic because 96 amino acids 105 , derivatives of C3 to C6 sugars 106 , N-heterocycles, hydroxy acids, aldehydes, ketones, aliphatic and aromatic hydrocarbons 107 have been detected. A very exciting discovery concerning these molecules are the reported ees: up to 18% for L-amino acids and up to 82% for D-sugar derivatives. As previously mentioned, this L ee corresponds to isovaline which is a particular amino acid in the analysis of extraterrestrial samples. Indeed, as it is a non-biological amino acid, contaminations can be discarded, and it has the advantage of being less prone to racemization 108 . It is therefore often targeted for the research of ees. Interestingly, the sign of these ees curiously tends to be the same as found in all present-day life forms. Other major biological building blocks, such as ribose, were also detected, but it is not yet known whether sugar molecules including ribose possess an ee 109 . Given these results, it is likely that these key chiral building blocks of life may have had an extraterrestrial origin. However, the analysis of meteorites is still a challenge in terms of terrestrial contaminations. For this reason, in situ measurements on asteroids or comets have been attempted to overcome this problem of biological contamination and will be discussed in the following sections.

Since one of the goals of the present work is to develop an analytical method to quantify amino acids and sugars and their possible ee in meteoritic samples, it is necessary to ensure that these biosignatures can be quantified as a function of the chosen sample mass. Thus, it is necessary to estimate how much can be expected in a sample like the Murchison meteorite. Murchison has been well studied since its fall and data on the amounts of amino acids and sugars are available as ppb values. The objective is to obtain an order of magnitude of the amino acid concentration to adapt and optimize the analytical procedure accordingly. Considering the analysis of a sample of 1 g of Murchison, the expected concentration in a final volume -before gas chromatographic analysis -of 50 µL can be expressed as follows in Equation 3.

Equation 3 [𝐴𝐴] 𝑠 =

𝑚 𝐴𝐴 𝑀 𝐴𝐴 × 𝑉 𝐹 = 𝑚 𝑆 × 𝑄 𝐴𝐴 × 10 -9 𝑀 𝐴𝐴 × 𝑉 𝐹 = 1 × 𝑄 𝐴𝐴 × 10 -9 𝑀 𝐴𝐴 × 50 × 10 -6

[𝐴𝐴] 𝑠 is the concentration of the amino acid in the sample (mol/L) 𝑚 𝐴𝐴 is the mass of the amino acid in the sample (g)

𝑀 𝐴𝐴 is the molar mass of the amino acid (g/mol)

𝑉 𝐹 is the final volume before injection in which the AA is solubilized (L)

𝑚 𝑆 is the mass of the sample (g)

𝑄 𝐴𝐴 is the concentration of the amino acid in the sample, in ppb (ng/g)

As can be seen in Table 3, the order of magnitude of amino acid concentration is in the 10 -5 -10 -4 M range for 1 g of a Murchison sample. If only 0.1 g of Murchison sample would be available, the amino acids are expected to be present in a concentration range of 10 -6 -10 -5 M. The amino acids presented here were selected from the data of T. Koga & H. Naraoka 110 . It is also interesting to note that the hydrolysis step, according to this data set, releases a significant proportion of oligomerized amino acids that would not otherwise be quantified. Thus, it appears as a basic and necessary step in the analytical method. Similarly, the expected amounts for sugars and sugar-related compounds in Murchison were calculated and are reported in Table 4. Compared to amino acids, the amounts are approximately 10 times lower within the range of 10 -5 -10 -6 M per 1 g of Murchison material. Some exceptions are low molecular weight compounds such as ethylene glycol, glycerol, and glyceric acid. As an overall trend, the abundances decrease with the number of sugar carbon. Other interesting compounds such as deoxy sugar acids including 2-deoxypentonic acid and 3-deoxyhexonic acid as well as dicarboxylic acids have also been reported 111 .

Both values for amino acids and sugars are essential for developing and selecting adequate analytical methods as they directly define the limits of detection and quantification. However, these limits must be as low as possible in order to successfully differentiate the compounds from the matrix noise involved in such a complex sample.

Comets

First described as "dirty snowballs" by Whipple in 1950 114 , comets are composed of a mixture of ices and dust originally formed outside the solar system. They are considered to be remnants from the time of solar system formation 115 . Several parts of a comet can be distinguished: the nucleus, which is a small solid part composed of rock, dust, and ices of H2O, CO, CO2, CH3OH, and H2CO 116 , the coma, which is a visible atmosphere of ice and dust 117 due to the outgassing process of the nucleus when it is heated, and the tail(s), which are the result of the pressure of the solar wind and the Sun's radiation on the coma. Two distinct tails are usually observed: a gas tail formed by ions following the (magnetic) streamline of the solar wind and a dust tail, often curved and left behind.

Comets are generally more primitive than asteroids and meteorites because the still-frozen ice shows less weathering and thermal processing 118 . Comets have a high orbital eccentricity and can therefore be studied near the Earth rather than in the outer solar system which would prevent their close-up investigation 118 . The Rosetta mission, for example, was designed to analyze the surface of comet 67P/Churyumov-Gerasimenko. Its Philae lander was supposed to reach the site of Agilkia but ended up at its final site of Abydos due to several bounces on the cometary surface 119 despite a sophisticated damping device to mitigate these bounces, an active descent system that was supposed to provide gaseous thrust as well as anchor harpoons to hold the lander to the surface. Unfortunately, the former did not activate while the latter did not deploy. As a result, the lander was left in an unfavorable position and orientation for the sampling and drilling device that was supposed to collect and deliver cometary surface material to the COSAC and Ptolemy analyzers. Nevertheless, the COSAC instrument operated several times in sniffing mode 120 , where the mass spectrometer analyzed compounds that passively entered the instrument without the aid of the sample-and-drill device. These analyses led to comparison studies with the National institute of standards and technology (NIST) database to find the best fit for all peaks. In the end, 16

species were putatively identified 120 , several of which, such as aldehydes, ketones, amines, and hydrogen cyanide, are thought to be molecules essential for abiotic amino acid synthesis.

Fortunately, the Rosetta orbiter spectrometer for ion and neutral analysis (ROSINA) on board the Rosetta parent probe revealed the presence of the simplest amino acid, glycine 121 , between 15 and 30 km from the core center. Two other nitrogen-bearing compounds, methylamine and ethylamine, were detected synchronously. K. Altwegg et al. 121 pointed out that glycine is the only amino acid that could form without liquid water, which would explain its only detection in the comet's coma. Given its low sublimation temperature, about 150 °C122 , they speculated that glycine is released from dust grain mantles that heat up in the coma. For comparison, alanine has a sublimation temperature in the same range, around 200 °C122 , but was not detected. Its absence was therefore attributed to the unlikely aqueous alteration of comets.

Another interesting finding is the presence of phosphorus, which is present in multiple forms in living organisms, one of which is the DNA shown in Figure 1. However, its origin is unknown because only the exact mass of the ionized phosphorus has been found. The parent molecules could be PO 123 , PN 124 , CP 125 or HCP 126 because they have already been detected in the ISM. The presence of elemental phosphorus was suspected in the comet Halley dust signature 127 but the lack of sufficient mass resolution prevented any differentiation with other ions such as CH2OH + with a m/z = 31.

Asteroids

Links between meteorites and asteroids were first established with the low albedo asteroid Bamberga 128 which was found to be similar to carbonaceous chondrites. It is now estimated that these carbonaceous C-type asteroids represent about 70% of all known asteroids 129 . The presence of organic and other compounds is typically studied in the visible and near-infrared at wavelengths between 0.5 and 4 µm, as Fe 2+ -Fe 3+ charge transfer absorption occurs at 0.7 µm, anhydrous silicates at 1 µm, water in hydrated minerals at 3 µm, and organic compounds between 3. 3 and3 Unfortunately, the masses collected by these sample return missions are small compared to the masses available from meteorites on Earth. Access to these valuable samples is therefore very limited.

Conclusion

Among extraterrestrial samples, meteorites are the most readily available but present a risk of terrestrial contamination. In situ analysis overcomes this difficulty but requires compromises in terms of analytical performance. Sample return missions are currently the best solution because they combine low risks of sample contamination and the use of high-performance analytical tools. Several biosignatures have been detected in these extraterrestrial samples and the following section aims at presenting the chemical reactions that could be responsible for their synthesis, especially for amino acids and sugars.

1.5 EXTRATERRESTRIAL SYNTHESIS OF BIOSIGNATURES

Amino acids

The pathway of amino acid synthesis in parent bodies has still not been resolved despite extensive studies of meteorites. The diversity of aldehydes and ketones found in these samples could have led to amino acids via a Strecker-type reaction involving ammonia and hydrogen cyanide, followed by hydrolysis via aqueous alteration on the parent body. This scenario is supported by the presence of α-hydroxy carboxylic and α-hydroxy dicarboxylic acids 138 . According to this hypothesis, α-methylated amino acids such as isovaline and 2-aminoisobutyric acid, abundant in meteorites, could also be formed. Other pathways such as reductive amination of α-keto acids 139 , carboxylation of amines using CO2 and proton irradiation 140 , and mineral-catalyzed reactions from H2, CO, and NH3 141 have also been suggested. Nevertheless, a Streckertype reaction cannot account for the generation of β-, γ-, and δ-amino acids thus questioning this scenario as plausible.

The synthesis of β-amino acids via a Michael addition to α,β-unsaturated nitriles coupled with reduction hydrolysis was considered 107 . Michael addition, discovered in the late 19 th century and shown in Figure 7, was adapted to the synthesis of β-alanine 142 and was considered as a prebiotic way to obtain β-amino acids. Finally, γ-and δ-amino acids could be the result of the decarboxylation of α-amino-dicarboxylic acids 143 or the hydrolysis of lactams 144 , an unlikely scenario given the unestablished source of the latter.

In addition, aqueous alteration of cyanide-based polymers by hydrolysis has also been proposed 145 , as well as radical mechanisms involving nitriles as precursors and gas-phase ion-molecule mechanisms 146 .

Nevertheless, Strecker's synthesis, discovered in 1850 147 and describing a route to obtain amino acids from an aldehyde (or ketone), ammonium chloride, and potassium cyanide, remains a good candidate for amino acid synthesis. The Strecker synthesis, shown in Figure 7, can be performed using ammonia and hydrogen cyanide. This synthesis is a 2-step reaction where the aldehyde reacts with ammonia via a condensation reaction to lead to an α-aminonitrile that is then hydrolyzed to form an amino acid. The use of a primary or secondary amine instead of ammonia leads to N-substituted amino acids. In addition, racemic mixtures are obtained using this Strecker synthesis. Asymmetric synthesis and asymmetric catalysis were mentioned previously in 1.2.4 Catalysis-based amplification. The former requires asymmetric amino reagents while the latter requires a chiral catalyst.

Sugars

The pathways for the synthesis of sugars under abiotic conditions have still not been solved, but several leads are being considered. The most famous is the Butlerov reaction 148 , also known as the formose reaction. Discovered in 1861, the formose reaction describes a pathway for obtaining a plethora of longchain sugars and sugar-related compounds from formaldehyde, as shown in Figure 8. The successive combination of isomerization and/or aldol reactions from formaldehyde leads to the complexification of the starting material to aldoses and ketoses. Basic conditions and a divalent metal such as calcium to coordinate the sugar oxygen atoms 149 were initially used as catalysts. The role of minerals has also been studied, and about half 150 of 34 tested minerals have been found to catalyze the formose reaction.

In addition, hydrothermal vent-like conditions using CaCO3 151 or interstellar-like 152 conditions have been studied for the formation of various sugar compounds. A major concern is the availability of formaldehyde in interstellar ices or on the early Earth, as it is easily hydrated or can undergo a Cannizzaro reaction leading to formic acid and methanol. In addition, the sugars synthesized by the formose reaction are known to have a short lifetime 153 (ribose half-life is 73 min at pH 7.0 and 100 °C and 44 years at pH 7.0 and 0 °C), which compromises the mentioned conditions. In the case of hydrothermal vents, Omran 151

proposed silica-CaCO3 chemical gardens that could potentially allow for the accumulation (in addition to catalysis) of free sugars as the precipitate membranes formed are kept in disequilibrium from the outside. HMT is usually obtained using formaldehyde and ammonia in liquid or vapor state by a condensation reaction. The search for pathways to form prebiotic molecules under abiotic conditions is still ongoing, but HMT is considered a key molecule to solve this problem because its acid hydrolysis can lead to several amino acids 155 . Indeed, laboratory-synthesized interstellar ice analogs can contain significant amounts of HMT and HMT derivatives, especially when methanol is used in the initial reagent mixture 156 .

Furthermore, methanol abundances in the ISM support this scenario where these molecules are likely to be incorporated into interstellar ices and lead to abiotic chemistries on the surface of grains 157,158 . The formose reaction and the inclusion of ammonia in the formose reaction are generally discussed to justify the role of HMT as a source of formaldehyde and ammonia 159 and as a feedstock for Maillard-type reactions. It cannot be reduced to these reactions alone as it is probably a complex network of chemical reactions 159 .

Conclusion

The conditions under which biosignatures are synthesized under interstellar conditions are still debated and not fully elucidated. Amino acids could be the result of multiple reactions such as Strecker synthesis and Michael addition, while sugars could probably originate from the formose reaction but suffer from rapid degradation. Other biosignatures such as HMT are another leads as intermediates to amino acids and a storage of formaldehyde for the formose reaction.

CONCLUSION

Numerous biosignatures have been found in extraterrestrial samples with ees of the same sign as found in living organisms. On the other hand, simulated cometary ice samples using CPL showed the presence of chiral biomolecules with small ees, that depend on the wavelength and helicity of the chiral photons used. Since the mechanisms and processes responsible for the synthesis of biosignatures and creation of extraterrestrial ees are not fully understood, additional analyses are required for understanding the origin of biomolecular homochirality. The following chapter aims to describe the materials and techniques that can be used to study such extraterrestrial ees.

CHAPTER II

Chiral resolution and analysis of biosignatures

COMPREHENSIVE TWO-DIMENSIONAL GAS CHROMATOGRAPHY (GC×GC)

Comprehensive two-dimensional gas chromatography is an advanced technology that evolved from conventional gas chromatography, which has been a common technique for analyzing volatile compounds for decades. The lack of full peak resolution in rather complex environmental samples has driven the evolution of gas chromatography through the use of heart-cut techniques that allow another separative run of an unresolved portion of the sample on a different column 163 . At this point, the idea of performing complete heart-cut analyses emerged, but the main concern was to preserve separation on each individual column 164 . The splitting devices used at that time have undergone many improvements and have been called modulators since 1991 165 .

General principle of operation

A few microliters of liquid sample are introduced into the injector, which is maintained at high temperature, allowing the sample to change from liquid to gaseous state. The first oven is maintained at a temperature at least 20 °C below the boiling point of the sample solvent -to condense and concentrate the analytes at the beginning of the first column. A preferably inert carrier gas passes the two columns during the analysis and allows the migration of the analytes via their partition between the mobile phase (gas) and the stationary phase (column). This migration is assisted by the temperature, the oven being progressively heated to release the less volatile compounds. Compared to conventional GC, GC×GC can exhibit two different heated elements: the modulator and the second oven. Both allow for the successful transfer of effluents, minimizing peak broadening, and optimize the separation on the second dimension.

The modulator traps the analytes between the columns and releases them periodically to preserve the separation in each dimension. The set of columns should preferably be chosen to be orthogonal, i.e. that the separation mechanism for each stationary phase is distinct to maximize peak capacity 166 . The length of the 2 nd column, which is approximately 1.5 m, is much shorter than the 1 st column which is typically 25 m, and hence, is commonly compared to as very fast GC analysis 167 . It should be noted that the addition of a second column does not change the total analysis time. The analytes are detected and quantified by a detector with a high acquisition frequency of at least 50 to 100 Hz, because of the resulting narrow peak widths of the secondary column. The typical width of these peaks is 100 to 200 ms, which corresponds to 10 to 5 Hz. Since about 10 data points are needed to reliably define each Gaussian peak, the acquisition rates of the detectors must be at least as high as those mentioned above. A simplified GC×GC scheme is shown in Figure 11.

Modulators

Principle of operation

The main roles of the modulator are to ensure a periodic re-injection of all the effluent from the first column into the second column as well as a conservation of the separation on each column, allowing a complete analysis defined by Giddings 168 in 1984. These criteria can be met through a multi-step modulator operating process. It collects, concentrates, focuses and re-injects effluents into the second column as shown in Figure 12. As a result, conventional GC peaks, typically 10-30 seconds wide on the first dimension, are sliced multiple times and arrive as 200 ms peaks after the second dimension at the detector. The time between each reinjection (pulse) is called the modulation period (PM) and corresponds to the elution time on the second dimension, which is typically between 2 and 6 seconds depending on the compound and secondary column interactions.

Technologic evolutions

Table 5 is intended to provide an overview of the operating principle and historical background of some well-known modulators. Two main categories stand out: thermal modulators and valve modulators.

Thermal modulators, based on thermal desorption, were the first modulators ever made but are nowadays mostly cryogenic due to their major performance 169 . The use of hot and cold jets with liquid nitrogen is in vogue because the low temperature cooling using liquid nitrogen with a boiling point of -196 °C is very efficient.

Valve modulators are based on pneumatic devices to cleverly reroute the effluent between columns.

However, they can suffer from peak disruption because a high flow rate from the secondary column is required. Rapid re-injection of analytes is of great importance as it minimizes peak broadening, maximizes detectability, and increases maximum peak capacity. Currently, none of them stand out as they each have their advantages and disadvantages 169 regarding robustness, reproducibility, universality, narrow injection pulse, fast modulation period, duty cycle, quality, and cost. The main disadvantages of thermal modulators are (i) an observed analyte breakthrough, i.e. analytes are not fully trapped by the modulator and pass into the second column during the PM and (ii) incomplete desorption, i.e. analytes are not fully released or not released quickly enough during the heating step at the end of PM 169 . The main drawbacks of valve modulators are the high flow rates required in the second dimension and low duty cycles, resulting in limited applicability and loss of detectability thresholds 169,170 .

The development of new thermal modulators may concern the use of cryogen-free modulators as this has a financial and logistical impact. In this regard, ceramic cooling blocks coupled with cooling fans have been designed 171 as well as the Peltier modulator 172 and have shown similar performance results compared to a commercially available N2 modulator. Moreover, there are commercially available consumable-free modulators, such as the ZX2 from Zoex. The development of new valve modulators may involve the use of thermally robust 173 and chemically inert materials, reduction of the 2 nd column flow rate 174 to be compatible with the MS and avoid disturbing the flow rate as well as higher duty cycles 169 .

Table 5. Evolution of early thermal and valve modulators used in GC×GC.

Modulator Principle Remarks Thermal desorption modulator 165 Modulator using conductive gold foil between the columns.

The periodic application of an electric current between electrodes E1 and E2, then E2 and E3 alternately, allows the successive release/ desorption of the analytes trapped in sections S1 and S2 thanks to an increase in temperature.

Detectability

The combination of peak compression, mass conservation, and inter-column focusing results in narrow, sharp peaks with 10-to 70-fold higher signal intensities compared to conventional GC 181 . However, the evaluation of better detectability needs to be correlated with the noise levels. Indeed, the noise level is proportional to the square root of the acquisition rate of the detector. High values of acquisition rate, around 100 Hz, are necessary for GC×GC to reliably define narrow peaks and thus reduce the effective detectability gain. According to Lee et al. 166 , this effective value -corrected enhancement -for isolated peaks is typically 4 to 5. Nevertheless, the detectability improvement is particularly valuable for the study of trace analytes 182 . Emphasis should therefore be placed on the development of modulators and column configurations to reduce the peak width on secondary columns. As demonstrated by Lee et al. 166 , there is a decreasing exponential function bounding the peak amplitude enhancement i.e., the ratio of secondary column peak amplitude over the first column peak amplitude, to the secondary column peak width. As the authors argued, a considerable increase in amplitude could be achieved if the width of the secondary peaks were about 100 ms or even 200 ms which is currently the case in most applications. Detectability is also influenced by the acquisition speed of the detector 183 , which will be briefly discussed below.

Time-of-flight mass spectrometry

As previously mentioned, high acquisition speed detectors are mandatory in GC×GC to ensure the acquisition of well-defined Gaussian peaks. Common detectors meeting this criterion are the microelectron capture detector (m-ECD), thermal conductivity detector (TCD), flame ionization detector (FID), and mass spectrometry (MS) with respective acquisition rates detailed in Table 6. R.C. Blase et al. 183 studied the effect of the acquisition rate at 10 Hz and 200 Hz using GC×GC-FID and found an increase in peak area by a factor of 20, an increase in peak height by a factor of 8, and a decrease in peak width by a factor of 5. Although it is possible to use detectors with low acquisition rates, analysts may risk missing or overlooking analytes with narrow band widths. It also implies overall poor GC×GC performances. Thus, high acquisition rates are generally preferred at the expense of a larger volume of data files. The advantage of m-ECD, despite its low acquisition rate, is its extreme selectivity towards halogenated compounds and thus its improved detectability which may justify its use depending on the application.

TCD and FID are also excellent and easy-to-use candidates with high acquisition rates, however, they suffer from their versatility because no distinction between compounds can be made besides the retention time. Finally, mass spectrometry is often the best compromise because it benefits from both, speed of acquisition and analysis of spectral data. Since a mass spectrometer consists of several components such as an ion source, a mass analyzer and a detector, a distinction is made between quadrupole (Quad) and time-of-flight (TOF) mass analyzers. Their difference in acquisition rate is due to their operating process:

a Quad is a scanning mass analyzer using rods to create electric fields allowing trajectory control of ions of a given m/z ratio, whereas a TOF is a non-scanning mass analyzer using electric fields to accelerate all ions with the same kinetic energy and separate them according to their time of flight needed to reach the detector 184 . The main advantage of a TOF over a Quad is the ability to acquire a full range of mass spectra without compromising speed and sensitivity. However, the sensitivity of the Quad can be increased by using the selected ion monitoring (SIM) mode for better quantitative analysis.

After elution from the secondary column, analytes are most often bombarded in the ion source of a mass selective detector with a 70 eV electron beam to ionize the molecules and further leads to their fragmentation as they exhibit high excess energies. Electron ionization (EI) is the most common ionization technique used in GC because it provides a high degree of fragmentation and detailed mass spectra. It is referred to as hard ionization mode as opposed to soft ionization. Once the ions are created, they are pushed into the mass analyzer with the help of a repeller and accelerated by an electric field. All ions have the same kinetic energy and will be sorted by their time of flight over a given path length, as their velocity depends on their mass through their m/z ratio 185 . The resolution can be improved and the kinetic energy of ions of the same m/z corrected by using a reflectron at the end of the flight path that increases the total distance 186 . After traveling through the field-free drift region, the ions are detected either using a microchannel plate detector, electron multiplier, Faraday cup, or channel photomultiplier 185 that also amplify the signal which is then recorded using a time-to-digital converter (TCD) or analog-to-digital converter (ADC).

The resulting mass spectrum of each analyte is characteristic and often called a fingerprint. Fragmentation patterns provide structural information about the molecule, as functional groups tend to show the same dissociation behavior after ionization. Other more specific rearrangements also occur and are related to the structure of the molecule.

Stationary phases

The heart of each gas chromatographic separation is the column and its respective stationary phase used to resolve a multitude of analytes in a mixture. A gas chromatographic column should therefore always be chosen very carefully and adapted to the given application. In the following, I will highlight the most prominent chiral columns used for the enantioseparation of chiral amino acids and sugar compounds. Because CDs are represented as shallow cones, it is interesting to highlight their dual hydrophilichydrophobic nature. The inner part of the cavity is represented as a surface (as a plane for each glucose unit) containing protons, H3 and H5, and solitary pairs of glycosidic oxygens conferring a hydrophobic behavior to the cavity 189 . On the other hand, the tapered ends have hydroxyl groups exhibiting hydrophilic behavior. Thus, CDs are described as "all-purpose molecular containers for organic, inorganic, organometallic, and metal organic compounds that may be neutral, cationic, anionic or even radical" 190 .

Some physicochemical parameters of different cyclodextrins are detailed in Table 7. Cyclodextrins are inherently chiral; they can form diastereomeric complexes with chiral guests. When this complex shows a higher stability for one enantiomer of the guest molecule, enantioselective recognition can be considered to occur. The difference in stability between the complex formed with one enantiomer compared to the other will define the efficiency of this chiral discrimination process.

The driving forces involved in cavity inclusion complexation of compounds are mainly van der Waals forces (Keesom, Debye and London) and hydrophobic interactions. To a lesser extent, hydrogen bonding and steric hindrance can impact the complexation behavior. The shape selectivity properties of CDs are suitable for all types of isomer separation, including enantiomers, which differ only in their stereochemistry. Indeed, although the size of the cavities allows for the inclusion of e.g., single (α) or multiple substituted phenyl rings (β and γ), it is the hydroxyl group at the edge of CDs that allow chiral recognition with the asymmetric center of the analyte. These properties have led to the development of modified CDs to control solubility, hydrophobicity, cavity outer diameter, and chiral recognition for example. Thousands of these derivatives currently exist with various ring sizes and chemical functionalizations 192 .

Modifications of CDs occur only at the hydroxyl groups which are nucleophilic, and these compete for electrophilic attack. Selective modification is therefore difficult but possible because these hydroxyl groups are not equivalent. The hydroxyls in position 2 are acidic, the hydroxyls in position 3 are sterically hindered, and the hydroxyls in position 6 are basic, which gives them increased nucleophilic behavior 191 .

Depending on the reactivity of the reagent, a single derivatization of the hydroxyl group in position 6 can be achieved. For example, a monosubstitution 193 using p-toluenesulfonyl chloride, a disubstitution 194 using arenedisulfonyl chloride and a persubstitution 195 have been reported.

Two stationary phases are described in more detail in the next sections as they were used for the amino acid and sugar analysis studies. Many CDs can be used for ee analysis but it should be noted that the separation strongly depends on the derivatization used, which will be further discussed in 2.1.4

Derivatization in gas chromatography. 13). The prefix octakis, oktákis in ancient Greek means eight times, which means that all glucose units of the γ-cyclodextrin are substituted with 3-O-butyryl-2,6-di-O-pentyl. The Lipodex E stationary phase was first introduced by König et al. 196 in 1989 as a highly versatile cyclodextrin derivative for the separation of proteinogenic and nonproteinogenic amino acids, α-and β-hydroxy acids, polyhydroxy compounds, alcohols, ketones, lactones, cyclopropane derivatives, cyclic acetals, amines and alkyl halides. Native γ-cyclodextrin was successively treated with 1-bromopentane under basic conditions (NaOH) followed by butyric anhydride under basic conditions (triethylamine) and 4dimethylaminopyridine as nucleophilic catalyst. The hydroxyl groups at position 2 and 6 are converted to ethers in the first step by an SN2 reaction while steric hindrance at position 3 prevents the substitution from occurring. In the second step, the hydroxyl at position 3 is acylated and gives an ester due to the nucleophilic attack of the hydroxyl on the anhydride. The ethers in position 2 and 6 do not react with the anhydride because they are less nucleophilic and because no inorganic catalyst is used.

2.1.3.1.3 Lipodex E Commercialized as Lipodex E, octakis-(3-O-butyryl-2,6-Di-O-pentyl)-γ-cyclodextrin is a fully substituted γ- cyclodextrin (Figure

New stationary phases for multidimensional gas chromatography

Ionic liquids have attracted a lot of interest due to their dual nature (polar and ionic), allowing optimized orthogonality between the stationary phases. Stationary phases based on ionic liquids may therefore find increased use as secondary columns in comprehensive two-dimensional gas chromatography to improve the general resolution and peak capacity of complex mixtures. They have better selectivity and thermal stability than conventional phases 199 . The first use of these ionic liquids in multidimensional gas chromatography was performed by G. R. Lambertus et al. 200 and many others have been designed since for targeted analytes 201 . Another important feature is the water and oxygen stability at high temperatures, allowing water to be used as an injection solvent and getting rid of tedious pretreatments. Ionic liquids were even used to perform a GC 3 (three-dimensional GC) 202 analysis where the column configuration was: 20 m apolar × 4 m ionic liquid × 1 m polar. As the authors pointed out, it is still difficult to exploit the third retention space because the two columns used are polar and the sample lacks polarity diversity.

Derivatization in gas chromatography

The derivatization process is a chemical modification of target compounds to obtain better properties for their analysis. The main purpose of this modification is to improve their volatility for GC analysis. However, this is not the only purpose and/or consequence of this procedure 203 , which are listed below. Volatility is improved by lowering the boiling point through the addition of functional groups that decrease the overall polarity of the target molecule. Indeed, polar groups such as -OH, -NH, or -SH can establish strong H-bond interactions and must be converted to -OR, -NR, or -SR groups which also protects the stationary phase.

Stability is improved by inhibiting intra and intermolecular reactions that may occur during heating in the injector and oven during gas chromatographic analysis. Detectability is improved by amplifying the intensity response through a better conversion of the compounds and/or the use of a specific detector.

Chromatographic behavior is improved as compounds with modified functional groups interact differently with the stationary phase, resulting in different retention times and separations/enantioseparations. Other improved benefits include minimized interferences with the matrix (robustness), separation of analytes from the matrix and shorter retention times. As previously stated, derivatization reagents must be reactive electrophiles to remove hydrogens holded by alcohols, carboxylic acids, or amines, as these functional groups are nucleophilic and often encountered. For convenience, the reagents must be stable, affordable, and available.

Enantioselective separation of chiral analytes can theoretically be achieved by two different routes. The first uses achiral reagents and a chiral stationary phase, while the second uses chiral agents and an achiral stationary phase to separate diastereomers. The latter requires several conditions to be used with confidence in terms of small ee determination: enantiomeric purity of the reagent, non-racemization of the reagent and reactants, equal reaction rates between the reagent and the two enantiomers, and equal detector response of the diastereomers 204,205 . Therefore, the use of achiral reagents in combination with a chiral stationary phase is often considered to be the better approach for efficient enantioseparation and precise ee determination, even though it may still suffer from racemization of the analytes during the derivatization reaction. Depending on the target molecules and derivatization method, however, racemization can be less critical as racemization of α-amino acids for example has been reported to be less than 0.1% (ee = 0.2%) from an enantiopure solution 205 using ethanol/trifluoroacetic anhydride derivatization. Racemization is derivatization-dependent and can therefore be more critical. Regarding sugars, the situation is slightly different because many epimerization reactions can be involved, including racemization. Cooper et al. 113 reported that there was no racemization but only oxidation of sugar molecules to sugar acids, with unchanged ee in the derivatization products using an alkyl/trifluoroacetic anhydride derivatization.

Finally, it is possible to combine a chiral reagent with a chiral stationary phase to improve chromatographic separation. Theoretically, the concerns regarding ee should remain the same, but depending on the derivative, chiral or achiral columns may be more appropriate.

Carbon number, stereochemistry & predominant forms

The number of carbon atoms used to build monosaccharides is another classification parameter.

Monosaccharides range from three carbons (triose) to nine carbons (nonose), as shown in Figure 15 and Furthermore, the appearance of a new stereogenic carbon due to an intramolecular nucleophilic attack between an alcohol group and the terminal carbonyl function is specific to carbohydrates. This carbon is called an anomeric carbon which is a special case of an epimeric hemiacetal carbon. The open-chain form of monosaccharides therefore leads to cyclic carbohydrates such as furanoses -5-carbon ring -and pyranoses -6-carbon ring. There is an equilibrium between these forms, which depends on temperature, concentration and solvent 214 . The cyclic forms, Table 8, are extremely predominant over the straight chain form in water and the pyranose form is in the majority over the furanose form 215 . As the nucleophilic attack can take place equally on both sides of the carbonyl, two possible anomers can be formed. The pair of anomers is designated as the α-anomer with the alcohol group pointing down and the β-anomer with the alcohol group pointing upwards at the anomeric carbon C-1 in the Haworth projections. Consequently, a total of 5 different stereoisomers of one single sugar enantiomer can be found in equilibrium due to intramolecular cyclization at the anomeric carbon and the competition of forming 5-and 6-carbon membered rings (Figure 17). This means that up to 10 forms (5 D and 5 L) can be encountered for each carbohydrate.

Carbohydrate derivatization strategies

Several disadvantages of the equilibrium mixtures of the straight and cyclic forms of monosaccharides on the enantioselective analysis can be pointed out. The first disadvantage is the decrease in intensity associated with the numerous existing forms. Considering the aldoses in Table 8, 2 to 5 forms in total are present for one sugar enantiomer. However, the derivatization procedure may have an impact on the equilibrium composition of these forms. The second disadvantage is the high number of chromatographic peaks that can lead to difficulties in resolving such a complex mixture as well as achieving sufficient enantioseparation, even when using a powerful separation technique like GC×GC. Several specific derivatization approaches have been developed to overcome these limitations, but each has its strengths and weaknesses. Below, different approaches are presented, and the strengths and weaknesses are discussed in order to carefully choose one of them for our applications.

