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Pulse EPR
This primer is an introduction to the basic theory and practice of Pulse EPR spectroscopy. It gives you sufficient background to understand the basic concepts. In addition, we strongly encourage the new user to explore some of the texts and articles located in the further reading and links sections of this webpage.
EPR has traditionally been a CW (Continuous Wave) spectroscopy, as described in the previous section of this webpage. The NMR spectroscopist enjoyed substantial gains in sensitivity with a correspondingly drastic reduction in measurement time by moving to a pulse FT technique because they have a large number of very narrow lines spread over a wide (compared to the linewidth) frequency range. In most cases, the EPR spectroscopist is unable to enjoy these sensitivity improvements because EPR spectra are usually broad and not as numerous. Why would EPR spectroscopists wish to switch to a pulse methodology without the promise of increased sensitivity? NMR spectroscopists soon discovered by measuring in the time domain and using multi-dimensional techniques, they were able to extract much more information than they ever could possibly imagine. We can enjoy these same advantages in EPR as well.
In its most basic form, pulse EPR spectroscopy entails applying a short (<20 ns) and intense (>300 W) microwave pulse and then measuring the microwave signals generated by the samples magnetization in the probehead. Upon Fourier transforming the signal, we obtain a frequency spectrum from the sample.
A common analogy for describing CW (Continuous Wave) and FT (Fourier Transform) techniques is in terms of tuning a bell. We are assigned the task of measuring the frequency spectrum of the bell. In one scheme for tuning the bell, we use a frequency generator and amplifier to drive the bell at one specific frequency. In order to obtain a frequency spectrum of the bell, we slowly sweep the frequency in order to detect any acoustic resonances in the bell. We essentially perform a similar experiment in CW EPR: the field is slowly swept and we detect any resonances in the sample. This does not seem like the best means for tuning because we know from everyday experience that if we strike a bell with a hammer, it will ring (i.e. resonate acoustically at multiple frequencies). So an alternative approach is to strike the bell, digitize the resultant sound, and Fourier transform the digitized signal to obtain a frequency spectrum. Only one short experiment is required to obtain the frequency spectrum of the bell. This fact is often called the multiplex advantage. In FT-EPR, we apply a short but very intense microwave pulse (analogous to a hammer strike) and digitize the signals coming from the sample. After Fourier transformation, we obtain our EPR spectrum in the frequency domain.
Perhaps one of the most common pulse EPR applications is ESEEM (Electron Spin Echo Envelope Modulation) in which you obtain information regarding interactions of the electron spin with the surrounding nuclei. Interpretation of the data yields important structural information, particularly for large metalloproteins for which no single crystals are available for X-ray diffraction and the molecules are too large to perform high resolution NMR experiments. Pulse experiments measure relaxation times more directly than CW techniques such as saturation. The relaxation time measurements offer you dynamical as well as distance information for the samples you are studying.
As interest in measuring longer distances between paramagnetic centers increases, the techniques of 2 plus 1, DEER (Double Electron Electron Resonance), and ELDOR (ELectron DOuble Resonance) are invaluable in measuring particularly long distances in very large molecules.
Quite often there are events that take place on time-scales that do not influence the relaxation times and hence the lineshapes. EXSY (EXchange SpectroscopY) measures rates for slow inter- and intramolecular chemical exchange, homogeneous electron transfer, and molecular motions.