Using complementary elucidation tools to solve for an unknown structure

MS and NMR are complementary tools for structure elucidation. Knowing when to apply which tool can assist an elucidator in solving for an unknown structure quickly and with less frustration. Although the sample data below is for a simple organic structure, it is working through many simple examples that one refines the skills needed for elucidation(s) on a bigger scale.

With the available spectral data below, the steps outlined for elucidating an unknown are as follows:

1. extract peak information from the spectral data,

2. piece together the fragments to build a complete structure,

3. finally, verify that the candidate structure(s) is consistent with all the spectral data. If more than one structure fits the data, then look into collecting additional data.

Below are three spectra for an unknown compound: an EI MS, a 1H NMR and a 13C NMR. Although additional information can be extracted from the spectral data, an explanation follows to some of the obvious bits of information that can be extracted at a first pass. The MS shows pairs of ion clusters at approximately equal intensity for m/z pairs 79/81, 93/95 and 133/135, thus indicating the presence of a bromine atom. The molecular ion is most likely the ion peak at m/z 133. The nitrogen rule indicates that the unknown contains an odd number of nitrogen atoms. The 1H NMR spectrum shows 2 triplets at a 1:1 ratio and coupled to each other. The 13C NMR shows 3 carbon peaks at 21, 24 and 117 ppm, thus indicating 3 carbon atoms.

The structural pieces are a bromine atom (79 Da), a nitrogen atom (14 Da), -CH2-CH2- group (28 Da) and a third carbon (12 Da). The candidate structure that fits the data is 3-bromopropanenitrile (133 Da).

Using complementary elucidation tools to solve for an unknown structure

MS and NMR are complementary tools for structure elucidation. Knowing when to apply which tool can assist an elucidator in solving for an unknown structure quickly and with less frustration. Although the sample data below is for a simple organic structure, it is working through many simple examples that one refines the skills needed for elucidation(s) on a bigger scale.

With the available spectral data below, the steps outlined for elucidating an unknown are as follows:

1. extract peak information from the spectral data,

2. piece together the fragments to build a complete structure,

3. finally, verify that the candidate structure(s) is consistent with all the spectral data. If more than one structure fits the data, then look into collecting additional data.

Below are three spectra for an unknown compound: an EI MS, a 1H NMR and a 13C NMR. Although additional information can be extracted from the spectral data, an explanation follows to some of the obvious bits of information that can be extracted at a first pass. The MS shows pairs of ion clusters at approximately equal intensity for m/z pairs 79/81, 93/95 and 133/135, thus indicating the presence of a bromine atom. The molecular ion is most likely the ion peak at m/z 133. The nitrogen rule indicates that the unknown contains an odd number of nitrogen atoms. The 1H NMR spectrum shows 2 triplets at a 1:1 ratio and coupled to each other. The 13C NMR shows 3 carbon peaks at 21, 24 and 117 ppm, thus indicating 3 carbon atoms.

The structural pieces are a bromine atom (79 Da), a nitrogen atom (14 Da), -CH2-CH2- group (28 Da) and a third carbon (12 Da). The candidate structure that fits the data is 3-bromopropanenitrile (133 Da).

Fragment loss of CF3 group

Although NMR offers some strategies for determining the presence of a fluorine atom(s), fragment loss of a CF3 group can be determined from a mass spectrum.

The EI MS for 2,2,2-trifluoro-N,N-dimethylacetamide (Mmi = 141 Da) is shown below. The ion peak at m/z 72 indicates the loss of a CF3 group. Although the ion peak at m/z 69 may not always be present, instead be on the lookout for this fragment loss of 69 Da.

Fragment loss of CF3 group

Although NMR offers some strategies for determining the presence of a fluorine atom(s), fragment loss of a CF3 group can be determined from a mass spectrum.

The EI MS for 2,2,2-trifluoro-N,N-dimethylacetamide (Mmi = 141 Da) is shown below. The ion peak at m/z 72 indicates the loss of a CF3 group. Although the ion peak at m/z 69 may not always be present, instead be on the lookout for this fragment loss of 69 Da.

