Racemate Resolution of Alanine and Leucine on Homochiral Quartz, and Its Alteration by Strong Radiation Damage

Homochiral proteins orchestrate biological functions throughout all domains of life, but the origin of the uniform l-stereochemistry of amino acids remains unknown. Here, we describe enantioselective adsorption experiments of racemic alanine and leucine onto homochiral d- and l-quartz as a possible mechanism for the abiotic emergence of biological homochirality. Substantial racemate resolution with enantiomeric excesses of up to 55% are demonstrated to potentially occur in interstitial pores, along grain boundaries or small fractures in local quartz-bearing environments. Our previous hypothesis on the enhanced enantioselectivity due to uranium-induced fission tracks could not be validated. Such capillary tubes in the near-surface structure of quartz have been proposed to increase the overall chromatographic separation of enantiomers, but no systematic positive correlation of accumulated radiation damage and enantioselective adsorption was observed in this study. In general, the natural l-quartz showed stronger enantioselective adsorption affinities than synthetic d-quartz without any significant trend in amino acid selectivity. Moreover, the l-enantiomer of both investigated amino acids alanine and leucine was preferably adsorbed regardless of the handedness of the enantiomorphic quartz sand. This lack of mirror symmetry breaking is probably due to the different crystal habitus of the synthetic z-bar of d-quartz and the natural mountain crystals of l-quartz used in our experiments.


Introduction
Homochiral polymers must have emerged rather early during abiogenesis, most probably in a pre-RNA/DNA/metabolism world [1][2][3]. Because of enantiomeric crossinhibition, the synthesis of polypeptides and the self-replication of polynucleotides is repressed or even impossible in a racemic environment [4][5][6]. Consequently, homochirality or at least a substantial enantiomeric enrichment (ee) in the chemical building blocks of the first functional biopolymers should have developed early during chemical evolution. While the chronological details are still unclear, it is well accepted that chemical evolution did not occur suddenly; instead, a gradual transition from simple organic building blocks, including chiral molecules toward polymeric and supramolecular assemblies, has built the fundaments for self-assembling, self-sustaining interactive systems with emerging patterns that have ultimately evolved into what could be considered as living entities [7,8].
Abiotic synthesis of asymmetric molecules, such as amino acids or sugars, usually produces racemic compositions. Several mechanisms capable to entail molecular symmetry breaking leading to biomolecular homochirality on Earth, so-called chirogenesis, have been proposed and investigated. The most popular hypotheses comprise symmetry breaking by crystallization [9], selective adsorption of enantiomers on mineral surfaces [10][11][12], Life 2021, 11, 1222 3 of 15 chiral pattern, and a granulated enantiomorphic mineral with similar handedness (either d or l) may have an enantioselective separation effect on a percolating racemic solution of chiral molecules.
Here, we experimentally demonstrate significant racemate resolution in percolating solutions of racemic alanine and leucine on homochiral quartz sand with randomly orientated surfaces (i.e., without well-defined crystallographic orientation). Moreover, our results partly disprove the amplifying role of etched nuclear particle tracks according to the hypothesis of Hejl on the enantioselective adsorption potential of enantiomorphic quartz [20].

Characterization and Preparation of Homochiral Quartz Samples
Homochiral quartz sand was produced in two different ways. A first sample was obtained by crushing and sieving a synthetic z-bar quartz from a commercial provider (Hausen GmbH, Telfs, Austria). Such quartz crystals grow under hydrothermal conditions on so-called seed plates that are produced by cutting already existing z-bar crystals in thin plates of well-defined crystallographic orientation. A commercial laboratory will, thus, always duplicate z-bars of the same handedness. They are not twinned and strictly homochiral lor d-quartz. Our z-bar quartz crystal had a mass of 1473 g (Figure 1a). Prior to crushing and sieving, we determined its sense of optical rotation for light propagation parallel to the crystallographic c-axis. For this purpose, we produced a quartz plate by cutting the crystal with a diamond saw perpendicular to the crystallographic c-axis. The plate was ground with silicon carbide, and polished with diamond paste down to a grain size of 1 µm. Its thickness of 3.16 mm was measured with a vernier calliper with a precision of ±0.01 mm. The sense and angle of optical rotation of the transparent plate were determined with an optical polarimeter (Modular Compact Polarimeter MCP 150, Anton Paar GmbH, Graz, Austria). The temperature of the sample during the measurement was kept constant using Peltier elements. Clockwise optical rotation corresponds to d-quartz, and counter-clockwise optical rotation corresponds to l-quartz when rotation is observed in the sense opposite to the light propagation [37,38]. Our z-bar crystal was found to be d-quartz with an optical rotatory power of +21.68 • mm −1 at 20 • C.
