2.1.1. 1D 1H-NMR
The 1H hydrogen isotope is NMR visible, has the highest gyromagnetic ratio (apart from tritium) of all of NMR active nuclei, and is combined with a vast natural abundance in organic chemical compounds. This makes the 1D-1H-NMR experiment the most commonly applied NMR approach. Moreover, many software databases [99,100,101,102] are well established for 1H-NMR spectra therefore assisting with processing, analyzing, and identifying the detected molecules automatically. Since almost all drug discovery and drug development studies are performed on samples dissolved in water, many different solvent suppression methods have been applied. The most common is presaturation [103,104]. The key point of this method is to use a low power induced field at the specific frequency of water. This effectively averages out any coherence of the water resonance. The experiment is simple for common hardware to perform and easy to set up; however, presaturation has a substantial disadvantage in that signals resonating close to the solvent signal will show decreased intensity [103,104] or may be lost entirely. This is due to the fact the even selective pulses or very low power pulses also excite some area around the water signal. Also suppressed hydrogen from H2O in solution can exchange with atoms of interest in the molecule and effectively bleed the suppressive spin state to any neighboring atoms. The water signal itself is usually broad, so a wider area of suppression is not necessarily undesirable but affects more of the molecule(s) of interest. More recent water suppression techniques have been developed such as those based on a scheme known as excitation sculpting [105,106]. The basic pulse sequence consists of a double pulsed field gradient echo (DPFGE) in each of which a selective component pulse is flanked by two pulsed field gradients [107]. The particular elements differ for different applications. In the case of water suppression known as WATER suppression by GrAdient Tailored Excitation (WATERGATE), this involves an initial encoding gradient along with the middle element; a combination of two selective 90° rotations on the water along with a central non-selective 180° excitation of all resonances [108]. This is predicated in that water experiences a 360° rotation (effectively nothing) while all other spins experience 180° rotation. The application of the second refocusing gradient does not rephase the water and therefore removes the signal. The reader is referred to the detailed literature [103,104,109] for further information. In principle, a water suppression element (or many elements combined) can be incorporated in any existing pulse sequence to enhance the performance, and it has been implemented in various 1D, 2D, and triple resonance 3D/4D experiments. Although 1H is the most sensitive nucleus for NMR yielding strong, sharp signals within a few minutes [110], chemical shift dispersion of 1H is quite narrow (only around 10 ppm). This has prompted the consideration of other nuclei such as 13C, 15N, or 31P for resolution improvements.