Conducting Miller-Urey Experiments

Summary

The Miller-Urey experiment was a pioneering study regarding the abiotic synthesis of organic compounds with possible relevance to the origins of life. Simple gases were introduced into a glass apparatus and subjected to an electric discharge, simulating the effects of lightning in the primordial Earth’s atmosphere-ocean system. The experiment was conducted for one week, after which, the samples collected from it were analyzed for the chemical building blocks of life.

Abstract

In 1953, Stanley Miller reported the production of biomolecules from simple gaseous starting materials, using an apparatus constructed to simulate the primordial Earth's atmosphere-ocean system. Miller introduced 200 ml of water, 100 mmHg of H₂, 200 mmHg of CH₄, and 200 mmHg of NH₃ into the apparatus, then subjected this mixture, under reflux, to an electric discharge for a week, while the water was simultaneously heated. The purpose of this manuscript is to provide the reader with a general experimental protocol that can be used to conduct a Miller-Urey type spark discharge experiment, using a simplified 3 L reaction flask. Since the experiment involves exposing inflammable gases to a high voltage electric discharge, it is worth highlighting important steps that reduce the risk of explosion. The general procedures described in this work can be extrapolated to design and conduct a wide variety of electric discharge experiments simulating primitive planetary environments.

Introduction

The nature of the origins of life on Earth remains one of the most inscrutable scientific questions. In the 1920s Russian biologist Alexander Oparin and British evolutionary biologist and geneticist John Haldane proposed the concept of a "primordial soup"^1,2, describing the primitive terrestrial oceans containing organic compounds that may have facilitated chemical evolution. However, it wasn't until the 1950s when chemists began to conduct deliberate laboratory studies aimed at understanding how organic molecules could have been synthesized from simple starting materials on the early Earth. One of the first reports to this end was the synthesis of formic acid from the irradiation of aqueous CO₂ solutions in 1951³.

In 1952, Stanley Miller, then a graduate student at the University of Chicago, approached Harold Urey about doing an experiment to evaluate the possibility that organic compounds important for the origin of life may have been formed abiologically on the early Earth. The experiment was conducted using a custom-built glass apparatus (Figure 1A) designed to simulate the primitive Earth. Miller's experiment mimicked lightning by the action of an electric discharge on a mixture of gases representing the early atmosphere, in the presence of a liquid water reservoir, representing the early oceans. The apparatus also simulated evaporation and precipitation through the use of a heating mantle and a condenser, respectively. Specific details about the apparatus Miller used can be found elsewhere⁴. After a week of sparking, the contents in the flask were visibly transformed. The water turned a turbid, reddish color⁵ and yellow-brown material accumulated on the electrodes⁴. This groundbreaking work is considered to be the first deliberate, efficient synthesis of biomolecules under simulated primitive Earth conditions.

Figure 1. Comparison between the two types of apparatuses discussed in this paper. The classic apparatus used for the original Miller-Urey experiment (A) and the simplified apparatus used in the protocol outlined here (B). Click here to view larger image.

After the 1953 publication of results from Miller's classic experiment, numerous variations of the spark discharge experiment, for example using other gas mixtures, were performed to explore the plausibility of producing organic compounds important for life under a variety of possible early Earth conditions. For example, a CH₄/H₂O/NH₃/H₂S gas mixture was tested for its ability to produce the coded sulfur-containing α-amino acids, although these were not detected⁶. Gas chromatography-mass spectrometry (GC-MS) analysis of a CH₄/NH₃ mixture subjected to an electric discharge showed the synthesis of α-aminonitriles, which are amino acid precursors⁷. In 1972, using a simpler apparatus, first introduced by Oró⁸ (Figure 1B), Miller and colleagues demonstrated the synthesis of all of the coded α-amino acids⁹ and nonprotein amino acids¹⁰ that had been identified in the Murchison meteorite to date, by subjecting CH₄, N₂, and small amounts of NH₃ to an electric discharge. Later, using this same simplified experimental design, gas mixtures containing H₂O, N₂, and CH₄, CO₂, or CO were sparked to study the yield of hydrogen cyanide, formaldehyde, and amino acids as a function of the oxidation state of atmospheric carbon species¹¹.

