Jimmy Castillo
email: jimmy.castillo@ciens.ucv.ve

Genesis Gonzalez
email: genesis.gonzalez@univ-pau.fr

Brice Bouyssiere
email: brice.bouyssiere@univ-pau.fr

Vicmary Vargas
email: vicmary.vargas@univ-pau.fr

Asphaltenes, subfractions A1 and A2 aggregation and adsorption onto RH-SiO 2 nanoparticles: solvent effect on the aggregate size

Keywords: asphaltene aggregation, adsorption, nanoparticle, asphaltene adsorption

Asphaltenes, subfractions A1 and A2 aggregation and adsorption onto RH-SiO 2 nanoparticles: solvent effect on the aggregate size

Jimmy Castillo, Genesis Gonzalez, Brice Bouyssière, Vicmary

Introduction

Asphaltenes comprise a series of molecules with a wide range of molecular weight distributions and heteroatoms, such as nitrogen, oxygen, sulfur, and metals, which modulate their properties.

From the operational viewpoint, asphaltenes are defined as the crude oil fraction soluble in light aromatic hydrocarbons (e.g., toluene, benzene) and insoluble in low molecular weight alkanes (e.g., n-pentane, n-hexane, n-heptane). Due to the complexity, their behavior is under study until this date [START_REF] Boussingault | Mémoire sur la composition des bitumes[END_REF].

The behavior of asphaltene aggregates and their association is under debate, and the existence of molecular units of natural asphaltenes has not been verified. Consequently, the aggregates have shown stability in solvents, such as toluene, and the original petroleum mixture.

The size of the heterogeneous population of aggregates has been measured with a range of analytical techniques, and the mean aggregate size is 2-20 nm [START_REF] Schuler | Unraveling the Molecular Structures of Asphaltenes by Atomic Force Microscopy[END_REF]. Evidence suggests a range of sizes in a polydisperse distribution [START_REF] Putman | Analysis of Petroleum Products by Gel Permeation Chromatography Coupled Online with Inductively Coupled Plasma Mass Spectrometry and Offline with Fourier Transform Ion Cyclotron Resonance Mass Spectrometry[END_REF]. Other hypotheses propose the existence of supramolecular assembly of molecules; according to the authors, this hypothesis presents a better context to understand, model, and predict the asphaltene behavior. The driving forces for this supramolecular assembly proposed by Gray et al. [START_REF] Gray | Supramolecular assembly model for aggregation of petroleum asphaltenes[END_REF] include a different type of interaction between polar and aromatic groups.

In this context, it is now widely accepted that asphaltenes form aggregates even in very dilute solutions of good solvents such as toluene or THF, but experimental data that describe the nature of the aggregates and aggregation process remain open to interpretation and are the subject of various disagreements. According to the literature, asphaltenes begin to form nanoscale aggregated structures at concentrations below 100 mg L -1 [START_REF] Goncalves | Absorbance and fluorescence spectroscopy on the aggregation behavior of asphaltene-toluene solutions[END_REF]. These nanoaggregates are considered to have a limited size because of steric inhibitions introduced by the alkyl chains attached to asphaltene aromatic cores. In a colloidal approach to the problem, a critical nanoaggregate concentration (CNAC) has been defined as the concentration at which the growth of nanoaggregates starts [START_REF] Evdokimov | Multi-step formation of asphaltene colloids in dilute solutions[END_REF]; at higher concentrations (2-10 g L -1 ), these nanoaggregates undergo additional association to form clusters or micelles. These aggregates and clusters have often been referred to and considered nanocolloidal and colloidal structures, respectively.

In a solid-liquid phase approach, asphaltene solutions can undergo liquid-solid phase separation to give a solvent-rich phase in equilibrium with asphaltene-rich droplets (clusters).

The asphaltene-rich particles formed upon phase separation can act as colloids, but thermodynamically, it is a liquid or glass that exhibits coarsening and coalescence instead of colloidal aggregation at elevated temperature or upon dilution. In discussing asphaltene aggregation, it is important to clearly define the type of aggregates under consideration. In some studies, particles in the size range of 5-10 nm are referred to as both aggregates and nanoaggregates, which of course they are. However, in scattering work, there is a clear distinction between an initial, limited association to form nanoaggregates approximately 2-3 nm in size and subsequent aggregation of these particles to form mass fractal clusters in the size range of 5-10 nm [START_REF] Painter | Asphaltene aggregation and solubility[END_REF].

As reported in previous works [START_REF] Acevedo | Investigation of physical chemistry properties of asphaltenes using solubility parameters of asphaltenes and their fractions A1 and A2[END_REF][START_REF] Castillo | Study of the aggregation and adsorption of asphaltene sub-fractions A1 and A2 by white light interferometry: Importance of A1 sub-fraction in the aggregation process[END_REF][START_REF] Castillo | New techniques and methods for the study of aggregation, adsorption, and solubility of asphaltenes. Impact of these properties on colloidal structure and flocculation[END_REF][START_REF] Acevedo | Trapping of metallic porphyrins by asphaltene aggregates: A size exclusion microchromatography with high-resolution inductively coupled plasma mass spectrometric detection study[END_REF], asphaltenes can be fractionated into two main subfractions A1 and A2 using the p-nitrophenol (PNP) method, where A1 has very low solubility (in toluene, under laboratory conditions) (approximately 90 mg L -1 ), and A2 has similar solubility to that observed for asphaltenes (5-12%, depending on the sample). This method has been tested numerous times and with different samples and always affords the two fractions with the main difference in solubility. The solubility parameters of asphaltenes A1 and A2 are consistent: A1 is less soluble than A2 in more than 50 different solvents [START_REF] Acevedo | Suppression of Phase Separation as a Hypothesis to Account for Nuclei or Nanoaggregate Formation by Asphaltenes in Toluene[END_REF].

Asphaltene adsorption governs many aspects of crude oil recovery and production.

Generally, the effects produced are undesirable, since they can lead to consequences such as deposition, wettability modification, and emulsion stabilization; thus, considerable efforts have been directed at understanding the behavior of crude oils and their constituents at interfaces. In general, the results from adsorption studies over the years have been fitted with classical adsorption models. The interpretation of these classical models does not give a real answer to all changes in the system, so new approaches to the problem remain under discussion [START_REF] Nassar | Development of a Population Balance Model to Describe the Influence of Shear and Nanoparticles on the Aggregation and Fragmentation of Asphaltene Aggregates[END_REF][START_REF] Hosseinpour | Asphaltene adsorption onto acidic/basic metal oxide nanoparticles toward in situ upgrading of reservoir oils by nanotechnology[END_REF][START_REF] Montoya | Kinetics and mechanisms of the catalytic thermal cracking of asphaltenes adsorbed on supported nanoparticles[END_REF][START_REF] Castillo | Evidence of selective asphaltene subfraction adsorption on SiO2 nanoparticles studied by UV-vis absorbance and fluorescence spectroscopy[END_REF][START_REF] Mirzayi | Adsorption kinetics and catalytic oxidation of asphaltene on synthesized maghemite nanoparticles[END_REF][START_REF] Castillo | Development of a Methodology Using GPC-ICP HR MS for Analysis of the Adsorption of Asphaltene Aggregates on SiO 2 Nanoparticles[END_REF][START_REF] Nassar | Asphaltene Adsorption onto Alumina Nanoparticles: Kinetics and Thermodynamic Studies[END_REF] In other researches, the use of nanoparticles (NPs) to reduce the size of asphaltene flocks formed in n-heptane:toluene mixtures has been reported [START_REF] Nassar | Asphaltene Adsorption onto Alumina Nanoparticles: Kinetics and Thermodynamic Studies[END_REF][START_REF] Guzmán | Importance of the Adsorption Method Used for Obtaining the Nanoparticle Dosage for Asphaltene-Related Treatments[END_REF][START_REF] Franco | Effects of Resin i on Asphaltene Adsorption onto Nanoparticles: A Novel Method for Obtaining Asphaltenes/Resin Isotherms[END_REF]. Large reductions in the average flock size of up to 50% were observed when NPs were added to an asphaltene-toluene solution. We propose that the removal of substantial amounts of aggregates by NPs disrupts the flocks by increasing their solubility, which results in large-sized reductions. The adsorption of asphaltenes on nanoparticles has been previously studied, and different behaviors have been found.

From a geochemical interpretation, asphaltenes are derived from geomacromolecules and generally have high molecular weights and complex chemical structures. Some conceptual structure models that represent the largest possible set of physicochemical analytical data for geomacromolecules have been used to represent structures of humic matter, kerogen, and asphaltenes. Kerogen, asphaltene, and solid bitumen are important for the geochemical information encoded with them. Their macromolecular structures can adsorb and even occlude other small molecules (e.g., biomarkers) [START_REF] Cheng | Geochemical Evolution of Occluded Hydrocarbons inside Geomacromolecules: A Review[END_REF]. In their works, Cheng et al. [START_REF] Cheng | Geochemical Evolution of Occluded Hydrocarbons inside Geomacromolecules: A Review[END_REF] present a different view, since asphaltenes are not formed from molecular systems but appear from the cracking of larger structures.

Combining experimental and theoretical studies, Moncayo-Riascos et al. [START_REF] Moncayo-Riascos | Physical Insights about Viscosity Differences of Asphaltene Dissolved in Benzene and Xylene Isomers: Theoretical-Experimental Approaches[END_REF] studied the effect of the solvent on the rheological behavior of asphaltenes dissolved in aromatic solvents such as benzene and the three isomers of xylene. Experimentally, differences in the viscosity of the solutions were obtained, being higher in p-xylene than in benzene. Likewise, a correspondence was observed by molecular dynamics simulations with the size of the aggregates obtained in the solutions being o-xylene > m-xylene > p-xylene ≈ benzene, which corresponds to the dipole moment and the solubility parameter due to the location of the -CH 3 substituent. Jiang et al. [START_REF] Jiang | Asphaltene Aggregation and Assembly Behaviors in Organic Solvents with Water and Inhibitor[END_REF] studied the aggregation behavior of a model polyaromatic asphaltene compound was evaluated. When heptane was added to a solution of the model in toluene, it increased the aggregation especially due to the possibility of establishing interactions via van der walls. When water was added to the mixture, an increase in aggregation and a different rearrangement was observed, in which the water molecules allowed the construction of a network to connect aggregates (via hydrogen bonding) and consequently the formation of aggregate clusters. In contrast, in the presence of an asphaltene inhibitor, it destroyed some interactions between the model and water, dispersing the conglomerates in solution. Xiong et al. [START_REF] Xiong | Adsorption of a Polyaromatic Compound on Silica Surfaces from Organic Solvents Studied by Molecular Dynamics Simulation and AFM Imaging[END_REF] studied the adsorption on protonated silica using molecular dynamics of a polyaromatic compound with fused rings similar to those reported for asphaltenes. They found that in heptane aggregates are found in the form of a long strip and these tend to adsorb rapidly on the surface. While in toluene the aggregates are more compact and tend to desorb from the silica surface remaining in the solvent phase.

