Identifying a monosubstituted benzene fragment in a 1H NMR spectrum

Although peak crowding can be a nuisance, a monosubstituted benzene fragment can be identified by a 1H NMR. A good marker for a monosubstituted benzene ring, and thus how an elucidator can clue in to its presence for an unknown, is whether the sum of the relative integrals for the aromatic resonances add up to 5.

Below are 6 1H NMR spectra illustrating the various patterns for a monosubstituted benzene fragment. Although other possibilities can exist, these are the typical patterns to be on the lookout for in the aromatic region.

Identifying a monosubstituted benzene fragment in a 1H NMR spectrum

Although peak crowding can be a nuisance, a monosubstituted benzene fragment can be identified by a 1H NMR. A good marker for a monosubstituted benzene ring, and thus how an elucidator can clue in to its presence for an unknown, is whether the sum of the relative integrals for the aromatic resonances add up to 5.

Below are 6 1H NMR spectra illustrating the various patterns for a monosubstituted benzene fragment. Although other possibilities can exist, these are the typical patterns to be on the lookout for in the aromatic region.

Examining the 12C and 13C ratio in a Mass spectrum – carbon isotopic abundance

On a mass spectrum, the carbon 13 isotope peak appears at approximately one mass unit higher (the actual mass delta 1.00335) than the carbon 12 ion peak. The intensity of these isotopes is proportional to the relative abundance of the naturally occurring isotopes. The relative abundance of the two isotopes is 12C ≈ 98.9% and 13C ≈ 1.1%.

Without any structural information, we can estimate a general ballpark figure for the number of carbons using the peak intensities for the 12C and 13C ion peaks.

For the 12C ion peak (m/z 386.4) shown below, the upper limit on the number of carbons is calculated at 386.4 / 12 = 32.2. Rounding down, we arrive at 32 carbons. Based on this information, the intensity of the 13C peak is expected at 32 * 1.1% = 35.2%.

Experimentally, the intensity of the 13C peak is 23.6% with the 12C peak at 100%. The calculation is (23.6 / 100 *100%) / 1.1% = 21.4.

Formula*:

To estimate the # of Carbons ≈ (Int13C/Int12C * 100%) / 1.1%

*Note: Instrument and the type of experiment can influence the intensity of the 13C peak and thus produce a less reliable estimate. Ideally, the result is best evaluated in conjunction with the carbon count from a 13C NMR.

Examining the 12C and 13C ratio in a Mass spectrum – carbon isotopic abundance

On a mass spectrum, the carbon 13 isotope peak appears at approximately one mass unit higher (the actual mass delta 1.00335) than the carbon 12 ion peak. The intensity of these isotopes is proportional to the relative abundance of the naturally occurring isotopes. The relative abundance of the two isotopes is 12C ≈ 98.9% and 13C ≈ 1.1%.

Without any structural information, we can estimate a general ballpark figure for the number of carbons using the peak intensities for the 12C and 13C ion peaks.

For the 12C ion peak (m/z 386.4) shown below, the upper limit on the number of carbons is calculated at 386.4 / 12 = 32.2. Rounding down, we arrive at 32 carbons. Based on this information, the intensity of the 13C peak is expected at 32 * 1.1% = 35.2%.

Experimentally, the intensity of the 13C peak is 23.6% with the 12C peak at 100%. The calculation is (23.6 / 100 *100%) / 1.1% = 21.4.

Formula*:

To estimate the # of Carbons ≈ (Int13C/Int12C * 100%) / 1.1%

*Note: Instrument and the type of experiment can influence the intensity of the 13C peak and thus produce a less reliable estimate. Ideally, the result is best evaluated in conjunction with the carbon count from a 13C NMR.

Complicating NMR data interpretation

Typically, structure elucidation via NMR can be ascribed by a stepwise workflow:

1. a sample is prepared for NMR, 2. the NMR instrument is optimized for data collection, 3. NMR data is acquired, 4. the spectral data is processed, 5. the spectral data is searched/compared to an internal database for possible hits or similarities, 6. the NMR data is pieced together to create a list of candidate structures, 7. the candidate structures are checked/verified against additional data.