Reduction

The most intuitive solution would be to reduce the number of forms by preventing cyclization from occurring 216 . This means inhibiting the reactivity of the carbonyl to constrain the carbohydrate to its linear form. Reduction can successfully fulfill this role but has strong drawbacks in terms of stereochemistry. It either leads to the loss of chiral information through the formation of a meso compound, or to the fact that the same sugar alcohol gives indistinguishable precursors, as shown in Figure 18. This phenomenon appears for some C4, C5 and C6 sugars as pointed out by Cooper et al. 217 . In the search for ee in extraterrestrial samples, this method cannot be employed despite its cyclization-preventing feature. The main advantages of this derivatization, Figure 20, concern the detection and enantioseparation of glyceraldehyde with good reproducibility. However, the C4, C5, and C6 sugars suffer from a lack of enantioseparation 223 , which is a significant drawback for the evaluation of potential ee values in extraterrestrial samples.

N,O-Bis(trimethylsilyl)trifluoroacetamide

As with amino acids, which will be discussed later, N,O-bis(trimethylsilyl)trifluoroacetamide derivatization (BSTFA), Figure 21, leads to tris(trimethylsilyl) derivatives by substituting the labile hydrogens of the hydroxyl and carboxyl groups of sugars. In contrast, MTBSTFA, which leads to tert-butyldimethylsilyl derivatives, should be avoided because steric hindrance does not allow for the detection of sugars 224 .

Indeed, silylation is a bimolecular nucleophilic substitution (SN2). Aldoses, ketoses, sugar alcohols, and sugar acids can be identified and resolved using CP-Chirasil-Dex CB coupled to DB-Wax columns (GC×GC) [START_REF] Meinert | Ribose and Related Sugars from Ultraviolet Irradiation of Interstellar Ice Analogs[END_REF] .

In addition, the cyclic forms pyranose and furanose can be distinguished based on their mass spectra.

Thus, this is a very versatile and convenient derivatization method. However, no enantioseparation was reported using a chiral stationary phase like CP-Chirasil-Dex CB [START_REF] Meinert | Ribose and Related Sugars from Ultraviolet Irradiation of Interstellar Ice Analogs[END_REF] . This major drawback therefore makes this method unsuitable for ee analysis and will not be considered.

Methylboronic acid/trifluoroacetic anhydride

Monosaccharides are constrained to a cyclic form via a condensation reaction involving vicinal or isolated diols and methylboronic acid (MBA) to form a methylboronic ester. Originally designed for C5 and C6

sugars, the unique feature of this derivatization is the need for several alcohols to be close enough to link them by removing labile hydrogens with MBA. However, as shown in Figure 22, when the monosaccharide -here glucose -has an odd number of alcohols or if they are too far apart to be derivatized, a second derivatization step is necessary. Silylation using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) is a typical route, as outlined by Von Donger et al. 225 . Acylation 226 , using trifluoroacetic anhydride would be an alternative. Rings with five and six atoms are favored and called dioxaborolanes and dioxaborinanes respectively. In the case of ribose, the only form found is that shown in Figure 22: a boronic ester cannot be formed between C1 (acetal) and C4 because the ring formed would be too constrained. Moreover, this addition could only occur between C2 and C3, leaving the hydroxyl groups of C1 and C4 free for a second derivatization step, but this compound was not observed. Using MBA only, arabinose, fucose, xylose, ribose and rhamnose were detected, whereas lyxose, fructose, galactose, mannose and glucose detection required a second derivatization step using BSTFA or acetic anhydride.

The use of TFAA to derivatize the remaining free hydroxyl groups significantly reduces the intensity of the C3 and C5 derivatized sugars with MBA alone. A significant advantage of the MBA/TFAA derivatization method is the formation of only one compound per enantiomer leading to less cluttered chromatograms which is especially important for trace analysis. Single carbon addition for two alcohols is also timely for isotopic studies. Mass spectra show similar fragments for all carbohydrates but can be distinguished based on their intensities.

(S)-2-Butanol/trifluoroacetic anhydride

Unlike other derivatizations that constrain monosaccharides into a linear or cyclic form, the first step of this (S)-BuOH/TFAA method aims to favor one or a few stereoisomers over the others 217 , followed by a second TFAA step to convert the alcohols into fluorinated esters, Figure 23. The introduction of (S)-2butanol as a chiral reagent to convert the hemiacetal of cyclic sugars to acetal generally favors only one anomer of pyranose as pointed out by Cooper et al. 217 . Some sugars such as xylose, arabinose, glucose, galactose, and allose, however, lead to the formation of two isomeric forms of equivalent intensity. This method has the advantage of leading to diastereoisomers and therefore allows enantioseparation on achiral columns. Efficient enantioseparation, especially for C6 sugars, was obtained on an achiral DB17 column in this study 217 . In addition, the mass spectra of the derivatives can be easily distinguished based on the crude formula of the starting sugars, i.e., the carbon number and the deoxy sugars. This feature could help to identify non-target sugars when analyzing real samples. Another strength is the detection limit of the method, which is in the picogram range, making it suitable for analyses of extraterrestrial samples and interstellar ice analogs. Unfortunately, glyceraldehyde was not detected by this method. Thus, each method represents a performance trade-off, so that a given application will guide the analyst in his choice of which derivatization will be best suitable.

Amino acids

Classification

Amino acids are chemically defined as organic compounds containing both an amine and carboxylic acid functional group. Thus, carbon, oxygen, hydrogen and nitrogen are their main atoms that compose them.

Other elements such as sulfur can also be found. They can be classified mainly according to their number of carbons, the side chain group, and especially according to the location of the functional groups. The location of the amine can be either in α, β, γ, or δ to the carboxylic acid. Hundreds 227 of amino acids have been discovered in nature, but life is built on 20 of them that are all α and L-configured except glycine being the only achiral amino acid. These are called proteinogenic because of their incorporation into proteins during RNA translation. Selenocysteine and pyrrolysine 228 are considered the 21 st and 22 nd proteinogenic amino acids because they have been reported to be incorporated into the proteins of some organisms. Of these proteinogenic amino acids, Table 9, only nine are considered essential because they cannot be synthesized by the human body. All other necessary amino acids can be synthesized from the nine essential amino acids.

Amino acid derivatization strategies

Unlike carbohydrates, amino acids do not suffer from the reduction in intensity of multiple stereoisomers.

However, the diversity of existing amino acids poses many challenges with respect to their separation and detection. Intensity, separation, and enantioseparation are essential to reliably calculate enantiomeric excesses in extraterrestrial samples. Since these compounds are not volatile -the melting point of glycine is about 230 °C -a derivatization step is necessary. In this regard, numerous attempts to develop an exceptional method have led to a wide variety of available techniques. Common derivatization procedures for meteorite analyses were reviewed by Simkus et al. 229 , highlighting 11 general amino acid derivatization routes. Of these, trifluoroacetic anhydride/alkyl (TFAA/alkyl) derivatization for GC and phthalaldehyde/NAC (OPA/NAC) derivatization for LC appear to be widely used to study amino acids from various meteorites. Volatility, intensity, enantioseparation, and versatility of all amino acids are still key factors in determining whether a derivatization is suitable for the analysis of a meteorite sample. The TFAA/alkyl functionalization meets some of these requirements. In particular, the high response of isovaline which is of great importance in finding clues to the origin of homochirality 108 in comparison to other methods is advantageous. Here, several amino acid derivatizations will be discussed.

N,N-dimethylformamide/dimethylacetal

Among amino acid derivatization reactions, N,N-dimethylformamide dimethylacetal (DMF/DMA), Figure 24, appears to be a rapid and simple route to successfully convert and detect most amino acids. Using this one-step method, 19 out of 20 proteinogenic amino acids were identified and 10 were enantioseparated on a CP-Chirasil-Dex CB (heptakis-(2,3,6-tri-O-methyl)-β-cyclodextrin) 230 column. However, it should be noted that for 6 out of 10 amino acids, the enantioresolution is less than 1. This method is considered an excellent candidate for in situ analyses in diverse space missions because it meets many requirements. It can be easily automated having a short reaction time of 3 minutes at 140 °C. Moreover, it produces relatively low weight compounds that require only limited m/z ranges for adequate detection which is beneficial for space missions and their restricted instrumental and analytical performances. Among these 10 enantioseparated amino acids, only asparagine, aspartic acid, proline and serine showed partial racemization while tyrosine showed complete racemization. Compromises had to be made to optimize yield and avoid as much as possible racemization that occurs at high temperature, as well as by-products.

This method is therefore not the best compromise for amino acid ee analysis of meteoritic samples.

Ethylchloroformate/heptafluorobutanol

The successive reaction of ethyl chloroformate and heptafluorobutanol on amino acids as a one-pot mixture leads to the formation of N(O,S)-ethoxycarbonylheptafluorobutylester derivatives. A by-product can be obtained after decarboxylation between the two reactions, Figure 26. This side-product corresponds to the compound that would have been obtained using ethanol instead of heptafluorobutanol. Overall good separation and enantioseparation 233 , Figure 27, are obtained with detection limits generally between 10 -6 and 10 -7 M, but several orders of magnitude higher for isovaline, sarcosine, and 2-aminoisobutyric acid. Nevertheless, the ees found in the Murchison meteorite using this method are reliable since the eeL of alanine is 3.51 ± 0.74 and 4.61 ± 0.83 for isovaline using three standard deviations. As isovaline is of great interest in understanding the origin of chiral biases in meteoritic samples (1.4.1 Meteorites), this method was not considered in my studies. This two-step derivatization involves successive heating steps and drying that are considered critical compared to other methods such as BuOH/TFAA or IPA/TFAA 235,236 . With respect to methodology, the drying steps have been reported to be performed using either a rotary evaporator, P2O5/NaOH under vacuum 237 or a nitrogen stream 238 . Studies using nitrogen stream have shown that a 10% loss can be induced on N-TFA methyl ester derivatives at room temperature. Equally 10% loss occurred at 100 °C when N-TFA n-butyl esters were dried under a nitrogen stream 238 . Using a rotary evaporator, up to 36% of alanine are lost 239 . Recently, careful drying using a nitrogen stream followed by drying at room temperature has been reported 240 . Complete drying was not performed to avoid evaporation of the amino acid derivatives. Instead, a relatively large amount of solvent was added for later analysis. It is likely that this quenches the TFAA-initiated reaction without losing the derivatized compounds.

Different conditions exist for each reaction in this two-step derivatization. Originally, HCl gas was used to perform the first acid-catalyzed esterification step by bubbling it into the chosen alcohol. Despite its easy removal under a nitrogen stream, HCl paved the way for another practical alternative using acetyl chloride and an alcohol to produce dry HCl in situ. It is interesting to note that although the alcohol/acetyl chloride ratio might have an optimal value for isopropanol 241 , various values for this esterification step depending on the alcohol are reported. As methanol is less sterically hindered than isopropanol, it might therefore be less sensitive to the importance of this ratio. Currently, a methanol/acetyl chloride ratio of 4:1 (v:v) is commonly used 242 . Along with this ratio, the reaction time and temperature of isopropanol have been studied and are critical for the amino acid tryptophan, and slightly for valine 241 . Indeed, care must be taken to ensure a good conversion without degradation due to overheating. To perform the second step, TFAA is rarely used as a pure reagent. Dichloromethane is the historical polar solvent for this reaction and is still widely used. However, nonpolar solvents such as hexane have also been used 240 or polar solvents such as trifluoroacetic acid 243 .

The derivatization procedure has also been modified to avoid problems during isotopic measurements of amino acids. A 1:2:5 (v:v:v) acetic anhydride/triethylamine/acetone mixture replaced the 4:1 (v:v) DCM/TFAA mixture to remove fluorine from the derivatives 244 because fluorine could be a toxic catalyst for the oxidation reactor. Among the modified procedures, the use of an insoluble sulfonated polystyrene resin in the hydrogen form as an acid catalyst 245 for the first step is noteworthy. This type of resin is now a common means of purifying extraterrestrial amino acids from impurities via ion exchange chromatography. At low and neutral pH, the amino acids bind to the resin due to their positive charge. It is interesting to keep in mind that during the purification process, resin breakthrough in the amino acid fraction could result in loss of compounds. Indeed, using a TFAA/solvent reaction, the amino acids could still be bound to the resin and not be derivatized due to the low pH. To overcome this problem, it should either be established that no resin breakthrough occurs or the derivatization should be adapted to be performed in alkaline medium, using for example a methyl trifluoroacetate/trimethylamine mixture 245 . Fox et al. 242 showed that using this derivatization, isovaline is baseline resolved on a Lipodex E column along with nearly 40 non-proteinogenic amino acid enantiomers being resolved. Unfortunately, no information on the detection limit of isovaline has been reported in this study. Thus, given the potential applicability for meteorite analyses, this method was selected for further study using non-proteinogenic and proteinogenic amino acids.

ION EXCHANGE CHROMATOGRAPHY

Prior to the derivatization step, meteoritic samples are usually purified using ion exchange chromatography (IEC) to remove salts and metal cations but can also be used to fractionate samples in two or several distinct fractions containing selected classes of compounds. It is also an additional filtering step to remove meteorites' solid particles.

Resin properties

The resin used throughout my studies was a sulfonated poly(styrene-co-divinylbenzene) copolymer obtained by post-sulfonation. The divinylbenzene leads to cross-linking and a three-dimensional network.

The pore size of the beads depends directly on the cross-linking rate of the resin. primarily in column chromatography for analytical and laboratory scale preparative applications. Smaller particles (200 -400 mesh / 75 -38 µm) are used for high resolution analytical separations 206 .

Relative selectivity of counter-ions

Depending on the counterion used, the ion exchange properties can vary. Conversion from one form to another is possible by saturating the resin beads with the chosen counterion. A low selectivity is usually chosen in order to easily exchange the sample ions when they are introduced. The H + form can be easily obtained using concentrated HCl and is quite versatile given the relative selectivity available in Table 10.

The higher the relative selectivity (RS), the stronger the binding between the resin and the counterion.

The RS gap is a performance indicator of how well the sample ions will bind. It also defines the ease of transition from one form to another.

Separation mechanisms

Several separation mechanisms can occur when using an ion exchange resin and are presented in Table 11. Resins can be used either as an HPLC stationary phase or as a preparative step for subsequent analysis 207 .

In the context of extraterrestrial samples, its main function is to remove salts and metal ions from the amino acid fraction 209 . It can be performed before 210 or after the derivatization step. However, it may pose some risks when performed after derivatization (GC) because the target molecules are volatile and must be concentrated after ion exchange chromatography. Desalting is an important step to remove contaminants that could potentially interfere with subsequent derivatization. In addition, it has also been used to fractionate compounds according to their chemical functions. Thus, in our case it is a convenient way to obtain multiple fractions: one fraction containing sugar molecules and another fraction containing amino acids from a single sample 144 . In this case, only a few mechanisms are prevalent and useful. Ion exchange is the main mechanism to retain only amino acids. Ion-moderated partitioning can be mentioned to some extent because some metals must be removed from the sample. Similarly, interactions with sulfonate groups and the matrix necessarily occur with amino acids and sugars. Finally, the flow rate of the column has a significant impact on the separation and must be tailored to the application. For example, an elution rate of 5 -10 cm/min is recommended for trace ion removal 206 . Since ion exchange chromatography is used here as a separation step rather than a fine separation tool, such an elution rate seems appropriate for the sample requirements.

Mechanism Description

Ion exchange

The cationic compounds compete on the copolymer surface through the sulfonate active site.

There is a relative selectivity for each counterion with respect to the H + counterion.

Ion exclusion

The anionic compounds are repelled from the negatively charged matrix. It is directly related to the degree of dissociation of the ionic species. This mechanism loses its intensity with increasing molecular weight.

Size exclusion

The compounds are physically separated according to their size. This exclusion depends on the degree of cross-linking. The percentage of cross-linking influences the pore size of the resin beads.

Ion moderated partition chromatography

Metal cations can be attached to the sulfonate group and create a coordination complex with other compounds. Chelates are formed by pyranose rings and metal cations, for example.

Interaction with sulfonate groups

Compounds are adsorbed onto the sulfonate groups through hydrogen bonding and dipoledipole forces or are separated by differences in water polarity induced by the sulfonate group.

The intraparticle water is more polar than the mobile phase water.

Interaction with matrix

Reverse phase partitioning can occur due to the apolar resin backbone and the polar mobile phase. This mechanism becomes more dominant when surfactant analytes are used.

Principle of ion exchange chromatography

Upon sample addition (Figure 30), positively charged species compete with H + ions at the sulfonate binding sites. As shown by the relative selectivity of counter ions in Table 10, This calculation provides an order of magnitude because the equation is for a dilute solution, between 10 - 6 M and 1 M. At this elevated pH, the ammonia group in the amino acids becomes mostly neutral when released from the exchange resin. This is unfortunately not often -if ever -stated in the literature. Once eluted, the amino acid fractions need to be concentrated using e. g. a rotary evaporator as several bed volumes are required to achieve their complete elution. Derivatization is usually performed after this "fractionation/purification" step. However, one has to keep in mind the basicity of the amino acid fraction which can have an impact on the efficiency of the derivatization reaction.

Resin conditioning

The ion exchange resin can potentially be a source of contamination and should therefore be cleaned before use. Equilibration is typically performed using 3-bed buffer volumes 206 . However, a successive elution of water-basic solution-water-acid solution-water to condition the resin can also be considered.

The basic solution is used to remove the H + ions already present and elute any contaminants bound to the resin. The acidic solution is used to regenerate the resin with H + ions. Water is used to gently raise or lower the pH of the solution in which the resin is located, and to equilibrate the resin before use. This is because pH shocks can lead to cracks in the resin beads, resulting in less efficient and/or less reliable separation. In addition, cracks can form due to the resin dry out during elution. Care must be taken to ensure that there is always sufficient solvent above the resin bed level.

EXTRACTION OF EXTRATERRESTRIAL ORGANIC MATTER

Solvent extraction of soluble organic compounds

Extraction is often the second step in the analysis of soluble organic matter of meteoritic samples and should not be neglected because subsequent sample handling and analyses depend on its success. The recovery rate after one extraction step is generally estimated to be about 80% for rock and soil samples 246 but must be adapted in the case of extraterrestrial samples. As Rodier et al. 246 pointed out, time, temperature, and solvent must be optimized to ensure maximum recoveries without experiencing compound degradation or side reactions between the target compounds or with the matrix. Compromises must be made regarding the solvent selected because its polarity must match the targeted compounds:

the broader the class of compounds studied, the less efficient the extraction step. Table 12 aims to summarize the different conditions employed to extract and study organic compounds in meteorites.

Note that the targeted compounds are highlighted for better extraction context.

As can be seen in the table below, past experiments favored the use of apolar solvents while water is currently often chosen due to its polarity and ability to solubilize amino acids and sugars. It is also used for less polar compounds such as aliphatic amines, carboxylic acids and hydroxy acids. Slightly concentrated HCl can be used as a support to facilitate the release of organic compounds from the meteorite matrix. However, depending on the conditions used, deleterious reactions may occur. Target compounds can be altered by the Maillard reaction 247 , Strecker degradation as well as by dimerization, oxidation, and oximation [START_REF] Glavin | The Search for Chiral Asymmetry as a Potential Biosignature in Our Solar System[END_REF] .

Interestingly, in the context of Mars soil analyses, A. Buch et al. 248 , demonstrated that isopropanol could be an alternative to water and achieved recoveries of 80% for alanine and glycine, and 25% for serine. The carboxylic acids also studied, showed recoveries ranging from 5% to 80%, making this solvent more versatile in terms of extraction than ethyl acetate, acetonitrile, dichloromethane, and ethanol. With these solvents, the recoveries of these amino acids are generally close to zero. Thus, a more extensive study using a 1:1 (v:v) water and isopropanol mixture to optimize the extraction of amino and carboxylic acids was performed, and showed a wider extraction range 249 . Because the targeted amino acids and sugars are water soluble, the use of such a mixture is not necessary, but could be justified if it could provide a protective effect during extraction, such as inhibition of degradation reactions. In addition, because ion exchange chromatography must be used after the extraction process, the use of a solvent other than water or a mixture of solvents raises questions about compatibility, purification performance, and fractionation, although isopropanol has the advantage of not dissolving salts and leading to cleaner samples 249 .

Another striking result is the efficiency of sonication on the extraction of all the compounds mentioned.

Indeed, without sonication, the best recoveries were obtained after 140 h at 60 °C of conventional shaking 248 . At the same temperature and using sonication, similar or even slightly higher recoveries were reported (generally > 80%) with a duration of only 15 min for alanine, glycine, and glutamic acid. The recovery of serine could be increased from 20 to 80%. Compared to the often-applied extraction temperature of 100 °C, shown in Table 12, this lower extraction temperature should be preferred to preserve carbohydrates and amino acids. Thus, sonication appears to be an excellent compromise to ensure mild extraction conditions. Nevertheless, it should be mentioned that sonication is not without risk for the sample. These pulsed ultrasonic waves involve a cavitation phenomenon that creates tiny bubbles followed by their implosive collapse. Local temperatures and pressures can reach values as high as 5200 K and 500 atm 265 , and 1900 K at the bulk-cavitation bubble interface. It is also reported that these hot spots can dissociate water, creating hydroxyl radicals 266 that can further oxidize organic compounds.

As a result, compounds can be degraded by thermolysis in the center of the cavitation bubble or oxidized by sonolysis via hydroxyl radicals at the edge of the bubble and in the bulk as these radicals escape the interface 267 . Fortunately, the efficiency of this sonolysis process is low for hydrophilic compounds compared to hydrophobic compounds, making the extraction of amino acids and sugars suitable using

Hydrolysis of meteoritic amino acids

In the case of amino acids, a hydrolysis step is required to cleave the oligomerized fraction and release the amino acids as monomers as mentioned in Table 3. This liquid hydrolysis is usually performed using concentrated 6 M HCl. Introduced by Tsugita et al. 270 , acid-vapor hydrolysis has notable features compared to traditional liquid hydrolysis of peptides. Contamination has been reported to be about 10times lower, with a shorter hydrolysis time but using a higher temperature. Indeed, about 22.5 minutes at 158 °C showed better yields than traditional hydrolysis at 106 °C for 72 h. Because direct contact is made between the sample and the acid in traditional hydrolysis, washing steps are required to remove the acid, which is not the case with acid-vapor hydrolysis. Moreover, trifluoroacetic acid was used in conjunction with hydrochloric acid to better recover hydrophobic amino acids 271 such as isoleucine and valine that are known to be less sensitive to bond cleavage in dipeptides. At first glance, acid-vapor hydrolysis appears attractive but suffers from increased racemization 272 . Racemization of amino acids under conventional hydrolysis conditions is known to occur and is dependent on pH and temperature 273 , but experiments with free amino acids and peptides have shown a more critical effect under acidic vapor conditions. Starting with the free L-enantiomer, the percentage of free D-alanine found in the acid-vapor analysis was 2.7 ± 0.1%, whereas it was only 0.6 ± 0.0% in the liquid-phase hydrolysis. For aspartic acid, 8.5 ± 0.3% is reported versus 3.2 ± 0.0%, respectively, and protein studies showed a similar trend.

In the end, water still appears to be the best extraction solvent for the simultaneous extraction of amino acids and sugars. It is also more appropriate to use it as a pure solvent to decrease the risk of contamination and potential disruption of ion exchange columns. Preserving the chemical structure and initial amount of analytes while optimizing their extraction is a major concern and leads to consider sonication to improve extraction under mild conditions (T < 60°C and t < 1 h). In the case of amino acids, a classical hydrolysis should be preferred to avoid increased racemization. If contamination occurs, it will be discussed and compared between proteinogenic and non-proteinogenic amino acids.

CONCLUSION

The investigation of chiral compounds in extraterrestrial samples can be conducted using a powerful tool like GC×GC. Improved separation and enantioseparation can be achieved through column coupling, and improved detectability is achieved through the modulation process, making this technique particularly suitable for trace analysis. The first column used is chiral to allow enantioseparation of derivatives, and cyclodextrins occupy an important place in the separation of amino acids and sugars due to their separation mechanism, which forms diastereomeric complexes with chiral guests. The efficiency of separation and enantioseparation also depends on the derivatization procedure, which has a great impact on other parameters such as volatility, detectability, and stability. It is therefore carefully selected, especially in the case of sugars that can lead to many isomers by intramolecular cyclization. The objective of the next chapter is to focus on the development of an analytical procedure to study ees in extraterrestrial samples through amino acid and sugar derivatization studies, as well as extraction and purification steps.

CHAPTER III

Toward the development of a complete analytical procedure to investigate extraterrestrial and laboratory-produced extraterrestrial samples The general procedure described in Figure 31 was inspired by various previously published papers on the extraction, fractionation, purification, derivatization and analysis of meteorite samples and have been further optimized to meet our research goals. Each step is briefly presented below to provide an overview of the protocol. Sampling -Approximately 1 g of fragment (i.e., meteorite, serpentine procedural blank, etc.) is finely ground using a ceramic mortar and pestle to increase the contact area and facilitate the release of organic compounds. Extraction -The resulting powder (or interstellar ice analog) is hydrolyzed with water at 100 °C for 24 h (for amino acid target analysis) or room temperature for 24 h in a flamesealed ampoule to recover the organic compounds. After cooling and centrifugation, the supernatant is separated from the insoluble solid material. It is then divided into two equal volumes to study free and oligomerized species such as amino acids and sugars separately. The free part does not undergo any additional step before fractionation while the bound part is hydrolyzed in 6 M HCl at 100 °C for 24 h to cleave any oligomeric bonds. Furthermore, the insoluble part is also hydrolyzed in HCl under the same conditions as mentioned above in order to better harvest the trapped organic compounds. Purification & fractionation -For each free, bound and insoluble part, sugars (S) and amino acids (AA) are separated by cation exchange chromatography via a sulfonated poly(styrene-co-divinylbenzene) resin. In its original H + form, the resin readily binds positively charged molecules such as amino acids, metal cations and other salts allowing purification of these fractions. Derivatization -Sugars and amino acids are made volatile and suitable for GC analysis through two-step derivatizations. GC×GC analysis -AAs are derivatized with the MeOH/TFAA method and analyzed on a Lipodex E column while sugars are derivatized with the MBA/TFAA method and analyzed on a CP-Chirasil-Dex CB column. These methods and columns were selected for their selectivity, separation, enantioseparation and reliability.

DERIVATIZATION & GC×GC ANALYSIS

3.2.1 MeOH/TFAA derivatization of amino acids

Materials and methods

Reagents

Standards and reagents used in this study were purchased from Sigma-Aldrich, Fluka, or Acros Organics.

The purity of DL and L amino acids was greater than 98% in all cases. Water used in all steps of the study -tool cleaning and sample processing -was obtained using a Milli-Q Direct 8 apparatus (18.2 MΩ.cm at 25 °C, < 5 ppb total organic carbon). All glassware was washed several times with ethanol and Milli-Q water, wrapped in aluminum foil, and then heated to 500 °C for at least 5 h to remove any organic contaminants. PTFE-lined lids and caps were washed in the same manner. Pipette tips and inserts of GC×GC vials were used without further cleaning.

To prepare the amino acid stock solution, individual amino acids were weighed, mixed, dissolved in Milli-Q water, and then merged into a single solution. A different amino acid stock solution was also prepared for the ee experiments using only 10 amino acids for simplicity. The concentration of amino acids was corrected depending on the purity of the standard.

Derivatization

Diluted series of the stock solutions were transformed into N-trifluoroacetyl-O-methyl ester derivatives following the procedure reported by Fox et al. 225 with some modifications. A volume of 50 µL of the aqueous amino acid solution was taken from a Reacti-vial® and dried under a gentle stream of nitrogen.

Then, 200 µL of a MeOH/AcOCl solution (4:1, v:v) was added and the reaction mixture was stirred for 10 seconds and heated to 110 °C for 1 hour. The mixture was then cooled and dried under a stream of nitrogen. At this point, the sample was not completely dried, but about 1 -2 µL remained to minimize the loss of the most volatile compounds after the first derivatization step. Next, 200 µL of a DCM/TFAA solution (4:1, v:v) was added, the reaction medium was stirred for 10 s, and then heated at 100 °C for 20 min. The solution was cooled again and then dried by a stream of nitrogen. Care must be taken to use a very gentle nitrogen stream to avoid over drying and losing important volatile compounds. Finally, the residue was dissolved in 50 µL of CHCl3 containing 10 -5 M methyl laurate as an internal standard and transferred to 1 mL GC vials equipped with 100 µL inserts for enantioselective GC×GC analyses.

GC×GC-TOFMS

Enantioselective analysis was performed using a GC×GC Pegasus IV D instrument coupled to a time-offlight mass spectrometer (LECO, St Joseph, Michigan, USA). The MS system was operated at a storage rate of 150 Hz, with a mass range of 50-400 amu. The detector voltage was set at 1650 V, or 1800 V for the method detection limit (MDL), with a solvent delay of 15 minutes. The temperature of the ion source and injector was 230 °C. A Lipodex E column (25 m × 0.25 mm, 0.12 µm film thickness, Agilent-Varian, Santa Clara, California, USA) in the first dimension coupled with a DB Wax column (1.4 m × 0.1 mm, 0.1 µm film thickness) in the second dimension were used. Modulation between the columns was provided by a twostage thermal jet modulator using liquid nitrogen. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. Samples were injected in splitless mode, and the MS transfer line was maintained at was held at 40 °C for 1 min, then increased to 80 °C at a rate of 10 °C/min and held for 10 min, then heated

to 125 °C at a rate of 1 °C/min, followed by a rate of 2 °C/min to 190 °C and held for 1 min. The secondary oven used a temperature offset of 30 °C. The modulator temperature was set 15 °C higher than the secondary oven. Depending on the purpose of the experiment, samples were injected multiple times to calculate peak areas with reliable statistical error bars. ChromaTOF TM software from LECO Corp. was used to quantify the analytes. The software integration was manually changed, when necessary, to correct the automatic data processing by the software.

Identification of amino acids

As derivatization has already been reported in the literature using the Lipodex E column, the elution orders allowed for the identification of several amino acids. However, identifying unreported peaks without access to their mass spectra is a more difficult task. In order to assign each peak to an amino acid, each amino acid was derivatized and injected independently to get access to its mass spectrum and retention time.

Mass spectrum & fragmentations

Unfortunately, mass spectral databases and articles were not very helpful in terms of availability of mass spectra to identify all of the investigated amino acids. After identifying each amino acid, a comprehensive list of their mass spectra was made and is available in Table S 1 (p. 157). The interpretation of the mass fragmentations of the methyl ester of N-trifluoroacetyl amino acids has been little described, although some unusual fragmentation pathways with this derivatization have been reported 258 . Figure 32 shows the mass spectrum and fragmentation of alanine. Since alanine is a well-studied amino acid, a detailed explanation of the fragmentation pattern is proposed.

Common fragmentation of derivatives involves

Overall resolution & enantioseparation

Figure 33 shows that enantioseparation is achieved for a large portion of the amino acids and that good overall resolution can be obtained using the chosen derivatization method in combination with enantio-GC×GC-TOFMS. Difficulties in resolving L-Ala from D-2-ABA as well as L-3-Aib/D-3-Aba/β-Leu and β-Leu/D-Ser are unavoidable even after program optimization. Each amino acid was injected individually to allow identification. The order of elution of the enantiomers is based on the work of Fox et al. 225 but was confirmed for Ala, Asp, Val, 2-Aba, Leu, Thr, Nva, Ile , Iva, and Pro using a solution with 5% eeL. Most of the time, the D-enantiomer elutes before the L-enantiomer. The α-methylated amino acids tend to have a reversed order as pointed out by Fox et al. 225 and verified for Iva. This reversed elution order was also observed for Pro -which is a secondary amine forming a pyrrolidine ring on the α-carbon. Coelutions of some analytes of interest can be avoided using a CP-Chirasil-Dex CB column, as shown in acids on the Chirasil-Dex compared with the Lipodex E column, it is most likely that they are hidden in the solvent delay. Overall, these 2 sets of columns appear to be complementary to achieve complete enantioseparation of the 30 amino acids investigated. A summary of N-trifluoroacetyl methyl ester amino acid derivatives in terms of characteristic fragment ions, retention time and method detection limit are reported Table 13. Asparagine turns into aspartic acid during the derivatization, as well as glutamine to glutamic acid.

