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).

Pure-Shift HSQC

Pure-shift NMR has become more and more common over the last few years.  A special issue of Magnetic Resonance in Chemistry has recently been dedicated to developments in these methods.  Pure-shift NMR methods offer simplified proton NMR spectra free of ¹H - ¹H coupling.  These methods have been extended to proton detected 2D NMR measurements, yielding 2D data sets with higher proton resolution compared to conventional 2D measurements.  The NMR Methodology Group at the University of Manchester has been a primary contributor to this technique and has kindly shared their efforts on-line.  The figure below compares the 600 MHz partial Pure-Shift HSQC spectrum of sucrose in DMSO-d6 to a more conventional HSQC spectrum acquired under similar conditions.  The projections on the spectra are independently collected high resolution ¹H NMR spectra.  Clearly, the  Pure-Shift HSQC data have higher ¹H resolution than the more conventional HSQC.  What may not be so obvious from the figure is that the sensitivity is also improved in the Pure-Shift HSQC.  The gain in signal-to-noise-ratio depends strongly on the degree of coupling collapsed.  For some signals in this spectrum, an improvement in the signal-to-noise-ratio as high as 72% was observed.

The the top and middle panels of the figure below show the 1D ¹H projections of the Pure-Shift HSQC and HSQC data from the above figure, respectively.  The bottom panel is the conventional high resolution ¹H spectrum for comparison.

Clearly, the Pure-Shift HSQC proton projection offers much improved resolution.   

13C Satellite Observation with 1D 1H - 13C HSQC

Two dimensional ¹H - ¹³C or  ¹H - ¹⁵N HSQC spectra are typically used to obtain one-bond heteronuclear correlation information between ¹H and ¹³C or ¹H and ¹⁵N.  The data are proton detected and typically employ pulsed field gradients for coherence selection.  The technique uses an incremented delay for chemical shift correlation and essentially discards the very intense signals from protons bound to ¹²C or ¹⁴N while enhancing the remaining doublet signals for protons bound to ¹³C or ¹⁵N.  Usually the ¹H FIDs are collected with ¹³C or ¹⁵N decoupling to collapse the doublets into singlets in the F2 domain leaving a single correlation between proton signals in F2 at the ¹³C or ¹⁵N chemical shifts in F1 of the nuclei to which the protons are bound.   One can use a 1D version of this experiment (without the incremented delay for chemical shift correlation) to collect ¹H NMR spectra exclusively of the protons bound to ¹³C or ¹⁵N.  If used without ¹³C or ¹⁵N decoupling, it allows easy observation of the ¹³C or ¹⁵N satellites in the absence of the very intense signals due to protons bound to ¹²C or ¹⁴N and allows the precise measurement of one-bond  ¹H - ¹³C  or  ¹H - ¹⁵N coupling constants.  The top panel of the figure below shows the 300 MHz 1D  ¹H - ¹³C HSQC spectrum of ethyl acetate.  Clearly the ¹³C satellites are observed and the  ¹H - ¹²C signals are suppressed.  The ¹H spectrum of ethyl acetate is shown in the bottom panel of the figure for comparison.

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.          

Non-uniform Sampling (NUS)

Collecting 2D or 3D NMR data can be very time consuming. The indirect dimension of a 2D experiment is sampled linearly via the t1 increments in the pulse sequence.  An FID must be collected for every single linearly spaced t1 increment. In the interest in collecting 2D or 3D NMR data in a more time efficient manner, a great deal of effort is made towards faster data collection techniques.  While some of these methods are based on spatial selectivity, others are based on sparse sampling techniques in the indirect dimensions of nD NMR sequences.  One such sparse sampling method, given the name non-uniform sampling (NUS), samples a sub-set of the indirect dimension in a random (or weighted random) manner and then predicts the uncollected data based on the data sampled, in much the same way data are predicted in the forward and backward linear prediction methods.  The reconstructed data is then used for the indirect Fourier transforms.  A comparison of the conventional and non-uniform data sampling methods is illustrated in the figure below.

