Will the correct structure please stand up? … Part 2

Part 1 presented a challenge to determine an experiment to distinguish two very similar products from each other, namely 3-methyl-5-(pyridin-2-yloxy)pyridine and 5'-methyl-2H-1,3'-bipyridin-2-one. The products have identical formula weights and the LC/MS and 1H NMR are too similar to draw any conclusion from.

 

The first step is to determine what is different between the two products and then identify an experiment specifically designed to focus on that difference. The obvious difference between the two products is the position of the oxygen atom—an ester group verse a carbonyl group. An FT-IR experiment, as commented by the reader Felipe A., can be used to sort out the products.

 

Other experiments can include the use of reducing agents, 15N NMR, 1H -13C HMBC, 1D NOE, 1H-1H TOCSY, MS2, etc. Note free water, acids and sample concentration can inhibit the use of some of these experiments.

 

A 13C NMR experiment may appear to be another good choice when trying to identify a carbonyl group. However, the carbonyl is part of a conjugated system and so the 13C chemical shift is expected around 160 ppm, which also happens to be expected for the 13C chemical shift of the O-C=N group on the other product.

Will the correct structure please stand up? … Part 2

Part 1 presented a challenge to determine an experiment to distinguish two very similar products from each other, namely 3-methyl-5-(pyridin-2-yloxy)pyridine and 5'-methyl-2H-1,3'-bipyridin-2-one. The products have identical formula weights and the LC/MS and 1H NMR are too similar to draw any conclusion from.

 

The first step is to determine what is different between the two products and then identify an experiment specifically designed to focus on that difference. The obvious difference between the two products is the position of the oxygen atom—an ester group verse a carbonyl group. An FT-IR experiment, as commented by the reader Felipe A., can be used to sort out the products.

 

Other experiments can include the use of reducing agents, 15N NMR, 1H -13C HMBC, 1D NOE, 1H-1H TOCSY, MS2, etc. Note free water, acids and sample concentration can inhibit the use of some of these experiments.

 

A 13C NMR experiment may appear to be another good choice when trying to identify a carbonyl group. However, the carbonyl is part of a conjugated system and so the 13C chemical shift is expected around 160 ppm, which also happens to be expected for the 13C chemical shift of the O-C=N group on the other product.

How to Interpret an HSQC-COSY Experiment

Where a COSY or TOCSY spectrum can be a challenge for a structure with severe spectral overlap, collecting an HSQC-TOCSY spectrum can be a better choice. An HSQC-TOCSY experiment stands for Heteronuclear Single Quantum Coherence-Total Correlation Spectroscopy and other variants include HMQC-TOCSY, HSQC-COSY, etc. Depending on the mixing time, the hybrid experiment generally offers information on both short-range and long-range coupled nuclei.

For the diol fragment below, 1H-13C HSQC correlations are expected for C-H atoms labeled 4, 5 and 9.

On the 1H-13C IDR-HSQC-COSY spectrum below, the three HSQC signals are phased negative (blue). (Note: the acronym IDR stands for Inverted Direct Response.) If a rectangle is draw connecting two HSQC signals, the COSY signals (phased positive) are located at the opposite corners of the rectangle. The spectrum shows a COSY correlation between protons 4 and 5, and between protons 4 and 9.

 

How to Interpret an HSQC-COSY Experiment

Where a COSY or TOCSY spectrum can be a challenge for a structure with severe spectral overlap, collecting an HSQC-TOCSY spectrum can be a better choice. An HSQC-TOCSY experiment stands for Heteronuclear Single Quantum Coherence-Total Correlation Spectroscopy and other variants include HMQC-TOCSY, HSQC-COSY, etc. Depending on the mixing time, the hybrid experiment generally offers information on both short-range and long-range coupled nuclei.

For the diol fragment below, 1H-13C HSQC correlations are expected for C-H atoms labeled 4, 5 and 9.

On the 1H-13C IDR-HSQC-COSY spectrum below, the three HSQC signals are phased negative (blue). (Note: the acronym IDR stands for Inverted Direct Response.) If a rectangle is draw connecting two HSQC signals, the COSY signals (phased positive) are located at the opposite corners of the rectangle. The spectrum shows a COSY correlation between protons 4 and 5, and between protons 4 and 9.

 

Stereochemistry Information from NOESY/ROESY data … Part 2

NOESY, ROESY, COSY and TOCSY are all 2D NMR experiments that sound so similar but offer different pieces of information about the puzzle. When interpreting the NMR data, it is important to understand how the nuclei interact with each other. For example, the presence of a cross peak (a correlation off the diagonal) on a COSY dataset is a result of nuclei coupling through a bond(s) whereas a NOESY dataset measures NOE’s (Nuclear Overhauser Effect) through space regardless of the number of bonds separating the nuclei. An NOE is typically observed for nuclei that are separated no farther than 5 Å apart.

