evaluating experimental artifacts in hydrothermal prebiotic synthesis experiments

EVALUATING EXPERIMENTAL ARTIFACTS IN HYDROTHERMALPREBIOTIC SYNTHESIS EXPERIMENTS�

ALEXANDER SMIRNOV∗ and MARTIN A. A. SCHOONENNASA Astrobiology Institute and Department of Geosciences, State University of New York, Stony

Brook, NY, U.S.A. (∗ author for correspondence, e-mail: [email protected],Fax: 1 631 632 8240)

(Received 17 July 2002; accepted in final form 24 September 2002)

Abstract. Control experiments with ultra pure deionized water were conducted to evaluate theorganic contamination in hydrothermal prebiotic experiments. Different combinations of reactionvessel material, sampling tubing and stirring were tested and the amounts of organic contaminantsdetermined. All tested types of polymer tubing were proven to introduce organic contaminants (form-ate, acetate and propionate ions) into the reacting solution. Stainless steel has a catalytic effect on thedecomposition of formate, consistent with earlier work at high temperatures and pressures.

Keywords: acetate, contamination, formate, hydrothermal, prebiotic synthesis

1. Introduction

Prebiotic synthesis experiments often yield organic compounds in low concentra-tions. Moreover, reaction pathways involving organic compounds are often verycomplicated with intermediate products at low concentrations. To study these com-plex systems it is often necessary to determine the solution composition at regulartime intervals. This can be accomplished by withdrawal of solution aliquots (Mc-Collom and Seewald, 2001), in situ analysis (Maiella and Brill, 1998), or by usinga closed microsampling system coupled with direct analysis (Schoonen and Xu,2001). The latter system has the advantage that heterogeneous systems can bestudied over long periods of time, with frequent sampling, and without significantlyaffecting the solid/solution ratio. However, such a system does require the use of acomplex plumbing system. Polymer tubing is often used in flow-through systems orclosed sampling systems. Polymer tubing is chosen mostly because of its flexibility,durability, thermal stability and chemical inertness. However, at higher temperat-ures (>100 ◦C) it may introduce organic contaminants into the reacting solution.Hence, it is imperative to conduct control experiments to determine contaminantlevels.

In this study, experiments employing various combinations of polymer, stainlesssteel and titanium tubing were conducted to evaluate which combination of mater-

� Paper presented at the Astrobiology Science Conference, NASA Ames Research Center, 7–11April, 2002.

Origins of Life and Evolution of the Biosphere 33: 117–127, 2003.© 2003 Kluwer Academic Publishers. Printed in the Netherlands.

118 A. SMIRNOV AND M. A. A. SCHOONEN

ials yields the lowest contaminant levels. Catalytic effect of the reaction vesselmaterial was also investigated in a preliminary experiment.

This study was conducted as a part of research into stability/reactivity of lowconcentration solutions of sodium formate (HCO2Na) at hydrothermal conditions.Formate ion (HCOO−), the hydration product of carbon monoxide and water, couldhave been a common aqueous species on the early Earth (Penfield, 2001; Kasting,1990; Van Trump and Miller, 1973). Formate/formic acid decomposition reactionsinclude: (1) disproportionation into formaldehyde, water and carbon dioxide (Mai-ella and Brill, 1998); (2) oxidation to bicarbonate (Shende and Mahajani, 1997)and (3) dehydration to carbon monoxide and water (Barham and Clark, 1951).Bicarbonate ion may then further react to form acetate and/or propionate ions.Formaldehyde, acetate and propionate would be synthetically useful compoundson the early Earth.