Collecting only a fraction of FID's reduces the experiment time by the same fraction.  The figure below shows a superposition of partial 600MHz ¹H-¹³C HSQC spectra of a D₂O solution of sucrose.

All of the spectra were collected with 2 scans per increment using a 1.5 second recycle time.  The lower spectrum in black was collected conventionally with 256 increments in 15 minutes. The middle spectrum in blue was collected conventionally with 64 increments in 3.75 minutes. The top spectrum in purple was collected using NUS with 25% of 256 increments (i.e. 64 increments) collected in 3.75 minutes.  A comparison of the two conventionally collected data sets shows the expected loss in F1 resolution with the 4-fold reduction in experiment time by reducing the number of increments by a factor of 4. The bottom (black) conventional spectrum and the top (purple) NUS spectrum are however virtually indistinguishable despite the 4-fold reduction in experiment time for the NUS spectrum.  NUS is a very valuable technique for reducing experiment times without sacrificing resolution.

NMR of the Christmas Tree

One of my fondest memories as a child is the colorful lights and especially the smell of a decorated Christmas tree.  The hot incandescent lights used years ago would heat up the tree evaporating the fragrant compounds in the needles producing the very memorable and wonderful smell of Christmas.  Although modern artificial Christmas trees and cool LED lights have made the holiday season safer with respect to fires, they have taken much of the magic out of Christmas.  Among many other compounds, it is pinene, bornyl acetate and citronellol that contribute to the Christmas smell of evergreen needles.

We can use NMR spectroscopy to look for these compounds and perhaps recover a bit of the Christmas magic.  The bottom panel of the figure below shows the ¹³C CPMAS spectrum of spruce needles.  One can easily identify the signals from cellulose in the CPMAS spectrum of the needles while some of the smaller peaks can be attributed to fragrant compounds.  Many of the fragrant compounds in the needles are likely to be in a liquid-like state and not cross polarize very well.  These will either be absent or under-represented in the CPMAS spectrum.  The top panel of the figure shows the ¹H - ¹³C HSQC spectrum of a benzene-d₆ extract prepared from crushed spruce needles.  The top and left-side projections are the high resolution ¹H and ¹³C NMR spectra, respectively.  This sample is expected to contain all of the benzene soluble compounds.  The spectrum is free of cellulose resonances and shows a mixture of fragrant compounds.

These data don't recover the childhood magic of Christmas but they do bring a little bit of joy to this NMR spectroscopist.

Merry Christmas  

TROSY

The chemical shift resolution and sensitivity of NMR generally benefit from an increase in magnetic field strength.  As a result, large sums of money are spent on magnets with higher and higher fields.  The boost in sensitivity means that smaller and smaller quantities of sample are needed and measurements can be completed in shorter periods of time.  There are particular cases however, where an increase in magnetic field can lead to a loss of sensitivity and resolution.  This is the case for ¹⁵N decoupled ¹H spectra of the ¹H-¹⁵N spin pairs in very large ¹⁵N labelled proteins.  The very long correlation times of the protein combined with the high resonance frequencies associated with high field strength lead to very short T₂ relaxation times and therefore broader lines.  The broad lines account for a significant loss in resolution and sensitivity.  One might then wonder why protein structural chemists spend so much money on very high field magnets.  What follows is one possible answer to this question.

The two main relaxation mechanisms for the ¹H and ¹⁵N in proteins are dipolar coupling and chemical shielding anisotropy.  These two mechanisms are also cross correlated with one another.  The cross correlation term is of different sign for each of the two peaks in a ¹H-¹⁵N doublet resulting in one of the peaks of the doublet having a shorter T₂ (and broader line) than the other one.  If ¹⁵N decoupling is applied, one sees a single resonance with a line width determined by the average of the two components of the doublet.  This is illustrated in the figure below for a ¹H-¹⁵N spin pair in a small and large molecule at high field .  The same is true in the ¹⁵N-¹H doublets in the ¹⁵N spectra of ¹⁵N-¹H spin pairs.

