# Photodegradation of the biomarker L-histidine induced by edge sites of a clay mineral in the Martian harsh UV environment

Cristina García-Florentino, Andrew Alberini, Sole Biancalani, Ophélie Mcintosh, John Brucato, Juan Manuel Madariaga, Maguy Jaber, Teresa Fornaro

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## To cite this version:

Cristina García-Florentino, Andrew Alberini, Sole Biancalani, Ophélie Mcintosh, John Brucato, et al. Photodegradation of the biomarker L-histidine induced by edge sites of a clay mineral in the Martian harsh UV environment. Icarus, 2025, 442, pp.116757. 10.1016/j.icarus.2025.116757. hal-05191635

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# Photodegradation of the biomarker L-histidine induced by edge sites of a clay mineral in the Martian harsh UV environment

Cristina García-Florentino<sup>a,b,*</sup>, Andrew Alberini<sup>a</sup>, Sole Biancalani<sup>a</sup>, Ophélie McIntosh<sup>c</sup>, John Brucato<sup>a</sup>, Juan Manuel Madariaga<sup>b</sup>, Maguy Jaber<sup>d</sup>, Teresa Fornaro<sup>a</sup>

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## ARTICLE INFO

### Keywords:

Astrobiology

Mars

Spectroscopy

## ABSTRACT

Clay minerals are thought to play a crucial role in preserving organics on Mars due to their high surface area and expandable interlayers, ideal for adsorbing and intercalating organic molecules. Our study simulated the adsorption of L-histidine, an amino acid potentially diagnostic of life, on the clay mineral saponite, in a slightly acidic aqueous environment as possibly present on Mars during the Hesperian era, followed by a desiccation event and then high UV irradiation typical of the subsequent Amazonian era. For comparison, the same experiments were performed also at alkaline pH. X-Ray Diffraction and Infrared and Deep Ultraviolet Raman and fluorescence spectroscopies were used to characterize the mineral-molecule interactions, indicating that molecules are partly intercalated in the interlayers of saponite at acidic pH and partly co-precipitated with saponite at alkaline pH, while the remainder is adsorbed on lateral edges in both cases. Surprisingly, UV irradiation experiments revealed faster degradation of L-histidine when adsorbed on saponite compared to its pure form at both pHs, suggesting a photocatalytic behaviour of this nano-sized clay mineral, driven by active surface edge sites, despite potential shielding by interlayer sites for the part of L-histidine molecules intercalated in the interlayers at acidic pH. Degradation/alteration of L-histidine adsorbed on saponite occurs in a few hours under Martian UV flux, suggesting that photocatalysis induced by active surface edge sites of clays might have contributed to the depletion of organics in clays at the highly UV-irradiated surface of Mars on very short timescales.

## 1. Introduction

The past and current exploration missions on Mars have already demonstrated its past habitability (Arvidson et al., 2014; Fornaro et al., 2018a; Grotzinger et al., 2014) and therefore, searching for biosignatures, such as the identification of organic molecules, has become one of the main goals of the exploration programs on Mars (Fornaro et al., 2020; Horneck et al., 2016). Low molecular weight hydrocarbons such as chloromethane and dichloromethane were detected for the first time by the NASA Viking twin landers as the result from the interaction of perchlorates and Martian indigenous organic compounds at the high temperatures reached in the instrument (Biemann et al., 1976; Guzman et al., 2018; Navarro-González et al., 2010). Since then, other organic compounds have been detected on Mars at Gale Crater by the Sample

Analyzer at Mars (SAM) instrumental suite onboard the NASA Mars Science Laboratory (MSL) Curiosity rover (Eigenbrode et al., 2018; Freissinet et al., 2015; Millan et al., 2022; Szopa et al., 2020). Since the SAM measurements can also modify the original Martian organics due to thermal decomposition or due to chemical reactions (Millan et al., 2016; Miller et al., 2016), non-destructive analyses are necessary to help to identify the original material, to select the most interesting samples for further analysis and to assist the identification with the more sensitive but destructive techniques. The NASA Mars 2020 Perseverance rover currently operating at Jezero Crater on Mars and the ESA ExoMars Rosalind Franklin rover mission scheduled to arrive on Mars in 2030, are both equipped with Near Infrared (NIR) and Raman vibrational spectroscopies (Vago et al., 2017; Williford et al., 2018), which are non-destructive techniques. Specifically, the SuperCam and Sherloc

<footnote><sup>a</sup> INAF- Astrophysical Observatory of Arcetri, L.go E. Fermi 5, 50125 Firenze, Italy</footnote>

<footnote><sup>b</sup> Department of Analytical Chemistry, University of the Basque Country UPV/EHU, 48080 Bilbao, Spain</footnote>

<footnote><sup>c</sup> LATMOS, UVSQ, 11 Boulevard d'Alembert, 78280 Guyancourt, France</footnote>

<footnote><sup>d</sup> Laboratoire d'Archéologie Moléculaire et Structurale (LAMS), Sorbonne Université, CNRS UMR 8220, 4 place Jussieu, 75005 Paris, France</footnote>

<footnote>* Corresponding author at: INAF- Astrophysical Observatory of Arcetri, L.go E. Fermi 5, 50125 Firenze, Italy.
E-mail address: cristina.garcia@inaf.it (C. García-Florentino).</footnote>
instruments currently onboard the Perseverance are multi-technique instruments to investigate mineralogy and identify possible organic compounds. SuperCam includes Laser Breakdown Spectroscopy (LIBS), passive visible-NIR spectroscopy, Raman spectroscopy (excitation laser 532 nm) and Time-Resolved Fluorescence (TRF) (Wiens et al., 2017). On the other hand, Sherloc is composed of a deep UV Raman (DUV Raman) and fluorescence spectrometer (Corpolongo et al., 2023; Fries et al., 2022). In the case of the future ExoMars mission, on the rover drill there will be the NIR spectrometer MaMISS (De Sanctis et al., 2017), and in the Analytical Laboratory Drawer (ALD) of the rover there will be the NIR spectrometer MicrOmega (Bibring et al., 2017) and the Raman Laser Spectrometer (RLS) (Rull et al., 2017).

Even if these techniques avoid the problem of sample alteration, the large variety of environmental factors and the interactions between the molecule and the mineral, may turn the interpretation of the IR and Raman spectral data very complicated. Vibrational spectroscopy depends on the vibrational modes of the bonds of the functional groups and if these bonds are affected by the interactions with the mineral, the vibrational bands corresponding to those affected functional groups will undergo significant change (Fornaro et al., 2020). For example, the vibrational bands corresponding to a functional group may be shifted to higher or lower wavenumbers if the corresponding bonds are strengthened or weakened due to the interaction with the mineral. Band intensity variations may also happen due to molecular spatial arrangements as imposed by surface selection rules and even new bands may appear due to the formation of chemical bonds as a consequence of chemisorption. The nature of the interactions between the mineral and the organic molecule depends on many factors such as the molecule and the mineral properties, the molecule-mineral relative concentration and the environmental conditions, temperature, pressure, pH, ionic strength and the presence of other reactive species (Fornaro et al., 2020). In particular, the pH is a very important parameter since it affects the protonation state of some molecules and the charge of the surface of the mineral and hence, the molecule-mineral interactions and spectroscopic features.

In addition to the complexity to interpret vibrational spectroscopic data, another important factor that makes the search for molecular biosignatures on Mars complicated is the current Martian atmosphere which allows ionizing radiations, such as energetic solar protons, galactic cosmic rays and ultraviolet (UV) photons, to reach the surface of the planet (Hassler et al., 2014). The effects of energetic solar protons and galactic cosmic rays usually occur in hundreds of millions of years, while UV-induced degradation can happen in just a few hours in the presence of photocatalytic minerals (Fornaro et al., 2018a). UV radiation can be absorbed by many astrobiologically relevant organic molecules, which are electronically excited and undergo photochemical reactions and degradations (Poch et al., 2014; Alberini et al., 2024). For example, amino acids considered important targets in future life-searching missions (Parnell et al., 2007) are known to degrade under UV radiation (Garry et al., 2006; Noblet et al., 2012; Stalport et al., 2019). Even if the penetration of UV radiation is limited, the aeolian erosion continuously exposes new material to the arriving radiation (Vago et al., 2017). In addition, UV light can drive the photochemical production of strong oxidants, such as perchlorates, already confirmed to be present in Martian soil (Glavin et al., 2013), which can penetrate deep into the soil and could cause the oxidative degradation of the organics present (Fornaro et al., 2020). However, at a certain depth with a constant temperature of -60 °C, the oxidation reactions are very slow. Taking this into account, it is considered that the most suitable conditions to preserve organic compounds can be found in the subsurface. At the surface, some preservation of organic compounds may be obtained in the presence of photoprotective minerals (Brucato and Fornaro, 2019; Fornaro et al., 2018a, 2018b; Poch et al., 2015), while photocatalytic minerals can seriously accelerate molecular degradation, and it might be questioned if any alteration of potential organic matter occurs when subsurface material is revealed by the Perseverance abrasion tool before

measuring with its payload instruments or will occur when the samples collected by ExoMars from the subsurface will be exposed to ambient Martian UV. Thus, it is crucial to study the impact of UV radiation of organics adsorbed in minerals that can represent Martian soils for detecting organic molecules in situ through robotic exploration missions. These investigations also provide insights into the preservation/degradation mechanisms of organics occurred throughout Martian history.

In this work, the likelihood of a biomarker to be preserved when embedded into a clay mineral, despite the Martian chemical weathering due to UV irradiation and, hence, to be detected by spectroscopic techniques like NIR and DUV Raman and fluorescence, was investigated. Specifically, adsorption of L-histidine, an amino acid potentially diagnostic of life, on the clay mineral saponite, was performed in a slightly acidic aqueous environment as possibly present on Mars during the Hesperian era, and then followed by desiccation and high UV irradiation typical of the subsequent Amazonian era. For comparison, the same experiments were performed also at alkaline pH. InfraRed and Raman spectroscopies and X-Ray Diffraction were used to characterize the mineral-molecule interactions.