The pieces of z-bar quartz were treated by alternating crushing and sieving, in order to produce sand with a grain size of 0.25-0.50 mm. The quartz pieces were first broken with a hammer on a steel plate, and then shortly crumbled with a disk mill for 5 s only. After each sieving step, the coarser fraction (>0.50 mm) was crushed and sieved again. Finally, we obtained about 400 g of homochiral d-quartz sand.
This quartz sand was washed with acetone, isopropanol, and double-distilled water, and dried in a drying chamber at 105 • C. The dried sand was purified with a Frantz magnetic separator to remove small steel particles that may have been produced by abrasion of the steel hammer or the mill. Afterwards, this purified sand was heated for 3 h at 500 • C in a muffle furnace to minimize biological contaminations. An aliquot of the glowed sand was then powdered in an agate mill and chemically analyzed by X-ray fluorescence, revealing the composition to be of 99.88% SiO 2 with minor contaminations of Fe and Cr, probably from abrasion of the steel hammer or the mill.
Unfortunately, we could not purchase synthetic l-quartz due to the method of production of z-bar quartz. We found only z-bar providers which offer either d-quartz or give no specification on the handedness of the quartz. Therefore, we decided to produce l-quartz sand by crushing and sieving crystallographically well-defined mountain crystals, which had grown under natural conditions. First, we evaluated 20 natural quartz crystals from an open tension joint in Madagascar (Figure 1b). These transparent crystals of fairly similar size and habitus had a total mass of 507 g with individual masses between 13.7 and 40.1 g, and individual lengths ranging from 5 to 9 cm. The longitudinal axes were found to be the crystallographic c-axis. Ten of these crystals were rejected because they were either twinned or intergrown with smaller quartz crystals. The remaining ten crystals were found to be single crystals without twinning. They had a total mass of 227 g and were chosen for further treatment. similar size and habitus had a total mass of 507 g with individual masses between 13.7 and 40.1 g, and individual lengths ranging from 5 to 9 cm. The longitudinal axes were found to be the crystallographic c-axis. Ten of these crystals were rejected because they were either twinned or intergrown with smaller quartz crystals. The remaining ten crystals were found to be single crystals without twinning. They had a total mass of 227 g and were chosen for further treatment. These single crystals were cut with a diamond saw perpendicular to their c-axis, to produce plates with a thickness of about 3 mm ( Figure 2). These plates were ground with silicon carbide and polished with diamond paste, in a similar way as for the plate from the z-bar. Six crystals were found to be l-quartz with optical rotatory powers between 21.26 and −22.74° mm -1 at 20 °C (Table 1). The small variations of the optical rotatory power are probably due to a slight imprecision of the cutting angle relative to the c-axis (ca. 90° +/− 2°). The other four crystals were found to be d-quartz with a positive optical rotatory power and, therefore, rejected. The material from the six l-quartz crystals was treated by alternating crushing and sieving, washed, purified, and glowed in a similar way as the z-bar sample. Finally, we obtained 44 g of homochiral l-quartz sand with a grain size of 0.25-0.50 mm. The specific surface area (SSA) of both kinds of quartz sand (l and d) can be deduced from their quasi equigranular grain size (0.25-0.5 mm), using published data of granulometric investigations [39]. Recovered amino acid quantities normalized to the specific surface area were calculated with a SSA of 60 m 2 g −1 and a porosity of 40% that is typical for equigranular sand with angular grains. These single crystals were cut with a diamond saw perpendicular to their c-axis, to produce plates with a thickness of about 3 mm ( Figure 2). These plates were ground with silicon carbide and polished with diamond paste, in a similar way as for the plate from the z-bar. Six crystals were found to be l-quartz with optical rotatory powers between 21.26 and −22.74 • mm −1 at 20 • C ( Table 1). The small variations of the optical rotatory power are probably due to a slight imprecision of the cutting angle relative to the c-axis (ca. 90 • ± 2 • ). The other four crystals were found to be d-quartz with a positive optical rotatory power and, therefore, rejected. The material from the six l-quartz crystals was treated by alternating crushing and sieving, washed, purified, and glowed in a similar way as the z-bar sample. Finally, we obtained 44 g of homochiral l-quartz sand with a grain size of 0.25-0.50 mm. The specific surface area (SSA) of both kinds of quartz sand (l and d) can be deduced from their quasi equigranular grain size (0.25-0.5 mm), using published data of granulometric investigations [39]. Recovered amino acid quantities normalized to the specific surface area were calculated with a SSA of 60 m 2 g −1 and a porosity of 40% that is typical for equigranular sand with angular grains.

