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.

11B Double Quantum - Single Quantum Correlation Spectroscopy

The 2D ¹³C INADEQUATE method provides double quantum - single quantum (DQ/SQ) correlations and enables one to determine the carbon - carbon skeleton of small organic molecules.  The method is quite insensitive for ¹³C since the natural abundance of ¹³C is only 1.1% and the chance of having two adjacent ¹³C nuclei is only 1 in 8264.  For spins other than ¹³C, for which the natural abundance is high, one expects the sensitivity of DQ/SQ correlation spectroscopy to be much higher.  ¹¹B has a natural abundance of 80.42% and the chance of having two adjacent ¹¹B nuclei in compounds with boron-boron bonds is 1 in 1.55.  ¹¹B NMR spectra are often sufficiently broad due to efficient quadrupolar relaxation such that homonuclear ¹¹B - ¹¹B J coupling is unresolved however the T₂'s are long enough to allow the collection of  2D ¹¹ B COSY and 2D DQ/SQ data.  The figure below shows the 2D ¹¹B DQ/SQ correlation spectrum of ortho-carborane.  The ¹¹B bonding connectivity can be determined easily from the spectrum.

2D 13C INADEQUATE

The 2D ¹³C INADEQUATE (Incredibe Natural Abundance DoublE QUAmtum Transfer Experiment) is undoubtedly one of the most definitive, yet under-used NMR techniques able to assign chemical structures of small organic molecules.  It gives a connectivity map for all carbon atoms in the molecule.  The reason that it is so under-used is that it relies on one bond ¹³C - ¹³C coupling therefore, adjacent carbon atoms must both be of the ¹³C isotope.  Since ¹³C is only 1.1% naturally abundant, the chance of having two adjacent ¹³C atoms is approximately 1 in 8300, reducing the sensitivity of the measurement drastically.  As a result, 2D INADEQUATE spectra can only be run on very concentrated samples.  The sensitivity afforded by high magnetic field strengths and cryogenically cooled probes has certainly made these measurements more accessible than they have been in the past and they may be within reach when sample quantity and solubility are not a problem.  The 2D ¹³C INADEQUATE spectrum of ~450 mg of limonene in benzene-d6 was acquired on a Bruker TCI H/C/N cryoprobe at 600 MHz and is shown in the figure below.

The spectrum was acquired with the gradient version of the INADEQUATE pulse sequence using a shaped refocusing pulse (Bruker pulse program inadgpqfsp).  It was acquired in 11.8 hours with 64 scans for each of 128 increments using a 5 second recycle delay. The proton decoupled ¹³C spectrum is in the horizontal F2 domain.  The spectrum is interpreted by locating the vertical cross peaks for each ¹³C resonance.  Each cross peak has a partner peak along the horizontal axis.  The partner peak lies on the same vertical axis as the carbon atom bonded to the initial carbon.  This is shown in the figure for C1 which is bonded to C2, C3 and C4.  The entire carbon skeleton of the molecule can be traced unambiguously in this manner to provide a complete assignment.  The same sample was run under the same conditions in 45 minutes with only 4 scans.  A comparison of the signal-to-noise ratio for both data sets is shown in the figure below.

It is clear that usable 2D INADEQUATE data can be acquired in less than an hour for extremely concentrated samples at high field with a cryoprobe.     

Fast 2D Data Collection - NOAH and NUS

One always strives to collect high quality 2D NMR data in a short period of time.  This is particularly important for samples of limited stability or perhaps for monitoring chemical reactions.  High magnetic fields and cryogenically cooled NMR probes have allowed for a higher signal-to-noise-ratio for a given quantity of sample, thereby reducing data collection time as a fewer number of scans are required.  Gradient enhanced 2D NMR data collection gained widespread use in the 1990s.  This represented a tremendous time saving as multi-step phase cycles required for coherence selection could be reduced or eliminated as they were replaced by pulsed field gradients.  Some pulse sequences which required 16 scans per increment to accommodate the necessary phase cycle could be run with a single scan for every increment with the use of pulsed field gradients, thus reducing the data collection time by a factor of 16.  Now, 2D data collection with coherence selection via pulsed field gradients is considered "conventional".  More recently, Non-Uniform Sampling (NUS) was introduced.  Data collection with this technique samples only a limited number of increments in the t1 domain.  The unsampled increments are calculated based on the sampled increments prior to Fourier transformation. The data collection time is reduced in accordance with the number of increments not sampled.  Recently, Kupce and Claridge^1,2 have developed a technique where multiple 2D methods are concatenated in a single super pulse sequence employing a single relaxation delay. They have called the technique NOAH (NMR by Ordered Acquisition using ¹H detection) The time saving of the NOAH technique compared to individually collected 2D spectra results from waiting a single relaxation delay for all experiments rather than a single relaxation delay for each separately acquired spectrum. The data for each spectrum is acquired in separate memory blocks which are separated after data collection allowing the data for each 2D method to be processed individually.  Very recently, both NUS and NOAH have been used together to further reduce data collection times³.  A comparison of the time saving is shown in the figure below for a sample of sucrose in DMSO-d6 collected on a Bruker AVANCE III HD 600 NMR spectrometer equipped with a cryoprobe.