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.

Interpreting a 1H-1H COSY spectrum with an Exchangeable Proton … Part 2

A 1H-1H COSY dataset provides information on coupled spin systems. A coupled spin system does not necessarily imply that the protons are bonded to only carbon atoms . An example of an exchangeable proton (OH) coupled to a proton from a CH group is shown below.

Three spectral datasets, 1H NMR, HMQC and a COSY-45 dataset, are shown below. The 1H NMR spectrum shows 2 multiplets: 4.12 and 5.29 ppm with both integrating to 1 proton each. For the time being, we will not consider the coupling constants of the multiplets. The HMQC spectrum shows that only the proton at 4.12 ppm has a correlation to a carbon peak and therefore the proton peak at 5.29 ppm is most likely an exchangeable proton. The COSY spectrum shows the off-diagonal correlations between protons 4.12 and 5.29 ppm.

Interpreting a 1H-1H COSY spectrum with an Exchangeable Proton … Part 2

A 1H-1H COSY dataset provides information on coupled spin systems. A coupled spin system does not necessarily imply that the protons are bonded to only carbon atoms . An example of an exchangeable proton (OH) coupled to a proton from a CH group is shown below.

Three spectral datasets, 1H NMR, HMQC and a COSY-45 dataset, are shown below. The 1H NMR spectrum shows 2 multiplets: 4.12 and 5.29 ppm with both integrating to 1 proton each. For the time being, we will not consider the coupling constants of the multiplets. The HMQC spectrum shows that only the proton at 4.12 ppm has a correlation to a carbon peak and therefore the proton peak at 5.29 ppm is most likely an exchangeable proton. The COSY spectrum shows the off-diagonal correlations between protons 4.12 and 5.29 ppm.

Interpreting a 1H-1H COSY spectrum

A component of structure elucidation involves the capability to interpret spectral data of an unknown compound. The interpretation of the data generally leads into a wide range of structural possibilities for the unknown. The goal of the elucidator is to narrow down the structural possibilities to a minimum set of fragments. Taking these fragments, the elucidator can piece together the parts that make structural sense.

Below is a portion of a 1H-1H COSY-45 spectrum for an unknown compound. The COSY data exhibits 5 correlations: 3 diagonal and 2 off-diagonal. The 2 off-diagonal correlations at (1.45, 2.36) and (2.36, 1.45) ppm indicate 4 possible 1H-C-1H structural configurations between the protons at 1.45 and 2.36 ppm. The issue is compounded by the fact that for each off-diagonal correlation, there are 4 possibilities to consider when trying to build a fragment(s). Keep in mind that COSY data by itself may not constitute adequate information to narrow down the possibilities. However, noting the multiple possibilities will ensure that nothing is overlooked.

Note: the intensity of the off-diagonal correlations, judged by the number of contours in respect to the diagonal correlations, may provide a clue in eliminating some of the possibilities. However, this is dependent on how the data is collected and/or on the dihedral angles of the coupled protons.

Interpreting a 1H-1H COSY spectrum

A component of structure elucidation involves the capability to interpret spectral data of an unknown compound. The interpretation of the data generally leads into a wide range of structural possibilities for the unknown. The goal of the elucidator is to narrow down the structural possibilities to a minimum set of fragments. Taking these fragments, the elucidator can piece together the parts that make structural sense.

Below is a portion of a 1H-1H COSY-45 spectrum for an unknown compound. The COSY data exhibits 5 correlations: 3 diagonal and 2 off-diagonal. The 2 off-diagonal correlations at (1.45, 2.36) and (2.36, 1.45) ppm indicate 4 possible 1H-C-1H structural configurations between the protons at 1.45 and 2.36 ppm. The issue is compounded by the fact that for each off-diagonal correlation, there are 4 possibilities to consider when trying to build a fragment(s). Keep in mind that COSY data by itself may not constitute adequate information to narrow down the possibilities. However, noting the multiple possibilities will ensure that nothing is overlooked.

