Structural Analysis of Organic Compound Using 2D - NMR Spectrum

In this column, we will explain the structural analysis of organic compound by using 2D-NMR spectrum.

Structural Analysis of one-dimensional NMR (1D-NMR)

One-dimensional NMR (1D-NMR) is the most basic measurement of NMR spectroscopy, in which the spectrum is displayed along a horizontal axis (chemical shift). As introduced in the previous column, information on chemical shift, integration ratio, and splitting pattern (coupling) is used to analyze the structure of an object. However, it can be difficult to perform structural analysis using only 1D-NMR data when the number of detected signals is large, when signals appear in overlapping positions, or when couplings are complex.
The two-dimensional (2D-NMR) spectral analysis method introduced in this column can be used extensively regardless of the object to be analyzed, since the basic sequence of steps is followed. In addition, by following the basic procedures, even beginners can mechanically perform structural analysis.

What is 2D-NMR?

2D-NMR is a method used to check a more detailed molecular structure than 1D-NMR.
Since there are many 2D-NMR measurement methods, here, we introduce representative measurement methods.

Structural Analysis Using J Coupling Correlation

This time we introduce an example of structural analysis using "J Coupling Correlation" which is an interaction via chemical bonding. Structural analysis of an object is conducted by following the 4 steps below.

In Step 1, we use a 1D-NMR spectrum of ¹³C and DEPT to determine and estimate the atomic group and functional group.

In Step 2, we use HMQC and 1D spectrum of ¹H to find ¹³C and ¹H that are directly bonded.

In Step 3, we use COSY to examine and locate which is the neighboring ¹Hs.

By using information obtained in Step 2 and 3, substructures can be determined to some extent.

In Step 4, we use HMBC to determine the structure of the remaining parts, by considering the connection of ¹³C and ¹H which are further apart.

Structural analysis example of C ₆H₁₀O₂

Step 1 ¹³C (1D-NMR) & DEPT

In Step 1, we use a 1D-NMR spectrum of ¹³C and DEPT to determine and estimate atomic group and functional group. First, as shown in Fig. 1, we put a symbol on each signal in 1D-NMR spectrum of ¹³C. Starting with the signal on the right side of the spectrum, from the high-field side, add symbols A, B, C... and so on. The chemical shift of each signal is read to one decimal place. The read information is summarized in Table 1.

Table 1 ¹³C Spectra Information

Next, we will confirm the results of the DEPT spectra. Since DEPT observes the carbon to which hydrogen is directly bonded, the series of the carbon atom of interest (the number of hydrogen directly bonded to the carbon of interest) is known. In other words, it is possible to identify atomic groups such as CH₃, CH₂, and CH. (DEPT cannot detect spectra of quaternary carbons.) DEPT provides three types of spectra, depending on the measurement parameters (DEPT135, DEPT90, and DEPT45).

Table 2 lists the signal appearance patterns in the three types of DEPT spectra. In DEPT135, the signals of CH₃ and CH are detected upward, while the signal of CH₂ appears downward (opposite phase). It is often possible to discriminate CH₃ or CH from the chemical shift. In many cases, the DEPT135 measurement alone is sufficient. If discrimination by DEPT135 alone is difficult, measure DEPT90, in which only the signal of CH is detected.

Table 2 DEPT Signal Appearance Patterns

We will use the DEPT spectra to determine the atomic groups of the sample. As shown in Figure 2, we will discriminate the atomic groups of each signal by comparing the signal appearance patterns of the 1D-NMR spectrum of ¹³C and the DEPT spectrum.

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DEPT135・・・Upward signal: CH₃ or CH, Downward signal: CH₂
DEPT90・・・ Detectable signal : CH
Signal detected only in the 1D-NMR spectrum of ¹³C and not detected in the DEPT spectrum: quaternary carbon
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Using the information so far, we were able to determine that the atomic groups of each signal are, from right to left, A (CH₃), B (CH₃), C (CH₂), D (CH), E (CH), and F (quaternary carbon).

The information so far is summarized in Table 3. This information is used to estimate the atomic groups and functional groups.

Table 3 ¹³C Spectra Information (2)

Figure 3 shows the chemical shift table for ¹³C. At an approximate border of 100 ppm, the saturated carbon signals are appeared on the right and the unsaturated carbon signals on the left. We will compare the information summarized in Table 3 with the chemical shift table. First, if we look at D (122.8 ppm) and E (144.0 ppm), we see that they are in the region of unsaturated carbon, indicating that this CH is derived from unsaturated carbon. Next, if we look at F, the chemical shift value is 166.2 ppm, which is in the ester region. The molecular formula of this substance is C₆H₁₀O₂, and since there are two Os, the quaternary carbon of F is presumed to be derived from the COO group.