At very high fields, One of the lines in the ¹H-¹⁵N doublet is very sharp and the other very broad.  If, in the ¹H spectrum of a protein, we could eliminate all of the broad doublet components leaving only the sharp ones, we would have a high resolution ¹H spectrum.  Further, if we could combine such a measurement with an HSQC, we would have a high resolution ¹H-¹⁵N HSQC at high field.  The combination of these two measurements is called transverse relaxation optimized spectroscopy (TROSY).  TROSY data collection employs an HSQC measurement with neither ¹H nor ¹⁵N decoupling elements (as described in a previous post) as well as other elements which suppress the broad lines of the doublets and retain the sharp lines.  The results of this are illustrated in the figure below for small and large proteins at high field.

Clearly, it is not advantageous to use the TROSY technique on small proteins rather than the conventional HSQC.  For large proteins at high field however, there is a significant sensitivity and resolution advantage compared to a conventional HSQC.  It should be noted that the TROSY cross peaks are shifted by ½ ¹J_HN in both the F2 and F1 domains.  The figure below shows a superposition of a conventional HSQC (black) and a TROSY (blue) for a protein at 500 MHz.

One can clearly see the ½ ¹J_HN shift in the F2 and F1 domains of the TROSY compared to the HSQC.  In this case, the conventional HSQC gives higher sensitivity than the TROSY.

Thank you to Adam Damry of Professor Roberto Chica’s research group at the University of Ottawa for providing the sample of ¹⁵N labelled protein.

Decoupling in 2D HSQC Spectra

HMQC and HSQC NMR data are commonly used to correlate the chemical shifts of protons and ¹³C (or ¹⁵N) across one chemical bond via the J coupling interaction.  The data are ¹H detected, with the ¹H chemical shift in the horizontal F2 domain and the ¹³C (or ¹⁵N) chemical shift in the vertical F1 domain.  In the case of ¹H and ¹³C, the technique depends on protons bonded to ¹³C.  ¹H–¹²C spin pairs provide no coupling information and are suppressed by the method.  If one is to observe the ¹H signal of a ¹H-¹³C spin pair, one expects to observe a doublet with splitting ¹J_H-C (i.e. the ¹³C satellites).  Likewise, if one is to observe the ¹³C signal of a ¹H-¹³C spin pair, one expects to observe a doublet with the same splitting.  2D HSQC spectra are normally presented with both ¹H and ¹³C decoupling yielding a simplified ¹H-¹³C chemical shift correlation map over one chemical bond.  The figure below shows one of the most commonly used gradient HSQC pulse sequences.  The ¹H and ¹³C decoupling elements of the sequence are highlighted in yellow and pink, respectively.

During the evolution time, t1, the ¹³C chemical shift and ¹H-¹³C coupling evolve.  The ¹H 180° pulse (color coded in yellow) in the center of the evolution time refocuses the coupling and as a result decouples protons in the F1 (¹³C) domain of the spectrum.  ¹³C is broadband decoupled from the F2 (¹H) domain by applying a GARP pulse train (color coded in pink) at the ¹³C frequency during the collection of the FID.  One can turn each of these elements “on” or “off” for data collection.  The figure below shows the ¹H-¹³C gradient HSQC spectrum of benzene with all possible combinations of ¹H and/or ¹³C decoupling.

In the top left panel both ¹H and ¹³C decoupling are turned “on” and one observes a singlet in both the F2 (¹H) and F1 (¹³C) domains.  In the top right panel, the ¹H decoupling element is “on” while the ¹³C decoupling element is “off”.  The result is a ¹H-¹³C doublet in the F2 (¹H) domain and a singlet in the F1 (¹³C) domain.  In the bottom left panel, the ¹H decoupling element is “off” while the ¹³C decoupling element is “on”.  The result is a ¹³C-¹H doublet in the F1 (¹³C) domain and a singlet in the F2 (¹H) domain.  In the bottom right panel, both the ¹H and ¹³C decoupling elements are “off”.  The result is a ¹H-¹³C doublet in both the F2 (¹H) and F1 (¹³C) domains.