For the enantiomers example shown below, the NOESY and COSY experiments differ in the presence or absence of the cross peaks. A clear difference between the two experiments is the information provided on the diastereotopic protons of the CH2 group.

The NOESY spectrum, also outlined in Part 1, shows 2 correlations at (4.29,1.28) and (4.29,3.13) ppm. There are no NOE's to the proton signal at 2.68 ppm. The DQF-COSY below shows two-bond and three-bond correlations at (4.29,3.13), (4.29,1.28) and (3.13,2.68) ppm. There are no four-bond correlations present as the 4J coupling constants are close to zero.

  

Stereochemistry Information from NOESY/ROESY data … Part 2

NOESY, ROESY, COSY and TOCSY are all 2D NMR experiments that sound so similar but offer different pieces of information about the puzzle. When interpreting the NMR data, it is important to understand how the nuclei interact with each other. For example, the presence of a cross peak (a correlation off the diagonal) on a COSY dataset is a result of nuclei coupling through a bond(s) whereas a NOESY dataset measures NOE’s (Nuclear Overhauser Effect) through space regardless of the number of bonds separating the nuclei. An NOE is typically observed for nuclei that are separated no farther than 5 Å apart.

For the enantiomers example shown below, the NOESY and COSY experiments differ in the presence or absence of the cross peaks. A clear difference between the two experiments is the information provided on the diastereotopic protons of the CH2 group.

The NOESY spectrum, also outlined in Part 1, shows 2 correlations at (4.29,1.28) and (4.29,3.13) ppm. There are no NOE's to the proton signal at 2.68 ppm. The DQF-COSY below shows two-bond and three-bond correlations at (4.29,3.13), (4.29,1.28) and (3.13,2.68) ppm. There are no four-bond correlations present as the 4J coupling constants are close to zero.

  

Stereochemistry Information from NOESY/ROESY data … Part 1

Several NMR experiments offer tools to help determine the stereochemistry of a structure. Some typical experiments are 1D NOE (Nuclear Overhauser Effect), 2D NOESY (NOE Spectroscopy) and ROESY (Rotating-frame Overhauser Effect Spectroscopy). These experiments will produce signals for nuclei that are close to each other through space independent of the number of bonds separating the nuclei.

A simplified 1H-1H NOESY spectrum is shown below. The spectrum shows 2 correlations at (4.29,1.28) and (4.29,3.13) ppm. There is no correlation to the proton signal at 2.68 ppm.

Based on NOESY data, there are 2 possible conformations. The enantiomers (partially drawn) are shown below.

Stereochemistry Information from NOESY/ROESY data … Part 1

Several NMR experiments offer tools to help determine the stereochemistry of a structure. Some typical experiments are 1D NOE (Nuclear Overhauser Effect), 2D NOESY (NOE Spectroscopy) and ROESY (Rotating-frame Overhauser Effect Spectroscopy). These experiments will produce signals for nuclei that are close to each other through space independent of the number of bonds separating the nuclei.

A simplified 1H-1H NOESY spectrum is shown below. The spectrum shows 2 correlations at (4.29,1.28) and (4.29,3.13) ppm. There is no correlation to the proton signal at 2.68 ppm.

Based on NOESY data, there are 2 possible conformations. The enantiomers (partially drawn) are shown below.

Distinguishing Impurities … Part 5

The past few blogs, Part 3 and Part 4, have examined impurity(ies) identification from short-range 2D NMR experiments without much success. If proton singlets, possibly attributed to impurities, are to be distinguished from the main unknown, then long-range 2D NMR experiments may help out by establishing long-range correlations to other atoms.

A 1H-1H TOCSY experiment with a mixing time of 30 ms is shown below. The correlations are colour-coded based on intensity, red for a high intensity and green for a low intensity. The black line spanning across the 2D NMR spectrum indicates the diagonal.

The alleged impurity singlets at 1.68 and 2.14 ppm do not show any long correlations to any of the remaining 1H signals as seen by a lack of any off-diagonal peaks. Although it is still possible that the singlets can pertain to the main unknown through a lack of any ‘neighbouring’ protons to couple to, more data is definitely needed to support/dismiss this claim.

Distinguishing Impurities … Part 5

The past few blogs, Part 3 and Part 4, have examined impurity(ies) identification from short-range 2D NMR experiments without much success. If proton singlets, possibly attributed to impurities, are to be distinguished from the main unknown, then long-range 2D NMR experiments may help out by establishing long-range correlations to other atoms.

A 1H-1H TOCSY experiment with a mixing time of 30 ms is shown below. The correlations are colour-coded based on intensity, red for a high intensity and green for a low intensity. The black line spanning across the 2D NMR spectrum indicates the diagonal.

