 Investigating the intimate behavior of organic and inorganic matter is the labor of general science. From this work come the basic scientific discoveries that precede all technological progress. Researchers busy in the various branches of general science are lately finding many of their problems simplified and some entirely new areas of investigation opened up by the application of a modern scientific tool, the radioisotope. The fundamentals of most radioisotope tracer experiments can be roughly diagrammed in terms of reactants, reaction, and products. The radioisotope is used to label one or more of the ingredients. Then in some cases the reaction is stopped after a desired period and examined to see where the label is located chemically or physically. More often, however, the products are tested for the presence or absence of the radioactive tracer. These techniques are exceedingly sensitive and specific. Quantities of some tracer atoms can be detected down to 10 to the minus 19 grams, even though mingled with stable atoms of the same element. A good example of the specificity of radioisotopes as tracers is found in metallic self-diffusion studies. Here in this laboratory, the rate of the self-diffusion of copper at high temperatures is being investigated. Since the only atoms involved are copper atoms, radioactive copper 64 is used as the tracer. Very little lead shielding is required for the safe handling of a typical 100 millicura unit of copper 64. However, for personnel safety, the operation is monitored by a cutie pie survey meter. And the workbench itself is especially designed for radioisotope work with air vents to carry off any possible radioactive spray or gases. In the experiment, a small amount of radioactive copper solution is added to an electrolytic cell, which already contains a copper solution. The cathode is a block of solid copper. The surface of the copper block is electroplated with a thin layer of copper containing the radioisotope. The specific activity of the copper in the plating solution is about 10 microcuries per milligram of copper. When approximately one microgram of copper has been plated onto the surface of the block to give an activity of 100 or so disintegrations per second per square centimeter, the block is taken from the cell. An initial measurement of the radiation from the plated surface is made before heating is begun. The block is counted with the thin walled Geiger tube having a density thickness of 3 milligrams per square centimeter. This absorbs only a small fraction of the radiation. Copper 64 emits beta particles and positrons of almost equal energy, about 0.6 MeV maximum, and emits a gamma ray in about 3% of the disintegrations. The gamma rays do not affect the count appreciably because of the rarity of their emission and the relative insensitivity of the GM tube. The initial radioactivity of the plated surface is recorded. It will be compared to the surface activity after the copper sample is subjected to a period of heating. After the block is removed from the counter, it is placed in an insulated electric furnace. The joints are then carefully sealed with dekatensky cement. The copper block will be kept in an argon atmosphere at a constant high temperature. Just under the experimental conditions, there is no other avenue of movement for the radioactive copper. A decrease in surface activity is evidence for the diffusion of the copper 64 into the block. After a heating period of several days, the cooled block is removed from the furnace. A second measurement is made of the surface activity. Since the activity decreases with physical decay, the 12.8 hour half-life of copper 64 must be taken into account and the readings corrected accordingly. By substituting the initial and final counting rates in the formula that relates the decrease in surface activity with a self-absorption coefficient for a beta radiation in copper, the diffusion coefficient D for a given temperature can be ascertained. The experiment is repeated with other samples to determine the rates of copper self-diffusion at various temperatures. The self-diffusion coefficients of other solid metals, impossible to determine by known chemical methods, may be obtained relatively simply by using radioisotope techniques. Diffusion between dissimilar metals may be investigated in a like manner. These experiments are particularly valuable in the study of alloys. For instance, much data can be brought to light through radioisotope work on the diffusion rates of copper into various copper alloys. This graph shows some of the data that can be obtained. Point X represents the diffusion rate of copper into copper. The other values show the diffusion rates of copper into various copper alloys. Thus, the radioisotope opens up a field of inquiry not readily approachable by any other means. The superior sensitivity of radioisotope measurement as an analytical method is well exemplified by a study dealing with vapor pressures over solid or liquid metals. With metals of low vapor pressure, in particular those with a high melting point, long periods of time are required to collect enough vapor to allow accurate chemical analysis. But the use of radioisotope techniques instead of chemical analysis can result in a great saving of time and labor. To illustrate, 100 milligrams of metallic silver containing four millicuries of radioactive silver 110 are placed in a crucible. And the crucible then placed in an apparatus especially designed to measure the vapor pressure of high melting point substances. The