Clays are of important relevance in astrobiology as they are evidence of past aqueous activity on Mars (Nazari-Sharabian et al., 2020). Moreover, clay smectite minerals like saponite tend to accumulate organic molecules due to their large surface area and optimal interlayer sites which also favour the preservation of organic molecules (Nazari-Sharabian et al., 2020). Indeed, smectites have expandable interlayers, optimal for organic intercalation, that may provide size-tailored microporosity for a variety of molecules, and the formation of clay particle aggregates with intra- and inter-porosity, which might constitute a shielded environment by limiting penetration of fluids and radiation in the inner part of the mineral. Fe/Mg smectites have already been identified by orbiters (Carter et al., 2013) and in situ by the Curiosity rover at Gale Crater (McLennan et al., 2014; Vaniman et al., 2014). Orbital data also shows that clays are present in the Mars 2020 (Salvatore et al., 2018; Schon et al., 2012) and ExoMars landing sites (Ivanov et al., 2020).

The L-histidine amino acid was selected as a possible target for life-search missions since it is an α-amino acid utilized in the biosynthesis of proteins and therefore, it is an organic molecule biotically relevant for life as we know it. Early Mars possessed Earth-like environmental characteristics, and received the same inputs of water and organics through meteoritic infall (Zhu et al., 2022; Chyba, 1987; Rotelli et al., 2016; Anders, 1989; Chyba et al., 1990). Under comparable geochemical conditions, it is likely that these abiotic organic compounds underwent a similar molecular evolution, bringing to a similar biochemistry and therefore is reasonable to assume that, if life ever emerged on Mars, it could possess analogous characteristics to those of terrestrial life (Committee on an Astrobiology Strategy for the Exploration of Mars, 2007).

This amino acid was also selected due to its different possible pH-dependent protonation states that may affect the kind of bonding with the mineral (Mesu et al., 2005). Due to the presence of an imidazole ring, L-histidine possesses three extra potential sites for (de)protonation and metal binding in addition to the two NH₂ and COOH end groups of other amino acids (Mesu et al., 2005) (see Fig. 1). This extra possible protonation sites in the imidazole group make this amino acid able to exhibit not only formal charges in the amino and carboxylic groups, but a total positive, neutral or negative charge depending on the pH (see Fig. 1). L-histidine can be present in five different pH-dependent ionic forms, H₄His²⁺, H₃His⁺, H₂His⁰, HHis⁻ and His²⁻ with the ionic structure H₂His⁰ and HHis⁻ occurring in different tautomeric forms as the nitrogen in the imidazole are not equivalent (Mesu et al., 2005) (see Fig. 1). This total charge may also affect the interaction with the mineral. When organic molecules are positively charged in water, the electrostatic interaction with the negatively charged surface of the mineral is expected to promote the adsorption of the molecule and even
<figure><img src="image_5.png" /><figcaption>Fig. 1. L-histidine in all its pH dependent ionic forms.</figcaption></figure>

its intercalation in the interlayer sites of the mineral (Hashizume and Fujii, 2016). Therefore, the effect of the pH is very important when studying the adsorption of organic compounds with acid-base properties on minerals. In the Hesperian era on Mars, the pH was slightly acid (pH = 2–5) due to the SO₂ exhalations from the Martian volcanic activity (Cockell, 2014). Nowadays however, in a variety of Martian settings, pH ranges are within the boundaries for life. For example, the pH measured in the subsurface of the Phoenix lander was almost neutral, slightly alkaline (pH = 7.7–7.9) (Cockell, 2014). In this work, the adsorption of L-histidine onto saponite was studied at pH = 4 (H₃His⁺) as possibly present during the Hesperian era on Mars and pH 7 (H₂His⁰), as well as at a more alkaline pH > 9 (HHis⁻), to check if the negative total charge of the molecule at this pH was preventing the molecule from going to the interlayer of the mineral and how the differences in molecular adsorption would affect the fate of L-histidine under UV irradiation.

## 2. Materials and method

### 2.1. Sample preparation

Synthetic saponite (surface area of 230 m²/g and volume of 0.2441 cm³/g) was employed in order to have precise control of the structure and the absence of any original organic contamination present on natural saponites that may affect to the data interpretation. For the synthesis of sodium saponite, a first solution of sodium acetate in water and sodium silicate was prepared. A second solution of deionized water, magnesium acetate, aluminum acetate and acetic acid was added to the first one under stirring. The composition corresponded to the following theoretical formula: Na₀.₃[(Si₃.₇Al₀.₃)(Mg₃)]O₁₀(OH)₂ (formula of Saponite per half unit cell for a substitution ratio of Silicon by Aluminum cations of 0.3). The hydrogels were aged under stirring at room temperature for 6 h and then were autoclaved for reaction at 300 °C and 90 bar for 6 h. The autoclave was cooled to room temperature and the solid product was washed thoroughly with distilled water and separated by centrifugation. The solids were then dried at 50 °C for 24 h.

L-histidine (purity >99.5 %, Sigma-Aldrich) was adsorbed onto saponite by mixing the saponite powder with aqueous solutions of L-histidine at different pH conditions and keeping the suspensions under rotation on a test-tube rotator for 24 h, using an equilibrium adsorption method as previously described by Fornaro et al. (2020). The pH was previously adjusted by adding several drops of a HCl 1 M or NaOH 1 M solutions until the desired pH was reached and at which the L-histidine is in its positively (pH 4.6) or negatively (pH 9.6) charged state, respectively. In the case of the acid pH adsorptions, the pH was monitored during the first hours and corrected by adding some more HCl drops due to the neutralising character of the saponite. It was also checked that the pH was lower than 6 at the end of the adsorption to

guarantee the positive charge of L-histidine during the entire process. The samples were dried at 50 °C in an oven. An initial set of samples was prepared containing 5 wt% of the organic molecule. Then, a second set of samples was prepared at 10 wt% organic concentration to better detect molecular bands by IR and easily follow the degradation kinetics by observation of changes of the molecular IR features during UV irradiation.

All the employed glassware was cleaned previously with dichloromethane and ethanol and heated up to 500 °C in a furnace to avoid any possible organic contamination.

### 2.2. Infrared characterization

The characterization of the samples was performed by Diffuse Reflectance InfraRed Fourier Transform Spectroscopy (DRIFTS) using a VERTEX 70v FT-IR spectrometer (Bruker) equipped with a Praying Mantis™ Diffuse Reflection Accessory (Harrick) which works in the Mid-Infrared (MIR) range (8000 to 400 cm⁻¹). The samples were measured in powder form using 200 scans for each spectrum with an instrumental resolution of 4 cm⁻¹. The samples were analysed with a constant flux of nitrogen (N₂) to avoid disturbances in the spectra from atmospheric CO₂ and O₂. Every day, an infragold standard was measured for the calibration of the instrument also under nitrogen flux.

### 2.3. Deep ultraviolet Raman and fluorescence characterization

Samples were characterized with a Photon Systems RPL200 Raman and fluorescence instrument analogous to the SHERLOC instrument on the Perseverance rover. The instrument utilizes Deep Ultraviolet (DUV) excitation (248.6 nm) from a NeCu pulsed laser. The spectra were acquired using 100 and 1–10 pulses per point, respectively, 20 A laser current and a repetition rate of 40 pulses per second, using a 20× microscope objective, and averaged over hundreds of points.

### 2.4. X-ray diffraction characterization

X-Ray Diffractograms were recorded at room temperature and under a vacuum between 2 and 3.10⁻³ mbar on a Panalytical Xpert Pro MPD (Materials Powder Diffractometer) operating at 45 kV and 40 mA (1800 W), equipped with a sealed Co-anode X-ray tube with Fe filter, an RTMS-type X' Celerator detector and an Anton Paar TTK-450 vacuum chamber. Acquisitions were carried out in Bragg-Brentano mode (θ - 2θ scan) in the angular range 3° ≤ 2θ ≤ 90° (increment = 0.0668° and exposure time 30 min with step time 180 s), in 1/2° split slit, antiscatter incident 1°; antiscatter diffracted 5.5 (=1.2°); soller 0.04 and a 15 mm mask.
## 2.5. Ultraviolet irradiation experiments

### 2.5.1. UV irradiation experimental set up

The UV irradiation experiments were carried out using a Newport Oriel 300 W Xenon arc discharge lamp with a spectral range between 200 and 930 nm. The UV light was focused directly on the sample through an optical fiber of 800 µm spot size inserted into the IR sample compartment to monitor the infrared spectra in situ during the UV irradiation. All the surface of the sample, which presents an area of 7.07 mm² in the IR sample holder, was irradiated. The calculated UV flux arriving on the sample is of 2.75·10¹⁷ photons/s·cm² in the 200–400 nm spectral range as measured through a single monochromator Spectro 320 scanning spectrometer. The sample was kept under a constant nitrogen flux during all the experiment. The degradation of the molecule along the irradiation time was monitored by the previously described Infrared spectrometer. All the samples were irradiated for a total time of 4200 s (1 h 10 min).

### 2.5.2. UV irradiation data treatment

All the bands corresponding to the L-histidine in the adsorbed samples and the corresponding ones in the pure molecule were integrated and normalized to the band area of the non-irradiated sample using MATLAB R2021b (The MathWorks Inc., 2021). The degradation curves obtained were fitted to the function shown in eq. 1.

$$ \frac{A(t)}{A_M} = a \cdot e^{-b \cdot t} + c \quad (1) $$

Where $A(t)$ is the area of the infrared band at irradiation time t, $A_M$ is the area of the band before irradiation, $b$ is the degradation rate, $a$ corresponds to the fraction of molecules affected by the UV flux and $c$ refers to the fraction molecules not affected by the UV flux. The $c$ term is needed because the beam of the IR laser is penetrating more than UV radiation of the lamp, and therefore part of the molecules detected on the IR spectrum are not affected by the UV of the lamp.

From the degradation rate obtained for each band ($b$) from the fitting to eq. 1, the half-lifetimes ($t_{1/2}$) of degradation for the different bands of the pure and adsorbed L-histidine were calculated according to eq. 2:

$$ t_{1/2} = \ln2/b \quad (2) $$

The degradation cross section ($\sigma$) was calculated according to eq. 3 from the degradation rate ($b$) and the total flux per area of irradiation of the UV lamp used in the experiment ($\varphi$):

$$ \sigma = b/\varphi \quad (3) $$

The degradation cross section ($\sigma$) obtained for each band of L-histidine in the different analysed samples is a constant characteristic parameter of the molecule, which can be used to give an estimation of the degradation half-lifetimes on Mars. Assuming a Martian flux per area ($\varphi_{Mars}$) of 1.4·10¹⁵ photons/s·cm² (Patel et al., 2002) in the 190–325 nm spectral range, the Martian $b$ can be calculated from eq. 3. Finally, from the obtained Martian $b_{Mars}$, the half-lifetimes of degradation on Mars ($t_{1/2_{Mars}}$) can be estimated for the L-pure histidine and the L-histidine adsorbed on saponite.

