Where is my INEPT signal? - Proton Exchange Issues

INEPT and DEPT sequences are routinely used to enhance the NMR signals for low γ nuclides such as ¹⁵N or ¹³C.  The enhancement relies on polarization transfer between the protons J-coupled and the low γ nuclide.  The pulse sequences incorporate delays based on the reciprocal of the J-coupling constant between the protons and the low γ nuclide.  In the case of ¹⁵N INEPT, the enhancement for each scan can be as much as γ_H/γ_N (~ 10) compared to that from a conventional one-pulse sequence with inverse gated decoupling.  Furthermore, the recycle delay for the INEPT sequence depends on the the ¹H T₁ relaxation time rather than that of ¹⁵N.  ¹H T₁'s are typically an order of magnitude (or more) less than those of ¹⁵N so the recycle delays required for ¹⁵N INEPT spectra are at least ten (and possibly 100 times) shorter than those required for one-pulse data collection.  These two factors mean that the true time saving for a ¹⁵N INEPT measurement compared to a one-pulse ¹⁵N measurement can be on the order of 100 - 1000 times.  There are, however cases where ¹⁵N INEPT signals are attenuated or entirely nonexistent.  Attenuated ¹⁵N INEPT signals are observed when the protons (with short T₁) coupled to ¹⁵N exchange with those of water (longer T₁) on a time scale of seconds.*  The problem arises because of saturation transfer during the inverse gated decoupling used during the acquisition time. The partially saturated protons are unable to transfer as much polarization to the ¹⁵N as they would were they fully polarized. The problem can be reduced if a recycle delay much greater than the T₁ relaxation time of the water protons is employed.  If the protons bound to ¹⁵N undergo exchange with other labile protons at a rate fast with respect to the ¹H-¹⁵N J coupling interaction, polarization transfer from ¹H to ¹⁵N is not possible and a ¹⁵N INEPT signal cannot be observed.  This is demonstrated in the figure below.

Concentrated solutions of the methyl ester of anthranilic acid and anthranilic acid were prepared in DMSO-d₆.  The ¹⁵N NMR data were collected on a 600 MHz instrument with a cryoprobe.  The left-hand panel of the figure compares the ¹⁵N one-pulse spectrum with inverse gated decoupling (bottom) to the INEPT spectrum (top) for the methyl ester.  The spectra were collected with the same number of scans. For the methyl ester, the ¹⁵N bound protons do not exchange with any other labile protons.  The enhancement in the ¹⁵N INEPT spectrum is clear.  Similar spectra for anthranilic acid are shown on the right-hand side of the figure.  In anthranilic acid, the ¹⁵N bound -NH₂ protons undergo intramolecular exchange with the acid proton at a rate fast with respect to the one-bond ¹⁵N-¹H coupling constant (~90 Hz).  As a result, polarization transfer is not possible and no INEPT signal is observed.  The same is true for the meta- and para- isomers (data not shown). 

Thank you to Jin Hong for sharing her experience with collecting ¹⁵N INEPT data for anthranilic acid and Mojmir Suchy for kindly providing the samples.

* G.D. Henry and B.D. Sykes, J. Magn. Reson. B, 102, 193 (1993).

Distortions due to Lock Saturation

The amplitude of the ²H lock signal provides information for an electronic feedback circuit which continuously corrects the magnetic field strength (by way of a B₀ shim) to compensate for environmental instability.   A poor ²H lock signal will provide unreliable input for the feedback circuit and B₀ compensation will be erratic.  This leads to undesirable effects in NMR spectra.  For example, noisy lock signals will lead to undesirable noise at the base of the observed NMR peaks.  If one uses too much lock power, the ²H lock signal gets saturated and the lock amplitude is unstable.  A saturated ²H lock will lead to problems in the NMR spectrum since the input to the B₀ compensation feedback circuit is unstable.  This is demonstrated in the figure below.

When one scan is collected, there are spectral distortions at the base of the NMR resonances.  When 16 scans are collected these artifacts average to produce a general broadening at the base of the NMR resonances.  Be careful not to saturate the ²H lock.