HSQC-TOCSY Analysis│Understanding 2D NMR Application in Comparison with TOCSY

What is HSQC-TOCSY?

HSQC-TOCSY is a two-dimensional NMR experiment that can simultaneously obtain the directly coupled ¹H-¹³C correlation by HSQC and the spin network information by TOCSY. As a result, TOCSY-type correlations are observed with ¹³C chemical-shift resolution.

HSQC-TOCSY is a combination of the two mechanisms:

• HSQC part: to detect a ¹H that is directly coupled with any given ¹³C
• TOCSY part: to relay magnetization to a ¹H in the same spin network starting from the ¹H.

As a result, for any given ¹³C,

• ¹H that is directly coupled with the ¹³C (HSQC correlation)
• Other ¹Hs connected via the same spin network as the ¹H's (TOCSY correlation) appear in the HSQC-TOCSY spectrum.

Comparison between TOCSY and HSQC-TOCSY Spectra

Fig.1 Schematic Diagram of TOCSY (left), Schematic Diagram of HSQC-TOCSY (right)

As shown in Fig. 1, when proton signals are severely overlapped, the corresponding TOCSY cross-peaks also overlap, making spectral analysis difficult. However, in HSQC-TOCSY, Y axis represents ¹³C chemical shift which have larger chemical-shift dispersion between chemical shifts for each signal. This additional dispersion enables more effective separation of correlation signals, thereby facilitating spectral analysis.

Fig.2 Schematic Diagram of HSQC-TOCSY Spectra

In addition, similar with the TOCSY, HSQC-TOCSY enables extended relay within a spin system by extending the mixing time. When paying attention to the correlation signal ¹³C_A in Fig. 2, when the mixing time is short, the result is the same as the HSQC spectrum. But as the mixing time is extended, the magnetization is transferred to ¹H_B, ¹H_C, and the correlation signal with ¹H_B, ¹H_C appears.

HSQC-TOCSY Analysis Example of Cedrol

Cedrol is a terpenoid compound, known as an aroma compound found in ceder and cypress. Fig. 3 shows the ¹H spectrum of cedrol. The OH proton signal is observed at 2.9 ppm, but other ¹H signals are all observed in a narrow range of about 1 ppm. This region is considered in detail in Fig. 4.

Fig. 3 ¹H Spectrum of Cedrol

Fig. 4 ¹H Spectrum of Cedrol

From the high field, protons of four methyl groups (-CH₃) are detected.
In the area circled in pink, protons from five methylene groups (-CH₂-) and three methine groups (-CH-) are overlapped. The reason that the area circled in pink has severe signals overlapping, is due to a lot of "non-equivalent methylene" which are present in the molecule.

The methylene groups in an alkyl chain which is freely rotating, are considered to be in NMR equivalent environments and chemical shifts of protons of such methylene groups are equal.
However, in the cyclic compound such as cedrol, the rotation is restricted. So, protons of the methylene group are in a non-equivalent environment and these signals have separate chemical shifts.
Methylene groups of this type are referred to as "non-equivalent methylene".

Fig.5 COSY Spectrum (left) and TOCSY Spectrum (right) of Cedrol

Fig. 5 shows the spectra of COSY and TOCSY (mixing time: 200 ms) with 400 MHz NMR spectrometer. In both spectra, there are regions where the signals are overlapping, complicating spectral analysis.

Even if analysis is attempted with methyl group protons of distant chemical shifts, spin network is interrupted because the adjacent carbon of these methyl groups are quaternary carbons, making it difficult to observe the relay.
Furthermore, when TOCSY spectrum of the methyl group detected at the highest field side (approx. 0.8 ppm) is examined, the correlation signals are observed, yet overlapped, making it difficult to determine to which ¹H the network forms.
In other words, sometimes it can be difficult to determine the spin network even if TOCSY is used. In such cases, HSQC-TOCSY becomes an effective experiment.