The information so far is summarized in Table 4.

Table 4 ¹³C Spectra Information (3)

Step 2 HMQC & ¹H

In Step 2, we will use HMQC and 1D-NMR spectra of ¹H to find directly bonded ¹³C and ¹H combinations. HMQC will tell you the combination of ¹H and ¹³C that are directly bonded. Spin couplings are written with a symbol, such as ¹J_CH. The number of bonds is written in the upper left corner of the J representing the spin coupling, and the nucleus to which it is bound is written in the lower right corner of the J.

Figure 4 shows an HMQC spectrum. In a 2D-NMR spectra, a high-resolution 1D-NMR spectrum is displayed at each axis; for HMQC, the ¹H spectrum is displayed on the X-axis and the ¹³C spectrum on the Y-axis. In Step 2, the same symbols that were attached to each signal in the ¹³C spectrum in Step 1 are attached to the signals in the ¹³C spectrum on the Y-axis, with the upper side of the Y-axis representing the high-field side (small chemical shift) and the lower side representing the low-field side (large chemical shift).

Next, draw a line from the ¹³C signal on the Y-axis to the HMQC correlation signal and confirm which ¹H is directly bonded to ¹³C. For example, if we focus on the ¹³C signal of C, we draw a line toward the side, and when the line hits the correlation signal, we draw a line up from there. The ¹H signal that we have reached here is the counterpart directly bonded to signal C of ¹³C. When the corresponding ¹H is found in this way, the ¹H is marked with the same symbol as the counterpart ¹³C. Since it is the counterpart of the signal C of ¹³C, we will also put the symbol C on this ¹H. All correlated combinations will be given a similar symbolization.

Step 3 ¹H-¹H COSY

Fig. 6 shows the actual COSY spectra. COSY shows ¹H spectra both on the X-axis and Y-axis.
In Step 1, we assigned a symbol to each ¹³C signal. In Step 2, we assigned a symbol to each ¹H signal that is directly coupled to ¹³C using HMQC. In Step 3, we first assigned the same symbols to the signals in the ¹H spectrum on the X and Y axes as we did in Step 2. We looked at the HMQC spectra we used earlier (Figure 4) and copied the symbols attached to the signals in the ¹H spectra on the X-axis as they were.

Note that in Figure 6, the symbols are alphabetically ordered from the high-field side, but this just happened to be the case for this sample. The symbols on the ¹H signal are not always in alphabetical order.
Also, in the COSY spectra, signals line up on the diagonal line (called diagonal signals) when the diagonal line is drawn. But these are not used as information for structural analysis. The off-diagonal signals are the COSY correlation signals.

We draw a line from the correlated signal toward the X- and Y-axes spectra and find the ¹Hs that are spin-coupled to each other. For example, if we focus on the correlation signal circled in green, we find the ¹H signal C on the X-axis and the ¹H signal A on the Y-axis. Therefore, we can see that signals C and A are spin-coupled (neighboring) to each other. Once the counterpart is found, we add a symbol to the correlated signal. For example, C/A, to specify that they are ¹H signals in a ³J_HH relationship. Since the correlated signals of COSY appear in a line symmetric position with respect to the diagonal, we find the ³J_HH counterpart in the same way for all correlated signals.

Once the information on the correlated signals of COSY is available, a correlation table for each signal is created.
Table 5 is the correlation table for COSY. Write the symbols for the ¹H signals in vertical and horizontal order, as per the spectrum of the data on the ¹H-¹HCOSY axis. (In the case of this sample, they happen to be arranged alphabetically, but you should look at the COSY spectra and list them in chemical shift order.) For example, if the correlated signals are C and A, mark the intersection of C and A. In this way, all correlated signals are entered in the COSY correlation table.

Fig.7 shows the diagram of correlation signal of COSY.
In this sample, COSY correlation signal was observed between ¹H of C and ¹H of A.
The HMQC spectra also indicated that ¹H of C and ¹³C of C are directly coupled. In the same way, ¹H of A and ¹³C of A are directly coupled.
Therefore, COSY correlation signals between C and A can induce that ¹³C of C and ¹³C of A are neighboring. In other words, using the COSY information, we can indirectly find the neighboring ¹³C connections from the neighboring ¹H connections.

The information that we have so far is the ¹³C spectrum information (Table 4) and the COSY correlation table (Table 5). From the COSY correlation table, we can see that the carbon atoms derived from the ¹³C signals A-C, B-E, and D-E are neighboring to each other. Combined with the information from the ¹³C spectrum, when we rewrite the symbols as atomic groups, we have A-C : "CH₃-CH₂-", B-E-D : "CH₃-CH=CH-", and F : "COO" in Step 1. Therefore, we know that this compound is composed of the following three substructures.