Repeatability

The repeatability of the derivatization was evaluated for nine replicate samples at 5×10 -5 M injected once.

The relative standard deviation (RSD) for each amino acid is shown in Table 14. For most amino acids the RSD was found to be between 3 and 7%, although high values, above 30%, were found for both enantiomers of 2,3-Dap, Thr and, in particular, for Met (118.54 & 114.07%). The average mean values (Ms)

of Thr and Met are rather small with 0.05 and 0.22, respectively, explaining the relative increased RSD values, whereas the high RSDs observed for 2,3-Dap are more puzzling. Indeed, 5-Ava with a Ms value of 0.64 similar to the one of 2,3-Dap exhibits a relative standard deviation of only 8.93%. 2,4-Dab, which also contains 2 amine groups like 2,3-Dap has a standard deviation of only 4.11%. In addition, Cys is detected randomly and is therefore not investigated further. This implies that the MeOH/TFAA derivatization for amino acids containing sulfur, hydroxyl group or 2 amino groups is not robust, and therefore less suitable.

Nevertheless, these results testify that for most amino acids, MeOH/TFAA derivatization seems reliable despite the risk of compound loss due to the high volatility of the synthesized derivatives. Indeed, Iva and 2-Aib are the first compounds to elute and show standard deviations of 3-4% and 8.68% respectively.

Linearity

The calibration curves for the amino acids show similar sensitivity given the slope values in Table 15.

Indeed, for each enantiomer except phenylalanine, the same order of magnitude is reported. This means Data obtained using 4 concentrations: 5×10 -5 M, 5×10 -6 M, 5×10 -7 M, and 5×10 -8 M. For each concentration, 3 samples were prepared and analyzed.

that derivatization does not discriminate amino acid conversion, which is convenient especially for Iva and 2-Aib, which are priority targets in the analysis of extraterrestrial samples. In addition, the coefficient of determination R 2 is generally greater than 0.999 although three orders of magnitude of concentration are used. This suggests a wide range of applications of the method. The R 2 is used as an indicator of linearity with visual observation of the residual plot. For each amino acid, although the number of data points is small -four concentrations injected each 3 times -the residual plot appears to

show uniformly scattered values around the x-axis and thus no bias in the method. The alanine residual plot is available in Figure S 1 (p. 162).

Limit of detection

Despite its clear definition, the practical way to determine the limit of detection (LOD) is controversial and ambiguous because several methods and terms can be confused. The LOD is often estimated from a single sample measurement where the peak intensity is three times above the background noise. However, because the detector response to an analyte is Gaussian in shape, it is necessary to account for measurement fluctuations and confidence intervals. The method detection limit (MDL) is therefore more appropriate for determining the LOD, especially when very low concentrations of the analytes are of interest. This is because by using this method, fluctuations in the response of the instrument and the derivatization procedure are taken into account. The MDL should not be confused with the instrument detection limit (IDL) where multiple re-injections of a sample are performed (IDL) instead of a single injection of multiple samples (MDL). Here, splitless mode is used to provide the best injection for trace analysis, and the use of an internal standard to correct for injection fluctuations. Statistically, since only a set of samples from the population is taken -meaning that only a few measurements are made -the mean and standard deviation values are only estimates. In the case of the detection limit where the fluctuations are of interest -which means that the standard deviation is of interest -one way to establish a confidence interval of the standard deviation is to use the values of the one-sided students' tdistribution. The MDL is expressed as follows in Equation 4. MDL = 𝒕 𝛂 𝑺 𝐬 (in counts or area) leading to MDL = 𝒕 𝛂 𝑹 𝐬 𝑸 𝐬 𝟏𝟎 -𝟐 (in g or in M) Equation 4tα is the n-1 one-sided Students' t-distribution value associated to the confidence interval α (no unit)

Ss is the standard deviation of the n analyzed samples (in counts or area)

Rs is the relative standard deviation of the n analyzed samples (in %)

Qs is the theoretical analyzed quantity (in g) or concentration (in M) Another method, using calibration curve data -based on the standard deviation extrapolated from the y-intercepts of the regression line -can provide an MDL value but also requires the preparation of several samples. Estimating a LOD using MDL is time consuming but more relevant for trace analysis. The concentration chosen to determine the MDL should be close to the MDL itself, with a peak intensity at least 10 times higher than the background. Since 30 amino acids are used in this study, the MDL of all these amino acids is chosen to be calculated without necessarily optimizing the intensity of each peak. It should be kept in mind that the MDLs displayed here may be overestimated or underestimated because it is not possible to optimize the concentration for all amino acids. The results of the MDLs are shown in Table 13. Reagent blanks were performed using TFAA ampoules before performing MDL injections. Only potential trace amounts of alanine were found when reagent blanks were analyzed at high detector voltage (1800 V), indicating that the TFAA ampoule is suitable for trace analysis.

A set of 8 separate experiments was performed to have a statistically relevant MDL. Thus, the tα value for 7 degrees of freedom and 99% confidence was set to 2.998. The values of Rs are summarized in Table 16.

Qs is the quantity -near the MDL -used to determine the MDL. As an example, injecting 1 µL of a 5×10 -

CONTAMINATION

In this study, experiments conducted to determine the detection limit of alanine showed that both the D and L forms of alanine were found, with a significant enantiomeric excess of the L enantiomer, as well as a very low detectability. This led to the consideration that contamination was likely. Free or polymerized alanine was investigated in methanol, acetyl chloride, dichloromethane and TFAA by derivatization of N(O,S)-ethoxy-carbonylheptafluorobutylester (ECHFBE). Since the detected alanine was fully derivatized, the first step reagents, methanol (MeOH) and acetyl chloride (AcOCl), were investigated for potential traces of free or polymerized alanine. 2 mL of each reagent were dried and derivatized to look for free alanine. Moreover, 2 mL of each reagent were dried and then hydrolyzed for 1 hour using a mixture of 200 µL H2O/AcOCl (4:1, v:v) at 110 °C to cleave potential traces of polyalanine followed by the ECHFBE derivatization. H2O/AcOCl, like MeOH/AcOCl, forms hydrochloric acid (HCl) in situ which is required for the first esterification step of the MeOH/TFAA derivatization. The ECHFBE derivatization, which has been

shown not to contain alanine (Table 17), was used. Indeed, the blank of the ECHFBE reagents showed no alanine presence, meaning that any alanine contamination from these reagents does not fall within the detection range of the instrument. Surprisingly, neither MeOH nor AcOCl revealed free or polyalanine after derivatization (Table 17). The same search for free and polyalanine was performed on DCM and TFAA. The results showed that hydrolyzed TFAA contains a huge excess of L-alanine while unhydrolyzed TFAA shows no trace of alanine.

As expected, other proteinogenic amino acids such as phenylalanine, methionine or valine were found while N-ethylglycine -a non-proteinogenic amino acid -for example was not detected. It is interesting to note, that it is in the second step of the MeOH/TFAA derivatization -at 100 °C for 20 minutes -that alanine is released. For this to happen, an acidic medium is required. Since the reaction medium from the first step is very difficult to remove -it contains methanol, acetyl chloride, HCl, and methyl acetate -it is likely that HCl is still trapped in the remaining droplets and/or on the glass walls of the reaction vial and capable to hydrolyze alanine present in TFAA. Another explanation would be the presence of trifluoroacetic acid due to the reaction of TFAA and water in the atmosphere.

QUANTIFICATION OF ALANINE IN TFAA

Estimation of TFAA contamination was performed using an ECHFBE calibration of alanine, Figure 35. Five standards containing 10 -5 , 5×10 -6 , 10 -6 , 5×10 -7 and 2×10 -7 M of each enantiomer of alanine were used. The calibration curve is shown below and includes hydrolyzed TFAA1. Three different grades of TFAA were studied and analyzed at a high detector voltage of 1800 V. A few milliliters of each TFAA were dried, hydrolyzed as described above, and derivatized with ECHFBE. The important information is the order of magnitude of the contamination, which ranges from 10 -8 to 10 -10 M depending on the grade, Table 18. It is interesting to note, that the lowest contamination is obtained using TFAA in ampoules. ,2,3,3,4,4,4-heptafluorobutanol), the actual concentration of L-alanine in TFAA1 is calculated by Equation 5.

[L-Ala] TFAA 1 = [L-Ala] TFAA sample × V Analyzed V TFAA = 3.7 × 10 -7 × 0.069 4.7 = 5.4×10 -9 M Equation 5
This contamination report is only an estimate of the actual alanine contamination because some of the concentrations were calculated by extrapolation to concentrations outside the linear range of the calibration curve.

OTHER PROTEINOGENIC AMINO ACID CONTAMINATION

Other amino acids were investigated, particularly in the most contaminated TFAA2 where they are easier to detect. The presence of proteinogenic amino acids such as alanine, glycine, phenylalanine, glutamic acid, isoleucine, leucine, aspartic acid, proline was confirmed. We believe it is similar for the TFAA3 (ampoule), because at least some of these amino acids including phenylalanine, alanine, glycine, and aspartic acid were found. Those amino acids are however not found when using the required 200 µL of DCM/TFAA (4:1) (v:v).

Enantiomeric excess conservation

The enantiomeric excess (ee) is defined as the excess of one enantiomer over the other in a mixture of enantiomers. Thus, racemic mixtures have an ee equal to 0% while enantiopure compounds have an ee equal to 100%. In practice, when determining an ee using GC×GC, two limitations can be highlighted. The first concerns the purity of the standard used, which may be due either to the manufacturer's ee determination or to the evolution of the standard's ee over time. The second concerns artifacts and instrument limitations. Response variations between enantiomers, co-eluting impurities, lack of enantioresolution, or peak tailing can lead to an inaccurate determination of the absolute ee of the sample. Thus, investigations of an apparent 5% eeL using GC×GC was conducted by comparing the response of the racemic standard against a standard solution doped with 5% of the L-amino acid enantiomer. To determine the ees reliably and account for procedural and measurement fluctuations, 3 samples were derivatized and injected 3 times each.

Generally speaking, increasing the concentration leads to increased accuracy of the determined ee, as measurement fluctuations and integration errors are reduced. Thus, the S/N ratio is an important parameter that correlates with the determination of ee accuracy. Figure 36, reports the evolution of eeL at four different concentrations and for 5 amino acids: 2,3-Dap, Ala, Glu, Iva and Pro. As expected, the standard deviation tends to be reduced at higher concentrations and ranges between ± 0.45% and ± 1.07%

for the amino acids studied at 10 -5 M. The accuracy of the ee at this concentration, on the other hand, is greater than 93.4% (Ala) for the amino acids studied except for Glu, with 82.8%. As calculated in Table 3 (p. 24), the concentrations of amino acids in 1 g of the Murchison meteorite are between 10 -5 and 10 -4 M and are therefore sufficient for the accurate and precise determination of potential ees in 2,3-Dap, Ala, Iva and Pro. The most striking results are the huge standard deviations and lack of precision at 10 -7 M, which is close to the detection limit of the method. Instead of obtaining eeL of about 5%, negative values can be obtained for Ala and Glu, as well as standard deviations of up to ± 9.20% for 2,3-Dap.

Stability

Stability regarding decomposition and/or evaporation of the derivatives was studied after 7 days. The derivatized sample was stored in a refrigerator at 4 °C. Three injections were performed before and after 7 days. Overall recoveries were generally between 80 and 100%, as shown in 5.9 a Recoveries: ratio between samples at day 7 over day 0. b Samples analyzed three times at day 0, and three times at day 7. ND -Not detected.

Conclusion

MeOH/TFAA derivatization proves to be a convenient and powerful procedure to separate and enantioseparate most of the 30 amino acids used in this study when coupled to a GC×GC apparatus using a Lipodex E column in the 1 st dimension. The utility of GC×GC is highlighted by the resolution of the 2 nd column which avoids coelution of target analytes, outlying derivatization byproducts and lowers the detection limits of the method. For most amino acids, the following data are reported: method detection limit in the picogram range, relative standard deviation between 3-9%, standard deviation of enantiomeric excess in the 1% range at 10 -5 M, and stability of derivatives after 7 days of 80-100%. Linearity is also evaluated over several orders of magnitude, from 5×10 -8 M to 5×10 -5 M. In addition, the first comprehensive listing of the mass spectrum of the N-trifluoroacetyl amino acid methyl ester allows for unambiguous identification of each amino acid. To compensate for the lack of resolution of a few targeted amino acids, a Chirasil-Dex column can be successfully used to complete the data set. As the derivatives have proven to be stable, it may be convenient and worthwhile to switch columns to obtain a thorough separation of targeted derivatives. This derivatization can find many applications in bioanalytical science but aims to be employed for extraterrestrial samples because of the previously mentioned advantages.

MBA/TFAA derivatization of sugars & sugar related compounds

Materials and methods

Reagents

Standards and reagents used in this study were purchased from Sigma-Aldrich, Fluka, or Acros Organics.

The purity of DL and L sugars was greater than 98% in all cases. Water used for tool cleaning and sample processing was obtained using a Milli-Q Direct 8 apparatus (18.2 MΩ.cm at 25 °C, < 5 ppb total organic carbon). All glassware was washed several times with ethanol and Milli-Q water, wrapped in aluminum foil, and then heated to 500 °C for at least 5 h to remove any organic contaminants. PTFE-lined lids and caps were washed the same way. Biopur pipette tips and GC×GC vial inserts were used without further cleaning. To prepare the sugar stock solution, individual sugars were weighed, mixed, dissolved in Milli-Q water, and then merged into a single solution. The concentration of the sugars was corrected depending on the purity of the standard.

Derivatization

Diluted series of the sugar stock solutions were dried under a gentle flow of nitrogen until the water was completely removed. 50 µL of a 10 mg/mL methylboronic acid solution in pyridine was added, stirred for 10 seconds, and held at 60 °C for 30 minutes. The samples were then allowed to cool to room temperature for 2 minutes and carefully dried under nitrogen for approximately 40 to 50 minutes. 50 µL of a 1:1 (v:v) mixture of trifluoroacetic anhydride and ethyl acetate was added, stirred for 10 seconds and held at room temperature for 1 h. The samples were finally thoroughly dried under nitrogen and dissolved in 50 µL of ethyl acetate containing 10 -6 M methyl myristate as an internal standard.

GC×GC-TOFMS

Enantioselective analysis was performed using a GC×GC Pegasus IV D instrument coupled to a time-offlight mass spectrometer (LECO, St Joseph, Michigan, USA). The MS system was operated at a storage rate of 100 Hz, with a mass range of 25 to 400 amu. The detector voltage was set to 1650 V with a solvent delay of 12 minutes. The temperature of the ion source and injector was 230 °C. A CP-Chirasil-Dex CB column (24.55 m × 0.25 mm, film thickness 0.25 µm, Agilent-Varian, Santa Clara, California, USA) in the first dimension coupled with a DB Wax column (1.55 m × 0.1 mm, film thickness 0.1 µm) in the second dimension were used. Modulation between the columns was provided by a two-stage thermal jet modulator using liquid nitrogen. Helium was used as the carrier gas at a constant flow rate of 1.2 mL/min.

Samples were injected in splitless mode, and the MS transfer line was maintained at 240 °C during analyses. A modulation period of 5 s was applied. The temperature of the primary column was held at 55 °C for 1 min, then increased to 85 °C at a rate of 10 °C/min and held for 8 min. It was then increased to 125 °C at a rate of 2 °C/min, then at a rate of 2 °C/min to 170 °C. It finally reached 190 °C at a rate of 10 °C/min and was held at that temperature for 5 min. The secondary oven used a constant temperature offset of 25 °C. The modulator temperature was set 15 °C higher than the secondary oven. Depending on the purpose of the experiment, samples were injected multiple times in order to calculate peak areas with reliable statistical error bars. ChromaTOF TM software from LECO Corp. was used to quantify the analytes.

The integration range during quantification was manually changed, when necessary, to correct the automatic data processing by the software.

Identification of sugars and sugar related compounds

Previous experiments performed in the team allowed to easily identify most sugars as methylboronic ester derivatives thanks to their mass spectra and retention times such as glyceraldehyde, arabinose, xylose, ribose and lyxose. However, several C6 sugars such as glucose and galactose are not detected because of their free hydroxyl group. Interestingly, this is not necessarily the case for erythrose and 2-deoxyribose which also have a free hydroxyl group. Both, however, have lower detection limits, by about an order of magnitude. One solution to overcome this difficulty is to use acylation as a second derivatization step.

This step significantly decreases the intensity of the derivatives using MBA only but could provide access to other classes of compounds such as sugar alcohols. However, this specific method has not been explored for this purpose, but it can be mentioned that S. Manhas et al. 260 successfully used phenylboronic acid on glycerol, xylitol, ribitol, and L-arabitol to selectively protect diols for selective Fischer esterification.

As pointed out by Van Dongen et al. 204 using MBA/BSTFA for sugars, the difference in yield between monosaccharides could be due to the distance between the hydroxyl groups involved by the stereochemistry of the molecules 261 . However, it seems that this stereochemistry leads to a unique sugar derivative because the authors report the appearance of several byproducts with prolonged reaction times with BSTFA. It then becomes clear that sugars and polyols with an even number of alcohols cannot necessarily be fully derivatized with MBA alone because the distance between the alcohols may be different depending on the alcohol conformer. They could be fully derivatized from MBA, fully derivatized from TFA, or lead to many MBA/TFA possibilities. This results in difficulties when trying to identify them using their mass spectra. Finally, sugar acids such as glyceric acid, threonic acid, erythronic acid, and gluconic acid were derivatized using MBA and MBA/TFAA but not detected. The mass spectra of the studied samples are available in Table S 2 (p. 163), and data on the characteristics ions, and retention times are reported in Table 20.

Sugars

Glyceraldehyde, ribose, arabinose, xylose, and lyxose were previously identified as MBA derivatives, whereas galactose, glucose, and mannose were previously identified as MBA/TFA derivatives.

Interestingly, lyxose showed two distinct peaks corresponding to either two MBA rings or an MBA ring with two TFA groups. A significant D-enantiomeric excess was used to confirm these two peaks. Van Dongen et al. 204 reported only the latter while both coexist. It would seem natural for xylose to have the same two peaks, but only one form with two MBA rings was observed. This reinforces the scenario in which the conformation of the molecule plays a major role for the derivatives formed. Erythrose and 2deoxyribose were identified as MBA derivatives, but the remaining alcohol would now be a TFA group thus changing the retention time and mass spectrum. Compared to the MBA derivative, 2-deoxyribose showed an intense m/z = 69 characteristic of TFA, but also two other ions, 140 and 224. The ion 85, on the other hand, has faded. In the case of erythrose, the fragmentation pathways changed significantly with the addition of TFA. The only remaining common ion is m/z = 85 and it is now faint compared to m/z = 43, 69 and 97.

Sugar alcohols

Glycerol, ethylene glycol, threitol, erythritol, arabitol, xylitol, adonitol, sorbitol, dulcitol, dihydroxyacetone, and glycolaldehyde dimer were studied. Ethylene glycol was not found but could elute very early, which explains its absence. Dihydroxyacetone and glycerol were only found as TFAA derivatives, which could be explained by the lack of conformational hindrance preventing MBA from derivatizing the two alcohol groups. Surprisingly, glycolaldehyde was found as a dimer and derivatized by MBA. As a dimer, its structure is very similar to that of glycerol, and one would expect similar reactivity.

Its mass spectrum is close to glyceraldehyde with major fragment ions 43, 113 and 85, but their m/z = 113 fragmentation is different.

The structure of the following sugar alcohol derivatives is not fully elucidated but can be described in sugar alcohols (the 88 ion is replaced by the 140 ion) but also show strong m/z = 84, 85, and 98 ions. Since the 85 and 97 ions are strongly represented in these C5 sugar alcohols, it can be assumed that these compounds are derivatized as two 5-atom MBA rings and a TFA group. Interestingly, adonitol revealed the presence of 2 peaks with a similar mass spectrum reminiscent of a pair of enantiomers. Since it is a meso compound, the presence of MBA rings connecting the hydroxyls is likely to result in optical stereoisomers. The C6 sugar alcohols, sorbitol and dulcitol, each show two peaks. The mass spectra of both sorbitol peaks are similar, while one of the dulcitol peaks resembles that of sorbitol and the other is faintly reminiscent of the C4 sugar alcohols. The major fragments are m/z = 85, 97 and 43 and no m/z = 69 is observed. This suggests that all hydroxyls are derivatives of MBA. It is not clear whether the derivative consists of two 6-atom MBA rings and one 5-atom MBA ring or three 5-atom MBA rings. However, in the case of sorbitol, the first peak has stronger m/z = 43 and 97 and is therefore more likely to be representative of two 6-atom MBA rings and one 5-atom MBA ring, as fragmentation would more easily lead to a charged 6-atom MBA ring with m/z = 97. In the case of dulcitol, these mass spectral characteristics are represented in the second peak. Concerning the first peak of dulcitol, no conclusion can be drawn on the structure of the derivative but the presence of at least 2 TFA groups given the intensity of m/z = 69 is suggested.

Interestingly, all sugar alcohols showed a significant tailing and peak width in the chromatogram, which increases with carbon number. At first glance, this should indicate the presence of underivatized hydroxyl(s) leading to undesired interactions with the capillary column. However, the relatively high intensities of m/z = 69 in some sugar alcohols (arabitol, erythritol, xylitol, adonitol) tend to consider the derivatives as TFA derivatives (apart from MBA) which is more consistent with the observed fragmentations. Then, the reasons why sugar alcohols tail remain unknown. Investigations to determine a more accurate structure could be performed at a lower ionization energy. Soft ionization could allow for better observation of higher m/z including the molecular ion.

Overall resolution and enantioseparation

Figure 37 shows that enantioseparation is achieved for all the compounds studied, and that a good overall separation can be obtained using the program provided. The only difficulty was found for the separation of fructose and dulcitol (peak 2), but this can be overcome by selecting an appropriate m/z for quantification. Regarding the order of elution, a certain trend can be noticed for erythrose, arabinose, ribose, and glucose, which share the same carbon configuration (except for the penultimate one) and whose L-enantiomer elutes before the D-enantiomer (Figure 15, p. 47). Similarly, xylose, lyxose (peak 1)

and galactose have the D-enantiomer eluting before the L-enantiomer. In addition, the two ketoses, fructose and ribulose, show the same elution order (Figure 16, p. 47). Unfortunately, more data would be needed to confirm such a trend. As mentioned earlier, sugar alcohols are generally tailing, which can generate additional coelutions with non-target compounds. Moreover, the detectability and accuracy of the integrated area is reduced. Calibration curve data is available Table S 3 (p. 167).

Conclusion

Although this method has not been studied as extensively as the MeOH/TFAA method described previously, several advantages can be highlighted. The separation and enantioseparation of sugars and sugar alcohols is efficient, except for fructose and dulcitol. The derivatization temperature is quite low with 60 °C and identical to the (S)-BuOH/TFAA method, which minimizes potential racemization and degradation of sugars. Sugars, sugar alcohols, and deoxy sugars can be detected and quantified with this method, which is not the case for sugar acids. However, many sugar alcohols suffer from a significant tail in the first dimension that could prevent detection and/or quantification. This method was specifically designed for the study of sugars and sugar alcohols synthesized under hydrothermal conditions in a formose-type reaction (4.2 Mineral-catalyzed formose reaction under hydrothermal conditions). It can also be applied to extraterrestrial samples but suffers from higher limit of detections for C3 to C5 sugars compared to other sugar derivatization protocols.

Materials & Reagents

The ion exchange chromatography (IEC) resin used was a BT AG® 50W-X8 biotech grade, 100-200 mesh in the H + form from Bio-Rad. A Supelco Visiprep 12 DL vacuum manifold, and a KNF Laboport N 86 pump were used to elute the IEC solvents through disposable PTFE flow control valve packings from Supelco.

Glass tubes (6 mL) and PTFE frits with 20 µm porosity from Supelco were used to retain the IEC resin.

Duran 10 mL pear-shaped vials, a splash-proof adapter with return hole from Aldrich were used to recover and concentrate the IEC eluates using a Büchi R-114 rotary evaporator and a B-480 water bath, as well as a Huber minichiller and a Vacuubrand PC 500 series pumping unit (and CVC 3000 vacuum regulator).

Hydrochloric acid (ACS reagent ≥ 37%) from Sigma, ammonia (Ph. Eur. reagent 25% or 28 -30%) from Supelco, and ethanol (Bioultra ≥ 99.8%) were used to condition the resin and/or elute the analytes.

Water used for tool cleaning and sample processing was obtained using a Milli-Q Direct 8 apparatus (18.2

MΩ.cm at 25 °C, < 5 ppb total organic carbon). All glassware was washed several times with ethanol and Milli-Q water, wrapped in aluminum foil, and then heated to 500 °C for at least 5 h to remove any organic contaminants. PTFE-lined lids and caps, plastic wire, and spatula were washed in the same manner.

Pipette tips and GC×GC vial inserts were used without further cleaning.

Column preparation & conditioning

About 1 g of resin was weighed into a clean beaker and poured with ethanol into a glass tube containing a teflon frit at the bottom. The beaker was rinsed several times with ethanol to recover as much of the resin particles as possible. The walls of the glass tube were cleaned, and excess ethanol was eluted under vacuum until the meniscus reached 0.5 cm above the resin bed. Approximately 10 ml of ethanol was used for the entire process. Bubbles in the resin were removed by stirring the resin/ethanol mixture for approximately 30 seconds with a spatula. The second Teflon frit was then added to the top of the resin to avoid disrupting the resin bed during further elution. Using a test tube, the top Teflon frit was lightly pressed toward the resin to remove bubbles.

The resin was then conditioned as follows: 4 bed volumes (BV) of 1 M HCl solution, 4 BV of Milli-Q water, 4 BV of 2 M NH4OH, 4 BV of Milli-Q water, 4 BV of 1 M HCl, and 4 BV of Milli-Q. The pH was monitored to be neutral after the last Milli-Q washing step. During the first HCl rinse, the recovered solution had a brown tone. The prepared columns were used only once for each experiment.

REMARKS

♦ In theory, performing the first two conditioning steps (HCl followed by Milli-Q water) should be sufficient to clean, regenerate and equilibrate the column before introducing the sample. However, it would be preferable to simulate a complete elution in order to remove any trapped compounds that would only elute with NH4OH.

♦ Ethanol is used instead of Milli-Q water to wet the Teflon frits and avoid additional bubbles, as Teflon is very hydrophobic. This also allows for additional washing with a less polar solvent.

♦ The bed volume is the volume of the cylinder containing the resin, calculated by the following equation: BV = π × r² × h with r and h the radius and height of the cylinder, respectively. Note that the resin beads are swollen with solvent, so BV is defined as the volume occupied by the resin plus the solvent.

♦ 4 BV are used to avoid any loss of compound during the process. The cleaning and conditioning were therefore performed in the same way. As the elution is performed manually, the reproducibility can be affected by several parameters such as flow rate, flow variations and the presence of fine bubbles.

♦As with all chromatographic techniques, injection plays an enormous role. A thin injection band is always sought after because it improves separation and detectability. Here, the additional aim was to avoid loss of analytes by dilution in the solvent fraction, Figure 38. Then, care must be taken to add the solvent dropwise during the elution process: the elution flow must be properly adjusted using the vacuum pump to allow dropwise addition without drying out the resin bed.

♦ Handmade columns were preferred over packaged columns because they are made of glass and can be washed and heated in a furnace to reduce any risk of contamination.

♦A slight color change of the resin from dark brown to light brown is observed when the elution solvent is NH4OH instead of water. It is likely that the counterion acts as an auxochrome and changes the chromophore properties of the resin.

♦Originally, a longer column (≈ 25 -30 cm) with an embedded glass frit (porosity 0: 160 -250 µm) was used, but the frit caused constantly column breakthroughs that could disrupt the subsequent derivatization. Moreover, this column size was not appropriate since only about 1 cm height was used for the resin. It is therefore less ergonomic and increases the risk of contamination. However, the use of smaller glass tubes usually requires a vacuum device to speed up the elution process.

♦ Calculations to compare the resin capacity and the content of the sample can be performed to ensure that the amino acids can be effectively filtered. Since the resin has a nominal capacity of 1.7 meq/mL 186 , 1 eq = 1 mole/valence, and assuming that amino acids have a valence equal to 1, 1 L of resin can hold up to 1.7 moles of amino acids, i.e., in our case, 1.25 mL (1 g resin with a nominal density of 0.8 g/mL) can hold up to about 2.13×10 -3 mole. If we consider the worst case for the resin, i.e., all amino acids studied (≈30) have a content of 10 -3 M (approximate concentration of glycine in 1 g Murchison) in 50 µL, the total amount is about 10 -3 ×50×10 -6 ×30 = 1.5×10 -6 M, which is about 3 orders of magnitude lower than the capacity of the resin. This is far from saturating the resin used, but it should be kept in mind that other positively charged compounds, such as ionic metals and nitrogen-bearing compounds from the extraterrestrial samples, will be retained and thus contribute to saturate the resin. Nevertheless, calculations are made using the concentration of glycine, which is by far the most abundant of all the meteoritic amino acids. Saturation of the resin should therefore not occur.

Sample purification and fractionation

After column conditioning, water was eluted until the meniscus was slightly above the level of the Teflon frit. A 10 mL pear-shaped flask was placed under the column to collect the upcoming sugar fraction. Then 50 µL of 5×10 -6 M sugar standard mixture and 50 µL of 5×10 -6 M amino acid standard mixture were gently added to the solvent section. Elution was performed using the vacuum pump, and the vacuum was manually controlled using a vent button. The elution rate was estimated to be between 10 and 20 cm/min, which is slightly faster than the 5 cm/min recommended in the column user manual 186 . After the dropwise addition of 4 BV of water to recover the sugar fraction, elution was stopped, and a new flask was placed to recover the amino acid fraction. After the dropwise addition of 4 BV of 2 M NH4OH, elution was stopped and both vials were stored and covered with aluminum foil after elution. As approximately 10 ml were recovered, a pre-concentration step of the fractions was performed using a rotary evaporator at 40 °C.

Several vacuum steps were arranged at 500, 300, 100, 50, 25 and 10 mbar to avoid boiling (bumping) and thus loss of analyte. Although a clean splash adapter was used so that any solvent splash could be collected, this still presents a risk of analyte loss and should be avoided. The eluate was dried but not to complete dryness to minimize sample loss and polymerization reactions. The analytes in the pear-shape flask were recovered with 3×150 µL of water and transferred to a Reactivial®. The water was then evaporated to complete dryness under a gentle flow of nitrogen. Derivatization of the amino acid fraction with MeOH/TFAA was performed after these steps.

Teflon frits

Solvent IEC Resin

Amino acid recoveries

The recovery values shown in Figure 39 are the result of 3 IEC experiments versus 3 reference comparisons at the same 5×10 -6 M concentration. Most recoveries are between 70 and 100%, which is an acceptable loss for this necessary purification and fractionation step. Methionine values are not reported because it was only detected in one sample. Amino acids such as Gly, β-Ala, L-Ala, D-2-Aba, 4-Aba and DL-2,4-Dab show recoveries above 100%. Particularly, β-alanine shows a recovery of 821.3%, which is consistent with an observed important contamination likely coming from the ammonia elution solvent (28 -30%) rather than the resin itself. Follow-up studies using a different grade of ammonia (Ph. Eur. 25% reagent) gave more satisfactory results in terms of contamination. The highest contamination observed was for glycine, in the order of 10 -6 M, while that for proline, phenylalanine, glutamic acid, serine, and threonine was less than 10 -7 M. The 25% grade ammonia is therefore considered to be appropriate for future analysis.

Leu, Ile, allo-Ile, beta-Leu and Nle, like Iva, tend to have lower recoveries than the other amino acids.

Those have the particularity of having a branched carbon side-chain (except Nle). Whether this influences the interaction with the resin and led to decreased recoveries is unsure given the relatively high errors bars in this experiment. Even if the addition of NH4OH releases the amine group from its ionic bond with the sulfonate group, these interactions with the resin could slow down and prevent their elution. Another striking result is the poor recovery of Phe and 2,3-Dap. In the case of Phe, this could be due to a matrix interaction as the aromatic backbone of Phe may involve π-stacking with the divinylbenzene polystyrene sulfonate resin. For 2,3-Dap, its recovery differs notably from that of the homologous diamino acid 2,4-Dab. 2,3-Dap low recovery should be due to the poor repeatability of the derivatization (Table 14, relative standard deviation of 38%) but interestingly shows a low standard deviation of 3.1% here. On the other hand, the standard deviation of 2,4-Dab is relatively high (31.7%) compared to the other amino acids (even in the references) and cannot be explained because the repeatability tests (Table 14) showed a standard deviation of 6.8%, in the same range as the other amino acids.

