what is wrong with miller urey experiment

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What the famous Miller-Urey experiment got wrong

• The famous experiment showed that a mixture of gases and water could produce amino acids and other biomolecular precursors.
• However, new research shows that an unexpected factor may have played a major part in the result: glassware.
• Complex experiments need good controls, and the Miller-Urey experiment failed in this regard.

Science in the early 20th century was undergoing many simultaneous revolutions . Radiological dating numbered the years of Earth’s existence in the billions, and eons of sediment demonstrated its geological evolution. The biological theory of evolution had become accepted, but mysteries remained about its selection mechanism and the molecular biology of genetics. Remnants of life dated far, far back, beginning with simple organisms. These ideas came to a head with the question of abiogenesis : could the first life have sprung from non-living matter?

In 1952, a graduate student named Stanley Miller, just 22 years old, designed an experiment to test whether the amino acids that form proteins could be created under the conditions thought to exist on the primordial Earth. Working with his Nobel Prize-winning advisor Harold Urey, he performed the experiment, which is now told time and again in textbooks all over the world.

The experiment mixed water and simple gases — methane, ammonia, and hydrogen — and shocked them with artificial lightning within a sealed glass apparatus . Within days, a thick colored substance built up at the bottom of the apparatus. This detritus contained five of the basic molecules common to living creatures. Revising this experiment over the years, Miller claimed to find as many as 11 amino acids. Subsequent work varying the electrical spark, the gases, and the apparatus itself created another dozen or so. After Miller’s death in 2007, the remains of his original experiments were re-examined by his former student . There may have been as many as 20-25 amino acids created even in that primitive original experiment.

The Miller-Urey experiment is a daring example of testing a complex hypothesis. It is also a lesson in drawing more than the most cautious and limited conclusions from it.

Did anyone consider the glassware?

In the years following the original work, several limitations curbed excitement over its result . The simple amino acids did not combine to form more complex proteins or anything resembling primitive life. Further, the exact composition of the young Earth did not match Miller’s conditions. And small details of the setup appear to have affected the results. A new study published last month in Scientific Reports investigates one of those nagging details. It finds that the precise composition of the apparatus housing the experiment is crucial to amino acid formation.

The highly alkaline chemical broth dissolves a small amount of the borosilicate glass reactor vessel used in the original and subsequent experiments. Dissolved bits of silica permeate the liquid, likely creating and catalyzing reactions . The eroded walls of the glass may also boost catalysis of various reactions. This increases total amino acid production and allows the formation of some chemicals which are not created when the experiment is repeated in an apparatus made of Teflon. But, running the experiment in a Teflon apparatus deliberately contaminated with borosilicate recovered some of the lost amino acid production.

Complex questions need carefully designed experiments

The Miller-Urey experiment was based on a complicated system. Over the years, many variables were tweaked, such as the concentration and composition of gases. For the purpose of demonstrating what might be plausible — that is, whether biomolecules can be created from inorganic materials — it was stunningly successful. But there wasn’t a good control. We now see that might have been a pretty big mistake.

One of the elements of art in science is to divine which of innumerable complexities matter and which do not. Which variables can be accounted for or understood without testing, and which ones can be cleverly elided by experimental design? This is a borderland between hard science and intuitive art. It is certainly not obvious that glass would play a role in the outcome, but it apparently does.

A more certain and careful form of science is to conduct an experiment that varies one and only one variable at a time. This is a slow and laborious process. It can be prohibitively difficult for testing complex hypotheses like, “Could life evolve from non-life on the early Earth?” The authors of the new work performed just such a single-variable test. They ran the entire Miller-Urey experiment multiple times, varying only the presence of silicate glass. The runs performed in as glass vessel produced one set of results, while those using a Teflon apparatus produced another.

Systematically marching through each potential variable, one at a time, might be called “brute force.” But there is art here too, namely, in deciding which single variable out of many possibilities to test and in what way. In this case, we learned that glass silicates played an important role in the Miller-Urey experiment. Perhaps this means that silicate rock formations on the early Earth were necessary to produce life. Maybe.

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Original protocol and results

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Miller-Urey experiment

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• Nature - The role of borosilicate glass in Miller–Urey experiment
• Frontiers - Frontiers in Physics - The spark of life: discharge physics as a key aspect of the Miller–Urey experiment
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Miller-Urey experiment , experimental simulation conducted in 1953 that attempted to replicate the conditions of Earth ’s early atmosphere and oceans to test whether organic molecules could be created abiogenically, that is, formed from chemical reactions occurring between inorganic molecules thought to be present at the time. The experiment—the results of which were published in the journal Science as “A Production of Amino Acids Under Possible Primitive Earth Conditions”—documented the production of amino acids and other organic molecules, thereby demonstrating that chemical evolution (that is, the formation of complex chemicals from simple ones) is possible. The Miller-Urey experiment is used as evidence to support hypotheses about the origins of life .

The Miller-Urey experiment was conducted by American chemist Stanley Miller under the supervision of American scientist Harold C. Urey at the University of Chicago . The experiment was designed to test ideas introduced independently in the 1920s by Russian biochemist Aleksandr Oparin and British physiologist J.B.S. Haldane , both of whom suggested that organic molecules, such as amino acids and sugars , could be formed from abiogenic materials when acted on by an external energy source within the context of a reducing atmosphere, that is, one with low levels of free oxygen ( see also oxidation-reduction reaction ). At the time, it was thought that the atmosphere of early Earth between 4 billion and 3.5 billion years ago was primarily composed of ammonia and water vapour. Oparin and Haldane noted that from this “primordial soup” of materials the first organic molecules arose, which became the precursors to molecules of ever-increasing complexity that resulted in the development of living cells ( see also abiogenesis ).

To test Oparin and Haldane’s ideas, Miller and Urey designed a closed experiment in a laboratory . They constructed an enclosed glass apparatus with two large boiling flasks connected to each other with glass tubing, in which water could pool, gases could mix, and matter could change phases between liquid and gas. A large lower chamber was filled with water (a boiling flask that stood in for the oceans), and the water was boiled to produce water vapour, which ascended into the large upper chamber (a boiling flask that simulated Earth’s early atmosphere). Additional tubing allowed material to descend from the upper chamber through a condenser, where water vapour would condense into liquid and fall into a collection trap from which samples could be taken. This trap was set slightly below the lower chamber, but it was also connected to the lower chamber above the water line. The researchers removed the air from the apparatus, replacing it throughout with ammonia, hydrogen , and methane gases, and they let the various materials cycle through liquid and gas phases.

In an early run of the experiment, Miller discovered that the energy produced from boiling water was not enough to drive the chemical reactions necessary to approximate the conditions of early Earth, so in a second version of the experiment (the one whose results were published in 1953) he added electrodes to the upper chamber. After the water was boiled, the mix of gases circulated through the system past electrodes that discharged sparks (which simulated lightning ), and a condenser converted some of the gas to liquid so that it could return to the lower chamber. This process ran continuously for one week. After this period, the contents of the apparatus had visibly changed colour. A red- and yellow-coloured solution had started to collect in the trap after running the experiment for a few days and became a broth of red and brown by the experiment’s end.

To determine the identity of the molecules that resulted from their procedure, Miller and Urey terminated the reaction, added chemicals that prevented the growth of microbes (which could be introduced to the closed system when samples were collected from the broth), extracted samples of the solution, and analyzed them using paper chromatography . They discovered several types of simple organic molecules in the samples, including amino acids, some of which were relevant as the building blocks of the proteins that are present in all living organisms. Miller was able to identify the amino acids glycine , alpha-alanine (α-alanine), and beta-alanine (β-alanine) confidently; however, he was less certain about the presence of aspartic acid and α-amino- n -butyric acid, whose signs in the analysis were weak.

Miller modified the original experiment several times, and each modification captured possible variations in Earth conditions that might influence the system’s products. Some of Miller’s subsequent experiments used different energy sources, such as an electrical source that produced a silent discharge instead of a spark, and improved gas circulation through the addition of glass tubing. Other researchers also repeated the experiment during the 1950s using different energy sources, such as ultraviolet light , and used different atmospheric gases (such as carbon dioxide , hydrogen sulfide , and nitrogen ) in various combinations, which also resulted in a mix of organic chemicals but only a handful of amino acids.

Miller and others would repeat the experiment several times in subsequent decades. He reran the experiment in the early 1970s using better analytical equipment, which revealed the presence of 33 different amino acids, including more than half of the 20 or so that appear in proteins present in living things. Researchers later criticized Miller for using what they considered to be the wrong gases in the experiment; carbon dioxide and nitrogen, not ammonia and methane, were shown later to be the primary gases in Earth’s early atmosphere, with ammonia and methane occurring only in minor amounts. Miller’s 1983 trial replaced methane and ammonia with carbon dioxide and nitrogen; however, fewer amino acids were produced than in the original published experiment. This result was attributed later to a buildup of nitrites in the system, which made the mixture more acidic and caused the amino acids to break down before they could be identified.

American chemist Jeffrey Bada of the Scripps Institution of Oceanography reran the experiment in 2007. In addition to simulating an atmosphere filled with carbon dioxide and nitrogen, he added iron and carbonates to the system (two materials that would have been present in large amounts on ancient Earth)—which neutralized both the nitrites and the acids in the system, thereby allowing the amino acids to persist. A group of Spanish and Italian researchers suggested in 2021 that materials in the glass apparatus itself may have also catalyzed the chemical reactions taking place within it; the various chemicals in the experiment were shown to have reacted with the interior surface of the glass to release silicates (which, in turn, reacted with other chemicals) while also leaving behind small imperfections and cracks on the interior surface that may have served as chambers for other chemical reactions.

Starting in the early 2000s, researchers examined archived vials containing samples of material collected from Miller’s experiments during the 1950s. Aided by modern analytical equipment, they discovered far more than the five amino acids Miller reported in his papers; Miller’s experiments conducted in 1953 and 1958 were each shown to have yielded more than 20 amino acids.

The experiment showed that amino acids, which are important components of proteins (which are critical to life on Earth), could have arisen from inorganic compounds during Earth’s prebiotic phase. It also demonstrated that the speculation that life could have originated through chemical reactions among nonliving materials is possible and that this hypothesis could be tested scientifically. The Miller-Urey experiment sparked research on how simple organic molecules might polymerize into more complex molecules, a process that may have produced the first living cells. Scientists have also considered the possibility that meteors brought the first organic molecules, formed in space, to Earth, and they continue to modify the Miller-Urey protocol to test new ideas about chemical reactions in primitive Earth conditions.

