INEPT pulse sequences

FIGURE 55. DOSY-INEPT spectrum of a mixture of cyclodimethylsiloxanes (OSiMe2) (n = 3, 4 and 5 CDCI3 solution, 23 °C, diffusion coefficients not calibrated, pulse sequence INEPT-DOSY391, 32 transients, 10 increments of gradient strength). Diffusion order agrees with the molecular size and the known chemical shifts of the siloxanes... 

Polarization transfer pulse sequences (INEPT or DEPT can be applied successfully if the approximate magnitude of coupling constants "/( Sn, H) is known (see Eigure 2.1.1). 

Schenker, K. V. and Von Philipsborn, W. Off-resonance effects and their compensation in the multiple-pulse sequences INEPT, DEPT, and INADEQUATE. J. Magn. Reson. 66 219-229, 1986. 

Figure 2.9 Pulse sequence for the INEPT experiment. (B) Effect of pulses on H magnetization. Application of the pulse sequence shown results in population inversion of one of the two proton vectors of the CH doublet and therefore causes an intensification of the corresponding C lines.

The INEPT experiment can be modified to allow the antiphase magnetization to be precessed for a further time period so that it comes into phase before data acquisition. The pulse sequence for the refocused INEPT experiment (Pegg et al., 1981b) is shown in Fig. 2.13. Another delay, A. is introduced and 180° pulses applied at the center of this delay simultaneously to both the H and the C nuclei. Decoupling during data acquisition allows the carbons to be recorded as singlets. The value of Z), is adjusted to enable the desired type of carbon atoms to be recorded. Thus, with D, set at V4J, the CH carbons are recorded at VsJ, the CH2 carbons are recorded and at VeJ, all protonated carbons are recorded. With D3 at %J, the CH and CH ( carbons appear out of phase from the CH2 carbons. 

The APT pulse sequence provides limited information about the number of hydrogens bonded to the carbons in a molecule, since it does not readily allow us to distinguish between the CH, and CH carbons or between CH and quaternary carbons. The INEPT spectrum not only can yield information about the multiplicity of all the carbons, but also affords sensitivity-enhanced C signals due to polarization transfer. 

The basic INEPT spectrum cannot be recorded with broad-band proton decoupling, since the components of multiplets have antiphase disposition. With an appropriate increase in delay time, the antiphase components of the multiplets appear in phase. In the refocussed INEPT experiment, a suitable refocusing delay is therefore introduced that allows the C spin multiplet components to get back into phase. The pulse sequences and the resulting spectra of podophyllotoxin (Problem 2.21) from the two experiments are given below ... 

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. 

INEPT (insensitive nuclei enhanced by polarization transfer) Polarization transfer pulse sequence used to record the NMR spectra of insensitive nuclei, e.g., C, with sensitivity enhancement may be used for spectral editing. 

J splittings cannot be directly resolved. In addition to the obvious advantage of providing a map of chemical bonds between the spins, /-based transfers do not require spin-locking and are not disturbed by molecular motions. The major drawback of polarization transfer through J coupling is that the delays involved in the pulse sequences, such as insensitive nuclei enhanced by polarization transfer (INEPT) [233] or heteronuclear multiple-quantum coherence (HMQC)... 

The kinetics of hydrogenation transfer is covered by the use of an exchange superoperator assuming a pseudo first-order reaction. Thereby, competing hydrogenations of the substrate to more than one product can also be accommodated. In addition, the consequences of relaxation effects or NOEs can be included into the simulations if desired. Furthermore, it is possible to simulate the consequences of different types of pulse sequences, such as PH-INEPT or INEPT+, which have previously been developed for the transfer of polarization from the parahydrogen-derived protons to heteronuclei such as 13C or 15N. The... 

Fig. 10.15. Pulse sequence for the multiplicity-edited gradient HSQC experiment. Heteronuclear single quantum coherence is created by the first INEPT step within the pulse sequence, followed by the evolution period, t . Following evolution, the heteronuclear single quantum coherence is reconverted to observable proton magnetization by the reverse INEPT step. The simultaneous 180° XH and 13C pulses flanked by the delays, A = l/2( 1 edits magnetization inverting signals for methylene resonances, while leaving methine and methyl signals with positive phase (Fig. 16A). Eliminating this pulse sequence element affords a heteronuclear shift correlation experiment in which all resonances have the same phase (Fig. 16B).

As in the case of the HN(CO)CA-TROSY scheme, the HN(CO)CANH-TROSY experiment can be readily expanded to a four-dimensional HN(CO)CANH-TROSY experiment without increasing the overall length of the pulse sequence. This can be accomplished by labelling the 13C (f) chemical shift during additional incremented time delay, implemented into the 13C — 13C INEPT delay. As a result, a well-dispersed 13C (i- 1), 13C (i- 1), 15N(i), Hn(0 correlation map is obtained with minimal resonance overlap albeit with the inherent sensitivity loss by a factor of y/2. 