A majority of amino acids tend to have an L-excess, which generally indicates contamination. However, Pro, and Ile, two proteinogenic amino acids tend to have an excess of D which challenges the contamination hypothesis. In addition, the large standard deviations make these assumptions difficult.

Further experiments would then be required to ensure that the ion exchange resin does not bias the ees.

Table 21 is intended to give an overview of the ee bias that can be observed employing IEC, using Ala and

Iva. Ala appears to be unaffected by the extraction and purification process, while Iva has a significant ee bias. Indeed, an eeL of -7.19% corresponding to a ΔeeL = 13.97% between the reference and IEC is observed for Iva, while the standard deviations are equal to 0.54% (reference) and 1.32% (IEC). This result is particularly important because isovaline is a priority target in meteorite analysis. In addition, large excesses of L-Iva have sometimes been reported by Murchison meteorite analysis 138,262 . A detailed investigation of the GCxGC chromatogram (Figure 40), however, revealed an abundant background noise from the derivatization reagents coeluting with the D-enantiomer of isovaline and thus induced this artificial bias. Sugars, on the other hand, were not studied here but were used in the first experiment (Figure 39). The estimation of the impact of sugars on amino acid recoveries is unfortunately difficult to assess in view of the data obtained. Indeed, using only amino acids, the recoveries of Ala (D: 97.9 ± 10.2%, However, as the recovery of Iva is greater than 100%, any conclusion is difficult to assess despite the reported standard deviations and background noise. Finally, sugars were not studied as thoroughly as amino acids with respect to IEC recoveries and ee bias. However, the following section provides preliminary results on the potential recoveries of sugars.

Early developments

The following section corresponds to previous research work in which alternative derivatization methods for amino acids and sugars were considered. For sugars, (S)-BuOH-TFAA derivatization was considered while for amino acids, ECHFBE was considered. Nevertheless, we assume that the results obtained can be transposed to other derivatizations, as long as this does not change the effect studied itself. The GC-FID was used with the old/larger IEC column configuration.

Recoveries of alanine and ribose

The required ammonia concentration and bed volumes were investigated to achieve high recoveries of alanine and ribose. As shown in Table 22, ribose recovery appeared to be slightly higher using 4 BV than 3 BV, although its value exceeded 100%. On the other hand, alanine was not detected using 3 BV, and only about 1.5% is reported using 4 BV. Thus, 4 BV will be retained, and the NH4OH concentration will need to be increased to elute alanine that is still bound to the resin after 4 BV. To confirm that the resin does indeed bind alanine, an amino acid derivatization was performed on a sugar fraction and revealed no presence or breakthrough of alanine. To ensure that the amino acids can be recovered, an experiment using 14.8 M NH4OH was conducted and revealed high recoveries of about 99.5%. Further experiments led to the final selection of 2 M N4OH as the optimal concentration (usually also used in the literature) and showed, using 4 BV (Table 23), for both compounds and both enantiomers, recoveries of around 95%. 3 references for alanine and 3 references for ribose were made to compare the results with 4 ion exchange chromatography experiments. However, the standard deviations (SD) are around 5% for alanine and 10% for ribose. The high SD for ribose is mainly due to the high SD of IEC, which is not the case for alanine. At this point, it is unclear whether this is from the derivatization itself or from the IEC treatment. Nevertheless, these results tend to be very satisfactory given the use of the IEC resin, rotary evaporator, and manual recovery in the pear-shaped flask.

Furthermore, the change in ees (ΔeeD, the difference in eeD between IEC and reference) induced by the resin for the racemic mixture was investigated but showed no significant difference as this difference is within the standard deviation range. Indeed, a -0.104 ± 0.152 is reported for alanine while 1.00 ± 1.21 is reported for ribose. However, this is not sufficient to conclude that there is no effect on ee. Studies using 5% ee on sugars should be performed to evaluate this effect on samples with effective ee. Interestingly, even though this column configuration led to resin breakthrough, it seems to have no impact on both derivatizations applied, but no general conclusions can be drawn as it could affect other compounds. GC-FID, old IEC setup, 10 -3 M.

Drying impact

The potential loss of sugars due to the drying step before derivatization was investigated. Each result shown in Table 24, corresponds to the relative recovery of a dried 50 µL standard (10 -3 M) of two samples with different drying times derivatized using (S)-BuOH/TFAA. The dried sample corresponds to a 50 µL standard plus 450 µL water, evaporated to complete dryness under a gentle nitrogen stream. The overdried sample corresponds to a dried sample plus a 30 minutes overdrying. Two conclusions can be drawn from these data: the drying step has no impact in terms of ribose degradation, and overdrying does not induce analyte loss due to its volatility.

Table 24. Ribose recovery depending on the drying procedure before derivatization.

Dried sample

Overdried sample Relative recovery (%) 102.6 ± 3.3 102.2 ± 3.9 GC-FID, (S)-BuOH/TFAA, 10 -3 M.

In addition, experiments of potential ribose loss using the rotary evaporator to concentrate the IEC eluate were performed but did not yield conclusive results and are therefore not reported here. However, the recovery results in Table 23 consider the entire IEC process and discards major degradation or loss of analytes.

Conclusion

Ion exchange chromatography, used to purify samples from salts, is also useful for separating a sample into two distinct fractions of amino acids and sugars. This reduces the risk of disruption of subsequent derivatization. Initial experiments have shown, after optimization of ammonia concentration and bed volumes, that recoveries of alanine and ribose, used as model compounds, are above 95 ± 10%.

Nevertheless, comprehensive studies on 30 amino acids have shown that the recovery values can be significantly modified depending on the amino acid, as is it the case for Phe or 2,3-Dap with recoveries of about 60%. Recoveries for other amino acids are generally between 70 and 100%. On the other hand, enantiomeric conservation tests using 5% doped L-amino acids showed no variation for Ala, but a serious reversal for Iva as a ΔeeL of 13.97% is observed due to an abundant background noise from the derivatization reagents. Thus, further method and temperature program optimization are required to avoid such coelution as Iva is a notable amino acid in meteorites and that IEC is used as a pretreatment step. Finally, comprehensive investigations on the recovery of sugars should be carried out with particular emphasis on the preservation of such 5% excess.

SAMPLING & EXTRACTION

Materials & methods

Serpentine was used as a meteorite analog to simulate liquid-solid interactions and adsorption. It is typically used to perform procedural blanks when analyzing extraterrestrial samples. A porcelain mortar and pestle were used to grind the serpentine into fine grains. Pre-scored 5 mL Duran ampoules were used to perform the samples' water extraction in the presence of serpentine, and flame-sealed. A Sigma centrifuge was used to recover the supernatant from the serpentine grains and operated at 6000 rpm.

Water used for tool cleaning and sample processing was obtained using a Milli-Q Direct 8 apparatus (18.2 MΩ.cm at 25 °C, < 5 ppb total organic carbon). All glassware was washed several times with ethanol and Milli-Q water, wrapped in aluminum foil, and then heated to 500 °C for at least 5 h to remove any organic contaminants. The serpentine was heated to 500 °C for at least 5 h to degrade and remove any contaminants before being crushed.

Degradation of amino acids during hydrolysis

Procedure

The degradation of amino acids during water hydrolysis (24 h at 100 °C or room temperature) with and without serpentine was studied. When used, approximately 500 mg of serpentine was crushed to a fine powder and placed in a pre-scored ampoule using a spatula. The powder was then doped with 50 µL of a 5×10 -5 M amino acid standard and allowed to dry for 2 h. For the remaining samples, 50 µL of this solution was placed to the bottom of the ampoule. Then, 3.5 mL of Milli-Q water to mimic the extraction of the powder from the mortar into the ampoule were added before flame-sealing and the hydrolysis. After the samples were cooled down, a centrifuge was used for 10 min, and the supernatant was recovered using a clean Pasteur pipette. 2 (or 3) additional 1 mL washes using water, vortex stirring, centrifugation and recovery of the supernatant were performed. The recovered extracts were purified on IEC detailed in

Results & discussion

As can be seen in Table 25, rinsing the powder 2 or 3 times with 1 mL after hydrolysis increases the recovery by 2% to no more than 4%, and should be used for meteorite analyses. In the presence of serpentine, amino acid recoveries after hydrolysis at 100 °C tend to be 5 -10% lower, which could be consistent with mineral-catalyzed degradation/secondary products. Curiously, D-Nva recoveries are significantly higher than L-Nva in all samples and should, at first glance, be consistent with contamination. Nevertheless, L-Nva is low in sample 4, which is also the case for Asp. In addition, high standard deviations greater than 10% are reported. Thus, questions about handling errors can be raised, but also about the repeatability of the derivatization, discussed in the next section. The main conclusion here, despite the variability of the results obtained, is that at least 60% of the amino acids can be recovered by the process of water hydrolysis followed by IEC fractionation. To obtain an estimate of the recovery of the whole process and to evaluate the potential reactions occurring at elevated temperatures between amino acids and sugars, a mixture of both compound classes was used. Approximately 500 mg of serpentine was crushed with a mortar and pestle and then doped, in the mortar, with 50 µL of a 5×10 -5 M sugar mixture and 50 µL of a 50×10 -5 M amino acid mixture. The sample was then allowed to dry for 2 h, allowing adsorption of the compounds onto the serpentine. The mineral powder was crushed again and recovered using 6×333 µL + 1 mL water, until the mortar appeared clean, and placed in an ampoule. The top of the ampoule was washed with 500 µL to recover any remaining powder. Flame sealing, water hydrolysis, IEC, and derivatization were performed as previously described. As can be seen in Table 26, lower recoveries are obtained than using only amino acids during hydrolysis.

The recoveries of Ala decreased from more than 90% to about 50%. However, the experimental conditions are not exactly the same. Here, the amino acids and sugars were deposited on the mineral, but in the mortar and significant loss could occur even with careful washing of the mortar. The material is different and could adsorb these compounds better. So far, on other experiments, standard deviations were in the range of 5 -15%, which gives a relatively high but acceptable uncertainty on the result obtained considering the number of measurements and steps performed. Here, standard deviations greater than 30% could, at first glance, correspond to poor repeatability of the mineral doping in the mortar. However, the reference standard deviations for Ala and Iva are in the range of 17 -25%, suggesting a problem with the repeatability of the derivatization. For this reason, this derivatization method was studied in detail in 3.2.1 MeOH/TFAA derivatization of amino acids. Therefore, it cannot be concluded that there is any impact of sugars. Unfortunately, no further experiments were performed using the full procedure because derivatization and IEC recoveries were studied preferentially.

Conclusion

Further analyses must be performed to study the behavior of amino acids and sugars throughout the procedure. The water hydrolysis in the presence of sugars and amino acids will be of great importance as both can react and decrease the recovered quantities. However, this should be studied using sonication to avoid their degradation and reaction. The presence of serpentine could also have an impact on their recoveries. HCl hydrolysis of recovered amino acids is also of great interest for these recoveries.

Racemization and ees should be studied in both cases as they will be investigated on a real sample.

CONCLUSION

Sampling, extraction, purification, fractionation, derivatization, and GC×GC analysis constitute numerous steps to be investigated, methods to be developed, and experience to be gained. Although, unfortunately, the development of a complete procedure to study ees in extraterrestrial samples has not yet been achieved, considerable progress has been made. A ready-to-use amino acid derivatization for trace analysis and ees studies of 30 amino acids is now available for extraterrestrial and simulated samples. A derivatization of sugars, based on the previous team's laboratory experiments, has been extended to sugar alcohols and the sugar data set has been enriched and can be further studied for application to extraterrestrial and simulated samples. Expertise in purification and fractionation steps using handmade IEC columns and glass reaction tubes was developed and showed recoveries of 70 -100% for amino acids. Sugars, on the other hand, were only studied through ribose in early developments but showed recoveries greater than 95%. As with amino acids, further studies are needed on recoveries and ees. The sources of contamination highlighted for amino acids are a major problem, but reagents and materials are now identified for further experiments. Sampling and extraction remain the least studied steps. Extraction will, in the future, be performed using sonication. The water extraction shown here, however, testifies that no major degradation occurred during this step and implies that water extraction by sonication should lead to better recoveries. In addition, the use of sonication could reduce the risk of mineral-catalyzed degradation/byproducts, compared to the conventional water extraction at 100 °C for 24 h.

CHAPTER IV

Applications of the optimized method to complex samples

AMINO ACID SYNTHESIS STARTING FROM HEXAMETHYLENETETRAMINE

This study is a follow-up to previously published work by V. Vinogradoff et al. 159 on the formation of amino acids from hexamethylenetetramine (HMT) under hydrothermal conditions, at pH 10 and 150 °C for 2, 7 or 31 days. Multiple parameters can have an impact on the synthesis of amino acids in parent bodies: composition of organic and mineral phases, pH, concentrations, temperature, oxidation state, relative water content and degree of aqueous alteration. The influence of the presence of phyllosilicates (Al-rich smectite or Fe-rich smectite) or carboxylic acids was studied and showed significant differences in the abundance for glycine, alanine and 4-aminobutyric acid compared to HMT alone. The highest abundance of amino acids reported was for the use of phyllosilicates, but the results for carboxylic acids led to consider the formose reaction with ammonia and Maillard-type reactions as possible synthetic routes.

Indeed, the decomposition of HMT leads to the initial reactants of these reactions such as formaldehyde and amines.

Here, only the sample containing HMT has been studied and two main objectives are highlighted. The first is to compare the results obtained -using ion exchange chromatography purification and MeOH/TFAA derivatization -with those reported by the authors. The second objective is to search for other amino acids not previously detected due to higher detection limits.

Materials and methods

The samples received from the Physique des Interactions Ioniques et Moléculaires (PIIM) laboratory at Aix-Marseille Université consisted of a liquid and a solid part of about 5-7 µL in total. The initial concentration of HMT was 2 M compared to 0.2 M in the previous work 159 . The degradation time of HMT was set to 31 days for both sample sets.

Each sample (2 µL for liquid 1 and liquid 2 samples, and the remaining solid portion) was subjected to 24 h of water hydrolysis at 100 °C in a flame-sealed glass ampoule. Each sample was then divided into 2 equal parts, the first untreated, and the second hydrolyzed with 6 M HCl for 24 h at 100 °C in a flame-sealed ampoule. Each part was then purified and fractionated using ion exchange chromatography,

Amino acids content

To compare the reported and obtained values, the amounts obtained here were divided by 10 to match the original 0.2 M experiment, Table 27. It is interesting to note, for the solid and both liquid 1 and liquid 2 samples, the difference between the free and hydrolyzed content obtained. Since each sample was equally fractionated into a free and HCl-hydrolyzed fraction, the difference between the two values corresponds either to variations in the cleavage of the oligomerized amino acids or to their degradation under the chosen hydrolysis conditions. It is also possible that the use of 6 M HCl, even after drying, may have an impact on MeOH/TFAA derivatization, even though the first derivatization step requires an acidic medium. However, no real trend can be established between the two liquid fractions and on the solid fraction. Therefore, it is difficult to know whether insufficient bond cleavage or degradation have occurred. Glycine, for example, appears to have an increased content between the free and hydrolyzed portion for the solid samples (690 µM to 831 µM) and the first liquid (584 µM to 711 µM). However, the second liquid sample only increased from 487 µM to 493 µM, which is not significant. In addition, glycine is known to be a contaminant present in ammonia that is required for IEC elution. Another hypothesis would be the lack of homogeneity of the sample, as the solid part is more like a gel and the second liquid sample was more difficult to sample as only liquid sample without any solid/gel fraction. As can be seen in Table 28, the trend in amino acid content previously reported 159 and obtained here is similar. The most abundant amino acids are C3 (Gly, Sar, α-Ala, β-Ala) compared to C4 (β-Aib, β-Aba, α-Aba, γ-Aba). However, differences in abundances can be noted. We found that Gly is about 1.5 times and γ-Aba 10 times less abundant in our experiments, while α-Ala is 4 times and, β-Ala 2 times more abundant compared to the previous experiments. These differences could originate from the difference in HMT concentration, which was 0.2 M for the reported study, and 2 M for the present work. As a general trend, α-(except glycine) and β-amino acids tend to be found in higher amounts using a more concentrated HMT. Conversely, the content of 4-Aba, a γ-amino acid, is much lower than reported in the original study.

Table 28. Comparison of the amino acids content between the work of V. Vinogradoff et al. 159 and the presented study.

Amino acids content

Reported

Gly >> α-Ala > β-Ala > γ-Aba > β-Aib > α-Aba = β-Aba Here Gly > Sar > α-Ala > β-Ala >> β-Aib > β-Aba > α-Aba > γ-Aba
As considered by V. Vinogradoff et al. 159 , these amino acids could be formed by a formose-type reaction in parallel with Maillard-type reactions, Figure 42. Under this assumption and using highly concentrated HMT, it is likely that the kinetics of the formose and Maillard-type reactions would be altered and lead to a higher abundance of low molecular weight amino acids. Because glycolaldehyde and glyceraldehyde would be more rapidly converted to amino acids (while assuming that tetrose formation is relatively slow), the equilibrium would have shifted to the production of glycolaldehyde and glyceraldehyde (Le Châtelier's principle), and thus of α-and β-amino acids rather than γ-amino acids. Thus, it would be interesting to study the sugar content of the 0.2 M and 2 M HMT samples to examine the relative proportions of tetroses and glyceraldehyde. Since IEC was employed, the sugar fractions were studied simultaneously using MBA derivatization. Surprisingly, no sugars were detected except for arabinose and xylose, which were also found in the procedural blank. From this perspective, one might assume that no formose-type reaction occurred. However, as we will point out in the next section, the lifetime of the sugars synthesized by the formose reaction is generally on the order of days. Indeed, after 45 days, no sugar content was found while the present experiment used 31 days of reaction time. Unfortunately, derivatization with MBA (used for the lower limit of detection) did not detect sugar alcohols, unlike the MBA/TFAA used in the next section. At the time these experiments were performed, the presence of these sugar alcohols and in particular glycerol at long reaction times was unknown. Thus, investigations of sugars in HMT samples should be performed using shorter reaction times or MBA/TFAA to identify sugar alcohols.

Other detected amino acids

Compared with previous work, several additional amino acids were successfully detected including Sar, DL-2,4-Dab, DL-Glu, DL-Pro, 2-Aib, DL-Val, DL-Asp, Etg, DL-2,3-Dap, DL-Thr, DL-Phe, 5-Ava, and DL-Leu. Eight of these were not investigated in the previous study and thus show that either a higher HMT concentration led to amino acid formation above instrumental detection limit or that our analytical device allowed for improved identification. The detection of the other five amino acids targeted but not detected in the previous work, Glu, Pro, Val, Asp and Thr, can mainly be explained by the lack of amino acid content in the original study, due to the use of 0.2 M HMT, which did not allow such detection. Nevertheless, the reported detection limits for the AccQ•Tag (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate)

derivatization used in the original study are in the 10 -7 M range using the UHPL-UV instrument 274 , whereas they are in the 10 -8 M range using MeOH/TFAA followed by GC×GC-TOFMS.

Conclusion

The analysis of the HMT samples indicates that ion exchange chromatography fractionation coupled with MeOH/TFAA derivatization is suitable for the analysis of complex samples. This notably allowed the identification of 13 additional amino acids not detected in the original study. The comparison with the original study is limited because the samples used different concentrations, were not derivatized with the same procedure nor on the same apparatus. Nevertheless, the same trend in amino acid abundance was measured. This first analysis of a complex sample serves mainly to justify the applicability of the developed protocol to other extraterrestrial-like samples. Future applications of this procedure would be the analysis of interstellar ice analogs that allows the simultaneous investigation of amino acid and sugar fractions.

MINERAL-CATALYZED FORMOSE REACTION UNDER HYDROTHERMAL CONDITIONS

In this study, several main objectives can be highlighted. The first is to investigate whether olivine is capable of initiating a formose-type reaction under hydrothermal conditions. The second is to compare the mineral-catalyzed synthesis of sugars with the classical calcium hydroxide catalyst in terms of sugar diversity and kinetics. Finally, the potential induction of ees into the sugars formed under hydrothermal conditions and the presence of the olivine mineral are investigated. This study focuses on longer reaction times, between 2 and 45 days, whereas classical experiments on the formose reaction are generally performed within minutes up to hours.

Materials and methods

The For the samples received, formose reaction experiments were performed in aqueous systems and under anoxic atmosphere at 80 °C. The mineral olivine was chosen as a model silicate given its abundance in the solar system, and as a catalyst for the formose reaction. As shown in Table 29, various experimental conditions and combinations of formaldehyde (F), glycolaldehyde (G), calcium hydroxide (Ca(OH)2 denoted by α), and olivine (O) were tested. Formaldehyde was introduced as paraformaldehyde (PFA, which is a polyoxymethylene with a low degree of polymerization, typically between 8 and 100 units) that was depolymerized during the experiments by heat treatment under acidic conditions. The weight ratios used in these experiments were set to 10:1 for F/(G or α) and 10:1 for O/F. The prepared mixtures were then loaded into closed cells in an argon glovebox and then heated to 80 °C. Once the experiments were started, three durations (day 2, day 7, and day 45) were studied for their sugar content.

Derivatization approach

At first glance, (S)-BuOH/TFAA derivatization would be an excellent option, as sugars, sugar alcohols and sugar acids could be derivatized and analyzed. However, it should be kept in mind that a formose reaction leads to a wide variety of sugars, sugar alcohols, sugar acids, and substituted sugars, and could potentially involve many co-elutions, even with a powerful separation technique such as GC×GC. Since this derivatization leads to many forms for each sugar, an alternative derivatization such as an MBA-based derivatization would offer more selectivity. MBA/TFAA seems to be a better choice than MBA because of the detection of sugar alcohols at the expense of sugar intensity. Nevertheless, the amount of sugars expected in this formose reaction are high (preliminary results from PIIM analyses) and may justify the use of MBA/TFAA derivatization. In addition, the TFA-derivatized compounds show an intense m/z = 69 peak, while the other peaks tend to be much smaller. Unfortunately, the m/z = 69 ion is associated with high noise and often an inability to integrate properly in case of coelution because it is a common mass for derivatives. Thus, alternative m/z ratios have to be used to reliably quantify the targeted molecules.

MBA/TFAA then appears to be a more appropriate choice as some MBA/TFAA derivatized sugars would only be MBA derivatives. Another consideration is the impact of the matrix on derivatization. Because the first (S)-BuOH/TFAA step is performed in an acidic medium (using acetyl chloride to form HCl in situ), this derivatization is less likely to be affected by the pH of the samples, which is between 3 and 7. On the other hand, the first step of MBA/TFAA derivatization is rather performed in basic medium (in pyridine) and could have an impact on the reactivity of MBA. A first test on FGO2 was performed and led to consider MBA/TFAA as a better choice. Indeed, as shown in Figure 43, MBA/TFAA shows a better distribution of compounds using m/z = 43 (characteristic of MBA ring fragmentation) than (S)-BuOH/TFAA using m/z = 57 (characteristic of (S)-BuOH fragmentation), Figure 44, with optimized temperature programs. The intensities of the highest peaks, in red, are of the same order of magnitude (more intense for (S)-BuOH/TFAA by a factor of 2.5). Therefore, MBA/TFAA could reduce the risk of coelution when integrating sugar peaks. In addition, glyceraldehyde is not detected with (S)-BuOH/TFAA and is of great interest because it is the simplest sugar and one of the first aldol reaction products from glycolaldehyde. Finally, ee will be studied and better results in terms of accuracy were found for the MBA (not MBA/TFAA) derivatization than for the (S)-BuOH/TFAA derivatization. This again led to the final decision to select MBA/TFAA as a better compromise for the overall analysis.

Since the MBA/TFAA derivatization was chosen to quantify sugars and study potential enantiomeric excesses, the BSTFA derivatization was used to complete the dataset of synthesized sugar acids, branched sugars, and other characteristic formose reaction products that could not be studied with MBA/TFAA.

Qualitative examination of the sugar acids and other relevant formose reaction products presented in Table 30 tends to confirm a formose reaction under the experimental conditions. In the case of FGO2, most of the listed compounds and especially 2-hydroxymethylglycerol (HMG) and 2-hydroxymethyltetritol (HMT*) were detected while this was not the case for FG2α. As discussed in 4.2.3 Evolution of sugars content, this could be due to kinetic differences as olivine and calcium hydroxide seem to catalyze the reaction at different rates. On the other hand, the presence of sugar acids is a good indicator of the formose reaction in terms of the diversity of products synthesized. This diversity is highlighted through

Evolution of sugars content

As can be seen, from Figure 46, the highest sugar abundancies are obtained for FG2α using the traditional calcium hydroxide catalyst of the formose reaction. Concentrations up to 1.16×10 -3 M were found for DLxylose. Using olivine, the highest concentration found was also for xylose with 3.90×10 -4 M in FGO2.

Unsurprisingly, the presence of glycolaldehyde in addition to formaldehyde leads to higher sugar content:

6.65×10 -4 M and 9.83×10 -4 M are reported for F7α and FG7α, respectively, whereas 2.97×10 -4 M and 3.90×10 -4 M are reported for FO2 and FGO2, respectively. Similarly, using Ca(OH)2, the presence of glycolaldehyde greatly impacts the kinetics of C5 and C6 sugar formation. Indeed, almost no sugar content was reported for F2α and 6.65×10 -4 M for F7α, whereas the highest reported values 1.16×10 -3 M and 9.83×10 -4 M are for FG2α and FG7α. Furthermore, comparing F2α (almost zero sugar content) and FO2

(2.97×10 -4 M for lyxose), it appears that olivine is a better catalyst than Ca(OH)2 when using only formaldehyde.

Both catalysts lead to a wide variety of compounds, as can be seen in Figure 45, including sugars and sugar-related compounds. Indeed, although we have focused here on quantifying 10 sugars and 6 sugar alcohols (and their enantiomers, when possible), many other chromatographic peaks could correspond to sugars formed in those samples. Using aldonitrile acetate (ANA) derivatization, the PIIM team estimated that about 35 peaks could be associated with oses or polyols in the highly concentrated samples (FO2, FO7, FGO2, FG2α, and FG7α) by comparing their mass spectra and the NIST database. As a reminder, the major drawback of ANA derivatization is the lack of enantioseparation of C4, C5, and C6 sugars.

Furthermore, the abundance of sugars formed is also an interesting result of this study. C5 sugars are found in highest amount in all samples compared to C4 or C6 sugars. It is interesting to note that between days 2 and 7, a decrease in C5 sugars is expected and obtained, while an increase in C6 sugars is expected but not obtained. It is therefore possible that aldopentoses (ribose, arabinose, lyxose, xylose) are more likely to isomerize into ketopentoses (e.g., ribulose) than to produce aldohexoses by aldol reaction (Figure 8, p. 30). Surprisingly, the ribulose content is barely detectable, which undermines this hypothesis.

Similarly, fructose should increase since aldohexoses (e.g., glucose and galactose) can isomerize to ketohexoses (e.g., fructose) but follows the same trend as glucose and galactose with a very high content compared to ribulose. In the end, it is likely that all the sugars studied here remain in equilibrium with each other and decrease overall with time, probably leading to branched sugars and secondary products before being degraded. It should be noted that fructose and ribulose were not directly quantified because no calibration curve was available for these compounds. Their concentration was approximated using the calibration curve of xylose but with a correction factor because they were injected at a single concentration. Therefore, the concentration values obtained must be considered as rough estimates, but the trend between samples should be accurate.

Another important result is the presence of 2-deoxyribose in several samples. Although this compound has been previously detected with other deoxysugars 275 under hydrothermal conditions, its presence indicates that formation of this compound could have been occurred during parent body processing of meteorites in the presence of phyllosilicates such as olivine. The synthesis of deoxy sugars would not be due to the formose reaction but could be the result of the combination of acetaldehyde and formaldehyde in the presence of a divalent metal oxide, or the combination of glyceraldehyde and acetaldehyde 276 . Here, this study reveals that a similar range of 2-deoxyribose concentration is achieved using calcium hydroxide (2.82×10 -6 M for FG2α and 1.09×10 -5 M for F7α) and olivine (2.73×10 -6 M for FGO2 and 1.01×10 -5 M for FGO7) catalysts.

The general trend is that C5 sugars are most abundant on days 2 and 7. This would, however, need to be correlated with the total amount of C6 sugars, as only glucose and galactose were quantified.

Unfortunately, mannose could not be identified because the chromatogram is too crowded in its retention

area. An intermediate time between day 7 and day 45 would have been interesting to study to follow the decrease in sugar content and to confirm if C5 and C6 sugars decrease in the same manner as previously reported. Among C5 sugars, xylose and lyxose are more abundant than ribose and arabinose in FG2α. On the other hand, xylose and arabinose are more abundant in F7α, FG7α, FO2, FO7, FGO2, and FGO7. This finding is very interesting because one would expect the same sugar content for each C5 sugar in all samples, or the same imbalance pattern between FG2α, F7α, and FO2 for example. Thus, it appears that the presence of glycolaldehyde in Ca(OH)2-containing samples promotes the synthesis of xylose and lyxose (FG2α) and subsequently improves the degradation and/or conversion kinetics of lyxose. Concerning the olivine-containing samples, the trend with and without glycolaldehyde remains the same but the relative intensities can be discussed, especially for FO2 where ribose is close to arabinose in terms of content. It would have been interesting to have data on threose to look for variations between threose and erythrose contents. Unfortunately, threose was not found in any samples, assuming that threose and erythrose share the same mass spectrum. The most plausible explanation would be that threose has a different MBA ring location, resulting in different fragmentation patterns. Unfortunately, no threose standard was available to test this hypothesis.

The lifetime of sugars under hydrothermal conditions is very limited as none were found in samples FGO45 or FO45. Unfortunately, data from F45α and FG45α are missing but should show the same result because the Ca(OH)2-catalyzed formose reaction is known to rapidly degrade sugars 277 . However, this shows that olivine does not provide better preservation of the synthesized sugars under these conditions.

Even in the absence of catalysts, the reaction occurs slowly, as the glyceraldehyde content is high in FG2, about 3.67×10 -4 M, and decreases to 5.53×10 -5 M in FG7 and 1.57×10 -5 M in FG45. At the same time, for these samples, the concentration of erythrose is relatively high compared to the other samples, and highest for FG45 at 1.48×10 -4 M. Higher carbon-containing sugars are only detected in samples FG45 and F45 samples with very low concentrations in the range of 10 -6 -10 -5 M.

Evolution of sugar alcohols content

As can be seen in Figure 47, the evolution of the sugar alcohol content is less obvious than in the case of sugars. However, the most important information is the considerable increase in glycerol concentration after 45 days under hydrothermal conditions. Concentrations ranging from 6.24×10 -3 M and 1.04×10 -2 M were found for F45, FG45, FO45, and FGO45 while the day 2 and 7 samples were, at best, in the 10 -5 M range. Glycerol can be obtained by the well-known Cannizzaro reaction of glyceraldehyde and formaldehyde 154 , reduction of glyceraldehyde and even dihydroxyacetone. Given the low concentrations of glyceraldehyde and dihydroxyacetone on days 2 and 7, it is likely that the oligomerized sugars were reduced and/or cleaved because they were not detected after 45 days. Indeed, if it formed from glyceraldehyde, its synthesis from formaldehyde would have resulted in the detection of another low molecular weight sugar such as erythrose. The higher content of C4 erythritol in FO45 and FGO45 tends to confirm this trend where sugars are degraded to lower molecular weight sugar alcohols. Interestingly, no such high values are reported for F45 and FG45 relative to glycerol. Thus, olivine could have an increased impact on the reduction and cleavage of sugars since the glycerol content is also higher at day 45 for samples containing olivine. A difference in pH can be noted between the day 45 samples, where the olivine-containing samples FO45 and FGO45 are less acidic (pHFO45 = 6, pHFGO45 = 7) than FG45 (pHFG45 = 4).

The role of olivine as a pH buffer may therefore alter the mechanism by which sugars are degraded and reduced along with different kinetics. Unfortunately, no data are available for F45α and FG45α to support these conclusions. The drop in pH of the formose reaction is an indicator of the Cannizzaro reaction which leads to alcohols and carboxylic acids 278 . The formation of various carboxylic acids in large quantities could be responsible of this drop as e.g., glyceraldehyde can lead to glyceric acid and glycerol, glycolaldehyde to glycolic acid and ethylene glycol, and formaldehyde to formic acid and methanol 154 . The detection of glycolic acid and especially glyceric acid, linked to glycerol formation, thus tend to support a Cannizzaro reaction. However, no standards were run at this point to investigate formic acid.