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Miller Urey Experiment: Hypothesis, Steps, Conclusions, and Limitations

The Miller Urey Experiment played a crucial role in investigating the origin of life on our planet. This comprehensive guide explores the experiment’s hypothesis, step-by-step process, key findings, and limitations, shedding light on its significance in unraveling the mysteries of life’s beginnings.

Oparin-Haldane Hypothesis

The Oparin-Haldane Hypothesis, proposed by Aleksandr Oparin and J.B.S. Haldane, postulates that life didn’t spontaneously emerge on early Earth due to different environmental conditions. It suggests that life gradually evolved from chemical reactions, starting with the combination of atoms into inorganic molecules and the subsequent formation of simple organic compounds. These compounds then assembled into complex organic structures, ultimately leading to the emergence of the first cell.

Steps of the Miller Urey Experiment

The Miller-Urey experiment, conducted in 1953 by Stanley L. Miller and Harold C. Urey, aimed to simulate early Earth’s conditions and test the Oparin-Haldane Hypothesis. Here are the key steps of the experiment:

Simulating Early Earth’s Atmosphere:  The researchers recreated early Earth’s atmosphere in a closed system using a mixture of gases believed to be present during that era. They used a mixture of gases, including methane (CH4), ammonia (NH3), water vapor (H2O), and hydrogen (H2).

Introduction of Energy:  Sparks or electric discharges were introduced to simulate the energy sources on early Earth, such as lightning strikes.

Circulation and Condensation:  The gas mixture and energy were circulated continuously, mimicking Earth’s water cycle and allowing for the formation of various organic compounds.

Collection and Analysis:  Samples were collected from the closed system and analyzed using chromatography and spectrometry to identify and characterize the organic compounds formed during the experiment.

Results and Findings:  The experiment produced a variety of organic molecules, including amino acids—the building blocks of proteins —supporting the notion that early Earth’s conditions could have facilitated the synthesis of organic compounds essential for life’s origin.

Conclusions of the Miller Urey Experiment

The Miller-Urey experiment yielded significant conclusions, including:

• Organic compounds, including amino acids, can be synthesized from inorganic materials under simulated early Earth conditions.
• Basic building blocks of life may have emerged spontaneously from non-living matter.
• The experiment demonstrated the potential for diverse organic compound formation, including rare amino acids.
• External energy sources played a crucial role in facilitating chemical reactions and organic compound synthesis.
• The experiment offered insights into the chemical reactions that might have occurred in early Earth’s atmosphere.
• The findings supported the concept of abiogenesis, where life can arise from non-living matter through natural processes.
• The Miller-Urey experiment laid the foundation for further research in prebiotic chemistry and the study of life’s origins.

Limitations of the Miller Urey Experiment

 It’s important to consider the limitations of the Miller-Urey experiment, which include:

• The experiment’s simulation of early Earth’s atmosphere may not perfectly represent the actual conditions.
• The specific gases used may not accurately reflect the true composition of early Earth’s atmosphere.
• The experiment’s short duration and scale may not fully capture the complexity and length of natural processes involved in life’s origin.
• While the experiment produced organic compounds, it didn’t address the assembly of complex biomolecules or replicating systems crucial for life’s origin.

Ongoing Debates and Significance

Critics argue that the experiment oversimplifies the interconnected nature of biochemical systems and may not fully represent the processes behind life’s origin. There is an ongoing debate regarding the specific conditions and pathways leading to life’s emergence, with the Miller-Urey experiment presenting one plausible scenario. While it doesn’t address the origin of genetic information or self-replicating systems, subsequent research has refined and expanded upon its findings, leading to revised interpretations. The Miller-Urey experiment remains a significant milestone in our understanding of prebiotic chemistry and contributes to unraveling the complex puzzle of life’s origin.

In conclusion, the Miller-Urey experiment’s hypothesis, steps, conclusions, and limitations provide valuable insights into the origin of life on Earth. It serves as a foundation for further research, stimulating ongoing debates and refining our understanding of life’s emergence from non-living matter.

Learn more:

Amino Acids: Types, Functions, Sources, and Differences between Essential and Non-Essential Amino Acids

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November 26, 2021

Redo of a Famous Experiment on the Origins of Life Reveals Critical Detail Missed for Decades

The Miller-Urey experiment showed that the conditions of early Earth could be simulated in a glass flask. New research finds the flask itself played an underappreciated, though outsize, role.

By Sarah Vitak

Chemist Stanley Miller re-creates the Miller-Urey experiment using the original laboratory equipment in the 1980s.

Roger Ressmeyer/Corbis/VCG/Getty Images

Sarah Vitak: This is Scientific American ’s 60 Second Science. I’m Sarah Vitak.

The question of how life came to be has captivated humans for millennia. The prevailing theory now is that, on a highly volatile early Earth, lightning struck mineral-rich waters and that the energy from lighting strikes turned those minerals into the building blocks of life: organic compounds like amino acids—something we often refer to as the “primordial soup.” The wide acceptance of this theory is in large part due to the very famous Miller-Urey experiment. You surely encountered this in a science textbook at some point. But to refresh your memory: in 1952 Stanley Miller and Harold Urey simulated the conditions of early Earth by sealing water, methane, ammonia and hydrogen in a glass flask. Then they applied electrical sparks to the mixture. Miraculously, amino acids came into existence amid the roiling mixture. It was a big deal.

But recently a team of researchers realized that—much like that first primordial soup sitting in a bowl of Earth—the experiment’s container played an underappreciated role—that perhaps it was also critical to the creation of organic building blocks inside their laboratory life soup.

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I talked to someone from the team.

Saladino: I am Raffaele Saladino from University of Tuscia in Italy.

Vitak: Then, much like today, when a researcher goes to start an experiment, often one of the first things they do is reach for their glassware. Well, today, actually, we use a lot of plastic as well.

Saladino:  But 20 years ago in the lab, only glass containers because, in the mind of the researcher, glass is inert.

Vitak: He said inert, meaning that it doesn’t react with the chemicals you put inside it. But in reality, that is not necessarily always the case.

Most of the time glass is pretty inert. When you’re baking with Pyrex (which is made of borosilicate glass, the same type of glass most labware is made out of) the cookware isn’t going into your brownies. But when you’re baking, whatever is in the pan is usually mostly water, so it will come at a pH of around 7 or so.

But the pH of the Miller-Urey experiment is much higher. In the original experiments, they used a pH of 8.7, which is more alkaline, or basic.

Saladino: Why alkaline environment is an important topic? Since under alkaline condition borosilicate can be impacted through blinds in the reaction menu, it is not inert it became a reagent. 

Vitak: In fact, this was actually noted by Miller in his original experiments--that the alkaline conditions caused the silica to dissolve. But it was overshadowed by the discovery of the synthesis of organic compounds. And as future researchers carried on they missed that point in Miller’s notes.

Saladino: The attention was concentrated on modifying the atmosphere, on modifying the energy, the intensity, and modifying the analytical tools.

Vitak: And the role of the silica got forgotten entirely. 

Dr. Saladino’s team wanted to see if the glass was doing anything in the reaction. To test this they set up three different versions of the original experiment where everything was the same except the containers. For comparison they chose teflon which does not dissolve when holding an alkaline solution, the way the glass does.

Saladino: There is the experiment only glass, the experiment only Teflon, and in the middle, there is the experiment in teflon with some pieces of glass added inside.

Vitak: Then they used a technique called mass spectrometry to analyze what each reaction produced. Mass spectrometry is great for figuring out what kinds of molecules are in a complex mixture.

They found that teflon produced very few organic compounds. There were more compounds in the teflon with glass pieces. But the glass container, by far, created the greatest number and largest variety of organic molecules.

The mechanism of exactly how the silica helps catalyze the reaction is not clear yet--but it is very clearly does.

The obvious question then is: Was there silica available in the early earth environment?

Saladino:  The water is not suspended in a vacuum. No? The water is in geochemistry, it is surrounded by minerals. Borosilicate and silica are the most abundant minerals surrounding the water.

Vitak: The team has two next major objectives in mind. First, to try updating the experiment to model more closely the amount of silica that would have been available in the early Earth.

Second, they want to try replacing the silica with extraterrestrial minerals like, pieces of meteorite or rocks from other planets. Apart from just being very cool, that could give a more concrete idea of how to look for life in space. 

But here on Earth, coming one step closer to fully understanding why we exist is that much more satisfying. Even after nearly 70 years, a key discovery in our complex origin story still carries new revelations. As the authors say in the paper: "The role of the rocks was hidden in the walls of the reactors."

Thanks for listening. For Scientific American’s 60 Second Science, I’m Sarah Vitak. 

[ The above text is a transcript of this podcast .]

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• 22 November 2021

Message in a bottle: revisiting the origin of life

• Andrea Gentile

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Artist's impression of the early Earth conditions that the Miller-Urey experiment tried to recreate. Credit: CSIC.

Some of the basic ingredients for life are well known: a dash of water, methane, ammonia, hydrogen and a spark. But a pinch of minerals is also needed, according to a new study 1 by Italian and Spanish researchers that recreated an experiment from 1952, paying attention to a detail that had been overlooked for all these years: the glass pot in which it was performed.

“In science you should take nothing for granted,” says Raffaele Saladino, a professor at Tuscia University and president of the Italian Society of Astrobiology. “Nobody would have guessed that a setting tested hundreds of times could tell us anything more.” In 1952 at the University of Chicago, Stanley Miller and Harold Urey simulated the Earth’s environment 4.6 billion years ago to study abiogenesis, the natural synthesis of organic molecules such as amino acids and nucleobases (the building blocks of proteins and DNA/RNA respectively). In a sealed flask, they recreated the primordial atmosphere along with water, while a spark simulated lightning. Later, they found several amino acids, demonstrating how the precursors of life could emerge in a prebiotic soup. “In some experiments Miller also noted the presence of silica [the main component of glass and some rocks],” says Saladino, “but he didn’t pay much attention to it.” And nobody else investigated its role until now.

In previous studies, the team found that silica and its minerals in a solution similar to Miller’s could facilitate the process. So they decided to test the idea that, in the original experiment, they had been diluted from the flask because of the causticity of the mixture. They repeated the experiment using three containers made of materials with different pHs: borosilicate glass or Pyrex (the same material used by Miller), Teflon, which is an inert material, and Teflon with some borosilicate bits in the solution. The results confirmed that organic matter emerged in every flask independently of the pH, but the Teflon container had the fewest products, followed by the one with glass pieces. The abundance of organic molecules in the Pyrex container – 56 different kinds, amino acids and nucleobases included – was staggering, with some molecules appearing only in the borosilicate glass, revealing the importance of minerals as hidden ingredients for the precursors of life. “It makes sense, if we want to simulate a realistic scenario,” explains Saladino, “because we would have the atmosphere, water, lightning, but what we missed was the rock containing the water.”

