Logic Puzzle #13: Extra Correlations on the HSQC Spectrum … Solution

The intensity of a correlation in relation to other correlations on a 2D NMR spectrum can be just as important in a structure elucidation as assessing the position and phase of a correlation. The example below presents one such case.

From the 1H-13C HSQC in the previous post, the correlations differ significantly in intensity. The weaker correlations can be attributed to long-range couplings, more specifically, 2J coupling responses. In addition to the 1H integrals, the carbons at 121 and 134 ppm are probably CH’s (4 CH’s in total) and probably not CH2’s. Two potential fragments are shown below and here.

 
I would like to send out a special thanks to Sarah T. for discussing the idea with me.

Logic Puzzle #13: Extra Correlations on the HSQC Spectrum

A 1H-13C HSQC is frequently used to assist in assigning a carbon as CH, CH2 or CH3 (additional blog here). The purpose of this puzzle is to examine a non-standard assignment.

The 1H-13C HSQC below shows 2 correlations for each carbon at 121 and 134 ppm. Given also the information on the 1H integrals on the F2 axis, are the carbons at 121 and 134 ppm really CH2’s?

 

Logic Puzzle #10: Deciphering the Fragment Pattern using 2D NMR Data … Solution

Like any new process, it takes some practice to extract, understand and convert the information presented from a set of experimental NMR datasets into a fragment.

The only fragment that can accommodate the set of restrictions from a 1H-13C HSQC and HMBC is 2,3-dimethylbutane-1,1-diyl. The green arrows illustrate the 2-3JCH coupling responses extracted from an HMBC experiment.

 

Logic Puzzle #4: Correlating to the Correct 13C Signal … Solution

The goal of this puzzle is to resolve the ambiguity exhibited within a 2D NMR spectrum and thus provide the correct signal correlation. Although this exercise may seem a trivial one, it is important to go over the rationale when correlating one signal to another.

For the following 1H-13C HSQC-DEPT NMR spectrum it is important to note that the 2 correlations are phased positively (red) and thus represent either a CH or CH3 group and not a CH2. Next, one must ensure that the carbons at 61.5 and 62.2 ppm are 1 carbon each. Although this detail is not certain, we will assume this to be the case. Finally, it is best to start with the easy part first. The 1H signal at 3.50 ppm is correlated to the 13C signal at 62.2 ppm. By process of elimination, one can conclude that the 1H signal at 2.73 ppm is correlated to the 13C signal at 61.5 ppm (see this post for more details).

Ambiguity in correlating 1D and 2D NMR data can routinely occur. Some extra steps that can help avoid this issue are re-aligning the data and/or re-processing the ‘raw’ data with different parameters.

 

Logic Puzzle #4: Correlating to the Correct 13C Signal … Solution

The goal of this puzzle is to resolve the ambiguity exhibited within a 2D NMR spectrum and thus provide the correct signal correlation. Although this exercise may seem a trivial one, it is important to go over the rationale when correlating one signal to another.

For the following 1H-13C HSQC-DEPT NMR spectrum it is important to note that the 2 correlations are phased positively (red) and thus represent either a CH or CH3 group and not a CH2. Next, one must ensure that the carbons at 61.5 and 62.2 ppm are 1 carbon each. Although this detail is not certain, we will assume this to be the case. Finally, it is best to start with the easy part first. The 1H signal at 3.50 ppm is correlated to the 13C signal at 62.2 ppm. By process of elimination, one can conclude that the 1H signal at 2.73 ppm is correlated to the 13C signal at 61.5 ppm (see this post for more details).

Ambiguity in correlating 1D and 2D NMR data can routinely occur. Some extra steps that can help avoid this issue are re-aligning the data and/or re-processing the ‘raw’ data with different parameters.

 

Logic Puzzle #4: Correlating to the Correct 13C Signal

The goal of this puzzle is to resolve the ambiguity exhibited within a 2D NMR spectrum and thus provide the correct signal correlation.

The following 1H-13C HSQC-DEPT NMR spectrum shows two one-bond correlations linked to the 1H signals 2.75 and 3.50 ppm and two closely spaced 13C signals at 61.5 and 62.2 ppm. Does the 1H signal at 2.75 ppm correlate to the 13C signal at 61.5 ppm or the one at 62.2 ppm?

Note: the blue line was added to help align the correlation to the F1 domain.

Logic Puzzle #4: Correlating to the Correct 13C Signal

The goal of this puzzle is to resolve the ambiguity exhibited within a 2D NMR spectrum and thus provide the correct signal correlation.