All spectra were collected with 2 scans and a 1 second recycle time.  Individually, both NOAH and NUS offer a significant time saving but when used together they permit very fast, high quality data collection.  A COSY, edited HSQC and HMBC can be collected in a total time of only 4 minutes and 8 seconds.  Other ultra-fast techniques have been developed by others where an entire 2D spectrum is collected in less than one second.

1. Eriks Kupce and Tim D. W. Claridge.  Chem. Commun. 54, 7139 (2018).
2. Eriks Kupce and Tim D. W. Claridge. Angew. Chem. Int. Ed., 56, 11779 (2017).
3. Maksim Mayzel, Tim D. W. Claridge and Ēriks Kupce. Bruker User Library (2018).

Dying iPhones and Liquid Helium Fills

As a habit I do not expose my iPhone to the large stray magnetic fields of high-field or unshielded NMR magnets.  I do however feel safe carrying it near low-field shielded magnets with 5 Gauss stray fields within the croyostat of the magnet. That is - until lately.  Last year, after topping up the liquid helium on a 300 MHz shielded magnet in a fairly small room, I noticed that my iPhone 8 had become completely unresponsive.  The only stimulus it appeared to respond to was gravity.  As it was under warranty, I sent it back to Apple.  After a week or so, they sent it back to me with a note saying that there was nothing wrong with it.  I found this very strange and did not make a connection between the helium fill and the problem with the phone.  I had done many helium fills in the past while carrying an older iPhone 5.  Approximately 9 months later, my iPhone 8 suffered a similar problem again after filling the same shielded 300 MHz magnet with liquid helium.  This time, I took it to a local Apple Store while it was dead.  The technician examined it, ran it through a software protocol, confirmed it was dead and issued me a new phone as it was in its last weeks of its warranty.  Shortly after this, I read about problems others have had with iPhones and Apple watches around helium gas and finally made the connection between the problem I was having and my helium fills. Some Apple iPhones (apparently, iPhone 6 and higher) will completely die when exposed to helium gas. As if in the spirit of Easter, however, they will resurrect themselves after the helium has dissipated from the phone and the battery has been allowed to discharge.  The problem is that in newer iPhones, Apple has swapped out a quartz oscillator, used in older versions of the phone, with a microelectromechanical systems chip (MEMS) which is sensitive to the presence of helium gas. This sensitivity is indeed mentioned in the User Guide of the iPhone.

Exposing iPhone to environments having high concentrations of industrial chemicals, including near evaporating liquified gasses such as helium, may damage or impair iPhone functionality. Obey all signs and instructions. 

Android phones apparently do not use MEMS and therefore are not vulnerable to the problem. Several weeks ago, I absent-mindedly entered a room with my iPhone 8 while a magnet was being filled with liquid helium.  Again, the same thing happened.  This time, I allowed the phone to sit for a week after which I was able to charge it.  After charging, it worked well with no loss of data.

Warning: If you see that a magnet is being filled with liquid helium, Do not enter the room with an iPhone 6 or higher.

Comparison of Broadband Decoupling Schemes

Many NMR measurements such as HSQC or HMQC rely on broadband X nucleus decoupling (X = ¹³C, ¹⁵N, ³¹P .... etc.)..  Broadband decoupling schemes, using conventional rectangular pulses (e.g GARP) require fairly high power levels leading to undesired sample heating.  They are also limited in their effective decoupling bandwidth.  Adiabatic decoupling schemes use shaped adiabatic pulses and have become more and more common over the last couple of decades due, in large part, to the flexibility of modern NMR instruments to generate shaped pulses.  Adiabatic decoupling schemes (e.g. WURST) use much less power than those using conventional rectangular pulses. thereby reducing or eliminating problems associated with sample heating.  Due to the lower power requirements and increased effectiveness over wider frequency ranges, adiabatic decoupling schemes are ideally suited for X nucleus decoupling at higher field strengths. The figure below shows 500 MHz ¹H [³¹P] NMR spectra measured with inverse gated decoupling for the P-CH₃ methyl resonance of dimethyl methylphosphonate  The single scan spectra were collected in a pseudo-2D fashion, as a function of the decoupler offset frequency from -256 ppm to +256 ppm from the ³¹P resonance frequency in 1 ppm steps.  The acquisition time and recycle time for each FID were 2 sec and 4 sec, respectively.   In the left-hand panel, broadband GARP decoupling was employed at a power of 1.22 W  (60 µsec 90° pulses).  In the right-hand panel, WURST decoupling was used at a peak power of 0.755 W (2 ms WURST pulses, bandwidth = 250 ppm).

Clearly, the data collected with WURST decoupling, at lower power, have a much larger decoupling range (250 ppm) compared to the data collected with GARP decoupling (98 ppm).  Furthermore, while the GARP data were collected, the sample temperature increased and had to be compensated for by the variable temperature unit.  No such temperature increase was observed while collecting  the WURST data.  It is also interesting to note that, in the case of GARP decoupling, distorted line shapes are observed just outside of the decoupling range, while for WURST decoupling, the spectra are fully coupled just outside of the decoupling range with a very sharp transition between being fully coupled and fully decoupled.  For broadband decoupling, WURST is best!