Structure elucidation is not as simple as it sounds. Collecting NMR data on an unknown sample and heading straight down the path to solve it is not a guarantee for success. Optimizing data collection is a critical step and one that is frequently overlooked. Here is a list of issues that may arise when step 2 is inadequately applied: peaks are poorly shimmed, a probe is poorly tuned and/or matched, the presence of solvent impurities, outside interferences on the instrument, the wrong experiment parameters are setup , and the list goes on. The major consequence of any of these actions is NMR information can be misinterpreted.

TIP: spending a few minutes to quickly check the processed NMR data before moving onto the next step can save you loads of time and anguish.

Complicating NMR data interpretation

Typically, structure elucidation via NMR can be ascribed by a stepwise workflow:

1. a sample is prepared for NMR, 2. the NMR instrument is optimized for data collection, 3. NMR data is acquired, 4. the spectral data is processed, 5. the spectral data is searched/compared to an internal database for possible hits or similarities, 6. the NMR data is pieced together to create a list of candidate structures, 7. the candidate structures are checked/verified against additional data.

Structure elucidation is not as simple as it sounds. Collecting NMR data on an unknown sample and heading straight down the path to solve it is not a guarantee for success. Optimizing data collection is a critical step and one that is frequently overlooked. Here is a list of issues that may arise when step 2 is inadequately applied: peaks are poorly shimmed, a probe is poorly tuned and/or matched, the presence of solvent impurities, outside interferences on the instrument, the wrong experiment parameters are setup , and the list goes on. The major consequence of any of these actions is NMR information can be misinterpreted.

TIP: spending a few minutes to quickly check the processed NMR data before moving onto the next step can save you loads of time and anguish.

Identifying fragments using a Neutral Loss spectrum

A calculated neutral loss spectrum is obtained from a mass spectrum by determining the mass differences between the precursor ion m/z and each of the other peaks in the spectrum and plotting the original intensity versus neutral mass. Neutral losses with small masses have limited possibilities for their composition and thus can facilitate the identification of specific species.

Common losses (based on the J.H. Beynon table) for fragments with C, H, O, N elements are reported below:

OH, NH3                                                     nominal mass of 17

CO, N2 C2H4, CH2N                                    nominal mass of 28

CHO2, CH3NO, CH5N2, C2H7N, C2H5O        nominal mass of 45

A positive ion EI mass spectrum of benzoic acid is shown below. The molecular ion (M+') appears at an m/z of 122. Two additional ion clusters appear at m/z of 77 and 105. The Beynon table reports 4 and 13 possible fragments for m/z 77 and 105, respectively. Examining the neutral loss spectrum, shown below, 3 basic ion clusters appear: m/z 17, 28 and 45. The Beynon Table (listed above) reports 2, 4 and 5 possible fragments for m/z 17, 28 and 45, respectively. When working with fewer fragment possibilities, one can reduce the time spent on an elucidation problem.

TIP: Knowing a partial fragment for an unknown can aid in narrowing down a molecular formula and limit the number of candidate structures.

Spectra courtesy of Joe DiMartino, M.Sc.

Identifying fragments using a Neutral Loss spectrum

A calculated neutral loss spectrum is obtained from a mass spectrum by determining the mass differences between the precursor ion m/z and each of the other peaks in the spectrum and plotting the original intensity versus neutral mass. Neutral losses with small masses have limited possibilities for their composition and thus can facilitate the identification of specific species.

Common losses (based on the J.H. Beynon table) for fragments with C, H, O, N elements are reported below:

OH, NH3                                                     nominal mass of 17

CO, N2 C2H4, CH2N                                    nominal mass of 28

CHO2, CH3NO, CH5N2, C2H7N, C2H5O        nominal mass of 45

A positive ion EI mass spectrum of benzoic acid is shown below. The molecular ion (M+') appears at an m/z of 122. Two additional ion clusters appear at m/z of 77 and 105. The Beynon table reports 4 and 13 possible fragments for m/z 77 and 105, respectively. Examining the neutral loss spectrum, shown below, 3 basic ion clusters appear: m/z 17, 28 and 45. The Beynon Table (listed above) reports 2, 4 and 5 possible fragments for m/z 17, 28 and 45, respectively. When working with fewer fragment possibilities, one can reduce the time spent on an elucidation problem.