In the present work, experiments of asphaltene aggregation and adsorption onto SiO 2 NPs are presented, and the results show that asphaltene solutions are composed of aggregates of different molecular weights in equilibrium with the solvent. The solvent properties produce a change in size distribution proportion in the solution. The tendency of asphaltenes to adsorb on surfaces is greater for large aggregates than for small aggregates. These results can be interpreted based on the existence of a solid-liquid equilibrium, which is consistent with the recent proposal of phase separation suppression, where crude oil or certain solvents act by stabilizing different asphaltene aggregates and preventing the phase separation. The stabilization of this solid phase depends on the quality of the environment and the type of asphaltenes.

Experimental Section

Materials and Methods

Reagents: toluene (99.8%, Scharlau), n-heptane (HPLC grade, 99% Scharlau), chloroform (synthesis grade, stabilized with ethanol, 99% Scharlau), cumene (99% Alfa Aesar), tetrahydrofuran [THF, GPC grade, ACS, stabilized with 250 mg L-1 of 2,6-di-tert-butyl 4methylphenol (BHT), 99%, Scharlau], sodium hydroxide (NaOH, synthesis grade, 98%, Scharlau) and p-nitrophenol (PNP, synthesis grade, 99%, Scharlau).

Asphaltenes Separation

Asphaltenes were separated from Hamaca and Cerro Negro (also known as Carabobo) Venezuelan crude oils, and their characteristics are shown in Table 1. The procedure for asphaltene separation is based on the ASTM D6560-17 standard and has been widely reported in different publications related to asphaltenes [START_REF] Castillo | Study of the aggregation and adsorption of asphaltene sub-fractions A1 and A2 by white light interferometry: Importance of A1 sub-fraction in the aggregation process[END_REF][START_REF] Castillo | New techniques and methods for the study of aggregation, adsorption, and solubility of asphaltenes. Impact of these properties on colloidal structure and flocculation[END_REF][START_REF] Acevedo | Trapping of metallic porphyrins by asphaltene aggregates: A size exclusion microchromatography with high-resolution inductively coupled plasma mass spectrometric detection study[END_REF][START_REF] Acevedo | Suppression of Phase Separation as a Hypothesis to Account for Nuclei or Nanoaggregate Formation by Asphaltenes in Toluene[END_REF]. N-heptane was added at a crude:heptane volume ratio of 1:40 to induce the precipitation of asphaltenes. After a resting time of 48-72 h and filtration by suction in two stages, Soxhlet extraction with n-heptane was performed for 6-8 days to remove the coprecipitated material. At this stage, the asphaltenes are separated from the maltenes (saturated, aromatics, and resins).

Asphaltenes Fractionation

Based on the fractionation by complex formation with PNP proposed for the first time by Gutiérrez et al. [START_REF] Gutiérrez | Fractionation of asphaltene by complex formation with p-nitrophenol. A method for structural studies and stability of asphaltene colloids[END_REF], subfractions A1 and A2 and the trapped compounds (TC) were obtained.

The asphaltenes were dissolved in cumene saturated with PNP and refluxed for 6 h. During the 8-day resting time at room temperature, subfraction A1 precipitates as an insoluble asphaltene-PNP complex, while in solution, subfractions A2 and TC remain in solution. After the filtration steps, reduced pressure distillation of the cumene, and successive washing with n-heptane, the solid subfractions were obtained. The PNP was removed by successive washing with a 5%

aqueous NaOH solution until colorless washing was obtained. The yields of subfractions A1 and A2 and TC (saved for further analysis) are shown in Table 1. The A1 and A2 subfractions have the same average composition and characteristic of previously reported [START_REF] Gutiérrez | Fractionation of asphaltene by complex formation with p-nitrophenol. A method for structural studies and stability of asphaltene colloids[END_REF] where the most important difference between these fractions is in the H/C ratio with 0.90 for A1 and 0.99 for A2

and the Double Bond Equivalent (DBE) of 56 for A1 and 52 for A2.

Asphaltene Solution Preparation

Solutions of the whole asphaltenes and their subfractions A1 and A2 were prepared in THF and CHCl 3 at a fixed concentration of 1000 mg L -1 and in the concentration range of 50-2000 mg L -1 .

All solutions were analyzed by GPC-ICP HR MS, where 51 V, 32 S, and 58 Ni isotopes were monitored.

SiO 2 nanoparticle preparation

SiO 2 nanoparticles from rice husks (RH-SiO 2 NPs) were prepared using a modification of a reported process [START_REF] Castillo | Evidence of selective asphaltene subfraction adsorption on SiO2 nanoparticles studied by UV-vis absorbance and fluorescence spectroscopy[END_REF]. In the first step, rice husks were leached with 100 mg/L HCl, washed to neutral pH, and heated at 120 °C to remove water. Then, with a heating ramp of 500 °C h -1 , the rice husks were heated at 500 °C for 1 h to burn carbonaceous residue and at 700 °C for 9 h to produce loosely interconnected amorphous SiO 2 . The amorphous silica size was reduced by a ball milling process for at least 24 h, and mesoporous nanoparticles were obtained with an average size of 40-60 nm, a porosity of 78%, and a specific surface of 234 m 2 g -1 . The BET and porosity plots are shown in Figs. S5 andS6, respectively, in the supporting information.

Asphaltenes Adsorption Experiment

The adsorption experiments were based on previous works of Castillo et al. [START_REF] Castillo | Evidence of selective asphaltene subfraction adsorption on SiO2 nanoparticles studied by UV-vis absorbance and fluorescence spectroscopy[END_REF][START_REF] Castillo | Development of a Methodology Using GPC-ICP HR MS for Analysis of the Adsorption of Asphaltene Aggregates on SiO 2 Nanoparticles[END_REF] and Acevedo et al. [START_REF] Acevedo | SiO 2 Biogenic Nanoparticles and Asphaltenes: Interactions and Their Consequences Investigated by QCR and GPC-ICP-HR-MS[END_REF]. In the first batch of experiments, the quantity of NPs was varied. For this purpose, 600 mg L -1 asphaltene solutions in toluene and CHCl 3 were prepared and placed in contact for 72 h with RH-SiO 2 NPs in the concentration range of 0-5 and 0-300 mg/L, respectively. The remaining solutions after adsorption were analyzed by GPC-ICP HR MS and UV-Visible spectroscopy.

Subsequently, the adsorption behavior of the asphaltenes was analyzed concerning their A1 and A2 subfractions. Then, 600 mg L -1 asphaltenes and their A1 and A2 subfraction solutions in CHCl 3 were placed in contact with 300 mg/L RH-SiO 2 NPs for 72 h. After this time, the remaining solution was separated from the solid phase by centrifugation (6000 rpm for 5 min).

From the experiments, the solutions before and after adsorption were analyzed, and the solid material (RH-SiO 2 nanoparticle NPs adsorbed with the asphaltene or subfraction) was extracted with THF to study the desorption of the material. From now on, the solution before adsorption is called "original", the one after adsorption "remains", and the one from the THF extracts "extract". All solutions were analyzed by GPC-ICP HR MS.

GPC-ICP HR MS Analysis

As reported in previous studies [START_REF] Castillo | Development of a Methodology Using GPC-ICP HR MS for Analysis of the Adsorption of Asphaltene Aggregates on SiO 2 Nanoparticles[END_REF][START_REF] Acevedo | SiO 2 Biogenic Nanoparticles and Asphaltenes: Interactions and Their Consequences Investigated by QCR and GPC-ICP-HR-MS[END_REF][START_REF] González | Study of Very High Molecular Weight Cluster Presence in THF Solution of Asphaltenes and Subfractions A1 and A2, by Gel Permeation Chromatography with Inductively Coupled Plasma Mass Spectrometry[END_REF], GPC-ICP HR MS analyses were performed using a Thermo Scientific Element XR sector field ICP HR MS instrument that operates at a resolution of 4000 (medium resolution) to access spectrally interfered isotopes of 58 Ni, 32 S, and 51 V. The spectrometer was equipped with a Fassel-type quartz torch shielded with a grounded Pt electrode and a quartz injector (1.0 mm i.d.). A Pt sampler (1.1-mm orifice diameter) and a Pt skimmer (0.8-mm orifice diameter) were used. An O 2 flow was continuously supplied to the nebulizer Ar gas flow. The introduction system was fitted with a modified DS-5 microflow total consumption nebulizer (CETAC, Omaha, NE) mounted on a laboratory-made, low-volume (8 mL), singlepass-jacketed glass spray chamber of total consumption. The spray chamber was thermostated to 60 °C. The ICP HR MS conditions were controlled and optimized daily using a 1.0 ng g -1 multielement tuning solution in THF delivered via a syringe pump at a flow rate of 15 µL min -1 .

Chromatographic separations were conducted using a Dionex HPLC 3000 with three Styragel styrene-divinylbenzene gel permeation columns connected in series (7.8 × 300 mm) that works at a flow rate of 0.7 mL min -1 in THF. These columns were HR4 (particle size, 5 µm; exclusion limit, 600 000 Da of polystyrene [PS] equivalent), HR2 (particle size, 5 µm; exclusion limit, 20 000 Da) and HR0.5 (particle size, 5 µm; exclusion limit, 1000 Da). A Styragel guard column (4.6 × 30 mm) was included before these three columns to protect them and prolong their lifetimes. The separation achieved by the columns was evaluated using a mixture of PS standards of 1,000,000-162 Da. According to the GPC column configuration, high-molecular-weight compounds elute at shorter times, and low-molecular-weight molecules elute at longer times, which allowed us to define integration intervals for super-high (SHMW), high (HMW), medium (MMW), and low (LMW) molecular weights of the eluted components, which correspond to ≈1,000,000-67,000 Da; ≈27,000-6,600 Da; ≈1,100 Da and ≈162 Da in equivalent polystyrene, respectively. For the calibration of chromatographic methods by GPC, polymers of known molar mass are used, and the retention time in the column is correlated with the size of the polymer and these values are used as a reference. In the case of asphaltenes the retention times of the aggregates are measured and these are associated with their size, but the size of these aggregates depends on the shape of the aggregates and the solvation, so the real size of the aggregates is smaller than what is determined by GPC, so the graphs give us the relative size.

UV-Vis Analysis

The fractions of each sample were measured in UV-Visible equipment from Mettler Toledo UV7 using a 1 cm cell. The absorbance signal is taken as a function of wavelength in the range of 350-700 nm. For each sample, the absorbance exponentially decays with wavelength showing a maximum at approximately 420 nm, which corresponds to the Soret band of the porphyrins.