In the 13C spectra, assignment of 0(4) and C2(3) rests not only on the larger intensity of the former but also on multiplicities found with the INEPT pulse sequence. (E)- and (Z)-methyl groups are distinguished in C2(3) CH3. 

The INEPT (Insensitive Nuclei Enhanced by Polarization Transfer) experiment [6, 7] was the first broadband pulsed experiment for polarization transfer between heteronuclei, and has been extensively used for sensitivity enhancement and for spectral editing. For spectral editing purposes in carbon-13 NMR, more recent experiments such as DEPT, SEMUT [8] and their various enhancements [9] are usually preferable, but because of its brevity and simplicity INEPT remains the method of choice for many applications in sensitivity enhancement, and as a building block in complex pulse sequences with multiple polarization transfer steps. The potential utility of INEPT in inverse mode experiments, in which polarization is transferred from a low magnetogyric ratio nucleus to protons, was recognized quite early [10]. The principal advantage of polarization transfer over methods such as heteronuclear spin echo difference spectroscopy is the scope it offers for presaturation of the unwanted proton signals, which allows clean spec-... 

Pig. 1. Pulse sequence for selective reverse INEPT. The time-shared homonuclear decoupling during acquisition is optional, and a variety of simplifications may be made to the sequence depending on the instrument used and on the spin system under investigation, as discussed in the text. A DANTE sequence is shown as the selective 90° carbon-13 pulse, but this may be replaced by a soft pulse or some other form of selective excitation. Phase cycling for this sequence is summarized in table 1. 

Phase cycling scheme for the selective reverse INEPT pulse sequence of fig. 1. Phases are shown in multiples of 90° subscripts indicate that a given phase or bracketed block of phases should be repeated the stated number of times, e.g., the notation (01)2 (13)2 indicates the sequence 0101 1313. Phases in the sequence of fig. 1 other than those listed above remain unchanged in successive transients. 

Fig. 2. Pulse sequence for selective reverse INEPT using pulsed field gradients to select the coherence transfer echo. The 180° pulse pair in the middle of the 2r delay is not normally needed for t < 50 ms, and the second proton 180° pulse and first t2 delay maybe omitted if a linear phase gradient in the resultant spectrum can be tolerated. The second field gradient pulse has an area (7c/th) times that of the first.

Fig. 1. Pulse sequences for determining spin-lattice relaxation time constants. Thin bars represent tt/2 pulses and thick bars represent tt pulses, (a) The inversion-recovery sequence, (b) the INEPT-enhanced inversion recovery, (c) a two-dimensional proton-detected INEPT-enhanced sequence and (d) the CREPE sequence. T is the waiting period between individual scans. In (b) and (c), A is set to (1 /4) Jm and A is set to (1 /4) Jm to maximize the intensity of IH heteronuclei and to (1/8) Jm to maximize the intensity of IH2 spins. The phase cycling in (c) is as follows 4>i = 8(j/),8(-j/) 3 = -y,y A = 2(x),2(-x) Acq = X, 2 —x), X, —X, 2(x), —x, —x, 2(x), —x, x, 2 —x),x. The one-dimensional version of the proton-detected experiment can be obtained by omitting the f delay. In sequence (d), the phase 4> is chosen as increments of 27r/16 in a series of 16 experiments.

A remaining limitation of the pulse sequence shown - the restriction of the accessible spectral range of Y nuclei which results as a consequence of the need to apply -pulses to the Y-channel - is circumvented by the sequence shown in Fig. lib43 which uses INEPT for the /X and a HMQC scheme for the... 

The J-MODulated (JMOD) C experiment, also known as Attached Proton Test (APT) was the first and simplest way to determine "C multiplicities. In contrast to DEPT no polarization transfer is included in the pulse sequence (Fig. 3.16) and as a consequence the signals of quaternary carbons are visible in the spectrum, but the sequence is far less sensitive than DEPT or INEPT. The value of D2 is used to differentiate between the different carbon multiplicities. The signal intensities of quaternary carbons are not influenced by the value of D2 for D2 equal to 1 CH and CH, groups have maximum negative intensity and CHj has maximum positive intensity. For D2 equal to 1 /C2 J, ) only the signals of quaternary carbons are visible. 

Pulse sequences for non-selective polarization transfer, not only useful for signal enhancement but also for multiplicity selection, are referred to as INEPT [54], abbreviated from Insensitive Nuclei Enhanced by Polarization Transfer . An improved method denoted as Distortionless Enhancement by Polarization Transfer or DEPT" [55] permits the cleanest multiplicity selection known so far, with full enhancement and low sensitivity to individual CH coupling constants. In addition, fully enhanced and undistorted coupled spectra can be recorded. Finally, subspectra for CH, CH2 and CH3 groups can be generated. 

Values of /CH were also determined by two-dimensional /-resolved spectroscopy using an INEPT-type pulse-sequence,29 with a BIRD pulse for the suppression of "/c H and folding in the F, dimension. Rules for the calculation of the correct values of /c H from the reduced splittings observed were given. The values were also obtained from a set of /-modulated, one-dimensional 13C spectra via the... 

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