The detailed requirements for the accurate determination of electron spin concentration by electron paramagnetic resonance spectroscopy are examined, and the use of the dual-sample cavity for measuring spin concentrations is reviewed.The lack of adequate solution-phase concentration standards is examined, and a new method based on photometric titration techniques is given for determining piperidine nitroxide concentrations in polar solvents. The titration procedure is used to determine the molar absorptivity of several common piperidine nitroxides in water and in water-methanol solutions. INTRODUCTIONOne important, but thorny, experimental problem in electron paramagnetic resonance (EPR) spectroscopy is the determination of the concentration or of the absolute number of electron spins in a sample. All methods for this require a standard or known sample (I, p. 443), yet no universal spin concentration standard is available. This problem was highlighted at the recent 14th International Symposium on EPR, at which an entire session was devoted to issues involved in arriving at standards in EPR (2).Methods to determine the number of electron spins in a sample by EPR have been in place since the discipline matured in the early 1960s (I, p. 443); however, many of these techniques and the associated lore have been lost to the expanded EPR community. Here we give a review of the principles of spin concentration determination, and we offer a new technique for the establishment of convenient solution concentration standards that is of special significance to biochemical applications. 145Kooser, Kirchmann, and Matkov GENERAL CONSIDERATIONSThe fundamental principle involved in determining the number of spins in a sample is the well-known fact that the integrated intensity of a spin resonance (the area under the resonance curve, or the first moment) is proportional to the spin density of the sample. The problem in EPR is that the intensity of a resonance is a complex function not only of the spin concentration of the sample, but it is also at the mercy of a host of instrumental and sample conditions (3, 4 ) . The general relationship can be given by Eq. [l] (3):[XI is the spin concentration, M is the first moment, G is the apparent amplifier gain as determined from the instrumental settings, A is a function of the true amplifier gain and the modulation field, and B is a factor related to the microwave bridge alignment. It is the terms A and B that present the,experimental difficulty. For instance, it is known that the sample tube causes a distortion of the microwave magnetic field seen by the sample inside (5) so that different sample containers will produce different microwave fields at the sample. Other variables affect the B term in Eq. 111, such as the cavity quality factor (I, p. 167) and the cavity filling factor ( 4 ) . The quality factor depends on what type of sample is inserted into the EPR cavity. Also, most EPR experiments are done using microwave cavities that have modulation coils that are embedded in their walls...