For each concentration, the absorbance signal at 450 nm is taken, where the dispersive component of the signal is negligible, and only the absorptive component is taken to construct the absorbance vs. concentration plots.

Methods

In all adsorption experiments, once the solution reached the contact time with the RH-SiO 2 NPs (72 h), aliquots of these solutions were removed and evaporated to dryness, redissolved in the same amount of THF, and analyzed using GPC-ICP HR MS techniques. The aggregate formation of different sizes depends on the chemical potentials of different aggregates present, their interaction potentials, and the functional form of this potential with the size of the aggregates. The chromatographic profile displays similar characteristics throughout the concentration range, which indicates that the aggregate fractions were in equilibrium. Fig. 1 shows a typical GPC-ICP HR MS chromatogram for the asphaltene solutions following the vanadium signal, and the SHMW, HMW, MMW, and LMW regions are detailed. This fact may be expressed as:

(

where is the potential for low-molecular-weight aggregates or monomers, is the potential for medium-molecular-weight aggregates, and is the potential for highmolecular-weight aggregates. k is the Boltzmann constant, T is temperature, and X HMW , X MMW , and X LMW correspond to the fraction of each region in THF solution. Assuming that a nanoaggregate of radius r can be represented as a simple spherical structure, number of units N is proportional to the volume (4/3πR 3 ), and is the interaction factor between the particle and the solvent:

(

From Equations 1 and 2, the concentration of aggregates of different sizes in equilibrium in the media can be expressed by:

(3) where r 0 is the average radius, and r is the radius of different aggregates. For this system of two immiscible components (X XMW ), parameter α corresponds to the free energy of transferring a solute molecule from the solute into the solvent phase. γ is the interfacial free energy per unit exposed area of the nanoaggregate, and r is the radius of the aggregate. For a set of nanoaggregates of different sizes ri > ri 0 , the fraction X XMW is the sum of the set of aggregates in that fraction that behave in an approximately similar manner.

(

) 4
The properties of the solvent play a very important role in the dissolution and stabilization of the aggregates and affect the aggregation process. The solvent interaction with the aggregates drives the formation of aggregates and their structural conformation, since depending on the properties of the solvent in which it is found, the aggregates may form structures with different geometrical forms. In the experiment, it is assumed that the aggregates behave as nanoparticles with defined sizes immersed in the solvent; despite being soluble in the medium, due to their interactions, the surface energy of the aggregates exceeds the dissolution energy.

Results and Discussion

GPC-ICP HR MS of asphaltenes, A1 and A2 subfractions in different solvents

Fig. 2 shows the vanadium GPC-ICP HR MS profiles that correspond to the hydrodynamic volume separation of asphaltenes and its subfractions A1 and A2 from Cerro Negro crude oil.

The samples showed a typical trimodal profile for the asphaltenes and small variations in the profile for the subfractions. Subfraction A1 presents a similar size distribution to asphaltenes with a shift toward high-molecular-weight compounds or larger hydrodynamic volume and a decrease in the low-molecular-weight section. The A2 subfraction shows a notorious shift toward high-molecular-weight compounds or larger hydrodynamic volumes and an evident decrease in the low-molecular-weight section. Similar profiles have been presented in previous works for different asphaltene samples [START_REF] Putman | Analysis of Petroleum Products by Gel Permeation Chromatography Coupled Online with Inductively Coupled Plasma Mass Spectrometry and Offline with Fourier Transform Ion Cyclotron Resonance Mass Spectrometry[END_REF][START_REF] Castillo | Development of a Methodology Using GPC-ICP HR MS for Analysis of the Adsorption of Asphaltene Aggregates on SiO 2 Nanoparticles[END_REF]. The profiles obtained by GPC-ICPMS are similar in most of the crude oils studied, in the present case, for the Hamaca crude oil, a greater definition of the areas is observed and an area of super high molecular weight appears in a very important way, corresponding to super-aggregates. All samples show that the A2 subfraction tends to form compounds with larger hydrodynamic volumes than asphaltenes. The A1 subfraction behaves similarly to the asphaltenes, which reinforces the previous results [START_REF] González | Study of Very High Molecular Weight Cluster Presence in THF Solution of Asphaltenes and Subfractions A1 and A2, by Gel Permeation Chromatography with Inductively Coupled Plasma Mass Spectrometry[END_REF], where the stabilization of the asphaltene aggregates is due the interaction of subfractions A1 and A2. Variations in the profiles show that the aggregation of different fractions can be modulated by geometrical parameters, the structure of the formed aggregates, and their fractal dimension [START_REF] Yarranton | On the size distribution of self-associated asphaltenes[END_REF][START_REF] Gray | Quantitative Modelling of Formation of Asphaltene Nanoaggregates[END_REF][START_REF] Chaisoontornyotin | Combined Asphaltene Aggregation and Deposition Investigation[END_REF].

Fig. 3 shows the GPC-ICP HR MS profiles for asphaltene and subfractions A1 and A2 in chloroform from Cerro Negro (A) and Hamaca (B) crude oils. The plots show the signals for vanadium, in the case of nickel, and sulfur isotopes the behavior is similar. Chloroform is an aprotic solvent with higher polarity than toluene and lower polarity than THF and a small molecular size, which allows it to more easily diffuse into the aggregate. Fig. 3B show the results

for Hamaca, where subfractions A1 and A2 show a tendency to form larger aggregates with a higher hydrodynamic volume than when they are mixed to form asphaltenes. This effect is much more marked for the solutions in CHCl 3 than in THF. In the case of the Cerro Negro crude oil (Fig. 4A-C), the differences are less noticeable, although the changes are still observed. These changes are due to the difference in solubility of the asphaltenes in both solvents and the solubility of the vanadyl compounds trapped in the asphaltene aggregate structure [START_REF] Hoepfner | The fractal aggregation of asphaltenes[END_REF][START_REF] Gascon | Size Distributions of Sulfur, Vanadium, and Nickel Compounds in Crude Oils, Residues, and Their Saturate, Aromatic, Resin, and Asphaltene Fractions Determined by Gel Permeation Chromatography Inductively Coupled Plasma High-Resolution Mass Spectrometry[END_REF][START_REF] Putman | Probing Aggregation Tendencies in Asphaltenes by Gel Permeation Chromatography. Part 1: Online Inductively Coupled Plasma Mass Spectrometry and Offline Fourier Transform Ion Cyclotron Resonance Mass Spectrometry[END_REF]. subfractions, probably because when the trapped vanadium is being liberated [START_REF] Acevedo | Trapping of metallic porphyrins by asphaltene aggregates: A size exclusion microchromatography with high-resolution inductively coupled plasma mass spectrometric detection study[END_REF][START_REF] Castillo | Metal porphyrin occlusion: Adsorption during asphaltene aggregation[END_REF], the asphaltene aggregates are redistributed, and this effect is more remarkable when a more polar solvent chloroform is used. In the case of Cerro Negro, the differences are less noticeable, although they persist, and the decrease in intensity for the subfractions is also present. The changes using CHCl 3 as the solvent are more noticeable than those using THF, and a tailing effect appears.

A B

Concentration Effects on the GPC-ICP HR MS Profiles

Fig. 5 shows the change in vanadium chromatogram as a function of the concentration for Cerro Negro asphaltenes in THF (A) and CHCl 3 (B) solutions the behavior its similar for A1 and A2.

In both cases, there is a linear decrease in the total area as a function of concentration, and the observed molecular mass distribution profile remains constant. This result shows that there are three distributions at equilibrium: HMW = MMW = LMW, and the dilution in the solvent only decreases the concentrations in all distributions without changing their proportions. This behavior was previously observed by González et al. [START_REF] González | Study of Very High Molecular Weight Cluster Presence in THF Solution of Asphaltenes and Subfractions A1 and A2, by Gel Permeation Chromatography with Inductively Coupled Plasma Mass Spectrometry[END_REF], who found that the behavior of asphaltene solutions and their subfractions in THF as a function of concentration does not follow the tendency of a micellar system; the behavior is similar to a dispersion of particles of different size distributions in a similar process to phase separation [START_REF] Acevedo | Suppression of Phase Separation as a Hypothesis to Account for Nuclei or Nanoaggregate Formation by Asphaltenes in Toluene[END_REF]. The most remarkable result is that in both solvents, the behavior is identical, and the redissolution of a determinate sample in THF for an injection in the GPC-ICP HR MS does not alter the aggregate characteristics.

Free surface energy of t h e aggregates

The fraction of asphaltene molecules is composed of multiple molecules of different molecular weights and composition, an approximation to understand their behavior and their interaction with nanoparticles can be made assuming an aggregate with average size and a size distribution of it [START_REF] Gray | Quantitative Modelling of Formation of Asphaltene Nanoaggregates[END_REF]. The adsorption experiments could not be interpreted from a classical thermodynamic point of view so we introduce a first approximation based on the calculation of the surface energy [START_REF] Yu | Correlations of surface free energy and solubility parameters for solid substances[END_REF][START_REF] Nategh | Impact of Asphaltene Surface Energy on Stability of Asphaltene-Toluene System: A Parametric Study[END_REF][START_REF] Samsonov | On applicability of Gibbs thermodynamics to nanoparticles[END_REF][START_REF] Samsonov | Size dependence of the surface energy and surface tension of metal nanoparticles[END_REF][START_REF] Vollath | Surface energy of nanoparticlesinfluence of particle size and structure[END_REF] for the interaction of the species, the surface energy of nanoparticles, and aggregate is the most important parameter because it provides information about the number of active sites on the surface of the nanoparticle and the type of preferential interaction between nanoparticle and aggregates. The free energy of the aggregate surface can be calculated from the fit of each distribution that composes the sample. The intensity of the signal is proportional to the mass fraction of the sample, and based on the experimental results, we propose the existence of three distributions of different aggregates. The fitting of the experimental data was made with 3

Gaussian distributions with the maximum approximately in the mean value of the regions defined as HMW, MMW and LMW. Small variations are observed in the maxima and semiwidths of the curves of the fits with respect to the experimental signal depending on the solvent. This is due to the ability of the medium to solvate and achieve a better definition of the aggregates present. Using Equation 3, the value of α is obtained using approximate radii of 8, 16

and 21 nm taken from the literature [START_REF] Castillo | Study of the aggregation and adsorption of asphaltene sub-fractions A1 and A2 by white light interferometry: Importance of A1 sub-fraction in the aggregation process[END_REF]. For the asphaltene aggregates using Equation 3, the interfacial tension γ is obtained. Fig. 6 shows the original curves for asphaltenes dissolved in toluene and CHCl 3 and their respective fits taking 3 distributions in each case. Table 2 shows the fit values: α is the fitting constant, γ is the surface free energy or the energy needed to transfer one molecule from the aggregate surface to the solution, and r is the radius of the aggregate. As observed in Table 2, the values of α grow with the size of the aggregate and γ, which indicates that a larger aggregate is less likely to dissolve, which increases its stability.