A renewed interest in abiogenesis could help the search for life on other planets. “The complexity of a molecule doesn’t guarantee that it was produced by biological processes,” notes Saladino. “If we were able to create such molecular richness with a single experiment, then finding molecules like glycine or phosphine on other planets wouldn’t necessarily imply that they were synthesized by a living organism.” Future studies will test which molecules can emerge in a Miller-Urey setting using different minerals and alien atmospheres. Then, when looking for life on different planets we will better know what molecules to expect and, more importantly, those that are truly unexpected.

doi: https://doi.org/10.1038/d43978-021-00144-0

Criado-Reyes J, Bizzarri BM, García-Ruiz JM, Saladino R & Di Mauro E, Sci. Rep. 11 , 21009 (2021)

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A is for abiogenesis —

Scientists recreated classic origin-of-life experiment and made a new discovery, 1952 miller-urey experiment showed organic molecules forming from inorganic precursors..

Jennifer Ouellette - Oct 28, 2021 6:59 pm UTC

In 1952, a University of Chicago chemist named Stanley Miller and his adviser, Harold Urey, conducted a famous experiment . Their results, published the following year, provided the first evidence that the complex organic molecules necessary for the emergence of life ( abiogenesis ) could be formed using simpler inorganic precursors, essentially founding the field of prebiotic chemistry. Now a team of Spanish and Italian scientists has recreated that seminal experiment and discovered a contributing factor that Miller and Urey missed. According to  a new paper published in the journal Scientific Reports, minerals in the borosilicate glass used to make the tubes and flasks for the experiment speed up the rate at which organic molecules form.

In 1924 and 1929, respectively, Alexander Oparin and J.B.S. Haldane had hypothesized that the conditions on our primitive Earth would have favored the kind of chemical reactions that could synthesize complex organic molecules from simple inorganic precursors—sometimes known as the " primordial soup " hypothesis. Amino acids formed first, becoming the building blocks that, when combined, made more complex polymers.

Miller set up an apparatus to test that hypothesis by simulating what scientists at the time believed Earth's original atmosphere might have been. He sealed methane, ammonia, and hydrogen inside a sterile 5-liter borosilicate glass flask, connected to a second 500-ml flask half-filled with water. Then Miller heated the water, producing vapor, which in turn passed into the larger flask filled with chemicals, creating a mini-primordial atmosphere. There were also continuous electric sparks firing between two electrodes to simulate lighting. Then the "atmosphere" was cooled down, causing the vapor to condense back into water. The water trickled down into a trap at the bottom of the apparatus.

That solution turned pink after one day and deep red after a week. At that point, Miller removed the boiling flask and added barium hydroxide and sulfuric acid to stop the reaction. After evaporating the solution to remove any impurities, Miller tested what remained via paper chromatography. All known life consists of just 20 amino acids. Miller's experiment produced five amino acids, although he was less certain about the results for two of them.

When Miller showed his results to Urey, the latter suggested a paper should be published as soon as possible. (Urey was senior but generously declined to be listed as co-author, lest this lead to Miller getting little to no credit for the work.) The paper appeared in 1953 in the journal Science. "Just turning on the spark in a basic pre-biotic experiment will yield 11 out of 20 amino acids," Miller said in a 1996 interview . The original apparatus has been on display at the Denver Museum of Nature and Science since 2013.

Miller died in 2007. Shortly before he passed, one of his students, Jeffrey Bada, now at the University of San Diego, inherited all his mentor's original equipment. This included several boxes filled with vials of dried residues from the original experiment. Those 1952 samples were re-analyzed the following year using the latest chromatography methods, revealing that the original experiment actually produced even more compounds (25) than had been reported at the time.

Miller had also performed additional experiments simulating conditions similar to those of a water-vapor-rich volcanic eruption, which involved spraying steam from a nozzle at the spark discharge. Bada and several colleagues re-analyzed the original samples from those experiments, too, and found this environment produced 22 amino acids, five amines, and several hydroxylated molecules. So the original experiments were even more successful than Miller and Urey realized.

There have been many, many more experiments on abiogenesis over the ensuing decades, but co-author Joaquin Criado-Reyes of the Universidad de Granada in Spain and his collaborators thought that one potential factor had been overlooked: the role of the borosilicate glass that comprised the flasks and tubes Miller had used. They noted that Miller's simulated atmosphere was highly alkaline, which should cause the silica to dissolve. "Therefore, it could be expected that upon contact of the alkaline water with the inner wall of the borosilicate flask, even this reinforced glass will slightly dissolve, releasing silica and traces of other metal oxides [into the vapor]," the authors wrote.

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Glass flask catalysed famous Miller-Urey origin-of-life experiment

By Jason Arunn Murugesu

25 October 2021

Harold Urey (1893-1981) was a pioneering chemist

US DEPARTMENT OF ENERGY/SCIENCE PHOTO LIBRARY

A famous origin-of-life experiment from the 1950s may have more accurately mimicked nature than we initially thought.

The influential Miller-Urey experiment showed that with just water, ammonia, hydrogen and methane – and electric sparks to mimic lightning – you could form several of the protein precursors necessary for life on Earth. Stanley Miller and Harold Urey’s aim was to recreate the chemical conditions of early Earth.

But what the researchers had never explicitly considered was whether the nature of the container used in the experiment had any effect on the outcome.

“We don’t know why no one looked at this before,” says Ernesto Di Mauro at the Institute of Molecular Biology and Pathology in Rome, Italy. “Sometimes it’s the simplest things that people miss.”

Di Mauro and his team repeated the experiment with the same type of borosilicate glass container used in the original experiment and also reran the study with a container made from Teflon. In a third rerun of the experiment, they added glass chips to the Teflon container mixture.

Read more: The epic hunt for the place on Earth where life started

The team speculated that the reactions performed in the presence of glass would generate more complex molecules because glass contains silicates. Silicate can get dissolved and reabsorbed on to the surface of a mixture and so affect what type of reactions occur, says Di Mauro.

Teflon on the other hand, which wasn’t widely used in the 1950s when Miller and Urey ran their experiment, is chemically inert and has no such effect.

Di Mauro’s team found that the glass beaker did indeed contain the most diverse mixture of complex organic reaction products. Meanwhile, the Teflon beaker with glass chips produced fewer complex compounds – probably because the glass chips had a lower combined surface area than the glass beaker itself. There were even fewer complex compounds when the experiment was run in a Teflon beaker with no glass present.

“The glass is like the rocks on Earth – it catalyses the reaction,” says Di Mauro.

More than 90 per cent of Earth’s crust is made up of silicates, and they are also common on planets like Mars, where they may also have helped to catalyse reactions that might be important for the origin of life.

“I’m surprised no one has looked at this before,” says Valentina Erastova at the University of Edinburgh, UK. “I think this study just confirms for me that the Miller-Urey experiments were even smarter than originally envisaged.”

Scientific Reports DOI: 10.1038/s41598-021-00235-4

• origins of life

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Miller-Urey Experiment

The Miller-Urey Experiment was a landmark experiment to investigate the chemical conditions that might have led to the origin of life on Earth. The scientist Stanley Miller, under the supervision of the Nobel laureate scientist Harold Urey conducted it in 1952 at the University of Chicago. They tried to recreate the conditions that could have existed in the first billion years of the Earth’s existence (also known as the Early Earth) to check the said chemical transformations.

Miller-Urey Experiment And The Primordial Soup Theory

The experiment tested the primordial or primeval soup theory developed independently by the Soviet biologist A.I. Oparin and English scientist J.B.S. Haldane in 1924 and 1929 respectively. The theory propounds the idea that the complex chemical components of life on Earth originated from simple molecules occurring naturally in the reducing atmosphere of the Early Earth, sans oxygen. Lightning and rain energized the said atmosphere to create simple organic compounds that formed an organic “soup”. The so-called soup underwent further changes giving rise to more complex organic polymers and finally life.

The Miller-Urey Experiment In Support Of Abiogenesis

From what was explained in the previous paragraph, it can undoubtedly be considered as a classic experiment to demonstrate abiogenesis. For those who are not conversant with the term, abiogenesis is the process responsible for the development of living beings from non-living or abiotic matter. It is thought to have taken place on the Earth about 3.8 to 4 billion years ago.

Miller-Urey Experiment Apparatus and Procedure

The groundbreaking experiment used a sterile glass flask of 5 liters attached with a pair of electrodes, to hold water (H 2 O), methane (CH 4 ), ammonia (NH 3 ) and hydrogen (H 2 ), the major components of primitive Earth. This was connected to another glass flask of 500 ml capacity half filled with water. On heating it, the water vaporized to fill the larger container with water vapor. The electrodes induced continuous electrical sparks in the gas mixture to simulate lightning. When the gas was cooled, the condensed water made its way into a U-shaped trap at the base of the apparatus.

After electrical sparking had continued for a day, the solution in the trap turned pink in color. At the end of a week, the boiling flask was removed, and mercuric chloride added to prevent microbial contamination. After stopping the chemical reaction, the scientist duo examined the cooled water collected to find that 10-15% of the carbon present in the system was in the form of organic compounds. 2% of carbon went into the formation of various amino acids, including 13 of the 22 amino acids essential to make proteins in living cells, glycine being the most abundant.

Though the result was the production of only simple organic molecules and not a complete living biochemical system, still the simple prebiotic experiment could, to a considerable extent, prove the primordial soup hypothesis.

Miller-Urey Experiment Animation

Chemistry of the miller and urey experiment.

The components of the mixture can react among themselves to produce formaldehyde (CH 2 O), hydrogen cyanide (HCN) and other intermediate compounds.

CO 2 → CO + [O] (atomic oxygen)

CH 4 + 2[O] → CH 2 O + H 2 O

CO + NH 3 → HCN + H 2 O

CH 4 + NH 3 → HCN + 3H 2

The ammonia, formaldehyde and HCN so produced react by a process known as Strecker synthesis to form biomolecules including amino acids.

CH 2 O + HCN + NH 3 → NH 2 -CH 2 -CN + H 2 O

NH 2 -CH 2 -CN + 2H 2 O → NH 3 + NH 2 -CH 2 -COOH (glycine)

In addition to the above, formaldehyde and water can react by Butlerov’s reaction to produce a variety of sugars like ribose, etc.