In addition to the exploration of alternative experimental designs over the years, significant analytical advances have occurred since Miller's classic experiment, which recently aided more probing investigations of electric discharge experimental samples archived by Miller, than would have been facilitated by the techniques Miller had access to in the 1950s. Miller's volcanic experiment¹², first reported in 1955⁴, and a 1958 H₂S-containing experiment¹³ were shown to have formed a wider variety, and greater abundances, of numerous amino acids and amines than the classic experiment, including many of which that had not been previously identified in spark discharge experiments.

The experiment described in this paper can be conducted using a variety of gas mixtures. Typically, at the very least, such experiments will contain a C-bearing gas, an N-bearing gas, and water. With some planning, almost any mixture of gases can be explored, however, it is important to consider some chemical aspects of the system. For example, the pH of the aqueous phase can have a significant impact on the chemistry that occurs there¹⁴.

The method described here has been tailored to instruct researchers how to conduct spark discharge experiments that resemble the Miller-Urey experiment using a simplified 3 L reaction vessel, as described in Miller's 1972 publications^9,10. Since this experiment involves a high voltage electric arc acting on inflammable gases, it is crucial to remove O₂ from the reaction flask to eliminate the risk of explosion, which can occur upon combustion of reduced carbon-bearing gases such as methane or carbon monoxide, or reaction of H₂ with oxygen.

There are additional details that should be kept in mind when preparing to conduct the experiment discussed here. First, whenever working with glass vacuum lines and pressurized gases, there exists the inherent danger of both implosion and over-pressuring. Therefore, safety glasses must be worn at all times. Second, the experiment is typically conducted at less than atmospheric pressure. This minimizes the risk of over-pressuring the manifold and reaction flask. Glassware may be rated at or above atmospheric pressure, however, pressures above 1 atm are not recommended. Pressures may increase in these experiments as water-insoluble H₂ is liberated from reduced gases (such as CH₄ and NH₃). Over-pressuring can lead to seal leakage, which can allow atmospheric O₂ to enter the reaction flask, making it possible to induce combustion, resulting in an explosion. Third, it should be borne in mind that modification of this protocol to conduct variations of the experiment requires careful planning to ensure unsafe conditions are not created. Fourth, it is highly recommended that the prospective experimenter read through the entire protocol carefully several times prior to attempting this experiment to be sure he or she is familiar with potential pitfalls and that all necessary hardware is available and in place. Lastly, conducting experiments involving combustible gases require compliance with the experimenter's host institution's Environmental Health and Safety departmental guidelines. Please observe these recommendations before proceeding with any experiments. All steps detailed in the protocol here are in compliance with the authors' host institutional Environmental Health and Safety guidelines.

Protocol

1. Setting Up a Manifold/Vacuum System

1. Use a glass manifold to introduce gases into the reaction flask. This manifold can be purchased or constructed by a glass-blowing facility, but must include vacuum-tight ports that can be connected to a vacuum system, gas cylinders, a vacuum gauge, and the reaction vessel.
   1. Use ground glass joints and glass plugs with valves on the manifold. Ensure that all O-rings on the plugs are capable of making the necessary seals. If using glass joints, a sufficient amount of vacuum grease can be applied to help make a seal, if necessary. Silicon vacuum grease can be used to avoid potential organic contamination.
   2. Use glass stopcocks on the manifold. Apply the minimum amount of vacuum grease necessary to make a seal.
   3. Measure the manifold volume. This volume will be used for calculations related to final gas pressures in the 3 L reaction flask and should be known as precisely as possible.
   4. Unless the manifold has enough connections to accommodate all gas cylinders simultaneously, connect one cylinder at a time to the manifold. Include in this connection, a tap allowing the manifold to be isolated from the ambient atmosphere.
   5. Use suitable, clean, inert, and chemical and leak resistant tubing and ultratorr vacuum fittings to connect the gas cylinders to the manifold. Ultratorr fittings, where used, are to be finger-tightened.
   6. Connect to the manifold, a vacuum pump capable of establishing a vacuum of <1 mmHg. The vacuum pump exhaust should be located within the fume hood, or properly vented by other means.
      1. To ensure rapid attainment of vacuum and to protect the pump, insert a trap between the manifold and the vacuum pump. A liquid nitrogen finger-trap is recommended as it will prevent volatiles such as NH₃, CO₂, and H₂O from entering the pump. Care should be taken, as trapped volatiles, upon warming, may overpressure the manifold and result in glass rupture.
   7. Connect to the manifold, a manometer or other vacuum gauge capable of 1 mmHg resolution or better. While various devices can be used, a mercury manometer, or MacLeod gauge, is preferable as mercury is fairly nonreactive.
   8. Measure and record the ambient temperature using a suitable thermometer.