Enantiomeric excesses investigation

One of the objectives of the study was to search for potential ees in sugars formed under mineralcatalyzed hydrothermal conditions. However, in most cases, coelutions of the enantiomers were observed, even after selecting the most appropriate m/z for integration and quantification. In addition, sugars and sugar alcohols were sometimes quantified close to the limit of quantification, resulting in high ee errors. Thus, the ee obtained cannot be considered reliable. As an example, 2-deoxyribose, Figure 48, shows a D-ee of -21% with a relatively low standard deviation of 3.5%. However, the detected signal is very low in abundance, noisy, and it is difficult to assess whether coelution is occurring. Therefore, this value cannot be considered for any interpretation. On the other hand, lyxose and glucose are opposite examples, i.e., they show high intensities (S/N is in the order of magnitude of 3000) and no visible significant coelution. The maximum ee values for lyxose and glucose are -4.48 ± 0.77 and 1.84 ± 0.29, respectively. The other reported ees for these compounds are in the same range and have the same sign.

Two conclusions can be drawn: (i) the number of analyses (4) to study these ees is small compared to the discussion in 3.2.1.7 Enantiomeric excess conservation (p. 85) and (ii) a reproducible bias may be included in the results as many peak slices are integrated (due to GC×GC and peak tailing). Ultimately, a more comprehensive study of the derivatization in terms of ee should be performed before evaluating any significant ee. Thus, since no major enantiomeric imbalances were found, this led to the consideration that the compounds were synthesized in racemic proportions because olivine is not a chiral mineral and Ca(OH)2 should not trigger ees.

Conclusion

Even with careful selection of the MBA/TFAA derivatization procedure, the use of a powerful resolution tool such as GC×GC, and selection of appropriate ions for quantification, coelutions of nontargeted compounds with the targeted enantiomers occurred and prevented accurate ee determination. The formose reaction was confirmed using well-known formose products with BSTFA derivatization, but it remains a complex sample that is difficult to analyze but may correspond in diversity to a true extraterrestrial sample. Nevertheless, the quantification of both sugar enantiomers and sugar alcohols led to interesting results concerning their evolution over a long period of time, up to 45 days, whereas studies on the formose reaction generally focus on shorter time scales. On the one hand, the sugars are rapidly synthesized over a period of at least 2 days and 7 days and are then probably transformed into branched or reduced sugars and degraded into lower molecular weight sugar alcohols as observed at day 45. In addition, olivine appears to be a better catalyst than Ca(OH)2 using formaldehyde only, despite lower molar concentrations of sugars have been reported. The detection of 2-deoxyribose is consistent with other studies conducted but is unlikely to be due to the formose reaction itself. It is nevertheless evidence that this life-required compound can be obtained under hydrothermal conditions in small quantities and gives clues to its plausible presence in extraterrestrial samples. Investigations on other timescales, i.e., first reaction hours, day 1 and between days 7 and 45 would be interesting to better understand the differences between calcium hydroxide and olivine catalysis. Besides, F45α and FG45α sample analyses would have provided valuable information on the stability of sugar alcohols using calcium hydroxide.

Unfortunately, these samples were not available, and no comparison can be made with olivine. Finally, significant standard deviations are reported for sugars and especially sugar alcohols. These errors may be due to extrapolation out of the linear range that was evaluated for the 5×10 -6 M -10 -4 M range. Values as high as 1.2×10 -3 M are however reported for sugars, and up to 10 -2 M for sugar alcohols. In addition, the presence of contaminants such as metals and other species could interfere with the derivatization, as no ion exchange chromatography purification was employed.

Where and how life emerged are still ongoing questions. All living organisms use proteins consisting of L-amino acids and DNA/RNA consisting of D-2-deoxyribose/D-ribose. Finding clues to the origin of this biological homochirality would help to understand how life evolved. Despite numerous theories about the emergence of enantiomeric excesses via a symmetry breaking event followed by amplification mechanisms, no conclusion can be drawn about which is responsible for life's enantiomeric selection.

Thus, it is still unclear whether this selection occurred via a deterministic or random mechanism. However, since meteorite analyses have shown the presence of amino acids and sugar derivatives, whose ees follows the same trend as life's homochirality, as well as of circularly polarized light, capable of inducing ees, in the interstellar medium, a plausible scenario would be that these biosignatures form on dust grains and that an enantiomer is selected by enantioselective synthesis or photolysis. It is therefore of interest to develop analytical procedures to investigate the organic content of meteorites and samples returned from asteroids or other astrophysical objects in our solar system to study the abiotic synthesis of chiral biosignatures. This manuscript has attempted to investigate this challenge.

Since amino acids and sugars are priority targets in this analysis, a suitable derivatization method must be employed. The methanol/trifluoroacetic anhydride derivatization for amino acids, which has been used especially for isovaline, showed suitable overall resolution and enantioseparation of about 30 amino acids by GC×GC-TOFMS. The lack of absolute resolution can be overcome by using a CP-Chirasil-Dex CB instead of a Lipodex E stationary phase as primary column. Repeatability, linearity, stability, mass fragmentation, and conservation of enantiomeric excess have been studied, and this method is now ready to be used for extraterrestrial samples. Sugars, on the other hand, are much more challenging, as 5 different stereoisomers of a single enantiomer can be found in equilibrium due to intramolecular cyclization, which leads to 5-and 6-carbon rings. Many derivatization procedures have been developed in the literature, but none of them is a remarkable compromise. It must be adapted to the purpose of the analysis. Thus, methylboronic acid/trifluoroacetic anhydride has been proposed as an alternative allowing the separation and enantioseparation of about 15 sugars and sugar alcohols. The main known drawbacks of this method are the non-detection of sugar acids as well as the tailing of high molecular weight sugar alcohols during GC×GC analysis. In addition, the use of trifluoroacetic anhydride is a compromise as the detection limits of C2 to C5 sugars are lowered using this second derivatization step but allows the detection of conformer compatible sugars, sugar alcohols and deoxy sugars.

Prior to derivatization of extraterrestrial or extraterrestrial-like samples, a purification and desalting step is usually performed to avoid subsequent derivatization disruption and allows for the selection of compounds by cationic filtering. Using such a configuration, and MeOH/TFAA derivatization, amino acid recoveries were studied and showed values between 61.2% and 115.8%. However, contamination was reported but was mainly due to the quality of the ammonia used as elution solvent in the fractionation step, which was particularly the case for β-alanine with recoveries above 800%. The know-how of handmade columns was developed, and early developments of ion exchange chromatography showed recoveries of alanine around 95 ± 5% as well as recoveries of ribose around 96 ± 10% which is promising for sugar recoveries.

Sampling and extraction were the last to be studied in the entire procedure, but the first tests on amino acids using water hydrolysis showed overall recoveries in the range of 61.7% to 120.7%. Unfortunately, no data are available for sugars and further experiments should be conducted on sugars with an amino acid/sugar mixture to discard or not degradation by the Maillard reaction. On the other hand, sonication is considered to be an efficient method to safely extract sugars and amino acids and will be used for further testing. Based on the derivatization methods studied or developed and using ion exchange chromatography, two types of simulated samples were analyzed.

The first, the formose reaction, is known to lead to a wide variety of sugars, sugar alcohols, and sugar acids, and has been shown to be mineral-catalyzed in the presence of olivine, a very abundant mineral in the solar system. Many minerals, including olivine, exhibit this property and this work shows that olivine can act as a formose reaction catalyst starting from formaldehyde under hydrothermal conditions. However, the data obtained do not allow us to conclude whether olivine produces more, or more diverse sugars compared to the traditional calcium hydroxide catalyst during the whole process, although the highest values reported are for calcium hydroxide. Although the abundances of sugars are known to decrease rapidly, another interesting result is the impressive lifetime of sugar alcohols that are still present after 45 days of hydrothermal alteration while sugars have not been detected. Furthermore, given the surprisingly high glycerol content after 45 days and the low content of potential precursors such as glyceraldehyde and dihydroxyacetone throughout days 2 and 7, it is likely that sugars at some point stop gaining in complexity and are instead degraded and reduced to smaller sugar alcohols and sugar acids via the Cannizzaro reaction. In some ways, this corroborates the results of meteorite analysis where high molecular weight sugars and sugar derivatives are not detected. The detection of 2-deoxyribose is consistent with other work, which gives clues to the possible presence of this life-required compound in extraterrestrial matter. The evolution of sugars from C5 to C6, however, did not show the expected trend. Indeed, the C5 and C6 aldoses and ketoses decrease in abundance in a similar way while an increase in C6 should theoretically be obtained.

The availability of interstellar formaldehyde for such a reaction to occur is often discussed. Hexamethylenetetramine (HMT), which was recently analyzed directly in an extraterrestrial sample, could be a feedstock for both formaldehyde and ammonia, and is the second application of this manuscript.

Interestingly, HMT is also known to potentially lead to amino acids during parent body alteration processes. The present work, using methanol/trifluoroacetic anhydride derivatization and ion exchange chromatography, detected 13 additional amino acids compared to previous works. Because a higher concentration of HMT led to a higher abundance of low-molecular-weight amino acids, it is likely that the formose reaction in the presence of ammonia (sugar model) favored the synthesis of α-and β-amino acids rather than γ-amino acids. However, this result should be correlated with the analysis of sugars to examine their relative proportions. Indeed, even if the analyzed sugar fractions did not show the presence of any sugars, they still could have been formed and be degraded as 31 days of hydrothermal treatment was used. Unfortunately, MBA/TFAA derivatization, which was not used, would have been a better alternative to detect sugar alcohols and discuss whether sugars and sugar derivatives were formed. Nevertheless, the HMT analysis represents our first analysis of a complex sample using the entire procedure (except for the sample extraction part) that proved to be effective.

The objectives defined at the beginning of this manuscript were to develop a procedure to extract, purify, fractionate, derivatize, and analyze extraterrestrial samples. Derivatization and GC×GC analysis of amino acids and sugars have been studied and are suitable for this purpose, but the MBA/TFAA derivatization for sugars would require more exhaustive experiments. Purification and fractionation by ion exchange chromatography showed good recoveries for amino acids and ribose. The main advantages are the simultaneous study of amino acids and sugars on a single sample and desalting. Further research is needed to estimate the recovery of other sugars. Sampling and extraction require extensive investigations, especially since sonication will be used for future sample processing, which has not been studied here.

Nevertheless, hydrolysis with water did not show any degradation of the amino acids. Future applications of this procedure concern the analysis of interstellar ice analogs irradiated by circularly polarized light, the analysis of hydrothermal-altered samples and the analysis of meteorites. Slope. b Standard error on S. Intercept was set to 0. Data obtained using 4 concentrations: 5×10 -5 M, 10 -5 M, 5×10 -6 M and 10 -6 M. For each concentration, 1 sample was prepared and analyzed 2 times.

Figure 3 .

 3 Figure 1. Structure of the deoxyribonucleic acid.Figure 2. Genetic material built from chiral molecules. Figure 3. Schematic representation of the construction of circularly polarized light.Figure 4. Induced ees as a function of circularly polarized light orientation and wavelength.Figure 5. Model of interstellar ice chemistry (grain model).Figure 6. Sublimation chamber used for interstellar ice analogs studies.Figure 7. Strecker synthesis of an α-amino acid from an aldehyde and Michael addition of ammonia to acrylonitrile leading to a β-amino acid under abiotic conditions. Figure 8. Formose reaction chemical network.Figure 9. Synthesis of hexamethylene tetranime (HMT) from formaldehyde and ammonia reagents.Figure 10. Plausible chemical pathway leading to HMT under abiotic conditions using methanol, ammonia and UV-light. Figure 11. Schematic of two-dimensional gas chromatography (GC×GC).Figure 12. Schematic of the modulation process.Figure 13. Chemical structure of cyclodextrins.Figure 14. Example of sugar acid, sugar alcohol, sugar aldehyde and sugar ketose for a three-carbon monosaccharide. Figure 15. Classification of aldoses (monosaccharides) according to their carbon number.Figure 16. Classification of ketoses (monosaccharides) according to their carbon number.Figure 17. Intramolecular cyclization of ribose into anomers in solution.Figure 18. Illustration of molecular duplication and loss of chiral information during the reduction of some

Figure 4 .

 4 Figure 1. Structure of the deoxyribonucleic acid.Figure 2. Genetic material built from chiral molecules. Figure 3. Schematic representation of the construction of circularly polarized light.Figure 4. Induced ees as a function of circularly polarized light orientation and wavelength.Figure 5. Model of interstellar ice chemistry (grain model).Figure 6. Sublimation chamber used for interstellar ice analogs studies.Figure 7. Strecker synthesis of an α-amino acid from an aldehyde and Michael addition of ammonia to acrylonitrile leading to a β-amino acid under abiotic conditions. Figure 8. Formose reaction chemical network.Figure 9. Synthesis of hexamethylene tetranime (HMT) from formaldehyde and ammonia reagents.Figure 10. Plausible chemical pathway leading to HMT under abiotic conditions using methanol, ammonia and UV-light. Figure 11. Schematic of two-dimensional gas chromatography (GC×GC).Figure 12. Schematic of the modulation process.Figure 13. Chemical structure of cyclodextrins.Figure 14. Example of sugar acid, sugar alcohol, sugar aldehyde and sugar ketose for a three-carbon monosaccharide. Figure 15. Classification of aldoses (monosaccharides) according to their carbon number.Figure 16. Classification of ketoses (monosaccharides) according to their carbon number.Figure 17. Intramolecular cyclization of ribose into anomers in solution.Figure 18. Illustration of molecular duplication and loss of chiral information during the reduction of some

Figure 5 .

 5 Figure 1. Structure of the deoxyribonucleic acid.Figure 2. Genetic material built from chiral molecules. Figure 3. Schematic representation of the construction of circularly polarized light.Figure 4. Induced ees as a function of circularly polarized light orientation and wavelength.Figure 5. Model of interstellar ice chemistry (grain model).Figure 6. Sublimation chamber used for interstellar ice analogs studies.Figure 7. Strecker synthesis of an α-amino acid from an aldehyde and Michael addition of ammonia to acrylonitrile leading to a β-amino acid under abiotic conditions. Figure 8. Formose reaction chemical network.Figure 9. Synthesis of hexamethylene tetranime (HMT) from formaldehyde and ammonia reagents.Figure 10. Plausible chemical pathway leading to HMT under abiotic conditions using methanol, ammonia and UV-light. Figure 11. Schematic of two-dimensional gas chromatography (GC×GC).Figure 12. Schematic of the modulation process.Figure 13. Chemical structure of cyclodextrins.Figure 14. Example of sugar acid, sugar alcohol, sugar aldehyde and sugar ketose for a three-carbon monosaccharide. Figure 15. Classification of aldoses (monosaccharides) according to their carbon number.Figure 16. Classification of ketoses (monosaccharides) according to their carbon number.Figure 17. Intramolecular cyclization of ribose into anomers in solution.Figure 18. Illustration of molecular duplication and loss of chiral information during the reduction of some

Figure 12 .

 12 Figure 1. Structure of the deoxyribonucleic acid.Figure 2. Genetic material built from chiral molecules. Figure 3. Schematic representation of the construction of circularly polarized light.Figure 4. Induced ees as a function of circularly polarized light orientation and wavelength.Figure 5. Model of interstellar ice chemistry (grain model).Figure 6. Sublimation chamber used for interstellar ice analogs studies.Figure 7. Strecker synthesis of an α-amino acid from an aldehyde and Michael addition of ammonia to acrylonitrile leading to a β-amino acid under abiotic conditions. Figure 8. Formose reaction chemical network.Figure 9. Synthesis of hexamethylene tetranime (HMT) from formaldehyde and ammonia reagents.Figure 10. Plausible chemical pathway leading to HMT under abiotic conditions using methanol, ammonia and UV-light. Figure 11. Schematic of two-dimensional gas chromatography (GC×GC).Figure 12. Schematic of the modulation process.Figure 13. Chemical structure of cyclodextrins.Figure 14. Example of sugar acid, sugar alcohol, sugar aldehyde and sugar ketose for a three-carbon monosaccharide. Figure 15. Classification of aldoses (monosaccharides) according to their carbon number.Figure 16. Classification of ketoses (monosaccharides) according to their carbon number.Figure 17. Intramolecular cyclization of ribose into anomers in solution.Figure 18. Illustration of molecular duplication and loss of chiral information during the reduction of some

Figure 13 .

 13 Figure 1. Structure of the deoxyribonucleic acid.Figure 2. Genetic material built from chiral molecules. Figure 3. Schematic representation of the construction of circularly polarized light.Figure 4. Induced ees as a function of circularly polarized light orientation and wavelength.Figure 5. Model of interstellar ice chemistry (grain model).Figure 6. Sublimation chamber used for interstellar ice analogs studies.Figure 7. Strecker synthesis of an α-amino acid from an aldehyde and Michael addition of ammonia to acrylonitrile leading to a β-amino acid under abiotic conditions. Figure 8. Formose reaction chemical network.Figure 9. Synthesis of hexamethylene tetranime (HMT) from formaldehyde and ammonia reagents.Figure 10. Plausible chemical pathway leading to HMT under abiotic conditions using methanol, ammonia and UV-light. Figure 11. Schematic of two-dimensional gas chromatography (GC×GC).Figure 12. Schematic of the modulation process.Figure 13. Chemical structure of cyclodextrins.Figure 14. Example of sugar acid, sugar alcohol, sugar aldehyde and sugar ketose for a three-carbon monosaccharide. Figure 15. Classification of aldoses (monosaccharides) according to their carbon number.Figure 16. Classification of ketoses (monosaccharides) according to their carbon number.Figure 17. Intramolecular cyclization of ribose into anomers in solution.Figure 18. Illustration of molecular duplication and loss of chiral information during the reduction of some

Figure 14 .Figure 15 .

 1415 Figure 1. Structure of the deoxyribonucleic acid.Figure 2. Genetic material built from chiral molecules. Figure 3. Schematic representation of the construction of circularly polarized light.Figure 4. Induced ees as a function of circularly polarized light orientation and wavelength.Figure 5. Model of interstellar ice chemistry (grain model).Figure 6. Sublimation chamber used for interstellar ice analogs studies.Figure 7. Strecker synthesis of an α-amino acid from an aldehyde and Michael addition of ammonia to acrylonitrile leading to a β-amino acid under abiotic conditions. Figure 8. Formose reaction chemical network.Figure 9. Synthesis of hexamethylene tetranime (HMT) from formaldehyde and ammonia reagents.Figure 10. Plausible chemical pathway leading to HMT under abiotic conditions using methanol, ammonia and UV-light. Figure 11. Schematic of two-dimensional gas chromatography (GC×GC).Figure 12. Schematic of the modulation process.Figure 13. Chemical structure of cyclodextrins.Figure 14. Example of sugar acid, sugar alcohol, sugar aldehyde and sugar ketose for a three-carbon monosaccharide. Figure 15. Classification of aldoses (monosaccharides) according to their carbon number.Figure 16. Classification of ketoses (monosaccharides) according to their carbon number.Figure 17. Intramolecular cyclization of ribose into anomers in solution.Figure 18. Illustration of molecular duplication and loss of chiral information during the reduction of some

Figure 16 .

 16 Figure 1. Structure of the deoxyribonucleic acid.Figure 2. Genetic material built from chiral molecules. Figure 3. Schematic representation of the construction of circularly polarized light.Figure 4. Induced ees as a function of circularly polarized light orientation and wavelength.Figure 5. Model of interstellar ice chemistry (grain model).Figure 6. Sublimation chamber used for interstellar ice analogs studies.Figure 7. Strecker synthesis of an α-amino acid from an aldehyde and Michael addition of ammonia to acrylonitrile leading to a β-amino acid under abiotic conditions. Figure 8. Formose reaction chemical network.Figure 9. Synthesis of hexamethylene tetranime (HMT) from formaldehyde and ammonia reagents.Figure 10. Plausible chemical pathway leading to HMT under abiotic conditions using methanol, ammonia and UV-light. Figure 11. Schematic of two-dimensional gas chromatography (GC×GC).Figure 12. Schematic of the modulation process.Figure 13. Chemical structure of cyclodextrins.Figure 14. Example of sugar acid, sugar alcohol, sugar aldehyde and sugar ketose for a three-carbon monosaccharide. Figure 15. Classification of aldoses (monosaccharides) according to their carbon number.Figure 16. Classification of ketoses (monosaccharides) according to their carbon number.Figure 17. Intramolecular cyclization of ribose into anomers in solution.Figure 18. Illustration of molecular duplication and loss of chiral information during the reduction of some

Figure 17 .

 17 Figure 1. Structure of the deoxyribonucleic acid.Figure 2. Genetic material built from chiral molecules. Figure 3. Schematic representation of the construction of circularly polarized light.Figure 4. Induced ees as a function of circularly polarized light orientation and wavelength.Figure 5. Model of interstellar ice chemistry (grain model).Figure 6. Sublimation chamber used for interstellar ice analogs studies.Figure 7. Strecker synthesis of an α-amino acid from an aldehyde and Michael addition of ammonia to acrylonitrile leading to a β-amino acid under abiotic conditions. Figure 8. Formose reaction chemical network.Figure 9. Synthesis of hexamethylene tetranime (HMT) from formaldehyde and ammonia reagents.Figure 10. Plausible chemical pathway leading to HMT under abiotic conditions using methanol, ammonia and UV-light. Figure 11. Schematic of two-dimensional gas chromatography (GC×GC).Figure 12. Schematic of the modulation process.Figure 13. Chemical structure of cyclodextrins.Figure 14. Example of sugar acid, sugar alcohol, sugar aldehyde and sugar ketose for a three-carbon monosaccharide. Figure 15. Classification of aldoses (monosaccharides) according to their carbon number.Figure 16. Classification of ketoses (monosaccharides) according to their carbon number.Figure 17. Intramolecular cyclization of ribose into anomers in solution.Figure 18. Illustration of molecular duplication and loss of chiral information during the reduction of some

Figure 18 .

 18 Figure 1. Structure of the deoxyribonucleic acid.Figure 2. Genetic material built from chiral molecules. Figure 3. Schematic representation of the construction of circularly polarized light.Figure 4. Induced ees as a function of circularly polarized light orientation and wavelength.Figure 5. Model of interstellar ice chemistry (grain model).Figure 6. Sublimation chamber used for interstellar ice analogs studies.Figure 7. Strecker synthesis of an α-amino acid from an aldehyde and Michael addition of ammonia to acrylonitrile leading to a β-amino acid under abiotic conditions. Figure 8. Formose reaction chemical network.Figure 9. Synthesis of hexamethylene tetranime (HMT) from formaldehyde and ammonia reagents.Figure 10. Plausible chemical pathway leading to HMT under abiotic conditions using methanol, ammonia and UV-light. Figure 11. Schematic of two-dimensional gas chromatography (GC×GC).Figure 12. Schematic of the modulation process.Figure 13. Chemical structure of cyclodextrins.Figure 14. Example of sugar acid, sugar alcohol, sugar aldehyde and sugar ketose for a three-carbon monosaccharide. Figure 15. Classification of aldoses (monosaccharides) according to their carbon number.Figure 16. Classification of ketoses (monosaccharides) according to their carbon number.Figure 17. Intramolecular cyclization of ribose into anomers in solution.Figure 18. Illustration of molecular duplication and loss of chiral information during the reduction of some D-aldopentoses.

Figure 19 .

 19 Figure 19. Oximation derivatization starting from ribose.Figure 20. Aldonitrile acetate derivatization starting from glyceraldehyde.Figure 21. N,O-Bis(trimethylsilyl)trifluoroacetamide derivatization on ribose.Figure 22. Methylboronic acid/trifluoroacetic anhydride derivatization on C3, C5 and C6 monosaccharides.Figure 23. (S)-2-butanol/trifluoroacetic anhydride derivatization on ribose.Figure 24. N,N-dimethylformamide/dimethylacetal derivatization (DMF/DMA) of amino acids.Figure 25. N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) derivatization of amino acids.Figure 26. Ethylchloroformate/heptafluorobutanol derivatization (ECHFBE) of amino acids.Figure 27. Two-dimensional chromatogram of ECHFBE derivatized amino acids and analyzed on Chirasil-Val coupled to DB-WAX columns. Figure 28. Methanol/trifluoroacetic anhydride derivatization (MeOH/TFAA) of amino acids.Figure 29. Structure and properties of a cation exchange resin.Figure 30. Principle of cation exchange chromatography for the separation of carbohydrates and amino acids.Figure 31. General scheme of sampling, extraction, fractionation and purification, derivatization and analysis of a meteorite sample.Figure 32. Mass spectrum & fragmentation of alanine as N-trifluoroacetyl methyl ester derivative.Figure 33. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a Lipodex E coupled to a DB-WAX column.Figure 34. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column. Figure 35. Calibration curve for D-Alanine and L-Alanine using ECHFBE derivatization.Figure 36. Evolution of ee over four concentrations of five 5% L-doped amino acids.Figure 37. Two-dimensional chromatogram of MBA/TFAA derivatized sugars analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column.

Figure 20 .

 20 Figure 19. Oximation derivatization starting from ribose.Figure 20. Aldonitrile acetate derivatization starting from glyceraldehyde.Figure 21. N,O-Bis(trimethylsilyl)trifluoroacetamide derivatization on ribose.Figure 22. Methylboronic acid/trifluoroacetic anhydride derivatization on C3, C5 and C6 monosaccharides.Figure 23. (S)-2-butanol/trifluoroacetic anhydride derivatization on ribose.Figure 24. N,N-dimethylformamide/dimethylacetal derivatization (DMF/DMA) of amino acids.Figure 25. N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) derivatization of amino acids.Figure 26. Ethylchloroformate/heptafluorobutanol derivatization (ECHFBE) of amino acids.Figure 27. Two-dimensional chromatogram of ECHFBE derivatized amino acids and analyzed on Chirasil-Val coupled to DB-WAX columns. Figure 28. Methanol/trifluoroacetic anhydride derivatization (MeOH/TFAA) of amino acids.Figure 29. Structure and properties of a cation exchange resin.Figure 30. Principle of cation exchange chromatography for the separation of carbohydrates and amino acids.Figure 31. General scheme of sampling, extraction, fractionation and purification, derivatization and analysis of a meteorite sample.Figure 32. Mass spectrum & fragmentation of alanine as N-trifluoroacetyl methyl ester derivative.Figure 33. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a Lipodex E coupled to a DB-WAX column.Figure 34. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column. Figure 35. Calibration curve for D-Alanine and L-Alanine using ECHFBE derivatization.Figure 36. Evolution of ee over four concentrations of five 5% L-doped amino acids.Figure 37. Two-dimensional chromatogram of MBA/TFAA derivatized sugars analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column.

Figure 21 .

 21 Figure 19. Oximation derivatization starting from ribose.Figure 20. Aldonitrile acetate derivatization starting from glyceraldehyde.Figure 21. N,O-Bis(trimethylsilyl)trifluoroacetamide derivatization on ribose.Figure 22. Methylboronic acid/trifluoroacetic anhydride derivatization on C3, C5 and C6 monosaccharides.Figure 23. (S)-2-butanol/trifluoroacetic anhydride derivatization on ribose.Figure 24. N,N-dimethylformamide/dimethylacetal derivatization (DMF/DMA) of amino acids.Figure 25. N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) derivatization of amino acids.Figure 26. Ethylchloroformate/heptafluorobutanol derivatization (ECHFBE) of amino acids.Figure 27. Two-dimensional chromatogram of ECHFBE derivatized amino acids and analyzed on Chirasil-Val coupled to DB-WAX columns. Figure 28. Methanol/trifluoroacetic anhydride derivatization (MeOH/TFAA) of amino acids.Figure 29. Structure and properties of a cation exchange resin.Figure 30. Principle of cation exchange chromatography for the separation of carbohydrates and amino acids.Figure 31. General scheme of sampling, extraction, fractionation and purification, derivatization and analysis of a meteorite sample.Figure 32. Mass spectrum & fragmentation of alanine as N-trifluoroacetyl methyl ester derivative.Figure 33. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a Lipodex E coupled to a DB-WAX column.Figure 34. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column. Figure 35. Calibration curve for D-Alanine and L-Alanine using ECHFBE derivatization.Figure 36. Evolution of ee over four concentrations of five 5% L-doped amino acids.Figure 37. Two-dimensional chromatogram of MBA/TFAA derivatized sugars analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column.

Figure 22 .

 22 Figure 19. Oximation derivatization starting from ribose.Figure 20. Aldonitrile acetate derivatization starting from glyceraldehyde.Figure 21. N,O-Bis(trimethylsilyl)trifluoroacetamide derivatization on ribose.Figure 22. Methylboronic acid/trifluoroacetic anhydride derivatization on C3, C5 and C6 monosaccharides.Figure 23. (S)-2-butanol/trifluoroacetic anhydride derivatization on ribose.Figure 24. N,N-dimethylformamide/dimethylacetal derivatization (DMF/DMA) of amino acids.Figure 25. N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) derivatization of amino acids.Figure 26. Ethylchloroformate/heptafluorobutanol derivatization (ECHFBE) of amino acids.Figure 27. Two-dimensional chromatogram of ECHFBE derivatized amino acids and analyzed on Chirasil-Val coupled to DB-WAX columns. Figure 28. Methanol/trifluoroacetic anhydride derivatization (MeOH/TFAA) of amino acids.Figure 29. Structure and properties of a cation exchange resin.Figure 30. Principle of cation exchange chromatography for the separation of carbohydrates and amino acids.Figure 31. General scheme of sampling, extraction, fractionation and purification, derivatization and analysis of a meteorite sample.Figure 32. Mass spectrum & fragmentation of alanine as N-trifluoroacetyl methyl ester derivative.Figure 33. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a Lipodex E coupled to a DB-WAX column.Figure 34. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column. Figure 35. Calibration curve for D-Alanine and L-Alanine using ECHFBE derivatization.Figure 36. Evolution of ee over four concentrations of five 5% L-doped amino acids.Figure 37. Two-dimensional chromatogram of MBA/TFAA derivatized sugars analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column.

Figure 23 .

 23 Figure 19. Oximation derivatization starting from ribose.Figure 20. Aldonitrile acetate derivatization starting from glyceraldehyde.Figure 21. N,O-Bis(trimethylsilyl)trifluoroacetamide derivatization on ribose.Figure 22. Methylboronic acid/trifluoroacetic anhydride derivatization on C3, C5 and C6 monosaccharides.Figure 23. (S)-2-butanol/trifluoroacetic anhydride derivatization on ribose.Figure 24. N,N-dimethylformamide/dimethylacetal derivatization (DMF/DMA) of amino acids.Figure 25. N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) derivatization of amino acids.Figure 26. Ethylchloroformate/heptafluorobutanol derivatization (ECHFBE) of amino acids.Figure 27. Two-dimensional chromatogram of ECHFBE derivatized amino acids and analyzed on Chirasil-Val coupled to DB-WAX columns. Figure 28. Methanol/trifluoroacetic anhydride derivatization (MeOH/TFAA) of amino acids.Figure 29. Structure and properties of a cation exchange resin.Figure 30. Principle of cation exchange chromatography for the separation of carbohydrates and amino acids.Figure 31. General scheme of sampling, extraction, fractionation and purification, derivatization and analysis of a meteorite sample.Figure 32. Mass spectrum & fragmentation of alanine as N-trifluoroacetyl methyl ester derivative.Figure 33. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a Lipodex E coupled to a DB-WAX column.Figure 34. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column. Figure 35. Calibration curve for D-Alanine and L-Alanine using ECHFBE derivatization.Figure 36. Evolution of ee over four concentrations of five 5% L-doped amino acids.Figure 37. Two-dimensional chromatogram of MBA/TFAA derivatized sugars analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column.

Figure 24 .

 24 Figure 19. Oximation derivatization starting from ribose.Figure 20. Aldonitrile acetate derivatization starting from glyceraldehyde.Figure 21. N,O-Bis(trimethylsilyl)trifluoroacetamide derivatization on ribose.Figure 22. Methylboronic acid/trifluoroacetic anhydride derivatization on C3, C5 and C6 monosaccharides.Figure 23. (S)-2-butanol/trifluoroacetic anhydride derivatization on ribose.Figure 24. N,N-dimethylformamide/dimethylacetal derivatization (DMF/DMA) of amino acids.Figure 25. N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) derivatization of amino acids.Figure 26. Ethylchloroformate/heptafluorobutanol derivatization (ECHFBE) of amino acids.Figure 27. Two-dimensional chromatogram of ECHFBE derivatized amino acids and analyzed on Chirasil-Val coupled to DB-WAX columns. Figure 28. Methanol/trifluoroacetic anhydride derivatization (MeOH/TFAA) of amino acids.Figure 29. Structure and properties of a cation exchange resin.Figure 30. Principle of cation exchange chromatography for the separation of carbohydrates and amino acids.Figure 31. General scheme of sampling, extraction, fractionation and purification, derivatization and analysis of a meteorite sample.Figure 32. Mass spectrum & fragmentation of alanine as N-trifluoroacetyl methyl ester derivative.Figure 33. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a Lipodex E coupled to a DB-WAX column.Figure 34. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column. Figure 35. Calibration curve for D-Alanine and L-Alanine using ECHFBE derivatization.Figure 36. Evolution of ee over four concentrations of five 5% L-doped amino acids.Figure 37. Two-dimensional chromatogram of MBA/TFAA derivatized sugars analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column.

