Sensitivity of NMR Experiments

The sensitivity of pulsed FT NMR experiments is given by the signal to noise ratio  

T2 = transverse relaxation time (determines the line width) 

SIN for less sensitive nuclei to excite one kind of nucleus and detect another kind with a better magnetogyric ratio in the same experiment. This is done in inverse experiments which are described in Chapters 5 and 6). 

A routine sample for proton NMR on a 300 MHz instrument consists of about 10 mg of the compound in about 0.5 ml of solvent in a 5-mm o.d. glass tube. Microprobes that accept a 1.0 mm, 2.5 mm, or 3 mm o.d. tube are available and provide higher sensitivity. Under favorable conditions, it is possible to obtain a spectrum on 100 ng of a compound of modest molecular weight in a 1.0 mm microtube (volume 5 pi) on a 600 MHz instrument. 

The development of cryogenically cooled probes has significantly decreased sample amount requirements for H NMR. Thermal noise in the probe and the first-stage receiver electronics dominate noise in NMR experiments. These new probes have built in first-stage receivers and rf coils that are cryogenically cooled ( 20°K), and have S/N improvements of 4 X standard probes. It is obvious to users that the highest field instrument available provides the best sensitivity. For fixed concentration (N), we would need 2.8 times as much material with a 300 MHz as on a 600 MHz system to obtain spectra with identical S/N  

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The gyromagnetic ratio, y, is defined as the ratio of magnetic moment to angular momentum of the nucleus and impacts on the sensitivity of NMRS experiments. Assuming that the coupling between the individual spins is weak, the relative populations of the two levels are given by a Boltzmaim distribution ... 

A higher-MHz NMR spectrometer is always a better choice, since the sensitivity of the experiment is proportional to the frequency of measurement. Moreover, with highly concentrated solutions, the presence of some solid particles can cause an increase in T) (FID will be short) and line broadening of the NMR signals will result. Therefore, an optimum concentration (say, 25-50 millimolar solution) is recommended. Of course, H-NMR spectra can be readily measured at much lower concentrations, though higher concentrations are necessary for recording - C-NMR spectra. 

The "decrease of the spin temperature means an increase of population difference between the upper and lower energy spin states and consequently an increased sensitivity of the NMR experiment. From Equation (25), the temperature of dilute spins has been lowered by a factor 7x/y1 h, that is, V4 when X = 13C. This means an increased sensitivity of the FID resonance experiment equal to about 4 for the 13C nuclei. Because the X signal is created from the magnetization of dilute nuclei, the repetition time of NMR experiment depends on the spin-lattice relaxation time of the abundant spin species, protons, which is usually much shorter than the spin-lattice relaxation times of the dilute nuclei. This, a further advantage of cross polarization, delay between two scans can be very short, even in the order of few tens of milliseconds. 

A key factor in the use of NMR for measuring dissociation constants is its sensitivity to the rate of chemical exchange . Complex formation necessarily involves the exchange of the nuclei or molecules being observed between (at least) two states - the free ligand or protein and the complex. The fact that the appearance of the NMR spectrum is sensitive not only to the position of this equilibrium but also to the rates involved has a major influence on the design of NMR experiments for measuring dissociation constants and on the analysis of such data. 

In the isotope edited/ filtered spectra of a protein-ligand complex, the species actually observed is generally the complex itself. This is an important difference from transferred NOE or saturation difference techniques, where the existence of an equilibrium between free and bound species - and a certain rate of exchange between them - is essential (Chapts. 13 and 16). The general conditions for isotope filtering/editing are therefore identical to those required for standard protein NMR sample concentrations are usually limited by availability and solubility of the components to the order of 1 mM. Considerably lower concentrations will reduce the sensitivity of the experiments to unacceptable levels,... 

