Training and Resources

standard NMR experiment to observe proton resonances.

standard NMR experiment to observe carbon resonances.

DistortionLess Enhancement by Polarization Transfer, transfers polarization from protons to carbon by carefully calibrating the decoupler power it is possible to distinguish CH3 (methyl), CH2 (methylene), and CH (methine) groups using three pulse widths of 45, 90, and 135 degrees. Since quaternary carbons have no attached protons they are not detected.

Attached Proton Test, is a simple experiment for assigning multiplicities in 13C NMR spectroscopy. In this experiment, CH and CH3 groups appear as positive peaks while those from CH2 and quaternary carbons are negative. Compared to the DEPT experiment all carbon nuclei are visible in one spectrum.

Spin-Lattice Relaxation Time ‘T1’, knowledge of the T1's of a molecule is important in the setup of many 1D and 2D experiments, in which the relaxation delay (d1) must be set according to the T1's of the signals of interest. In some instances, the T1 values themselves are used as structural parameters in the characterization of a variety of compounds. Knowledge of the T1's of a molecule is important in the setup of many 1D and 2D experiments, in which the relaxation delay (d1) must be set according to the T1's of the signals of interest. In some instances, the T1values themselves are used as structural parameters in the characterization of a variety of compounds. For example, a quantitative NOESY one needs a d1 set to 3-5xT1, while a HMBC or HMQC requires 1.5xT1 for its’ d1 relaxation delay.

The underlying principle behind monitoring a reaction over time by NMR is the ability to repeatedly acquire spectra at controlled time intervals. This is achieved with an arrayed experiment using the pre - acquisition delay ( pad ) as the arrayed variable.

Homo-nuclear Decoupling, is most effective for relatively simple spectra where the couplings are, at least, somewhat resolved (i.e. there is a minimal amount of spectral congestion). The experiment consists of irradiating a single selected resonance with a low power decoupler pulse eliminating any couplings to that resonance. By comparing the resulting spectrum to that without decoupling, it is easily determined which resonance(s) are coupled to the irradiated peak.

Nuclear Overhauser Effect Difference, is used to assess the spatial proximity of protons in a molecule. One resonance is selectively irradiated at low power for several seconds. The effect of this irradiation is to saturate the target resonance. Perturbing the populations of this spin have subtle effects on the population of spins that are close in space (< ~5 Å).

1H-1H through bond correlation - The first and most popular two-dimension NMR experiment, which is used to identify coupled spins. The COSY pulse sequence consists of an initial delay (d1) during a period that allows the magnetization to return to an equilibrium steady-state. This is followed by a pulse (pw) and delay (d2) that generates the spin-coupling information and allows that coupling to evolve. Finally, the resulting magnetization is examined with the pulse (pw) and the data acquired during time (at). Important parameters in the COSY sequence are shown in the following table.

TOtal Correlation SpectroscopY, same as COSY , but also able to generate cross peaks via intermediate spins (mix). Uses a spin lock that produces rf heating of the sample and hence requires many steady state scans (ss).

Nuclear Overhauser Effect SpectroscopY, allows one to see through-space effects, useful for investigating conformation and for determining proximity of adjacent spin systems. The NOESY experiment offers a simple way to obtain 1H-1H distances in a molecule without prior knowledge of the spectral assignment or molecular structure. The basic scheme of this experiment involves an initial delay (d1) during a period that allows the magnetization to return to an equilibrium steady-state. After an excitation 90o 1H pulse (pw), transverse magnetization evolves during a free variable evolution t1 period (d2). A 90o 1H pulse (pw) creates longitudinal magnetization and during the NOE mixing time magnetization transfer via cross- relaxation or chemical exchange can take place. A final 90o 1H proton pulse (pw) creates transverse magnetization which is detected as usual.

Rotational Overhauser Effect SpectroscopY, same as NOESY, but works for all molecular weights. Has the disadvantage of producing more rf heating, hence it requires more steady state scans (ss).

Diffusion-Ordered SpectroscopY, seeks to separate the NMR signals of different species according to their diffusion coefficient. Great for determining if you have a mixture of molecules.

Heteronuclear Multiple Quantum Correlation, allows one to pair NH or CH resonances. Often uses X-nucleus decoupling and hence gives rise to rf heating, requires reasonably well calibrated pulses and many steady state scans (ss). Similar to 13C-1H HSQC; however, the HMQC spectrum also shows through-bond 1H-1H couplings along the indirect (13C) dimension. These 1H-1H couplings are often small and only obvious when the resolution is good along the indirect dimension. Some peak broadening along the 13C dimension is expected in a HMQC. Also, several couplings may be involved, leading to a complex peak profile and rendering this method less useful in measuring 1H-1H J couplings in many situations.

Heteronuclear Single Quantum Correlation, provides the same information as HMQC , but gives narrower resonances for 1H-13C correlations. Also requires X-decoupling and hence a large number of steady state scans and is also more sensitive to pulse imperfections.The HSQC experiment is a highly sensitive 2D-NMR experiment is applicable to many nuclei such as 1H - 13C system. The basic scheme of this experiment involves an initial delay (d1) during a period that allows the magnetization to return to an equilibrium steady- state. This is followed by the transfer of magnetization on the proton to the second nucleus, which may be 15N or 13C, via an INEPT (Insensitive nuclei enhanced by polarization transfer) step. After a time delay, the magnetization is transferred back to the proton via a retro-INEPT step and the signal is then recorded. CH and CH3 are positive and CH2 are negative, so you get the equivalent information of a DEPT experiment at the same time. Can also be used to obtain 15N chemical shifts at natural abundance. HSQC gives higher resolution and is preferred over HMQC.

Heteronuclear Multiple Bond Correlation, a variant of the HMQC pulse sequence that allows one to correlate X-nucleus shifts that are typically 2-4 bonds away from a proton. The HMBC experiment correlates chemical shifts of two types of nuclei separated from each other with two or more chemical bonds. For example 1H,13C-HMBC correlates chemical shifts of protons with carbons separated with two or three bonds, in some cases even with the more distant ones. 1H,13C-HMBC is frequently used for assigning of quaternary and carbonyl carbons. In some cases it can be used for assigning -OH protons, and protons bonded to other heteroatoms also. HMBC experiment is quite prone to different artifacts, like are the 1JCH coupling artifacts, missing of crosspeaks for some C/H pairs, etc.