Figure 25 .

 25 Figure 19. Oximation derivatization starting from ribose.Figure 20. Aldonitrile acetate derivatization starting from glyceraldehyde.Figure 21. N,O-Bis(trimethylsilyl)trifluoroacetamide derivatization on ribose.Figure 22. Methylboronic acid/trifluoroacetic anhydride derivatization on C3, C5 and C6 monosaccharides.Figure 23. (S)-2-butanol/trifluoroacetic anhydride derivatization on ribose.Figure 24. N,N-dimethylformamide/dimethylacetal derivatization (DMF/DMA) of amino acids.Figure 25. N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) derivatization of amino acids.Figure 26. Ethylchloroformate/heptafluorobutanol derivatization (ECHFBE) of amino acids.Figure 27. Two-dimensional chromatogram of ECHFBE derivatized amino acids and analyzed on Chirasil-Val coupled to DB-WAX columns. Figure 28. Methanol/trifluoroacetic anhydride derivatization (MeOH/TFAA) of amino acids.Figure 29. Structure and properties of a cation exchange resin.Figure 30. Principle of cation exchange chromatography for the separation of carbohydrates and amino acids.Figure 31. General scheme of sampling, extraction, fractionation and purification, derivatization and analysis of a meteorite sample.Figure 32. Mass spectrum & fragmentation of alanine as N-trifluoroacetyl methyl ester derivative.Figure 33. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a Lipodex E coupled to a DB-WAX column.Figure 34. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column. Figure 35. Calibration curve for D-Alanine and L-Alanine using ECHFBE derivatization.Figure 36. Evolution of ee over four concentrations of five 5% L-doped amino acids.Figure 37. Two-dimensional chromatogram of MBA/TFAA derivatized sugars analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column.

Figure 26 .

 26 Figure 19. Oximation derivatization starting from ribose.Figure 20. Aldonitrile acetate derivatization starting from glyceraldehyde.Figure 21. N,O-Bis(trimethylsilyl)trifluoroacetamide derivatization on ribose.Figure 22. Methylboronic acid/trifluoroacetic anhydride derivatization on C3, C5 and C6 monosaccharides.Figure 23. (S)-2-butanol/trifluoroacetic anhydride derivatization on ribose.Figure 24. N,N-dimethylformamide/dimethylacetal derivatization (DMF/DMA) of amino acids.Figure 25. N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) derivatization of amino acids.Figure 26. Ethylchloroformate/heptafluorobutanol derivatization (ECHFBE) of amino acids.Figure 27. Two-dimensional chromatogram of ECHFBE derivatized amino acids and analyzed on Chirasil-Val coupled to DB-WAX columns. Figure 28. Methanol/trifluoroacetic anhydride derivatization (MeOH/TFAA) of amino acids.Figure 29. Structure and properties of a cation exchange resin.Figure 30. Principle of cation exchange chromatography for the separation of carbohydrates and amino acids.Figure 31. General scheme of sampling, extraction, fractionation and purification, derivatization and analysis of a meteorite sample.Figure 32. Mass spectrum & fragmentation of alanine as N-trifluoroacetyl methyl ester derivative.Figure 33. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a Lipodex E coupled to a DB-WAX column.Figure 34. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column. Figure 35. Calibration curve for D-Alanine and L-Alanine using ECHFBE derivatization.Figure 36. Evolution of ee over four concentrations of five 5% L-doped amino acids.Figure 37. Two-dimensional chromatogram of MBA/TFAA derivatized sugars analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column.

Figure 27 .Figure 28 .

 2728 Figure 19. Oximation derivatization starting from ribose.Figure 20. Aldonitrile acetate derivatization starting from glyceraldehyde.Figure 21. N,O-Bis(trimethylsilyl)trifluoroacetamide derivatization on ribose.Figure 22. Methylboronic acid/trifluoroacetic anhydride derivatization on C3, C5 and C6 monosaccharides.Figure 23. (S)-2-butanol/trifluoroacetic anhydride derivatization on ribose.Figure 24. N,N-dimethylformamide/dimethylacetal derivatization (DMF/DMA) of amino acids.Figure 25. N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) derivatization of amino acids.Figure 26. Ethylchloroformate/heptafluorobutanol derivatization (ECHFBE) of amino acids.Figure 27. Two-dimensional chromatogram of ECHFBE derivatized amino acids and analyzed on Chirasil-Val coupled to DB-WAX columns. Figure 28. Methanol/trifluoroacetic anhydride derivatization (MeOH/TFAA) of amino acids.Figure 29. Structure and properties of a cation exchange resin.Figure 30. Principle of cation exchange chromatography for the separation of carbohydrates and amino acids.Figure 31. General scheme of sampling, extraction, fractionation and purification, derivatization and analysis of a meteorite sample.Figure 32. Mass spectrum & fragmentation of alanine as N-trifluoroacetyl methyl ester derivative.Figure 33. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a Lipodex E coupled to a DB-WAX column.Figure 34. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column. Figure 35. Calibration curve for D-Alanine and L-Alanine using ECHFBE derivatization.Figure 36. Evolution of ee over four concentrations of five 5% L-doped amino acids.Figure 37. Two-dimensional chromatogram of MBA/TFAA derivatized sugars analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column.

Figure 29 .

 29 Figure 19. Oximation derivatization starting from ribose.Figure 20. Aldonitrile acetate derivatization starting from glyceraldehyde.Figure 21. N,O-Bis(trimethylsilyl)trifluoroacetamide derivatization on ribose.Figure 22. Methylboronic acid/trifluoroacetic anhydride derivatization on C3, C5 and C6 monosaccharides.Figure 23. (S)-2-butanol/trifluoroacetic anhydride derivatization on ribose.Figure 24. N,N-dimethylformamide/dimethylacetal derivatization (DMF/DMA) of amino acids.Figure 25. N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) derivatization of amino acids.Figure 26. Ethylchloroformate/heptafluorobutanol derivatization (ECHFBE) of amino acids.Figure 27. Two-dimensional chromatogram of ECHFBE derivatized amino acids and analyzed on Chirasil-Val coupled to DB-WAX columns. Figure 28. Methanol/trifluoroacetic anhydride derivatization (MeOH/TFAA) of amino acids.Figure 29. Structure and properties of a cation exchange resin.Figure 30. Principle of cation exchange chromatography for the separation of carbohydrates and amino acids.Figure 31. General scheme of sampling, extraction, fractionation and purification, derivatization and analysis of a meteorite sample.Figure 32. Mass spectrum & fragmentation of alanine as N-trifluoroacetyl methyl ester derivative.Figure 33. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a Lipodex E coupled to a DB-WAX column.Figure 34. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column. Figure 35. Calibration curve for D-Alanine and L-Alanine using ECHFBE derivatization.Figure 36. Evolution of ee over four concentrations of five 5% L-doped amino acids.Figure 37. Two-dimensional chromatogram of MBA/TFAA derivatized sugars analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column.

Figure 30 .

 30 Figure 19. Oximation derivatization starting from ribose.Figure 20. Aldonitrile acetate derivatization starting from glyceraldehyde.Figure 21. N,O-Bis(trimethylsilyl)trifluoroacetamide derivatization on ribose.Figure 22. Methylboronic acid/trifluoroacetic anhydride derivatization on C3, C5 and C6 monosaccharides.Figure 23. (S)-2-butanol/trifluoroacetic anhydride derivatization on ribose.Figure 24. N,N-dimethylformamide/dimethylacetal derivatization (DMF/DMA) of amino acids.Figure 25. N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) derivatization of amino acids.Figure 26. Ethylchloroformate/heptafluorobutanol derivatization (ECHFBE) of amino acids.Figure 27. Two-dimensional chromatogram of ECHFBE derivatized amino acids and analyzed on Chirasil-Val coupled to DB-WAX columns. Figure 28. Methanol/trifluoroacetic anhydride derivatization (MeOH/TFAA) of amino acids.Figure 29. Structure and properties of a cation exchange resin.Figure 30. Principle of cation exchange chromatography for the separation of carbohydrates and amino acids.Figure 31. General scheme of sampling, extraction, fractionation and purification, derivatization and analysis of a meteorite sample.Figure 32. Mass spectrum & fragmentation of alanine as N-trifluoroacetyl methyl ester derivative.Figure 33. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a Lipodex E coupled to a DB-WAX column.Figure 34. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column. Figure 35. Calibration curve for D-Alanine and L-Alanine using ECHFBE derivatization.Figure 36. Evolution of ee over four concentrations of five 5% L-doped amino acids.Figure 37. Two-dimensional chromatogram of MBA/TFAA derivatized sugars analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column.

Figure 31 .

 31 Figure 19. Oximation derivatization starting from ribose.Figure 20. Aldonitrile acetate derivatization starting from glyceraldehyde.Figure 21. N,O-Bis(trimethylsilyl)trifluoroacetamide derivatization on ribose.Figure 22. Methylboronic acid/trifluoroacetic anhydride derivatization on C3, C5 and C6 monosaccharides.Figure 23. (S)-2-butanol/trifluoroacetic anhydride derivatization on ribose.Figure 24. N,N-dimethylformamide/dimethylacetal derivatization (DMF/DMA) of amino acids.Figure 25. N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) derivatization of amino acids.Figure 26. Ethylchloroformate/heptafluorobutanol derivatization (ECHFBE) of amino acids.Figure 27. Two-dimensional chromatogram of ECHFBE derivatized amino acids and analyzed on Chirasil-Val coupled to DB-WAX columns. Figure 28. Methanol/trifluoroacetic anhydride derivatization (MeOH/TFAA) of amino acids.Figure 29. Structure and properties of a cation exchange resin.Figure 30. Principle of cation exchange chromatography for the separation of carbohydrates and amino acids.Figure 31. General scheme of sampling, extraction, fractionation and purification, derivatization and analysis of a meteorite sample.Figure 32. Mass spectrum & fragmentation of alanine as N-trifluoroacetyl methyl ester derivative.Figure 33. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a Lipodex E coupled to a DB-WAX column.Figure 34. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column. Figure 35. Calibration curve for D-Alanine and L-Alanine using ECHFBE derivatization.Figure 36. Evolution of ee over four concentrations of five 5% L-doped amino acids.Figure 37. Two-dimensional chromatogram of MBA/TFAA derivatized sugars analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column.

Figure 32 .

 32 Figure 19. Oximation derivatization starting from ribose.Figure 20. Aldonitrile acetate derivatization starting from glyceraldehyde.Figure 21. N,O-Bis(trimethylsilyl)trifluoroacetamide derivatization on ribose.Figure 22. Methylboronic acid/trifluoroacetic anhydride derivatization on C3, C5 and C6 monosaccharides.Figure 23. (S)-2-butanol/trifluoroacetic anhydride derivatization on ribose.Figure 24. N,N-dimethylformamide/dimethylacetal derivatization (DMF/DMA) of amino acids.Figure 25. N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) derivatization of amino acids.Figure 26. Ethylchloroformate/heptafluorobutanol derivatization (ECHFBE) of amino acids.Figure 27. Two-dimensional chromatogram of ECHFBE derivatized amino acids and analyzed on Chirasil-Val coupled to DB-WAX columns. Figure 28. Methanol/trifluoroacetic anhydride derivatization (MeOH/TFAA) of amino acids.Figure 29. Structure and properties of a cation exchange resin.Figure 30. Principle of cation exchange chromatography for the separation of carbohydrates and amino acids.Figure 31. General scheme of sampling, extraction, fractionation and purification, derivatization and analysis of a meteorite sample.Figure 32. Mass spectrum & fragmentation of alanine as N-trifluoroacetyl methyl ester derivative.Figure 33. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a Lipodex E coupled to a DB-WAX column.Figure 34. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column. Figure 35. Calibration curve for D-Alanine and L-Alanine using ECHFBE derivatization.Figure 36. Evolution of ee over four concentrations of five 5% L-doped amino acids.Figure 37. Two-dimensional chromatogram of MBA/TFAA derivatized sugars analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column.

Figure 33 .

 33 Figure 19. Oximation derivatization starting from ribose.Figure 20. Aldonitrile acetate derivatization starting from glyceraldehyde.Figure 21. N,O-Bis(trimethylsilyl)trifluoroacetamide derivatization on ribose.Figure 22. Methylboronic acid/trifluoroacetic anhydride derivatization on C3, C5 and C6 monosaccharides.Figure 23. (S)-2-butanol/trifluoroacetic anhydride derivatization on ribose.Figure 24. N,N-dimethylformamide/dimethylacetal derivatization (DMF/DMA) of amino acids.Figure 25. N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) derivatization of amino acids.Figure 26. Ethylchloroformate/heptafluorobutanol derivatization (ECHFBE) of amino acids.Figure 27. Two-dimensional chromatogram of ECHFBE derivatized amino acids and analyzed on Chirasil-Val coupled to DB-WAX columns. Figure 28. Methanol/trifluoroacetic anhydride derivatization (MeOH/TFAA) of amino acids.Figure 29. Structure and properties of a cation exchange resin.Figure 30. Principle of cation exchange chromatography for the separation of carbohydrates and amino acids.Figure 31. General scheme of sampling, extraction, fractionation and purification, derivatization and analysis of a meteorite sample.Figure 32. Mass spectrum & fragmentation of alanine as N-trifluoroacetyl methyl ester derivative.Figure 33. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a Lipodex E coupled to a DB-WAX column.Figure 34. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column. Figure 35. Calibration curve for D-Alanine and L-Alanine using ECHFBE derivatization.Figure 36. Evolution of ee over four concentrations of five 5% L-doped amino acids.Figure 37. Two-dimensional chromatogram of MBA/TFAA derivatized sugars analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column.

Figure 34 .Figure 35 .

 3435 Figure 19. Oximation derivatization starting from ribose.Figure 20. Aldonitrile acetate derivatization starting from glyceraldehyde.Figure 21. N,O-Bis(trimethylsilyl)trifluoroacetamide derivatization on ribose.Figure 22. Methylboronic acid/trifluoroacetic anhydride derivatization on C3, C5 and C6 monosaccharides.Figure 23. (S)-2-butanol/trifluoroacetic anhydride derivatization on ribose.Figure 24. N,N-dimethylformamide/dimethylacetal derivatization (DMF/DMA) of amino acids.Figure 25. N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) derivatization of amino acids.Figure 26. Ethylchloroformate/heptafluorobutanol derivatization (ECHFBE) of amino acids.Figure 27. Two-dimensional chromatogram of ECHFBE derivatized amino acids and analyzed on Chirasil-Val coupled to DB-WAX columns. Figure 28. Methanol/trifluoroacetic anhydride derivatization (MeOH/TFAA) of amino acids.Figure 29. Structure and properties of a cation exchange resin.Figure 30. Principle of cation exchange chromatography for the separation of carbohydrates and amino acids.Figure 31. General scheme of sampling, extraction, fractionation and purification, derivatization and analysis of a meteorite sample.Figure 32. Mass spectrum & fragmentation of alanine as N-trifluoroacetyl methyl ester derivative.Figure 33. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a Lipodex E coupled to a DB-WAX column.Figure 34. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column. Figure 35. Calibration curve for D-Alanine and L-Alanine using ECHFBE derivatization.Figure 36. Evolution of ee over four concentrations of five 5% L-doped amino acids.Figure 37. Two-dimensional chromatogram of MBA/TFAA derivatized sugars analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column.

Figure 36 .

 36 Figure 19. Oximation derivatization starting from ribose.Figure 20. Aldonitrile acetate derivatization starting from glyceraldehyde.Figure 21. N,O-Bis(trimethylsilyl)trifluoroacetamide derivatization on ribose.Figure 22. Methylboronic acid/trifluoroacetic anhydride derivatization on C3, C5 and C6 monosaccharides.Figure 23. (S)-2-butanol/trifluoroacetic anhydride derivatization on ribose.Figure 24. N,N-dimethylformamide/dimethylacetal derivatization (DMF/DMA) of amino acids.Figure 25. N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) derivatization of amino acids.Figure 26. Ethylchloroformate/heptafluorobutanol derivatization (ECHFBE) of amino acids.Figure 27. Two-dimensional chromatogram of ECHFBE derivatized amino acids and analyzed on Chirasil-Val coupled to DB-WAX columns. Figure 28. Methanol/trifluoroacetic anhydride derivatization (MeOH/TFAA) of amino acids.Figure 29. Structure and properties of a cation exchange resin.Figure 30. Principle of cation exchange chromatography for the separation of carbohydrates and amino acids.Figure 31. General scheme of sampling, extraction, fractionation and purification, derivatization and analysis of a meteorite sample.Figure 32. Mass spectrum & fragmentation of alanine as N-trifluoroacetyl methyl ester derivative.Figure 33. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a Lipodex E coupled to a DB-WAX column.Figure 34. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column. Figure 35. Calibration curve for D-Alanine and L-Alanine using ECHFBE derivatization.Figure 36. Evolution of ee over four concentrations of five 5% L-doped amino acids.Figure 37. Two-dimensional chromatogram of MBA/TFAA derivatized sugars analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column.

Figure 37 .Figure 38 .

 3738 Figure 19. Oximation derivatization starting from ribose.Figure 20. Aldonitrile acetate derivatization starting from glyceraldehyde.Figure 21. N,O-Bis(trimethylsilyl)trifluoroacetamide derivatization on ribose.Figure 22. Methylboronic acid/trifluoroacetic anhydride derivatization on C3, C5 and C6 monosaccharides.Figure 23. (S)-2-butanol/trifluoroacetic anhydride derivatization on ribose.Figure 24. N,N-dimethylformamide/dimethylacetal derivatization (DMF/DMA) of amino acids.Figure 25. N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) derivatization of amino acids.Figure 26. Ethylchloroformate/heptafluorobutanol derivatization (ECHFBE) of amino acids.Figure 27. Two-dimensional chromatogram of ECHFBE derivatized amino acids and analyzed on Chirasil-Val coupled to DB-WAX columns. Figure 28. Methanol/trifluoroacetic anhydride derivatization (MeOH/TFAA) of amino acids.Figure 29. Structure and properties of a cation exchange resin.Figure 30. Principle of cation exchange chromatography for the separation of carbohydrates and amino acids.Figure 31. General scheme of sampling, extraction, fractionation and purification, derivatization and analysis of a meteorite sample.Figure 32. Mass spectrum & fragmentation of alanine as N-trifluoroacetyl methyl ester derivative.Figure 33. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a Lipodex E coupled to a DB-WAX column.Figure 34. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column. Figure 35. Calibration curve for D-Alanine and L-Alanine using ECHFBE derivatization.Figure 36. Evolution of ee over four concentrations of five 5% L-doped amino acids.Figure 37. Two-dimensional chromatogram of MBA/TFAA derivatized sugars analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column.

Figure 39 .Figure 40 .Figure 41 .

 394041 Figure 38. Schematic of glass tubes containing IEC resin and Teflon frits.Figure 39. Recoveries of amino acids after ion exchange chromatography and MeOH/TFAA derivatized. Figure 40. Two-dimensional chromatograms of Iva retention time area highlighting derivatization reagents background noise induced by IEC. Figure 41. Schematic of the HMT sample processing. Figure 42. Proposed pathway for the formation of amino acids from the decomposition of HMT followed by the formose reaction in the presence of ammonia. Figure 43. Two-dimensional chromatogram of MBA/TFAA derivatized FGO2 sample analyzed on CP-Chirasil-Dex CB coupled to DB-WAX columns. Figure 44. Two-dimensional chromatogram of (S)-BuOH/TFAA derivatized FGO2 sample analyzed on CP-Chirasil-Dex CB coupled to DB-WAX columns. Figure 45. Two-dimensional chromatograms of FGO2, FG2α, FGO7 and FG7α.Figure 46. Evolution of sugar content using calcium hydroxide and olivine as catalysts for the formose reaction on days 2, 7 and 45. Figure 47. Evolution of sugar alcohols and dihydroxyacetone content using calcium hydroxide and olivine as catalysts for the formose reaction on days 2, 7, and 45.Figure 48. D-Enantiomeric excess in calcium hydroxide and olivine catalyzed formose reaction on day 2.

Figure 42 .Figure 43 .Figure 44 .Figure 45 .

 42434445 Figure 38. Schematic of glass tubes containing IEC resin and Teflon frits.Figure 39. Recoveries of amino acids after ion exchange chromatography and MeOH/TFAA derivatized. Figure 40. Two-dimensional chromatograms of Iva retention time area highlighting derivatization reagents background noise induced by IEC. Figure 41. Schematic of the HMT sample processing. Figure 42. Proposed pathway for the formation of amino acids from the decomposition of HMT followed by the formose reaction in the presence of ammonia. Figure 43. Two-dimensional chromatogram of MBA/TFAA derivatized FGO2 sample analyzed on CP-Chirasil-Dex CB coupled to DB-WAX columns. Figure 44. Two-dimensional chromatogram of (S)-BuOH/TFAA derivatized FGO2 sample analyzed on CP-Chirasil-Dex CB coupled to DB-WAX columns. Figure 45. Two-dimensional chromatograms of FGO2, FG2α, FGO7 and FG7α.Figure 46. Evolution of sugar content using calcium hydroxide and olivine as catalysts for the formose reaction on days 2, 7 and 45. Figure 47. Evolution of sugar alcohols and dihydroxyacetone content using calcium hydroxide and olivine as catalysts for the formose reaction on days 2, 7, and 45.Figure 48. D-Enantiomeric excess in calcium hydroxide and olivine catalyzed formose reaction on day 2.

Figure 46 .Figure 47 .

 4647 Figure 38. Schematic of glass tubes containing IEC resin and Teflon frits.Figure 39. Recoveries of amino acids after ion exchange chromatography and MeOH/TFAA derivatized. Figure 40. Two-dimensional chromatograms of Iva retention time area highlighting derivatization reagents background noise induced by IEC. Figure 41. Schematic of the HMT sample processing. Figure 42. Proposed pathway for the formation of amino acids from the decomposition of HMT followed by the formose reaction in the presence of ammonia. Figure 43. Two-dimensional chromatogram of MBA/TFAA derivatized FGO2 sample analyzed on CP-Chirasil-Dex CB coupled to DB-WAX columns. Figure 44. Two-dimensional chromatogram of (S)-BuOH/TFAA derivatized FGO2 sample analyzed on CP-Chirasil-Dex CB coupled to DB-WAX columns. Figure 45. Two-dimensional chromatograms of FGO2, FG2α, FGO7 and FG7α.Figure 46. Evolution of sugar content using calcium hydroxide and olivine as catalysts for the formose reaction on days 2, 7 and 45. Figure 47. Evolution of sugar alcohols and dihydroxyacetone content using calcium hydroxide and olivine as catalysts for the formose reaction on days 2, 7, and 45.Figure 48. D-Enantiomeric excess in calcium hydroxide and olivine catalyzed formose reaction on day 2.

Figure S 1 .

 1 Figure S 1. Alanine residual plot calculated from the calibration curve.

Figure 1 .

 1 Figure 1. Structure of the deoxyribonucleic acid. In blue, the D-2-deoxyribose is highlighted and in black, the phosphate. Both constitute the skeleton of the DNA. In color, the nucleobases are highlighted and establish Hbonds between the pairs.

Figure 2 .

 2 Figure 2. Genetic material built from chiral molecules.

Figure 3 .

 3 Figure 3. Schematic representation of the construction of circularly polarized light. Vertically and horizontally polarized waves are combined with a λ/4 wavelength shift and lead to CPL which has a helical path.

Figure 4 .

 4 Figure 4. Induced ees as a function of circularly polarized light orientation and wavelength. (Left) Anisotropy spectrum of D-alanine (gold) and L-alanine (blue) in the solid state. (Right) Enantioselective GC×GC analysis of initially racemic alanine irradiated with the l-CPL anisotropic maximum (λ=200 nm, top) and the r-CPL anisotropic maximum (λ=184 nm, bottom).

Figure 5 .

 5 Figure 5. Model of interstellar ice chemistry (grain model). From C. Meinert et al.[START_REF] Meinert | Photochemical Models on the Absolute Asymmetric Formation of Amino Acids in Interstellar Space[END_REF]

Figure 6

 6 Figure6shows a typical experimental setup for experiments to simulate the complex chemistry of interstellar ice. Vacuum pumps and a cryostat ensure that interstellar conditions (≈ 10 K and ≈ 10 -8 Torr) are reproduced while gaseous compounds condense on a window for subsequent irradiation with an energy source to trigger chemical reactions. An IR source and detector can be used to monitor the in situ evolution of the ice composition.

Figure 6 .

 6 Figure 6. Sublimation chamber used for interstellar ice analogs studies.

Figure 7 .

 7 Figure 7. Strecker synthesis of an α-amino acid from an aldehyde (left) and Michael addition of ammonia to acrylonitrile leading to a β-amino acid (right) under abiotic conditions.

Figure 8 .

 8 Figure 8. Formose reaction chemical network. Inspired by M.Haas et al. 154

Figure 9 .

 9 Figure 9. Synthesis of hexamethylenetetramine (HMT) from formaldehyde and ammonia reagents.

Figure 10 .

 10 Figure10. Plausible chemical pathway leading to HMT under abiotic conditions using methanol, ammonia and UVlight. Inspired by M.Caro et al. 161 and M.P.Bernstein et al. 162 .

Figure 11 .

 11 Figure 11. Schematic of two-dimensional gas chromatography (GC×GC).

Figure 12 .

 12 Figure 12. Schematic of the modulation process. (Top left) Successive steps from A to E showing the periodic trapping and release of the modulator between the two columns. (Bottom left) Response of the detector as a function of time. Shaded lines represent the detector response if no second column and no modulator were installed. (Bottom right) Schematic software reconstruction of the chromatogram as a stack of slices. (Top right) Three-dimensional view of the two-dimensional chromatogram.

  ) are cyclic oligosaccharides that possess a conical structure allowing non-covalent hostguest inclusion in its cavity. CDs are constructed from D-glucopyranose units in chair conformation and obtained by treatment of starch with amylase enzymes called cyclodextrinases 187 . The hydrolysis of starch by amylases is not very specific and ends up forming a mixture of α-, β-, γ-, δ-cyclodextrins as well as CDs containing up to 13 glucose units through α-1,4 linkages 188 . From these mixtures, α-, β-, γ-, δ-cyclodextrins were successfully isolated by selective precipitation using organic compounds. Smaller CDs, containing less than 6 glucose units, however, are not encountered because steric hindrance would stretch the glucose units. Examples of native and modified cyclodextrins are shown in Figure13.

Figure 13 .

 13 Figure 13. Chemical structure of cyclodextrins. (Left) Chemical structure of native α (n=6) cyclodextrins composed of D-glucopyranose units in chair conformation. The primary alcohols, in blue, represent the narrowest tapered ends while the secondary alcohols, in red, represent the widest tapered end. (Right) Chemical structure of octakis-(3-Obutyryl-2,6-Di-O-pentyl)-γ-cyclodextrin.

2. 1

 1 .3.1.4 CP-Chirasil-Dex CB Commercialized as CP-Chirasil-Dex CB, heptakis-(2,3,6-tri-O-methyl)-β-cyclodextrin is a fully methyl substituted β-cyclodextrin containing seven glucose units bound to a polysiloxane backbone. In the search of best enantiomeric separation of volatile organics for in situ analysis, C.Freissinet et al. 197 showed that this stationary phase is the most suitable compared to Chiraldex β-PM which is the same as CP-Chirasil-Dex CB but is not bound to a polysiloxane backbone as well as CP-Chirasil L-Val which is typically used for amino acid analysis. Indeed, among the 14 chiral compounds investigated of four different chemical families, 11, 7 and 3 of them showed an enantiomeric separation on CP-Chirasil-Dex CB, Chiraldex β-PM and CP-Chirasil L-Val, respectively. This stationary phase has already been employed for meteorite analyses of amino acids, hydroxy acids, amines 198 and sugars 113 .

Figure 14 .

 14 Figure 14. Example of sugar acid, sugar alcohol, sugar aldehyde and sugar ketose for a three-carbon monosaccharide.

Figure 16 .

 16 Figure16. In terms of stereochemistry, they can be found in their D or L form depending on the configuration of the penultimate stereogenic carbon. Other configurations of stereogenic carbons have led to compounds with new names.

Figure 15 .

 15 Figure 15. Classification of aldoses (monosaccharides) according to their carbon number. In green is highlighted the aldehyde function and in red the enantiomeric configuration for which the D is obtained. From top to bottom, each line represents the addition of one carbon from triose (aldotriose) to hexose (aldohexose).

Figure 16 .

 16 Figure16. Classification of ketoses (monosaccharides) according to their carbon number. In green is highlighted the ketone function and in red the enantiomeric configuration for which the D is obtained. From top to bottom, each line represents the addition of one carbon from tetrose (ketotetrose) to hexose (ketohexose).

Figure 17 .

 17 Figure 17. Intramolecular cyclization of ribose into anomers in solution.

Figure 18 .

 18 Figure 18. Illustration of molecular duplication (left and right) and loss of chiral information (middle) during the reduction of some D-aldopentoses.

Figure 19 .

 19 Figure 19. Oximation derivatization starting from ribose. 2.2.1.2.3 Aldonitrile acetate Introduced by Guerrant & Moss 221 , monosaccharides are constrained in their straight form because the nitrile function prevents any cyclization reaction. As with oximes, the first step involves the transformation of the carbonyl of the monosaccharide using hydroxylamine. Moreover, during this step, the presence of methanol and pyridine increases the basicity and thus the nucleophilic behavior of the hydroxylamine. The heating during the first step allows the formation of the nitrile function by the elimination of a molecule of water. The addition of acetic anhydride in the second step converts the hydroxyl groups to esters.

Figure 20 .

 20 Figure 20. Aldonitrile acetate derivatization starting from glyceraldehyde.

Figure 21 .

 21 Figure 21. N,O-Bis(trimethylsilyl)trifluoroacetamide derivatization on ribose.

Figure 22 .

 22 Figure 22. Methylboronic acid/trifluoroacetic anhydride derivatization on C3, C5 and C6 monosaccharides.

Figure 23 .

 23 Figure 23. (S)-2-butanol/trifluoroacetic anhydride derivatization on ribose.

Figure 24 .Figure 25 ,

 2425 Figure 24. N,N-dimethylformamide/dimethylacetal derivatization (DMF/DMA) of amino acids.

Figure 25 .

 25 Figure 25. N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) derivatization of amino acids.

Figure 26 .

 26 Figure 26. Ethylchloroformate/heptafluorobutanol derivatization (ECHFBE) of amino acids.

Figure 27 .

 27 Figure 27. Two-dimensional chromatogram of ECHFBE derivatized amino acids and analyzed on Chirasil-Val coupled to DB-WAX columns 233 .

Figure 28 .

 28 Figure 28. Methanol/trifluoroacetic anhydride derivatization (MeOH/TFAA) of amino acids.

  Low cross-linking values, as shown in Figure 29, are associated with open and permeable structures with respect to high molecular weight compounds. They are also less resistant in terms of swelling and shrinkage, and less efficient/selective in separating compounds.

Figure 29 .

 29 Figure 29. Structure and properties of a cation exchange resin. (Left) Sulfonated poly(styrene-co-divinylbenzene) copolymer used as ion exchange resin in the H + form. (Right) Approximate equivalence between cross-linking valuesand molecular weight exclusion limit.

= 11 . 8

 118 cations generally have a greater affinity for the sulfonate binding site than H + ions and are therefore exchanged and retained on the polymer resin beads. The use of water as the first elution solvent allows neutral and negatively charged species to elute directly, without being retained. Carbohydrates have pKA values around10-12 and are therefore neutral. Then, selective elution of amino acids can be achieved using appropriate pH and ionic strength. The use of ammonium hydroxide NH4OH as a second elution solvent outperforms the R-NH3 + amino acids due to its high NH4 + concentration despite their similar affinity. It should also be noted that the introduction of ammonium hydroxide raises the pH above the pKA of the ammonium ion with 𝑝𝐾 𝐴 𝐴 ≈ 9 -10, 𝑝𝐾 𝐴 𝐶 ≈ 2 -2.5 being the pKA of the ammonium and carboxylate ions, respectively. Indeed, at 2 M NH4OH, 𝑝𝐻 = 7 for a weak base.