HCO2H → 12CO2 + 1

2H2CO + 122H2O (1)

HCO−2 + O2 → HCO−

3 (overall oxidation) (2)

HCO2H → CO + H2O . (3)

1.1. EXPERIMENTAL PROCEDURES

Two sets of experiments were conducted using two different reaction vessels: (1) a316 stainless steel (SS) AE MagneDrive II� Autoclave (Figure 1); (2) a custom-made 6AL/4V titanium vessel (Figure 2). Both vessels were equipped with quartzglass inserts; working volume of reaction vessels was ∼1 L (MagneDrive) and∼500 mL (Ti-vessel). 6AL/4V titanium vessel was pre-treated by oxidative heatingat ∼300 ◦C for 4 hr to form a protective, chemically inert Ti-O layer (Ulmer andBarnes, 1987; Vaquila et al., 1999). Differences in chemical composition of 316SS and 6AL/4V Titanium are summarized in Table I.

Both reaction vessels were equipped with multiple ports enabling liquid/gasinput, liquid/gas sampling, pressure relief and temperature monitoring. Materialsused include: (P)TFE – (poly)tetrafluoroethylene; PEEK – polyetheretherketoneand Grade #2 titanium. Stirring was performed using an internal motorized TFEstirrer (MagneDrive) or an orbital shaker (Ti-vessel).

All experiments were conducted using DI UF/UV water in a closed system withN2 atmosphere (∼400 psi). Nitrogen gas was checked for purity using gas chroma-tography prior to pressurizing the vessel. The temperature range of experimentsvaried between 136–220 ◦C with fluctuations within ±3 ◦C.

Experiments lasted between 5–24 days. For sample collection, 1/16′′ coiledPEEK tubing submerged in a water bath at ∼0 ◦C was used. This provided in-stant quenching of the sample and minimized the loss of sample (and subsequent

ARTIFACTS IN HYDROTHERMAL PREBIOTIC SYNTHESIS EXPERIMENTS 119

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Figure 2. A custom-made 6AL/4V titanium reaction vessel stirred by an orbital shaker.

change in concentration) through evaporation. To avoid ‘dead volume’ contamina-tion of samples, the sampling tubing was dismantled and thoroughly cleaned beforecollecting the next sample. At each sample period, duplicate aliquots were collec-ted. All samples were collected into 5 mL Dionex PolyVials� . After collection,samples were stored at ∼1 ◦C to inhibit the spontaneous decomposition reactionsof carboxylic ions (Penfield, 2001). In control experiments, Dionex PolyVials� didnot introduce any contamination into the sample.

Liquid samples were analyzed for the presence of total formate (HCOO−) andacetate (CH3COO−) using DIONEX DX-500� Ion Chromatograph with 4 mmAS4A-SC IonPac� columns (5 mM Na2B4O7 eluent) and 100 µL sample loop.The presence of propionate (CH3CH2COO−) was detected, but not quantified.Formaldehyde (H2CO) was analyzed colorimetrically using a HACH-DR2000�

spectrophotometer with the MBTH (3-methyl-2-benzothiazoline hydrazone) me-thod. Gas samples were analyzed on single column SRI 8610C� Gas Chromato-graph with FID detector and 4% CarboWax� column.

The role of the vessel material as a potential catalyst was evaluated by determin-ing the stability of low-concentration (250–280 µmol) solutions of sodium formate.


TABLE I

Comparison of chemical composition of 316 stainless steel, grade 2and 6AL/4V titanium

Element 316 Stainless Grade 2 6AL/4V (Gr. 5)

(%) steel titanium titanium

(UNS S31600) (UNS R50400) (UNS R65400)

C 0.08 0.1a –

Fe 65 0.3a 0.5a

H – 0.015a –

N – 0.03a –

O – 0.25a 0.2a

Ti – 99.2 90

Al – – 6

V – – 4

Cr 17 – –

Mn 2 – –

Mo 2.5 – –

Ni 12 – –

P 0.045 – –

S 0.03 – –

Si 1 – –

a Denotes the maximum content; UNS number denotes Uni-fied Numbering System for Metals and Alloys. Compiled fromwww.matweb.com

Sodium formate solution was prepared from 99% pure Na-formate salt (NaHCO2)and DI UV/UF water. The pH of starting formate solutions was ∼5.8.