Adsorption Experiment in Toluene

Asphaltene adsorption experiments are focused on determining the amount of asphaltenes that are adsorbed onto nanoparticles. These experiments are usually performed to determine the efficiency of these nanoparticles to be used as adsorbents, or many reports in the literature focus on associating the efficiency with properties of the nanoparticles such as their size and composition [START_REF] Mirzayi | Adsorption kinetics and catalytic oxidation of asphaltene on synthesized maghemite nanoparticles[END_REF][START_REF] Tarboush | Adsorption of asphaltenes from heavy oil onto in situ prepared NiO nanoparticles[END_REF][START_REF] Vargas | Surface modification of SiO2 nanoparticles to increase asphaltene adsorption[END_REF][START_REF] Montes | Effect of Textural Properties and Surface Chemical Nature of Silica Nanoparticles from Different Silicon Sources on the Viscosity Reduction of Heavy Crude Oil[END_REF][START_REF] Montoya | A Novel Solid-Liquid Equilibrium Model for Describing the Adsorption of Associating Asphaltene Molecules onto Solid Surfaces Based on the "Chemical Theory[END_REF][START_REF] Mejia | Sorption of Asphaltenes onto Nanoparticles of Nickel Oxide Supported on Nanoparticulated Silica Gel[END_REF][START_REF] Kashefi | Asphaltene Adsorption using Nanoparticles with Different Surface Chemistry: Equilibrium and Thermodynamics Studies[END_REF]. In our experiment, we focused on studying the interaction of the nanoparticles with different aggregates that are in equilibrium in an asphaltene dispersion [START_REF] Castillo | Evidence of selective asphaltene subfraction adsorption on SiO2 nanoparticles studied by UV-vis absorbance and fluorescence spectroscopy[END_REF]. Fig. 7A shows the vanadium GPC-ICP HR MS profiles of Hamaca asphaltene solutions after being in contact with different percentages of silica nanoparticles. As shown in plot A, for percentages below 0.5%, a homogeneous decrease in the signal is observed; at higher percentages of nanoparticles, a significant decrease in the high-molecular-weight fraction and a relative increase in the low-molecular-weight fraction are observed. Plot 7B

shows the UV-visible spectrum for each sample, where a decrease in magnitude of the spectrum is observed when the amount of adsorbent increases. Plot 7C shows the variation in the absorbance at 450 nm, which indicates a typical exponential decay for these systems. Both plots 7B and 7C show the typical adsorption behavior of asphaltenes, as presented in the literature.

This result is the average behavior of the system without considering the individual behavior of different distributions that compose the sample. The curves in plot A show how the areas corresponding to the high-molecular-weight zone decrease, while the low-molecular-weight zone has a relative increase.

Adsorption Experiment in CHCl 3

Fig. 8A shows the vanadium GPC-ICP HR MS chromatograms of Hamaca crude asphaltene solutions in chloroform after being in contact with different percentages of silica nanoparticles. As shown in plot 8A, for percentages below 0.5%, a homogeneous decrease in the signal is observed; at higher percentages of nanoparticles, a significant decrease in the high-molecular-weight fraction and a relative increase in proportion of the low-molecularweight fraction are observed. Plot B shows the UV-visible spectrum for each sample, where a decrease in magnitude of the spectrum is observed when the adsorbent amount increases.

Conclusions and Final Comments

The results in this work show that asphaltenes are present in the form of aggregates of different hydrodynamic volumes in different solvents. The measurements with different solvents show that there is no critical aggregation concentration, as formally defined, since different molecular distributions proportionally dilute, which suggests that the interaction forces responsible for stabilizing them in the medium are greater than solvation and solubilization. The results show that for different solvents, there are different aggregate volume distributions. A shift to a larger hydrodynamic volume is observed when the solvency power of the solvent is lower, which indicates the increase in interaction forces between the aggregates. Our results demonstrate that the nanoparticles preferentially adsorb the larger or heavier fractions of the asphaltenes. The adsorption process is primarily mediated by interparticle (asphaltene nanoaggregatesilica nanoparticle) interactions instead of being a typical adsorption process mediated by the active center adsorption type. The adsorption experiments in different solvents for asphaltenes and their subfractions A1 and A2 show that for polar solvents such as THF, the stabilization of the solvent on the aggregates is more important than the interaction energies, while the same experiments in toluene or CHCl 3 exhibit a different behavior. Toluene can dissolve them because it can enter the structure of the aggregates and enhance the stabilization.

Fig. 1 .

 1 Fig. 1. Asphaltene vanadium GPC-ICP HR MS profile at 1000 mg L -1 in THF for Hamaca crude

Fig. 2 .

 2 Fig. 2. Vanadium GPC-ICP HR MS profiles of asphaltenes, subfractions A1 and A2 at 1000 mg L -1 in THF for Hamaca crude oil.

Fig. 3 .

 3 Fig. 3. GPC-ICP HR MS profiles of vanadium, for Hamaca (A) and Cerro Negro (B) asphaltenes and subfractions A1 and A2 in CHCl 3 at 1000 mg L -1 .

Fig. 4 .

 4 Fig. 4. Vanadium GPC-ICP HR MS profiles for asphaltenes and their subfractions A1 and A2 dissolved in two solvents (THF and CHCl 3 ) at 1000 mg L -1 for Hamaca and Cerro Negro crude

Fig. 5 .

 5 Fig. 5. Vanadium GPC-ICP HR MS profiles for Cerro Negro asphaltenes in THF (A) and in CHCl 3 (B) by varying the concentration (50-2000 mg L -1 ).

Fig. 6 .

 6 Fig. 6. Vanadium GPC-ICP HR MS normalized profiles in THF (left) and CHCl 3 (right) and the

Fig. 7 .

 7 Fig. 7. Hamaca asphaltenes adsorption onto silica nanoparticles in toluene. Vanadium GPC-ICP

Fig. 8 .

 8 Fig. 8. Hamaca Asphaltene adsorption onto silica nanoparticles in chloroform. Vanadium GPC

Fig. 9 .

 9 Fig. 9. Vanadium GPC-ICP HR MS chromatogram obtained during adsorption experiment in

Fig. 10 .

 10 Fig. 10. Adsorption model of asphaltenes aggregates on NPs.

Table 1 .

 1 Crude oil characteristics and yields of the asphaltene separation and fractionation.

	Crude oil	°API Stability % Asphaltenes % A1 % A2 % TC
	Hamaca	9.0 Stable	13.7	38	58	3
	Cerro Negro	8.5 Stable	8.0	20	72	6

Table 2 .

 2 Fitting parameters for asphaltenes in both solvents, where α is the fitting parameter, γ is the surface energy or interfacial tension calculated from Equation 3 and using ratio r

	Solvent		LMW	MMW	HMW
		α	2.7	13.88	60.52
	CHCl 3	γ (mJ/m 2 )	0.0124	0.0168	0.044
		r (nm)	8.52	16.49	21.14
		α	2.31	7.85	76.09
	THF	γ (mJ/m 2 )	0.0130	0.0089	0.0530
		r (nm)	7.44	17.04	21.71

Acknowledgements

The financial support of the Conseil Régional d'Aquitaine (20071303002PFM) and the Fonds Européen de Développement Economique et Regional (FEDER) (31486/08011464) is acknowledged.


Carlos Javier Villagómez

Fernando Buendía

Lauro Oliver Paz-Borbón

Bernardo Fuentes

Tomaso Zambelli

Xavier Bouju

Surface Vacancy Genera0on by STM Tunneling Electrons in the Presence of Indigo Molecules on Cu(111)

Herein, we invesOgate the consequence of local voltage pulses on the adsorpOon state of single indigo molecules on the Cu(111) surface as well as on the atomic structure underneath the molecule. With a scanning tunneling microscope, at 5 K, intact molecules are imaged as two lobes corresponding to the electron density of each indoxyl moiety of the molecule which are connected by a carbon double bond. Then, two short successive voltage pulses with the Op placed above the molecule generate irreversible modificaOons, as revealed by consecuOve scanning tunneling microscopy (STM) imaging. Density-funcOonal theory calculaOons coupled to STM image calculaOons indicate the creaOon of a double surface vacancy of copper surface atoms below the oxygen atom of the indigo molecule as the most plausible scenario. These extracted copper atoms are stabilized as adatoms by the indigo oxygens, oxidizing each copper adatom to 0.32 electron.

Introduc0on

 Briefly, ajer light irradiaOon: (a) indigo is excited to the electronic state S 1 (1π π*) with a different potenOal energy surface than the S 0 state; (b) ajer

relaxaOon of the molecule, the intramolecular proton transfer occurs at the femtosecond scale; (c) then, there is a fast non-radiaOve decay by moOon of the molecule in the S 1 minimum toward the conical intersecOon of the S 1 /S 0 states, and internal conversion is reached; and (d) the molecule returns to the S 0 ground state minimum from the enol to keto form. The fast non-radiaOve decay of indigo via hopping is the most accepted mechanism of photostability. This process explains why indigo is robustly photostable in the gas phase. [START_REF] Brode | The RelaOon between the AbsorpOon Spectra and the Chemical ConsOtuOon of Dyes. XXVII. Cis-Trans Isomerism and Hydrogen Bonding in Indigo Dyes1[END_REF][START_REF] Yamazaki | Molecular Mechanisms of the Photostability of Indigo[END_REF][START_REF] Kobayashi | On the picosecond kineOcs and photostability of indigo and 6,6′-dimethoxyindigo[END_REF][START_REF] Iwakura | Why Is Indigo Photostable over Extremely Long Periods?[END_REF][START_REF] Chaierley | On the Intrinsic Photophysics of Indigo: A Time-Resolved Photoelectron Spectrosco-py Study of the Indigo Carmine Dianion[END_REF][START_REF] Seixas De Melo | Photophysical and Spectroscopic Studies of Indigo DerivaOves in Their Keto and Leuco Forms[END_REF][START_REF] Seixas De Melo | The Triplet State of Indigo[END_REF][START_REF] Moreno | A TheoreOcal Study of the Photochemistry of Indigo in Its Neutral and Dianionic (leucoindigo) Forms[END_REF][START_REF] Demelo | Spectral and Photophysical Studies of SubsOtuted Indigo DerivaOves in Their Keto Forms[END_REF][START_REF] Ngan | The Triplet State of Indigo: Electronic Structure CalculaOons[END_REF][START_REF] Haggmark | Evidence for CompeOng Proton-Transfer and Hydrogen-Transfer ReacOons in the S1 State of Indigo[END_REF][START_REF] Pina | Excited-State Proton Transfer in Indigo[END_REF][START_REF] Volkov | Indigo Chromophores and Pigments: Structure and Dynamics[END_REF][START_REF] Fang | CalculaOon of Free-Energy Barriers with TD-DFT: A Case Study on Excited-State Proton Transfer in Indigo[END_REF]

Figure 1. Le&, representa-on of the diketo indigo molecule (simply named indigo in the following). Bo?om, representa-on of the monoenol indigo a&er a single proton transfer. Right, representa-on of the dienol indigo molecule a&er a double proton transfer mechanism. No-ce that the double central C-C bond for indigo changes to a single bond for the monoenol indigo, whereas it remains double for the dienol indigo.

