1. Fundamentals of NMR Spectroscopy
    1. Chapter 1. INEPT and Signal Enhancement
    2. Chapter 2. Refocused INEPT, Delayed Decoupling and In-Phase Spectra
      1. 2.1. Theory
        1. 2.1.1. Methine, Amide and the AX Spin System
        2. 2.1.2. Cosine and Sine Modulation
        3. 2.1.3. Methylene, Methyl, AX2 and AX3 Spin Systems

2 Refocused INEPT, Delayed Decoupling and In-Phase Spectra

Justin Lorieau

The refocused INEPT sequenceBurum D., Ernst R. Net polarization transfer via a J-ordered state for signal enhancement of low-sensitivity nuclei. J Magn Reson. 1980 Apr;39(1):163–168. (Fig. 2.1) converts the anti-phase magnetization of the INEPT sequence into in-phase magnetization, while still benefiting from the signal enhancement of the INEPT sequence. This approach has the advantage that the spectrum can be decoupled to produce singlet peaks.

2.1. Theory

Fig. 2.1. The refocused INEPT experiment.
Fig. 2.2. Comparison of 13C spectra for the INEPT sequence and refocused INEPT sequence with 1H decoupling during 13C acquisition.

The INEPT sequence produces anti-phase magnetization (e.g. ) with peaks of opposite sign, and the refocused INEPT produces in-phase magnetization (e.g. ) with peaks of the same sign. When 1H decoupling is applied to the anti-phase magnetization of the INEPT sequence, the peaks cancel each other to produce a null spectrum. The objective of the refocused INEPT and DEPT experiments is to produce in-phase magnetization that can be decoupled to produce singlet peaks.

2.1.1. Methine, Amide and the AX Spin System

The refocused INEPT sequence produces in-phase magnetization that can 1H decoupled to produce high intensity singlet peaks.

In this example, we’ll use a 1H spin bonded to a 13C spin. If we use a delay, , the first step of the sequence is simply an INEPT sequence (Fig. 2.3).

Fig. 2.3. The first step of the refocused INEPT sequence highlighted in red.

In the second step, we’ll only propagate the JCH-coupling since the `' pulse sequence block refocuses the 13C chemical shifts (Fig. 2.4). Thereafter, we’ll apply the two 180○ pulses.

Fig. 2.4. The second step of the refocused INEPT sequence highlighted in red.

The final and third step (Fig. 2.5) propagates the magnetization with another period.

Fig. 2.5. The third step of the refocused INEPT sequence highlighted in red.

The term is maximum when . As with the INEPT sequence, the magnetization is enhanced by a factor over the unenhanced version.

For a 13C-INEPT between for a 1H spin bonded to a 13C (JCH = 145 Hz), the magnetization after the is . The refocused INEPT experiment produces in-phase magnetization, , suitable for 1H decoupling (Fig. 2.6).

a. Magnetization after INEPT b. Magnetization after ref-INEPT c. Comparison of Spectra
Fig. 2.6. Comparison of the INEPT sequence and the refocused INEPT sequence for a CH group.

2.1.2. Cosine and Sine Modulation

2.1.3. Methylene, Methyl, AX2 and AX3 Spin Systems

Different periods emphasize different types of spin systems. Varying periods is commonly used to differentiate between CH, CH2 and CH3 groups. This principal applies to other X spins, such as 15N, but in the case of NH2 and NH3 groups, rapid hydrogen exchange with the solvent may impede the discrimination between these groups.

The initial INEPT period behaves the same for CH, CH2 and CH3 groups. This is because each 1H spin is only bonded to one 13C spin.

Once the magnetization is converted to transverse magnetization for the 13C spin, the magnetization evolves with J-couplings to multiple 1H spins during the rest of the refocused INEPT. This is because, from the 13C spin’s perspective, a CH group appears as a doublet, a CH2 group appears as a triplet and a CH3 group appears as a quartet.

The conversion to magnetization follows different time dependencies for the CH (AX), CH2 (AX2) and CH3 (AX3) groups.