# 3. Results and discussion

## 3.1. Infrared, XRD and DUV-Raman characterizations and L-histidine adsorption mechanisms onto saponite at different pHs

Understanding the kind of adsorption between the mineral and the amino acid is of great importance as it affects the stability of the amino acid against the UV irradiation and therefore the survival of the molecule on Mars (Fornaro et al., 2018b).

In order to understand the complexity of the adsorption interaction between a mineral and an organic molecule such as an amino acid, it is

necessary to know the structure of the mineral. Saponite is a trioctahedral 2:1 smectite and its ideal composition is $M_xMg_3Al_xSi_{4-x}O_{10}(OH, F)_2 \cdot 2H_2O$ where M is the interlayer cation ($Na^+, k^+, Mg^2+, Rb^+$ etc). In the synthetic saponite used in this work, the ideal formula is $Na_{0.3}[(Si_{3.7}Al_{0.3})(Mg_3)]O_{10}(OH)_2$. A single 2:1 layer is composed of a central sheet of $Mg^{2+}$ with an octahedral coordination placed between two layers of $Si^{4+}$ with a tetrahedral coordination. The partial substitution of some of the $Si^{4+}$ by $Al^{3+}$ results in an overall negative charge in the tetrahedral layers, which is compensated by the interlayer cations (Kloprogge and Ponce, 2021) ($Na^+$ in our synthetic saponite). While basal surfaces (surfaces facing the interlayer) interact with adsorbates, mostly through electrostatic interaction, the edge surfaces have amphoteric properties and can bind the organics through surface complexation (Gao et al., 2023). The edges consist on $-O$, $-OH$ and $-OH_2$ groups and their relative amounts depend on the pH of the aqueous environment where is the mineral (Gao et al., 2023) (see Fig. 2 for a schematic visualization of saponite).

Amino acids are bound to the minerals by cation exchange and/or complexation. The positively charged amino acids can be adsorbed in the interlayer by cation exchange while the negatively charged amino acids may be adsorbed at the edges. A further mechanism of adsorption is the complexation of the interlayer cations by the carboxylate groups of the zwitterion and the anionic form (Lagaly et al., 2006; Schoonheydt and Johnston, 2006). The degree of ion exchange and complexation in an acidic medium depends on the interlayer cation, being stronger the complexation with transition metal cations than with alkali and earth alkali cations (Lagaly et al., 2006). There is a competition between the acid-base chemistry and the complexation and as a result is a complex adsorption (Schoonheydt and Johnston, 2006).

The vibrational bands corresponding to a functional group may be shifted to higher or lower wavenumbers if the corresponding bonds are strengthened or weakened due to the interaction with the mineral and therefore this can help identifying the functional groups involved in the interaction helping to understand the adsorption mechanisms.

### 3.1.1. Infrared characterization

All the bands of L-histidine that can be appreciated when adsorbed on saponite and their corresponding vibrational mode assignments and shifts with respect to the original positions in the pure L-histidine are listed in Table 1. The Infrared spectra can also be checked in Fig. 3. Note that only the shifts >4 cm⁻¹ are shown in the table, the rest are under the resolution of the instrument. Therefore, only bands with higher shifts are indicated and discussed as related to chemical interactions. These shifts of the molecule infrared bands observed when they are adsorbed onto the mineral with respect to the position of the pure molecule, along with the changes in the mineral bands, can be used for interpreting the kind of interactions between the mineral and the amino acid (Deacon and Phillips, 1980; Dong et al., 2018; Madejová et al., 2017; Nkoh et al., 2022; Ramos and Huertas, 2013). In Table 2, the bands of the mineral that presented a shift after the adsorption are shown. The observed IR shifts are not very big in none of the cases but are corroborated by complementary data afterwards.

Amino acids adsorbed inside the interlayer through electrostatic attraction or hydrogen bond between the negatively charged surface of the mineral and the $-NH_3^+$ will undergo a shift in the bands corresponding to the vibrational modes of $-NH_3^+$. In our case of study, it is not possible to identify the band of L-histidine corresponding to the N–H stretching of the amino group due to the overlap with the broad band of water (See Fig. 3). However, the bands related to the in-plane deformation of the N–H bond that can be appreciated in the pure amino acid as two separated bands at 1506 and 1489 cm⁻¹, appear as one broader band when the L-histidine is adsorbed indicating that some interactions occur though the amino group in the imidazole ring in all the pHs. The band at 1350 cm⁻¹ for L-histidine is referred in bibliography (Dammak et al., 2007; Deplazes et al., 2008; Mesu et al., 2005) to be related to the cation $NH_3^+$ with a contribution δ out of plane $CH_2$. However, in our case
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Scheme with the suggested interactions between L-histidine and saponite at pH 4 and pH 9.6. Note that the L-histidine is represented as a green box with the functional groups implied at each interaction highlighted. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

it seems that the band is completely due to the cation $NH_3^+$, because this band disappears for both 5 and 10 wt% concentrations when the adsorption was done at pH = 9.6 and the L-histidine is mainly as $HHis^-$ where the amino group is as $NH_2$. Therefore, the shifts of this band to higher wavenumbers observed for the L-histidine adsorbed at acidic pH at both concentrations and also for the zwitterionic form of the L-histidine observed at pH 7.7 suggest an interaction between the positively charged $-NH_3^+$ and the negatively charged basal surface of saponite. In addition, in Table 2, the bands of saponite in the interval 400 and 1000 $cm^{-1}$, related to the internal bending and stretching motions of the $SiO_4^{2-}$ tetrahedra (Schoonheydt and Johnston, 2006) show some shift also indicating there might be an interaction of these groups with the amino acid. This may imply an intercalation of the molecule in the interlayer to react with the negatively charged basal surface of saponite in a cation exchange type of interaction, as observed in several studies (Dong et al., 2018; Nkoh et al., 2022; Ramos and Huertas, 2013) of amino acids adsorbed onto another smectite like montmorillonite, or just an electrostatic interaction with the outermost basal surface of the L-histidine or a combination of both.

Moreover, the band at 1406 $cm^{-1}$, related to the symmetric stretching of the $COO^-$ group, presents a shift to slightly higher wavenumbers only for the acidic adsorptions, being the highest shift for the

most concentrated at 10 wt% and a slightly smaller shift for the lower concentration 5 wt%. This observation indicates that the interaction with the edges becomes more important as increasing the concentration of L-histidine. In both acidic adsorptions, the pH interval 3.5–4 is low enough to protonate some of the saponite surface edge groups as $AlOH_2^-$ and $SiOH_2^-$ allowing an interaction with the $COO^-$ group the carboxylic group present in this pH interval. Therefore, the shift observed for the symmetric stretching of the $COO^-$ at acidic conditions suggests the presence of another adsorption mechanism on the edge surface of saponite through interaction between the $COO^-$ and the $AlOH_2^-$ and $SiOH_2^-$ This edge interaction has already been described for other amino acids such lysine and glycine when adsorbed onto montmorillonite competing with the adsorption in the interlayer between the $NH_3^+$ and the basal negative surface (Dong et al., 2018; Ramos and Huertas, 2013). In Fig. 2, the interactions suggested are presented in a schematic visual way to help in the comprehension.

At pH 7.7 and 9.6, the carboxylic group is also in the deprotonated state as $COO^-$ (see Fig. 1), thus the corresponding IR bands are observed in the spectrum of the L-histidine adsorbed at these pHs (see Figs. 2 and Table 2). However, the adsorptions of L-histidine at these basic pHs do not show a shift for the $COO^-$ IR bands indicating the carboxylate group of L-histidine is not involved in an interaction in this case. Probably, in
<caption>**Table 1**</caption>

Infrared bands of L-histidine and L-histidine adsorbed onto saponite at 5 wt% and 10 wt% at pH 4 and 9.6. Vibrational shifts with respect to the pure molecule are indicated in brackets.

<table><thead><tr><th>Pure<br>L-histidine<br>(cm<sup>-1</sup>)</th><th>5 wt%<br>L-histidine on<br>saponite<br>pH 3.5 (cm<sup>-1</sup>)</th><th>5 wt%<br>L-histidine on<br>saponite<br>pH 7.7 (cm<sup>-1</sup>)</th><th>5 wt%<br>L-histidine on<br>saponite<br>pH 9.6 (cm<sup>-1</sup>)</th><th>10 wt%<br>L-histidine on<br>saponite<br>pH 4 (cm<sup>-1</sup>)</th><th>10 wt%<br>L-histidine on<br>saponite<br>pH 9.6 (cm<sup>-1</sup>)</th><th>Vibrational mode assignment</th></tr></thead><tbody><tr><td>6164</td><td>6178 (+14)</td><td>6176 (+12)</td><td>6176 (+12)</td><td>6178 (+14)</td><td>6164 (0)</td><td>-</td></tr><tr><td>5871</td><td>-</td><td>-</td><td>-</td><td>-</td><td>5863 (-8)</td><td>-</td></tr><tr><td>5819</td><td>-</td><td>-</td><td>-</td><td>-</td><td>5818 (-1)</td><td>-</td></tr><tr><td>5763</td><td>-</td><td>-</td><td>-</td><td>5759 (-4)</td><td>5762 (-1)</td><td>-</td></tr><tr><td>5668</td><td>-</td><td>-</td><td>-</td><td>-</td><td>5658 (-10)</td><td>-</td></tr><tr><td>4627</td><td>-</td><td>-</td><td>-</td><td>-</td><td>4621 (-6)</td><td>-</td></tr><tr><td>4102</td><td>-</td><td>-</td><td>-</td><td>-</td><td>4102 (0)</td><td>-</td></tr><tr><td>4056</td><td>-</td><td>-</td><td>-</td><td>4052 (-4)</td><td>4058 (+2)</td><td>-</td></tr><tr><td>1506</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>δ in plane N-H imidazole (Kumar et al., 2010)</td></tr><tr><td>1489</td><td>1497 (+8)</td><td>1497 (+8)</td><td>1497 (+8)</td><td>1495 (+6)</td><td>1496 (+7)</td><td>δ in plane N-H and ν C=N imidazole (Mesu et al., 2005)</td></tr><tr><td>1431</td><td>1437 (+6)</td><td>1437 (+6)</td><td>1437 (+6)</td><td>1437 (+6)</td><td>1437 (+6)</td><td>δ CH<sub>2</sub> (scissoring) (Deplazes et al., 2008; Mesu et al., 2005)</td></tr><tr><td>1406</td><td>1412 (+6)</td><td>1410 (+4)</td><td>1408 (+2)</td><td>1414 (+8)</td><td>1410 (+4)</td><td>ν<sub>s</sub> CO<sub>2</sub> (Deplazes et al., 2008; Mesu et al., 2005)</td></tr><tr><td>1350</td><td>1366 (+16)</td><td>1366 (+16)</td><td>-</td><td>1360 (+10)</td><td>-</td><td>δ NH<sub>3</sub> (Deacon and Phillips, 1980; Mesu et al., 2005)/ δ out of plane CH<sub>2</sub> (wagging) (Dammak et al., 2007; Deplazes et al., 2008; Mesu et al., 2005)</td></tr><tr><td>1335</td><td>1344 (+9)</td><td>1342 (+7)</td><td>1342 (+7)</td><td>1344 (+9)</td><td>1340 (+5)</td><td>δ CH<sub>2</sub> (Mesu et al., 2005)</td></tr><tr><td>1317</td><td>1321 (+4)</td><td>1321 (+4)</td><td>1321 (+4)</td><td>1319 (+2)</td><td>1317(0)</td><td>δ CH<sub>2</sub> and δ = C-H (Mesu et al., 2005)</td></tr><tr><td>1273</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1271 (-2)</td><td>ν = C-N and δ C-H (Mesu et al., 2005)</td></tr><tr><td>825</td><td>-</td><td>-</td><td>-</td><td>829 (+4)</td><td>829 (+4)</td><td>ν<sub>sym</sub> of the ring (Dammak et al., 2007)</td></tr><tr><td>629</td><td>627 (-2)</td><td>-</td><td>629 (0)</td><td>627 (-2)</td><td>633 (+4)</td><td>Ring deform. (Kumar et al., 2010)</td></tr></tbody></table>