Though later studies have indicated that the reducing atmosphere as replicated by Miller and Urey could not have prevailed on primitive Earth, still, the experiment remains to be a milestone in synthesizing the building blocks of life under abiotic conditions and not from living beings themselves.

https://www.bbc.co.uk/bitesize/guides/z2gjtv4/revision/1

https://www.juliantrubin.com/bigten/miller_urey_experiment.html

Article was last reviewed on Thursday, February 2, 2023

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One response to “Miller-Urey Experiment”

This experiment is currently seen as not sufficient to support abiogenesis. See Stephen C. Meyer, James Tour.

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Conducting Miller-Urey Experiments

Eric t. parker.

1 School of Chemistry and Biochemistry, Georgia Institute of Technology

James H. Cleaves

2 Earth-Life Science Institute, Tokyo Institute of Technology

3 Institute for Advanced Study

Aaron S. Burton

4 Astromaterials Research and Exploration Science Directorate, NASA Johnson Space Center

Daniel P. Glavin

5 Goddard Center for Astrobiology, NASA Goddard Space Flight Center

Jason P. Dworkin

Manshui zhou, jeffrey l. bada.

6 Geosciences Research Division, Scripps Institution of Oceanography, University of California at San Diego

Facundo M. Fernández

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 H 2 , 200 mmHg of CH 4 , and 200 mmHg of NH 3 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 CO 2 solutions in 1951 3 .

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 elsewhere 4 . After a week of sparking, the contents in the flask were visibly transformed. The water turned a turbid, reddish color 5 and yellow-brown material accumulated on the electrodes 4 . 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 CH 4 /H 2 O/NH 3 /H 2 S gas mixture was tested for its ability to produce the coded sulfur-containing α-amino acids, although these were not detected 6 . Gas chromatography-mass spectrometry (GC-MS) analysis of a CH 4 /NH 3 mixture subjected to an electric discharge showed the synthesis of α-aminonitriles, which are amino acid precursors 7 . In 1972, using a simpler apparatus, first introduced by Oró 8 ( Figure 1B ), Miller and colleagues demonstrated the synthesis of all of the coded α-amino acids 9 and nonprotein amino acids 10 that had been identified in the Murchison meteorite to date, by subjecting CH 4 , N 2 , and small amounts of NH 3 to an electric discharge. Later, using this same simplified experimental design, gas mixtures containing H 2 O, N 2 , and CH 4 , CO 2 , 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 species 11 .

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 experiment 12 , first reported in 1955 4 , and a 1958 H 2 S-containing experiment 13 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 there 14 .

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 O 2 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 H 2 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 H 2 is liberated from reduced gases (such as CH 4 and NH 3 ). Over-pressuring can lead to seal leakage, which can allow atmospheric O 2 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.

1. Setting Up a Manifold/Vacuum System

• 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.
• Use glass stopcocks on the manifold. Apply the minimum amount of vacuum grease necessary to make a seal.
• 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.
• 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.
• 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.
• 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 NH 3 , CO 2 , and H 2 O from entering the pump. Care should be taken, as trapped volatiles, upon warming, may overpressure the manifold and result in glass rupture.
• 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.
• Measure and record the ambient temperature using a suitable thermometer.

2. Preparation of Reaction Flask

• Clean the tungsten electrodes by gently washing with clean laboratory wipes and methanol, and drying in air.
• Introduce a precleaned and sterilized magnetic stir bar, which will ensure rapid dissolution of soluble gases and mixing of reactants during the experiment.
• 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.
• Insert an adapter with a built-in stopcock into the neck of the 3 L reaction flask and secure with a clip.
• Lightly grease all connections to ensure a good vacuum seal.
• 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.
• 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.
• 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 NH 3

• Calculate the necessary pressure of gaseous NH 3 to introduce into the manifold such that 200 mmHg of NH 3 will be introduced into the reaction flask. Details on how to do this are provided in the Discussion section.
• Close Valves 1 and 6, and Stopcock 1 ( Figure 4 ) before introducing any gas into the manifold. Leave the other valves and stopcock open.
• Introduce NH 3 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.
• Introduce NH 3 into the manifold to reach the pressure determined in step 3.1.
• Open Stopcock 1 ( Figure 4 ) to introduce 200 mmHg of NH 3 into the 3 L reaction flask. The NH 3 will dissolve in the water in the reaction flask and the pressure will fall slowly.
• 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.
• Open Valve 1 ( Figure 4 ) to evacuate the manifold to a pressure of <1 mmHg.
• Close Valve 2 ( Figure 4 ) and disconnect the NH 3 gas cylinder from the manifold.

4. Introduction of CH 4

• Calculate the necessary pressure of CH 4 to be introduced into the manifold such that 200 mmHg of CH 4 will be introduced into the 3 L reaction flask. Example calculations are shown in the Discussion section.
• Connect the CH 4 gas cylinder to the manifold.
• Open all valves and stopcocks, except Valve 6 and Stopcock 1 ( Figure 4 ), and evacuate the manifold to a pressure of <1 mmHg.
• Close Valve 1 once the manifold has been evacuated ( Figure 4 ).
• Introduce CH 4 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.
• Introduce CH 4 into the manifold until the pressure calculated in step 4.1, is reached.
• Open Stopcock 1 ( Figure 4 ) to introduce 200 mmHg of CH 4 into the 3 L reaction flask.
• Close Stopcock 1 once the intended pressure of CH 4 has been introduced into the 3 L reaction flask ( Figure 4 ) and record the pressure measured by the manometer.
• Open Valve 1 (Figure 4 ) to evacuate the manifold to <1 mmHg.
• Close Valve 2 ( Figure 4 ) and disconnect the CH 4 cylinder from the manifold.

5. Introduction of Further Gases ( e.g.  N 2 )

• At this point, it is not necessary to introduce additional gases. However, if desired, it is recommended to add 100 mmHg of N 2 . In this case, calculate the necessary pressure of N 2 to be introduced into the manifold such that 100 mmHg of N 2 will be introduced into the 3 L reaction flask. Example calculations are shown in the Discussion section.
• Connect the N 2 gas cylinder to the manifold.
• Introduce N 2 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.
• Introduce N 2 into the manifold until the pressure calculated in step 5.1 is reached.
• Open Stopcock 1 ( Figure 4 ) to introduce 100 mmHg of N 2 into the reaction flask.
• Close Stopcock 1 once the intended pressure of N 2 has been introduced into the reaction flask, ( Figure 4 ) and record the pressure using the manometer.
• Open Valve 1 ( Figure 4 ) to evacuate the manifold to <1 mmHg.
• Close Valve 2 ( Figure 4 ) and disconnect the N 2 cylinder from the manifold.

6. Beginning the Experiment

• 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.
• 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).
• Disconnect the vacuum pump and carefully remove the cold trap and allow venting inside a fully operational fume hood.
• Secure the Tesla coil connected to the high frequency spark generator.
• Connect the opposite tungsten electrode to an electrical ground to enable the efficient passage of electrical current across the gap between the two electrodes.
• Set the output voltage of the spark generator to approximately 30,000 V, as detailed by documents available from the manufacturer.
• 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

• Stop the experiment by turning off the Tesla coil.
• 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

• 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.
• 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

• 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.
• 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.
• 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).
• Cover all open orifices of the reaction flask with aluminum foil and wrap the adapter and its components in aluminum foil.
• Once all the glassware has been wrapped in aluminum foil, pyrolyze for at least 3 hr in air at 500 °C.
• 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 elsewhere 15 , 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.

• 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.

Representative 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-L-cysteine (OPA/NAC) 16 , a chiral reagent pair that tags primary amino groups, yielding fluorescent diastereomer derivatives that can be separated on an achiral stationary phase. Figure 2 shows a chromatogram of an OPA/NAC-derivatized amino acid standard obtained by HPLC coupled to fluorescence detection and QqQ-MS. The amino acids contained in the standard include those typically produced in Miller-Urey type spark discharge experiments. The identities of these amino acids are listed in Table 1 . Representative fluorescence traces of a typical sample and analytical blank are shown in Figure 3 , demonstrating the molecular complexity of Miller-Urey type electric discharge samples. The sample chromatogram in Figure 3 was produced from a spark discharge experiment using the following starting conditions: 300 mmHg of CH 4 , 250 mmHg of NH 3 , and 250 ml of water.

Figure 2. The 3-21 min region of the HPLC-FD/QqQ-MS chromatograms produced from the analysis of an OPA/NAC-derivatized amino acid standard . Amino acid peak identities are listed in Table 1 . The fluorescence trace is shown at the bottom and the corresponding extracted mass chromatograms are shown above. The electrospray ionization (ESI) QqQ-MS was operated in positive mode and monitored a mass range of 50-500 m/z. The ESI settings were: desolvation gas (N 2 ) temperature: 350 °C, 650 L/hr; capillary voltage: 3.8 kV; cone voltage: 30 V. The unlabeled peaks in the 367 extracted ion chromatogram are the 13 C 2 peaks from the 365 extracted ion chromatogram, as a result of the approximately 1% natural abundance of 13 C. Click here to view larger image .

Table 1. Peak identities for amino acids detected in the standard and that are typically produced in Miller-Urey type spark discharge experiments.

Figure 3. The 3-21 min region of the HPLC-FD chromatograms representative of Miller-Urey type spark discharge experiments. Peaks were identified and quantitated by retention time and mass analysis of target compounds compared to a standard and analytical blank. All target analytes with coeluting fluorescence retention times can be separated and quantitated using mass spectrometry, except for α-AIB and L-β-ABA (peaks 13 and 14), and D/L-norleucine, which coelutes with D/L-leucine (peak 23), under the chromatographic conditions used. D/L-norleucine was added as an internal standard to samples and analytical blanks during sample preparation. Amino acid separation was achieved using a 4.6 mm x 250 mm, 5 μm particle size Phenyl-Hexyl HPLC column. The mobile phase was composed of: A) ultrapure water (18.2 MΩ cm, <5 ppb TOC), B) methanol, and C) 50 mM ammonium formate with 8% methanol, at pH 8. The gradient used was: 0-5 min, 100% C; 5-15 min, 0-83% A, 0-12% B, 100-5% C; 15-22 min, 83-75% A, 12-20% B, 5% C; 22-35 min, 75-35% A, 20-60% B, 5% C; 35-37 min, 35-0% A, 60-100% B, 5-0% C; 37-45 min, 100% B; 45-46 min, 100-0% B, 0-100% C 46-55 min, 100% C. The flow rate was 1 ml/min. Click here to view larger image .