Thermal Neutron Irradiation of Homochiral Quartz Sand Mixed with Uraniferous Zircon Powder
Synthetic z-bar quartz nor mountain quartz contain any uraniferous inclusions. In order to produce fission tracks on the fractured surfaces of quartz grains, aliquots of the homochiral sand samples were mixed with uraniferous zircon powder. For this purpose, zircon sand from a placer deposit in New South Wales (Australia) was ground to fine powder in an agate mill. The chemical composition of the zircon powder was analyzed by X-ray fluorescence using a Bruker Pioneer S4 crystal spectrometer at the Department for Chemistry and Physics of Materials (University of Salzburg). Obtained net count rates on single X-ray lines were recast into concentration values (wt% and ppm) based on an in-house calibration routine that involves measurements of ca. 30 international geo-standards (USGS and GSJ). The calibration was based on the Bruker AXS software SPECTRAplus FQUANT (v1.7) which corrects absorption, fluorescence, and line overlap effects. Concentrations of U and Th were 0.049 and 0.018 wt%, respectively (Table 2). , 11, x FOR PEER REVIEW Figure 2. Some non-twinned mountain crystals with ca. 3-mm-thi ular to the crystallographic c-axis. ble 1. Polarimetric measurements on quartz slices perpendicular to the crystallographic c-a were found to be l-quartz. The corresponding crystals were used to produce l-quartz sand 3.17 not measurable 1 Figure 2. Some non-twinned mountain crystals with ca. 3-mm-thick sheets that were cut perpendicular to the crystallographic c-axis. Table 1. Polarimetric measurements on quartz slices perpendicular to the crystallographic c-axes. Slices 4, 7, 11, 12, 14 and 20 were found to be l-quartz. The corresponding crystals were used to produce l-quartz sand.  The zircon powder was glowed in a muffle furnace at 500 • C for 3 h to reduce the risk of biological contaminants, such as natural L-amino acids. Afterwards, it was mixed with aliquots of either homochiral dor l-quartz sand. Thus, 125 g of d-quartz sand were mixed with 25 g of zircon powder, and 12.5 g of l-quartz sand were mixed with 2.5 g of zircon powder corresponding to an identical mass ratio of 88.33% quartz and 16.67% zircon. Both mixtures were filled in polyethylene tubes. When the surface of quartz grains is completely covered by zircon powder with a uranium concentration of 0.049 wt%, a thermal neutron fluence of 5 × 10 15 neutrons cm −2 will induce a fission-track density of about 10 7 tracks per cm 2 onto the quartz surface [40]. The addition of zircon powder alone would not lead to enough fission tracks within an adequate time scale as the spontaneous fission of 238 U is a very rare event. It would take several million years to produce a significant quantity of fission tracks on the surface of quartz. We, thus, induced the fission of 235 U by subsequent neutron irradiation of the mixed quartz sand/zircon powder.

Quartz Slices Normal to C Axis
Thermal neutron irradiation of the filled tubes was performed in the TRIGA Mark-II Reactor of the TU Vienna (Atominstitut der TU Vienna, Austria). This research reactor of the swimming pool type has a nominal power of 250 kW. The reactor is controlled by three control rods which contain boron carbide as an absorber material. The thermal neutron flow rate is in the order of 10 13 cm −2 s −1 in a central position. Desired neutron fluences of about 5 × 10 15 neutrons cm −2 were obtained after an irradiation time of about 1 h, with irradiation position at the border of the reactor core. After a decay time of two months, the capsules possessed total activities of up to 200 kBc-mainly due to the activities of 46 Sc, 95 Zr, 95 Nb, 181 Hf, and 175 Hf. Besides uranium fission tracks, the samples must have accumulated a rather high radiation damage by beta decay and gamma radiation.