The following 1H-13C HSQC-DEPT NMR spectrum shows two one-bond correlations linked to the 1H signals 2.75 and 3.50 ppm and two closely spaced 13C signals at 61.5 and 62.2 ppm. Does the 1H signal at 2.75 ppm correlate to the 13C signal at 61.5 ppm or the one at 62.2 ppm?

Note: the blue line was added to help align the correlation to the F1 domain.

Signals can simply disappear on a DEPT-135 experiment

There are many advantages in working with a 1H-13C HSQC-DEPT spectrum over a 13C DEPT-135 and a 1H-13C HSQC (see Post 1 & Post 2). In most cases, a 1H-13C HSQC-DEPT is more valuable than either one of those experiments.

The aliphatic region of a 1H-13C HSQC-DEPT is spectrum below. Two coincidental carbon signals are overlapping at 38.51 ppm, one pertaining to a CH while the other a CH2 group. The DEPT-135, attached to the F1 domain, exhibits a weak 13C signal that can easily be misconstrued, for example as an impurity, if not for the extra information from the 2D NMR experiment.

Signals can simply disappear on a DEPT-135 experiment

There are many advantages in working with a 1H-13C HSQC-DEPT spectrum over a 13C DEPT-135 and a 1H-13C HSQC (see Post 1 & Post 2). In most cases, a 1H-13C HSQC-DEPT is more valuable than either one of those experiments.

The aliphatic region of a 1H-13C HSQC-DEPT is spectrum below. Two coincidental carbon signals are overlapping at 38.51 ppm, one pertaining to a CH while the other a CH2 group. The DEPT-135, attached to the F1 domain, exhibits a weak 13C signal that can easily be misconstrued, for example as an impurity, if not for the extra information from the 2D NMR experiment.

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.

 

Distinguishing Impurities … Part 3

In the series Distinguishing Impurities, Part 1 pointed to certain signs in which an elucidator can differentiate a signal as pertaining to an impurity and not to the main unknown. Part 1 also made reference to using 2D NMR data as a practical approach to ascertain whether a signal from a 1H NMR was an impurity. With so many choices for 2D NMR experiments, the question is which one(s) will offer the best chance to assist with this method.

The diagram below is a 1H -13C HSQC spectrum related to the 1H NMR spectrum shown in Part 1. Originally flagged as possible impurities based on the 1H NMR data, the singlets at 1.68 and 2.14 ppm show a H-C correlation as do the remaining 1H signals. Based on this example, the HSQC data cannot differentiate any impurities from the main unknown.

Distinguishing Impurities … Part 3

In the series Distinguishing Impurities, Part 1 pointed to certain signs in which an elucidator can differentiate a signal as pertaining to an impurity and not to the main unknown. Part 1 also made reference to using 2D NMR data as a practical approach to ascertain whether a signal from a 1H NMR was an impurity. With so many choices for 2D NMR experiments, the question is which one(s) will offer the best chance to assist with this method.

The diagram below is a 1H -13C HSQC spectrum related to the 1H NMR spectrum shown in Part 1. Originally flagged as possible impurities based on the 1H NMR data, the singlets at 1.68 and 2.14 ppm show a H-C correlation as do the remaining 1H signals. Based on this example, the HSQC data cannot differentiate any impurities from the main unknown.

Identifying Peak Overlap on an HMBC Spectrum … Part 2

One of the trickiest parts of interpreting a 1H-13C HMBC is deciding whether a 13C resonance is coinciding with another 13C resonance, i.e. overlapping 13C peaks. A past blog, Part 1, describes a specific case where the possibility of two coinciding 13C resonances can be deciphered based on a high carbon correlation count. Herein, we present a specific case for a 1H-13C HMBC with paired 1J responses to distinguish peak overlap.

The 1H -13C HMBC spectrum below illustrates 2 protons multiplets at 1.89 and 2.37 ppm and 2 carbon resonances at 26.8 and 43.0 ppm. The paired 1J responses at 43.0 ppm indicate a CH2 group with the proton multiplet at 1.89 ppm. Similarly, the carbon at 26.8 ppm shows a paired 1J response with the proton multiplet at 2.37 ppm (CH group). In addition, a single correlation at exactly 2.37, 26.8 ppm indicates a quaternary carbon coinciding with the carbon resonance for the CH group.

The quaternary carbon and CH group are in the vicinity of each other.

