L. H. GRAY 



where AT = molecular weight, p — density, .V = Avogadro's number, the 

 paramagnetic term [i^ilSkT for a material consisting entirely of free radicals 

 \vould be 1260 x 10~^ c.g.s. units at 20 °C. Since the diamagnetic term ao for 

 most organic materials is not more than 10 per cent of this, paramagnetism 

 is a characteristic property of free radicals. The Theorell balance is said to be 

 capable of measuring a 10~^ molar concentration of radicals in a 0-02 ml. 

 sample with 10 per cent accuracy^*. If, therefore, all the free radicals formed 

 by radiation in a given material were permanent, and the yield of free radi- 

 cals was of the order of 1 per 30 eV, then exposure to dose levels of the order 

 of 1 megarad would, in the absence of any other change in the magnetic 

 conditions of the sample, produce sufficient paramagnetism to be measured 

 by the Theorell balance. In fact the conditions for such a measurement 

 are not likely often to be fulfilled, even in non-living materials, so other 

 methods of detection have to be employed. The method of electron spin 

 resonance spectroscopy offers considerable promise. 



Though first applied to the detection of radicals by Zavoisky^^ in 1945, 

 some 10 years elapsed before this technique was pressed into the service of 

 radiation chemistry. In 1954 and 1955 papers appeared by Combrisson and 

 Uebersfeld^^ and by Gordy, Ard, and Shields^', on paramagnetism induced 

 by irradiation of organic materials. The latter paper in particular, which 

 was concerned with electron spin resonance studies of X irradiated amino 

 acids and proteins, marks the beginning of a development in radiation 

 chemistry which is of the greatest interest to radiobiologists. 



Detection of radicals by electron spin resonance depends on the fact that 

 the two orientations of a free electron in a magnetic field H, namely with and 

 against the field, differ in energy by an amount E = g^H, so that if an electron 

 which is precessing undisturbed around the direction of a uniform magnetic 

 field H is exposed to an electromagnetic field of frequency v such that hv = 

 g^H, transitions to the higher energy state will occur as a result of resonant 

 absorption of energy from the electro-magnetic wave. On introducing 

 numerical values for the spectroscopic splitting factor, g = 2 -0023, and for ^ 

 (the value of the Bohr magneton) = 0-927 10"^° erg/gauss, it becomes evident 

 that resonant absorption will occur when a 3 cm wave passes through a 

 material containing free radicals situated in a magnetic field of about 

 3600 gauss. 



In general, there may be some interaction, and therefore some coupling, 

 between the spin magnetic moment of the electron, its orbital magnetic 

 moment, and the nuclear magnetic moment, if any, of the nuclei with which 

 the unpaired electron is associated. The latter interaction gives rise to hyper- 

 fine structure of the energy levels whereby a particular type of free radical 

 may be recognized. The absorption spectrum of the radical is usually 

 presented as a function of magnetic field at fixed frequency, or as the first 

 differential of this function. 



This method of measuring free radicals is not only very much more sensi- 

 tive than the Theorell balance and similar methods, but has a number of 

 features of particular interest to radiobiologists. Chief of these are perhaps : 



(1) That it is applicable to the study of radicals formed in living cells, 

 suspended if necessary in an aqueous medium. 



(2) Quantitative work is not in general confused either by the diamagnetism 



293 