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 preparing samples for analysis, care must be taken to avoid organic contamination. The risk of contamination can be minimized by wearing nitrile gloves, a laboratory coat, and protective eyewear. Be sure to work with samples away from one's body as common sources of contamination include finger prints, skin, hair, and exhaled breath. Avoid contact with wet gloves and do not use any latex or Nylon materials. Second, thorough degassing of the reaction flask prior to gas addition into the reaction flask is critical. The presence of even small amounts of molecular oxygen in the reaction flask poses an explosion risk when the spark is discharged into inflammable gases such as CH 4 . While degassing the flask, the water inside the flask will boil, which will prevent a stable reading. At this stage there are two options: 1) degas the flask via freeze-thaw cycles (typically 3 are used), or 2) simply degas the liquid solution. In the latter case, some water will be lost, however, the amount will be relatively minor compared to the remaining volume. Third, a well-equipped and efficient setup must be carefully constructed to establish a consistent spark across the electrodes throughout the entirety of the experiment. BD-50E Tesla coils are not designed for prolonged operation, as they are intended for vacuum leak detection. Intermittent cooling of the Tesla coil is thus recommended for extended operational lifetime. There are multiple ways of achieving this. One simple way is to attach a timer in-line between the spark tester and its power supply and program the timer such that it alternates in 1 hour on/off cycles. Cooling the Tesla coil with a commercial fan may also be necessary to prolong the life of the Tesla coil. The Tesla coil tip should be touching or almost touching one of the tungsten electrodes; a distance between the two of approximately 1 mm or less. Additionally, an intense discharge can be achieved using a length of conductive metal wire with a loop in one end draped lightly over the electrode opposite the one touching the Tesla coil to avoid breaking the seal to the contents. It is also recommended to have a second spark generator available in case the primary spark generator fails due to extended use.

There are many additional notes worth keeping in mind when carrying out various steps in the protocol outlined here. When preparing the manifold system for an experiment and using a mercury manometer, it is generally conceded that a precision of 1 mmHg is the best achievable, due to the resolution of the human eye. Some gases may present conductivity problems with resistance-based gauges. Mercury manometers present potential spill hazards, which should be prepared for in advance.

While assembling the 3 L reaction flask, the use of silicon vacuum grease can mitigate potential organic contamination, but care should be taken to remove this thoroughly between runs. Failure to do so will result in the accumulation of silica deposits during high-temperature pyrolysis, which can interfere with vacuum seals. Additionally, the tungsten electrodes are commercially available as 2% thoriated tungsten and should be annealed into half-round ground glass fittings . Do not pyrolyze the glass-fitted tungsten electrodes in an oven. The coefficients of thermal expansion of tungsten and glass are different and heating above 100 °C may weaken the seal around the glass annealed electrodes and introduce leaks to the system. Also, ultrapure water can be introduced into the 3 L reaction flask by pouring, using care to avoid contact with any grease on the port used, or by pipetting, using a prepyrolyzed glass pipette. The aqueous phase in the reaction flask can be buffered, if desired. For example, Miller and colleagues 9 buffered the solution to pH ~8.7 with an NH 3 /NH 4 Cl buffer. To do this the aqueous phase is made 0.05 M in NH 4 Cl prior to introducing it into the reaction flask. NH 4 Cl of 99.5% purity, or greater, should be used. The remainder of the NH 3 is then added to the reaction flask as a gas.

In preparation for gas introduction into the 3 L reaction flask, the flask can be secured onto the manifold by placing the flask on a cork ring, set atop a lab jack and gently raising the flask assembly until a snug connection is achieved. When checking for leaks, it is worth noting that likely sources of leaks include poor seals at the junctions of the half-round ground glass joints, which attach the tungsten electrodes to the reaction flask, and the stopcock of the adapter attached to the neck of the 3 L reaction flask. If leaks from these sources are detected, carefully remove the 3 L reaction flask from the manifold, wipe these areas with clean laboratory tissue, reapply a fresh coating of vacuum grease and reattach the flask to the manifold to search for leaks. If no leaks are found, proceed to introduce gases into the reaction flask.

While introducing gases into the apparatus, gas cylinders should be securely fastened to a support. Care should be taken to introduce gases slowly. Valves on gas cylinders should be opened slowly and carefully while monitoring the manometer to avoid over-pressuring the glassware and attached fittings. It is important to note that while adding NH 3 into the reaction flask, because NH 3 is appreciably soluble in water below the pK a of NH 4 + (~9.2), essentially all of the NH 3 gas introduced into the manifold will dissolve in the aqueous phase, rendering the final pressure in the flask and manifold as the vapor pressure of water at the ambient temperature. Once this pressure is attained, one may assume the transfer is complete. The following are examples of the calculations that must be executed in order to precisely introduce gases into the reaction flask at their desired pressures:

Introduction of Gaseous NH 3

Due to the solubility of NH 3 , essentially all of it will transfer from the manifold to the reaction flask and dissolve in the aqueous phase as long as the NH 3 in the manifold is at a higher pressure than the vapor pressure of water in the reaction flask. Therefore, the ambient temperature should be noted and the vapor pressure of water at that temperature should be referenced prior to introducing NH 3 into the manifold. The target pressure of NH 3 to be introduced into the reaction flask should be equal to the target pressure of NH 3 in the 3 L reaction flask, plus the vapor pressure of water in the reaction flask, at the recorded ambient temperature. For example, at 25 °C, the vapor pressure of water is approximately 24 mmHg. Thus, in order to introduce 200 mmHg of NH 3 into the reaction flask, load roughly 225 mmHg of NH 3 into the manifold prior to transferring NH 3 from the manifold and into the reaction flask. This will result in approximately 200 mmHg of NH 3 being introduced into the reaction flask.

Introduction of CH 4

After NH 3 addition and its dissolution in the aqueous phase, the pressure in the headspace of the reaction flask will be equal to the vapor pressure of water at 25 °C, approximately 24 mmHg. This value will be used, in conjunction with the example manifold shown in Figure 4 , to carry out a calculation for how much CH 4 to introduce into the manifold such that 200 mmHg of CH 4 will be introduced into the reaction flask:

P 1 = total pressure desired throughout the entire system, including the reaction flask V 1 = total volume of the entire system, including the reaction flask

P 2 = pressure of CH 4 needed to fill manifold volume prior to introduction into reaction flask V 2 = volume of manifold used for gas introduction

P 3 = pressure already in the headspace of the reaction flask V 3 = volume of the reaction flask

P 1 = 200 mmHg of CH 4 + 24 mmHg of H 2 O = 224 mmHg V 1 = 3,000 ml + 100 ml + 300 ml + 40 ml + 20 ml + 3,000 ml + 40 ml + 500 ml = 7,000 ml

P 2 = pressure of CH 4 being calculated V 2 = 100 ml + 300 ml + 40 + 20 + 3,000 ml+ 40 ml + 500 ml = 4,000 ml

P 3 = 24 mmHg of H 2 O V 3 = 3,000 ml

Introduction of N 2

After introduction of CH 4 , the headspace of the reaction flask is occupied by 200 mmHg of CH 4 and 24 mmHg of H 2 O for a total of 224 mmHg. This value will be used, along with the dimensions of the example manifold shown in Figure 4 , to calculate the N 2 pressure that needs to be introduced into the manifold such that 100 mmHg of N 2 will be introduced into the reaction flask:

P 2 = pressure of N 2 needed to fill manifold volume prior to introduction into reaction flask V 2 = volume of manifold used for gas introduction

P 1 = 24 mmHg of H 2 O + 200 mmHg of CH 4 + 100 mmHg of N 2 = 324 mmHg V 1 = 3,000 ml + 100 ml > + 300 ml + 40 ml + 20 ml + 3,000 ml + 40 ml + 500 ml = 7,000 ml

P 2 = pressure of N 2 being calculated V 2 = 100 ml + 300 ml + 40 ml + 20 ml + 3,000 ml + 40 ml + 500 ml = 4,000 ml

P 3 = 200 mmHg of CH 4 + 24 mmHg of H 2 O = 224 mmHg V 3 = 3,000 ml

Figure 4. Manifold/vacuum system used to introduce gases into the 3 L reaction flask. Valves controlling gas flow are labeled as V 1 - V 8 , while stopcocks controlling gas flow are labeled as S 1 and S 2 . It is worth noting that while Valves 1, 2, and 6, and Stopcock 1 are referred to explicitly in the protocol, the other valves and stopcock in the manifold shown here are useful for adding or removing volume ( i.e.  holding flasks) to or from the manifold. For example, when introducing gases into the manifold at relatively high pressures (approximately 500 mmHg or greater), it is advised that the experimenter makes use of all purge flasks attached to the manifold to increase the accessible volume of the manifold and help minimize the risk of over-pressuring the manifold.

After initiating the experiment, the system must be checked on regularly to ensure the experiment is running properly. Things to check include: 1) the spark generator is producing a spark, and 2) the spark is being generated across the tungsten electrodes in a continuous manner. If the above conditions are not met, disconnect the Tesla coil from its power supply and replace it with the backup Tesla coil. Meanwhile, repairs to the malfunctioning Tesla coil can be made. Often times, the contact plates inside the spark generator housing can become corroded from extended use and should be polished, or replaced.

Upon completion of the experiment, the gases in the head-space may be irritating to the respiratory system. Harmful gases, such as hydrogen cyanide 4 can be produced by the experiment. If the experimenter is not collecting gas samples for analysis, it may be helpful to connect the apparatus to a water aspirator to evacuate volatiles for approximately one hour after completion of the experiment, while the apparatus remains in the fume hood, prior to collecting liquid samples. For safety reasons, it is advised that the apparatus is vented in a fully-operational fume hood. Sample collection should be performed in an operational fume hood and sample handling in a positive-pressure HEPA filtered flow bench is recommended.

Among the numerous types of products formed by spark discharge experiments, amino acids are of significance. Amino acids are synthesized readily via the Strecker synthesis 17 . The Strecker synthesis of amino acids involves the reaction of aldehydes or ketones and HCN generated by the action of electric discharge on the gases introduced into the reaction apparatus, which upon dissolving in the aqueous phase, may react with ammonia to form α-aminonitriles that undergo hydrolysis to yield amino acids. This is, of course, but one mechanism of synthesis, and others may also be operative, such as direct amination of precursors including acrylonitrile to give β-alanine precursors, or direct hydrolysis of higher molecular weight tholin-like material to give amino acids directly, by-passing the Strecker mechanism.

