Where is my INEPT signal? - Proton Exchange Issues
INEPT and
DEPT sequences are routinely used to enhance the NMR signals for low ? nuclides such as 15N or 13C. The enhancement relies on polarization transfer between the protons J-coupled and the low ? nuclide. The pulse sequences incorporate delays based on the reciprocal of the J-coupling constant between the protons and the low ? nuclide. In the case of 15N
INEPT, the enhancement for each scan can be as much as ?H/?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).
Source:
University of Ottawa NMR Facility Blog
Where is my INEPT signal? - Proton Exchange Issues
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