Tocsy 2d Nmr Assignment

2D-NMR

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Types of 2D NMR

Two dimensional (2D) NMR spectroscopy includes:-

Homonuclear

Heteronuclear correlation

Examples of 2D spectral assignment

Assignment of 12,14-ditbutylbenzo[g]chrysene

Assignment of cholesteryl acetate

The basis of 2D NMR

In a 1D-NMR experiment the data acquisition stage takes place right after the pulse sequence. This order is maintained also with complex experiments although a preparation phase is added before the acquisition. However, in a 2D-NMR experiment, the acquisition stage is separated from the excitation stage by intermediate stages called evolution and mixing. The process of evolution continues for a period of time labeled t1. Data acquisition includes a large number of spectra that are acquired as follows: the first time the value of t1 is set close to zero and the first spectrum is acquired. The second time, t1 is increased by Δt and another spectrum is acquired. This process (of incrementing t1 and acquiring spectra) is repeated until there is enough data for analysis using a 2D Fourier transform. The spectrum is usually represented as a topographic map where one of the axes is f1 that is the spectrum in the t1 dimension and the second axis is that which is acquired after the evolution and mixing stages (similar to 1D acquisition). The intensity of the signal is shown by a stronger color the more it is intense.

In the resulting topographic map the signals are a function of two frequencies, f1 and f2. It is possible that a signal will appear at one frequency (e.g., 20 Hz) in f1 and another frequency (e.g., 80 Hz) f2 that means that the signal's frequency changed during the evolution time. In a 2D-NMR experiment, magnetization transfer is measured. Sometimes this occurs through bonds to the same type of nucleus such as in COSY, TOCSY and INADEQUATE or to another type of nucleus such as in HSQC and HMBC or through space such as in NOESY and ROESY.

The various 2D-NMR techniques are useful when 1D-NMR is insufficient such when the signals overlap because their resonant frequencies are very similar. 2D-NMR techniques can save time especially when interested in connectivity between different types of nuclei (e.g., proton and carbon).

The basic 2D NMR experiment(fig. 1) consists of a pulse sequence that excites the nuclei with two pulses or groups of pulses then receiving the free induction decay (fid). The groups of pulses may be purely radiofrequency (rf) or may include magnetic gradient pulses. The acquisition is carried out many times, incrementing the delay (evolution time - t1) between the two pulse groups. The evolution time is labeled t1 and the acquisition time, t2.

Fig. 1. Basic pulse sequence for 2D acquisition

2D Fourier transform

The FID is then Fourier transformed in both directions (fig. 2) to yield the spectrum. The spectrum is conventionally displayed as a contour diagram. The evolution frequency is labeled f1 and the acquisition frequency is labeled f2 and plotted from right to left.

Fig. 2. 2D Fourier transform

The 2D spectrum is usually plotted with its 1D projections for clarity. These may be genuine projections or the equivalent 1D spectra. In a homonuclear spectrum there is usually a diagonal (with the exception of 2D-INADEQUATE) that represents the correlation of peaks to themselves and is not in itself very informative. The signals away from the diagonal represent correlations between two signals and are used for assignment. For example in the homonuclear COSY spectrum in Fig. 3, the 1H signal at 1.4 ppm correlates with the 1H signal at 2.8 ppm because there are cross-peaks but they do not correlate with the signals at 7.3 ppm.

Fig. 3. 2D COSY spectrum of ethylbenzene

In a heteronuclear spectrum there are no diagonal signals and all the signals represent correlations. For example in the heteronculear HSQC short-range correlation spectrum in fig. 4, the 1H signal at 1.4 ppm correlates with the 13C signal at 15.7 ppm, the 1H signal at 2.8 ppm correlates with the 13C signal at 29.0 ppm, etc.

Fig. 4. 2D HSQC spectrum of ethylbenzene

The signals in a 2D spectrum are not always pure phase. Sometimes, the phase cannot be expressed simply as in HMBC and 2D-INADEQUATE, in which case a magnitude spectrum is plotted. However, magnitude spectra sacrifice resolution as compared to pure phase spectra (and unlike window functions that broaden lines, do not yield sensitivity gains). Therefore, wherever possible, the 2D spectrum should be phased. The resulting signals may be pure phase, anti-phase or negatively phased as in the fig. 5. Negative signals are conventionally represented by dotted or red contours.