Amino acid contamination of the samples produced by Miller-Urey experiments can occur if the precautions mentioned earlier are not followed explicitly. During sample analysis, it is important to search for signs of terrestrial contamination that may have originated from sample handling or sample storage. The use of OPA/NAC 16 in conjunction with LC-FD techniques allows for the chromatographic separation of D- and L-enantiomers of amino acids with chiral centers and their respective, individual quantitation. Chiral amino acids synthesized by the experiment should be racemic. Acceptable experimental error during the synthesis of amino acids with chiral centers is generally considered to be approximately 10%. Therefore chiral amino acid D/L ratios suggestive of enrichment in one enantiomer by more than 10% is a good metric by which to determine if the sample has been contaminated.

The methods presented here are intended to instruct how to conduct a Miller-Urey type spark discharge experiment; however, there are limitations to the technique described here that should be noted. First, heating the single 3 L reaction flask ( Figure 1B ), will result in condensation of water vapor onto the tips of the electrodes, dampening the spark, and reducing the generation of radical species that drive much of the chemistry taking place within the experiment. Furthermore, the use of a heating mantle to heat the apparatus is not necessary to synthesize organic compounds, such as amino acids. This differs from Miller's original experiment where he used a more complex, custom-built, dual flask apparatus ( Figure 1A ) 5 and heated the small flask at the bottom of the apparatus, which had water in it ( Figure 1A ). Heating the apparatus helped with circulation of the starting materials and aimed to mimic evaporation in an early Earth system. Second, the protocol detailed here recommends a 1 hr on/off cycle when using the Tesla coil, which effectively doubles the amount of time an experiment takes to complete, compared to the experiments conducted by Miller, as he continuously discharged electricity into the system 4 . Third, as spark generators are not intended for long-term use, they are prone to malfunction during prolonged use and must be regularly maintained and sometimes replaced by a back-up unit, if the primary spark generator fails during the course of an experiment. Last, the protocol described here involves the use of glass stopcocks, which require high vacuum grease to make appropriate seals. If desired, polytetrafluoroethylene (PTFE) stopcocks can be used to avoid vacuum grease. However, if examining these stopcocks for potential leaks with a spark leak detector, be cautious to not overexpose the PTFE to the spark as this can compromise the integrity of the PTFE and lead to poor seals being made by these stopcocks.

The significance of the method reported here with respect to existing techniques, lies within its simplicity. It uses a commercially available 3 L flask, which is also considerably less fragile and easier to clean between experiments than the original design used by Miller 5 . Because the apparatus is less cumbersome, it is small enough to carry out an experiment inside a fume hood.

Once the technique outlined here has been mastered, it can be modified in a variety of ways to simulate numerous types of primitive terrestrial environments. For example, more oxidized gas mixtures can be used 14,18,19 . Furthermore, using modifications of the apparatus, the energy source can be changed, for example, by using a silent discharge 4 , ultraviolet light 20 , simulating volcanic systems 4,12,21 , imitating radioactivity from Earth's crust 22 , and mimicking energy produced by shockwaves from meteoritic impacts 23 , and also cosmic radiation 18,19 .

The classic Miller-Urey experiment demonstrated that amino acids, important building blocks of biological proteins, can be synthesized using simple starting materials under simulated prebiotic terrestrial conditions. The excitation of gaseous molecules by electric discharge leads to the production of organic compounds, including amino acids, under such conditions. While amino acids are important for contemporary biology, the Miller-Urey experiment only provides one possible mechanism for their abiotic synthesis, and does not explain the origin of life, as the processes that give rise to living organisms were likely more complex than the formation of simple organic molecules.

Disclosures

The authors declare no competing financial interests.

Acknowledgments

This work was jointly supported by the NSF and NASA Astrobiology Program, under the NSF Center for Chemical Evolution, CHE-1004570, and the Goddard Center for Astrobiology. E.T.P. would like to acknowledge additional funding provided by the NASA Planetary Biology Internship Program. The authors also want to thank Dr. Asiri Galhena for invaluable help in setting up the initial laboratory facilities.

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• NOT EXACTLY ROCKET SCIENCE

Scientists finish a 53-year-old classic experiment on the origins of life

In 1958, a young scientist called Stanley Miller electrified a mixture of simple gases, designed to mimic the atmosphere of our primordial lifeless planet. It was a sequel to one of the most evocative experiments in history, one that Miller himself had carried five years earlier. But for some reason, he never finished his follow-up. He dutifully collected his samples and stored them in vials but, whether for ill health or dissatisfaction, he never analysed them.

The vials languished in obscurity, sitting unopened in a cardboard box in Miller’s office. But possessed by the meticulousness of a scientist, he never threw them away. In 1999, the vials changed owners. Miller had suffered a stroke and bequeathed his old equipment, archives and notebooks to Jeffrey Bada , one of his former students. Bada only twigged to the historical treasures that he had inherited in 2007. “Inside, were all these tiny glass vials carefully labeled, with page numbers referring Stanley’s laboratory notes. I was dumbstruck. We were looking at history,” he said in a New York Times interview .

By then, Miller was completely incapacitated. He died of heart failure shortly after, but his legacy continues. Bada’s own student Eric Parker has finally analysed Miller’s samples using modern technology and published the results, completing an experiment that began 53 years earlier.

Miller conducted his original 1953 experiment as a graduate student, working with his mentor Harold Urey. It was one of the first to tackle the seemingly insurmountable question of how life began. In their laboratory, the pair tried to recreate the conditions on early lifeless Earth, with an atmosphere full of simple gases and laced with lightning storms. They filled a flask with water, methane, ammonia and hydrogen and sent sparks of electricity through them.

The result, both literally and figuratively, was lightning in a bottle. When Miller looked at the samples from the flask, he found five different amino acids – the building blocks of proteins and essential components of life.

The relevance of these results to the origins of life is debatable, but there’s no denying their influence. They kicked off an entire field of research, graced the cover of Time magazine and made a celebrity of Miller. Nick Lane beautifully describes the reaction to the experiment in his book, Life Ascending : “Miller electrified a simple mixture of gases, and the basic building blocks of life all congealed out of the mix. It was as if they were waiting to be bidden into existence. Suddenly the origin of life looked easy.”

Over the next decade, Miller repeated his original experiment with several twists. He injected hot steam into the electrified chamber to simulate an erupting volcano, another mainstay of our primordial planet. The samples from this experiment were among the unexamined vials that Bada inherited. In 2008, Bada’s student Adam Johnson showed that the vials contained a wider range of amino acids than Miller had originally reported in 1953.

Miller also tweaked the gases in his electrified flasks. He tried the experiment again with two newcomers – hydrogen sulphide and carbon dioxide – joining ammonia and methane. It would be all too easy to repeat the same experiment now. But Parker and Bada wanted to look at the original samples that Miller had himself collected, if only for their “considerable historical interest”.

Using modern techniques, around a billion times more sensitive than those Miller would have used, Parker identified 23 different amino acids in the vials, far more than the five that Miller had originally described. Seven of these contained sulphur, which is either a first for science or old news, depending on how you look at it. Other scientists have since produced sulphurous amino acids in similar experiments, including Carl Sagan . But unbeknownst to all of them, Miller had beaten them to it by several years. He had even scooped himself – it took him till 1972 to publish results where he produced sulphur amino acids!

The amino acids in Miller’s vials all come in an equal mix of two forms, each the mirror image of the other. You only see that in laboratory reactions – in nature, amino acids come almost entirely in one version. As such, Parker, like Miller before him, was sure that the amino acids hadn’t come from a contaminating source, like a stray bacterium that had crept into the vials.

Imagine then, a young and violent planet, wracked with exploding volcanoes, noxious gases and lightning strikes. These ingredients combined to brew a “primordial soup”, fashioning the precursors of life in pools of water. On top of that, meteorites raining down from space could have added to the accumulating molecules. After all, Parker found that the amino acid cocktail in Miller’s samples is very similar to that found on the Murchison meteorite , which landed in Australia in 1969.

These are powerful images, so why aren’t people more excited? Echoing many sources I spoke to, Jim Kasting , who studies the evolution of Earth’s atmosphere, said, “I am underwhelmed by it.” The main problem with the study is that Miller was probably wrong about the conditions on early Earth.

By analysing ancient rocks, scientists have since found that Earth was never particularly teeming in hydrogen-rich gases like methane, hydrogen sulphide or hydrogen itself. If you repeat Miller’s experiment with a more realistic mixture – heavy in carbon dioxide and nitrogen, with just trace amounts of other gases – you’d have a hard time finding amino acids in the resulting brew.

Parker accepts the problem, but he suggests that a few specific places on the planet may have had the right conditions. Exploding volcanoes, for example, throw up masses of sulphurous compounds, as well as methane and ammonia. These gases, belched forth into lightning storms , could have produced amino acids that rained out and gathered in tidal pools. But Kasting still isn’t convinced. “Even then the reduced gases would not be as concentrated as they are in this experiment.”

Even if our young planet had the right conditions to produce amino acids, that’s a less impressive feat than it appeared in the 1950s. “Amino acids are old hat and are a million miles from life,” says Nick Lane. Indeed, as Miller’s experiments showed, it’s not difficult to create amino acids. The far bigger challenge is to create nucleic acids – the building blocks of molecules like RNA and DNA. The origin of life lies in the origin of these “replicators”, molecules that can make copies of themselves. Lane says, “Even if you can make amino acids (and nucleic acids) under soup conditions, it has little if any bearing on the origin of life.”

The problem is that replicators don’t spontaneously emerge from a mixture of their building blocks, just as you wouldn’t hope to build a car by throwing some parts into a swimming pool. Nucleic acids are innately “shy”. They need to be strong-armed into forming more complex molecules, and it’s unlikely that the odd bolt of lightning would have been enough. The molecules must have been concentrated in the same place, with a constant supply of energy and catalysts to speed things up. “Without that lot, life will never get started, and a soup can’t provide much if any of that,” says Lane.

Deep-sea vents are a better location for the origins of life. Deep under the ocean’s surface, these rocky chimneys spew out superheated water and hydrogen-rich gases. Their rocky structures contain a labyrinth of small compartments that could have concentrated life’s building blocks into dense crowds, and minerals that would have catalysed their get-togethers. Far away from visions of languid soups, these churning environments are the current best guess for the site of life’s hatchery.

So Miller’s iconic experiment, and its now-completed follow-ups, probably won’t lay out the first steps of life. As Adam Rutherford, who is writing a book on the origin of life, says, “It’s really a historical piece, like finding that Darwin had described a Velociraptor in one of his notebooks.”