Note: the intensity of the off-diagonal correlations, judged by the number of contours in respect to the diagonal correlations, may provide a clue in eliminating some of the possibilities. However, this is dependent on how the data is collected and/or on the dihedral angles of the coupled protons.

How to apply the Nitrogen rule to organic compounds … Part 3

Without correctly identifying the molecular ion on a mass spectrum, the nitrogen rule generally cannot be applied to any arbitrary ion peak.

The chemical structure for N-[(1E)-1-phenylethylidene]methanamine and its EI mass spectrum are shown below. As expected, the odd value for the molecular ion at m/z 133 dictates an odd number of nitrogen atoms. However, the even value for the fragment ion peak at m/z 118 corresponds to the C8H8NO fragment with an odd number of nitrogen atoms. As such, the nitrogen rule is best reserved for the molecular ion peak as not to risk an incorrect assessment of the number of nitrogen atoms for an unknown compound.

How to apply the Nitrogen rule to organic compounds … Part 3

Without correctly identifying the molecular ion on a mass spectrum, the nitrogen rule generally cannot be applied to any arbitrary ion peak.

The chemical structure for N-[(1E)-1-phenylethylidene]methanamine and its EI mass spectrum are shown below. As expected, the odd value for the molecular ion at m/z 133 dictates an odd number of nitrogen atoms. However, the even value for the fragment ion peak at m/z 118 corresponds to the C8H8NO fragment with an odd number of nitrogen atoms. As such, the nitrogen rule is best reserved for the molecular ion peak as not to risk an incorrect assessment of the number of nitrogen atoms for an unknown compound.

How to apply the Nitrogen rule to organic compounds … Part 2

For MS data, the ionization method will dictate how the nitrogen rule is applied.

For example, if the ionizer is Electron Impact (EI) ionization, then the nitrogen rule is to be applied to the molecular ion [M]+* as follows:

-an odd nominal mass indicates an odd number of nitrogen atoms, e.g. 1,3,5

-an even nominal mass indicates an even number of nitrogen atoms, e.g. 0,2,4.

If the particles are charged using ElectroSpray Ionization (ESI), for example, then the nitrogen rule is to be applied to the molecular ion [M+H]+ or [M-H]- as follows:

-an odd nominal mass indicates an even number of nitrogen atoms

-an even nominal mass indicates an odd number of nitrogen atoms.

The chemical structure for Etifenin is shown below; the differences between the structures are displayed in red. The nominal mass for the [M+H]+ structure, shown on the left side, is odd and therefore the nitrogen rule dictates an even number of nitrogen atoms. For the M+* structure shown on the right side, the even nominal mass dictates an even number of nitrogen atoms.

How to apply the Nitrogen rule to organic compounds … Part 2

For MS data, the ionization method will dictate how the nitrogen rule is applied.

For example, if the ionizer is Electron Impact (EI) ionization, then the nitrogen rule is to be applied to the molecular ion [M]+* as follows:

-an odd nominal mass indicates an odd number of nitrogen atoms, e.g. 1,3,5

-an even nominal mass indicates an even number of nitrogen atoms, e.g. 0,2,4.

If the particles are charged using ElectroSpray Ionization (ESI), for example, then the nitrogen rule is to be applied to the molecular ion [M+H]+ or [M-H]- as follows:

-an odd nominal mass indicates an even number of nitrogen atoms

-an even nominal mass indicates an odd number of nitrogen atoms.

The chemical structure for Etifenin is shown below; the differences between the structures are displayed in red. The nominal mass for the [M+H]+ structure, shown on the left side, is odd and therefore the nitrogen rule dictates an even number of nitrogen atoms. For the M+* structure shown on the right side, the even nominal mass dictates an even number of nitrogen atoms.

How to apply the Nitrogen rule to organic compounds

The purpose of the nitrogen rule is to assist with deciphering how many nitrogen atoms are present without any prior information on the molecular formula. Depending on the ionization mode, an odd nominal mass indicates an odd number of nitrogen atoms, e.g. 1,3,5, whereas an even nominal mass indicates an even number of nitrogen atoms, e.g. 0,2,4.