2. Preparation of Reaction Flask

1. Heat all glassware at 500 °C for at least 3 hr in air prior to use, to remove organic contaminants.
   1. Clean the tungsten electrodes by gently washing with clean laboratory wipes and methanol, and drying in air.
2. Pour 200 ml of ultrapure water (18.2 MΩ cm, <5 ppb TOC) into the 3 L reaction flask.
   1. Introduce a precleaned and sterilized magnetic stir bar, which will ensure rapid dissolution of soluble gases and mixing of reactants during the experiment.
3. Attach the tungsten electrodes to the 3 L reaction flask using a minimal amount of vacuum grease, with tips separated by approximately 1 cm inside the flask. Fasten with clips.
4. Insert an adapter with a built-in stopcock into the neck of the 3 L reaction flask and secure with a clip.
5. Attach the 3 L reaction flask to the gas manifold via the adapter. Use a clip or clamp to help secure the flask.
   1. Lightly grease all connections to ensure a good vacuum seal.
6. Open all valves and stopcocks on the manifold, except Valve 6 and Stopcock 1 (Figure 4), and turn on the vacuum pump to evacuate the manifold. Once a stable vacuum reading of <1 mmHg has been attained, close Valve 1 and allow the manifold to sit for ~15 min to check for vacuum leaks. If none are detected, proceed to step 2.8. Otherwise troubleshoot the various connections until the leaks can be identified and fixed.
7. Apply magnetic stirring to the reaction vessel. Open Valve 1 and Stopcock 1 (Figure 4) to evacuate the headspace of the 3 L reaction flask until the pressure has reached <1 mmHg.
8. Close Valve 1 (Figure 4) and monitor the pressure inside the 3 L reaction flask. The measured pressure should increase to the vapor pressure of water. To ensure that no leaks exist, wait ~5 min at this stage. If the pressure (as read on the manometer) increases while Valve 1 is closed during this step, check for leaks in Stopcock 1 and the various reaction flask connections. If no leak is found, proceed to the next step.

3. Introduction of Gaseous NH₃

1. Calculate the necessary pressure of gaseous NH₃ to introduce into the manifold such that 200 mmHg of NH₃ will be introduced into the reaction flask. Details on how to do this are provided in the Discussion section.
2. Close Valves 1 and 6, and Stopcock 1 (Figure 4) before introducing any gas into the manifold. Leave the other valves and stopcock open.
3. Introduce NH₃ into the manifold until a small pressure (approximately 10 mmHg) is reached and then evacuate the manifold to a pressure of <1 mmHg by opening Valve 1 (Figure 4). Repeat 3x.
4. Introduce NH₃ into the manifold to reach the pressure determined in step 3.1.
5. Open Stopcock 1 (Figure 4) to introduce 200 mmHg of NH₃ into the 3 L reaction flask. The NH₃ will dissolve in the water in the reaction flask and the pressure will fall slowly.
6. Once the pressure stops dropping, close Stopcock 1 (Figure 4) and record the pressure read by the manometer. This value represents the pressure inside the flask and will be used to calculate the pressures for other gases that will be introduced into the manifold later.
7. Open Valve 1 (Figure 4) to evacuate the manifold to a pressure of <1 mmHg.
8. Close Valve 2 (Figure 4) and disconnect the NH₃ gas cylinder from the manifold.