If anything, the analysis of Miller’s vials is a testament to the value of meticulous scientific work. Here was a man who prepared his samples so cleanly, who recorded his notes so thoroughly, and who stored everything so carefully, that his contemporaries could pick up where he left off five decades later.

Reference: Parker, Cleaves, Dworkin, Glavin, Callahan, Aubrey, Lazcano & Bada. 2011. Primordial synthesis of amines and amino acids in a 1958 Miller H2S-rich spark discharge experiment. PNAS http://dx.doi.org/10.1073/pnas.1019191108

Photos by Carlos Gutierrez and Marco Fulle

More on origins:

• A possible icy start for life
• Tree or ring: the origin of complex cells
• The origin of complex life – it was all about energy

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Does the Miller-Urey experiment explain the origin of life?

According to the Miller-Urey experiment, the early earth atmosphere could have supported the formation of amino acids. This could provide an explanation for the origin of life. However, I do not understand how life could have started from amino acids (or proteins) which do not self replicate. Certain types of RNA do self-replicate, and it is plausible how life could have evolved from there.

Does it matter if amino acids came before RNA if they did? Is it correct to assume that in the RNA world, life somehow learnt how to use amino acids?

• biochemistry
• molecular-evolution
• protein-evolution

3 Answers 3

This is a common question. I think the experiment and its conclusions are often misunderstood.

Originally, the null hypothesis was that the ingredients for life could not have come about spontaneously, from inert, inorganic molecules. There was no evidence of strictly physical, non-biological processes being able to produce compounds that were necessary for the proteinaceous life as we know it. In other words, abiogenesis was therefore not supported by any line of positive evidence... until the Miller-Urey experiment. The experiment showed that the ingredients could arise through plausible, natural conditions prior to the existence of life! It bridges the gap between inert, inorganic, non-biological, and the rich soup of complex molecules that would - arguably - be necessary for anything like abiogenesis to even be a consideration based on empirical observation.

Of course the presence of amino acids is not evidence for the abiogenic origin of life. Life is not protein nor amino acid alone. But demonstrating that complex biological ingredient chemistry occurs spontaneously in large abundance - that sounds like a great take-off point for an abiogenic origin of life! That's really all the experiment achieved. In its historical context, it is a very impressive discovery, but it is certainly not a complete explanation, merely something that makes myriad biochemical explanations possible (and perhaps even plausible!). Perhaps you can now better appreciate why it excited and continues to excite biochemists working on trying to understand the chemical origins of life. The Miller-Urey experiment is foundational.

As for the transition from RNA to an RNA-protein origin of life, I quote briefly from another answer elsewhere here:

Regarding the transition from RNA-only to RNA-protein world, peptides function as cofactors for some ribozymes. Amino acids and peptides are known to have existed in the prebiotic environment and have been found in space (glycine has been found in comets, along with other 70 amino acids).

• $\begingroup$ The actual problem with this experiment is that the environmental conditions implied in it might have never existed on Earth - at least, this is what the geologists say. $\endgroup$ –  Roger V. Commented Mar 24, 2021 at 14:07
• 6 $\begingroup$ The discussion about the historical validity of the conditions assumed in the experiment has been ongoing and rich and full of disagreement, even among experts; similar experiments have been performed with many adjustments since. It's worth taking a look into it if interested! $\endgroup$ –  S Pr Commented Mar 24, 2021 at 14:09
• $\begingroup$ This is reaching back several decades, but has the Miller Urey experiment ever been replicated? I thougt not... am I mistaken? I'd love to know. $\endgroup$ –  anongoodnurse Commented Mar 24, 2021 at 16:41
• 1 $\begingroup$ @anongoodnurse thousands of times, with thousnds of variations. Miller Urey is just the first such experiment which is why it famous. The wiki has a nice summary of follow up work en.wikipedia.org/wiki/Miller%E2%80%93Urey_experiment $\endgroup$ –  John Commented May 11 at 13:02
• $\begingroup$ @John - I didn't know that! Thanks! $\endgroup$ –  anongoodnurse Commented May 11 at 13:23

Answers gets more precise but conclusions are far stretched.

Going from amino acids to proteins or peptides (small proteins) is an unsolved problem. In water where they are supposed to be stored, they cannot combine. Water molecule occupies the link necessary to produce a valid amino-acid chain by which all amino-acids are linked into a protein.

Also, it is well less known that some mechanism must protect amino acids from linking by side chains, which is tough to avoid, and destroys the achievement of a correct peptide or protein. Chemists know how to do it with specialized chemicals and specialized laboratory material.

Nobody ever described a simple chemical formula to bring correctly amino acid together to form proteins, from chemical material that exists in a pre-biotic world.

Needless to say, that in the four classes of the building block of the building block of the life, amino acid, nucleotic acid, sugars, lidips, nobody knows how to pre-biotically control chemistry to assemble more complex molecules. It is almost impossible to bring a correctly linked chain of nucleotic acid in proper order (they must be obeying precise linkage (links to 3' and 5' ends). Ribose that is part of it, has still no explanation for its coming into existence in a prebiotic world.

The idea that time helps all this by failures and restart is misleading. Misaligned molecules (which is so highly inevitable) remain as they are. Source material is lost. Accumulation of chemical impurities impairs the success of having something useful. Delicate molecules like ARN have a short half-life. An extended explanation of difficulties of abiogenesis is available at https://www.youtube.com/watch?v=71dqAFUb-v0 . This a seriously challenging 12 episodes series about the chemistery required for abiogenesis.

We have been raised by an education system far outdated about the difficulties of abiogenesis (that make appear it so much more possible that is was in reality). This is why no significant success were made since the 60 about this subject.

• $\begingroup$ except self forming nucleic acid chian have been done, it is actually remarkably easy if you not keep purifiying out your samples. nature.com/articles/nature08013 and pubs.acs.org/doi/10.1021/jacs.0c12955 you may want to update your information A LOT has changed since the 60's $\endgroup$ –  John Commented May 11 at 12:17
• $\begingroup$ You may want to learn prebiotic chemistry from soneone serious not a creationist shill who openly lies. youtube.com/… as an example 7 years is not a short half-life, it is more than enough time to get billions of reactions, James just lies about the relevent chemistry or worse does not understand it. $\endgroup$ –  John Commented May 11 at 12:55

The Miller-Urey experiment does not explain the origin of life. It just propose a plausible way to develop life from organic matter, as part of the abiogenesis model of the origin of life. A better approach to understand the theories of life origin is looking for a context.

We already know, by the cellular theory, that cell are the main component of organisms and that just cells could originate others cells. So, the research is focused on finding a way to create at least one cell. From knowed compounds, cells could be modeled as a set of autoregulated biomolecules. Just two of them are used explicitly for replication: nucleic acid and proteins. If you think about this two components, the Miller-Urey experiment just propose a way to develop simple proteins from aminoacids, and aminoacids from inorganic matter. Amazing, but not enough for explaining the complexity of actual proteins and enzymes.

But there are other theories and facts that works well with this experiment. Some non-translated olygopeptides have intracellular functions. The PAHs world theory proposes a way to develop nitrogenated bases from aromatic hydrocarbons. This bases are precursors for RNAs and DNAs. The RNAs world hypothesis establish a way to develop life from RNA and not DNA or proteins. The phospholipid bilayer have some common patterns with micelles and all this mechanisms would be part of the Last Universal Common Ancestor (LUCA). But there is no evidence of the relationships between this theories and maybe there are just too simple for explaining life.

Maybe the first cell had come from the space, where a pre-term version the krebs cycle have been demostrated to work. We could think on it as "biogenesis" until "abiogenesis" could be demonstrated.

• 2 $\begingroup$ "first cell from space" does not solve the problem it just side steps it, also you may be interested is some of the more recent work on RNA synthesis. pubmed.ncbi.nlm.nih.gov/33547911 $\endgroup$ –  John Commented Mar 24, 2021 at 16:17

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Reflections on Stanley Miller’s Death and Life

Chemist Stanley L. Miller died in late May (March 7, 1930-May 20, 2007).

Even if you don’t recognize the name, you are probably familiar with his famous experiment. Virtually every introductory biology text describes the work Miller performed in the early 1950s. Miller showed that the primitive atmosphere of early Earth could, in principle, generate amino acids, one of the key building blocks of life.

Miller’s work was the first experimental validation of the Oparin-Haldane hypothesis and launched origin-of-life studies as an experimental research program. His success prompted scientists to conduct similar experiments in the quest to discover chemical routes to other critical biomolecules.

Implications of the Miller-Urey Experiment

Beyond its scientific impact, Miller’s work has had profound philosophical and even theological consequences. For many people, the generation of amino acids from simple chemical compounds thought to be present in early Earth’s atmosphere meant that life could originate all on its own without the need for a Creator. I’ve met many people who have struggled with their faith as Christians after learning about this experiment in high school and college biology courses. And I’ve known many nontheists who use this experiment as part of the rationale to reject belief in a personal God.

It’s probably safe to say that next to Charles Darwin, Stanley Miller has probably been one of the most disliked and maligned scientists within the Christian community. His work and, unfortunately, his reputation have been the subject of attack and ridicule by Christian apologists for the last half-century. For many Christians, Miller—like Darwin—embodied atheistic materialism. By attacking Miller—his character and his work—Christians saw themselves striking back against the threat they perceived from science.

Miller’s death has prompted me to reflect on how Christians often inappropriately view and treat scientists, particularly evolutionary biologists. We must reconsider the way we engage in apologetics and evangelism. The bottom line: If we ever want to effectively engage skeptics within the scientific community, we must avoid alienating them with our apologetics methodology. We cannot treat them as the enemy of the Christian faith, but rather must regard scientists as people occupying an important mission field. We need to look for opportunities to build bridges, not erect walls, as we present the Christian faith to them.

An Amazing Story

The behind-the-scenes story of the Miller-Urey experiment is remarkable in many respects, and provides an opportunity for Christians to affirm Stanley Miller and Harold Urey, in spite of the profound philosophical and scientific differences on life’s origin. These points of affirmation represent shared values and common ground that many scientists and Christians can stand on.

Miller conducted his famous experiment as a young graduate student at the University of Chicago. After hearing Nobel Laureate Harold Urey lecture on the current ideas about the early Earth’s atmosphere, he approached the eminent scientist and asked if he could join his lab and attempt to verify the Oparin-Haldane hypothesis. Urey initially declined out of concern for Miller’s future, viewing the work as too risky for a graduate student to pursue.