system will be evacuated and the crucible will be heated by induction to a given temperature. The vapor from the heated silver is allowed to stream through a pinpoint orifice in the crucible lid and condense on a cold tantalum target disk for a given time. The target is then ejected for a later measurement of the accumulated radioactive silver. A new target is exposed for measurement of vapor pressure at another temperature. Measurement of the radioactivity of the metallic silver deposited on the target is possible after a few minutes' exposure. The targets are removed and counted with a conventional Geiger-Mueller tube and scaler. Radioactive silver 110 is a beta and gamma emitter with a half-life of 225 days and has a specific activity of four millicuries per gram of silver. One can easily determine vapor pressure from a deposit of less than a microgram of silver on a target. Knowing such constants as the hole and target geometry, time of evaporation and the amount of metal deposited on the target, one can calculate the vapor pressure of silver at a given temperature. This method provides one of the best means of calculating thermodynamic activities. It is now widely used to obtain more exact data on solids of extremely low vapor pressure. In the field of chemistry, radioisotopes are proving valuable in the study of exchange phenomena, in which there is an interchange of atoms or groups of atoms of identical chemical nature, even though they exist in different chemical or physical states. A typical study involves the heterogeneous exchange between chloride ions in solution and chloride ions in the solid silver chloride lattice. One-thousandth of a mole of chloride ions containing radioactive chlorine-36 is added to a solution containing freshly precipitated solid silver chloride by means of a microvolumetric pipette. With chlorine-36 having a half-life of approximately a million years, no correction for decay in the radioisotope during the experiment need be applied. The mixture of solid silver chloride and the solution containing the newly added tracer, after stirring for a brief interval, is then centrifuged. A known aliquot of the supernatant is then drawn off for analysis. It is carefully mounted and evaporated to dryness under an infrared lamp before counting. Chlorine-36 is a beta-emitter exhibiting no-gamma activity. It is measured with an n-window type of geiger tube and a conventional scaler. After a very short time, the disappearance of activity from the solution is evident. This indicates that the chloride ions in solution are changing places with the chloride ions in the solid lattice. In the basic diagram, this method corresponds to the technique of labeling one of the reactants and then searching for the label in the products. In this case, the radioactivity in the initial solution is found to decrease, showing that some of the activity has entered the solid by exchange. As the experiment is continued, the rate of the exchange is found to decline. This is attributed to the changing nature of the solid silver chloride surface as it ages, resulting in a decrease of surface area and of the energy of the solid lattice remaining. Thus, the radioisotope makes it relatively easy to differentiate between like atoms in different chemical species. Another ingenious application of the radioisotope to biological exchange phenomena has led to significant disclosures in the dynamics of physiological processes. These experiments employed squid, which have a giant main nerve cell called the axon nerve. It is known that under normal conditions, the concentration of potassium within a living cell is about 20 times greater than that outside the cell wall. Conversely, the concentration of sodium is 10 to 1 in the opposite direction. These relationships are known to remain constant under normal conditions, but to change with alteration in the extracellular environment. In a typical experiment, a bath is prepared containing sodium and potassium mixed with microcurie amounts of potassium 42 of 12.4 hour half-life. This extracellular fluid will ultimately flow through a small chamber in which the squid nerve cell is suspended. The radioactive solution is introduced into the shielded lead tank, which protects the workers from radiation. Small amounts of the radioactive fluid bathe the giant axon, and exchange is given an opportunity to take place. After a regulated interval, the axon is removed and washed. Its interior fluids are very carefully wrung out and weighed precisely on a semi-microbalance prior to counting. The intracellular fluid is found to contain some of the radioactive potassium 42 from the extracellular bath. Continuous exchange is taken place across the cell membrane. When radioactive sodium 24 is added to the bath, instead of radioactive potassium, a similar mobility is found to exist for the sodium ions. This permeability of the membrane is not believed to be a simple diffusion process, but an exchange of atoms on and off the molecules of the cell wall. To study the effects of stimuli on cell equilibrium and the rate of exchange, electrodes are connected to the axon nerve to produce an action potential on the oscilloscope. During electrical stimulus, exchange of sodium and potassium ions is found by this tracer technique to increase in rapidity due to increased permeability of the membrane. Sodium enters at a high rate during the ascending phase of the action potential, while potassium breaks through during the descending phase. This opening of the barrier is the source of the