Sand Etching and Subsequent Infiltration with Aqueous Solutions of dl-alanine and dl-Leucine
Irradiated dand l-quartz sand, as well as nonirradiated dand l-quartz, were etched with 10% HF. This etching transforms superficial latent fission tracks into capillary tubes [24,40]. Identical etching conditions were applied to the nonirradiated dand lquartz samples to avoid any differences in the adsorption properties caused by radiation damage vs. etching, allowing for a better comparison of the enantioselective adsorption with the irradiated samples.
The sand fractions were filled in plastic Erlenmeyer flasks together with 10% HF and were etched at 20 • C for 2 h under permanent agitation on a shaking table. Afterwards, the sand was rinsed several times with double-distilled water. A second etching with 10% HCl was applied for 20 min, to dissolve potential fluorine components. While repeatedly rinsing the etched sand with double-distilled water, the zircon powder was removed by decantation. Finally, the sand was desiccated at 95 • C in a drying chamber.
Subsequent adsorption experiments of both d-(synthetic) and l-quartz (natural), respectively, with diluted aqueous solutions of individual racemic amino acids (DL-Ala and DL-Leu) were conducted as follows. The glassware (Erlenmeyer flasks, beakers, watch glasses, and volumetric flasks) as well as needles, spatulas, and aluminum foil were cleaned with acetone and isopropanol, and then glowed for 3 h in a muffle furnace at 500 • C. Solutions of racemic amino acids with concentrations of 10 −3 M were prepared in volumetric flasks. This amino acid concentration was chosen to allow for accurate quantitation of recovered amino acids. Glass funnels were placed on the Erlenmeyer flasks, and conically folded aluminum foils were placed in the funnels. These foils were checked for impermeability prior to utilization. First, 4 mL of 10 −3 M racemic amino acid solutions (either DL-Ala or DL-Leu) were dropwise added to the funnels, followed by homochiral quartz sand (either d-quartz or l-quartz) up to the liquid surface. This procedure avoided the formation of air bubbles, and the interstices between the sand grains became entirely filled with the racemic solutions. Eight funnels were prepared in this way, i.e., DL-Ala + nonirradiated d-quartz (1), DL-Ala + irradiated d-quartz (2), DL-Leu + nonirradiated d-quartz (3), DL-Leu + irradiated d-quartz (4), DL-Ala + nonirradiated l-quartz (5), DL-Ala + irradiated l-quartz (6), DL-Leu + nonirradiated l-quartz (7), and DL-Leu + irradiated l-quartz (8). In addition, four blanks with nonirradiated dand l-quartz as well as irradiated dand l-quartz were filled with 4 mL of double-distilled water.
The funnels filled with DL-amino acids and homochiral sand were covered with watch glasses and were allowed to stand for 72 h at 20 • C. Afterwards, a clean needle-with a thickness smaller than the grain size of the sand-was inserted vertically in the sand body and pushed downward to the bottom of the foil to perforate it. Then, 8 mL of doubledistilled water was dropwise added to the top of the sand. In this way, the aqueous solution containing the diluted amino acid depleted in one adsorbed enantiomer was rinsed out from the pores between the sand grains and recovered in a small beaker and then filled in vials with silicon screw caps.
In a previous experiment, thin glass tubes were used instead of funnels. The lower end of these tubes was closed with aluminum foil and silicone. Mixed solutions containing 10 −3 M DL-Ala and 10 −3 M DL-Leu were used in the following manner: DL-Ala + DL-Leu + nonirradiated d-quartz (9) and DL-Ala + DL-Leu + irradiated d-quartz (10). In addition, two blanks with nonirradiated and irradiated d-quartz, both with 4 mL of double-distilled water, were prepared.