ν = stretching; δ = bending.

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Infrared spectra for L-histidine, saponite and L-histidine adsorbed on saponite at pH 4 and 9.6. Whole spectra range (A) and different zoom regions (B, C and D). The wavenumber positions of the bands of L-histidine that can be appreciated in the adsorbed samples are marked.</figcaption></figure>

the adsorptions of L-histidine at pH 7.7 and 9.6, the edge surface groups of saponite are not protonated, so the interaction with them is not possible in slightly basic or basic conditions. This kind of adsorption has already been described in the study by Ramos and Huertas, 2013 about adsorption of glycine on montmorillonite, observing that the adsorption of glycine was dominated by the complexation of the carboxylate group of the zwitterionic glycine onto the edge surface followed by cation

exchange in the interlayer when the edge was saturated. However, they studied the zwitterionic adsorption below pH 7 when the montmorillonite developed a positive charge due to the protonation of the aluminum sites (Ramos and Huertas, 2013). In our case, the study of the adsorption of the zwitterionic form of L-histidine onto saponite was done at a higher pH, probably too high to find the edge surface groups of the saponite protonated, and therefore this kind of edge adsorption
<caption>**Table 2**</caption>

Infrared bands of saponite that undergo vibrational shifts when L-histidine is adsorbed at different pHs and concentrations. Vibrational shifts with respect to the pure saponite blank are indicated in brackets.

<table><thead><tr><th>Pure saponite (cm<sup>-1</sup>)</th><th>Saponite 5 wt% L-histidine pH 3.5</th><th>Saponite 5 wt% L-histidine pH 7.7</th><th>Saponite 5 wt% L-histidine pH 9.6</th><th>Saponite 10 wt% L-histidine pH 4</th><th>Saponite 10 wt% L-histidine pH 9.6</th><th>Vibrational mode assignment</th></tr></thead><tbody><tr><td>1242</td><td>1215 (-27)</td><td>1215(-27)</td><td>1209 (-33)</td><td>1209 (-33)</td><td>1190 (-52)</td><td>Combination band</td></tr><tr><td>978</td><td>974</td><td>978</td><td>976</td><td>974</td><td>972 (-6)</td><td>ν Si-O (Madejová et al., 2017)</td></tr><tr><td>704</td><td>698 (-6)</td><td>698 (-6)</td><td>696 (-8)</td><td>696 (-8)</td><td>694 (-10)</td><td>ν Si-O and δ MgOH (Madejová et al., 2017)</td></tr><tr><td>590</td><td>582 (-8)</td><td>580 (-10)</td><td>584 (-6)</td><td>580 (-10)</td><td>580 (-10)</td><td>δ SiO (Madejová et al., 2017)</td></tr></tbody></table>

$$\nu = stretching; \delta = bending.$$

together with the cation exchange was only observed for the acidic pHs. Carboxylates can also bind by complexation with metal cations forming an inner sphere complex (Deacon and Phillips, 1980). However, we do not think this could be the case, first of all because the formation of this kind of complexes is stronger with metal transition cations than with alkali and earth alkali cations (Lagaly et al., 2006), as the Na⁺ present in our saponite. In addition, in our study at pHs 7.7, the carboxylate does not present a shift that might indicate the formation of a complex between the zwitterionic form and the cations at this pH.

For the adsorption of the L-histidine at basic pH of 9.6, the only thing that we can appreciate in the IR the transformation of the two bands related to the deformation of the N-H bonding at 1506 and 1489 cm⁻¹ into a single one. This may also indicate an interaction through the imidazole NH group of the L-histidine present at this pH. As the L-histidine is negatively charged at pH 9.6, the interlayer interaction is improbable due to electrostatic repulsion with the basal negative charge of the saponite. However, in this case it might be another edge surface interaction but in this case through the imidazole NH that may form a hydrogen bond interaction with the non-charged edge surface groups like the silanol and other OH groups capable of H-bonding. In Fig. 2, the interactions are shown in a schematic way.

Other small shifts that were observed are related to the CH₂ group (at 1431 and 1335 cm⁻¹) in adjacent position to the carboxylic group involved in the interactions with the mineral, especially important in the acid and neutral states. This can also be observed in the shift for the band at 1335 cm⁻¹, which is higher when the L-histidine is adsorbed at acid or neutral pHs than when adsorbed in basic conditions. It seems that the adjacent CH₂ to the amino group vibration modes are also affected and this may suggest an interaction between the amino group and the edge of the mineral via another hydrogen bond at basic pH even if the band for the NH₂ cannot be observed.

### 3.1.2. XRD characterization

The XRD results confirm the observations done by IR. In Fig. S1 from Supplementary Material, the X-Ray diffraction patterns for raw saponite and its hybrid derivatives are depicted. All reflexions typical of saponite are present as already reported in the literature. The d(001) in the pristine clay is about 13.5 Å corresponding to a hydrated interlayer space. After histidine adsorption, the interlayer space increased with a measured d(001) of 13.6 Å at pH 4. However, the d(001) decreased for the adsorption at pH 9.6 to 13.4 Å. It is worth noting that in adsorbed sample in alkaline medium, the peaks corresponding to crystalline L-histidine are observed in the corresponding sample (indicated with asterisks in Fig. S1). However, in acidic medium no peaks of the crystalline amino acid were observed. These observations can be directly correlated to the speciation of the amino acid as a function of the pH: indeed in the acidic medium, L-histidine is positively charged and the intercalation probably occurred via cationic exchange with sodium cations as observed in IR by the shift in the band related to the NH₃⁺ group reacting with the negative surface of the saponite once is intercalated and increasing the size of the interlayer as indicated by the distance d(001).

The interlayer surface is two times bigger than the basal surface and can be evaluated approximatively from the d₀₀₁ (10.5 Å) and the density

(2.85 g·cm⁻³) for an anhydrous clay, as 668.3 m²/g. If we assume that each basal surface is occupied by a monolayer of L-histidine molecules lying parallel to the surface, the amount of molecules that can enter inside the interlayer space is 1.29·10²¹ for 1 g of mineral, considering the area occupied by a single molecule being 8.5·6.1 Å = 5.185·10⁻¹⁹ m². In our 10 wt% samples, per 1 g of mineral there are 3.88·10²⁰ molecules of L-histidine. This means that all molecules might potentially enter in the interlayer space in a parallel arrangement. Nevertheless, infrared evidence at acidic pH (where the molecule exhibits a total positive charge) indicates that some molecules interact through the COO⁻ group. This interaction is precluded within the interlayer space due to repulsion with the negatively charged interlayer. However, it can occur with the positively charged edges of the mineral. Furthermore, the observed shift for the COO⁻ band is more pronounced when the concentration is increased from 5 to 10 wt%, indicating that the interaction with the edges becomes more significant as the concentration increases. The data suggest the existence of two distinct and competitive adsorption mechanisms at acidic pH. This is not observed at basic pH (as confirmed by XRD results) because the molecule, having a total negative charge, cannot penetrate the interlayer. This suggests that even though there is enough space within the interlayer, not all molecules are intercalated.

This behaviour may be attributed to competitive adsorption in the acid case, as observed by Ramos and Huertas, 2013 in the adsorption of glycine into montmorillonite. In this process, the edge surfaces are initially saturated, followed by cation exchange with the interlayer. Alternatively, the observed behaviour may result from the molecules arranging themselves in the interlayer spaces in a non-parallel orientation, which may prevent the entry of amino acid molecules into the interlayer. Moreover, in the acid adsorption, we cannot observe a crystallized L-histidine over the saponite, as we can for the basic adsorption where we can observe the peaks of the pure L-histidine (see Fig. S1). This suggests that the rest of the L-histidine that does not go inside the interlayer has to be reacting on the protonated edges (AlOH₂⁺ and SiOH₂⁻) as suggested by the IR results with the observed shift for the band at 1406 cm⁻¹, related to the symmetric stretching of the COO⁻ group (see Fig. 2). The observed IR shift for the COO⁻ due to the interaction between the carboxylate and the Na⁺ by complexation with metal cations forming an inner sphere complex is not considered a highly probable outcome. This is because the formation of such complexes is stronger with metal transition cations than with alkali and earth alkali cations (Lagaly et al., 2006) as is the case with the Na⁺ in saponite. The observation of an interaction between the mineral and the acid adsorption, as evidenced by corresponding shifts in the mineral bands (Table 2) and the NH₃⁺ IR band of the amino acid (Table 1), suggests the involvement of cation exchange and electrostatic interactions between the protonated amino group of L-histidine and the negatively charged interlayer surface. These interactions appear to be the primary mechanism governing the adsorption process within the interlayer. If we consider the possibility that some of the sodium ions may react outside the interlayer after undergoing cation exchange with another amino acid that remains outside the interlayer, and if this were a primary reaction, the presence of the salt precipitated outside the
mineral would be expected and detectable by XRD. Similarly, the precipitation of the amino acid on the mineral at basic pH is also observed. Nevertheless, no evidence of salt or pure amino acid crystallization in the acid adsorption was discerned by XRD (see Fig. S1).