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Substructure 1：CH₃-CH₂-　・・・ A-C
Substructure 2： CH3-CH=CH-　・・・ B-E-D
Substructure 3： COO　・・・ F
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Next, we will consider what kind of molecule these will make by connecting each substructure (Figure 8). We will list all possible molecular structures from the combination of the three substructures. When considering molecular structures, it is easier to focus on the substructure containing CH₃ because CH₃ is located at the end of the structure. In this case, there are two possible structures. Firstly, Substructures 1 and 2 are found to be the ends of the molecular structure because they contain CH₃.And since 1 and 2 are located at the ends, we can predict that substructure 3 is sandwiched between 1 and 2. The different orientations of substructure 3 allowed us to create two different inferred structures (I, II). The orientation of the COO can be used to determine which inferred structure is more reasonable. Therefore, HMBC measurements are performed to confirm the long-range spin coupling with respect to the COO.

Step 4 HMBC

Fig. 10 is the actual spectra of HMBC. The HMBC axes are the same as HMQC's case, ¹H spectra on X-axis and ¹³C spectra on Y-axis.
In Step 4, we will put the same symbol that we assigned for HMQC spectra in Step 2, for each signal of a 1D-NMR spectrum on the X and Y axes.

Next, we draw lines from the correlation signals to the X- and Y-axis spectra to find the spin-coupled ¹³C and ¹H combinations. Here, the two horizontally-lined up signals circled in orange are the signals of ¹J_CH, a direct coupling of ¹H and ¹³C. Since these are remnant signals, they are not observed in all direct couplings. As the direct couplings have already been observed by HMQC, it is not necessary to focus on them here.
In the HMBC spectrum, only the long-range information is read, ignoring the direct coupling signal.
For example, if we focus on the correlation signal circled in green, we can see that ¹³C of C and ¹H of A are long-range spin coupled to each other, since tracing to Y-axis results in C and to X-axis results in A.

Once the counterpart is known, add a symbol to the correlation signal, e.g., A/C, to indicate that it is a long-range spin-coupled ¹H and ¹³C of ²J_CH or ³J_CH.
For all correlation signals, do the same to find a long-range spin-coupling counterpart and mark the correlation signal with a symbol. Once you have written out the information for the HMBC correlation signals, create a correlation table.

Table 6 is the correlation table for HMBC. We write the symbol for the ¹H signal horizontally and the symbol for the ¹³C signal vertically, as per the spectrum. (Note that the symbols for the ¹H signals are not necessarily in alphabetical order.) Correlation signals for the HMBC spectra will be filled in. For example, if the correlation signal is ¹³C for C and ¹H for A, mark the intersection of C for ¹³C and A for ¹H in the correlation table. In this way, all correlation signals are entered in the HMBC correlation table.

We use the information in the HMBC to connect the substructures.
Look at the HMBC correlation table (Table 7) that you filled out earlier. In the correlation signal marks, bonds that have already been analyzed by COSY in Step 3 are marked with ○, and bonds that were found for the first time in HMBC are marked with ●. The bond that was found for the first time in HMBC is the COO correlation for F. This indicates that F is a long-range spin couplings with C, D, and E.

We will look at the long-range correlations of F-C, F-D, and F-E to consider which direction is correct for the COO to be attached, among the inferred structures I and II. (Figure 11)
First, in inferred structure I, F's ¹³C and C's ¹H have three bonds "³J_CH", ; F's ¹³C andD's ¹H have two bonds "²J_CH", ; and F's ¹³C and E's ¹H have three bonds "³J_CH".
Next, let us look at the inferred structure II. The ¹H of ¹³C and C in F is "²J_CH" with two bonds, the ¹H of ¹³C and D in F is "³J_CH" with three bonds, and the ¹H of ¹³C and E in F is "⁴J_CH" with four bonds.
Since ⁴J_CH is not likely to be observed, we can expect that the inferred structure I is a reasonable structure.

Finally, the inferred structure I is checked against the information in the 1D-NMR spectrum of ¹³C to confirm if it is really correct. In the inferred structure I, CH₂ of the symbol C is bonded to oxygen. And the ¹³C chemical shift of this C was 59.8 ppm (Table 1).
The ¹³C chemical shift table for CH₂ (Table 8) shows that normal CH₂ is observed at 20-45 ppm, while CH₂ bonded to oxygen is observed at 40-70 ppm with a lower field shift.
From this fact, we can conclude that "Structure I is correct".

Table 8 ¹³C Chemical Shift for CH₂

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