GC×GC-TOFMS Analysis-Sample Preparation and Derivatization
All sample-handling glassware was wrapped in aluminum foil and heated at 500 • C for 3 h prior to usage. Eppendorf tips ® were sterile, and the water used for standard solutions, reagent solutions, and blanks was prepared with a Direct-Q ® water purification system (<3 ppb total organic carbon). All solvents, reagents, and amino acid standards were purchased from Sigma-Aldrich and stored according to the respective instructions. Specific volumes of the eluate of each infiltration experiment described above were transferred into a 1 mL of Reacti-Vial TM and dried under a gentle flow of nitrogen. These volumes were chosen to reach final concentrations that resulted in adequate peak intensities and areas for reliable enantiomeric excess quantification. The chosen volumes were as follows: 75 µL of (1) DL-Ala + nonirradiated d-quartz, 100 µL of (2) DL-Ala + irradiated d-quartz, 75 µL of (3) DL-Leu + nonirradiated d-quartz, 100 µL of (4) DL-Leu + irradiated d-quartz, 75 µL of (5) DL-Ala + nonirradiated l-quartz, 200 µL of (6) DL-Ala + irradiated l-quartz, 75 µL of (7) DL-Leu + nonirradiated l-quartz, 200 µL of (8) DL-Leu + irradiated l-quartz, 50 µL of (9) DL-Ala + DL-Leu + nonirradiated d-quartz, and 50 µL of (10) DL-Ala + DL-Leu + irradiated d-quartz.
All samples were re-dissolved in 50 µL of 0.1 M HCl, and individually derivatized to form N-ethylchloroformate heptafluorobutyryl esters (ECHFBE) by adding first 6 µL of pyridine and 19 µL of heptafluorobutanol followed by 5 µL of ethylchloroformate. The reaction mixtures were vortexed for 10 s, and the derivatives were extracted and transferred into GC vials by adding 50 µL of chloroform containing methyl myristate as an internal standard at a concentration of 10 −6 M. Three aliquots of each incubated quartz sample (1) to (10) were derivatized, and each derivatized sample was analyzed (1 µL) as triplicates by enantioselective two-dimensional gas chromatography coupled to a time-of-flight mass spectrometer GC×GC-TOFMS [41,42], i.e., each quartz sample was analyzed nine times by preparing three derivatized aliquots with n = 3 to obtain sufficient replicates for the statistical analysis.
Racemic standard solutions of alanine and leucine-identical to the standards used for the adsorption experiments-with approximately the same concentration than the final quartz samples were used to determine the enantiomeric bias of the commercially available racemic standards. These reference standards were injected three times in between each triplicate injections of the three aliquots of the same quartz infiltration experiment to follow any chromatographic bias over time. In total, nine injections of the alanine and leucine reference standard, respectively, were performed for one adsorption sample, i.e., three individually derivatized reference standards with n = 3 injections. To ensure that no contamination occurred during the analytical protocol, nonirradiated and irradiated dand l-quartz blanks were separately dried and derivatized following the same procedure, as described for the above samples and analyzed (1 µL) by GC×GC-TOFMS. The volume of the blank aliquots was the same as for the corresponding infiltration experiment. No contamination was observed.

GC×GC-TOFMS Analysis-Instrumental Conditions
The enantioselective multidimensional analysis was carried out by a GC×GC Pegasus instrument For the analyses of samples containing alanine, the temperature of the primary oven was held at 40 • C for 1 min, increased to 80 • C at a rate of 10 • C min −1 , and held for 5 min, followed by an increase to 120 • C at 2 • C min −1 and finally to 190 • C at 10 • C min −1 and held for 6 min. The temperature of the secondary oven was held at 65 • C for 1 min, increased to 105 • C at a rate of 10 • C min −1 with a 5-min hold time, followed by an increase to 145 • C at 2 • C min −1 and, finally, to 190 • C at 8 • C min −1 .
For the analyses of samples containing leucine, the temperature of the primary column was held at 40 • C for 1 min, then increased to 105 • C at a rate of 10 • C min −1 and held for Life 2021, 11, 1222 9 of 15 5 min, followed by an increase to 140 • C at 2 • C min −1 and finally to 190 • C at 10 • C min −1 and held for 5 min. The secondary oven used the same temperature program with a constant temperature offset of 20 • C.
For the mixed alanine-leucine samples, the temperature of the primary column was held at 40 • C for 1 min, increased to 80 • C at a rate of 10 • C min −1 and held for 5 min, then by an increase to 170 • C at 2 • C min −1 , and finally to 190 • C at 10 • C min −1 and held for 5 min. The secondary oven used the same temperature program with a constant temperature offset of 25 • C. The modulator used a temperature offset of 40 • C compared to the primary oven temperature and a modulation period of 5 s was applied to all samples.