Because the photostability is triggered by the proton transfer in the gas phase and in soluOon, one can ask what happens when molecules are adsorbed on a substrate. Indeed, since a long Ome ago, the photostability of indigo has been assigned to the moiety of the molecule with the hydrogen bonding between the carbonyl and N-H groups, and the presence of a surface may affect this mechanism. Nevertheless, most of the studies on adsorpOon of indigo on novel materials for applicaOons in semiconductor organic electronic devices have focused on the ambipolar charge transfer mobility and the fronOer molecular orbital alignment (HOMO-LUMO) with respect to the Fermi level on metal electrodes. TheoreOcal calculaOons for understanding the chemistry of the molecule also show that the reacOvity of indigo is highly affected by subsOtuents of the N-H group. [START_REF] Cervantes-Navarro | Density Func-Oonal Study of the Effects of the SubsOtuents on the Chemical ReacOvity of the Indigo Molecule[END_REF][START_REF] Cervantes-Navarro | DFT Study of the Effect of SubsOtuents on the AbsorpOon and Emission Spectra of Indigo[END_REF] However, there are few studies on indigo adsorbed onto surfaces tackling the opOcal and electronic properOes of the dye. STM experiments carried out by Honda et al. [START_REF] Honda | Surface Structures Formed by a Copper(II) Complex of Alkyl-DerivaOzed Indigo[END_REF] studied the alkyl derivaOves of the indigo copper complex deposited on the o-dichlorobenzene/graphite interface; they found out that the molecules exhibit a mordanOng effect on the surface (addiOon of metal atoms to stabilize the adsorpOon of dye molecules) in comparison to the molecules without the metal ions and observed the formaOon of 2D and 3D chiral structures for larger alkyl chains. In our previous work, [START_REF] Villagomez | Self-Assembly of EnanOopure Domains: The Case of Indigo on Cu(111)[END_REF] we studied the indigo molecules by STM at different deposiOon rates on Cu(111). We have reported that molecules assemble in linear chains at low coverages presenOng chirality due to the symmetry reducOon on the surface. Indigo forms almost homochiral chains by hydrogen bonding. At higher coverages, the molecules presenOng the other chirality compared to the ones in the almost homochiral chains are expelled, and indigo molecules form enanOopure domains.

The stabilizaOon of the mono-and dienol forms seems to be challenging in the gas phase and soluOon, but it remains achievable for molecules adsorbed on a surface. On the other hand, electron excitaOon carried out by the STM Op pulses is expected to induce isomerizaOon or intramolecular proton transfer (single or dual). [START_REF] Alemani | Electric Field-Induced IsomerizaOon of Azobenzene by STM[END_REF][START_REF] Auwärter | A Surface-Anchored Molecular Four-Level Conductance Switch Based on Single Proton Transfer[END_REF] In comparison to azobenzene where the transcis isomerizaOon can be carried out by opOcal excitaOon or even induced by electric-field excitaOon of STM on molecules adsorbed on the surface, indigo does not show such an isomerizaOon of the C═C bond by photoexcitaOon. [START_REF] Wyman | The Cis-Trans IsomerizaOon of Conjugated Compounds[END_REF][START_REF] Kobayashi | On the picosecond kineOcs and photostability of indigo and 6,6′-dimethoxyindigo[END_REF] The moOvaOon of the present study was not to study the opOcal properOes of the molecule adsorbed on the metallic surface but to address the possible ESIP transfer as a response of the indigo molecule to a local electron sOmulus instead of photoexcitaOon. Once adsorbed onto a surface, a localized electron injecOon could generate the permanent monoenol state from indigo by ESIP transfer due to the molecule/surface interacOon. Then, a second sOmulus would lead to the dienol form. An STM Op can be used for such a manipulaOon of individual adsorbates. It is now well established that STM manipulaOons can be performed according to three different strategies. First, by using mechanical lateral forces between the Op and the adsorbate, one can pull, slide, or push a molecule or an atom between definite posiOons on the surface. [START_REF] Hla | Atom-by-Atom Assembly[END_REF][START_REF] Moresco | ManipulaOon of Large Molecules by Low-Temperature STM: Model Systems for Molecular Electronics[END_REF][START_REF] Grill | Large Molecules on Surfaces: DeposiOon and Intramolecular STM ManipulaOon by DirecOonal Forces[END_REF][START_REF] Bouju | Mechanics of(Xe) N atomic Chains under STM ManipulaOon[END_REF][START_REF] Bartels | Basic Steps of Lateral ManipulaOon of Single Atoms and Diatomic Clusters with a Scanning Tunneling Microscope Tip[END_REF][START_REF] Moresco | Recording Intramolecular Mechanics during the ManipulaOon of a Large Molecule[END_REF][START_REF] Alemani | Recording the Intramolecular DeformaOon of a 4-Legs Molecule during Its STM ManipulaOon on a Cu(211) Surface[END_REF][START_REF] Chen | Breaking a DaOve Bond with Mechanical Forces[END_REF][START_REF] Villagómez | Boiom-up Assembly of Molecular Wagons on a Surface[END_REF][START_REF] Grill | Rolling a Single Molecular Wheel at the Atomic Scale[END_REF][START_REF] Saywell | ManipulaOng the ConformaOon of Single Organometallic Chains on Au(111)[END_REF] Second, the localized electric field, enhanced by the Op effect, can induce a controlled moOon of the adsorbate, [START_REF] Alemani | Electric Field-Induced IsomerizaOon of Azobenzene by STM[END_REF][START_REF] Alemani | AdsorpOon and Switching ProperOes of Azobenzene DerivaOves on Different Noble Metal Surfaces: Au(111), cu(111), and Au(100)[END_REF][START_REF] Dri | SpaOal Periodicity in Molecular Switching[END_REF][START_REF] Borca | Electric-Field-Driven Direct DesulfurizaOon[END_REF] as exemplified by the recent nanocar race. [START_REF] Simpson | How to Build and Race a Fast Nanocar[END_REF][START_REF] Simpson | How to Control Single-Molecule RotaOon[END_REF][START_REF] Pawlak | Design and CharacterizaOon of an Electrically Powered Single Molecule on Gold[END_REF][START_REF] Soe | ConformaOon ManipulaOon and MoOon of a Double Paddle Molecule on an Au(111) Surface[END_REF][START_REF] Rapenne | The First Nanocar Race[END_REF][START_REF] Jacquot De Rouville | From the Synthesis of Nanovehicles to ParOcipaOon in the First Nanocar Race-View from the French Team[END_REF] Finally, the current of energeOc tunnel electrons can chemically modify the conformaOon of a molecular adsorbate. [START_REF] Auwärter | A Surface-Anchored Molecular Four-Level Conductance Switch Based on Single Proton Transfer[END_REF][START_REF] Sope | Single-Molecule DissociaOon by Tunneling Electrons[END_REF][START_REF] Hahn | Orbital Specific Chemistry: Controlling the Pathway in Single-Molecule DissociaOon[END_REF][START_REF] Sprodowski | DissociaOon of Oxygen on Ag(100) Induced by InelasOc Electron Tunneling[END_REF][START_REF] Lauhon | Control and CharacterizaOon of a MulOstep Unimolecular ReacOon[END_REF][START_REF] Kudernac | Electrically Driven DirecOonal MoOon of a Four-Wheeled Molecule on a Metal Surface[END_REF] Thus, by triggering the excitaOon strength, a local-probe Op is able to prompt conformaOonal changes [START_REF] Borca | Bipolar Conductance Switching of Single Anthradithiophene Molecules[END_REF][START_REF] Choi | ConformaOonal Molecular Switch of the Azobenzene Molecule: A Scanning Tunneling Microscopy Study[END_REF] and even breaking/formaOon of chemical bonds. [START_REF] Chen | Breaking a DaOve Bond with Mechanical Forces[END_REF][START_REF] Van Der Lit | Suppression of Electron-Vibron Coupling in Graphene Nanoribbons Contacted via a Single Atom[END_REF][START_REF] Schuler | Reversible Bergman CyclizaOon by Atomic ManipulaOon[END_REF][START_REF] Mohn | Reversible Bond FormaOon in a Gold-Atom-Organic-Molecule Complex as a Molecular Switch[END_REF][START_REF] Kaiser | An Sp-Hybridized Molecular Carbon Allotrope, cyclo[18]carbon[END_REF][START_REF] Hla | Inducing All Steps of a Chemical ReacOon with the Scanning Tunneling Microscope Tip: Towards Single Molecule Engineering[END_REF][START_REF] Ho | Single-Molecule Chemistry[END_REF][START_REF] Wang | Chemical Synthesis at Surfaces with Atomic Precision: Taming Complexity and PerfecOon[END_REF][START_REF] Kim | AcOon spectroscopy for singlemolecule reacOons -Experiments and theory[END_REF][START_REF] Stone | A Single-Molecule Blueprint for Synthesis[END_REF] Applying localized voltage pulses with an STM Op on the keto group of molecules on Cu(111) at 5 K, we reproducibly observed the irreversible transformaOon of the molecules. We considered two possible mechanisms to explain these facts: (i) a stable keto-enol transfer stabilized by the surface and (ii) the formaOon of a vacancy on the copper surface with consequent formaOon of a complex between the oxygen atoms of the molecule and the metallic adatoms extracted from the surface.