On the other hand, in the alkaline adsorption, the interlayer is not expanded. The d(001) of 13.4 Å (see Fig. S1) corresponding to the interlayer distance is slightly smaller than the one observed for the pure saponite. This could be due to the 1 h heating procedure to which all the samples were subjected before the XRD measurements, being the dehydration of the interlayer more effective for the sample with the adsorbed L-histidine at pH 9.6. The interaction of the negatively charged L-histidine species with the clay layers that also bear a negative charge is repulsive; thus, it is hard to envisage a sufficient driving force for L-histidine intercalation. The only possibility is that L-histidine is adsorbed on the edge of the clay particles, which do not bear any significant electric charge in our pH range but exhibit silanol and other OH groups capable of H-bonding. As shown in the IR transformation of the two bands related to the deformation of the imidazole N-H bonding at 1506 and 1489 cm⁻¹ into a single one, the NH group could be the one interacting with the edges via hydrogen bond (see Fig. 2) and the shift of the adjacent CH2 to the amino NH₂ may suggest also this amino interacting with the edge. However, it is very important to highlight that the presence of many peaks of crystalline L-histidine can be appreciated in the adsorption done at basic pH showing the presence of part of L-histidine not adsorbed into the mineral. This could also be the reason of the more IR bands observed for the L-histidine in the adsorption done at pH 9.6 than at pH 4 (see Fig. 3, S1 and Table 1).

### 3.1.3. DUV Raman characterization

In Table 3, the DUV Raman data are shown for pure L-histidine, L-histidine adsorbed on saponite at acid and basic pHs and their corresponding shifts. For the DUV-Raman analyses, the resolution varies between 10 and 25 cm⁻¹. In order to be sure of a real shift of the bands due to interactions, only the shifts above 25 cm⁻¹ have been indicated on the table and are considered as indicative of an intercation/change in the corresponding functional group. The Raman analyses confirmed the edge interaction at acid pH between the -CO₂ functional group of the amino acid and the protonated edge of saponite (AlOH₂ and SiOH₂). At acid pH, the band at 1572 cm⁻¹ assigned to vₐ₃ₘₐ CO₂ in pure solid L-histidine, exhibits a big shift indicating again the interaction through this functional group at acid pH. In addition, in the adsorption at acid pH, a new band appears at 3656 cm⁻¹ which is assigned to the stretching of a new O-H bond probably due to the new interaction between the CO₂ amino acid and the edge groups of L-histidine. The electrostatic interaction in the interlayer in this case through NH₃⁺ cannot be confirmed, however the shift in the band 678 cm⁻¹ related to the deformation of the imidazole ring and to the out of plane N-H bending indicates that the electrostatic interaction between the molecules inside the interlayer may happen also between the charged NH⁺ in the imidazole ring (see Fig. 1) and the negative interlayer. The same as observed in IR for the bands at 1506 and 1489 cm⁻¹.

In the adsorption at basic pH, however, none of these shifts are observed. What is more, no fundamental bands show a shift with respect to the pure. It seems that the average Raman spectrum is dominated by the pure L-histidine precipitated at basic pH as observed in the XRD (see Fig. S1 in Supplementary Material). Some combination bands do present some shifts indicating that there are some differences with respect to the pure L-histidine, and this can only be due to some L-histidine interacting with the mineral edges as shown by IR.

### 3.2. Detection of L-histidine adsorbed onto saponite at different pHs and implications for space exploration missions

The main problem of the Infrared identification of L-histidine adsorbed onto saponite is the loss of most of the characteristic bands of the amino acid even at the high concentration of 10 wt% as it can be

<caption>**Table 3**</caption>

Deep UV Raman bands for pure L-histidine, L-histidine adsorbed on saponite at acid and basic pHs and at 10 wt% concentration.

<table><thead><tr><th>Pure L-histidine</th><th>L-histidine in saponite acid pH</th><th>L-histidine in saponite basic pH</th><th>Vibration mode assignment</th></tr></thead><tbody><tr><td>x</td><td>3656 w</td><td>x</td><td>νOH (Hadjiivanov et al., 2021)</td></tr><tr><td>3119 int</td><td>x</td><td>3127 int</td><td>νC-H imidazole ring (De Sousa et al., 2013; Jenkins et al., 2005)</td></tr><tr><td>3021 w. broad</td><td>x</td><td>2963 (-58) int</td><td>νs C-H (De Sousa et al., 2013)</td></tr><tr><td>2930 int</td><td>2942 broad</td><td>2884 (-46) int</td><td>νs CH₂ (De Sousa et al., 2013)</td></tr><tr><td>2858 w</td><td>x</td><td>2838 w</td><td></td></tr><tr><td>2750 w</td><td>x</td><td>2740 w</td><td></td></tr><tr><td>2634 w</td><td>x</td><td>2627 w</td><td></td></tr><tr><td>2556 w</td><td>x</td><td>2561 w</td><td></td></tr><tr><td>2495 w</td><td>x</td><td>2494 w</td><td></td></tr><tr><td>2422 w</td><td>x</td><td>2421 w</td><td></td></tr><tr><td>2354 w</td><td>x</td><td>2340 w</td><td></td></tr><tr><td>1700 m</td><td>x</td><td>1665 (-35) int</td><td></td></tr><tr><td>1645 m</td><td>x</td><td>1634 int</td><td></td></tr><tr><td>1572 v. int</td><td>1621 (+49) v. int</td><td>1561 v. int</td><td>νₐ₃ₘₐ CO₂ (De Sousa et al., 2013)/ imidazole bending mode (Razzell Hollis et al., 2021)</td></tr><tr><td>1489 v. int</td><td>1478 v. int</td><td>1492 v. int</td><td>imidazole bending mode (Razzell Hollis et al., 2021)</td></tr><tr><td>x</td><td>x</td><td>1464 int sh</td><td></td></tr><tr><td>1405 w</td><td>1415 v. int</td><td>1408 m</td><td>νs CO₂ (De Sousa et al., 2013)</td></tr><tr><td>1345 sh</td><td>x</td><td>x</td><td>Deform. C-H (De Sousa et al., 2013)</td></tr><tr><td>1313 v. int</td><td>x</td><td>1316 v. int</td><td>Rocking NH₂ (Razzell Hollis et al., 2021; Wang et al., 2025)</td></tr><tr><td>1267 int</td><td>1267 m</td><td>x</td><td></td></tr><tr><td>1235 v. int</td><td>x</td><td>1256 v. int</td><td></td></tr><tr><td>1178 v. int</td><td>1185 v. int</td><td>1170 v. int</td><td>Twisting CH₂ (De Sousa et al., 2013)</td></tr><tr><td>1092 m</td><td>1085 w</td><td>1091 m</td><td>Rocking NH₃ (De Sousa et al., 2013)/CNH₂ bending (Wang et al., 2025)</td></tr><tr><td>1060 sh</td><td>x</td><td>x</td><td>νs C-N</td></tr><tr><td>991 m</td><td>994 vw</td><td>x</td><td>In plane deform. C-H imidazole ring (De Sousa et al., 2013)</td></tr><tr><td>962 m</td><td>x</td><td>972 int</td><td>νC-C* (De Sousa et al., 2013)</td></tr><tr><td>923 m</td><td>x</td><td>925 int</td><td>νC-C* (De Sousa et al., 2013)</td></tr><tr><td>853 int</td><td>x</td><td>838 int</td><td>Wagging of Imidazole ring H atoms (De Sousa et al., 2013)</td></tr><tr><td>795 int</td><td>x</td><td>775 int</td><td>Deform. CO₂ (De Sousa et al., 2013)</td></tr><tr><td>722 m</td><td>x</td><td>721 m</td><td></td></tr><tr><td>648 m</td><td>678 (+30)</td><td>672 m broad</td><td>Deform. Imidazole ring + δ out of plane N-H imidazole (De Sousa et al., 2013)</td></tr><tr><td>618 sh</td><td>x</td><td>621 sh</td><td></td></tr><tr><td>541 w</td><td>x</td><td>540 w</td><td>Rocking CO₂ (De Sousa et al., 2013)</td></tr><tr><td>447 w</td><td>x</td><td>432 w</td><td>Vibration of the skeletal (De Sousa et al., 2013)</td></tr></tbody></table>

In Table 4, a summarize of the most important data and the corresponding interpretation given to it can be observed.

* Tentative assignments.

appreciated in Fig. 3. The bands that can still be detected are weak and a careful check of the spectrum is needed to avoid many of them going unnoticed. This is even worse at lower concentration such as at 5 wt% (see Fig. S2 in Supplementary Material).

From the infrared spectra range measured, the Near Infrared region is of special interest because the SuperCam infrared spectrometer on board the Perseverance rover from the Mars 2020 mission is analyzing rocks at Jezero crater in the 7692-3846 cm⁻¹ spectral range (Fouchet et al., 2022). In addition, the instruments Ma_MISS and MicrOmega on board the Rosalind Franklin rover in the upcoming ESA ExoMars mission will work in the 25,000-4545 cm⁻¹ and 10,526-2740 cm⁻¹ spectral ranges respectively. Among the bands of the pure L-histidine that can still be observed when adsorbed onto saponite at 5 wt% concentration,
<caption>**Table 4**</caption>

Summarize of the data and the adsorption mechanisms of L-histidine adsorbed into saponite at acid and basic pHs.