Enantiomeric Excess Determination
The enantiomeric excess ee which expresses the excess of one enantiomer over the other is defined as ee = (E 1 − E 2 )/(E 1 + E 2 ) where E 1 and E 2 are the quantities of the enantiomers. In practice, ee is often quoted as a percentage, defined as %ee = 100 × (E 1 − E 2 )/(E 1 + E 2 ). Thus, a racemate is expressed as ee = 0%. While amino acid standards are commercially available as racemic mixtures, their expected equimolar ratios determined by chromatographic techniques are generally unequal to 0% (ee = 0%) due to (i) intrinsic manufacture effects of the racemic standards itself; (ii) differential instrument responses for each enantiomer such as stereoselective signal suppression; or (iii) erroneous chromatographic quantitation caused by the co-elution of chemical impurities with only one enantiomer spuriously increasing peak areas, poor enantioresolution (R S < 1.5), or peak tailing. To best account for any non-racemic composition measurements for analytes which are, in fact, racemic and vice versa, the non-zero ee of the racemic alanine and leucine standard, respectively, were determined as follows: where A is the peak area of the GC×GC signal and AA is the corresponding amino acid.

Results
A summary of the results of the GC×GC-TOFMS analyses of recovered interstitial liquids is given in Table 3 and Figure 3. Both amino acids, Ala and Leu, were enriched in the D-enantiomer (ee L < 0), regardless of the handedness of the quartz sand. Moreover, the original hypothesis that capillary tubes on the quartz surface, produced by etching of uranium fission tracks, may enhance the overall enantioselective adsorption is partly disproved by our experiments. Table 3. Enantiomeric excesses %ee L of alanine and leucine after adsorption on nonirradiated and irradiated enantiomorphic dand l-quartz. The recovered amino acid quantity is specified for each adsorption experiment as well as the recovered quantity normalized to the estimated total specific surface area of quartz (954 m 2 ).  Comparing the ratios of the peak areas of the amino acid and the internal standard (AAA/AIS) before and after the adsorption experiments on enantiomorphic quartz allowed us to evaluate the adsorption efficiencies of each experiment (Table 3). Starting from an initial amino acid concentration of 10 -3 M (sum of both enantiomers), the recovered quantities were found to range from 1.6 × 10 -4 M for the experiments using irradiated l-quartz -4 First, we examined the absolute values of %ee L , regardless of their algebraic sign. We measured six %ee L values for Ala, and additional six %ee L values for Leu. The |%ee| for Ala in six independent experiments ranges from 1.1 to 15.2%. These %ee values are significantly above the 3σ standard deviations (n = 3). They demonstrate that substantial racemate resolution can be expected in interstitial pores, along grain boundaries or small fractures in a local quartz-bearing environment. The |%ee| for Leu in six independent experiments ranges from 3.5 to 55.6%. These values are even higher than those for Ala. They substantiate the foregoing conclusion that random quartz fractures may produce significant racemate resolution on a local scale. This statement holds true for polygranular quartz occurrences with a chiral enrichment of either dor l-quartz.

Amino Acid %ee L -d-Quartz %ee L -l-Quartz
Second, we examined the influence of thermal neutron irradiation, induced fission tracks, and radiation damage, in general, on the observed ee values. In the case of Ala, the mean %ee L value in the three experiments using nonirradiated quartz is −4.0% (spread between −2.3% and −6.8%), and the mean %ee L value with irradiated quartz is −9.7% (spread between −1.1 and −15.2%). These results indicate an overall stronger enantioselective adsorption of L-Ala on irradiated quartz compared with nonirradiated quartz. One exception is the experiment of the pure racemic alanine solution absorbed on d-quartz were the %ee L of −2.92% for nonirradiated quartz decreased to −1.09% for irradiated quartz. Thus, the results for Ala are ambiguous and will be discussed later.