Figure 30 .

 30 Figure 30. Principle of cation exchange chromatography for the separation of carbohydrates and amino acids.

  sonication. Generation of hydroxyl radicals is also frequency dependent 266 , making low frequencies less likely to lead to degradation. Intensity is also an important parameter because the extraction of the analytes increases with the amplitude of vibration, until a maximum value is reached 268 . High values are not necessarily required because they could promote premature degradation of analytes.In addition to the degradation pathways mentioned above, another extraction risk would be the polymerization of sugar molecules i.e., caramelization, which occurs at high temperature and involves complex sets of reactions such as dehydration and polymerization. This reaction can occur in solution or in the dry state and is dependent on pH and concentration. Ratsimba et al.269 studied the caramelization process of D-glucose, D-fructose, and sucrose by GC-MS and showed that for D-glucose, 1,6-anhydro-β-Dglucopyranose and 1,6-anhydro-β-D-glucofuranose as well as complex disaccharides were synthesized.Another striking finding was the presence of D-mannose and D-fructose, which could result in the Bruyn-Alberda van Ekenstein Lobry enolization process of D-glucose. It is essential to point out that the caramelization of D-glucose was performed under concentrated conditions (2.5 kg D-glucose in 250 ml water) at 170 °C for 2.8 h. Obviously, the amounts expected in extraterrestrial samples are much lower than the amounts used in this study. It does, however, testify that prolonged treatment at high temperature can induce structural changes and oligomerization of sugars. Apart from simple degradation, isomerization is even more critical because it can lead to false detection of sugars not initially present in a sample.

Figure 31 .

 31 Figure 31. General scheme of sampling, extraction, fractionation and purification, derivatization and analysis of a meteorite sample. S -Sugars fraction, AA -Amino acids fraction.

Figure 32 .

 32 Figure 32. Mass spectrum & fragmentation of alanine as N-trifluoroacetyl methyl ester derivative.

Figure 34 .

 34 Using the same temperature program, the derivatives have a shorter elution time, about 15 minutes. Ala and 2-Aba are well resolved from each other and Phe is enantioseparated. The L-3-Aib/D-3-Aba/β-Leu/D-Ser area is well spread using CP-Chirasil-Dex CB: 3-Aba, Ser, and 3-Aib are well resolved and enantioseparated whereas β-Leu is not enantioseparated. 2,4-Dab is still not enantioseparated. Moreover, 2-Aib and one Ala enantiomer are not observed in the chromatogram. Given the shorter retention times of all amino

Figure 33 .

 33 Figure 33. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a Lipodex E coupled to a DB-WAX column.

Figure 34 .

 34 Figure 34. Two-dimensional chromatogram of MeOH/TFAA derivatized amino acids analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column.

8 M

 8 solution of D-alanine corresponds to Qs = 5.10 -8 × 10 -6 × 89.09 g/mol = 4.5 pg. Then, the D-Ala LOD = 2.998 × 18.2 × 4.5 × 10 -12 × 0.01 ≈ 2.46 pg corresponding to 2.73 × 10 -8 M.

  standard deviation. b Only detected in two samples. c Unexpected coelution with unknown compound on one sample. d Peak absence on one sample. ND -Not detected.

Figure 35 .

 35 Figure 35. Calibration curve for D-Alanine and L-Alanine using ECHFBE derivatization. The small diamond on the calibration curve represents the concentration of alanine found in the TFAA1 sample, 3.7×10 -7 M.

Figure 36 .

 36 Figure 36. Evolution of ee over four concentrations of five 5% L-doped amino acids.

  terms of MBA rings and TFA-derivatized hydroxyl numbers. Threitol and erythritol have identical mass spectra and correspond to a single MBA ring and TFA groups. It is likely that the MBA ring is located on centered hydroxyls because no m/z = 85 or 84 is observed. The major fragments are m/z = 97, 69, 43 and m/z = 88, 110 and 182 are characteristic of these C4 sugar alcohols. Arabitol and xylitol have identical and similar mass spectra to adonitol. The mass spectra show the same major and characteristic ions as the C4

Figure 37 .

 37 Figure 37. Two-dimensional chromatogram of MBA/TFAA derivatized sugars analyzed on a CP-Chirasil-Dex CB coupled to a DB-WAX column.

Figure 38 .

 38 Figure 38. Schematic of glass tubes containing IEC resin and Teflon frits.

Figure 40 .

 40 Figure 40. Two-dimensional chromatograms of Iva retention time area highlighting derivatization reagents background noise induced by IEC. On the left, the total-ion current is displayed. On the right, the m/z = 168 characteristic ion of Iva is displayed.

3. 3 . 1 . 3

 313 Sample purification and fractionation (p. 97) and derivatized MeOH/TFAA, detailed in 3.2.1.1.2 Derivatization (p. 73).

3. 4 . 3

 43 Degradation of amino acids in the presence of sugars3.4.3.1 Procedure

Figure 41 ,

 41 detailed in 3.3.1.3 Sample purification and fractionation (p. 97). The amino acid fractions were derivatized with the MeOH/TFAA procedure and analyzed by GC×GC-TOFMS, the conditions of which are detailed 3.2.1.1.2 Derivatization (p. 73) and 3.2.1.1.3 GC×GC-TOFMS (p. 73). Each sample was injected twice. Concentrations were corrected using ion exchange chromatography recoveries of amino acids.

Figure 41 .

 41 Figure 41. Schematic of the HMT sample processing. S represents the sugar fraction and AA the amino acid fraction.

Figure 42 .

 42 Figure 42.Proposed pathway for the formation of amino acids from the decomposition of HMT followed by the formose reaction in the presence of ammonia. From V.Vinogradoff et al. 159

Figure 45

 45 Figure45 showing chromatograms of FGO2, FG2α, FGO7 and FG7α. Ion 43, characteristic of polyols, is displayed. Trimethylsilyl derivatives were identified and searched according to the work of C. Meinert et al.[START_REF] Meinert | Ribose and Related Sugars from Ultraviolet Irradiation of Interstellar Ice Analogs[END_REF] , providing exhaustive information on characteristic ions and retention times.

Figure 43 .

 43 Figure 43. Two-dimensional chromatogram of MBA/TFAA derivatized FGO2 sample analyzed on CP-Chirasil-Dex CB coupled to DB-WAX columns. Ion 43, characteristic of MBA fragmentation, is displayed.

Figure 44 .Table 30 .✓Figure 45 .

 443045 Figure 44. Two-dimensional chromatogram of (S)-BuOH/TFAA derivatized FGO2 sample analyzed on CP-Chirasil-Dex CB coupled to DB-WAX columns. Ion 57, characteristic of (S)-BuOH fragmentation, is displayed.

  Figure 46. Evolution of sugar content using calcium hydroxide and olivine as catalysts for the formose reaction on days 2, 7 and 45.

  Figure 47. Evolution of sugar alcohols and dihydroxyacetone content using calcium hydroxide and olivine as catalysts for the formose reaction on days 2, 7, and 45. In red are highlighted samples not received.

  Figure 48. D-Enantiomeric excess in calcium hydroxide and olivine catalyzed formose reaction on day 2.

  sample "100-Glycolaldehyde -Erythrose -Sorbitol 5x10-4 M MM 10-5 M (MBA-TF AA):1", peak 41, at 10:55.00 , 2.300 min:sec , sample "122-DL-Sugar mix (x25) 10-4M MM 10-5M :1", peak 18, at 21:45.00 , 2sample "122-DL-Sugar mix (x25) 10-4M MM 10-5M :1", peak 23, at 23:50.00 , 1sample "122-DL-Sugar mix (x25) 10-4M MM 10-5M :1", peak26, at 24:20.00 , 3

  sample "122-DL-Sugar mix (x25) 10-4M MM 10-5M :1", peak 118, at 42:20.00 sample "122-DL-Sugar mix (x25) 10-4M MM 10-5M :1", peak 124, at 44:20.00 , 3sample "122-DL-Sugar mix (x25) 10-4M MM 10-5M :1", peak 138, at 49:05.00 , 3sample "122-DL-Sugar mix (x25) 10-4M MM 10-5M :1", peak 154, at 54:55.00 , 2. 420 min:sec , sec

  sample "122-DL-Sugar mix (x25) 10-4M MM 10-5M :1", peak 157, at 55:25.00 sample "122-DL-Sugar mix (x25) 10-4M MM 10-5M :1", peak 137, at 48:05.00 , 2sample "323-D-Fructose 10^-4M MM 10-^5M:1", peak 69, at 47:05.00 , 2.440 min :sec , sec

  Structure of the deoxyribonucleic acid. Figure 2. Genetic material built from chiral molecules.

	3.2.2.1 3.2.2.1.1 Reagents Materials and methods 3.2.2.1.2 Derivatization 3.2.2.1.3 GC×GC-TOFMS 3.2.2.2 Identification of sugars and sugar related compounds 3.2.2.2.1 Sugars LIST OF ABBREVIATIONS Deoxyribonucleic acid Ribonucleic acid PAH LIST OF FIGURES DNA RNA Figure 1.
		3.2.2.2.2 Sugar alcohols
		3.2.2.3	Overall resolution and enantioseparation
		3.2.2.4	Conclusion
	3.3	PURIFICATION & FRACTIONATION
	3.3.1 Materials & methods
		3.3.1.1	Materials & Reagents
		3.3.1.2	Column preparation & conditioning
		3.3.1.3	Sample purification and fractionation
	3.3.2 Amino acid recoveries
	3.3.3 Early developments
		3.3.3.1	Recoveries of alanine and ribose
		3.3.3.2	Drying impact
	3.3.4 Conclusion
	3.4	SAMPLING & EXTRACTION
	3.4.1 Materials & methods
	3.4.2 Degradation of amino acids during hydrolysis
		3.4.2.1	Procedure
		3.4.2.2	Results & discussion
	3.4.3 Degradation of amino acids in the presence of sugars
		3.4.3.1	Procedure
	3.4.4 Conclusion
	3.5	CONCLUSION
	CHAPTER IV -APPLICATIONS OF THE OPTIMIZED METHOD TO COMPLEX SAMPLES
	4.1	AMINO ACID SYNTHESIS STARTING FROM HEXAMETHYLENTETRAMINE
	LABORATORY-PRODUCED EXTRATERRESTRIAL SAMPLES 4.1.1 Materials and methods
	3.1 4.1.2 Amino acids content GENERAL APPROACH
	3.2 4.1.3 Other detected amino acids DERIVATIZATION & GC×GC ANALYSIS
	3.2.1 MeOH/TFAA derivatization of amino acids 4.1.4 Conclusion
	4.2	3.2.1.1 MINERAL-CATALYZED FORMOSE REACTION UNDER HYDROTHERMAL CONDITIONS Materials and methods
	3.2.1.1.1 Reagents 4.2.1 Materials and methods
	3.2.1.1.2 Derivatization 4.2.2 Derivatization approach
	3.2.1.1.3 GC×GC-TOFMS 4.2.3 Evolution of sugars content
	3.2.1.2 4.2.4 Evolution of sugar alcohols content Identification of amino acids
	3.2.1.2.1 Mass spectrum & fragmentations 4.2.5 Enantiomeric excesses investigation
	3.2.1.3 4.2.6 Conclusion Overall resolution & enantioseparation
	3.2.1.4 CONCLUSION & PERSPECTIVES Repeatability
	3.2.1.5 REFERENCES	Linearity
	3.2.1.6 SUPPLEMENTARY MATERIALS Limit of detection
		3.2.1.7	Enantiomeric excess conservation
		3.2.1.8	Stability
		3.2.1.9	Conclusion
	3.2.2 MBA/TFAA derivatization of sugars & sugar related compounds
			XV XVII

Table S 1

 S . Mass spectrum & fragmentation patterns of N-trifluoroacetyl amino acid methyl ester derivatives. Table S 2. Mass spectrum of MBA/TFAA derivatized sugars and sugar related compounds. Table S 3. Regression data of the MBA/TFAA calibration curve, used for days 2 and 7. Table S 4. Regression data of the MBA/TFAA calibration curve, used for day 45.

  In summary, among all deterministic symmetry breaking processes, CPL is a plausible candidate able to induce ees in many chiral biomolecules, hence initiated further studies of the interstellar medium and related chemical reactivities. On the other hand, the amplification mechanisms are various and different in nature. Only catalysis-based mechanisms create ees permanently, while alternative mechanisms concentrate and separate enantiomers. The amplification process will not be developed further in this manuscript; however, it could have occurred either on Earth under primitive Earth conditions or in space under interstellar conditions.

	1.2.6 Conclusion

Table 1 .

 1 Components of the interstellar medium. From P. Ehrenfreund et al.[START_REF] Ehrenfreund | Cosmic Carbon Chemistry: From the Interstellar Medium to the Early Earth[END_REF] and adapted from D.H.Wooden et al.

	ISM component	Designation	Temperature (K)	Density [cm -3 ]	Hydrogen state
	Hot ionized medium	Coronal gas	10 6	0.003	H +
	Warm ionized medium	Diffuse ionized gas H II	10 4	>10	H +
	Warm neutral medium	Intercloud H I	10 4	0.1	H 0
	Atomic cold neutral medium	Diffuse clouds	100	10-100	H 0 + H2
	Molecular cold neutral medium Dark, molecular, and dense clouds	<50	10 3 -10 5	H2
	Molecular hot cores	Protostellar cores	100-300	>10 6	H2

group are briefly reviewed in Table 2. Carbonaceous chondrites can contain up to

	1.4 BIOSIGNATURES IN EXTRATERRESTRIAL MATTER
	1.4.1 Meteorites
	Meteorites are solid fragments of extraterrestrial matter such as comets and asteroids that have survived
	the entry into a planet's atmosphere. During the atmospheric entry phase, these objects are called
	meteors because friction, pressure and chemical reactions increase their temperature until a fireball,
	commonly called a shooting star, is observed. They can be considered either as falls if they can be linked
	to an actual observation of a fall, or as finds if they cannot. In terms of classification, they are divided into
	3 categories: chondrites (undifferentiated meteorites), achondrites (differentiated meteorites) or
	primitive achondrites. Chondrites come from bodies that have preserved the original matter of the solar
	system and contain chondrules (spheres of 1 to 2 mm) while achondrites are igneous rocks from
	differentiated parent bodies (asteroids, planets), which means that they were huge enough to melt and
	organize themselves in several layers. Primitive achondrites possess partial characteristics of both
	categories: an achondritic texture (without chondrules) and a primitive chemical signature of their original
	chondritic parent body. Among chondrites, three major classes are distinguished: carbonaceous (C),
	ordinary (O) and enstatite (E). Each class can be divided into groups and furthers subdivided into
	subgroups.
	The nomenclature of carbonaceous chondrites such as CI, CM, CR, CV, CO, CH, CB, CK are related to the
	type of specimen 101 . For example, CM stands for Carbonaceous Mighei-like meteorite. Parameters such
	as average chondrule diameter, matrix/chondrule ratios, metal abundance, mineral content, and
	composition of each 5%
	carbon in the form of organic matter, carbonates, and other materials such as graphite, diamond, and
	silicon carbide 102 . Of the organic matter, about 25% are free and available, i.e. soluble compounds that
	are easily extracted with solvents (SOM), while the remaining 75% are bound, complex, insoluble
	compounds such as polyaromatic hydrocarbons 102 (IOM). Carbonaceous chondrites represent only a small

Table 2 .

 2 Average petrological characteristics of carbonaceous chondrites. From and inspired by M.K.

	Weisberg et

Table 3 .

 3 Amino acid amounts in Murchison (in ppb) and their calculation in mol/L for our analyses. Data taken from T.Koga & H. Naraoka 110 . Amino acids were derivatized as N-trifluoroacetyl O-methyl esters and analyzed by GC-MS equipped with a Chirasil-L-Val column. Standard deviations are not reported in this table for clarity but are available in T. Koga & H. Naraoka 110 .

	Amino acid	H2O (ppb)	H2O + HCl (ppb)	M (g/mol)	H2O (M)	H2O + HCl (M)
	Glycine	1373	3566	75.1	3.7 x 10 -4	9.5 x 10 -4
	D-Alanine	318	570	89.1	7.1 x 10 -5	1.3 x 10 -4
	L-Alanine	398	1130	89.1	8.9 x 10 -5	2.5 x 10 -4
	Sarcosine	201	298	89.1	4.5 x 10 -5	6.7 x 10 -5
	D-Serine	267	394	105.1	5.1 x 10 -5	7.5 x 10 -5
	L-Serine	318	1261	105.1	6.1 x 10 -5	2.4 x 10 -4
	β-Alanine	1064	1721	89.1	2.4 x 10 -4	3.9 x 10 -4
	2-Aib	863	1015	103.1	1.7 x 10 -4	2.0 x 10 -4
	D-2-Aba	532	873	103.1	1.0 x 10 -4	1.7 x 10 -4
	L-2-Aba	541	944	103.1	1.0 x 10 -4	1.8 x 10 -4
	D-Aspartic acid	108	301	133.1	1.6 x 10 -5	4.5 x 10 -5
	L-Aspartic acid	267	1048	133.1	4.0 x 10 -5	1.6 x 10 -4
	D-Threonine	tr.	tr.	119.1	-	-
	L-Threonine	173	691	119.1	2.9 x 10 -5	1.2 x 10 -4
	D-3-Aba	147	200	103.1	2.9 x 10 -5	3.9 x 10 -5
	L-3-Aba	146	193	103.1	2.8 x 10 -5	3.7 x 10 -5
	D-3-Aib	150	235	103.1	2.9 x 10 -5	4.6 x 10 -5
	L-3-Aib	131	204	103.1	2.5 x 10 -5	4.0 x 10 -5
	4-Aba	1244	1882	103.1	2.4 x 10 -4	3.7 x 10 -4
	D-Valine	58	87	117.2	9.9 x 10 -6	1.5 x 10 -5
	L-Valine	129	548	117.2	2.2 x 10 -5	9.4 x 10 -5
	D-Norvaline	75	129	117.2	1.3 x 10 -5	2.2 x 10 -5
	L-Norvaline	67	119	117.2	1.1 x 10 -5	2.0 x 10 -5
	DL-Isovaline	1418	1668	117.2	2.4 x 10 -4	2.8 x 10 -4
	D-Glutamic acid	172	314	147.1	2.3 x 10 -5	4.3 x 10 -5
	L-Glutamic acid	426	1214	147.1	5.8 x 10 -5	1.7 x 10 -4
	D-Leucine	17	80	131.2	2.6 x 10 -6	1.2 x 10 -5
	L-Leucine	170	1058	131.2	2.6 x 10 -5	1.6 x 10 -4
	D-Isoleucine	101	273	131.2	1.5 x 10 -5	4.2 x 10 -5
	L-Isoleucine	251	947	131.2	3.8 x 10 -5	1.4 x 10 -4

Table 4 .

 4 Example of amounts of sugars and sugar-related compounds in Murchison (in pmol/g or ppb) and their calculation for our analyses. Data taken from Y.Furukawa et al. 112 and G. Cooper et al.

113 Compounds pmol/g or ppb M (g/mol) Content (M)

		Ethylene glycol	320000	62.07	6.4 x 10 -3
		Glycerol	160000	92.09	3.2 x 10 -3
	alcohols	Erythritol DL-Threitol Adonitol	14* 14* 0	122.12 122.12 152.15	2.8 x 10 -7 2.8 x 10 -7 0
	Sugar	DL-Arabinitol Xylitol	0 0	152.15 152.15	0 0
		Glyceric acid	80000	106.08	1.6 x 10 -3
		Threonic acid	4000	136.1	8.0 x 10 -5
	acids Sugar	Erythronic acid Ribonic acid Arabinonic acid	4000 589 963	136.1 166.13 166.13	8.0 x 10 -5 1.2 x 10 -5 1.9 x 10 -5
		Gluconic acid	273	196.16	5.5 x 10 -6
		Mannonic acid	743	196.16	1.5 x 10 -5
		Ribose	25	150.13	3.3 x 10 -6
	Sugars	Arabinose Xylose	120 180	150.13 150.13	1.6 x 10 -5 2.4 x 10 -5
		Lyxose	6.7	150.13	8.9 x 10 -7
	*Values reported for another meteorite, ALH 85013, which is lower than the quantities in Murchison. Maximum values are 26
	pmol/g for erythritol & 32 pmol/g for threitol. pmol/g values were converted to ppb using molar mass and a 1000 factor for
			further calculation.

Table 6 .

 6 Acquisition rate of GC×GC compatible detectors.

		ECD	TCD	FID	MS
	Acquisition rate (Hz)	≤ 50	≤ 300	≤ 500	Quad: ≤ 50* & TOF: 100-500

*In selected ion mode (SIM) which restrains spectral data acquisition. Full spectrum acquisition is in the 10 Hz range.

Table 7 .

 7 Physical and chemical properties of native cyclodextrins. Derived and inspired byDai et al. 191 .

	Properties	α-CD	β-CD	γ-CD
	Number of glucose units	6 (n=1)	7 (n=2)	8 (n=3)
	Formula (anhydrous)	C36H60O30	C42H70O35	C48H80O40
	Molecular weight	972.85	1134.99	1297.12
	Approximate cavity volume (nm 3 )	1.74	2.62	4.27
	Cavity external diameter (nm)	0.49	0.62	0.79
	αD (deg.)	+150.5	+162.0	+177.4
	pKa (25 °C)	12.33	12.20	12.08
	Solubility (g/100 mL water, 25 °C)	14.5	1.85	23.2

2.1.3.1.2 Chiral recognition principle

  Compoundssuch as acetic acid, formaldehyde or glycolaldehyde meet these requirements but are excluded. The first way to classify carbohydrates is to distinguish them according to their degree of polymerization, ranging from 1 to about a million for amylopectins211 . Three main categories are used: sugars (mono-and disaccharides), oligosaccharides and polysaccharides 212 . Monosaccharides are the fundamental monomers and the smallest unit used to construct di-, oligo-, and polysaccharides. A monosaccharide unit is polyhydroxylated (alcohol functions) and may be called an aldose (aldehyde) or ketose (ketone) depending on the chemical function of the carbonyl 213 . Sugar acids and sugar alcohols also exist and contain a carboxylic acid and an alcohol terminal group, respectively, as shown in Figure14. The bonding between monomers is called a glycosidic bond and involves the hemiacetal group of one monosaccharide and the hydroxyl group of another.

	2.2 TARGET COMPOUNDS CONSIDERATIONS
	2.2.1 Carbohydrates
	2.2.1.1 Classifications
	2.2.1.1.1 Degree of polymerization & functions

Carbohydrates (CHs) were originally chemically defined as compounds of formula Cm(H2O)n (m is generally equal to n) but are now considered as carbohydrates when m,n > 2 and inherit an "ose" suffix.

Table 8 .

 8 Anomer ratios for a series of aldoses in aqueous solution. Taken from215

	Monosaccharide	α-p	β-p	Total p	α-f	β-f	Total f
	D-Ribose	20	56	76	6	18	24
	D-Arabinose	63	34	97			3
	D-Allose	18	70	88	5	7	12
	D-Altrose	27	40	67	20	13	33
	D-Glucose	36	64	100			< 1
	D-Mannose	67	33	100			< 1
	D-Galactose	27	73	100			< 1
			p: pyranose form, f: furanose form

  Figure19, can be formed by a condensation reaction between hydroxylamine and an aldehyde or ketone of a carbohydrate. In this case, the chiral information is not lost, and the number of stereoisomers for one enantiomer is reduced to 2 instead of 5 because the C=N bond involves an E/Z configuration. Subsequent derivatization of the remaining hydroxyl groups to trimethylsilyl 218 (TMS) ethers or trifluoroacetyl 219 (TFA) esters can be performed. The method is suitable for C3 to C7 sugars and disaccharides 219 . Despite the reported good resolution of C3 and C4 sugars 220 , both E and Z compounds are not always resolved 219 .

	2.2.1.2.2 Oximation
	Carbohydrate oximes,

Table 9 .

 9 20 to 22 (in yellow) proteinogenic amino acids including the 9 essential amino acids (in grey).

	Alanine	Arginine	Asparagine	Aspartic Acid	Cysteine	Glutamic Acid
	Glutamine	Glycine	Histidine	Isoleucine	Leucine	Lysine
	Methionine	Phenylalanine	Proline	Serine	Threonine	Tryptophan
		Tyrosine	Valine	Selenocysteine	Pyrrolysine

Table 10 .

 10 Relative counter ion selectivity for AG 50W-X8 resin. From the instruction manual 206 .

	Li +	H +	Na +	NH4 +	Mg 2+	Fe 2+	Zn 2+	Cu 2+	Cu +
	0.85	1	1.5	1.95	2.5	2.55	2.7	2.9	5.3

Table 11 .

 11 List of mechanisms involved in ion exchange chromatography. Inspired byHuber et al. 208 and Huck et al. 207

Table 12 .

 12 Extraction methods of various targeted compounds in meteoritic samples.

	Sample	Mass (mg)	Solvent	T (°C) Time (h)	Targeted compounds	Reference
	11	< 1400	Dichloromethane 2 mL	100	24	Aldehydes Ketones	Aponte 250 2019
	10-15	< 350	H2O 1 mL	100	24	Monocarboxylic acids	Aponte 251 2019
	1	100	H2O / mL	100	20	Amino-& hydroxy-acids Amines & carboxylic acids	Pizzarello 252 2018
	5	400-1100	HCl 1 mL (0.1 M) H2O 1 mL	100	24	Amines Amino acids	Aponte 253 2016
	1	900	0.5 M HCl 1 mL	100	24	Aliphatic amines	Aponte 254 2014
	2	2000	1N NaOH 10 mL	RT	2.5	Monocarboxylic acids	Aponte 255 2014
	1	10	H2O 2 mL Dichloromethane/methanol	100	24	Aliphatic compounds	Pizzarello 256 2013
	8	50-250	H2O / mL	100	24	Amino acids, hydroxyacids, polyols, amines, monocarboxylic acids	Pizzarello 257 2012
	1-2	3994	H2O 2 mL Dichloromethane/methanol 2 mL	100	24	Amino acids, ammonia, amines	Pizzarello 258 2011
	3	1000, 485, 165	H2O 1 -4 mL	100	20	Hydroxy acids	Pizzarello 259 2010
	4	500	H2O 5mL HCOOH 5 mL HF/HCl 10 mL	60 60 80	2 2 6	Nucleic acids	Shimoyama 260 1990
	4	5000-18 350	Benzene 20 -73 mL H2O 20 -73 mL HCOOH 40 -146 mL	60	0.5	Pyrimidines & N-heterocyclic compounds	Stoks 261,262 1979 & 1981
	1	1000	HCOOH 8 mL	60	1	Nucleobases	Martins 263 2008
	1	1200	H2O 3×300 mL	RT	46	Carboxamides	Cooper 144 1995
	3	2000	HCl 10 mL (2 %)	RT	-	Sugars	Furukawa 2019
	14	320-1611	Benzene/acetone 100 mL (8/2) H2O / mL HCl (5 N) / mL	-Reflux Reflux	24	Amino acids & sugars	Kaplan 264 1962

Table 13 .

 13 Characterization of N-trifluoroacetyl methyl ester amino acid derivatives by mass spectra, retention time and method detection limit.

	#	Compound	[M] +•	Characteristic ions, m/z	𝑹 𝒕 𝟏 [min:sec] a	𝑹 𝒕 𝟐 [sec] b	Rs 1 st Dim. c	MDL (nM ; pg)
	Glycine (Gly)		185	126, 69, 78, 56, 59, 88, 50, 106	45:20	0.27	-	43 ; 3.9
	Sarcosine (Sar)		199	140, 69, 60, 78, 74, 90, 102, 199	44:45	2.91	-	33 ; 2.9
	D-α-Alanine (D-Ala) L-α-Alanine (L-Ala)	199	140, 69, 70, 92, 59, 102, 93, 66	33:10 35:15	3.46 3.27	2.8	27 ; 2.4 32 ; 2.9
	β-Alanine (β-Ala)	199	55, 69, 139, 70, 126, 167, 98, 59	57:50	3.99	-	21 ; 1.9
	D-Serine (D-Ser) L-Serine (L-Ser)	311 ** 69, 138, 59, 110, 139, 153, 70, 96	55:20 57:25	3.32 3.12	4.2	28 ; 3.0 22 ; 2.3
	D-Cysteine d (D-Cys) L-Cysteine d (L-Cys)	327 ** 69, 117, 59, 96, 61, 138, 129, 70	66:55 67:15	3.14 3.08	1	NF
	D-2,3-Diaminopropionic acid (D-2,3-Dap) L-2,3-Diaminopropionic acid (L-2,3-Dap)	310 ** 69, 153, 185, 126, 78, 125, 96, 138	84:50 85:15	4.10 4.01	1.25	45 ; 4.7 44 ; 4.5
	N-Ethylglycine (Etg)	213	126, 154, 69, 56, 140, 78, 74, 116	50:25	2.69	-	31 ; 3.2
	2-Aminoisobutyric acid (2-Aib)	213 * 154, 59, 69, 114, 84, 166, 138, 73	28:10	2.97	-	28 ; 2.9
	D-2-Aminobutyric acid (D-2-Aba) L-2-Aminobutyric acid (L-2-Aba)	213 * 154, 69, 126, 59, 56, 84, 96, 116	35:25 40:00	3.17 2.82	6.9	40 ; 4.1 37 ; 3.8
	D-3-Aminoisobutyric acid (D-3-Aib) L-3-Aminoisobutyric acid (L-3-Aib)	213	69, 88, 57, 153, 56, 126, 59, 84	53:10 54:35	3.44 3.32	3.4	23 ; 2.4 24 ; 2.5
	D-3-Aminobutyric acid (D-3-Aba) L-3-Aminobutyric acid (L-3-Aba)	213	69, 140, 59, 70, 153, 74, 156, 102	54:30 58:00	3.16 2.90	7	45 ; 4.6 e 18 ; 1.8
	4-Aminobutyric acid (4-Aba)	213	74, 69, 126, 59, 57, 182, 56, 78	72:05	3.49	-	13 ; 1.3
	D-Aspartic acid (D-Asp) L-Aspartic acid (L-Asp)	257 * 156, 59, 198, 69, 166, 85, 61,71	67:20 70:30	4.04 3.40	6.3	41 ; 5.5 41 ; 5.4
	D-2,4-Diaminobutanoic acid (D-2,4-Dab) L-2,4-Diaminobutanoic acid (L-2,4-Dab)	324 ** 152, 69, 126, 153, 57, 56, 78, 185	88:10 88:25	4.33 4.27	NA	36 ; 4.2
	D-Threonine (D-Thr) L-Threonine (L-Thr)	325 ** 69, 152, 57, 153, 59, 185, 141, 96	54:05 54:50	1.88 1.87	1.3	28 ; 3.3 25 ; 3.0
	DL-Asparagine (DL-Asn)	-	Aspartic Acid	-	-	-	-
	D-Isovaline (D-Iva) L-Isovaline (L-Iva)	227 * 55, 69, 168, 166, 114, 138, 59, 110	31:20 27:55	2.39 2.56	5.9	62 ; 7.2 e 61 ; 7.1 e
	D-Valine (D-Val) L-Valine (L-Val)	227 * 55, 153, 168, 69, 114, 125, 59, 56	33:00 37:15	2.85 2.59	7.3	29 ; 3.4 42 ; 4.9
	D-Norvaline (D-Nva) L-Norvaline (L-Nva)	227 * 55, 168, 126, 69, 114, 153, 59, 56	39:20 46:30	3.62 2.97	12.3	31 ; 3.6 43 ; 5.1
	D-Proline (D-Pro) L-Proline (L-Pro)	225	166, 69, 96, 71, 128, 68, 53, 167	67:10 65:25	3.09 3.34	3	24 ; 2.8 24 ; 2.8
	5-Aminopentanoic acid (5-Ava)	227 * 55, 126, 74, 69, 59, 139, 78, 82	77:15	3.68	-	NF
	D-Glutamic acid (D-Glu) L-Glutamic acid (L-Glu)	271 * 152, 69, 180, 212, 57, 59, 55, 82	71:50 73:25	0.16 4.66	3.2	29 ; 4.2 28 ; 4.1
	D-Methionine (D-Met) L-Methionine (L-Met)	259	61, 69, 153, 75, 185, 59, 152, 62	71:40 72:45	4.84 4.53	6.5	NF NF
	D-Isoleucine (D-Ile) L-Isoleucine (L-Ile)	241 * 69, 153, 185, 57, 182, 125, 126, 59	38:25 43:15	3.12 2.78	8.3	25 ; 3.3 32 ; 4.2
	D-allo-Isoleucine (D-allo-Ile) L-allo-Isoleucine (L-allo-Ile)	241 * 69, 153, 185, 57, 182, 125, 126, 59	37:40 42:35	3.06 2.70	8.4	NA 28 ; 3.6
	D-Leucine (D-Leu) L-Leucine (L-Leu)	241 * 69, 140, 182, 153, 70, 185, 55, 59	40:55 44:10	3.83 3.48	5.6	36 ; 4.8 25 ; 3.3
	D-Norleucine (D-Nle) L-Norleucine (L-Nle)	241 * 69, 182, 126, 153, 114, 70, 55, 59	43:45 47:15	4:00 3.61	7	28 ; 3.7 30 ; 3.9
	D-β-Leucine (D-β-Leu) L-β-Leucine (L-β-Leu)	241 * 156, 69, 198, 55, 59, 166, 139, 70	54:40 55:05	3.30 3.27	1	43 ; 5.6 22 ; 2.9
	D-Phenylalanine (D-Phe) L-Phenylalanine (L-Phe)	275	91, 162, 69, 65, 131, 103, 77, 51	74:40 74:55	0.09 0.01	NA	23 ; 3.8

Table 14 .