Identifying Peak Overlap on an HMBC Spectrum … Part 2

One of the trickiest parts of interpreting a 1H-13C HMBC is deciding whether a 13C resonance is coinciding with another 13C resonance, i.e. overlapping 13C peaks. A past blog, Part 1, describes a specific case where the possibility of two coinciding 13C resonances can be deciphered based on a high carbon correlation count. Herein, we present a specific case for a 1H-13C HMBC with paired 1J responses to distinguish peak overlap.

The 1H -13C HMBC spectrum below illustrates 2 protons multiplets at 1.89 and 2.37 ppm and 2 carbon resonances at 26.8 and 43.0 ppm. The paired 1J responses at 43.0 ppm indicate a CH2 group with the proton multiplet at 1.89 ppm. Similarly, the carbon at 26.8 ppm shows a paired 1J response with the proton multiplet at 2.37 ppm (CH group). In addition, a single correlation at exactly 2.37, 26.8 ppm indicates a quaternary carbon coinciding with the carbon resonance for the CH group.

The quaternary carbon and CH group are in the vicinity of each other.

Hampering Data Interpretation

A common misinterpretation of 2D NMR data can occur when dealing with weak correlations. Weak correlations are commonly introduced in how the sample is prepared or how the data is collected or processed. Examining the spectrum down to level of the density matrix can ensure all correlations are picked up.

The 1H-13C HMQC below shows a protonated carbon at 3.2 and 32 ppm. Since the proton multiplet at 3.6 ppm has no carbon correlation, the proton is most likely from an exchangeable group such as NH or OH group.

When the spectrum threshold is lowered to 1 % relative to the most intense correlation, a weak correlation at 2.6 and 41.9 ppm is seen. Therefore, the proton at 3.6 ppm is actually a CH group and not an exchangeable one.

Hampering Data Interpretation

A common misinterpretation of 2D NMR data can occur when dealing with weak correlations. Weak correlations are commonly introduced in how the sample is prepared or how the data is collected or processed. Examining the spectrum down to level of the density matrix can ensure all correlations are picked up.

The 1H-13C HMQC below shows a protonated carbon at 3.2 and 32 ppm. Since the proton multiplet at 3.6 ppm has no carbon correlation, the proton is most likely from an exchangeable group such as NH or OH group.

When the spectrum threshold is lowered to 1 % relative to the most intense correlation, a weak correlation at 2.6 and 41.9 ppm is seen. Therefore, the proton at 3.6 ppm is actually a CH group and not an exchangeable one.

Extracting Proton Information from 13C NMR data

In addition to using chemical shift information to ascertain a carbon’s proton count (i.e. C, CH, CH2 or CH3), 13C NMR experiments can be set up in a variety of ways to assist with this process.

The following simulated spectra compare a variety of 13C NMR experiments for aspartame. Please note that there are variations to the list below that are not being shown. These include such experiments as DEPTQ-135 and DEPT-HSQCSE (sensitivity enhanced).

 

   

 

   

 

 

Extracting Proton Information from 13C NMR data

In addition to using chemical shift information to ascertain a carbon’s proton count (i.e. C, CH, CH2 or CH3), 13C NMR experiments can be set up in a variety of ways to assist with this process.

The following simulated spectra compare a variety of 13C NMR experiments for aspartame. Please note that there are variations to the list below that are not being shown. These include such experiments as DEPTQ-135 and DEPT-HSQCSE (sensitivity enhanced).

 

   

 

   

 

 

Missing the Big Picture?

When peak picking a 2D NMR experiment, past weblogs have advocated zooming in on correlations especially in cases dealing with ambiguity. Depending on the data collection parameters, a 1H-13C HMBC experiment can contain paired 1J coupling responses. Without careful scrutiny of the data, these extra responses can be misinterpreted as long-range correlations (2J or longer).

The 1H -13C HMBC spectrum below indicates two peaks picked, thus, correlating proton 1.24 ppm to carbon 20.47 ppm and proton 1.35 ppm to carbon 23.07 ppm. This is an easy interpretation that cannot possibly be wrong, or is it?

    

Before accepting the peak picking, it is best to take a step back—actually zoom out a little further to get the bigger picture. The correlations from the 1H -13C HMBC spectrum are 1J coupling responses and thus not long-range correlations. The blue lines indicate the paired 1J coupling responses. When assigning long-range correlations, 1J coupling responses are best not to be picked.

    

NOTE: Although it is possible for a long-range correlation to overlap with a 1J coupling response, it may be wise to examine the volumes of the responses for any significant differences.