TIP: Knowing a partial fragment for an unknown can aid in narrowing down a molecular formula and limit the number of candidate structures.

Spectra courtesy of Joe DiMartino, M.Sc.

Technical details on NMR instrumentation and acquisition

Every analytical instrument will give an answer; whether that answer is right or wrong or the power is off is a different story. Knowing how to troubleshoot an instrument or setup a specific experiment can be just as important as knowing how to interpret the data resulting from that instrument.

A great way to learn about NMR instrumentation is from the NMR spectroscopists who have the extensive experiences and who are willing to share their knowledge with others. One such example is the NMR blog belonging to Glenn Angus Facey Ph.D. a.k.a. Bubbles (the NMR facility manager at the University of Ottawa). I admit I know the individual personally so my opinion is biased.

Click here to view Bubbles’ blog.

Technical details on NMR instrumentation and acquisition

Every analytical instrument will give an answer; whether that answer is right or wrong or the power is off is a different story. Knowing how to troubleshoot an instrument or setup a specific experiment can be just as important as knowing how to interpret the data resulting from that instrument.

A great way to learn about NMR instrumentation is from the NMR spectroscopists who have the extensive experiences and who are willing to share their knowledge with others. One such example is the NMR blog belonging to Glenn Angus Facey Ph.D. a.k.a. Bubbles (the NMR facility manager at the University of Ottawa). I admit I know the individual personally so my opinion is biased.

Click here to view Bubbles’ blog.

Interpreting a Mass Spectrum with the aid of a calculated Neutral Loss Spectrum

As a first pass, a good approach to extracting information from a mass spectrum (MS) is to look for two things: intense peaks with a recognizable m/z and losses (or gains) between peaks. A mass-to-charge peak at 77 is an example of a recognizable peak; it is most likely attributed to a C6H5+’ fragment in an electron impact (EI) MS. As for losses (or gains) of neutral fragments, subtracting a mass-to-charge peak from the molecular ion will produce a calculated neutral loss spectrum. The purpose of the calculated neutral loss spectrum is to facilitate the calculation of the losses and thus assist in fragment interpretation.

The EI mass spectrum of Bisphenol A (aka BPA - an environmentally unfriendly agent) and its calculated neutral loss spectrum are shown below. The loss of 15 (demethylation) is calculated by subtracting the molecular ion for BPA at 228 from the most intense signal at m/z 213.

Thank you to Graham McGibbon Ph.D. for suggesting the compound.

Interpreting a Mass Spectrum with the aid of a calculated Neutral Loss Spectrum

As a first pass, a good approach to extracting information from a mass spectrum (MS) is to look for two things: intense peaks with a recognizable m/z and losses (or gains) between peaks. A mass-to-charge peak at 77 is an example of a recognizable peak; it is most likely attributed to a C6H5+’ fragment in an electron impact (EI) MS. As for losses (or gains) of neutral fragments, subtracting a mass-to-charge peak from the molecular ion will produce a calculated neutral loss spectrum. The purpose of the calculated neutral loss spectrum is to facilitate the calculation of the losses and thus assist in fragment interpretation.

The EI mass spectrum of Bisphenol A (aka BPA - an environmentally unfriendly agent) and its calculated neutral loss spectrum are shown below. The loss of 15 (demethylation) is calculated by subtracting the molecular ion for BPA at 228 from the most intense signal at m/z 213.

Thank you to Graham McGibbon Ph.D. for suggesting the compound.

Methoxy groups just stick out

Methoxy groups have a distinct NMR signature that make them easy to spot in virtually every case. The basic pattern is a singlet integrating to 3 and ranging between 2.4 to 4.4 ppm on a 1H NMR spectrum (assuming no overlap with other resonances). On the 13C end, a 13C resonance is seen between 46 and 69 ppm. In addition, when an elucidator is working on narrowing down a molecular formula (MF) for an unknown sample, the prospect of OCH3 information helps in the minimum oxygen count.

The 1H -13C DEPT-HSQC spectrum below shows two OCH3 groups. The singlet integrating to 3 in the 1H NMR spectrum and the carbon information from the DEPT-HSQC at ~56 ppm. With this information on hand, the minimum atom count for the MF is C2H6O2.