For instance, previous manipulaOons of molecules induced by electrons from STM pulses showed tautomerizaOon and proton transfer of a free base tetraphenyl-porphyrin (TPP) on Ag(111) between two states for 2H-TPP or among four states ajer removing one internal hydrogen atom of the TPP molecular core. [START_REF] Auwärter | A Surface-Anchored Molecular Four-Level Conductance Switch Based on Single Proton Transfer[END_REF] Molecular orientaOon induced by tunneling electrons has been shown to switch azobenzene molecules adsorbed on the Au(111) surface at the threshold voltage applied to the molecules. [START_REF] Hao | OrientaOon Switching of Single Molecules on Surface Excited by Tunneling Electrons and Ultrafast Laser Pulses[END_REF] Then, a similar quesOon arises for the indigo molecule about the possible reacOon induced by the excitaOon of the molecule adsorbed on the metal surface by the tunneling electrons of the STM Op. In this way, it is improbable that indigo adsorbed on the surface carries out isomerizaOon by the central C═C bond, but uncertainty exists as to whether the pulses can induce a permanent isomeric tautomerizaOon of the molecule. In this work, we concentrate on the adsorpOon at the single-molecule level to understand the individual adsorpOon of the molecule and the manipulaOon by the STM Op pulses.

Experimental and Computa0onal Methods

The Cu(111) crystal surface (Goodfellow Inc., UK) was cleaned by standard repeated procedures of Ar + spuiering, followed by annealing of the sample to 600 °C at a UHV pressure of 10 -10 Torr. The indigo molecules were deposited by sublimaOon using an external filament with molecules adsorbed onto it, the evaporator was posiOoned poinOng the sample placed inside a lowtemperature cryostat by an access hole, while the surface crystal was kept at a low temperature in order to have single molecules during the adsorpOon process and to avoid diffusion of the molecules for autoassembling. All the measurements were carried out at a low temperature of 5 K and at an UHV chamber pressure of 10 -11 Torr.

The tungsten STM Ops were fabricated by electrochemical aiack of a 0.25 mm diameter wire immersed in a soluOon of NaOH, and ajer the fabricaOon, the STM Ops were annealed by Joule heaOng inside the preparaOon chamber to desorb the possible oxidaOon product formaOon in air. The cleanliness of the surface and the STM Op was verified by STM image measurements in situ showing larger flat terraces and monoatomic step edges. Density funcOonal calculaOons were performed using Vienna ab iniOo simulaOon package 5.4 (VASP 5.4). [START_REF] Kresse | Ab iniOomolecular-dynamics simulaOon of the liquid-metal-amorphoussemiconductor transiOon in germa-nium[END_REF][START_REF] Kresse | Efficient iteraOve schemes forab iniOototal-energy calculaOons using a plane-wave basis set[END_REF][START_REF] Kresse | Ab iniOomolecular dynamics for liquid metals[END_REF] The gradient-corrected Perdew-Burke-Ernzerhof (PBE) exchange and correlaOon funcOonal [START_REF] Perdew | Generalized Gradient ApproximaOon Made Simple[END_REF] was used in combinaOon with the Tkatchenko-Scheffler van der Waals (vdW) dispersion. [START_REF] Tkatchenko | Accurate Molecular Van Der Waals InteracOons from Ground-State Electron Density and Free-Atom Reference Data[END_REF] Projected-augmented wave potenOals were used for different species (Cu, C, N, O, and H atoms). The Brillouin zone was sampled in a k-mesh of 3 × 3 × 1 employing an energy cutoff of 400 eV for the plane waves. The copper substrate was a Cu(111) fcc 3 × 3 slab with three atomic layers in the non-periodic direcOon. The lauce parameter employed was 3.63 Å, obtained ajer slab relaxaOon without restricOons and this value is similar to those inferred in previous experimental studies. [START_REF] Smura | High-Spin Cobalt(II) Ions in Square Planar CoordinaOon: Structures and MagneOsm of the Oxysulfides Sr2CoO2Cu2S2 and Ba2CoO2Cu2S2 and Their Solid SoluOon[END_REF] On the other hand, for the adsorpOon process of the indigo molecule, the deepest atomic layer was kept fixed to mimic the behavior of the inner atomic layers of the solids. The convergence criterion for the electronic self-consistent field was 1 × 10 -4 eV, whereas the energeOc threshold was 1 × 10 -3 eV for the relaxaOon process. Finally, STM images were calculated with the extended Hückel molecular orbital-elasOc scaiering quantum chemistry (EHMO-ESQC) code. [START_REF] Sautet | CalculaOon of the Benzene on Rhodium STM Images[END_REF]86 Here, the STM juncOon comprising the substrate, the adsorbates, the Op apex, and the Op support is fully described at the atomic level. A set of semiempirical extended Hückel orbitals is assigned on each atom site. The scaiering of tunnel electrons through the juncOon is calculated to allow the evaluaOon of the tunneling current with the Landauer formula. This method has already proven its reliability with small and large molecular systems. [START_REF] Sautet | CalculaOon of the Benzene on Rhodium STM Images[END_REF][START_REF] Villagómez | AdsorpOon of Single 1,8-Octanedithiol Molecules on Cu(100)[END_REF][START_REF] Yu | Self-Assembly of Hydrogen-Bonded Chains of Molecular Landers[END_REF][START_REF] Xu | Exploring the Transferability of Large Supramolecular Assemblies to the Vacuum-Solid Interface[END_REF][START_REF] Xie | Switching the Spin on a Ni Trimer within a Metal-Organic MoOf by Controlling the On-Top Bromine Atom[END_REF]

Experimental Results

On STM images, an isolated molecule consistently appears like a dumbbell with two nearly circular lobes (Figure 2a). [START_REF] Villagomez | Self-Assembly of EnanOopure Domains: The Case of Indigo on Cu(111)[END_REF] This defines the main axis of the molecule relying on the direcOon of the two rings. The bright spots have a relaOve height of about 0.7 Å and a distance separaOon of 1.1 nm. As previously menOoned, STM pulses were used in experiments in the past to induce different conformaOons of individual molecules on the surface due to tautomerizaOon or mechanical movement on the terraces and step edges. Ajer imaging the intact indigo molecule, a voltage pulse of 2.4 V was applied on one lobe of the molecule (blue spot in Figure 2a) for a Ome of 400 ms. We ensured that the regulaOng loop was switched off before the pulse and restored ajer the pulse Ome. To obtain quanOtaOve informaOon, we applied systemaOc other pulses at various bias voltages on indigo molecules to induce conformaOonal changes. We have chosen the Op-surface distance corresponding to the setpoint (10 pA, -300 mV) as the iniOal point Zo, and we applied pulses with varying duraOons unOl the changes were observed. In Figure 3a, experimental data are shown corresponding to the duraOon threshold to successfully change the appearance of the molecule for each bias voltage. The curve follows an exponenOal decrease that can be fiied by A•exp(-eV/B) with A = 1.26 × 10 15 ms and B = 83 meV. From that, one can deduce that the dose, that is to say, the number of tunneling electrons impinging on the molecule and the surface, varies as

Figure 2. (a) Three indigo molecules on Cu(111) prior to any voltage pulse; (b) one molecule is turned into a new form a&er a first posi-ve voltage pulse with the -p posi-oned between the two lobes, marked by the blue spot in (a); (c) a second pulse is applied, marked by the red spot in (b) changing the conforma-on of the targeted molecule; (d,e) the same manipula-on process is done on the other two molecules (STM imaging: 10 pA, -300 mV).

Figure 3. (a) Quan-ta-ve informa-on about the switching mechanism: dura-on threshold from which a conforma-onal change is obtained as a func-on of the pulse voltage. The fiSng red curve has the form A•exp(-eV/B) with

N = N 0 •exp[-e(V -V 0 )/B],
with N 0 = I 0 A/e (I 0 = 10 pA) and V 0 = -300 mV. The dose varies exponenOally, meaning that the more the voltage bias, the more energeOc the electrons, and the more the efficiency change. AddiOonally, the manipulaOon rate R versus the tunneling current I follows a power-law dependence R ∝ I α using a model for bond breaking by inelasOc electron tunneling, [START_REF] Sope | Single-Molecule DissociaOon by Tunneling Electrons[END_REF][START_REF] Eigler | An Atomic Switch Realized with the Scanning Tunnelling Microscope[END_REF][START_REF] Wang | Electron SOmulated DesorpOon of Vanadyl-Groups from Vanadium Oxide Thin Films on Ru(0001) Probed with STM[END_REF] which is reported in Figure 3b for a bias voltage of 2.4 V (for each Op-sample distance, one deduces a set point current and measures the transformaOon rate). One can deduce that the parameter α = 1.85 ± 0.05, indicaOng that one deals with a double-electron process. [START_REF] Sope | Inducing and Viewing the RotaOonal MoOon of a Single Molecule[END_REF][START_REF] Yamazaki | Interplay between Switching Driven by the Tunneling Current and Atomic Force of a Bistable Four-Atom Si Quantum Dot[END_REF] The STM image exhibits a contrast change as shown in Figure2b (molecule at the boiom) corresponding to a conformaOonal change of the molecule. Ajer this first bias voltage pulse was applied above a targeted molecule, the dumbbell feature disappears (Figure 3c): a bright lobe is switched off (relaOve height diminished to 0.3 Å, Figure 3c), and a lateral depression becomes much darker (a contrast of about 0.2 Å with respect to the central part of the molecule is measured as one can see with the blue profile in Figure 3d). Then, ajer a second pulse of 3 V (red spot in Figure 2b), the remaining bright spot is turned off to 0.3 Å, and a pronounced dark trace appears perpendicularly to the main axis (Figure 2c) with a depression around 0.2 Å relaOve to the central molecule contrast similar to the one shown in Figure 3d. NoOce that this double process does not apparently modify the locaOon of the molecule like a rotaOon or a translaOon onto the surface, as shown in Figure 2d,e ajer several STM manipulaOons proceeded on two other molecules. We observed that the second voltage pulse has to be higher than the first one, meaning that the second manipulaOon requires a higher electron energy. Moreover, only posiOve bias voltage pulses are efficient to induce a manipulaOon, a fact that we are not able to raOonalize.

Analysis and Discussion

The goal of this work is thus to determine which phenomenon is responsible for the observed change of contrast of the indigo molecule upon STM pulses. For this purpose, calculaOons are a necessary complement to the experiments, and the first step is the determinaOon of the most stable adsorpOon site of the indigo molecule on the Cu(111) substrate by means of DFT calculaOons. Once this has been determined, we conOnue to calculate the energy barrier of the hydrogen transfer and the conformaOonal structure of the monoenol and dienol chemical structures on the surface. Then, calculated STM images with the electronic scaiering quantum chemistry (ESQC) method are obtained for each structure for comparison with experimental images.