In the case of Leu, the mean %ee L value of three independent experiments with nonirradiated quartz is −28.8% (spread between −6.8 and −55.6%), and the mean %ee L value with irradiated quartz is −9.0% (spread between −3.5 and −12.6%). The L-enantiomer of Leu adsorbs distinctly stronger on nonirradiated quartz than on irradiated quartz, without any exception. In all experiments with either dor l-quartz, the adsorption of the L-enantiomers on nonirradiated quartz was about two or even four times higher than on irradiated quartz. Strong radiation damage clearly reduces enantioselective adsorption of L-Leu onto the quartz surface or in any superficial structures. Thus, the hypothesis of Hejl is disproved for leucine [20]. This observed reduction in enantioselective adsorption on irradiated quartz might be due to metamictization of the crystal lattice. Radiation dose-dependent destruction of the crystalline structure, up to amorphous consistency, is well known for zircon [43]. Atomic scale observations by transmission electron microscopy (TEM) have shown that the core zone of latent fission tracks in zircon is amorphous [44]. If this amorphous domain is not completely removed by etching, the walls of the fission track capillary tube will keep a certain metamictization after etching. Although the atomic structure of latent fission tracks in quartz has not yet been investigated, a superficial metamictization by heavy ion bombardment from adjacent uraniferous minerals, such as zircon, cannot be excluded. This kind of radiation damage would explain the alteration of enantioselective adsorption properties.
Comparing the ratios of the peak areas of the amino acid and the internal standard (A AA /A IS ) before and after the adsorption experiments on enantiomorphic quartz allowed us to evaluate the adsorption efficiencies of each experiment (Table 3). Starting from an initial amino acid concentration of 10 −3 M (sum of both enantiomers), the recovered quantities were found to range from 1.6 × 10 −4 M for the experiments using irradiated l-quartz up to 4.3 × 10 −4 M for the nonirradiated dand l-quartz samples. Thus, less than 15% to 25% of the amino acids were recovered from irradiated land d-quartz, respectively, whereas almost 50% of amino acids were desorbed from the nonirradiated enantiomorphic quartz samples when using individual amino acid solutions. While these results indicate an increased adsorption affinity of irradiated over nonirradiated enantiomorphic quartz due to irradiation-induced fission tracks-that was not found to directly correlate with enhanced enantioselective adsorption, no significant adsorption selectivity between alanine and leucine was observed. The results of the competitive adsorption experiments are less obvious with very similar adsorption affinities of the irradiated and nonirradiated d-quartz but inverse trends in enantioselectivity for alanine and leucine. While the %ee L of alanine significantly decreased to −15% on irradiated d-quartz, i.e., enhanced adsorption of D-alanine on irradiated d-quartz, D-leucine adsorbed more efficiently on nonirradiated d-quartz compared to irradiated d-quartz. It is currently uncertain whether these differences in enantioselectivity of alanine and leucine in the combined mixture are due to systematic or pure random effects. We note, however, that, in preparation of the incubation experiments of the alanine-leucine mixtures, the quartz sand was filled using thin tubes, whereas small funnels were used to prepare the adsorption experiments of the individual amino acids. This may have eventually entailed a difference in the packing density of the grains and thus differences in the cumulative pore volume. The grain packing in the funnels corresponds much better to the textural conditions in natural sediments and will be chosen as a preferred experimental design for future adsorption experiments. Further competitive enantioselective adsorption experiments are required to study the profound impact of intimate binding between enantiomorphic quartz and amino acids of different size, charge, and polarizability, as well as mutual effects between different molecular species during competitive adsorption.

Discussion
In our experimental investigation, the measured %ee values of both amino acids, Ala and Leu, were always enriched in the D-enantiomer (L-%ee < 0). Originally, we had assumed that sand grains have a nearly isometric form corresponding approximately to a sphere. With regard to the crystal lattice, all possible spatial orientations of crystalline planes occur with similar likelihood on the surface of the sphere. Even irregular planar orientations which cannot be denominated by Miller-Bravais indices would have the same frequency than any other plane. Indeed, sand grains of aeolian dunes, beach sands, or fluvial sediments are often well rounded [45]. Most quartz grains exhibit a spherical or ellipsoidal shape. If two monocrystalline quartz grains consist of quartz with opposite handedness (d and l, respectively), their overall chiral adsorption properties of their surfaces are expected to produce bulk-ee with opposite handedness on both grain surfaces. This assumption is still valid when a quasi-infinite number of homochiral grains with irregular forms is considered-provided that these irregular forms have no preferential orientation relative to the crystal lattice. Quartz has no preferential cleavage planes and mainly produces conchoidal fragments at rupture. This well-known fact seems to support the foregoing assumption.