Fig.6 ¹³C Spectrum of Cedrol

Prior to HSQC-TOCSY analysis, the ¹³C spectrum and HSQC spectra of cedrol are collected.
Fig. 6 shows the ¹³C spectrum of cedrol.
Although the signal D coming from the methyl group is overlapping with the solvent signal, all 15 signals of ¹³C are separated and observed.

Fig. 7 HSQC Spectrum of Cedrol

Next, the HSQC spectrum of cedrol is shown in Fig. 7. The horizontal axis corresponds to the ¹H chemical-shift axis, while the vertical axis represents the ¹³C spectrum data. HSQC correlates each ¹³C with its directly bonded ¹H. Therefore, the correlation of two ¹H signals appears for one carbon in the case of the methylene group.

When focusing on "methylene at B", two ¹H correlation signals are observed, indicating that two protons of methylene group B, have different chemical shifts.
Thus, HSQC enables easy confirmation of non-equivalent methylene. In cedrol, four out of five methylene groups (B, G, H, J) are identified as non-equivalent methylene.

Fig. 8 HSQC-TOCSY Spectra of Cedrol

The HSQC-TOCSY spectrum of cedrol is shown in Fig. 8. The leftmost shows HSQC spectrum. When analyzing the HSQC-TOCSY spectra, the signal observed in the HSQC spectrum (signal arising from the directly bonded ¹H-¹³C correlation) is treated as the starting point of the relay.
Then, as in conventional TOCSY, by comparing with spectra with mixing time varied, it is possible to confirm the relay along the spin network.

For example, comparing the areas circled in dotted lines indicate that the correlation signals increase with the relay. Now let's enlarge these areas and observe in more detail.

Fig.9 HSQC-TOCSY Spectra of Cedrol (Correlation of M)

Fig. 9 shows an enlarged comparison of the correlation signals for the methine carbon labeled M.
At the mixing time of 20 ms, a correlation with the non-equivalent methylene B appears, indicating an M-B connectivity. Moreover, at mixing time of 40 ms, a correlation signal with non-equivalent methylene H appears, indicating that M-B-H are connected in this order.
In HSQC-TOCSY, the signals of non-equivalent methylene typically appear as two separate ¹H correlation signals for a single carbon. This provides the advantage that the assignment of magnetically nonequivalent methylene protons becomes easier.

Fig. 10 HSQC-TOCSY Correlation of Cedrol

By applying the same analytical procedure, we obtained three spin networks as shown in Fig. 10. The regions where no further magnetization transfer is observed correspond to positions at which the ¹H-¹H spin relay is terminated by intervening quaternary carbons.
To proceed with the structural analysis to the next step, it will be necessary to analyze the ¹H and ¹³C long-range correlation (HMBC spectrum) and complete the planar structure determination.

Summary of COSY,TOCSY, and HSQC-TOCSY

Finally, we summarize the characteristics of the COSY, TOCSY, and HSQC-TOCSY.
COSY is a fundamental and essential 2D NMR spectrum as it can be easily measured with high sensitivity.
A first step for molecular structure analysis, which is widely used from beginners to experts.

On the other hand, TOCSY and HSQC-TOCSY are useful when ¹H signals overlap, or when complicated spin networks need to be analyzed. TOCSY allows for confirmation of the entire spin network, but sometimes it is necessary to measure multiple spectra with different mixing times. In addition, HSQC-TOCSY has a great advantage to be able to avoid overlapping of signals since the correlation analysis of TOCSY is performed by way of ¹³C. However, as the sensitivity is lower than COSY and TOCSY, the measurement time is longer than COSY and TOCSY, which needs attention.

Each measurement method has advantages and disadvantages. It is important to select the most suitable 2D NMR measurement method according to complexity of the target molecule, degree of signal overlapping, and the spin network range to be analyzed.

Related Application

Spectroscopic separation of mixed organic fluorine compounds by 2D ¹⁹F-¹⁹F TOCSY --To extract 1D ¹⁹F NMR spectrum of each compound--

Contact

If you are interested in the details of spin network analysis using TOCSY and HSQC-TOCSY and NMR spectrometer, please contact JEOL. We will support your molecular structure analysis capability with our abundant application examples and technical support.

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