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).

NOAH - Faster 2D Data Collection

NMR users typically run ¹H, ¹³C, COSY, HSQC, HMBC and NOESY spectra to elucidate the structures of small molecules. Even with cryogenically cooled probes and pulsed field gradient accelerated methods, collecting 2D spectra can be quite time consuming. For concentrated samples, each 2D experiment will typically take minutes to tens of minutes to collect. Much of this time is the result of waiting for T₁ relaxation in each of the experiments. Recently, Kupce and Claridge^1,2 have developed a technique using standard NMR hardware 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. The data can also be processed in automation. The authors have kindly made this method accessible to all Bruker users through the Bruker User Library which contains pulse sequences, parameter sets, automation scripts and detailed instructions. The left-hand panel of the figure below shows the 600 MHz HMBC, Ed-HSQC and COSY spectra obtained from the NOAH-3 BSC (HMBC, HSQC, COSY) pulse sequence for sucrose in DMSO-d6.  The right-hand panel shows separately acquired 2D data sets for comparison.

The NOAH spectra were obtained from the raw concatenated data with the automation script provided. The high quality NOAH-3 data using 2 scans, 256 increments and a 2 second relaxation delay, took only 24 minutes to acquire in comparison to the separately acquired 2D spectra obtained with similar parameters, which took a total of 59 minutes to acquire. This represents a time saving of 35 minutes or 59%. It should also be noted that the data from the NOAH-3 BSC sequence is of comparable quality to that of the individually collected spectra.

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).

HMBC vs. H2BC

NMR spectroscopy is an indispensable tool for assigning the structure of organic compounds.  One very useful method in the NMR toolbox is the Heteronuclear Multiple Bond Correlation (HMBC) experiment.  HMBC data are ¹H detected and provide a 2D correlation map between ¹H and ¹³C similar to HMQC or HSQC except that the correlations are between protons and carbons separated by two, three and sometimes even four bonds.  This long range information is very helpful in elucidating chemical structures, especially those with non-protonated carbons.  The problem, however with HMBC data is that the correlations depend only on the magnitude of the long-range ¹H-¹³C coupling constants.  Two- or three- bond coupling constants are very similar in magnitude to one another and therefore it is not possible to distinguish between two- and three- bond correlations.  Also, since many long range ¹H-¹³C coupling constants (including two-bond coupling constants) are near zero, some correlations may be absent.  These problems may make structure elucidation frustrating or impossible.  The Heteronuclear 2 Bond Correlation (H2BC) experiment¹ provides an HMBC-like correlation map with (almost) exclusively two-bond ¹H-¹³C correlations.  Unlike the correlations in the HMBC measurement, which rely exclusively on long range ¹H-¹³C coupling constants, the ¹H-¹³C correlations in the H2BC experiment rely on three-bond ³J_H-H coupling between the protons on adjacent carbons.  It is a combined HMQC-COSY experiment.  The size of the H2BC correlations depends on the magnitude of the ³J_H-H coupling constant.  Three-bond ¹H-¹³C correlations are possible only if four-bond ⁴J_H-H coupling is significant.  One disadvantage to the H2BC experiment is that all correlations between protons and non-protonated carbons are necessarily absent because of the absence of H-H coupling.  In general, two-bond ¹H-¹³C correlations that are weak or absent in HMBC spectra are strong in H2BC spectra and three-bond ¹H-¹³C correlations which are strong in HMBC spectra are absent or very weak in H2BC spectra.  The techniques are very complimentary.  The figure below illustrates  the complimentary nature of the two methods for styrene.

The HMBC spectrum in the left panel was scaled up until some of the HMQC artifacts (color coded in blue) were visible.  The data show only one 2-bond ¹H-¹³C correlation (color coded in pink). The three-bond ¹H-¹³C correlations are color coded in yellow.  In comparison, the H2BC spectrum in the right panel shows exclusively two-bond ¹H-¹³C correlations with the exception of those involving the C1 non-protonated carbon.

1. Nyberg, Duus, Sorensen.  J. Am. Chem. Soc. 127, 6154 (2005).

HMQC Responses in HMBC Data

Despite the built in low pass J filters written into the HMBC pulse sequences, the much larger one-bond 1H - 13C coupling responses are not fully suppressed. These one-bond HMQC responses show up as a pair of responses in the acquisition domain separated by the one-bond 1H - 13C coupling. The responses are doubled since 13C decoupling is not typically used in HMBC sequences. An example of these artifacts is shown below.

Measurement of 13C-19F Coupling in a 1H-13C HMBC

It is often difficult to find the 13C signals in fluorinated organics by direct 13C observation. It may sometimes be easier to find these signals by way of a 1H-13C HMBC spectrum if there are nearby protons in the molecule. As shown below, it is even possible to measure the 13C-19F coupling constants.

Nyquist Fold-Backs in HMBC Spectra

The digital filters of modern NMR spectrometers have not only improved the signal-to-noise specification quoted by instrument companies but they have also eliminated Nyquist fold-backs for signals outside of the spectral window. This is not the case however for the indirect dimension of 2D experiments. In the left panel of the figure below is a properly recorded HMBC spectrum of 3-heptanone. In the right panel is a spectrum acquired with the 13C spectral window set too small. The carbonyl correlations are folded into the low frequency end of the 13C axis.
If you inadvertently collect a spectrum with a Nyquist fold-back you can still calculate the correct chemical shift, as the signal will be the same number of ppm away from the wrong end of the axis as it is outside of the correct end of the axis.

Missing Signals in HMBC Data

The HMBC sequence is very useful at establishing long range correlations between protons and carbon. It is particularly useful at finding carbonyl or quaternary carbons when only small amounts of sample are available. The HMBC spectrum has the same appearance as an HMQC or HSQC spectrum except that the responses are between protons and carbon separated by 2, 3 or possibly even 4 bonds. People often ask why are some signals missing from the data? Like the DEPT experiment discussed yesterday, the HMBC sequence is optimized for a particular coupling constant (usually about 10 Hz). If the long range proton-carbon coupling is close to zero then the signal will be absent from the spectrum. Such absences can give you valuable structural information as 3 bond proton-carbon coupling follows a Karplus type relationship.