The nitrogen rule can only be applied under the following conditions:

1. the m/z value is the molecular ion,

2. the unknown is an organic compound with any combination of hydrogen, carbon, oxygen, nitrogen, phosphorous, silicon, sulfur, fluorine, chlorine, bromine, iodine, and

3. the MS data is nominal data.

For a better way to tell if nitrogen is present, please visit this link to Fiehn’s Lab.

How to apply the Nitrogen rule to organic compounds

The purpose of the nitrogen rule is to assist with deciphering how many nitrogen atoms are present without any prior information on the molecular formula. Depending on the ionization mode, an odd nominal mass indicates an odd number of nitrogen atoms, e.g. 1,3,5, whereas an even nominal mass indicates an even number of nitrogen atoms, e.g. 0,2,4.

The nitrogen rule can only be applied under the following conditions:

1. the m/z value is the molecular ion,

2. the unknown is an organic compound with any combination of hydrogen, carbon, oxygen, nitrogen, phosphorous, silicon, sulfur, fluorine, chlorine, bromine, iodine, and

3. the MS data is nominal data.

For a better way to tell if nitrogen is present, please visit this link to Fiehn’s Lab.

Does my unknown structure contain Bromine?

MS and NMR are complementary elucidation tools. Knowing when to apply the correct tool can facilitate the elucidation process.

Compounds with bromine atoms exhibit a distinct ion pattern on a mass spectrum. The A+2 peak for a monobrominated compound appears at almost identical intensity to the 79Br peak due to the presence of 81Br (~49.3% natural isotope abundance). A compound with two bromines shows a distinct A+4 peak with an approximate ratio of 1:2:1.

The following EI mass spectra are for 3-bromopropanenitrile and (1Z)-1,2-dibromobut-1-ene with nominal masses at 133 and 212 Da, respectively. The top MS shows 3 identified fragments containing bromine (m/z 79/81, 93/95 and 133/135) as noted by the ~1:1 intensity of the ion clusters. The ion peak at m/z 54 does not show a 1:1 ratio since the fragment does not contain a bromine atom.

The MS for (1Z)-1,2-dibromobut-1-ene shows 4 identifiable fragments containing bromine (m/z 117/119, 133/135, 197/199/201 and 212/214/216). In particular, ion clusters at m/z 197/199/201 and 212/214/216 show the distinct ~1:2:1 intensity. This is a result of the varying amounts of the isotopes 79Br and 81Br, that is, the 3 peaks represent 79Br/79Br : 79Br/81Br : 81Br/81Br.

TIP: Check the fragment ion peaks too for the distinct pattern especially when the molecular ion peak is not visible.

Does my unknown structure contain Bromine?

MS and NMR are complementary elucidation tools. Knowing when to apply the correct tool can facilitate the elucidation process.

Compounds with bromine atoms exhibit a distinct ion pattern on a mass spectrum. The A+2 peak for a monobrominated compound appears at almost identical intensity to the 79Br peak due to the presence of 81Br (~49.3% natural isotope abundance). A compound with two bromines shows a distinct A+4 peak with an approximate ratio of 1:2:1.

The following EI mass spectra are for 3-bromopropanenitrile and (1Z)-1,2-dibromobut-1-ene with nominal masses at 133 and 212 Da, respectively. The top MS shows 3 identified fragments containing bromine (m/z 79/81, 93/95 and 133/135) as noted by the ~1:1 intensity of the ion clusters. The ion peak at m/z 54 does not show a 1:1 ratio since the fragment does not contain a bromine atom.

The MS for (1Z)-1,2-dibromobut-1-ene shows 4 identifiable fragments containing bromine (m/z 117/119, 133/135, 197/199/201 and 212/214/216). In particular, ion clusters at m/z 197/199/201 and 212/214/216 show the distinct ~1:2:1 intensity. This is a result of the varying amounts of the isotopes 79Br and 81Br, that is, the 3 peaks represent 79Br/79Br : 79Br/81Br : 81Br/81Br.

TIP: Check the fragment ion peaks too for the distinct pattern especially when the molecular ion peak is not visible.