2. Organic Contamination During Experiments

Figure 3 and Table II depict the evolution of originally ultra pure UF/UV DI waterin the stainless steel autoclave in contact with TFE tubing, stirring rod and stainlesssteel thermocouple sheath covered with PTFE heat shrink tubing. After 48 hr ofreaction time, one can observe the presence of formate, propionate and acetatepeaks with their subsequent increase in concentrations over the reaction time. Thisindicates leaching of low-molecular weight organic compounds (and/or precursorsfor their formation) from polymers in contact with the solution and/or its gaseousphase. Organic compounds released into solution may subsequently react and thesolution may evolve into a complex mixture of organic compounds. Concentrationsof formaldehyde, a common formate decomposition product, in our experiments


Figure 3. Experiment 5/2002. Contamination occurring in blank experiment in the 316 stainless steelreaction vessel. For clarity, the chromatogram traces are offset along the y-axis.

TABLE II

Evolution of organic contamination instainless steel vessel with polymertubing in contact with the solution.Formate and acetate concentrations arein µmol L−1. Experiment 5/2002

Day Formate Acetate

0 0 0

2 5 9

3 5 15

5 5 14

7 5 13

9 5 13

10 6 14

12 7 17

18 7 19


TABLE III

Summary of all control experiments with the outline of materials used. Formate and acetateconcentrations shown are the highest observed

Vessel Tubing Stirring Thermocouple Formate Acetate

sheath (µ mol) (µ mol)

316 SS TFE Mechanical (TFE) PTFE covered 316 SS 6.83 19.26

6AL/4V Ti Gr #2 Ti, PEEK No stirring PTFE covered 316 SS 13.14 22.5

6AL/4V Ti Gr #2 Ti, PEEK Orbital shaking Gr #2 Ti 4.3 16.62

6AL/4V Ti Gr #2 Ti Orbital shaking Gr #2 Ti 1.1 6.88

did not exceed 2 µM. All control experiments conducted follow the same pattern;the results are summarized in Table III.

3. Catalytic Properties of the Reaction Vessel

Experiments 1/2001 (Figure 4) and 15/2002 (Figure 5) were conducted in 316Stainless Steel vessel at 136±3 ◦C. The initial 250 µmol formate concentration wasobserved to decrease to ∼32% of the initial concentration, while the acetate andpropionate concentrations had increased over the course of 24 days (Table IV). Thedata suggests that the formate ion is not stable at these conditions and decomposes;while acetate- and propionate-forming reactions progress.

Previous experiments (1/2001 and 15/2002) were then replicated (7/2002) atapproximately the same conditions (165±3 ◦C) in the 6AL/4V titanium reactionvessel. Despite the higher temperature of this experiment, formate decompositionkinetics appears to be much slower in this experiment (Table IV; Figure 6). Theoriginal 280 µmol sodium formate solution decomposes slowly, thus limiting theamount of reactants for acetate, propionate and formaldehyde formation reactions.The fluctuations in the formate concentration (initial increase) in both reactionvessels are caused by volume changes resulting from sampling. Each sample hasa volume of 5 mL. The sample withdrawal decreases the volume of the solution,thus increasing the headspace volume. This causes more water to partition intothe vapor phase. As a result the concentration of formate increases. The largestincrease is directly after the start of the experiment as the water is heated up andequilibrates with the N2 atmosphere in the headspace. The concentration change isthus directly related to the temperature and the headspace volume.


Figure 4. Experiment 1/2001. Formate decomposition in the 316 stainless steel reaction vessel. No-tice newly formed peaks of acetate and propionate. For clarity, the chromatogram traces are offsetalong the y-axis.

Figure 5. Experiment 15/2002. Formate decomposition in the 316 stainless steel reaction vessel. Forclarity, the chromatogram traces are offset along the y-axis.