cell's electromotive force. Detection of the radiation of the sodium and potassium radioisotopes is no problem in view of the energetic beta and gamma rays emitted. And so, by the use of radioisotopes, for the first time, it has become possible to explore the very dynamic processes taking place in the cell walls and other membrane structures of living systems. For many years, chemists suspected without convincing proof that under some conditions, atoms will rearrange within the same molecule. With the advent of the radioisotope, an ideal weapon has been provided with which to attack this type of problem. The wolf rearrangement of a diazo ketone is a striking illustration of this phenomenon. In wolf's reaction, an azo halide and diazomethane yield it a diazo ketone. Hydrolysis of the ketone in the presence of a silver oxide catalyst produces an organic acid containing the same number of carbon atoms. Does the carbon atom which holds the oxygen of the diazo ketone rearrange with the terminal carbon during hydrolysis to become the new carboxyl carbon? Or does it remain in its original position? The diazo ketone is synthesized to make its carbonyl carbon radioactive. The vacuum system used in C14 syntheses prevents contamination of the laboratory and protects workers against inhalation and ingestion of radioactive materials. The carbonyl carbon of the diazo ketone is tagged in this manner. If we trace the radioactive carbon to the product, we can see whether the carbonyl carbon of the diazo ketone has rearranged to become the terminal carbon. The terminal carbon of the acid is chemically taken off to see if it contains the label. This decarboxylation of the acid illustrates how organic compounds may be broken down stepwise. After heating a known weight of sample, the evolved carbon dioxide is introduced directly into an ion chamber. This ion chamber is then connected to a vibrating reed electrometer. The magnitude of the observed ion current is a measure of the amount of radioactive carbon 14 present. A standard is used to check the performance of this electrometer which employs AC amplification. The introduction of radioactive carbon dioxide into an ion chamber is a widely used technique, since a greater sensitivity of detection is possible than through the counting of solid samples. The carbon dioxide derived from the terminal carbon is found to contain all of the radioactive carbon 14 label. This indicates that what was originally the carbonyl carbon atom of the diazo ketone is now the terminal carbon of the acid. Rearrangement has occurred within the molecule during hydrolysis. This type of investigation has already yielded a wealth of significant information concerning the mode of reaction of organic molecules. The unusual sensitivity of the radioisotope tracer technique is well demonstrated in a difficult biochemical investigation concerned with the metabolism of complex chemical compounds in living organisms. Here, the surviving liver tissue of young rats is used to find out whether or not sodium acetate is involved in the synthesis of cholesterol by the liver. The rate of synthesis in the organ is often very slow. Consequently, the increment resulting is so small that it cannot be measured by ordinary analytical means. For this experiment, carbon 14 labeled sodium acetate, the suspected metabolite, is added to 3 grams of surviving liver tissue of young rats, cut into thin slices, and suspended in a phosphate buffer. Only a few micro curies of C14 label sodium acetate are required. With such low activities, only limited safety precautions are necessary. After incubating for three hours, the organic fraction containing cholesterol is separated from the mixture. Following this separation, an isotope dilution technique is used since only a trace amount of cholesterol is present in this fraction. Non-radioactive cholesterol is added to carry off the trace amount of cholesterol formed during incubation. This isotope dilution technique is used widely and yields quantitative data, and yet employs only qualitative techniques for the isolation of the tag compound. This technique does require, however, careful purification of the final fraction. After isolation and purification, the cholesterol sample is prepared for counting and its specific activity is determined. The radioactivity detected in the cholesterol isolated from the tissue slices suggests that acetic acid plays a key role in the synthesis of cholesterol. As a useful byproduct of this research, the extremely complicated compound, cholesterol, has been biosynthesized with radioactive carbon. By this same biosynthetic method, other complicated compounds such as penicillin, hormones, vitamins, proteins, and digitoxin may be labeled isotopically for further tracer research. In addition to use in in vitro studies, radioisotopes have proved invaluable for investigations conducted in vivo. Using glycine labeled with C14 and a dog as the experimental animal, the metabolic fate of the principal atoms in a molecule of this important amino acid is elucidated to a remarkable degree. In biochemical studies with animal subjects, it has been determined, using the stable isotope nitrogen 15 as a tracer, that the nitrogen in glycine appears in the hemen. Hemen with globin is one of the fundamental constituents of blood. But what is the fate of the two carbon atoms of glycine? To answer this question, the carboxyl carbon must first be traced. The glycine used here has been synthesized with its carboxyl carbon labeled with carbon-14. This labeled glycine is