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).

Ultra-Fast 1H COSY

It cannot be disputed that the introduction of routine 2D NMR spectroscopy in the 1980's revolutionized the way in which NMR measurements are made. Now, with literally thousands of 2D methods available, the quantity of accessible information has dramatically increased. One cannot imagine a modern NMR lab without a 2D NMR toolbox.  One of the main drawbacks to traditional 2D NMR spectroscopy has always been the time required to collect the data.  Data collection can take anywhere from a few minutes to tens of hours.  Many 1D FIDs (typically more than 128) must be acquired as a function of evolution time to construct the 2D data matrix.  The measurement of each of these signals may require multiple scans as a result of necessary phase cycling between which a relaxation delay must be employed.  Once all of the data have been collected, each of the FID's is Fourier transformed followed by a second Fourier transform with respect to the evolution time.  Typical data collection and processing are illustrated here. The introduction of pulsed field gradients for coherence selection has reduced the time required to collect 2D spectra by reducing or  eliminating the need for phase cycling however, one still has to collect many FID's as a function of evolution time.  Even when multiple scans are not required for sensitivity, data collection can take minutes to hours.

Ultra-fast 2D measurements, employing an entirely different method of data collection, were introduced in 2002 and subsequently improved.  In this method, z-field gradients combined with linearly swept chirp pulses are used to phase encode spins linearly along the z axis of the sample according to specific evolution times.  The dephasing depends on both the position along the z axis of the sample and the resonance frequency of each spin.  After this encoding scheme is applied, each slice element of the sample has experienced a different evolution time as a function of its position in the sample.  After a conventional mixing period dictated by the type of 2D measurement, the site specific, spatially phase encoded spins must be read.  This is accomplished by applying a series of bipolar gradient pulse pairs while the receiver is collecting data.  During each gradient pulse (lasting typically 250 μsec) echos are collected.  The position of each echo during a single gradient pulse is related in a one-to-one fashion to the frequency of each of the spins in the sample thus mimicking a mini NMR spectrum whose frequency axis is replaced by a linearly related time axis.  The "spectra" collected during the negative gradient pulses are mirror images to those collected during the positive gradient pulses and must be reversed during data processing.  A series of typically 128 bipolar gradients are applied with the receiver open thus all of the data are acquired in a single scan.  Each "spectrum" collected is a function of the z slice position in the sample, which in turn is linearly related to the evolution time. The collection of "spectra" represent the ultra-fast domain and is Fourier transformed point by point as a function of evolution time (or z position).  The entire data collection sequence takes approximately 100 msec.

The left panel of the figure below shows a conventional 300 MHz gradient enhanced COSY-45 spectrum for a concentrated sample of menthol in CDCl₃ collected in 4.5 minutes.  The panel on the right shows a 300 MHz ultra-fast COSY spectrum of the same sample collected in only 100 msec - a time saving factor of 2700!  Both spectra were collected on a Bruker AVANCE II 300 NMR spectrometer equipped with a standard BBOF probe.  Both data sets were symmetrized.  Although the ultra-fast data set has noticeably lower resolution and sensitivity, one can see that it is very similar to the conventional COSY.

There are, of course, a number of drawbacks to the ultra-fast scheme including low sensitivity, limited resolution and limited accessible spectral widths.  Some of these drawbacks can be overcome  with the use of cryoprobes and strong pulsed field gradients. Molecular diffusion over the course of the measurement may also cause problems.  Despite the drawbacks however, the method is extremely well suited to time studies of chemical reactions where conventional 2D data collection would simply take too long.

The references below are a good place to start in order to find out more about this technique.  There is also a very well documented setup procedure available on the Bruker User Library, provided by Patrick Giraudeau, including pulse sequences and processing scripts.

Annual Rev. Anal. Chem. 7, 129-161 (2014).
Mag. Res. Chem. 53, 986-994 (2015).
J. Am. Chem. Soc. 125, 9204–17 (2003).
J. Am. Chem. Soc. 125, 12345–50 (2003).

E.COSY and the Relative Signs of Coupling Constants

Spin-spin coupling constants can have values greater than or less than zero. The absolute sign of the coupling constants cannot be discerned from the simple examination of a ¹H NMR spectrum. The E.COSY¹ (Exclusive COrrelation SpectroscopY) technique is one method which can be used to determine the relative signs of coupling constants. E.COSY is a phase sensitive COSY variant which produces off-diagonal signals showing only the active coupling (i.e. the coupling directly responsible for the cross-peak) as 2x2 antiphase square tetrads displaced in both the F2 and F1 domains by an amount equal to the passive coupling constants (i.e. the couplings not directly responsible for the cross peak). The slope of a line drawn through the cross-peaks is used to determine the relative signs of the passive coupling constants. The sign of the slope depends on whether the signs of the passive couplings are the same or whether they differ. The figure below shows the gradient E.COSY spectrum² (using the Bruker pulse program,"ecosygpph" ) for the ABX three-spin system in phenylalanine.The cross-peaks highlighted in yellow in the top panel of the figure are expanded in the bottom panel. Black contours are positive and red contours are negative. The panel on the bottom left shows the cross-peaks for AX and BX. In the case of the AX cross-peak, the line drawn through the cross peak has a positive slope indicating that the passive couplings J_AB and J_BX are of opposite sign. The line drawn through the BX cross peak also has a positive slope indicating that the passive couplings J_AB and J_AX are of opposite sign. From this we can deduce that J_AX and J_BX are of the same sign. The panel on the bottom right shows the cross-peak for AB. In this case, the line drawn through the cross peak has a negative slope confirming that the passive couplings J_AX and J_BX are of the same sign. In conclusion, the geminal and vicinal coupling constants are of opposite sign.