4. Introduction of CH₄

1. Calculate the necessary pressure of CH₄ to be introduced into the manifold such that 200 mmHg of CH₄ will be introduced into the 3 L reaction flask. Example calculations are shown in the Discussion section.
2. Connect the CH₄ gas cylinder to the manifold.
3. Open all valves and stopcocks, except Valve 6 and Stopcock 1 (Figure 4), and evacuate the manifold to a pressure of <1 mmHg.
4. Close Valve 1 once the manifold has been evacuated (Figure 4).
5. Introduce CH₄ into the manifold until a small pressure (approximately 10 mmHg) is obtained. This purges the line of any contaminant gases from preceding steps. Open Valve 1 (Figure 4) to evacuate the manifold to <1 mmHg. Repeat 2x more.
6. Introduce CH₄ into the manifold until the pressure calculated in step 4.1, is reached.
7. Open Stopcock 1 (Figure 4) to introduce 200 mmHg of CH₄ into the 3 L reaction flask.
8. Close Stopcock 1 once the intended pressure of CH₄ has been introduced into the 3 L reaction flask (Figure 4) and record the pressure measured by the manometer.
9. Open Valve 1 (Figure 4) to evacuate the manifold to <1 mmHg.
10. Close Valve 2 (Figure 4) and disconnect the CH₄ cylinder from the manifold.

5. Introduction of Further Gases (e.g. N₂)

1. At this point, it is not necessary to introduce additional gases. However, if desired, it is recommended to add 100 mmHg of N₂. In this case, calculate the necessary pressure of N₂ to be introduced into the manifold such that 100 mmHg of N₂ will be introduced into the 3 L reaction flask. Example calculations are shown in the Discussion section.
2. Connect the N₂ gas cylinder to the manifold.
3. Open all valves and stopcocks, except Valve 6 and Stopcock 1 (Figure 4), and evacuate the manifold to a pressure of <1 mmHg.
4. Close Valve 1 once the manifold has been evacuated (Figure 4).
5. Introduce N₂ into the manifold until a small pressure (approximately 10 mmHg) is obtained. Open Valve 1 (Figure 4) to evacuate the manifold to <1 mmHg. Repeat 2x more.
6. Introduce N₂ into the manifold until the pressure calculated in step 5.1 is reached.
7. Open Stopcock 1 (Figure 4) to introduce 100 mmHg of N₂ into the reaction flask.
8. Close Stopcock 1 once the intended pressure of N₂ has been introduced into the reaction flask, (Figure 4) and record the pressure using the manometer.
9. Open Valve 1 (Figure 4) to evacuate the manifold to <1 mmHg.
10. Close Valve 2 (Figure 4) and disconnect the N₂ cylinder from the manifold.

6. Beginning the Experiment

1. Detach the reaction flask from the manifold by closing Stopcock 1 and Valve 1 (Figure 4) once all gases have been introduced into the reaction flask, so that ambient air may enter the manifold and bring the manifold up to ambient pressure.
2. After carefully disconnecting the reaction flask from the manifold, set the flask somewhere it will not be disturbed (e.g. inside an empty fume hood).
3. Disconnect the vacuum pump and carefully remove the cold trap and allow venting inside a fully operational fume hood.
4. Secure the Tesla coil connected to the high frequency spark generator.
5. Connect the opposite tungsten electrode to an electrical ground to enable the efficient passage of electrical current across the gap between the two electrodes.
6. Set the output voltage of the spark generator to approximately 30,000 V, as detailed by documents available from the manufacturer.
7. Prior to initiating the spark, close the fume hood sash, to serve as a safety shield between the apparatus and the experimenter. Turn the Tesla coil on to start the experiment, and allow sparking to continue for 2 weeks (or other desired period) in 1 hr on/off cycles.

7. End of Experiment

1. Stop the experiment by turning off the Tesla coil.
2. Open Stopcock 1 (Figure 4) to slowly introduce ambient air into the reaction flask and facilitate the removal of the adapter and the tungsten electrodes so samples can be collected. If desired, a vacuum can be used to evacuate the reaction flask of noxious reaction gases.

8. Collecting Liquid Sample

1. Using a pyrolyzed glass pipette, remove liquid samples from the reaction flask, being careful to minimize exposure to contaminants, such as those that might be introduced by touching the pipette to the vacuum grease or other nonsterile surfaces.
   1. Transfer the sample to a sterile plastic or glass receptacle. Plastic receptacles are less prone to cracking or breaking upon freezing, compared to glass receptacles.
2. Seal sample containers and store in a freezer capable of reaching temperatures of -20 °C or lower, as insoluble products may prevent the sample solution from freezing at 0 °C.