Methoxy groups just stick out

Methoxy groups have a distinct NMR signature that make them easy to spot in virtually every case. The basic pattern is a singlet integrating to 3 and ranging between 2.4 to 4.4 ppm on a 1H NMR spectrum (assuming no overlap with other resonances). On the 13C end, a 13C resonance is seen between 46 and 69 ppm. In addition, when an elucidator is working on narrowing down a molecular formula (MF) for an unknown sample, the prospect of OCH3 information helps in the minimum oxygen count.

The 1H -13C DEPT-HSQC spectrum below shows two OCH3 groups. The singlet integrating to 3 in the 1H NMR spectrum and the carbon information from the DEPT-HSQC at ~56 ppm. With this information on hand, the minimum atom count for the MF is C2H6O2.

Recognizing the NMR pattern for morpholine

An integral part of an elucidation is to recognize and associate an NMR spectral pattern to a structure or fragment. A challenging elucidation can be simplified by being aware of as many as possible NMR patterns. One example is morpholine; it typically shows a distinct NMR pattern.

A portion of the 1H-13C DEPT-HSQC spectrum for 4-phenylmorpholine is shown below. The 2 negative correlations on the DEPT-HSQC experiment indicate the presence of CH2 groups. In addition, the carbon at 67 ppm must be closer to an electron-withdrawing group such as an oxygen atom. The attached 1H NMR also shows the resonances having approximately equal integrals and tilted towards each other.

Recognizing the NMR pattern for morpholine

An integral part of an elucidation is to recognize and associate an NMR spectral pattern to a structure or fragment. A challenging elucidation can be simplified by being aware of as many as possible NMR patterns. One example is morpholine; it typically shows a distinct NMR pattern.

A portion of the 1H-13C DEPT-HSQC spectrum for 4-phenylmorpholine is shown below. The 2 negative correlations on the DEPT-HSQC experiment indicate the presence of CH2 groups. In addition, the carbon at 67 ppm must be closer to an electron-withdrawing group such as an oxygen atom. The attached 1H NMR also shows the resonances having approximately equal integrals and tilted towards each other.

How do I know if my unknown contains a fluorine atom(s)? … Part 4

2D NMR experiments such as 1H-13C HMQC, HSQC, and HETCOR spectra, offer the elucidator the opportunity to assess the presence of a fluorine atom(s).

The 1H -13C HSQC spectrum below is another case where a protonated carbon that experiences 13C-19F coupling shows a unique correlation pattern. The CH carbon projected on the F1 domain at 34 pm correlates with the 1H multiplet (ddt) at 2.4 ppm. The 1H multiplet overlaps with another multiplet (dd) for the CH2 resonance at 30 ppm.

The second spectrum is a close-up on the region around the 1H multiplets at 2.4 ppm. Notice the unique pattern whereby the correlations do not line-up.

For both spectra, the F1 trace is a projection of all the 13C slices and a 1D 1H NMR spectrum is attached to the F2 domain, hence the better resolution. The chemical shifts for the CH and CH2 group are shown in blue on the steroid-type fragment.

TIP: To avoid missing any extra bit of information, zoom-in on each individual HSQC correlation for a closer look at the data.

How do I know if my unknown contains a fluorine atom(s)? … Part 4

2D NMR experiments such as 1H-13C HMQC, HSQC, and HETCOR spectra, offer the elucidator the opportunity to assess the presence of a fluorine atom(s).

The 1H -13C HSQC spectrum below is another case where a protonated carbon that experiences 13C-19F coupling shows a unique correlation pattern. The CH carbon projected on the F1 domain at 34 pm correlates with the 1H multiplet (ddt) at 2.4 ppm. The 1H multiplet overlaps with another multiplet (dd) for the CH2 resonance at 30 ppm.

The second spectrum is a close-up on the region around the 1H multiplets at 2.4 ppm. Notice the unique pattern whereby the correlations do not line-up.

For both spectra, the F1 trace is a projection of all the 13C slices and a 1D 1H NMR spectrum is attached to the F2 domain, hence the better resolution. The chemical shifts for the CH and CH2 group are shown in blue on the steroid-type fragment.

TIP: To avoid missing any extra bit of information, zoom-in on each individual HSQC correlation for a closer look at the data.