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).

13C-13C Connectivity via 1H-13C 1,1-ADEQUATE

One of the most valuable pieces of information one could obtain in elucidating the structure of a small organic molecule is carbon-carbon connectivity information.  This information can sometimes be indirectly deduced from HMBC and/or H2BC data with reasonable sensitivity.  The same information can be determined directly, albeit with dramatically less sensitivity, using the ¹³C INADEQUATE technique.  Another option for obtaining carbon-carbon connectivity information is the 1,1-ADEQUATE technique (Adequate sensitivity DoublE QUAnTum spEctroscopy).  This method is proton detected and relies on a 1-bond INEPT transfer between ¹H and ¹³C.  One-bond ¹³C-¹³C double quantum coherence between the carbon bound to the proton used for the initial INEPT transfer and adjacent carbons is allowed to evolve in much the same way as in the INADEQUATE technique.  Magnetization is transferred back to single quantum coherence for proton detection.  The 2D NMR data show correlations between the proton resonances and the double quantum frequencies between the carbon attached to the proton and those carbons bound to that carbon.  The carbon-carbon connectivity information is provided in the double quantum carbon frequencies.  One drawback to the 1,1-ADEQUATE technique is that connectivity cannot be established between two quaternary carbon atoms not attached to protonated carbons.  Connectivity information between a quaternary carbon bound to a protonated carbon can however be established.  The sensitivity advantage of the 1,1-ADEQUATE technique compared to the ¹³C INADEQUATE technique arises from ¹H rather than ¹³C detection and that the recycle delay depends on the proton T₁'s rather than the ¹³C T₁'s.  Here is an example of how one could use the 1,1-ADEQUATE technique with other methods to unambiguously assign the structure of a small organic molecule.  The edited HSQC spectrum of the unknown molecule with separately acquired ¹H and ¹³C NMR spectra as projections is shown in the figure below.

 The ¹³C spectrum provides all of the ¹³C frequencies, while the edited HSQC signals provide the ¹H-¹³C one-bond connectivity and multiplicities for each protonated carbon.  Note that the carbon frequencies could also be determined from a high resolution HMBC spectrum if insufficient material is available for a direct ¹³C measurement.  From the carbon frequencies, one can determine all of the double quantum frequencies as shown in the table below, taking into account the ¹³C offset frequency expressed in ppm, 'o1p'.

Those  highlighted in pink are those that are present in the 1,1-ADEQUATE spectrum which is shown below.

The spectrum was acquired on a concentrated sample at 600 MHz with a cryoprobe using the standard 'adeq11etgpsp' Bruker pulse program .  The total data collection time was less than 1 hour.  The carbon-carbon connectivity is labelled on the spectrum based on the double quantum frequencies using the numbering scheme from the ¹³C spectrum presented as the projection on the edited HSQC spectrum above.  From these connectivities, the structure of the compound can unambiguously be assigned to limonene.

Exchange Effects in HSQC Spectra

The effects of chemical or dynamic exchange on NMR spectra are very well known.  Exchange is often studied by observing line shape changes as a function of temperature, by 2d EXSY, inversion transfer or saturation transfer methods.  Effects due to exchange can also be observed in ¹H - ¹³C HSQC spectra.  The HSQC method works by transferring ¹H magnetization to ¹³C magnetization via an INEPT transfer through the one-bond J coupling across the ¹H - ¹³C chemical bond.  The ¹³C magnetization evolves during the incremented delay, t1, of the 2D pulse sequence according to its chemical shift.  The ¹³C magnetization is then transferred back to ¹H magnetization where is observed during t2.  HSQC spectra thus exhibit cross peaks between ¹H resonances and the resonances of their attached carbons.  If there is exchange between nonequivalent carbon sites during t1, some ¹H resonances may appear to be correlated to two carbon resonances.  An example of this is shown in the figure below.