9. Cleaning the Apparatus

1. Use clean laboratory wipes to carefully remove vacuum grease from the neck of the apparatus, the adapter and stopcock, and the glass surrounding the tungsten electrodes.
2. Thoroughly clean the same surfaces described in step 9.1 with toluene to fully remove organic vacuum grease from the glassware. If using silicon grease, the high vacuum grease may remain on the glassware after pyrolysis, creating future problems, as detailed in the Discussion section.
3. Thoroughly clean the reaction flask with a brush and the following solvents in order: ultrapure water (18.2 MΩ cm, <5 ppb TOC), ultrapure water (18.2 MΩ cm, <5 ppb TOC) with 5% cleaning detergent, methanol, toluene, methanol, ultrapure water (18.2 MΩ cm, <5 ppb TOC) with 5% cleaning detergent, and finally ultrapure water (18.2 MΩ cm, <5 ppb TOC).
4. Cover all open orifices of the reaction flask with aluminum foil and wrap the adapter and its components in aluminum foil.
5. Once all the glassware has been wrapped in aluminum foil, pyrolyze for at least 3 hr in air at 500 °C.
6. Gently clean electrodes with methanol and let air dry.

10. Sample Analysis

Note: When preparing samples for analysis, the use of an acid hydrolysis protocol such as has been described in detail elsewhere¹⁵, is useful for obtaining more amino acids. Hydrolysis of a portion of the recovered sample provides the opportunity to analyze both free amino acids as well as their acid-labile precursors that are synthesized under abiotic conditions.

1. For amino acid analysis, use a suitable technique (such as liquid chromatography and mass spectrometry-based methods, or other appropriate approaches). Such analytical techniques include high performance liquid chromatography with fluorescence detection (HPLC-FD)¹⁴, and ultrahigh performance liquid chromatography with fluorescence detection in parallel with time-of-flight positive electrospray ionization mass spectrometry (UHPLC-FD/ToF-MS)^12,13. This manuscript describes analysis using mass spectrometric analyses via a triple quadrupole mass spectrometer (QqQ-MS) in conjunction with HPLC-FD.

Results

The products synthesized in electric discharge experiments can be quite complex, and there are numerous analytical approaches that can be used to study them. Some of the more commonly used techniques in the literature for analyzing amino acids are discussed here. Chromatographic and mass spectrometric methods are highly informative techniques for analyzing the complex chemical mixtures produced by Miller-Urey type spark discharge experiments. Amino acid analyses can be conducted using o-phthaldialdehyde/N-acetyl...

Discussion

Numerous steps in the protocol described here are critical for conducting Miller-Urey type experiments safely and correctly. First, all glassware and sample handling tools that will come in contact with the reaction flask or sample need to be sterilized. Sterilization is achieved by thoroughly rinsing the items in question with ultrapure water (18.2 MΩ cm, <5 ppb TOC) and then wrapping them in aluminum foil, prior to pyrolyzing at 500 °C in air for at least 3 hr. Once the equipment has been pyrolyzed and while prepa...

Materials

Name	Company	Catalog Number	Comments
Glass Plugs for Manifold	Chemglass	CG-983-01
High Vacuum Grease	Apiezon	N/A	Type M/N
Silicon High Vacuum Grease	Dow Corning	1597418
Teflon PFA Tubing	McMaster-Carr	51805K54
Ultra-Torr Vacuum Fittings	Swagelok	SS-4-UT-6
Dry Scroll Vacuum Pump	Edwards	A72401905
U-Tube Manometer	Alta-Robbins	100SS
Tungsten Electrodes	Diamond Ground Products	TH2-1/16	2% thoriated
Methanol	Alfa Aesar	N/A	Ultrapure HPLC Grade
Teflon-Coated Magnetic Stir Bar	McMaster-Carr	5678K127
Gaseous NH3	Airgas	AMAHLB	99.99% purity
Gaseous CH4	Airgas	ME UHP300	99.99% purity
Gaseous N2	Airgas	NI UHP300	99.999% purity
Tesla Coil	Electro-Technic Products	15001	Model BD-50E
24 hr Plug-in Basic Timer	General Electric Company	15119
Cleaning Detergent	Alconox	1104
Toluene	Thermo Fisher Scientific	N/A	Optima Grade
Luna Phenyl-Hexyl HPLC Column	Phenomenex	00G-4257-E0	Brand: Luna
Formic Acid	Sigma-Alrich	F0507	Used to make 50 mM ammonium formate