1H J-Resolved Spectroscopy to Evaluate 1H-1H and 1H-19F Coupling Constants

2D ¹H J-RESolved spectroscopy (JRES) is able to separate the ¹H chemical shift and J coupling interactions in the F2 and F1 domains of the 2D data, respectively. The F2 projection represents the pure-shift ¹H decoupled ¹H NMR spectrum while the individual F1 slices at each chemical shift reveal the ¹H - ¹H J coupling for each resonance.  When this technique is applied to a spin system with both homonuclear ¹H-¹H coupling and heteronuclear coupling, it has the ability to provide both the homonuclear and heteronuclear coupling constants.  This is demonstrated in the figure below for 2,3-difluoro pyridine which has both ¹H-¹H and ¹H-¹⁹F coupling.

The top trace in the figure is the ¹H NMR spectrum showing the complex resonances due to both the homonuclear and heteronuclear coupling.  The 2D JRES spectrum is highlighted in grey.  The ¹H-¹H coupling is shown in the F1 slices which were summed to produce the blue, red and green vertical traces in the figure for ¹H resonances A, C and B, respectively.  These traces are identical to the resonances in the separately collected ¹H spectrum with ¹⁹F decoupling shown in the bottom trace of the figure.  The F2 projection of the JRES spectrum is shown in the trace directly on top of the 2D spectrum, colour coded in yellow.  The F2 projection represents the ¹H decoupled ¹H spectrum showing only the ¹H- ¹⁹F coupling.  It can be compared to the separately collected PSYCHE pure-shift ¹H spectrum, colour coded in orange which is very nearly identical.  Clearly this very simple, often overlooked, technique can provide a great deal of both homonuclear and heteronuclear coupling information. 

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).

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.

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).

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.

Phasing a 2D NMR Spectrum - Video Tutorial

The following video demonstrates how to phase a 2D NMR spectrum in TOPSPIN 3.

19F NOESY

Two-dimensional ¹H NOESY data are routinely used to assign specific stereo-isomers based on the proton nuclear Overhauser effects (NOE's) which are strongly correlated to inter-proton distances through space.  For example, NOE's may be observed for cis- protons across a double bond but not observed for trans- protons.  The same technique can be used with ¹⁹F in fluorinated compounds to gauge the inter-fluorine distance and assign stereochemistry.  The figure below shows the ¹⁹F NOESY spectrum of a fluorine containing cobalt complex.

From the 1D-¹⁹F NMR spectrum, it is not clear which fluorine atoms are on the same or opposite sides of the four membered cobalt containing ring.  The 2D-¹⁹F NOESY spectrum, on the other hand, shows strong NOE cross peaks between fluorine C and both A and E indicating that C, A and E are on the same side of the ring.  There are also strong cross peaks between fluorine D, and both B and F indicating that D, B anf F are on the same side of the ring.

Thank you to Graham Lee (of Dr. R.T. Baker's research group at the University of Ottawa) for kindly providing the sample and sharing his data. 

The Effect of Viscosity on 1H NOESY Spectra

For small molecules, ¹H 2D NOESY spectra exhibit positive NOE's between protons close to one another in space and the off-diagonal correlations are opposite in phase to those of the diagonals. As molecules become larger and larger the motional correlation times become longer and longer. As the correlation times become longer and longer, the NOE's become less positive, cross zero and then become negative. For large molecules, like proteins, the NOE's are negative and the off-diagonal correlations between close protons are of the same phase as the diagonal peaks. When the correlation times are extremely long, for example in rigid macromolecules or solids, the dipolar coupling among all of the protons is inefficiently averaged by molecular motions and spin diffusion becomes efficient. Spin diffusion allows all of the protons in a dipolar coupled network to exhibit correlations with one another. The sign of the correlations is similar to that observed for chemical exchange or negative NOE's. This phenomenon is demonstrated in the figure below. In the figure, positive contours are represented in black and negative contours are represented in red. The NOESY spectrum on the left is that of a solution of menthol in CDCl₃. The off-diagonal correlations between proximate protons is opposite in phase compared to the diagonal responses, typical of small molecules with short correlation times. The NOESY spectrum on the right is that of the same solution of menthol dissolved in a very viscous fluorinated oil. The extreme viscosity of the sample is sufficient to make the correlation time for the molecules very long such that the menthol behaves like a very large macromolecule where spin diffusion is efficient. As a result, off-diagonal responses are of the same phase as those of the diagonal and are observed between all protons in the molecule.This technique has been cleverly applied* to mixtures of molecules immersed in viscous oils where intra-molecular correlations are observed whereas inter-molecular correlations are not observed. The data allow for the observation of the constituent components of complex mixtures.

* Andre J. Simpson, Gwen Woods, and Omid Mehrzad, Anal. Chem, 80, 186-194 (2008).