Fig. 5. Possible phases for a correlation between two doublets

1H-1H TOCSY

1H-1H TOCSY (TOtal Correlated SpectroscopY also known as HOHAHA – HOmonuclear HArtmann HAhn) is useful for dividing the proton signals into groups or coupling networks, especially when the multiplets overlap (have very similar chemical shifts) or there is extensive second order coupling. A TOCSY spectrum yields through bond correlations viaspin-spin coupling. Correlations are seen throughout the coupling network and intensity is not related in a simple fashion to the number of bonds connecting the protons. Therefore a five-bond correlation may or may not be stronger than a three-bond correlation. TOCSY is usually used in large molecules with many separated coupling networks such as peptides, proteins, oligosaccharides and polysaccharides. If an indication of the number of bonds connecting the protons is required, for example in order to determine the order in which they are connected, a COSY spectrum is preferable.

The pulse sequence (fig. 1) used in our laboratory is the gradient enhanced TOCSY that means that during the pulse sequence, magnetic field gradients are applied. The spin-lock is a composite pulse and should be applied for between 20 and 200 ms with a pulse power sufficient to cover the spectral width. A short spin-lock makes the TOCSY more COSY-like in that more distant correlations will usually be weaker than short-range ones. A long spin-lock holds the magnetic vector in the x-y plane allowing correlations over large coupling networks. The length of the spin-lock is roughly related to the distance through the coupling network that correlations are seen. However, too long a spin-lock will heat the sample causing signal distortion and can damage the electronics of the spectrometer. Therefore care should be taken.

The attenuation should be set so that the 90° pulse width will be less than 1/(4SWH) (SWH is the spectral width in Hz) and typically 1/(6SWH). An attenuation of 12 dB with a 50 W amplifier yielding a 90° pulse width of 35 μs is typical for a 5 mm high resolution probe.

Fig. 1. Pulse sequence for gradient TOCSY

The TOCSY spectrum as shown in fig. 2 for ethylbenzene (fig. 3) contains a diagonal and cross-peaks. The diagonal consists of the 1D spectrum with single peaks suppressed. The cross-peaks indicate couplings between two mutliplets. The cross-peaks correspond to correlations through bonds, the most apparent of these is between H1' and H2' at 2.65 and 1.24 ppm. All the desired signals (that correspond to connectivity) are pure phase, albeit with minor symmetrical phase distortion. In addition, artifacts (undesired signals) also appear in the spectrum as vertical streaks (interference and f1 noise, fig. 4). These artifacts are rarely in phase with the desired signals and appear in specific locations.

Fig. 2. 2D TOCSY spectrum of ethylbenzene

Fig. 3. Structure of ethylbenzene

Fig. 4. Artifacts in the TOCSY spectrum of ethylbenzene

For example, in 12,14-ditbutylbenzo[g]chrysene (fig. 5), only a partial analysis of the regular 1H-NMR spectrum is possible. TOCSY (fig. 6) confirms the initial division of protons intofour groups or coupling networks. No correlations (cross-peaks) are seen to the tbutyls (at 0.92 and 1.40 ppm) because they are too many bonds away from the ring system.

Fig. 5. Structure of 12,14-ditbutylbenzo[g]chrysene

Fig. 6. Artifacts in the COSY spectrum of ethylbenzene

The aromatic region of the spectrum (fig. 7) shows all the correlations within each coupling network. However, unlike COSY, these cannot be used to determine which protons are neighbors. For example the proton at 8.62 ppm is next to the proton at 7.55 ppm but its correlation is weaker than with the four-bond correlation to the signal at 7.59 ppm which is in turn weak than the five-bond correlation to the proton at 8.56 ppm.

Fig. 7. Aromaric region of the 2D TOCSY spectrum of 12,14-ditbutylbenzo[g]chrysene

Using horizontal and vertical lines, it is possible to separate each group and follow its connectivity (fig, 8). The blue group of four protons consist of the signals at 8.62 ppm, 7.55, 7.59 to 8.56, the green group of four protons at 8.54 to 7.34 to 7.44 to 8.17 and the red group or two protons, that correspond to H9 and 10 because they are the only group of two protons expected to have a three-bond coupling constant (8.9 Hz), are at 7.76 and 8.32 ppm. The yellow group of two protons correspond to H11 and 13 because the coupling constant is small (1.9 Hz) and consistent with a four bond correlation.

Fig. 8. Aromaric region of the 2D TOCSY spectrum of 12,14-ditbutylbenzo[g]chrysene showing connectivity and separation into four color-coded proton groups

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