INEPT sequence

Both experiments are based on polarization transfer from sensitive nuclei to insensitive nuclei, and therefore the mjyor portions of their pulse sequences are common. The INEPT experiment, without refocusing and decoupling, however, yields spectra with distorted" multiplets. For instance, the two lines of a doublet appear in antiphase with respect one another. Similarly, the central line of a triplet may be too small to be visible, while the outer two lines of the triplet will be antiphase to one another. Introducing a variable refocusing delay A and broadband decoupling in the INEPT sequence can convert this experiment into a more useful one. 

In a heteronuclear nOe experiment, the first step may be identical to the homonuclear nOe experiment (i.e., involving an incoherent transfer of coherence from H to H/), while the second step could involve a coherence transfer from H to C nucleus by an INEPT sequence. These methods suffer from poor sensitivity and have therefore not been used extensively. 

To understand the pulse sequence, we will try to get an overview of what is happening and then look at some simplified product operator analysis. Consider first the CH case in the DEPT-90 experiment. Ignoring the 180° pulses, the DEPT-90 sequence can be viewed as an INEPT sequence in which the coherence transfer is split up into two steps (Fig. 7.41) the two 90° pulses are no longer simultaneous and between them we have an intermediate state in coherence transfer multiple-quantum coherence (ZQC and DQC). 

Let s pick a concrete example we are familiar with the INEPT transfer from ll to 13C. The simplest INEPT sequence is just 90°(1H) — x — 90°(1H)/90°(13C), where the x delay is for /-coupling evolution of the ll doublet into antiphase and the simultaneous 90° pulses give coherence transfer from ll to 13C. Normally, we would set this delay to 1/(2 J) to get complete conversion from in-phase to antiphase ll coherence (I 2IVS-), and we would put... 

Mixing an INEPT sequence converts the magnetization into antiphase magnetization with respect to its attached 13C nucleus, and then transfers the magnetization... 

A short example will illustrate the usefulness (and complexity) of density matrix notation. Consider the INEPT sequence for a13C-1 H (S-I) spin system. The pulse sequence is simply... 

It is not necessary that the evolving 13C coherences be detected immediately. As shown in Section 9.6, they can be allowed to precess until they are in phase, then detected while protons are decoupled to provide a single enhanced signal. Alternatively, the entire INEPT sequence can be treated as the preparation period of a 2D experiment. The coherences then evolve during a period t, and can be manipulated in various ways by further pulses. One of the most commonly used methods is to apply a second INEPT sequence, without the initial 90v pulse, after the evolution period to convert the 13C coherences back into H coherences, which can be observed. As we mentioned in Chapter 10, this method, heteronuclear single quantum coherence (HSQC), is widely employed to obtain... 

FIGURE 12.16 Pulse sequence for the triple resonance 3D NMR experiment HNCO. H and N denote H and 15N, C denotes 13C=0, and K denotes 13C . Pulses at times 1, 2, and 3 constitute an INEPT sequence that transfers coherence from H to. V, where it precesses during q. Pulses at times 6, 7, and 8 represent an HMQC sequence that creates multiple quantum coherence in C (where it precesses during and transfers coherence back to N. Pulses 10 and 11 are an inverse INEPT sequence that transfers coherence back to H for detection during f3.The other 180° pulses refocus heteronuclear spin couplings. Note that coherence is not transferred to spin K. 

N magnetization, which evolves to in-phase N magnetization at the end of the second A period. Finally, a reverse INEPT sequence converts this to H magnetization that is antiphase in N, and it evolves during the period r = 1/2JNH to in-phase H magnetization for detection. A summary of this last portion of the coherence pathway is... 

Figure 12 The z-filtered INEPT pulse sequence. The delay A4 is set to 0.5//s,x and the other delays are as in the standard INEPT sequence. Dec. = decoupling...

HSQC. The Heteronudear Single Quantum Correlation (HSQC) experiment is an alternative to HMQC that accomplishes a similar objective. The experiment generates, via an INEPT sequence, single quantum (or " N) coherence, which evolves and then is transferred back to the proton frequency by a second INEPT sequence, this time in reverse. The main difference from the HMQC result is that HSQC spectra do not contain H- H couplings in the C ( Jj) dimension. As a result, HSQC cross peaks tend to have improved resolution over analogous HMQC cross peaks. HSQC is preferred when there is considerable spectral overlap. 

Fig. 7.2.32 [Dum3] Imaging with homonuclear polarization transfer based on the INEPT sequence. Slice selection and space encoding are incorporated into the filter.

Figure 4.21. (a) the INEPT sequence and (b) the refocused INEE sequence. 

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