<table><thead><tr><th>Data observation</th><th>Interpretation</th></tr></thead><tbody><tr><td>IR shift 1350 cm<sup>-1</sup> δ NH<sub>3</sub><sup>+</sup> at acid pH</td><td><strong>At acid pH:</strong> Electrostatic interaction NH<sub>3</sub><sup>+</sup> negatively charged basal surface—partial intercalation of the molecule</td></tr><tr><td>IR shift 1406 cm<sup>-1</sup> ν<sub>s</sub> COO<sup>-</sup> at acid pH</td><td><strong>At acid pH:</strong> Electrostatic edge interaction COO<sup>-</sup> with protonated edge AlOH<sub>2</sub><sup>+</sup> and SiOH<sub>2</sub><sup>+</sup>—not all the molecules are intercalated in the interlayer even if there is space for the L-histidine molecules at the adsorbed concentration</td></tr><tr><td>Shift and trasnformation of IR bands 1506 and 1489 cm<sup>-1</sup> related to NH imidazole group vibrational modes at all pHs</td><td><strong>At acid pH:</strong> electrostatic interaction NH<sup>+</sup> imidazole group and negatively charged basal surface—intercalation of the molecule</td></tr><tr><td>IR shift 1335 cm<sup>-1</sup> vibrational modes of the CH2 group adjacent to carboxylate and amino group at all pHs</td><td><strong>At basic pH:</strong> Edge interaction via hydrogen bond with the NH imidazole group</td></tr><tr><td>XRD interlayer expansion at acid pH</td><td><strong>At acid pH:</strong> CH2 affected by the adjacent COO<sup>-</sup> electrostatic edge interactions</td></tr><tr><td>XRD no interlayer expansion and pure L-histidine crystals at basic pH</td><td><strong>At basic pH:</strong> CH2 affected by the adjacent amino NH<sub>2</sub> group hydrogen bond interaction with the edge</td></tr><tr><td>Raman shift ν<sub>asym</sub> COO<sup>-</sup> at acid pH</td><td><strong>At acid pH:</strong> Partial intercalation of the molecule</td></tr><tr><td>Raman shift 678 cm<sup>-1</sup> bending NH<sup>+</sup> imidazole and deform imidazole ring at acid pH</td><td><strong>At basic pH:</strong> No intercalation of the molecule and pure L-histidine precipitated on top with no interaction with the mineral</td></tr><tr><td>Raman spectrum almost identical to pure L-histidine at basic pH</td><td><strong>At acid pH:</strong> Electrostatic edge interaction COO<sup>-</sup> with protonated edge AlOH<sub>2</sub><sup>+</sup> and SiOH<sub>2</sub><sup>+</sup>—no total intercalation of the molecules</td></tr><tr><td><strong>At acid pH:</strong> Electrostatic interaction NH<sup>+</sup>—negatively charged basal surface—partial intercalation of the molecule</td></tr><tr><td><strong>At basic pH:</strong> Pure precipitated L-histidine crystals predominate in the average Raman spectrum</td></tr></tbody></table>

only one band of L-histidine adsorbed at all the different studied pHs, 3.5, 7.7 and 9.6, falls in the Near Infrared region relevant for interpreting data acquired by the infrared spectrometers onboard the Perseverance and ExoMars rovers, corresponding to non-fundamental vibrational modes. When the concentration of L-histidine is increased to 10 wt%, three bands can be detected in this region when it was adsorbed onto saponite at pH 4 and up to eight bands can be observed when the L-histidine was adsorbed at pH 9.6. The detection of L-histidine in saponite can be however corroborated by DUV fluorescence measurements, as L-histidine exhibits a characteristic band at 258 nm (See Fig. S3 in Supplementary Material), which is within the range of SHERLOC, and can be appreciated when adsorbed on saponite at 10 wt% at both pHs. The DUV Raman measurements can also help identifying the L-histidine at 10 wt% (See Fig. S4 in Supplementary Material and Table 3), however in this case careful must be taken with the spectrum change and band shifts present specially at acid pH which as previously discussed, the interaction with the mineral is much more important than at basic pH. It is important to highlight the difficulties to identify an adsorbed amino acid and the importance of the combined information analysis.

### 3.3. UV irradiation experiments

The UV irradiation experiments were performed for the L-histidine 10 wt% adsorbed at pH 4 and at pH 9.6 to study the possible effect of the different adsorption mechanisms occurring at the two different pH conditions on the preservation of the amino acid against the UV radiation. In order to see if the interactions with the mineral favour photo-protection or photodegradation of the L-histidine, the pure L-histidine was also irradiated with UV.

All the bands of pure L-histidine detected in the adsorbed samples

presented a degradation tendency under the irradiation of the UV lamp, which fitted the mathematical model stated in eq. 1 (see Fig. S5). In Fig. S5 of Supplementary material, all the degradation curves fitted to eq. 1 are shown for all the bands. In Table 5, the calculated degradation cross section (σ) and Martian half-lifetimes (t<sub>1/2</sub>) are shown. In Table S1, the calculated degradation coefficient (b), half-lifetime (t<sub>1/2</sub>), degradation cross section (σ) under the lab conditions and the extrapolated

<caption>**Table 5**</caption>

Degradation Cross sections (σ) and Comparison of the Martian half-life times (t<sub>1/2</sub>(h)) for the pure L-histidine and the L-histidine adsorbed onto saponite at pH 4 and 9.6.

<table><thead><tr><th rowspan="2">Wavenumbers<br>Pure/pH 4/pH 9.6<br>(cm<sup>-1</sup>)</th><th colspan="3">σ (cm<sup>2</sup>) (·10<sup>-21</sup>)</th><th colspan="3">Martian t<sub>1/2</sub> (h)</th></tr><tr><th>L-hist.</th><th>10 wt% L-hist.</th><th>10 wt% L-hist.</th><th>L-hist.</th><th>10 wt% L-hist.</th><th>10 wt L-hist.</th></tr></thead><tbody><tr><td>6164/6183/6164</td><td>12<br>± 1</td><td>Incr.<br>Bck</td><td>Spr.<br>data<br>S-B.</td><td>11<br>± 1</td><td>Incr.<br>Bck</td><td>Spr.<br>data S.<br>B.</td></tr><tr><td>5871/x/5861</td><td>6.2<br>±<br>0.8</td><td>No<br>appr. B.</td><td>Spr.<br>data S.<br>B.</td><td>22<br>± 2</td><td>No<br>appr. B.</td><td>Spr.<br>data S.<br>B.</td></tr><tr><td>5819/x/5821</td><td>5.2<br>±<br>0.8</td><td>No<br>appr. B.</td><td>Spr.<br>data S.<br>B.</td><td>26<br>± 4</td><td>No<br>appr. B.</td><td>Spr.<br>data S.<br>B.</td></tr><tr><td>5765/x/5761</td><td>9 ±<br>1</td><td>No<br>appr. B.</td><td>Spr.<br>data S.<br>B.</td><td>15<br>± 2</td><td>No<br>appr. B.</td><td>Spr.<br>data S.<br>B.</td></tr><tr><td>5668/x/5651</td><td>6.7<br>±<br>0.8</td><td>No<br>appr. B.</td><td>Spr.<br>data S.<br>B.</td><td>20<br>± 3</td><td>No<br>appr. B.</td><td>Spr.<br>data S.<br>B.</td></tr><tr><td>4627/x/4623</td><td>8.0<br>±<br>0.8</td><td>No<br>appr. B.</td><td>Spr.<br>data S.<br>B.</td><td>17<br>± 2</td><td>No<br>appr. B.</td><td>Spr.<br>data S.<br>B.</td></tr><tr><td>4102/x/4102</td><td>4.8<br>±<br>0.8</td><td>No<br>appr. B.</td><td>Incr.<br>Bck</td><td>29<br>± 5</td><td>No<br>appr. B.</td><td>Incr.<br>Bck</td></tr><tr><td>4058/x/4056</td><td>7.0<br>±<br>0.8</td><td>No<br>appr. B.</td><td>Incr.<br>Bck</td><td>20<br>± 2</td><td>No<br>appr. B.</td><td>Incr.<br>Bck</td></tr><tr><td>1506 and 1489/<br>1497/1497<br>(δ<sub>l,p</sub>N<sup>-</sup>H and δ<sub>l,p</sub>N<sup>+</sup>H and ν<sub>C=N</sub>)</td><td>4.4<br>±<br>0.7</td><td>Atm.<br>water</td><td>Atm.<br>water</td><td>31<br>± 5</td><td>Atm.<br>water</td><td>Atm.<br>water</td></tr><tr><td>1429/1437/1435<br>δ CH<sub>2</sub> scissor.</td><td>6.1<br>±<br>0.8</td><td>Atm.<br>water</td><td>Atm.<br>water</td><td>22<br>± 3</td><td>Atm.<br>water</td><td>Atm.<br>water</td></tr><tr><td>1406/1412/1408<br>ν sym. COO<sup>-</sup></td><td>8 ±<br>1</td><td>220 ±<br>20</td><td>35 ± 4</td><td>17<br>± 2</td><td>0.62 ±<br>0.06</td><td>3.9 ±<br>0.4</td></tr><tr><td>1350/1360<br>β NH<sup>3+</sup>/δ<sub>l,p</sub> CH<sub>2</sub><br>(wagg.)</td><td>10<br>± 1</td><td>Diff.<br>effects.</td><td>No B. at<br>basic<br>pH</td><td>13<br>± 1</td><td>Diff.<br>effects.</td><td>No B.<br>basic<br>pH</td></tr><tr><td>1335/1344/1340<br>β CH<sub>2</sub></td><td>5.1<br>±<br>0.8</td><td>49 ± 6</td><td>46 ± 6</td><td>27<br>± 4</td><td>2.8 ±<br>0.3</td><td>3.0 ±<br>0.4</td></tr><tr><td>1317/1321/1319<br>δ CH<sub>2</sub> and δ = C-<br>H</td><td>10<br>± 1</td><td>66 ± 6</td><td>10 ± 2</td><td>14<br>± 1</td><td>2.1 ±<br>0.2</td><td>14 ± 4</td></tr><tr><td>1273/x/1271<br>ν = C-N and δ C-<br>H</td><td>11<br>± 1</td><td>No<br>appr. B.</td><td>75 ± 6</td><td>13<br>± 1</td><td>No<br>appr. B.</td><td>1.8 ±<br>0.2</td></tr><tr><td>825/829/829<br>ν sym. Ring</td><td>7.2<br>±<br>0.9</td><td>S.B.</td><td>S.B.</td><td>19<br>± 2</td><td>S.B.</td><td>S.B.</td></tr><tr><td>629/627/633<br>ring deform.</td><td>10<br>± 1</td><td>Spr.<br>data S.<br>B.</td><td>Spr.<br>data S.<br>B.</td><td>14<br>± 1</td><td>Spr.<br>data S.<br>B.</td><td>Spr.<br>data S.<br>B.</td></tr></tbody></table>

**Incr.Bck:** Increasing tendency due to exchange in the background; **Spr. data S. B.:** Spread data due to a small band, low intensity band; **No appr. B.:** No appreciable band in the adsorbed sample; **Atm. Water:** Band affected due to exchange in atmospheric water; **Diff. effects:** different effects; **No B. basic pH:** the band does not exist in the basic form of L-histidine. **S.B:** Small band to be integrated.
Martian degradation coefficient (b) and half-lifetime ($t_{1/2}$) for all the bands of pure L-histidine are reported for more detailed results. The bands at 1506 and 1489 cm⁻¹ were integrated together to be able to compare the results with the adsorbed L-histidine onto saponite at both pHs, where only one broad band could be appreciated in both cases.