Baseline Correction in 2D NMR Spectra

Sometimes a 1D NMR spectrum will have a baseline roll. One way of correcting this is to fit the baseline of the spectrum (between two limits) to a polynomial and then subtract the polynomial from the spectrum to produce a flat baseline. Baseline roll is not an exclusive problem to 1D data. It can also be present in each domain of a 2D data set. Baseline orrection can be applied to each of the dimensions. The figure below shows a proton NOESY spectrum which is uncorrected (top left), baseline corrected in the rows (top right), baseline corrected in the columns (bottom left) and baseline corrected in both domains (bottom right).

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.

What are Those Positive Peaks in My NOESY Spectrum?

Students will sometimes ask me, "What are those positive off-diagonal peaks in my NOESY spectrum?". Since the NOESY pulse sequence is exactly the same as the EXSY sequence, a NOESY spectrum will show all possible correlations due to cross relaxation. These correlations include those from NOE's, chemical exchange and conformational (or rotational) exchange. For small molecules, if one phases the diagonal responses such that they are positive, the NOE's correlations will be negative and the exchange correlations will be positive. The answer to the question is therefore that the positive peaks are due to exchange. The figure below shows an example where three different types of cross peaks are visible. The molecule in the figure has two distinct rotational conformations in slow exchange with one another and gives a proton spectrum with every resonance doubled. Each resonance for one rotational conformation gives a positive (black) cross peak correlating it to the corresponding resonance of the other conformation. There is also a positive cross peak correlating the -NH- proton in the molecule to the residual water in the DMSO-d6 solvent. These protons exchange chemically with one another. Finally, the negative (red) correlations in the figure are due to NOE's.
Thank you to Jean-Gregoire Roveda of Dr. Beauchemin's group for giving me permission to use his spectrum as an example.

2D EXSY

The 2D EXchange SpectroscopY (EXSY) technique is exactly the same as the same as the 2D NOESY technique. The pulse sequences are identical. The method provides off-diagonal responses for spins which exchange slowly with one another (either conformationally or chemically) and also between spins with NOE's. The EXSY method is useful for showing exchange when the rate of the exchange is greater than or of the same order as the T₁ relaxation rate (1/T₁) but less than the frequency difference between the two spins (in the absence of exchange). Depending on the experimental conditions, the responses due to exchange are often much more intense than those due to NOE's. The figure below shows the 300 MHz 2D ¹H EXSY spectrum of N,N-dimethylacetamide at room temperature. At this temperature, the molecule exhibits slow rotation about the (CH₃)₂N - C bond such that both methyl groups exchange with one another rotationally yet are distinct in the spectrum. This is evident by the cross peaks in the spectrum between the two methyl groups on the nitrogen. Note that the off diagonal peaks are of the same phase as the diagonal peaks.

Apodization of 2D Data

In the interest of data collection time and disk storage space, 2D data sets are often collected with short t2 acquisition times and as few as possible t1 slices. In such cases, the FID's in the t2 domain do not decay into the noise and the interferograms in the t1 domain do not decay completely. Applying a 2D Fourier transform to such truncated data will cause ripples in the 2D frequency domain spectrum analogous to those observed in 1D spectra where the acquisition time is too short. The application of an appropriate apodization function (and/or forward linear prediction) to the t2 and t1 domains is important to produce high quality spectra. The figure below illustrates the effect of the more common apodization functions on the cross peak in the phase sensitive COSY spectrum of ethyl acetate. The panel on the upper left shows the appearance of the cross peak when no apodization is applied. One can easily see the ripples in both the F2 and F1 frequency domains. The panel on the upper right shows the effect of applying a 2 Hz exponential line broadening function to each domain. The data are improved but the ripples are still visible. The panel on the lower left shows the effect of applying a sine bell weighting function with the maximum at the midpoint of each of the t2 and t1 domains. This apodization function is suitable for magnitude mode data where the phase is irrelevant. In this example of a phase sensitive data set, one can see a major distortion in the cross peak. The panel on the lower right shows the effect of using a sine squared weighting function with the maximum at the beginning of the t2 and t1 signals. The spectrum is free of ripples and clearly shows the phase information.

Artifacts Due To Setting the Receiver Gain Too High in 2D Homonuclear Experiments

Setting the receiver gain too high leads to very characteristic artifacts in 1D NMR spectra. If the receiver gain is set too high in 2D experiments, one can also expect artifacts in the 2D Fourier transformed data. In homonuclear experiments, setting the receiver gain too high will lead to parallel diagonal signals. This is illustrated in the COSY data in the figure below. In the left hand panel, the receiver was set correctly while in the right hand panel it was set too high. Dotted lines were drawn through the artifacts.

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.

1H - 11B HMQC

HMQC and HSQC experiments are very commonly applied to ¹H - ¹³C and ¹H - ¹⁵N. These techniques can also be applied to other isotopes. The figure below shows the ¹H - ¹¹B HMQC spectrum or ortho-carborane. The top and side traces are separately acquired ¹H [¹¹B] and ¹¹B [¹H] spectra, respectively. These data, combined with the ¹¹B COSY data, allow the complete assignment of the ¹H [¹¹B] spectrum.
Thank you to Dominique Duguay for providing the sample of ortho-carborane.