Mass–energy equivalence
Radioactivity and nuclear energy

It was quickly noted after the discovery of radioactivity in 1897 that the total energy due to radioactive processes is about one million times greater than that involved in any known molecular change, raising the question of where the energy comes from. After eliminating the idea of absorption and emission of some sort of Lesagian ether particles, the existence of a huge amount of latent energy, stored within matter, was proposed by New Zealand physicist Ernest Rutherford and British radiochemist Frederick Soddy in 1903. Rutherford also suggested that this internal energy is stored within normal matter as well. He went on to speculate in 1904: "If it were ever found possible to control at will the rate of disintegration of the radio-elements, an enormous amount of energy could be obtained from a small quantity of matter."

Einstein's equation does not explain the large energies released in radioactive decay, but can be used to quantify them. The theoretical explanation for radioactive decay is given by the nuclear forces responsible for holding atoms together, though these forces were still unknown in 1905. The enormous energy released from radioactive decay had previously been measured by Rutherford and was much more easily measured than the small change in the gross mass of materials as a result. Einstein's equation, by theory, can give these energies by measuring mass differences before and after reactions, but in practice, these mass differences in 1905 were still too small to be measured in bulk. Prior to this, the ease of measuring radioactive decay energies with a calorimeter was thought possibly likely to allow measurement of changes in mass difference, as a check on Einstein's equation itself. Einstein mentions in his 1905 paper that mass–energy equivalence might perhaps be tested with radioactive decay, which was known by then to release enough energy to possibly be "weighed," when missing from the system. However, radioactivity seemed to proceed at its own unalterable pace, and even when simple nuclear reactions became possible using proton bombardment, the idea that these great amounts of usable energy could be liberated at will with any practicality, proved difficult to substantiate. Rutherford was reported in 1933 to have declared that this energy could not be exploited efficiently: "Anyone who expects a source of power from the transformation of the atom is talking moonshine."

This outlook changed dramatically in 1932 with the discovery of the neutron and its mass, allowing mass differences for single nuclides and their reactions to be calculated directly, and compared with the sum of masses for the particles that made up their composition. In 1933, the energy released from the reaction of lithium-7 plus protons giving rise to two alpha particles, allowed Einstein's equation to be tested to an error of ±0.5%. However, scientists still did not see such reactions as a practical source of power, due to the energy cost of accelerating reaction particles. After the very public demonstration of huge energies released from nuclear fission after the atomic bombings of Hiroshima and Nagasaki in 1945, the equation E = mc became directly linked in the public eye with the power and peril of nuclear weapons. The equation was featured on page 2 of the Smyth Report, the official 1945 release by the US government on the development of the atomic bomb, and by 1946 the equation was linked closely enough with Einstein's work that the cover of Time magazine prominently featured a picture of Einstein next to an image of a mushroom cloud emblazoned with the equation. Einstein himself had only a minor role in the Manhattan Project: he had cosigned a letter to the U.S. president in 1939 urging funding for research into atomic energy, warning that an atomic bomb was theoretically possible. The letter persuaded Roosevelt to devote a significant portion of the wartime budget to atomic research. Without a security clearance, Einstein's only scientific contribution was an analysis of an isotope separation method in theoretical terms. It was inconsequential, on account of Einstein not being given sufficient information to fully work on the problem.

While E = mc is useful for understanding the amount of energy potentially released in a fission reaction, it was not strictly necessary to develop the weapon, once the fission process was known, and its energy measured at 200 MeV (which was directly possible, using a quantitative Geiger counter, at that time). The physicist and Manhattan Project participant Robert Serber noted that somehow "the popular notion took hold long ago that Einstein's theory of relativity, in particular his equation E = mc, plays some essential role in the theory of fission. Einstein had a part in alerting the United States government to the possibility of building an atomic bomb, but his theory of relativity is not required in discussing fission. The theory of fission is what physicists call a non-relativistic theory, meaning that relativistic effects are too small to affect the dynamics of the fission process significantly." There are other views on the equation's importance to nuclear reactions. In late 1938, the Austrian-Swedish and British physicists Lise Meitner and Otto Robert Frisch —while on a winter walk during which they solved the meaning of Hahn's experimental results and introduced the idea that would be called atomic fission—directly used Einstein's equation to help them understand the quantitative energetics of the reaction that overcame the "surface tension-like" forces that hold the nucleus together, and allowed the fission fragments to separate to a configuration from which their charges could force them into an energetic fission. To do this, they used packing fraction, or nuclear binding energy values for elements. These, together with use of E = mc allowed them to realize on the spot that the basic fission process was energetically possible.
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