The iniOal DFT calculaOons show that the indigo molecule is adsorbed parallel to the surface of the Cu(111) slab. The preferred orientaOon of the molecule was analyzed ajer the relaxaOon of the molecule adsorbed on the slab in 12 different orientaOons, each of them rotated 15° with respect to the previous one (n × 180°/12, with n = 0, ..., 11), as shown in Figure 4. The rotaOon axis is located above a bridge posiOon of the Cu(111) surface. This posiOon was found to be more energeOcally favored at the top or hollow posiOons. The C 2h symmetry of the molecule and the locaOon of the rotaOon axis enabled the angles greater than 165° to generate equivalent geometric configuraOons. The inclusion of the dispersion energy is crucial for these systems due to the interacOon between the aromaOc rings of the molecule and the metal substrate, and it was addressed using the Tkatchenko-Scheffler vdW dispersion correcOon. [START_REF] Tkatchenko | Accurate Molecular Van Der Waals InteracOons from Ground-State Electron Density and Free-Atom Reference Data[END_REF] We have measured various orientaOons of adsorbed molecules and have checked that they are compaOble with the metastable and stable orientaOons in Figure 4. Once the preferred orientaOon of the indigo ground state molecule (GS) is determined, the possible reacOons generated by the voltage pulse were explored. The proton transfer from some other possible reacOons, such as the double-hydrogen transfer or the generaOon of Cu vacancies was taken into account to raOonalize the phenomenon observed in the experimental STM images. The single proton transfer barrier was obtained by means of the nudge elasOc band (NEB) method, and five images between the iniOal and final states were employed. The final state was determined in an independent search, and it is corroborated as a local minimum in the potenOal energy surface. Bader analysis was performed for different systems using the method developed by the Henkelman group [START_REF] Tang | A Grid-Based Bader Analysis Algorithm without Lauce Bias[END_REF] to get a deeper insight of the electronic structure of the molecule-substrate complex in various conformaOons. As menOoned already, due to the C 2h symmetry and a similar interacOon of the indoxyl groups with the Cu(111) surface slab, the following explanaOons are limited for half of the molecule and can be applied for both sides. Previously, semiempirical calculaOons were performed without relaxaOon of the surface atoms and have provided similar qualitaOve results compared to the present DFT ones. [START_REF] Villagomez | Self-Assembly of EnanOopure Domains: The Case of Indigo on Cu(111)[END_REF] However, for accurate determinaOon of transient states, DFT calculaOons were preferred in the following. Here, DFT calculaOons presented in Figure 4 show that the central C═C bond sits above a bridge site of the (111) surface. The most stable conformaOon (GS) is found at 105° with respect to the reference, and it possesses an adsorpOon energy of -2.92 eV, its geometric structure is at the boiom of Figure 4. The less stable system (15°) has an adsorpOon energy of -2.46, and it is 0.46 eV higher than that of the most stable molecule-substrate conformaOon. Another adsorpOon conformaOon with a high stability is the molecule orientated at 75° from the reference, and it is only 0.01 eV less stable than GS. For all cases, the molecule strongly interacts with the surface by means of the oxygen atom, and this bond generates the distorOon of the molecule and the substrate. For the most stable geometry, the interacOon of the oxygen atom with the substrate generates a decrease in the C-O interacOon compared with the gas-phase molecule, which is reflected in the elongaOon of the C-O bond length by 0.09 Å. The C-O chemical bond decreases from a double to a single bond; for heterocyclic aromaOc compounds with a carbonyl group, it is achievable to bond metals through a lone pair of electrons. [START_REF] Baia | Molecules with AnObacterial ProperOes[END_REF] With regard to the Cu atom beneath the oxygen atom, it experiences a slight lij of 0.14 Å with respect to its original height. This phenomenon occurs on the two indoxyl groups of the molecule. In accordance with our results, other DFT calculaOons of indigo molecules adsorbed on silver nanoparOcles show the formaOon of a complex between the molecule and the substrate via the O-Ag interacOon for different silver clusters of Ag 2, Ag 14 , and Ag 16 . [START_REF] Nardo | SERS and DFT Study of Indigo Adsorbed on Silver Nanostructured Surface[END_REF][START_REF] Ricci | The Raman and SERS spectra of indigo and indigo-Ag2 complex: DFT calculaOon and comparison with experiment[END_REF][START_REF] Ricci | Chemical Enhance-ment in the SERS Spectra of Indigo: DFT CalculaOon of the Raman Spectra of Indigo-Ag14 Complexes[END_REF] In addiOon to the flat-lying adsorpOon preference of the molecule on the silver substrate, [START_REF] Nardo | SERS and DFT Study of Indigo Adsorbed on Silver Nanostructured Surface[END_REF] it is also worth noOcing that surface-enhanced Raman spectroscopy measurements of indigo molecules adsorbed on silver nanoparOcles irradiated at 514 nm involve intramolecular electronic excitaOons, intercluster excitaOons, and charge-transfer effects. These effects are visualized in the intensity enhancement and appearance of new vibraOonal Raman spectra, but it is not the case for other laser excitaOon wavelengths of lower energy where just the charge-transfer effect is involved. [START_REF] Ricci | Chemical Enhance-ment in the SERS Spectra of Indigo: DFT CalculaOon of the Raman Spectra of Indigo-Ag14 Complexes[END_REF] This energy value is very close to our electronic tunneling excitaOon of 2.4 eV pulses with the copper surface.

On the other hand, the vdW interacOon between the aromaOc rings of the molecule and the substrate is essenOal for the adsorpOon process. 100 The total energy values including a vdW interacOon range from -2.46 to -2.92 eV, while without the vdW interacOon they vary from -0.31 to 0.64 eV. This difference reflects the importance of taking into account the dispersion interacOons for complexes formed by organic molecules and metallic substrates. For the most stable system, the distances between the center of the rings and the surface are 2.54 and 2.56 Å. On the other hand, the N-H bond length (1.01 Å) shows a similar value compared to the gas phase (1.02 Å). The hydrogen bond between C-O and N-H is similar to the gas phase, but the distance between the two groups is slightly increased by 0.04 Å due to the introducOon of the substrate. The charge analysis corroborates the ionic character of the O-Cu bond with a charge transfer of 0.19 electron (e -) from the Cu toward the oxygen. However, the charge in the oxygen atom is similar to that in the gas phase due to the charge received from the C atom in the gas phase. As a result of the new Cu-O interacOon, the C═O double bond is weakened to a single bond, this is confirmed by the change in the bond length. These results show that the vdW dispersion and C-O interacOons are responsible for the geometric configuraOon of the molecule-Cu(111) substrate complex.

The finding of the most stable posiOon and orientaOon of the molecule on the Cu(111) substrate is crucial as a starOng point in the search for the possible reacOons that could occur in the system ajer the STM pulse. Various mechanisms can be explored to explain the resulOng molecular contrast change in the STM images ajer the pulse, such as keto-enol tautomerizaOon, trans-cis isomerizaOon, fragmentaOon, dissociaOon, and surface vacancy generaOon. In the following, only an intact molecule without bond breaking is considered because no fragments have been observed. Moreover, a parOal molecular dissociaOon consisOng of the detachment of the oxygen atom that could be adsorbed nearby the molecule on a hollow site has been esOmated and appeared largely unfavorable compared to the mechanisms discussed ajer.

The first explored mechanism is the possibility of a permanent proton transfer process on the surface due to the electronic and chemical interacOons of the indigo molecule with the substrate in contrast to soluOon irradiaOon experiments. Such a keto-enol tautomerizaOon mechanism has already been proposed for the Au/isophorone complex, where the surface and the presence of gold adatoms play a crucial role in the appearance of an enol tautomer. 101 The trans-cis isomerizaOon was not explored because this process requires higher energy due to the strong interacOon of the indoxyl group with the copper surface. The switching of other aromaOc double bond compounds lying flat on the surface such as azobenzene on Au(111) was possible by adding chemical spacers such as tert-butyl in order to separate the phenyl rings from the surface interacOon. [START_REF] Alemani | Electric Field-Induced IsomerizaOon of Azobenzene by STM[END_REF] This is not the case for the indigo compound that is directly adsorbed on the surface.

The chemical process of one hydrogen transfer from nitrogen to oxygen is presented in Figure 5, showing that the symmetry constraint of the indigo molecule is broken once the hydrogen is transferred. The reacOon was analyzed using the most stable configuraOon such as the iniOal state N-H, shown in Figure 5, and the molecule ajer the hydrogen transfer that has been obtained in an independent search such as the final state O-H, shown in Figure 5. On the other hand, the TS (in Figure 5) was found using the NEB method. In the gas phase, once the proton has been transferred, the C═O double bond goes from 1.24 to 1.28 Å, showing the weakening of the bond due to the new O-H bond. On the other hand, the distance between N and C atoms close to the center of the molecule is shortened, from 1.37 Å in the iniOal state to 1.30 Å once the hydrogen has been transferred. The reacOon on the Cu(111) substrate presents some differences with respect to the gas phase case. The Cu-O bond is broken due to the new O-H interacOon, and it is reflected in the Cu-O bond length that increases from 2.12 to 2.64 Å ajer the hydrogen transfer. Moreover, the nitrogen atom does not generate a new bond with a Cu surface atom, in contrast with the oxygen atom before the hydrogen transfer. The rings slightly change their geometry, but the distances between the aromaOc rings and the substrate are similar. On the other hand, the Cu atom of the substrate that was bound with the oxygen atom before the proton transfer returns to its original posiOon. The new system is 0.28 eV higher in energy than the iniOal state. Bader analysis exhibits a charge transfer of 0.14 e -from the substrate toward the nitrogen of the molecule ajer the hydrogen transfer. This value is smaller than the charge received by the oxygen before the hydrogen transfer, and this difference is related to the higher electronegaOvity of the oxygen atom. The double hydrogen-transfer system was also analyzed, and the geometric changes are similar to both sides of the molecule. This system shows lower stability by 0.79 eV compared with the most stable system, and it is 0.51 eV higher in energy than the single hydrogen transfer. The C-O bond distance on both sides of the molecule (1.30 Å) is increased compared with the one hydrogen-transfer system (1.28 Å) and also with respect to the gas phase (1.24 Å). On the other hand, the two Cu atoms close to the nitrogen atoms do not change their original posiOon, while the two Cu atoms close to the oxygen atoms return to the original height of the remaining Cu atoms in the slab. The minimum energy path (MEP) of the hydrogen transfer was analyzed by using eight intermediate geometries between the iniOal and the final state. The TS is 0.65 eV higher in energy than the iniOal state as can be seen in Figure 5. This value is lower than the one observed for the system in the gas phase. The geometry of the TS locates the hydrogen atom at 2.94 Å from the closest Cu atom of the substrate, 1.35 Å from the nitrogen and 1.40 Å from the oxygen atom. The oxygen atom is located at 2.49 Å from the closer Cu atom of the surface, while the nitrogen one is 2.90 Å from the closer Cu atom of the surface. The copper atom close to the oxygen atom presents a lij from the surface of 0.10 Å compared with their neighbors. This fact reflects the contribuOon of the substrate to stabilize the intermediate state due to a Cu-O interacOon, while the transferred hydrogen atom seems to be interacOng very weakly with the substrate.