Weak One-bond or Multiple Bond Correlations in 1H / 13C HMQC / HSQC Spectra

Many people are quite surprised to see either unusually weak one-bond correlations or weak multiple bond correlations in their ¹H / ¹³C HMQC / HSQC spectra. These people must be reminded that there is nothing "magic" about these experiments - the responses are based solely on an assigned delay proportional a reciprocal coupling constant. The large scale success of the ¹H / ¹³C HMQC / HSQC techniques can be attributed to the fact that most one-bond ¹H - ¹³C coupling constants are very similar ( ~ 145 Hz). The pulse sequences are therefore run with a delay based on a 145 Hz coupling constant. When one-bond coupling constants are significantly different than 145 Hz then the correlation will be either very weak or absent in the spectrum. Also, if multiple bond couplings are unusually large then those multiple bond correlations may be present in the spectrum. The figure below is an example. In the 500 MHz HMQC spectrum of an alkyne (optimized for 145 Hz coupling), one can see an unusually small one-bond correlation between the terminal alkyne proton and its attached carbon. There is also a weak two-bond correlation between the terminal alkyne proton and the other alkyne carbon.

HMQC vs HSQC

Proton detected Heteronuclear Multiple Quantum Coherence (HMQC) and Heteronuclear Single Quantum Coherence (HSQC) are both NMR techniques used to correlate the chemical shift of the protons in a sample to a heteronucleus such as ¹³C or ¹⁵N via the J coupling interaction between the nuclei. Since both techniques essentially provide the same information - a correlation map between the coupled spins - students sometimes ask which of these two methods is better and which should they use routinely. The difference between the two techniques is that during the evolution time of an HMQC both proton and X magnetization (eg: X = ¹³C ) are allowed to evolve whereas in an HSQC only X magnetization is allowed to evolve. This means that an HMQC is affected by homonuclear proton J coupling during the evolution period while an HSQC is not affected as there is no proton magnetization during the evolution time. The homonuclear proton J coupling manifests itself as broadening in the X dimension. The top panel of the figure below shows the 7.05 T ¹H /¹³C HMQC and HSQC spectra of menthol with an expansion of one of the resonances highlighted in yellow. One can see that the expanded cross peak of the HMQC is broader in the ¹³C dimension than that of the HSQC. The bottom panel of the figure shows the corresponding ¹³C projection spectra. One can see that the resolution is better in the projection of the HSQC compared to the HMQC. One might conclude that, due to the higher ¹³C resolution, it is always better to run an HSQC rather than an HMQC. This is definitely the case if all of the pulses are calibrated well, however since there are many more pulses in an HSQC compared to an HMQC, it is more susceptible to losses in signal-to-noise-ratio due to poor probe tuning or poor pulse calibration. My advice to students is that, if high ¹³C resolution is required, then make sure the pulses are calibrated well on a well tuned and matched probe and run an HSQC. If high ¹³C resolution is not critical then run an HMQC.

HSQC and Edited HSQC Spectra

Many of you use a simple magnitude HMQC sequence to establish heteronuclear one-bond 1H -13C correlations. One can also use the phase sensitive HSQC sequences to obtain the same information. Although the data must be phased by the user, the phase sensitive sequences will provide data with higher resolution as absorption line shapes are much narrower than the magnitude signals obtained in non-phase-sensitive sequences. There is also an edited HSQC sequence available which provides multiplicity information similar to that of a 13C DEPT-135 sequence where CH and CH3 signals are phased up and CH2 signals are phased down. In the figure below is the HSQC and edited HSQC spectra of 3-heptanone. The red CH2 signals in the edited HSQC are negative with respect to the black CH3 signals. Using this sequence may save you time (and money) as there will be no need to run a 13C DEPT spectrum.