The ¹³C spectrum of cannabidiol has equally intense broad, resolved aromatic resonances for non-protonated carbons 2 and 6 (not shown) as well as for the protonated carbons 3 and 5.  The ¹H spectrum has broad resolved resonances for both aromatic protons.  This indicates that either the aromatic ring undergoes 180° flips about the 1 - 4 axis or it has two equally probable rotomers defined by a rotation about the 1 - 4 axis.  In either case, the dynamic exchange is slow enough on the NMR time scale to produce resolved resonances yet fast enough to cause significant line broadening.  For each of the two aromatic protons, the HSQC spectrum shows correlations to both C3 and C5; a strong correlation to the carbon to which it is chemically bonded and a weaker correlation to the carbon site in exchange with its attached carbon.          

INEPT

The sensitivity of a low γ, spin I = ½ nucleus is determined by the difference in populations between the low energy and high energy states, governed by the Boltzmann distribution. If the low γ, spin I = ½ nucleus is coupled to a proton the energy level diagram is more complicated than simply two levels and is shown in the figure below where a ¹³C-¹H spin pair is used as an example.

The populations of the states involved in the ¹³C transitions and hence the sensitivity of the ¹³C signal can be altered by inverting the H1 or H2 ¹H transitions with 180° pulses. This is illustrated in the figure below.

In the left panel, the H1 transition of a ¹³C-¹H spin pair is inverted (i.e. the populations of the two energy levels of the H1 transition are swapped). This also affects the populations of the energy levels involved in the C1 and C2 ¹³C transitions. After inversion of the H1 ¹H transition, the intensities of the C1 and C2 ¹³C transitions have changed from their equilibrium value of 1:1 to an enhanced value of 5:-3. If the H2 transition is inverted (right-hand panel), the C1:C2 intensity ratio is -3:5. In both cases the sensitivity of the ¹³C doublet has been enhanced compared to its equilibrium value. This enhancement is called INEPT (Insensitive Nuclei Enhanced by Polarization Transfer) and is one of the most common sensitivity enhancement techniques used in NMR pulse sequences. The simplest implementation of INEPT is shown in the figure below along with the vector diagrams.

Phase cycling can be employed to obtain a -4:4 anti-symmetric doublet, rather than doublets with components of unequal magnitude. This is represented in the figure below.

A refocusing element can be added to the end of the sequence to refocus the anti-symmetric doublets and data can be collected with proton decoupling.

The result is a singlet with 4 times  (i.e. γ_H/γ_C) the intensity of the singlet one would expect under equilibrium conditions without an NOE.  For ¹⁵N, one obtains a sensitivity gain of ~10. The results of these implementations of INEPT are compared to the equilibrium situation in the figure below.

INEPT has the additional advantage that its repetition rate is determined by the ¹H T₁ rather than the ¹³C T₁.  This is a tremendous additional sensitivity improvement when multiple scans are collected because the ¹H T₁ is often shorter than the ¹³C T₁ by an order of magnitude.  One can collect approximately ten times as many scans per unit time.  This advantage is even more significant for ¹⁵N.  Reverse INEPT is used in the collection of ¹H data for  carbon-proton pairs to suppress the protons bound to ¹²C. 

Proton Probe Tuning for 13C Detected Experiments

I am sometimes asked: Do I have to tune the proton channel of the probe for my 13C detected experiments? The answer is yes. If you are just running a simple 13C with proton decoupling then the efficiency of the decoupler will depend on the tuning of the proton channel. If the proton channel of the probe is very badly tuned, you may see broadening and even splittings in your carbon signals. The signal-to-noise ratio will also suffer. For experiments like DEPT or INEPT, the proton tuning is critical as these sequences require proton pulses of specific flip angles. If the proton channel of the probe is not tuned and matched then the flip angles for the proton pulses in the sequence will be less than they should be and you will not get the results you expect. In the figure below is an example. The bottom trace is a 13C DEPT-135 spectrum of menthol with the proton channel of the probe properly tuned. The spectrum in the upper trace is also a DEPT-135 spectrum of menthol run under the same conditions except that the proton channel of the probe was detuned. It looks more like a DEPT-90 as the proton pulses are closer to 90 degrees than 135 degrees.

Remember to tune the proton channel!