However, the interstitial liquid of both enantiomorphic quartz crystals was always enriched in the D-enantiomer and no mirror effect was observed. Moreover, both L-Ala and L-Leu enantiomers tend to adsorb much stronger on l-quartz (natural) than on d-quartz (synthetic z-bar). We suppose that this phenomenon is due to different frequency distributions of the crystallographic orientation of fracture planes in the z-bar and the natural mountain quartz. The longitudinal axis of the z-bar is perpendicular to the crystallographic c-axis, while the longitudinal axis of the mountain crystals is parallel to the c-axis (Figure 4). These differences in the crystal habitus may have caused a dissimilar behavior at rupture. When a longitudinal homogenous material without preferential cleavage is broken with a hammer, one would expect that the predominant factures are mainly perpendicular to the longitudinal axis. These first fractures may have influenced the further ruptures in course of crumbling with the disk mill.
With regard to future adsorption experiments, we suggest and intend the use of dand l-quartz with similar habitus for the production of homochiral sand-i.e., natural quartz crystals from the same location, with similar size and form. By this procedure, similar frequency distributions of crystallographic fractures orientations in both dand l-quartz could be obtained. Random effects will be minimized, and the resulting bulk adsorption on the grain surfaces of dor l-quartz will produce rather 'symmetrical' ee with opposite sign.  With regard to future adsorption experiments, we suggest and intend the use o and l-quartz with similar habitus for the production of homochiral sand-i.e., natu quartz crystals from the same location, with similar size and form. By this procedure, s ilar frequency distributions of crystallographic fractures orientations in both d-and quartz could be obtained. Random effects will be minimized, and the resulting bulk sorption on the grain surfaces of d-or l-quartz will produce rather 'symmetrical' ee w opposite sign.
Overall, leucine showed higher racemate resolution on quartz with a maximum % of 55.6% compared with alanine in our experiments. Molecular size-related effects may responsible for the observed trend and future investigations on various individual am acid solutions covering different molecular sizes or branching, and different functio groups (aliphatic, aromatic, acidic, basic, etc.) are required to study size-and polarizati related effects on the enantioselective adsorption behavior. Moreover, additional comp itive adsorption experiments are required for investigating the interplay of amino a mixtures and the enantioselective adsorption forces of enantiomorphic minerals to illu nate prebiotically plausible systems.

Conclusion
The enantioselective adsorption of alanine and leucine with %ee values ranging fr 1% to 55% was observed to occur on homochiral quartz sand. The L-enantiomer of b investigated amino acids was preferably adsorbed regardless of the handedness of enantiomorphic quartz. We currently explain the lack of mirror symmetry breaking in o experiments with the different crystal habitus of the synthetic z-bar of d-quartz and natural mountain crystals of l-quartz. Future experiments using d-and l-quartz with s Overall, leucine showed higher racemate resolution on quartz with a maximum %ee of 55.6% compared with alanine in our experiments. Molecular size-related effects may be responsible for the observed trend and future investigations on various individual amino acid solutions covering different molecular sizes or branching, and different functional groups (aliphatic, aromatic, acidic, basic, etc.) are required to study size-and polarization-related effects on the enantioselective adsorption behavior. Moreover, additional competitive adsorption experiments are required for investigating the interplay of amino acid mixtures and the enantioselective adsorption forces of enantiomorphic minerals to illuminate prebiotically plausible systems.

Conclusions
The enantioselective adsorption of alanine and leucine with %ee values ranging from 1% to 55% was observed to occur on homochiral quartz sand. The L-enantiomer of both investigated amino acids was preferably adsorbed regardless of the handedness of the enantiomorphic quartz. We currently explain the lack of mirror symmetry breaking in our experiments with the different crystal habitus of the synthetic z-bar of d-quartz and the natural mountain crystals of l-quartz. Future experiments using dand l-quartz with similar habitus to minimize random effects are required to conclude on the possible mirror symmetry breaking stimulus through amino acid adsorption on enantiomorphic minerals with opposite handedness.
Despite the absence of %ee values of the opposite sign, there are consistent trends in our collected data, such as natural l-quartz, showed stronger enantioselective adsorption affinities than synthetic d-quartz for both amino acids. Moreover, strong radiation damage clearly reduced the enantioselective adsorption of leucine, while no clear conclusion can be drawn for the results on alanine regarding nonirradiated vs. irradiated quartz. Finally, the Data Availability Statement: All data analyzed during this study are included in this published article. The datasets generated or analysed during the current study are available from the corresponding authors upon request.