1. C. Griesinger, O.W. Sorensen & R.R. Ernst, J. Magn. Reson. 75, ; 474 - 492 (1987).

2. The ecosygpph pulse program produces spectra similar to those described in reference 1 as "complimentary" E.COSY spectra. The slope of the lines through the cross peaks in "complementary" E.COSY spectra are of opposite sign to those obtained from the E.COSY spectra described in reference 1.

Double Quantum Filtered COSY

COSY spectra are very useful in structure elucidation as they provide correlations between coupled spins. Often, NMR spectra have large singlet signals from uncoupled protons (such as t-butyl methyls, methoxy protons, excess water or a solvent signal) which provide no information in the COSY spectrum and perhaps even get in the way of looking for smaller coupled spins. In such cases one can use a double quantum filtered COSY sequence rather than a standard COSY 90 or COSY 45 sequence. Double quantum filtered COSY spectra filter out uncoupled singlets. A comparison of a standard COSY 90 and a double quantum filtered COSY sequence for ethyl acetate is shown below. One can see that the singlet is present in the COSY 90 spectrum but absent in the double quantum filtered COSY spectrum.

Magnitude COSY-90 vs. Phase Sensitive COSY-90

Most commonly, chemists run simple COSY spectra in magnitude mode. A magnitude COSY provides positive peaks for both the diagonal and off-diagonal responses due to the magnitude calculation. The responses in a standard COSY-90 sequence have lines with a phase twist shape. The magnitude calculation is necessary to provide positive responses and the time domain data are usually treated with a sine bell weighting function (or something similar) to enhance the resolution lost in the phase twist lineshape. There are however, several versions of the COSY experiment. One such version is a phase sensitive COSY-90. This version provides off-diagonal responses that can be phased. Furthermore, the coupling giving rise to the cross peak (the active coupling) will be antiphase. The disadvantage to this method is that the diagonal responses are 90 degrees out of phase and can obscure off-diagonal responses close to the diagonal. The figure below compares a magnitude COSY-90 to a phase sensitive COSY-90 for ethyl acetate. In the figure, black is positive and red is negative.

COSY- 90 vs COSY- 45

Aside from the standard ¹H and ¹³C NMR 1D experiments, ¹H COSY experiments are among the most commonly used NMR techniques by organic chemists. There are many different modifications to the standard two pulse COSY experiment and often the organic chemist does not even know which one they are using. Two of the most common experiments for routine work are the gradient magnitude COSY- 90 and COSY- 45 experiments. The only difference between the two methods is the flip angle of the second pulse (90 degrees for the COSY- 90 and 45 degrees for the COSY- 45). For a concentrated sample, these experiments can be acquired in a matter of minutes. Although the signal to noise ratio is higher for a COSY- 90, the COSY- 45 is usually the preferred experiment because the diagonal signals are smaller and less intense allowing correlations between close resonances to be resolved more easily. The figure below shows magnitude gradient COSY- 90 and COSY- 45 spectra for 3-heptanone. Note the smaller diagonal responses in the COSY- 45.

COSY vs TOCSY

COSY (COrrelation SpectroscopY) and TOCSY (TOtal Correlation SpectroscopY) experiments are very common. Although both experiments provide a diagonally symmetric two dimensional contour plot with the one dimensional spectrum on the diagonal and correlations off of the diagonal, they have different information content. A COSY spectrum will have off-diagonal correlations between coupled spins whereas a TOCSY spectrum will have off-diagonal correlations between all spins in a spin system. For example, consider a spin system where A is coupled to B, B is coupled to C, and C is coupled to D. A COSY spectrum will have correlations between A and B, B and C, and C and D, whereas a TOCSY spectrum will have correlations between all of the spins. This is illustrated in the figure below for 3-heptanone. In this case, there are two spin systems (one on either side of the carbonyl group). The COSY spectrum shows correlations between adjacent spins whereas the TOCSY spectrum shows correlations between all of the spins in each of the two spin systems.

11B COSY

Many NMR users are very familiar with the ¹H COSY experiment. It is used extensively by organic chemists. Many people do not realize that the COSY method can be used for many other abundant isotopes as well. The figure below shows the ¹¹B COSY spectrum of ortho-carborane. The data were collected with ¹H decoupling and allow the complete assignment of the ¹¹B NMR spectrum. Note that in this case, the ¹¹B - ¹¹B J couplings are unresolved in the 1D spectrum. Thank you to Dominique Duguay for providing the sample for the figure.