Relaxometry measurements can be performed in water solutions of diamagnetic or paramagnetic unlabeled molecules or on unlabeled macromolecules dissolved in D2O. The solubility of the molecules in water at room temperature should be checked to be at least in the millimolar range. In the case of molecules dissolved in water solution, and for paramagnetic systems in particular, the concentration should be accurately determined. For the analysis of the paramagnetic relaxation enhancements, a diamagnetic reference should also be available.
The acquisition of a relaxation profile requires some hours for measurements in water solutions, depending on the concentration of the paramagnetic species (if present) and on the molecular weight (the higher the faster). The acquisition of a relaxation profile of macromolecules dissolved in D2O requires several days.
Once the sample is placed in the probe of the relaxometer and the desired temperature has been reached, the times defined in the pulse sequence must be set. The main times to be defined by the user are the polarization time and the recycle time (both of them not less than four times the proton relaxation time at the polarization field), and the proton relaxation time at the largest field of measurement, which can be estimated from a few preliminary measurements.
The relaxometer provides a list of the relaxation times/rates measured at the different proton Larmor frequencies (from 0.01 to 45 MHz), called a relaxation dispersion profile. The relaxation times are calculated through a monoexponential fit of the magnitude of the magnetization as a function of the time at which the sample has been kept at the field corresponding to the proton Larmor frequency. In the presence of systems for which the monoexponential fit is not appropriate (the quality of the fit can be checked in real time), the magnitude of the magnetization as a function of the time at which the sample has been kept at the field corresponding to the proton Larmor frequency can also be obtained for each field, for further analysis that the user can perform. Such analysis may provide a collective proton relaxation dispersion profile.
The relaxation dispersion profiles can then be fitted with available software to obtain the reorientation time of the system and other parameters providing information on its structure and dynamics.
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