Miller persisted, however, and Urey reluctantly agreed. But in Miller’s best interest, Urey gave him a time limit to show progress on the project. The rest is history. Miller was able to generate amino acids and alpha-hydroxy acids from a simple mixture of gases in short order and later determined that the reaction mechanism was closely related to the Strecker Reaction.

In an act of selflessness, Urey insisted that Miller publish the work as the sole author, contrary to standard academic practices. (Usually the research advisor is listed as the author on all papers generated within his or her laboratory.) Urey’s name rightfully belonged on the paper submitted to Science, but Urey recognized the significance of Miller’s work and wanted him to be the full beneficiary. If Urey’s name had appeared on the paper, he would have taken all the attention away from Miller.

And what attention Miller received! When published in Science , Miller’s results met with instantaneous and worldwide excitement and fanfare. Both the New York Herald Tribune and New York Times wrote about Miller and his discovery on the same day that his paper appeared in Science. A short time later Time , Newsweek , and Life wrote about his work. At twenty-three years of age, Stanley Miller was suddenly propelled to worldwide fame.

Most graduate students are drawn to science because of their fascination with nature and a deep desire to understand how it all works. This allure provides the motivation to work long, hard hours in the laboratory. I am sure that this was true for Miller. Still, in the back of the minds of most young scientists resides the hope that their research will lead to a breakthrough so significant that it will propel them to worldwide fame. More often than not, this great expectation never happens.

But Stanley Miller lived the dream.

This story endears Miller and Urey to me. As a scientist, I can’t help but marvel at Miller’s accomplishments and impact on science as a young chemist. And as a Christian, I am impressed with the compassion and generosity Urey displayed toward Miller, and his sincere concern for the young scientist’s interests and career. This painfully reminds me that sometimes non-Christians do a better job at living out these Godly qualities than many Christians, including me.

Miller’s persistence and courage are to be admired as well, whether one is a Christian or not. As a young scientist, Miller pursued a question that many established scientists shied away from, because it was too risky. He was not dissuaded by a Nobel Laureate’s protests. As Christians, persistence and courage are two virtues that we need to strive for as we seek to live out our faith, particularly in pursuit of God’s calling on our lives. Miller’s life reminds me that sometimes non-Christians manifest these types of virtues to a greater extent than Christians.

A Poignant Encounter

The first time I saw Miller was at the 1999 meeting of the International Society for the Study of the Origin of Life (ISSOL ’99) held at the University of California, San Diego. Even though I disagree with the views Miller held about the explanation for the origin of life, I was thrilled to be in the presence of such an important figure. I saw him at a poster session, walking around the room reading and discussing the presentations with the participants.

It was clear that Miller was a mentor to many younger scientists, offering his insights and constructive feedback about their work and encouraging them. Miller willingly and selflessly invested himself into the lives of others, a quality that merits admiration.

The next time I saw him was at ISSOL ’02 held in Oaxaca, Mexico. My reaction was very different this time around. Instead of excitement, I felt a strong sense of sorrow and compassion for him. Miller was confined to a wheelchair and was clearly suffering from a debilitating sickness. He appeared feeble and required the constant attention of a caretaker. While other conference participants made their way to the veranda of the conference hotel to enjoy a coffee break or have lunch, Miller remained behind.

During the sessions, a special place was reserved at the front of the room for him. It was sad to see him wheeled in right before each session started, a constant reminder to all of us of the struggle he faced. Miller appeared to be in the last years of his life.

One particularly heartrending moment came during a session on prebiotic chemistry, when the session chairman pointed out during the introduction that Miller’s work was no longer relevant. He was quick to extend respect to Miller and qualified his assessment by emphasizing the work’s historical value, but the harm had been done. The painful reality was that Miller had devoted his life to understanding the origin of life and, at the end of his life, his most important contribution was no longer regarded as genuinely significant to the current paradigm.

At that time, I truly saw Miller as a human being, not as a caricature to be ridiculed or an embodiment of atheism to be assaulted. He was someone like me, confronted with disappointments and frustrations that arise from life’s challenges and difficulties so severe that they bring the ultimate questions about life’s meaning and purpose to the forefront.

Stanley Miller and the Gospel

As Miller reached the end of his life, was he troubled about his fate after death? Was he concerned about his life’s meaning and purpose? I have no way of knowing if he asked questions like this. But if he did, it would be hard for me to imagine that he would have turned to the Christian faith to see if it had any answers, if for no other reason than the vitriol spewed at him by Christians for the last half century.

Peter reminds us (1 Peter 3:15) that when we raise apologetics issues with non-Christians, it should always be with gentleness and respect. Tragically that has not been how Christians have treated Miller and other skeptics in the scientific community. I wish I could say that I have never been guilty of these offenses. But I am.

Stanley Miller’s death deeply saddens me.

For more detailed discussions on problems confronting the evolutionary paradigm for the origin of life and the astounding evidence for a Creator’s role in the life’s beginnings, see the article “Origins-of-Life Predictions Face Off: Evolution Vs. Biblical Creation” and the book Origins of Life: Biblical and Evolutionary Models Face Off

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Miller-Urey Revisited

Members of NAI ’s Carnegie Institution of Washington, Indiana University, and NASA Goddard Space Flight Center Teams and their colleagues have revisited the Miller-Urey experiments, and found some surprising results.

A classic experiment proving amino acids are created when inorganic molecules are exposed to electricity isn’t the whole story, it turns out. The 1953 Miller-Urey Synthesis had two sibling studies, neither of which was published. Vials containing the products from those experiments were recently recovered and reanalyzed using modern technology. The results are reported in this week’s Science .

One of the unpublished experiments by American chemist Stanley Miller (under his University of Chicago mentor, Nobelist Harold Urey) actually produced a wider variety of organic molecules than the experiment that made Miller famous. The difference between the two experiments is small — the unpublished experiment used a tapering glass “aspirator” that simply increased air flow through a hollow, air-tight glass device. Increased air flow creates a more dynamic reaction vessel, or “vapor-rich volcanic” conditions, according to the present report’s authors.

“The apparatus Stanley Miller paid the least attention to gave the most exciting results,” said Adam Johnson, lead author of the Science report. “We suspect part of the reason for this was that he did not have the analytical tools we have today, so he would have missed a lot.”

Johnson is a doctoral student in IU Bloomington’s Biochemistry Program. His advisor is biogeochemist Lisa Pratt, professor of geological sciences and the director of NASA ’s Indiana-Princeton-Tennessee Astrobiology team.

In his May 15, 1953, article in Science, “A Production of Amino Acids Under Possible Primitive Earth Conditions,” Miller identified just five amino acids: aspartic acid, glycine, alpha-amino-butyric acid, and two versions of alanine. Aspartic acid, glycine and alanine are common constituents of natural proteins. Miller relied on a blotting technique to identify the organic molecules he’d created — primitive laboratory conditions by today’s standards. In a 1955 Journal of the American Chemical Society paper, Miller identified other compounds, such as carboxylic and hydroxy acids. But he would not have been able to identify anything present at very low levels.

Johnson, Scripps Institution of Oceanography marine chemist Jeffrey Bada (the present Science paper’s principal investigator), National Autonomous University of Mexico biologist Antonio Lazcano, Carnegie Institution of Washington chemist James Cleaves, and NASA Goddard Space Flight Center astrobiologists Jason Dworkin and Daniel Glavin examined vials left over from Miller’s experiments of the early 1950s. Vials associated with the original, published experiment contained far more organic molecules than Stanley Miller realized — 14 amino acids and five amines. The 11 vials scientists recovered from the unpublished aspirator experiment, however, produced 22 amino acids and the same five amines at yields comparable to the original experiment.

“We believed there was more to be learned from Miller’s original experiment,” Bada said. “We found that in comparison to his design everyone is familiar with from textbooks, the volcanic apparatus produces a wider variety of compounds.”

Johnson added, “Many of these other amino acids have hydroxyl groups attached to them, meaning they’d be more reactive and more likely to create totally new molecules, given enough time.”

The results of the revisited experiment delight but also perplex.

What is driving the second experiment’s molecular diversity? And why didn’t Miller publish the results of the second experiment?

A possible answer to the first question may be the increased flow rate itself, Johnson explained. “Removing newly formed molecules from the spark by increasing flow rate seems crucial,” he said. “It’s possible the jet of steam pushes newly synthesized molecules out of the spark discharge before additional reactions turn them into something less interesting. Another thought is that simply having more water present in the reaction allows a wider variety of reactions to occur.”

An answer to the second question is relegated to speculation — Miller, still a hero to many scientists, succumbed to a weak heart in 2007. Johnson says he and Bada suspect Miller wasn’t impressed with the experiment two’s results, instead opting to report the results of a simpler experiment to the editors at Science.

Miller’s third, also unpublished, experiment used an apparatus that had an aspirator but used a “silent” discharge. This third device appears to have produced a lower diversity of organic molecules.

Research on early planetary geochemistry and the origins of life isn’t limited to Earth studies. As humans explore the Solar System, investigations of past or present extra-terrestrial life are inevitable. Recent speculations have centered on Mars, whose polar areas are now known to possess water ice, but other candidates include Jupiter’s moon Europa and Saturn’s moon Enceladus, both of which are covered in water ice. The NASA Astrobiology Institute, which supports these investigations, has taken a keen interest in the revisiting of the Miller-Urey Synthesis.

“This research is both a link to the experimental foundations of astrobiology as well as an exciting result leading toward greater understanding of how life might have arisen on Earth,” said Carl Pilcher, director of the NASA Astrobiology Institute, headquartered at NASA Ames Research Center in Mountain View, Calif.

Henderson Cleaves (Carnegie Institution for Science) also contributed to the report. It was funded with grants from the NASA Astrobiology Institute, the Marine Biological Laboratory in Woods Hole, Mass., and Mexico’s El Consejo Nacional de Ciencia y Tecnologia.

Scripps Institution of Oceanography is a research center of the University of California at San Diego.

The NASA Astrobiology Institute ( NAI ), founded in 1998, is a partnership among NASA , 16 U.S. teams and five international consortia. NAI ’s goal is to promote, conduct and lead interdisciplinary astrobiology research and to train a new generation of astrobiology researchers. For more information, see http://astrobiology.nasa.gov/nai.

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The Miller-Urey Experiment – Chemical Evolution

The Miller-Urey experiment was a simulation of conditions on the early Earth testing the idea that life, or more specifically organic molecules, could have formed by nothing more than simple chemical reactions. Miller’s success validated the theoretical ideas of A.I. Oparin and is considered to be the classic experiment investigating the concept of abiogenesis.

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