When L-histidine was adsorbed onto saponite at pH 4, the band at 6178 cm⁻¹ showed an increasing tendency due to a change in the background continuum (See Fig. S6, zoom indicated for band at 6178 cm⁻¹). This effect may be due to the desorption of water with the time; specifically, the change in the background with the diminution of the water band can cause the increase of intensity in a band. This effect is usually higher in this part of the spectrum at high wavenumbers, and in this particular case, it is higher than any other changes in the band (e.g. degradation of the band due to the UV irradiation). The data collected for the bands at 1495 cm⁻¹ related to N-H in plane bending and C-N stretching and 1437 cm⁻¹ related to CH₂ scissoring are difficult to interpret also due to the effect of the slight presence of atmospheric water in this range especially at the beginning of the irradiation. All the spectra are divided by the infragold background after pumping nitrogen gas to the chamber until the CO₂ and water are more or less constant. However, it seems that the presence of atmospheric water in the chamber continues decreasing very slightly for a long time and this makes small changes at the beginning of the irradiation experiment in the bands overlapping the position of the atmospheric water bands. In Fig. S6, it can be seen how there are random ups and downs for the area of these two bands at the beginning of the experiment and afterwards it seems there is a very slight decrease. However, due to the problems at

the beginning of the irradiation the points were not fitting to the mathematical model. However, if we look at the degradation curves (see Fig. S6), when the atmospheric water seems to have been stabilized, the area measurements seem to be very stable with almost no change, which would mean that the band is not affected by the UV radiation. The problem with the presence of the atmospheric water does not appear for the bands of the pure L-histidine because the bands for the pure amino acid are very big in comparison to the small changes observed for the atmospheric water. Therefore, the changes in the band are very slightly affected by the presence of the atmospheric water and the decreasing of the area due to the irradiation is highly dominant in this case and the small effect of the atmospheric water is negligible. For the band at 1360 cm⁻¹ related to NH³⁺ bending and CH₂ wagging, the first points after the UV irradiation present a higher area than the point before irradiation in a random way too. After the first minutes of irradiation, it seems that this band has a small degradation and then stays constant along the rest of the irradiation (see Fig. S6). The bands at 1414 cm⁻¹ related to symmetric stretching of CO²⁻, 1344 cm⁻¹ related to CH₂ bending and 1319 cm⁻¹ related to CH₂ and C-H deformations present a degradation (See Fig. 4 and S6) that could be fitted to the mathematical model (Eq. 1) and the calculated degradation cross section (σ) and Martian half-lifetimes ($t_{1/2}$) are shown in Table 5. The rest of the parameters are collected in Table S2 from Supplementary Material. Finally, the small bands at 825 and 627 cm⁻¹ related to the stretching and deformation of the ring showed a dispersion of points (see Fig. S6) probably because the bands were too small to distinguish the changes due to the UV radiation.

Finally, when the L-histidine was adsorbed onto saponite at pH 9.6,

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Degradation of the bands related to $v_{sym}$ COO⁻, β CH₂, δ CH₂ + δ = C-H and $v = C-N + δ$ C-H vibrational modes in L-histidine (red), L-histidine adsorbed at pH 4 (green) and L-histidine adsorbed at pH 9.6 (black). Errors for the data points are smaller than 3·10⁻⁴, this is, smaller than the dimension of the plotted points on these graphics. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
the bands at 5863, 5818, 5762, 5658 and 4621 cm⁻¹ showed no degradation but spread data due to the low intensity of all these bands (see Fig. 3A and Fig. S7). The band at 6164 cm⁻¹ showed spread data, the noise in this part of the spectrum is the highest and it may be the reason of the spread areas. The last two bands in the near infrared region 4102 and 4058 cm⁻¹ were more intense and in a less noisy part of the spectrum and they showed an increasing tendency, for the same reason of exchange in the background continuum explained previously for the acid adsorption (See Fig. S7). The same as for pH 4, the bands at 1496 (δᵢ, p N-H and ν C=N) and 1437 (δ CH₂ scissoring) are affected by the atmospheric water. However, in this case, the bands showed an increasing tendency and not random ups and downs due to the appearance of new bands related to atmospheric water (See Figs. S4 and S5 in Supplementary Material). On the opposite, the bands at 1410 (νsym CO²⁻), 1340 (β CH₂), 1317 (δ CH₂ and δ = C-H) and 1271 (ν = C-N and δ C-H) cm⁻¹ showed degradation curves (Fig. 4), which were able to be fitted to the expected degradation mathematical model. The calculated degradation cross section (σ) and Martian half-lifetimes (t₁/₂) for these bands are shown in Table 5. To check the rest of the calculated parameters see Table S3 in Supplementary Material. Finally, the two bands related to the ring were too small to be integrated.

In Table 5, a comparison of the Martian half-lifetimes t₁/₂(h) for the bands of pure L-histidine and the L-histidine adsorbed onto saponite at pH 4 and 9.6 is collected to try to describe the photocatalytic or photoprotective behaviour of saponite for L-histidine against the UV radiation. As it can be observed, in the Near Infrared Region (>4000 cm⁻¹) almost no bands were detected when L-histidine was adsorbed at acidic pH, while only few bands were detected when L-histidine was adsorbed at basic pH and these were too small to assess any change during the UV irradiation. In other cases, some bands in the adsorbed L-histidine presented different effects higher than the degradation effect as previously explained and therefore these bands cannot be used for comparison.

However, for the bands related to vsym COO⁻, β CH₂, δ CH₂ + δ = C-H and ν = C-N + δ C-H a comparison between the degradation in the pure molecule and in the adsorbed samples can be done (See Fig. 4 and Table 5). Pure L-histidine presented higher half-lifetimes in the pure state than when it was adsorbed onto saponite in both acid and basic pHs (see Table 5 and Fig. 4). It seems in this case, according to the calculated half-life times that the mineral saponite is acting as a photocatalyzer of the L-histidine degradation under UV light. However, as it can be appreciated in Fig. 4, the slope of degradation is always less steep in the pure amino acid (red degradation curve in Fig. 4) than for adsorbed L-histidine at pH 9.6. However, the degradation observed for the basic pH is more similar to the degradation of pure L-histidine than the degradation observed for the L-histidine adsorbed at acid pH. This can be specially well appreciated for the bands related to ν = C-N + δ C-H and vsym COO⁻ vibrational modes (see Fig. 4). For the band related to ν = C-N + δ C-H, the degradation of the pure L-histidine and adsorbed at basic pH is almost the same. For the band related to vsym COO⁻ vibrational mode, the kinetics of the degradation for the basic is faster than for the pure, but still slower than for the one adsorbed at acid conditions. This can be due to the combined effect of the presence of crystallized pure L-histidine together with a part of L-histidine reacting with the edges of the saponite.

According to these observations, the reaction with the edges of the mineral are causing the photocatalization of the L-histidine at both pHs likely promoted by the metals in the edge groups of the saponite. Indeed, as already reported in the literature, smectites catalyse several reactions implying organic molecules such as condensation reactions, Diels-Alder, Isomerization reactions, and Oxidation reactions, esterification reactions, Friedel-Crafts and related. The effect of UV light can accelerate and modify some of these reactions, especially when generating some active radicals such as HO• due to the presence of water in the system (Nagendrappa, 2011). The photocatalization for the L-histidine adsorbed at basic pH is a bit lower and could be due to the fact that part of the molecule is crystalized as pure molecule as seen by XRD (see Fig. S4) and

therefore that a part of the molecule behaves as pure L-histidine in the degradation reducing the effect of the final observed photocatalization.

The observation of a plateau after some irradiation time could be due to the formation of photoresistive compounds. However, in the IR spectra, we could not observed the formation of any new bands attributable to photoproducts. This could be due to the different penetration depths of IR and UV radiations. In fact, the UV radiation only penetrates few microns and degrades only the first layers of the sample, while the IR intercepts also a deeper portion of the sample that is not UV-irradiated. For this reason we cannot have any hints of the degradation process going on, however, decarboxylation induced by UV radiation has been proposed as one of the main destruction pathways of amino acids (Bertrand et al., 2015; Ehrenfreund et al., 2001; Johns and Seuret, 1970; dos Santos et al., 2016; Ten Kate et al., 2005). As it can be appreciated in Table 5, the band at 1406 cm⁻¹ related to the symmetric stretching of the COO⁻ is one with the smallest half-lifetimes in the pure L-histidine together with some bonds related to the N-H bonds. This band of COO⁻ is the fastest and the most efficient to be degraded when L-histidine is adsorbed onto saponite at acidic pH. It seems that the likely electrostatic interaction of this COO⁻ group with the edge protonated groups of the mineral at acid pH makes the carboxylate group less stable against the UV radiation. Ten Kate et al. (2005) in a similar study of the photo-degradation of glycine and D-alanine under UV radiation suggested that the observed difference in the obtained Martian half-lifetimes 22 ± 5 h and 3 ± 1 h for glycine and D-alanine, respectively, could be explained by the decarboxylation mechanism previously proposed by Ehrenfreund et al. (2001). This mechanism would start with the separation of the carboxyl group and the subsequent rearrangement of the proton on the formed radical leading to the formation of the corresponding amine and the liberation of CO₂.

$$h \cdot v \quad H_2N-CHR-COOH \rightarrow H_2N-CHR \cdot + COOH \cdot \rightarrow H_2NCH_2R + CO_2$$

In this way, the much faster degradation observed for D-alanine by Ten Kate et al. (2005) would be due to the higher stability of the radical formed in the degradation of D-alanine in respect to the one formed in the glycine. Due to the higher substitution of the carbon atom, secondary and tertiary radicals are more stable than primary ones and as R for glycine is another hydrogen atom, its radical is the least stable. This would also explain our higher Martian half-lifetimes observed for L-histidine than the one calculated by Ten Kate et al. (2005) for glycine and more similar to the ones that they obtained for the D-alanine.