The alleged impurity singlets at 1.68 and 2.14 ppm do not show any long correlations to any of the remaining 1H signals as seen by a lack of any off-diagonal peaks. Although it is still possible that the singlets can pertain to the main unknown through a lack of any ‘neighbouring’ protons to couple to, more data is definitely needed to support/dismiss this claim.

Distinguishing Impurities … Part 4

Certain NMR experiments offer clues to differentiate a signal from the main unknown and from the impurity(ies). Some clues are not as obvious as others and so it takes a little practice to understand what to look for in a dataset.

A 1H-1H double quantum filter (DQF) COSY experiment (shown below) is used to filter out uncoupled systems. The alleged impurity singlets at 1.68 and 2.14 ppm do not show any diagonal and off-diagonal correlations. As such, this 2D NMR experiment is a bad choice in cases where the impurities exhibit uncoupled proton signals.

Distinguishing Impurities … Part 4

Certain NMR experiments offer clues to differentiate a signal from the main unknown and from the impurity(ies). Some clues are not as obvious as others and so it takes a little practice to understand what to look for in a dataset.

A 1H-1H double quantum filter (DQF) COSY experiment (shown below) is used to filter out uncoupled systems. The alleged impurity singlets at 1.68 and 2.14 ppm do not show any diagonal and off-diagonal correlations. As such, this 2D NMR experiment is a bad choice in cases where the impurities exhibit uncoupled proton signals.

Is it possible to elucidate an unknown with just a 1H NMR spectrum? … Part 3

Misconstrued coupling patterns on a 1H NMR spectrum can halt an elucidation process in its track. As such, structural connectivity is best validated with additional data such as a 1H-1H COSY.

A good use of coupling patterns and coupling constants is at the end of the elucidation process. Candidate structures can be verified or eliminated by the use of coupling information.

The following coupling patterns are some examples where the observed coupling pattern is not as simple as tallying the number of observed lines.

 

 

Is it possible to elucidate an unknown with just a 1H NMR spectrum? … Part 3

Misconstrued coupling patterns on a 1H NMR spectrum can halt an elucidation process in its track. As such, structural connectivity is best validated with additional data such as a 1H-1H COSY.

A good use of coupling patterns and coupling constants is at the end of the elucidation process. Candidate structures can be verified or eliminated by the use of coupling information.

The following coupling patterns are some examples where the observed coupling pattern is not as simple as tallying the number of observed lines.

 

 

Is it possible to elucidate an unknown with just a 1H NMR spectrum? … Part 2

Peak shape, and subsequently the observed coupling pattern, is highly dependent on a range of factors such as temperature, concentration, pH, FID processing, etc. Relying solely on a 1H NMR spectrum for structure determination can lead into the risky realm of misinterpreting a coupling pattern. One of the simplest solutions to most elucidations is to acquire a 1H-1H COSY in addition to a 1H NMR spectrum and thus validate the coupled systems.

The 1H NMR spectrum below shows 3 multiplets at 3.1 (CH3), 3.5 (CH2) and 4.0 ppm (CH) with identical J-coupling constants. Does the coupling pattern for the CH2 group at 3.5 ppm indicate a coupling to the methine at 4.0 ppm or the methyl group at 3.1 ppm? To rephrase the question: does the 1H NMR spectrum indicate the presence of a CH-CH2 group or a CH2-CH3 group?

The peak shape for the multiplet at 3.5 ppm (CH2) appears to be a doublet and so can be considered to be coupled to the CH triplet at 4.0 ppm. However, upon closer examination of the doublet, it appears to be a poorly-resolved quartet, and thus, be considered coupled to the CH3 triplet at 3.1 ppm. In conclusion, further spectral data is needed to confirm the coupling system.

Is it possible to elucidate an unknown with just a 1H NMR spectrum? … Part 2

Peak shape, and subsequently the observed coupling pattern, is highly dependent on a range of factors such as temperature, concentration, pH, FID processing, etc. Relying solely on a 1H NMR spectrum for structure determination can lead into the risky realm of misinterpreting a coupling pattern. One of the simplest solutions to most elucidations is to acquire a 1H-1H COSY in addition to a 1H NMR spectrum and thus validate the coupled systems.

The 1H NMR spectrum below shows 3 multiplets at 3.1 (CH3), 3.5 (CH2) and 4.0 ppm (CH) with identical J-coupling constants. Does the coupling pattern for the CH2 group at 3.5 ppm indicate a coupling to the methine at 4.0 ppm or the methyl group at 3.1 ppm? To rephrase the question: does the 1H NMR spectrum indicate the presence of a CH-CH2 group or a CH2-CH3 group?