The new metastable configuraOons present lower stability compared with the indigo molecule adsorbed in the ground state; however, they could be accessible configuraOons ajer the pulse introducOon. In order to verify if the proposed systems are observed experimentally ajer the pulse, ESQC calculaOons of the calculated DFT geometries were carried out: (1) keto, (2) monoenol, and (3) dienol, in Figure 6a-c, respecOvely. The STM calculated images in Figure 6 show a surprisingly negligible change of the STM contrast. By comparing Figure 6b,a, for a single proton transfer, almost insignificant modificaOon is observed, and only a Ony indentaOon near a lobe is visualized. For the double proton transfer, there is slight diminuOon in the intensity surrounding the center of the lobes, Figure 6c. However, the experimental STM images (Figure 2b,c) differ greatly with respect to the calculated STM images with one or two proton transfers: there are no dark depressions in the middle of the molecule. The hydrogen-transfer process has been analyzed in depth as a possible path in view of the fact of previously reported studies and also because the STM voltage pulses applied on the molecule on the substrate could help induce the reacOon. Nevertheless, the single and double proton transfers are discarded. As a second alternaOve mechanism, we focused on the creaOon of vacancies in the copper substrate sOmulated by the STM pulses, which could be favored by the strong interacOon of the oxygen with the copper atoms. Previous experiments of oxygen atoms adsorbed on Cu(111) at room temperature showed that the oxygen atoms upon adsorpOon release 3-fold copper atoms on the hollow site, producing a preliminary roughness and disorder at the early formaOon of the Cu2O layer surface. [102][103][104] On the other hand, the energy for single surface vacancy formaOon has been calculated using the embedded-atom method and is about 0.72 eV, 105 whereas DFT-based calculaOons provided values in the same range: theoreOcal DFT-D2 calculaOons with the Grimme method esOmated the surface vacancy formaOon energy on prisOne Cu(111) at 0.82 eV, 106 this energy value is higher than 0.69 eV obtained using the DFT-PW91 funcOonal, 107 and it is lower and reasonably similar to other calculated surface vacancy formaOon energies of 0.92 eV employing the DFT-LDA funcOonal 108 and 1.05 eV using the DFT-PBE funcOonal. 109 The effect of vacancies on the prisOne Cu(111) is visualized in the electron charge density map by a depression or hole calculated using the DFT-PBE funcOonal. 109 NoOce that once the adatom is on the (111) terrace, it experiences a small diffusion energy: in the literature, one can find calculated hopping barrier values of 28, 105 34, 110 41, 111 44, 112 50, 113 53, 114 59, 115 and 64 115 meV that are comparable to experimental values of 37 ± 5 and 40 ± 1 meV. [116][117][118] In our model, using the GS geometry as reference, the energy to completely remove the Cu atom below one oxygen atom of the molecule is 2.33 eV, while the energy necessary to remove one Cu atom below a nitrogen atom is 2.73 eV. This energy difference comes from the energy necessary to break the Cu-O bond formed in the first case. The ESQC image of the copper vacancies below the oxygen atom is presented in Figure 7. Now, the calculated image reproduces qualitaOvely well the experimental image features: the dark depression at the lateral moiety of the molecule and a slight diminuOon of the bright lobe belonging to the indoxyl part, where the copper atom of the surface is missing.

tension pulse (it was rotated for comparison). The pink arrow shows the vacancy and the appearance of a lateral dark spot between two bright spots with different intensi-es in the calculated image.

In the same way as the single-vacancy calculaOon, the second Cu vacancy generates a handle fanshaped dark depression on the remaining part of the calculated STM contrast that is expanded at the middle of the molecule, as shown in Figure 8. It is also remarkable to noOce that the remaining bright lobe reduces the surrounding bright intensity from the center of the lobe. The experimental STM image ajer the second voltage pulse is added to the right for comparison in Figure 8. It is observed that both calculated and experimental images match very well. Another invesOgated mechanism was the creaOon of a vacancy and the formaOon of an adatom/ molecule complex. Indeed, as menOoned before, the diffusion energy of a single Cu adatom located on the Cu(111) surface is rather small, and consequently the adatom would tend to stay bound to the molecule once extracted from the surface due to a stronger adatom/molecule interacOon. DFT calculaOons assessing this case show that the geometry corresponds to a Cu adatom siung in a bridge site, bound to two copper surface atoms and interacOng with the oxygen atom of the molecule (Figure 9a). The minimal energy geometry of these systems is 1.05 eV higher in energy of the single adsorbed indigo. This new geometry shows a reducOon of the Cu-O bond length from 2.14 to 1.90 Å ajer the total lij of the Cu atom. Bader analysis exhibits an increase of the Cu oxidaOon, being 0.32 e -transferred from Cu to the oxygen atom. The interacOon of the molecule with the copper substrate generates a new stable configuraOon that is not possible to find in the gas phase, and it is reached when the Cu adatom of the surface is completely oxidized by the oxygen atom. This state is only possible to be obtained once the Cu atom is pulled out from the surface, and this new state is 0.77 eV higher in energy than the hydrogen transfer. Nevertheless, the oxidaOon of the copper atom seems to be the most favorable ajer the introducOon of the pulse. These electronic characterisOcs give the system good energeOc stability that in combinaOon with the low-temperature experimental condiOons suggest that this configuraOon could be stable and observed in the STM image. From the geometrical point of view, the N-H, C-N, and C-O bond lengths are 1.02, 1.39, and 1.30 Å, respecOvely, these values being similar to those of GS, showing that these groups of molecules do not change. 7). Right, the calculated STM-ESQC image with two vacancies and two adatoms, and the depression is extended at the middle, but has less width than for vacancies alone (Figure 8 ).

The system with a double lijing of the copper atoms was also analyzed (Figure 9b), and this geometry is less stable than the one lijing, the calculated energy of which is 2.22 eV higher in energy than the ground state. In this case, the two copper atoms present a similar charge transfer toward the oxygen atoms of 0.32 e -, and the Cu-O bond lengths decreased from 2.14 to 1.90 Å as in the one vacancy case.

The calculated STM image of one vacancy and the copper adatom bound to the oxygen atom in Figure 9a also shows the dark depression at the side of the vacancy. The adatom does not seem to show a significant change of the contrast in comparison to the vacancy alone. In the same way, the calculated image of two vacancies at the boiom of the oxygen atoms that are bound to one copper adatom each, as shown in Figure 9b, exhibits the handle-fan-shaped dark feature. The comparison between the two cases, that is to say vacancies and vacancies + adatoms, does not allow us to definiOvely conclude regarding experimental images. However, DFT calculaOons would rather show an energeOcally favorable scenario with the formaOon of a vacancy generated by an STM voltage excitaOon with an adatom remaining bound to the molecule.

Conclusions

We performed STM imaging followed by applicaOon of voltage pulses on single indigo molecules adsorbed on Cu(111) at a low temperature, complemented by DFT and ESQC calculaOons. Experimental STM images showed an irreversible change of contrast with handle-fan-shaped dark depression at the middle of the molecule induced by two successive pulses of 2.4 and 3 V in lateral parts of the molecule. In order to explain the experimental results, two main scenarios are suggested. The first one considers proton transfer from the N to O atom induced by the voltage pulses. The TS barrier from the diketo to monoenol forms was calculated by DFT, and it reached about 0.65 eV for a single proton transfer and 0.28 eV for the final geometry. The permanent tautomerizaOon of the single and double proton transfer of N-H•••O ajer two consecuOve STM voltage pulses on the molecule is discarded because the comparison between experimental and ESQC calculated images disagrees, and the laier do not show a considerable change of contrast with respect to the indigo adsorbed on the Cu(111) reference. The second scenario is related to the extracOon of copper surface atoms by applying successive voltage pulses and creaOng two vacancies beneath the molecule. Here, experimental and calculated STM images show a remarkable qualitaOve agreement whether adatoms remain bound to the molecule or not. However, DFT calculaOons suggest that the adatoms stay close to the molecule and are oxidized by the oxygen atom with 0.32 e -transfer.

  Figure 3. (a) Quan-ta-ve informa-on about the switching mechanism: dura-on threshold from which a conforma-onal change is obtained as a func-on of the pulse voltage. The fiSng red curve has the form A•exp(-eV/B) with A = 1.26 × 10 15 ms and B = 83 meV. (b) Manipula-on rate as a func-on of tunneling current at 2.4 V. (c) STM images of two individual molecules a&er a single manipula-on. (d,e) Scan lines along the red and blue lines in (b), respec-vely.

Figure 4 .

 4 Figure 4. Adsorp-on energy of the indigo molecule with respect to its orienta-on above the Cu(111) surface.

Figure 5 .

 5 Figure 5. Top and side views of the molecule conforma-on during the proton transfer from the nitrogen atom to the oxygen atom. The energy barrier to cross the transient state (TS) is 0.65 eV with a final state at + 0.28 eV compared to the ini-al state.

Figure 6 .

 6 Figure 6. Calculated STM images and the corresponding models. (a) Adsorbed molecule, (b) a&er one proton transfer, and (c) a&er a double proton transfer (the scanning was displaced 2 Å to the right with respect to (a) and (b) STM-calculated images).

Figure 7 .

 7 Figure 7. Le&, the relaxed conforma-on of the molecule with a single surface vacancy beneath the oxygen atom. Middle, the corresponding calculated STM image. Right, the experimental STM image of a single molecule a&er a tension pulse (it was rotated for comparison). The pink arrow shows the vacancy and the appearance of a lateral dark spot between two bright spots with different intensi-es in the calculated image.

Figure 8 .

 8 Figure 8. Le&, the relaxed conforma-on of the molecule with two surface vacancies beneath the oxygen atoms. Middle, the corresponding calculated STM image. Right, the experimental STM image of a single molecule a&er a double tension pulse (it was rotated for comparison). Two bright spots are separated by a black channel.

Figure 9 .

 9 Figure 9. Le&, the calculated STM-ESQC image and the DFT atomic structure of the molecule with one vacancy and the copper adatom extracted from the Cu(111) first layer of the slab. The image shows the depression and almost indis-nguishable contrast with respect to the calculated image with a vacancy and without the adatom on the surface (Figure7). Right, the calculated STM-ESQC image with two vacancies and two adatoms, and the depression is extended at the middle, but has less width than for vacancies alone (Figure8).

Acknowledgments

C.J.V. acknowledges the project "Estudio de máquinas moleculares y transporte electrónico a la escala atómica" PAPIIT-UNAM IN116320. Part of this work was granted access to the HPC resources of CALMIP SupercompuOng Center under the allocaOon 2021-P0832.

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