For the amino group due to experimental limitations, we cannot affirm the same effect. However, the CH₂ adjacent to the amino group (band at 1335 cm⁻¹), which as explained in the previous section presented some shifts probably due to the effect of the amino interaction with the mineral, exhibits a similar behaviour under the UV radiation. The degradation is faster when the L-histidine is adsorbed onto saponite than in the pure. The band at 1360 cm⁻¹ related to the protonated amino group, presents first random points with higher areas than the initial ones, followed by a small degradation (from Aₜ/A₀ = 1 to Aₜ/A₀ = 0.93) and then the band seems to stay stable for the rest of the irradiation time (approx t > 100 s). The highly variable behaviour of this band makes impossible to calculate any degradation coefficients and, thus, makes complicated to assess the stability of the amino group through its band. However, as it can be observed in Table 5, according to bands 1317 and 1273 cm⁻¹ related to δ = C-H and ν = C-N vibration modes respectively, the imidazole ring is also degraded faster when the L-histidine is adsorbed onto saponite than when L-histidine is in its pure solid state.

In the work by Garry et al. (2006), they also observed a photocatalytic effect when the amino acids they studied were adsorbed to the Martian regolith analogues they used. This may be due to some minerals scattering and absorbing the arriving UV radiation rather than reflecting it. Therefore, the UV-absorbing mineral surfaces coated with water will be more likely to present UV driven processes such as mobilization of
electrons at a surface and the subsequent formation of trapped radicals that will enhanced the degradation of the organic compounds (Garry et al., 2006; Gil-Lozano et al., 2020; Shkrob et al., 2010; Yen et al., 2000). If we compare the photodegradation between the L-histidine adsorbed at acid and basic pHs, the fastest and most efficient degradation occurs when the L-histidine is adsorbed onto saponite at acid pH. In addition, in the work by Gil-Lozano et al. (2020) a higher photodegradation of glycine in the acid-treated nontronite triggered by decarboxylation and deamination processes was also observed due to exchanges in the interlayer of nontronite due to the acid treatment and the presence of Fe and Fenton reactions.

In another study performed by Poggiali et al. (2020) on the degradation of glycine in different minerals the calculated half-life was of 0.5–2 h depending on the mineral such as forsterite, antigorite, spinel, or pyrite. This relatively rapid degradation of glycine may also indicate that these minerals on Mars may be involved in photo-Fenton catalytic reactions. The general trend emerging from these experiments is that the interaction with metals in solution can considerably increase the degradation due to iron and non-iron metals forming Fenton-type reaction pathways (Fornaro et al., 2018a). However, caution should be taken when generalizing, as evidenced by the detection of nontronite in multiple locations on Mars. This mineral has been demonstrated to exhibit remarkable stability under the Martian surface due to its high ferric (Fe3+) iron content, which serves as a potent UV absorber and is therefore regarded as a potential organic preservative (Gil-Lozano et al., 2020). The outcome depends on the nature of the interaction between the mineral and the molecule, as well as the role that the metals involved may play in either protecting or degrading the organic matter. Additionally, the oxidation states of the metals and their impact on the mineral crystalline structure must be considered.

dos Santos et al. (2016) analysed the stability of several amino acids in different minerals and stated that sulphates and smectites were the best minerals protecting the amino acids against the UV and therefore these minerals should be the targets to find organic molecules. However, among these minerals, saponite showed the lowest concentration of the non-degraded amino acid after the UV radiation of all the smectites and sulphates (dos Santos et al., 2016). In addition, in this work was not compared the stability of the amino acids adsorbed in the minerals versus the pure amino acids or at different pHs. Therefore, the amino acid that was recovered may have been the one undergoing the interlayer, but not the part of amino acid adsorbed onto the edges.

In addition, Liu and Kounaves, 2021 studied that the UV degradation of glycine and proline was increased in the presence of sodium chloride (NaCl), sodium chlorate (NaClO<sub>3</sub>), and sodium perchlorate (NaClO<sub>4</sub>) salts. However, the same experiments conducted for tryptophan showed no difference depending on the presence of the salt and therefore showing a different degradation mechanism. All this showing the complexity of making a generalisation for stating targets for detecting organics such as amino acids on Mars and the importance of studying specific cases.

## 4. Conclusions

The shifts present in the fingerprint region of the molecule may help to understand the kind of interactions between the mineral and the biomolecule and therefore to understand better the stability of the organic molecule in different kinds of minerals against the UV radiation. This is of great importance in order to establish mineral targets in the Mars missions as more probable to have preserved the organic molecules. In this case, the L-histidine IR and DUV-Raman shifts in the main bands showed different possible adsorption mechanisms depending on the adsorption pH. The IR shifts observed for acid and neutral pHs adsorptions suggest an electrostatic interaction between the -NH<sub>3</sub><sup>+</sup> and the basal surface SiO<sub>4</sub><sup>2-</sup> with an intercalation of the molecule as confirmed afterwards by XRD. The IR shift observed for the NH imidazole bands also suggest that the interaction electrostatic interaction in the

interlayer may also occur through the charged imidazole amino group. The DUV-Raman shift for the charged NH<sup>+</sup> in the imidazole ring confirmed that the electrostatic interaction in the interlayer may also be through the imidazole charged amino group. For the acid adsorption, the IR and DUV-Raman shifts observed for the COO<sup>-</sup> group suggests a second adsorption mechanism between the carboxylate and the protonated edge groups AlOH<sub>2</sub><sup>+</sup> and SiOH<sub>2</sub><sup>+</sup> (see Fig. 2) competing with the adsorption in the interlayer. Finally, for the adsorption at basic pH, only the two bands related to the in plane deformation of the imidazole N-H, converted into one broader and shifted, suggest a possible interaction through the imidazole amino group -NH probably in this case through hydrogen bonds with the deprotonated edge groups (see Fig. 2). The shift for the CH2 band adjacent to the NH<sub>2</sub> may suggest that the NH<sub>2</sub> is also interacting with the edges via another hydrogen bond. The rest of the molecule appeared as crystallized pure L-histidine as shown by XRD.

This work showed the importance of studying the adsorption of the amino acids at different pHs, not only to be able to identify them correctly but also because due to the different interactions, the degradation of the molecule under UV can be very different. According to our UV results, the interactions of L-histidine with the edge groups of the saponite mineral makes the functional groups less stable against the UV radiation than when they are in their pure solid structure, especially when the adsorption occurs at acid pH. This is in contrast to the photoprotective behaviour expected through mechanical shielding when molecules intercalate in the interlayer of saponite at acid pH. It was found that the fastest degradation occurred when the adsorption of L-histidine was done at this pH due to the competing interaction with the saponite edges of the molecules of L-histidine that do not go inside the interlayer, even when the interlayer is not saturated. Similar processes might have occurred in the past slightly acidic Martian conditions, due to the existence of this competitive second adsorption mechanism with the protonated edges of saponite, which is responsible for a faster degradation of the organics due to the photocatalization promoted by the metals on the edges. Degradation/alteration of L-histidine adsorbed on saponite occurs in a few hours under Martian UV flux, suggesting that photocatalysis induced by active surface edge sites of clays might have contributed to the depletion of organics in clays at the highly UV-irradiated surface of Mars on very short timescales. From this work it can also be differ the complexity of the UV degradation mechanisms depending on the amino acid and the mineral interactions and the difficulty to make a generalisation for stating targets for detecting organics such as amino acids on Mars and the importance of studying specific cases.

Finally, this work showed also the importance of studying the IR characteristic bands of biomolecules when they are adsorbed onto minerals as most of the characteristic bands of the pure molecules are not present anymore, especially in the case of the expected low concentrations on Mars. In addition, for the specific case studied in this work of L-histidine adsorbed onto saponite, the bands of the amino acid in the Near Infrared region are very scarce and weak. Moreover, at 5 wt % concentration of L-histidine, only one weak band can be appreciated in this range for all the adsorption pHs studied. At 10 wt% concentration, up to 3 and 8 bands were detected for acid and basic adsorptions, respectively. It seems that the number of bands observed could also be indicative of the adsorption type of the amino acid to the mineral. Apart from the number of bands, it is also important to notice that some of the combination bands when L-histidine is adsorbed onto saponite present shifts in their position respect to their original position on the pure amino acid. This may lead to an incorrect identification of the biomolecule on Mars if the comparison is not done with the correct IR database taking into account the mineral-organic molecule interaction. DUV Raman and fluorescence measurements could assist the identification of L-histidine even if the Raman bands may also present important shifts when the interaction of the molecule with the mineral is important.
## CRediT authorship contribution statement

**Cristina García-Florentino:** Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization.
**Andrew Alberini:** Writing – review & editing, Software, Methodology, Conceptualization.
**Sole Biancalani:** Writing – review & editing, Visualization, Conceptualization.
**Ophélie McIntosh:** Investigation, Formal analysis.
**John Brucato:** Writing – review & editing, Supervision, Resources, Project administration, Methodology, Funding acquisition, Conceptualization.
**Juan Manuel Madariaga:** Writing – review & editing, Supervision, Project administration, Funding acquisition.
**Maguy Jaber:** Writing – review & editing, Investigation, Formal analysis, Conceptualization.
**Teresa Fornaro:** Writing – review & editing, Supervision, Resources, Project administration, Methodology, Funding acquisition, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

Part of this work has been financially supported through the the PAM-MAT project (Grant No. PID2022-1427500B-100), funded by the Spanish AEI, MCINN and the European Regional Development Fund. CGF acknowledges her postdoctoral contract from the Basque Government. TF, AA and JB were funded through the ASI/INAF agreement 2023-3-HH.0. TF is also funded by the European Union – Next Generation EU through the PRIN MUR 2022 "Experimental and computational analog studies to support identification of organics on Mars by the NASA Mars 2020 Perseverance rover" and by INAF through Mini Grant Ricerca Fondamentale INAF 2022.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.icarus.2025.116757.

## Data availability

Data will be made available on request.

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