The peak shape for the multiplet at 3.5 ppm (CH2) appears to be a doublet and so can be considered to be coupled to the CH triplet at 4.0 ppm. However, upon closer examination of the doublet, it appears to be a poorly-resolved quartet, and thus, be considered coupled to the CH3 triplet at 3.1 ppm. In conclusion, further spectral data is needed to confirm the coupling system.

How to reference 1D and 2D NMR spectra? … Part 2

When working with multiple NMR datasets, it is key to set a reference point(s) in order to align all the datasets. The elucidation purpose -- as opposed to standard NMR purposes of referencing to a standard such as TMS -- behind referencing the spectral data to a common point(s) is to facilitate the interpretation and thus minimize any incorrect grouping of data. For heteronuclear 2D NMR experiments, such as 1H-13C HMQC and HMBC experiments, two reference points are needed. Typically, an elucidator selects a peak from a 1H and a 13C NMR spectrum as the reference points and the 2D NMR data is adjusted accordingly. If a 13C NMR spectrum is not available, as is the case sometimes, then a correlation on an HSQC or HMQC experiment can serve as the stand-in reference point.

From the 1H NMR spectrum below, the resonance at 5.90 ppm is selected as the reference point for the 1H dimension. The resonance at 5.90 ppm is a good choice for a reference point, as it is isolated from other signals and thus is clearly visible. In the case where there are multiple choices for a reference point, I typically lean towards an intense singlet as the 2D counterpart tend to be easy to locate on a 2D NMR spectrum.

Checking the COSY data, the diagonal correlation at 5.9, 5.9 ppm is properly aligned to the 1D resonance at 5.9 ppm. Therefore, no further data manipulation is needed. The HMQC spectrum also shows good alignment with the 1H resonance. Since there is no 1D 13C NMR spectrum available, the correlation at 122.2 ppm in the HMQC is selected as the 13C reference point for the HMBC. The F1 dimension of the HMBC spectrum needs to be referenced as the carbon at 123.3 ppm is off by 1.1 ppm.

How to reference 1D and 2D NMR spectra? … Part 2

When working with multiple NMR datasets, it is key to set a reference point(s) in order to align all the datasets. The elucidation purpose -- as opposed to standard NMR purposes of referencing to a standard such as TMS -- behind referencing the spectral data to a common point(s) is to facilitate the interpretation and thus minimize any incorrect grouping of data. For heteronuclear 2D NMR experiments, such as 1H-13C HMQC and HMBC experiments, two reference points are needed. Typically, an elucidator selects a peak from a 1H and a 13C NMR spectrum as the reference points and the 2D NMR data is adjusted accordingly. If a 13C NMR spectrum is not available, as is the case sometimes, then a correlation on an HSQC or HMQC experiment can serve as the stand-in reference point.

From the 1H NMR spectrum below, the resonance at 5.90 ppm is selected as the reference point for the 1H dimension. The resonance at 5.90 ppm is a good choice for a reference point, as it is isolated from other signals and thus is clearly visible. In the case where there are multiple choices for a reference point, I typically lean towards an intense singlet as the 2D counterpart tend to be easy to locate on a 2D NMR spectrum.

Checking the COSY data, the diagonal correlation at 5.9, 5.9 ppm is properly aligned to the 1D resonance at 5.9 ppm. Therefore, no further data manipulation is needed. The HMQC spectrum also shows good alignment with the 1H resonance. Since there is no 1D 13C NMR spectrum available, the correlation at 122.2 ppm in the HMQC is selected as the 13C reference point for the HMBC. The F1 dimension of the HMBC spectrum needs to be referenced as the carbon at 123.3 ppm is off by 1.1 ppm.

Interpreting a 1H-1H COSY spectrum … Part 3

A 1H-1H COSY dataset of an unknown structure with protons offers many combinations of atom connectivity. The goal for the elucidator is to assess the correlations and narrow down a set of fragments that support the data.

Below is a structural list for 1H to C to 1H connectivities. Absent from the list, heteroatoms such as O, N and S should also be considered. The 2J coupling, the 3J coupling, outlined with a blue box, the 4J coupling, outlined in a green box, and the 5J coupling, outlined with a purple box, offers 1, 3, 6 and 14 possible combinations, respectively.

Interpreting a 1H-1H COSY spectrum … Part 3

A 1H-1H COSY dataset of an unknown structure with protons offers many combinations of atom connectivity. The goal for the elucidator is to assess the correlations and narrow down a set of fragments that support the data.

Below is a structural list for 1H to C to 1H connectivities. Absent from the list, heteroatoms such as O, N and S should also be considered. The 2J coupling, the 3J coupling, outlined with a blue box, the 4J coupling, outlined in a green box, and the 5J coupling, outlined with a purple box, offers 1, 3, 6 and 14 possible combinations, respectively.