then injected into the dog. Later, a blood sample is taken from the animal and the red blood corpuscles separated from the plasma. The red blood corpuscles are broken down in the globin and hemen isolated separately. These two fractions are prepared for counting. These fractions are repeatedly purified until their specific activity becomes constant. Our experiment reveals that the radioactivity introduced into the carboxyl carbon of glycine is found only in the globin. When the number 2 carbon of glycine is labeled with carbon-14, the activity is found predominantly in the hemen. The experiment demonstrates that the nitrogen and the number 2 carbon of glycine remain together and end up in the hemen of the animal's blood. The carboxyl carbon, however, is destined for the globin. In addition, this demonstrates that during the metabolism of glycine, a bond rupture occurs at the 1-2 position of the molecule. Thus, radioisotopes permit study in vivo of the metabolism of individual atoms of biologically important compounds. The manner in which plants synthesize their chemical compounds during the process of photosynthesis has been a problem of great interest since the early days of science. The first work on this problem was hindered because investigators were unable to follow the paths of ordinary carbon dioxide once it had entered into the photosynthetic reaction. The work of the early investigators is now being continued with the most modern of research tools, the radioisotope. This versatile tool enables present-day investigators to follow a tagged molecule through the many paths which exist in photosynthesis. Here an experiment is being prepared to trace the chemical path of radioactive carbon from carbon dioxide through the complex reaction sequence of photosynthesis in algae. A solution containing radioactive carbon dioxide as soluble carbonate is made available to the growing plants. The algae cells in suspension are allowed to photosynthesize for a definite period in the presence of the labeled CO2. Photosynthesis under strong artificial illumination continues for 30 seconds. The algae are removed from the photosynthesis cell, called a lollipop, and are then killed by the addition of boiling ethanol to stop all metabolic reactions. The extract of the algae suspension is then prepared for analysis by paper chromatography. This technique of paper chromatography supplies a means for separating the compounds in the algae in order to find out which ones have been synthesized from the radioactive carbon dioxide. After evaporation to a small volume, the extract of the alcoholic suspension containing the dead algae is applied to filter paper. The total radioactivity applied to the paper is measured with a Geiger counter. One edge of the paper containing the extract is immersed in the first solvent, and separation is accomplished by partition and absorption down the paper in one dimension. Time must be allowed for the slow movement of the compounds down the paper. This movement varies for different compounds and results in their partial separation. The paper must then be hung up to dry. After drying at room temperature, the paper is removed and prepared for the second step in chromatography. The adjacent edge of the filter paper is immersed in butanol. This second solvent distributes the compounds on the paper at right angles to the first direction. This double separation places the different compounds at various coordinates of the filter paper. To summarize this technique, one edge of the filter paper on which the mixture of tagged compounds exists as a spot is immersed in a solvent solution A. The slow movement of this solvent up the paper is accompanied by the movement of the tagged compounds in the same direction. Each compound moves with a characteristic velocity in this particular solvent. After being dried, the other edge of the filter paper is immersed in a second solvent solution B, in which the tagged compounds have different relative rates of movement. These different rates of movement at right angles to each other result in the localization of each compound at a characteristic spot when the same two solvents are employed. Control tests establish the characteristic location of each compound on the paper. X-ray film is then exposed by contact with the filter paper. The soft beta radiation from Carbon-14, 0.15 MeV, are very effective for producing successful radio autographs on X-ray film. This graph shows the effect of different beta energies for identical exposure times. The maximum film blackening occurs at 0.15 to 0.2 MeV. Density measurement of the photographic image can be used to supply quantitative data. With such test runs for various periods of photosynthesis, it is possible to obtain a step-by-step itinerary of the radioactive carbon. Since the process is a very rapid one, it is important that data be obtained at closely spaced intervals. Otherwise, some brief stage of chemical development might be missed entirely. The resulting radio autograph film indicates the location of the separated compounds on the paper chromatogram. After matching the film and paper chromatogram, the radioactivity of these spots can then be measured individually. This type of approach is proving exceedingly useful in the study of many obscure metabolic problems. In all areas of general science, the radioisotope is opening up new highways of research, cutting corners on other difficult paths of investigation, and widening the horizon of man's knowledge, helping scientific development toward new frontiers.