NOAH sequences for fast Small Molecule Analysis implemented on a Spinsolve benchtop NMR spectrometer

NOAH: NMR by Ordered Acquisition using 1H detection implemented on a Spinsolve benchtop NMR spectrometer

 

To elucidate or confirm molecular structure by NMR, a series of homo- and hetero-nuclear experiments are typically required. Besides standard 1D spectra, powerful 2D methods like COSY, HSQC, and HMBC are acquired in order to make the signal assignment possible. The measurement of these 2D experiments is a time consuming process where each raw of the 2D data set is acquired in an individual experiment and a generally long recovery time needs to be included between steps. Recently, Kupce and Claridge [1] proposed a powerful strategy to reduce the time required to measure a set of 2D experiments by acquiring the steps of the different 2D experiments without any waiting time between them. They called this super-sequence, NOAH, which stands for NMR by Ordered Acquisition using 1H detection. In this way, instead of acquiring the 2D data sets of the different 2D experiments just one after the other (including the recovery time between each raw), the NOAH [1] approach acquires the raw number n of the different matrices one after the other, without recovery time in between, and adds a recovery time only before raws n+1 are queried. As this recovery or relaxation period is much longer than the duration of the pulse sequences, a substantial time saving can be achieved. Figure 1 shows a scheme that illustrates the implementation of what typically is called a NOAH-3, where three sequences are acquired per recovery time.

Figure 1: NOAH supersequences nest a series of experiments in order to acquire them using a single relaxation delay. As the duration of each sequence is typically much shorter than this recovery delay required between scans, NOAH can acquire a set of experiments in the time it usually takes to acquire a single one in a conventional way.

The key that allows one to measure these experiments one after the other relies on the fact that heteronuclear experiments detect the signal of about 1% of the molecules in the sample leaving the rest of the molecules available for the homonuclear experiments. Figure 1 shows a scheme where an HSQC is first applied to acquire the signal of the protons directly attached to a 13C, leaving the signal of the protons attached to 12C unaffected. Then, the HMBC acquires the signal of the protons long range coupled to 13C and leaves the magnetization prepared for the COSY sequence included at the end of the loop.

For low concentrated samples, the acquisition of the heteronuclear experiments is the most time consuming. While an HSQC or and HMBC can take an hour or more to acquire, the homonuclear experiments make take just some minutes. In this blog post, we demonstrate the implementation of a NOAH-2 sequence, where an HSQC and an HMBC are acquired in a single experiment.  The pulse sequence has been adapted from ref. [1] and is shown in the picture below. This sequence has been optimized to minimize the effect of the HSQC on the protons that are not directly coupled to a 13C. In this way, the HMBC is acquired without appreciable sensitivity loss.

 

Figure 2: NOAH-2 supersequence where an HSQC and an HMBC are nested using a single relaxation delay.

NOAH2-SB sequence implemented on Spinsolve expert

 

This sequence has been implemented on our Spinsolve Expert software, where besides programming the sequence, the user can define the Graphical unit interface and processing steps to get the data displayed automatically even during the data is being acquired.

Figure 3: print screen of the graphical unit interface of the NOAH-2 sequence, where the HSQC and HMBC are simultaneously displayed.

NOAH sequences performance

 

To demonstrate that NOAH delivers the same signals as the single sequences, a neat propyl benzoate sample was measured. A highly concentrated sample makes it possible to identify potential artifacts caused by the fact that the HMBC is acquired after the HSQC, which may have affected the protons that are long range couplet to 13C. The spectra were collected using 128 steps along the indirect dimension, 4 scans, and a recovery time of 1.5 seconds. As the acquisition of the signal takes another 0.5 seconds per sequence the single HSQC is acquired in 17 minutes, while it takes 21 minutes to the NOAH-2 to collect both experiments. The time saving in this case is the difference between the 34 minutes that are required to collect the two experiments individually and the 21 minutes required by the NOAH sequence. Figure 4 shows a comparison between the standard and the NOAH versions of the HSQC. The signals showing the correlation between 1H and 13C have similar amplitudes and even the cross peaks between the groups 11 and 10 are of comparable amplitude. The vertical zoom is set large enough to see the spurious signals in both data sets, which are much smaller than the signals indicating the correlation between directly coupled nuclei. Figure 5 shows the comparison of the HMBC spectra acquired with the standard and the NOAH versions of the sequences. The signals showing the long range correlation between 1H and 13C have similar amplitudes. In the case of the HMBC, artifacts can be observed in the form of satellites around the position corresponding to the directly coupled 1H and 13C. As in the NOAH sequence, the HSQC acquires this signal first, the amplitude of the artifacts is smaller for the NOAH-2 HMBC. The vertical zoom is set large enough to see the spurious signals in both data sets, which are much smaller than the signals indicating the true long correlation between 1H and 13C nuclei.

 

Figure 4: Comparison of the HSQC spectra collected for a neat sample of propyl benzoate with a classical sequence (left) and the NOAH-2 supersequence (right).

 

Figure 5: Comparison of the HMBC spectra collected for a neat sample of propyl benzoate with a classical sequence (left) and the NOAH-2 supersequence (right).

Finally, a lidocaine sample dissolved in CDCl3 at a concentration of 250 mMolar was chosen to demonstrate that the sensitivity of the NOAH-2 sequence offers a similar sensitivity compared to the standard versions of the HSQC and HMBC sequences. The experiments were acquired with the same parameters used for propyl benzoate.  In this way, individual spectra were acquired in 17 minutes each, and the NOAH sequences in 21 minutes. The comparison between single and NOAH spectra is shown in Fig. 6 and 7 for the HSQC and HMBC, respectively. As it can be observed, both the resolution and the sensitivity of the single spectra are similar to the one delivered by the NOAH sequence. Moreover, it is important to notice that the sensitivity of the Spinsolve 80 MHz allows one to collect 2D heteronuclear experiments of samples dissolved at 250 mMolar in just 21 minutes when a NOAH sequence is implemented.

 

Figure 6: Comparison of the HSQC spectra collected for a 250 mMolar lidocaine sample with a classical sequence (left) and the NOAH-2 supersequence (right).

 

Figure 7: Comparison of the HMBC spectra collected for a 250 mMolar lidocaine sample with a classical sequence (left) and the NOAH-2 supersequence (right).

 

[1] Dr. Ēriks KupčeProf. Tim D. W. Claridge DOI: 10.1002/anie.201705506