 14 Repeatability tests on 9 samples analyzed once for each amino acid at 5×10 -5 M. Not detected. a Relative standard deviation. b Ratio of amino acid over internal standard area. Temperature program slightly different than the one used in Table13.

	Compound	S a (%)	MS b	Compound	S a (%)	MS b	Compound	S a (%)	MS b
	Gly	4.28	8.64	D-Glu	6.75	1.96	D-3-Aba	5.86	2.42
	Sar	4.95	8.33	L-Glu	4.43	2.06	L-3-Aba	4.9	2.46
	D-α-Ala	5.08	10.68	D-Met	118.54	0.22	4-Aba	6.67	1.43
	L-α-Ala	4.42	10.48	L-Met	114.07	0.22	D-Asp + Asn	3.95	2.81
	β-Ala	5.13	3.49	D-Ile	4.45	2.34	L-Asp + Asn	3.69	2.83
	D-Cys	ND	ND	L-Ile	4.34	2.32	DL-2,4-Dab	6.84	4.11
	L-Cys	ND	ND	D-allo-Ile	4.83	1.54	D-Thr	34.85	0.05
	D-2,3-Dap	38.14	0.80	L-allo-Ile	4.71	1.47	L-Thr	38.45	0.05
	L-2,3-Dap	31.6	0.86	D-Leu	4.98	1.91	D-Iva	5.99	3.99
	Etg	4.84	4.49	L-Leu	2.93	1.91	L-Iva	5.98	3.28
	2-Aib	4.94	8.68	D-Nle	4.4	2.31	D-Val	4.51	3.70
	D-2-Aba	4.17	6.66	L-Nle	5.16	2.33	L-Val	4.15	3.76
	L-2-Aba	5.15	6.92	D-β-Leu	5.23	1.88	D-Nva	4.2	3.36
	D-3-Aib	4.99	2.25	L-β-Leu	5.9	1.88	L-Nva	5.16	3.32
	L-3-Aib	6.13	2.35	DL-Phe	5.25	5.22	D-Pro	5.91	4.87
	5-Ava	8.93	0.64				L-Pro	4.99	4.95
	ND -

Table 15 .

 15 Regression data of the MeOH/TFAA calibration curve.

	#	Compound	S a	ΔS b	I c	ΔI d	R 2
	1 Glycine		7.79 × 10 4	210	3.80 × 10 -2	5.27 × 10 -3	0.9999
	2 Sarcosine		7.76 × 10 4	187	-2.38 × 10 -5	4.70 × 10 -3	0.9999
	3 D-α-Alanine	8.69 × 10 4	86.4	-4.49 × 10 -3	2.17 × 10 -3	0.9999
	4 L-α-Alanine	8.74 × 10 4	102	-2.42 × 10 -3	2.56 × 10 -3	0.9999
	5 β-Alanine		2.20 × 10 4	214	-6.42 × 10 -3	5.37 × 10 -3	0.9998
	6 D-Serine		4.67 × 10 4	634	-1.98 × 10 -2	1.59 × 10 -2	0.9996
	7 L-Serine		4.13 × 10 4	307	-7.22 × 10 -3	7.71 × 10 -3	0.9998
	8 D-Cysteine e 9 L-Cysteine e	NA	NA	NA	NA	NA
	10 D-2,3-Diaminopropionic acid 11 L-2,3-Diaminopropionic acid	5.70 × 10 4	308	-1.08 × 10 -2	7.75 × 10 -3	0.9999
	12 N-Ethylglycine	4.69 × 10 4	122	-4.42 × 10 -3	3.06 × 10 -3	0.9999
	13 2-Aminoisobutyric acid	6.80 × 10 4	60.7	-3.6 × 10 -3	1.53 × 10 -3	0.9999
	14 D-2-Aminobutyric acid	5.80 × 10 4	152	-5.97 × 10 -3	3.81 × 10 -3	0.9999
	15 L-2-Aminobutyric acid	6.28 × 10 4	436	8.23 × 10 -3	1.10 × 10 -2	0.9999
	16 D-3-Aminoisobutyric acid	2.64 × 10 4	65.8	-2.4 × 10 -3	1.65 × 10 -3	0.9999
	17 L-3-Aminoisobutyric acid	3.33 × 10 4	523	-1.46 × 10 -2	1.31 × 10 -2	0.9995
	18 D-3-Aminobutyric acid	3.50 × 10 4	479	-1.31 × 10 -2	1.20 × 10 -2	0.9996
	19 L-3-Aminobutyric acid	3.25 × 10 4	439	-1.22 × 10 -2	1.10 × 10 -2	0.9996
	20 4-Aminobutyric acid	2.68 × 10 4	192	-5.69 × 10 -3	4.82 × 10 -3	0.9998
	21 D-Aspartic acid + Asparagine	5.09 × 10 4	274	-9.3 × 10 -3	6.87 × 10 -3	0.9999
	22 L-Aspartic acid + Asparagine	4.21 × 10 4	623	1.47 × 10 -2	1.56 × 10 -2	0.9995
	23 D-2,4-Diaminobutanoic acid f 24 L-2,4-Diaminobutanoic acid f	9.54 × 10 4	6540	-1.52 × 10 -1	1.64 × 10 -1	0.9906
	25 D-Threonine	3.87 × 10 4	56.3	-2.67 × 10 -3	1.41 × 10 -3	0.9999
	26 L-Threonine	4.58 × 10 4	402	-1.18 × 10 -2	1.01 × 10 -2	0.9998
	27 DL-Asparagine
	28 D-Isovaline	2.57 × 10 4	110	1.11 × 10 -3	2.75 × 10 -3	0.9999
	29 L-Isovaline	2.99 × 10 4	71.5	1.59 × 10 -4	1.80 × 10 -3	0.9999
	30 D-Valine		3.30 × 10 4	24.7	-1.37 × 10 -3	6.21 × 10 -4	0.9999
	31 L-Valine		3.26 × 10 4	180	3.73 × 10 -3	4.51 × 10 -3	0.9999
	32 D-Norvaline	3.72 × 10 4	30.5	-1.18 × 10 -3	7.66 × 10 -4	0.9999
	33 L-Norvaline	3.79 × 10 4	197	-6.25 × 10 -3	4.94 × 10 -3	0.9999
	34 D-Proline		6.60 × 10 4	275	-9.34 × 10 -3	6.92 × 10 -3	0.9999
	35 L-Proline		6.91 × 10 4	326	-1.05 × 10 -2	8.20 × 10 -3	0.9999
	36 5-Aminopentanoic acid	1.71 × 10 4	177	-4.95 × 10 -3	4.44 × 10 -3	0.9997
	37 D-Glutamic acid	5.78 × 10 4	982	-2.71 × 10 -2	2.47 × 10 -2	0.9994
	38 L-Glutamic acid	5.03 × 10 4	526	-1.4 × 10 -2	1.32 × 10 -2	0.9997
	39 D-Methionine g 40 L-Methionine g	-	-	-	-	-
	41 D-Isoleucine	2.65 × 10 4	54.8	4.06 × 10 -4	1.38 × 10 -3	0.9999
	42 L-Isoleucine	2.68 × 10 4	40.6	2.32 × 10 -4	1.02 × 10 -3	0.9999
	43 D-allo-Isoleucine	1.82 × 10 4	105	-3.54 × 10 -3	2.64 × 10 -3	0.9999
	44 L-allo-Isoleucine	1.61 × 10 4	52.8	6.35 × 10 -4	1.33 × 10 -3	0.9999
	45 D-Leucine		2.41 × 10 4	93.1	-3.29 × 10 -3	2.34 × 10 -3	0.9999
	46 L-Leucine		2.40 × 10 4	87.3	-3.15 × 10 -3	2.19 × 10 -3	0.9999
	47 D-Norleucine	3.41 × 10 4	340	-1.03 × 10 -2	8.55 × 10 -3	0.9998
	48 L-Norleucine	3.40 × 10 4	449	-1.26 × 10 -2	1.13 × 10 -2	0.9996
	49 D-β-Leucine	3.43 × 10 4	501	-1.43 × 10 -2	1.26 × 10 -2	0.9995
	50 L-β-Leucine	3.27 × 10 4	647	-1.82 × 10 -2	1.63 × 10 -2	0.9992
	53 D-Phenylalanine 54 L-Phenylalanine	2.11 × 10 5	393	-1.16 × 10 -2	9.86 × 10 -3	0.9999

a Slope. b Standard error on S. c Intercept. d Standard error on I. e Not detected. f One concentration missing. g Only found at highest concentration.

Table 16 .

 16 Relative standard deviation of 8 samples analyzed once using 5×10 -8 M of each amino acid.

	Compound	RS a (%)	Compound	RS a (%)	Compound	RS a (%)
	Gly	29.0	L-3-Aib	16.3	L-3-Aba	11.8
	Sar	22.0	D-Glu	19.2	4-Aba	8.4
	D-α-Ala

Table 17 .

 17 Investigation

	Blank (Intensity 373)	MeOH (Intensity 570)	AcOCl (Intensity 466)
	DCM (Intensity 290)	TFAA (Intensity 1068)	10 -7 M DL-Ala (Intensity 896)

of alanine contamination using 2 mL of dried reagent, hydrolyzed with H2O/AcOCl and derivatized as ECHFBE derivatives. The ion m/z = 116, corresponding to the most intense alanine fragment, is displayed. A blank and a 10 -7 M DL-alanine sample are also provided. The maximum intensity is also reported.

Table 18 .

 18 Alanine contamination in three different grades of TFAA.

	Since 4.7 mL were dried and analyzed in an approximately total volume of 69 µL (50 µL chloroform + 19
	µL 2

Table 19 .

 19 The most volatile derivatives, i.e., the first third of the chromatogram, are closer to a recovery value of 80-90% than the other derivatives. The less volatile derivatives are closer to a recovery value of 90-100%. There are exceptions, however. Methionine has a recovery of only 50-55% despite its late elution time. This suggests, as mentioned in 3.2.1.4 Repeatability, that the sulfur-containing derivatives, methionine, and cysteine, tend to decompose. Indeed, this may be particularly the case for cysteine because it is known that the O-trifluoroacetyl group is less stable than the N-trifluoroacetyl group towards hydrolysis 259 , meaning that the S-trifluoroacetyl group of cysteine is even more sensitive. Cysteine can form a dimer, cystine, through a disulfide bond that can prevent its detection as cysteine. However, tests on both cysteine and cystine standards did not show any dimerization of cysteine. Another example, serine,

containing an O-trifluoroacetyl group, showed a good recovery of 100-110% but with a significant standard deviation of 20-25%.

Table 19 .

 19 Recovery tests after 7 days using 5×10 -5 M for each amino acid.

	Compound	R a (%)	± (%) b	Compound	R a (%)	± (%) b	Compound	R a (%)	± (%) b
	Gly	85.0	2.4	L-3-Aib	88.3	2.0	L-3-Aba	87.6	2.0
	Sar	73.1	2.6	D-Glu	99.4	1.5	4-Aba	97.9	1.6
	D-α-Ala	80.2	3.8	L-Glu	99.4	2.2	D-Asp	97.2	2.3
	L-α-Ala	82.2	4.3	D-Met	52.4	3.2	L-Asp	97.4	1.8
	β-Ala	97.8	2.3	L-Met	56.3	4.2	DL-2,4-Dab	103.8	7.3
	D-Ser	99.8	19.6	D-Ileu	88.0	3.7	D-Thr	91.2	2.0
	L-Ser	110.1	24.1	L-Ileu	93.7	3.7	L-Thr	80.3	4.6
	D-Cys	ND	ND	L-allo-Ileu	82.2	2.7	D-Iva	79.8	5.1
	L-Cys	ND	ND	D-Leu	86.5	3.7	L-Iva	86.6	5.1
	D-2,3-Dap	104.1	6.7	L-Leu	79.1	2.5	D-Val	82.5	4.5
	L-2,3-Dap	97.7	5.6	D-Nle	89.3	4.7	L-Val	86.4	4.4
	Etg	86.5	3.1	L-Nle	88.1	3.5	D-Nva	86.7	3.0
	2-Aib	77.6	4.5	DL-β-Leu	86.8	9.4	L-Nva	90.7	2.5
	D-2-Aba	82.8	3.9	DL-Phe	100.5	2.7	D-Pro	89.6	2.9
	L-2-Aba	82.7	4.1	D-3-Aba	80.1	5.9	L-Pro	98.9	2.8
	D-3-Aib	93.1	3.3				5-Ava	80.1

Table 20 .

 20 Characterization of MBA/TFAA sugar derivatives by mass spectra and retention time.

	#	Compound	[M] +•	Characteristic ions, m/z	𝑹 𝒕 𝟏	a [min:sec] 𝑹 𝒕 𝟐	b [sec]
		Glycerol	380	69 (99.8) 153 (17.2) 43 (13.1) 97(12.1) 59 (11.7) 125 (6.7) 267 (1.0)		19:00	2.36

  : 115.8 ± 19.0%) and Iva (D: 81.0 ± 9.3%, L: 77.7 ± 10.6%) are somewhat different from those obtained using both 5% eeL amino acids and sugars. In the latter case, recoveries of Ala (D: 93.2 ± 12.1%, L: 93.2 ± 11.4%) and Iva (D: 119.1 ± 10.6%, L: 89.3 ± 13.0%) are slightly lower for Ala and significantly higher for Iva.

L

Table 21 .

 21 Comparison of 5% eeL doped amino acids at 10 -5 M with and without ion exchange chromatography. Standard deviation of eeL. In blue are highlighted the averages obtained for the reference and ion exchange chromatography experiments. In green are highlighted the standard deviations.

			Reference	IEC 1	IEC 2		IEC 3	IEC 4
	Alanine	eeL (%) σeeL (%)	4.87 0.21	4.54 0.11	5.40 0.07	4.87 0.32	4.76 0.13	4.81 0.10
	Isovaline	eeL (%) σeeL (%)	6.78 0.54	-6.28 0.17	-9.04 0.51	-7.19 1.32	-7.79 0.61	-5.65 0.88
	σeeL -

Table 22 .

 22 Recovery of DL-alanine and DL-ribose by ion exchange chromatography for 3 and 4 bed volumes and with 1 M NH4OH.

			Alanine (ECHFBE)	Ribose ((S)-BuOH/TFAA)
	3 BV	D Recovery (%) L Recovery (%)	0 0	98.52 ± 4.48 98.46 ± 4.84
	4 BV	D Recovery (%) L Recovery (%)	1.56 ± 6.63 1.75 ± 6.44	109.14 ± 4.48 105.77 ± 4.84
	Standard deviations correspond to the 3 references, recoveries are based on only one sample.
			GC-FID, old IEC setup, 10 -3 M.

Table 23 .

 23 Recoveries of DL-alanine and DL-ribose using ion exchange chromatography for 4 BV and using 2 M NH4OH.

		Alanine (ECHFBE)	Ribose ((S)-BuOH/TFAA)
	D Recovery (%)	96.69 ± 5.11	96.75 ± 10.31
	L Recovery (%)	96.88 ± 5.26	94.71 ± 9.24
	ΔeeD (%)	-0.104 ± 0.152	1.00 ± 1.21
	In parentheses, the derivatization used is detailed.

Table 25 .

 25 Amino acids recoveries after water extraction.

		1	2	3	4
		Serpentine	Serpentine	-	-
		100 °C, 24 h	100 °C, 24 h	100 °C, 24 h	RT, 24 h
		Recovery 2×1 mL	Recovery 3×1 mL	Recovery 2×1 mL	Recovery 2×1 mL
	D-Nva	115.7 ± 13.3	120.7 ± 13.4	92.7 ± 14.8	100.9 ± 13.6
	L-Nva	96.0 ± 9.8	98.5 ± 9.8	80.5 ± 11.6	87.6 ± 10.0
	D-Ala	93.1 ± 12.5	93.6 ± 12.6	97.0 ± 14.3	93.1 ± 12.8
	L-Ala	94.4 ± 12.0	98.8 ± 12.1	93.8 ± 14.2	99.7 ± 12.3
	D-Pro	83.2 ± 9.4	85.5 ± 9.4	92.5 ± 10.0	98.4 ± 9.5
	L-Pro	82.9 ± 9.0	84.4 ± 9.1	94.6 ± 10.1	102.2 ± 9.1
	D-Asp A	61.7 ± 11.5	64.0 ± 11.3	91.8 ± 11.4	77.8 ± 11.6
	L-Asp A	70.9 ± 14.7	63.2 ± 11.0	94.2 ± 10.8	80.8 ± 10.7
		New column, GC×GC-TOFMS, MeOH/TFAA, 5×10 -5 M.

Table 26 .

 26 Recoveries of Ala and Iva after mortar-doped serpentine, water hydrolysis and IEC.

		Recovery (%)
	D-Ala	49.92 ± 30.45
	L-Ala	50.54 ± 28.21
	D-Iva	41.51 ± 48.28
	L-Iva	32.69 ± 37.31
	New column, GC×GC-TOFMS, MeOH/TFAA, 5×10 -5 M.

Table 27 .

 27 Comparison of the amino acid content reported in the original study159 and in the present work. Total amino acid content obtained through the sum of the solid and liquid values. **This content was divided by 10 to obtain an equivalent of 0.2 M HMT for comparison with data reported in the original study.

	Amino acids	#C	Reported 0.2 M (µM)	Obtained 0.2 M** (µM)	Obtained 2 M* (µM)	Free	Solid hydrolysis HCl	Free	Liquid 1 HCl hydrolysis	Free	Liquid 2 HCl hydrolysis
	Gly		573.44 ± 15.15	379.69	3796.90	689.81	830.71	584.24	711.47	487.21	493.45
	β-Ala		15.35 ± 0.38	33.02	330.24	65.47	63.74	54.82		51.92	46.90	47.40
	D-α-Ala L-α-Ala		30.31 ± 2.11	128.92	1289.18	158.05 141.21	135.84 126.94	92.02 77.75		115.88 96.44	113.23 89.59	73.32 68.91
	Sarc		-	159.94	1599.37	314.12	385.82	122.87	318.77	223.68	234.12
	D-3-Aib L-3-Aib		2.39 ± 0.77	10.17	101.73	10.56 8.40	11.24 8.98	9.84 7.57		8.91 7.11	7.98 6.13	8.37 6.65
	D-3-Aba L-3-Aba		1.22 ± 1.05	3.79	37.87	3.52 4.02	3.70 3.81	3.11 3.33		2.62 2.72	2.59 2.83	2.80 2.82
	D-2-Aba L-2-Aba		1.22 ± 0.27	1.71	17.15	-2.44	2.48 2.61	-1.64		1.42 1.53	-1.75	1.53 1.76
	4-Aba		11.01 ± 2.88	1.05	10.53	1.84	2.23	1.45		1.62	1.47	1.93
	DL-2,4-Dab		-	0.57	5.73	1.25	0.98	1.05		0.73	0.91	0.81
	D-Glu L-Glu		< 0.1	1.26	12.60	0.04 1.28	0.12 2.08	0.03 3.01		0.08 1.47	0.02 1.36	0.11 3.00
	D-Pro L -Pro		< 0.1	0.58	5.83	0.35 0.58	0.38 0.81	0.35 0.85		0.27 0.51	0.27 0.48	0.30 0.67
	2-Aib		-	0.26	2.57	-	0.83	0.65		0.55	-	0.54
	D-Val L -Val		< 0.1	0.36	3.64	0.29 0.35	0.30 0.61	0.22 0.36		0.19 0.29	0.16 0.29	0.18 0.40
	D-Asp L-Asp		< 0.1	0.36	3.62	-0.20	0.30 0.80	0.18 0.76		0.09 0.41	0.02 0.27	0.27 0.32
	Etg			0.27	2.74	0.52	0.60	0.44		0.37	0.38	0.43
	DL-2,3-Dap			0.16	1.56	-	0.75	0.00		0.24	-	0.56
	D-Thr L-Thr		< 0.1	0.09	0.92	-0.06	-0.31	-0.17		-0.10	-0.09	-0.18
	DL-Phe		-	0.16	1.55	0.23	0.18	0.49		0.23	0.26	0.17
	5-Ava		-	0.03	0.33	0.06	0.06	0.18		0.04	-	-
	D-Leu L-Leu			0.11	1.06	0.00 0.10	0.00 0.25	0.00 0.25		0.00 0.15	0.00 0.13	0.00 0.18

*

  samples received (about 200 µL each) from the Physique des Interactions Ioniques et Moléculaires (PIIM) laboratory, Aix-Marseille Université were used without any treatment before the drying step and subsequent derivatization. 20 µL of the supernatant of each sample was placed in a Reactivial® and dried

under a gentle stream of nitrogen. MBA/TFAA derivatization and GC×GC-TOFMS conditions were as detailed in 3.2.2.1.2 Derivatization (p. 89) and 3.2.2.1.3 GC×GC-TOFMS (p. 89). Quantification of sugars and sugar alcohols is based on the calibration curves provided in this same section. Due to GC×GC maintenance between the analyses of samples from day 7 and day 45 that altered the sensitivity of the instrument, another calibration curve dedicated to day 45 was performed to reliably quantify the sugars in these samples. The calibration curve data is available in Table

S

4 (p. 168)

.

Table 29 .

 29 Overview of samples studied in this work. Samples derivatized once and analyzed once, -Samples not supplied. Other samples were derivatized twice and analyzed twice.

	Composition	Day 2	Day 7	Day 45
	Formaldehyde + Glycolaldehyde	FG2 (pH = 4 -5)	FG7 (pH = 3 -4)	FG45 (pH = 4)
	Formaldehyde + Olivine	FO2 (pH = 6 -7)	FO7 (pH = 6 -7)	FO45 (pH = 6)
	Formaldehyde + Olivine + Glycolaldehyde	FGO2 (pH = 7)	FGO7 (pH = 6 -7)	FGO45 (pH = 7)
	Olivine	O2 *	O7 *	-
	Formaldehyde	F2 *	-	F45 *
	Formaldehyde + Calcium hydroxide	F2α (pH = 5 -6)	F7α (pH = 5)	-
	Formaldehyde + Glycolaldehyde + Calcium hydroxide FG2α (pH = 6 -7)	FG7α (pH = 5)	-

*

Table S 2

 S . Mass spectrum of MBA/TFAA derivatized sugars and sugar related compounds.

	Molecular ion: 198	Glycolaldehyde (dimer)
	TFAA group(s):
	MBA ring(s):
	Molecular ion: 380	Glycerol
	TFAA group(s):
	MBA ring(s):
	Molecular ion: 114	Glyceraldehyde
	TFAA group(s):
	MBA ring(s):
	Molecular ion: 240	Erythrose
	TFAA group(s):
	MBA ring(s):
	Molecular ion: 282	1,3-Dihydroxyacetone
	TFAA group(s):
	MBA ring(s):
	Molecular ion: 338	Threitol
	TFAA group(s):
	MBA ring(s):

Table S 2

 S . Continued.Peak True -sample "122-DL-Sugar mix (x25) 10-4M MM 10-5M :1", peak 60, at 28:55.00 , 2.4 20 min:sec , sec

	Molecular ion: 338										Erythritol
		1000				69	97
	TFAA group(s): MBA ring(s):	500	43	55	88	110		182
	Molecular ion: 254	50 Peak True -sample "122-DL-Sugar mix (x25) 10-4M MM 10-5M :1", peak 70, at 32:20.00 , 2.2 100 150 200 250 300 350 400 2-Deoxyribose 30 min:sec , sec
		1000	43		69
	TFAA group(s): MBA ring(s):	500			57 84	97	140	166	195	224
		50 Peak True -sample "122-DL-Sugar mix (x25) 10-4M MM 10-5M :1", peak 89, at 36:00.00 , 2.6 100 150 200 250 300 350 400
	Molecular ion: 366	00 min:sec , sec				Lyxose (peak 1)
		1000				69
	TFAA group(s): MBA ring(s):	500	43	55	85	97	139	182	223	253
		50 Peak True -sample "122-DL-Sugar mix (x25) 10-4M MM 10-5M :1", peak 146, at 51:40.00 , 4. 100 150 200 250 300 350 400
	Molecular ion: 198	340 min:sec , sec				Lyxose (peak 2)
		1000					97
			43
	TFAA group(s): MBA ring(s):	500			57	84		110	139 168
		50 Peak True -sample "122-DL-Sugar mix (x25) 10-4M MM 10-5M :1", peak 107, at 39:20.00 , 4. 100 150 200 250 300 350 400
	Molecular ion: 296	290 min:sec , sec					Xylitol
		1000					97
	TFAA group(s): MBA ring(s):	500	43 69 57			140	182
		50 Peak True -sample "122-DL-Sugar mix (x25) 10-4M MM 10-5M :1", peak 109, at 39:45.00 , 2. 100 150 200 250 300 350 400
	Molecular ion: 296	640 min:sec , sec					Adonitol
		1000	43			85
	TFAA group(s): MBA ring(s):	500			57	69		110	140	182	236
			50		100	150	200	250	300	350	400

Table S 2

 S . Continued.

	Molecular ion: 296	Arabitol
	TFAA group(s):
	MBA ring(s):
	Molecular ion: 198	Arabinose
	TFAA group(s):
	MBA ring(s):
	Molecular ion: 198	Xylose
	TFAA group(s):
	MBA ring(s):
	Molecular ion: 198	Ribose
	TFAA group(s):
	MBA ring(s):
	Molecular ion: 324	Mannose
	TFAA group(s):
	MBA ring(s):
	Molecular ion: 324	Galactose
	TFAA group(s):
	MBA ring(s):

Table S 2

 S . Continued.Peak True -sample "122-DL-Sugar mix (x25) 10-4M MM 10-5M :1", peak 150, at 54:20.00 , 3.

	Molecular ion: 324	250 min:sec , sec					Glucose
		1000	43		97
	TFAA group(s): MBA ring(s):	500		57	84	126 113 139	167	240	295
	Molecular ion: 254	50 Peak True -sample "122-DL-Sugar mix (x25) 10-4M MM 10-5M :1", peak 142, at 50:55.00 , 3. 100 150 200 250 300 350 400 Sorbitol (peak 1) 250 min:sec , sec
		1000			85
	TFAA group(s): MBA ring(s):	500	43	69	97			169
		50 Peak True -sample "122-DL-Sugar mix (x25) 10-4M MM 10-5M :1", peak 140, at 50:50.00 , 0. 100 150 200 250 300 350 400
	Molecular ion: 254	076 min:sec , sec				Sorbitol (peak 2)
		1000			85
	TFAA group(s): MBA ring(s):	500	43	57	97 110		169 194
			50	100	150	200	250	300	350	400
	Molecular ion: 254							Dulcitol (peak 1)
		1000			85
	TFAA group(s): MBA ring(s):	500	43		97
			50	100	150	200	250	300	350	400
	Molecular ion: 422							Dulcitol (peak 2)
	TFAA group(s):
	MBA ring(s):
	Molecular ion: 324								Fructose
	TFAA group(s):
	MBA ring(s):

Table S 2

 S . Continued.

	Molecular ion: 198	Ribulose
	TFAA group(s): 0
	MBA ring(s): 2

Table S 3

 S . Regression data of the MBA/TFAA calibration curve, used for days 2 and 7. Intercept was set to 0. Data obtained using 3 concentrations: 10 -4 M, 10 -5 M and 5×10 -6 M. For each concentration, 2 samples were prepared and analyzed 3 times.Peak True -sample "333-Dee Threitol + Ribulose MM 10^-5 M:1", peak 51, at 29:40.00 , 2.090 min:sec , sec

		1000		97
		500	43
			55	84	126	168
			50	100	150		200	250	300	350	400
	#	Compound		S a (M -1 )	ΔS b (M -1 )	R 2
	1	D-Glyceraldehyde		6.51 × 10 3	231		0.99748
	2	L-Glyceraldehyde		6.09 × 10 3	211		0.99756
	3	D-Erythrose		2.33 × 10 3	78.2		0.99774
	4	D-Ribose		1.02 × 10 4	272		0.99858
	5	L-Ribose		1.03 × 10 4	271		0.99863
	6	D-Arabinose		1.31 × 10 5	3327		0.99871
	7	L-Arabinose		1.29 × 10 5	2963		0.99895
	8	D-Xylose		5.34 × 10 4	1301		0.99881
	9	L-Xylose		5.00 × 10 4	1121		0.99900
	10	D-Lyxose		1.36 × 10 3	15		0.99976
	11	L-Lyxose		1.42 × 10 3	17		0.99972
	12	D-2-Deoxyribose		1.86 × 10 3	20		0.99976
	13	L-2-Deoxyribose		2.14 × 10 3	35		0.99947
	14	D-Glucose		3.74 × 10 4	830		0.99901
	15	L-Glucose		3.26 × 10 4	480		0.99956
	16	D-Galactose		1.88 × 10 5	3199		0.99942
	17	L-Galactose		2.25 × 10 5	4961		0.99902
	18	Glycerol		1.01 × 10 4	534		0.99441
	19	D-Threitol		1.14 × 10 3	50		0.99623
	20	L-Threitol		1.04 × 10 3	42		0.99677
	21	Erythritol		8.10 × 10 2	13		0.99952
	22	D-Arabitol		2.01 × 10 3	64		0.99797
	23	L-Arabitol		1.84 × 10 3	62		0.99774
	24	Xylitol		1.64 × 10 4	417		0.99871
	25	Adonitol		1.76 × 10 3	53		0.99821
	26	Dihydroxyacetone		8.67 × 10 2	18		0.99914

a Slope. b Standard error on S.

Table S 4

 S . Regression data of the MBA/TFAA calibration curve, used for day45.

	#	Compound	S a (M -1 )	ΔS b (M -1 )	R 2
	1	D-Glyceraldehyde	1.63 × 10 3	49	0.99732
	2	L-Glyceraldehyde	1.58 × 10 3	50	0.99698
	3	D-Erythrose	2.59 × 10 2	46	0.94090
	4	D-Ribose	4.96 × 10 3	145	0.99743
	5	L-Ribose	4.80 × 10 3	121	0.99810
	6	D-Arabinose	2.79 × 10 4	1606	0.99012
	7	L-Arabinose	2.90 × 10 4	1369	0.99334
	8	D-Xylose	1.82 × 10 4	272	0.99933
	9	L-Xylose	1.78 × 10 4	273	0.99929
	10	D-Lyxose	8.68 × 10 2	13	0.99933
	11	L-Lyxose	8.75 × 10 2	12	0.99941
	12	D-Glucose	2.79 × 10 4	936	0.99662
	13	L-Glucose	2.59 × 10 4	668	0.99801
	14	D-Galactose	1.52 × 10 5	4822	0.99699
	15	L-Galactose	1.70 × 10 5	4571	0.99783
	16	Glycerol	2.95 × 10 3	605	0.88776
	17	D-Threitol	1.09 × 10 3	29	0.99861
	18	L-Threitol	9.66 × 10 2	24	0.99872
	19	Erythritol	2.02 × 10 2	8	0.99720
	20	D-Arabitol	1.34 × 10 3	49	0.99737
	21	L-Arabitol	1.28 × 10 3	58	0.99591
	22	Adonitol	8.69 × 10 2	15	0.99941
	23	Dihydroxyacetone	8.93 × 10 1	21	0.86171
	a

SUPPLEMENTARY MATERIALS