J-Modulated Spin Echo Experiments

While the SPT experiment has obvious utility, it is cumbersome to use unless the selective nature of the experiment is specifically being exploited for structure elucidation purposes. A group of experiments that may be categorized as J-modulated spin echo experiments allows the simultaneous investigation of the entire spectrum. As the name of this group of experiments implies, they utilize a spin echo of the type shown in Eq. (1), over which a scalar coupling driven process (using jcH most commonly) is superimposed. 

Experiments that fall into this category include INEPT, DEPT, and APT. These experiments are discussed in considerable detail in any of the monographs cited in 

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APT Attached proton test, a modification of the J-modulated spin-echo experiment to determine C//multiplicities, a less sensitive alternative to DEPT... 

The combination of these two spectra then allows direct identification of XH,XH2 and XH3 signals in even the most complex molecules in a similar manner as in the J modulated spin echo experiments described above. 

Fig. 8.6 Pulse sequence for the amplitude modulated 2DJ-resolved NMR experiment. The experiment is based on a J-modulated spin echo. The first 90° pulse tips magnetization into the xy-plane where it evolves during the first half of the evolution period, t]/2. The 180° pulse is applied and the decoupler is simultaneously gated off for the second half of evolution.

As shown in Fig. 3 a, spin coherence is manifested in the optically detected transient nutation signal for [Rh(bpy)3] (0)04)3 the phosphorescent triplet state. In this experiment, one observes that the phosphorescence intensity becomes modulated as the pulse length of microwave pulses, resonant with the D - transition, is gradually increased. The modulation is evidence that the micro-wave excitation induces a spin coherence in the ensemble of molecules in the photoexcited triplet state [44]. Moreover, from the transient nutation experiment one obtains the information about the duration of the pulses needed in a spin echo experiment. In the case of the example, the n/2 pulse is 100 ns and the 71 pulse has a length of 200 ns. Similarly, transient nutation signals for the other zero-field spin resonances could be obtained. The optically detected spin echo decay as measured for the D - j j zero-field transition for [Rhlbpylj](004)3... 

D J-resolved NMR experiments are a conceptual amalgamation of two topics discussed above, the /-modulated spin echo and the two-dimensional characteristic of the spin—lattice relaxation experiments. As the name of these experiments implies, scalar coupling information, /, will be displayed in the one frequency domain chemical shift information will be presented in the second frequency domain. The simplest 2D/ experiments sort chemical shift information in the detected time domain, labeled by convention, while the heteronuclear scalar couplings of each carbon are sorted into the indirectly determined time domain, tj (do not be confuse lower case h with the spin—lattice relaxation time, Tj). 

In heteronuclear y-resolved spectra chemical shifts of an arbitrary nucleus X which couples with protons (this is mostly are presented on one axis and proton-X J couplings on the other. The information content is equivalent to that in a proton-coupled "C spectrum (Fig. 12) but without the severe overlap of multiplets which is usually encountered in the latter. In common with off-resonance proton decoupling, y-modulated spin echo, and DEPT experiments, it facilitates multiplicity determination. In addition, it enables proton-X coupling constants to be measured. 

The basis of the method can be illustrated by the following simple example. In proton NMR spectroscopy, the lines in the individual patterns are phase modulated by the spin-spin coupling during the spin-echo experiment by using variations in For example, the two components in a first-order doublet of a two-spin system are phase modulated at a frequency of J/2. The phase modulation of the resonance intensity results from the fact that the spin states of the protons producing the multiplet pattern are different. If a methyl proton is interacting with a methine proton, a doublet is observed because there are two possible energy orientations of the methine proton relative to Hq. 

Fig. 10.12. Pulse sequence for amplitude modulated 2D J-resolved spectroscopy. The experiment is effectively a spin echo, with the 13C signal amplitude modulated by the heteronuclear coupling constant(s) during the second half of the evolution period when the decoupler is gated off. Fourier transformation of the 2D-data matrix displays 13C chemical shift information along the F2 axis of the processed data and heteronuclear coupling constant information, scaled by J/2, in the F1 dimension.

From the above discussion, it is clear that observation of ESEEM requires that the microwave pulses affect branching of the EPR transitions. This places a quantum mechanical constraint on the ESEEM experiment, in that each energy level must be involved in at least two different microwave transitions, and an experimental constraint that requires the microwave pulse bandwidth to cover the spread in frequencies needed to fully excite the branching . The experimentally observed ESEEM function is a product of the quantum mechanically derived modulation function and a decay function that describes the loss of magnetization due to spin relaxation. These decay functions are typically modeled with exponential forms exp(—t/Tq)" where n = 1,2 or 0.5. For a 90° — x — 180° or two-pulse echo experiment, Tq = T, a time that is typically on the order of 1 J,s, as evidenced by the data shown for the Cu(II) center in Figure 1. This... 

The measurement of one-bond coupling constants from the distance between the multiplet components in HSQC spectrum becomes unreliable for large proteins due to the differential relaxation that can severely broaden one of the components, and even make it undetectable. Luy and Marino proposed to overcome this problem by introduction of J-modulation to the sharp TROSY component. The JE-TROSY experiment starts with a spin-echo J-evolution period, which is followed by a traditional TROSY detection sequence. As the result the resonances are independently modulated by the single-bond coupling, which leads to the displacement of the cross-peak in the independent spectral dimension after the 3D Fourier transformation. This displacement is measured either relative to zero frequency if real FT is used in the J-dimension, or as the splitting between the pseudo multiplet components if the complex FT is applied. The spectral width in the J-dimension is normally set to twice the value of the... 

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