TABLE IV

Formate and acetate concentrations (in µmol L−1) from the experiment conducted in two differentreaction vessels. 316 SS experiment compiled from experiments 15/2002 (days 0–5) and 1/2000(days 20–24)

Day 316 SS 316 SS 6Al4V Ti

(15/2000) (1/2001) (7/2002)

Formate Acetate T Formate Acetate T Formate Acetate T

0 250 0 136 250 0 136 280 0 166

1 297 n.a. 136 – – – 299 n.a. 166

2 309 n.a. 136 – – – 301 n.a. 166

3 292 n.a. 136 – – – 301 n.a. 166

4 n.a. n.a. 136 – – – 300 n.a. 166

5 265 23.4 136 – – – 309 4 166

6 – – – – – – 306 5 220

7 – – – – – – 224 4 220

8 – – – – – – 209 10 220

9 – – – – – – 186 4 220

10 – – – – – – 181 3 220

20 – – – 109 82 136 – – –

22 – – – 89 91 136 – – –

23 – – – 86 97 136 – – –

24 – – – 79 104 136 – – –

4. Discussion and Conclusions

All experiments in which polymer tubing was used introduced contamination intothe reacting solution. Low-molecular weight carboxylic acids or compounds thatreact to form them are leached from the polymer tubing, which is in contact withthe solution and its gaseous phase. The amount of contaminants released appears tobe dependent on the reaction time, temperature and the amount of polymer tubingused. The choice of reaction vessel material also appears to be crucial, since tworeaction vessels, running the same experiment, can yield significantly differentresults. 316 stainless steel contains admixtures of various transition elements (toenhance corrosion resistance properties) (Table I), which can act as ‘unaccountedfor’ reaction catalysts. Oxidation (corrosion) of 316 stainless steel may signific-antly affect the composition of the reacting solution (Ulmer and Barnes, 1987).6AL/4V titanium contains 4% admixture of vanadium, but the alloy is protectedfrom the reacting solution by the chemically inert Ti-O layer created by oxidativeheating. The ‘passivation’ of titanium metal surface is essential, since unoxidized


Figure 6. Experiment 7/2002. Formate decomposition in the 6AL/4V titanium vessel. For clarity, thechromatogram traces are offset along the y-axis.

(untreated) titanium metal may act as a catalyst and influence the redox state ofsystem by reducing water to hydrogen gas.

The results of the preliminary stability experiment in the 316 stainless steelvessel are consistent with those reported by Maiella and Brill (1998) at 280–330 ◦Cand 275 bar. They observed different rate constants for decarboxylation reactionsof sodium formate for titanium and 316 stainless steel reaction vessels (see alsoBrill, 2000). Similar results were reported for the decomposition of sodium acetate(CH3COONa) and acetic acid (CH3COOH) by Bell et al. (1994) at 200–355 ◦C,who observed the half-life difference of a factor 1010 between 316 stainless steeland titanium reaction vessels.

Given the fact that 316 Stainless Steel promotes decarboxylation reaction andmay promote other decomposition reactions, it is unlikely that the stability ex-periments conducted in 316 Stainless Steel (Bjerre and Sorrensen, 1992) yieldhomogenous reaction rates.

Based on experiments carried out, we highly recommend the exclusive use oftitanium alloys for any prebiotic synthesis experiments, especially if conducted athigh (>100 ◦C) temperatures, low reactant/product concentration and/or prolongedreaction time.


Acknowledgements

This research project has been supported by a grant from NASA’s AstrobiologyInstitute to Pennsylvania State University (NASA Cooperation Agreement NCC2– 1057) and by a grant from NASA’s Exobiology Program to M. A. A. Schoonen.Brian Hahn (SUNY Stony Brook) is thanked for proofreading the manuscript. Wethank Dr. T. McCollom (University of Colorado) and an anonymous reviewer fortheir valuable comments on an earlier version of this article. Dr. Alan Schwartz isthanked for handling the manuscript.

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evaluating experimental artifacts in hydrothermal prebiotic synthesis experiments

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