A variety of sequences exist, which differ with respect to the detected interaction ( J, or Jx ) and the mode of detection ( C or H detected, magnitude or phased mode, phase cycling or gradients for coherence selection). In view of the reduced sensitivity of heteronudear experiments with respect to homonuclear COSY experiments and the steadily decreasing sample amounts submitted for NMR experiments, there is no doubt that the inverse ( H) detected, gradient enhanced experiments are currently the best methods to apply. However on older type spectrometers, not equipped for inverse detection the old-fashioned direct C detected experiments are still in use. 

Inverse-detected experiments have had the greatest effect in making 15N NMR experiments feasible for small samples. These experiments take advantage of the higher sensitivity of NMR to facilitate the observation of insensitive nuclei like 13C and 15N. The H-13C heteronuclear multiple quantum coherence (HMQC) and the related heteronuclear multiple-bond correlation (HMBC) experiments are important in contemporary natural products... 

Nuclear magnetic resonance (NMR) is another spectroscopic technique that has many formal similarities to EPR. It is widely used for product analysis, as in studies leading to the development of electrosynthetic methods. The intrinsic sensitivity of NMR is several orders of magnitude lower than that of EPR, making simultaneous NMR-electrochemical experiments unattractive. 

The time required for each type of NMR experiment depends on many factors, including sample concentration and complexity, shimming, magnetic field strength, and the sensitivity of the individual experiments. Indication of time requirements of individual 1-D and 2-D experiments are given in the legends of Figures F. 1.4.2, F. 1.4.3, F. 1.4.5, F. 1.4.6, and F. 1.4.7, as well as Table Fl.4.9. 

The study of nucleic acid bases by NMR has been reported in a number of monographs (/), but very little data is available on the, 3C and, 5N NMR chemical shift tensors in these compounds. The low sensitivity of NMR spectroscopy and the long relaxation times exhibited by many of these compounds have posed the main impediments for these studies. The use of sample doping with free radical relaxation reagents, to reduce the relaxation times facilitating 2D multiple pulse experiment (2, 3), enables one to measure and analyze the principal values of the chemical shift tensors in natural abundance samples. In previous papers from this laboratory we have presented, 5N NMR chemical shift principal values for adenine, guanine, cytosine, thymine and uracil (4, 5). 

Due to the intrinsically low sensitivity of NMR spectroscopy when compared to UV or MS, LC-NMR measurements typically can take an hour or more to complete. Therefore, it makes no sense for an operator to sit and wait for completion of the NMR experiment to activate another chromatographic separation - this has to be done automatically. 

In the biochemical field, proteins are prepared for NMR analysis by synthesizing them with bN labeled amino acid residues. Since 15N has a natural abundance of 0.368%, labeling increases the sensitivity of the experiment by a factor of about 270, which, when combined with the HSQC type experiment, yields about an 84,000-fold sensitivity increase over natural abundance directly detected methods. Thus, it is possible to obtain quality spectra on proteins up to 50 kDa. 

For example, at 7.05 T magnetic field (a 300 MHz NMR instrument) and 25 °C, the population difference for protons is 0.00064% of the number of nuclei N. This equilibrium population difference is a constant throughout the NMR experiment and, as we perturb the equilibrium, the spins will always try to return to this equilibrium population distribution. Because the measureable signal from a nucleus in the ft state is exactly cancelled by the signal from a nucleus in the a state, it is this population difference that is the only material we have to work with and to detect in the NMR experiment. Because the difference is so small, the sensitivity of NMR is in many orders of magnitude lower than all other analytical techniques so low, in fact, that NMR is not considered a branch of analytical chemistry but rather a tool used by organic chemists and biologists. 

Two-dimensional NMR experiments are sometimes thought to be much less sensitive than their one-dimensional analogs. However, the sensitivity of 2D experiments can be quite high with well-executed experimental procedures. In this section we discuss briefly several of the general factors that determine... 

The development of pulsed Fourier-transform NMR spectrometers has greatly increased the sensitivity of NMR measurements, allowing spectra to be obtained for and other nuclei at natural abundances and low sample concentrations. In this experiment this enhanced capability is utilized in a low-density gas-phase measurement of the equilibrium constant Kp for H-D exchange in the reaction... 

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