The power of solvent suppression and 13C decoupling forsynthesis and reaction control

Figure 1. Spinsolve NMR spectrometer used in the chemistry lab for quick analysis of products. The typical presenceof protonated solvents in the sample requires the use of solvent suppression and carbon decoupling methods toacquire the spectra of analytes of interest.

Solvent suppression
Benchtop NMR systems installed in chemistry labs are typically applied to identify or quantify products
dissolved in protonated instead of deuterated solvents. The presence of protonated solvents in thesample introduces two main challenges at the time of acquiring an NMR spectrum. The first challenge isdue to the large signal from the protonated solvent which can saturate the receiver of the spectrometer.
This forces a reduction of the receiver gain which very quickly leads to losses in the signal-to-noise ratio(SNR), or sensitivity in the spectrum. The issue particularly affects high-field NMR systems. A common
solution to this problem is to apply solvent suppression methods to attenuate the solvent signal before
the measurement is performed. Although the use of these methods is mandatory for high-field systems,they are not required on benchtop spectrometers. At the lower field strengths of these systems, the NMR
signals have a smaller amplitude, and the receiver amplifier is designed with a large enough dynamic range
to acquire the signals of pure solvents without saturation. This enables the optimum SNR in the spectrum
to be maintained. To demonstrate the large dynamic range of the receiver in a Spinsolve spectrometer,
the spectrum of histidine dissolved at a concentration of 10 millimolar in neat water was acquired without
solvent suppression. The neat water has a concentration of about 55 Molar, which defines an effective
concentration of 110 Molar of 1H contributing to the singlet observed for water. The full-scale and zoomed
spectra ( red spectra in Fig. 2a and 2b) show no distortion caused by saturation of the receiver. Taking
into account that the solute has a concentration that is four orders of magnitude smaller than the solvent,a large zoom of the vertical scale is required to see the signals of the solute (see Fig. 2b).

At this point we can identify the second challenge introduced by the presence of a large signal in thespectrum. The small signals from histidine in the standard spectrum shown in Fig. 2b (red) appear mounted
on the tails (or sides) of the water peak, which covers a big fraction, if not all, of the spectrum andmakes quantification almost impossible. Solvent suppression can be very useful for benchtop systems
to remove the solvent peak and its tails, but because these methods are based on selective frequency
excitation, they can only work efficiently if the magnetic field of the magnet is highly homogeneous. In ahomogeneous magnetic field all nuclei of a particular chemical group have the same resonance frequency
and the shape of the NMR peak in the spectrum is described by a Lorentzian function defined only by
the relaxation time of the sample. If this criteria is fulfilled it is said that the NMR signal is homogeneously
broadened and all nuclei have actually the same resonance frequency.
a)Standard
Solvent Suppressionb) zoom x 2000
Standard
Solvent Suppression

Figure 2. a) Spectra of histidine dissolved at a concentration of 10 mM in water acquired with a standard pulse
and acquire sequence (red) and using a solvent suppression sequence (cyan). Shown in full vertical scale, only thewater signal is visible as it has a concentration of 55 Molar (effective 110 Molar of 1H). b) The spectra from a) shownwith a x 2000 vertical zoom. In this scale it can be observed how the signals of histidine overlap with the tails of
the huge water peak in the standard experiment (red), but can be all very well resolved on the spectrum acquiredwith a solvent suppression method (cyan). It can be observed that the water peak is attenuated to the point where
the tails are efficiently removed without affecting the signals of histidine as they appear at resonance frequencies
different than the one of water. The spectra in b) were acquired using 64 scans and a repetition time of 10 seconds
on a Spinsolve 60 MHz Carbon ULTRA.

Although the peak may extend over a wide region in the spectrum it can be attenuated as a whole byapplying continuous excitation at the central frequency of the line. In this situation, solvent suppression
can considerably reduce the tails of the solvent peaks and allow the signals of the products to be moreaccurately quantified. The cyan spectrum shown in Fig. 2a and 2b was obtained when a selective
excitation pulse is applied at the resonance frequency of the water peak for a period of two seconds prior
to the acquisition of the NMR signal. In this experiment the water peak was attenuated a factor of about
100, while the signals corresponding to histidine remained unaffected.
As a second example, Fig. 3 shows the performance of the solvent suppression method on a 25mM sample of sucrose dissolved in water. This sample is used as a standard for testing the selectivity ofthe solvent suppression to saturate the water peak without affecting the signal of the anomeric protons at
5.4 ppm. Note that the sample does not require any D2O as Spinsolve benchtop NMR spectrometers areequipped with an external hardware lock system that works independent of the sample being measured.
While this peak completely overlaps with the tail of the water peak in the standard measurement (red), itis fully resolved on the spectrum measured with solvent suppression (cyan).

Standard
Solvent Suppression

Figure 3. Spectra of sucrose dissolved at a concentration of 25 mM in neat water acquired with a standard pulse
sequence (red) and with a solvent suppression method (cyan). The spectra were acquired using 64 scans and arepetition time of 10 seconds on a Spinsolve 60 MHz Carbon ULTRA.

This high performance solvent suppression can only be achieved if the magnetic field is highly
homogeneous, which is a challenge for benchtop systems. Permanent magnet geometries designed forbenchtop spectrometers are required to be as small as possible, generate the strongest magnetic field,
and keep the sample volume constrained to use standard 5 mm NMR tubes. In such an optimization
process the specification that suffers the most is the homogeneity of the magnetic field. In general, whilea reasonable homogeneity can be achieved in the center of the tube, it tends to become inhomogeneousas the volume approaches the wall of the tube, leading to a line shape that is relatively narrow at halfheight, but that can be very broad in the tails at the bottom of the peak. When the tails are defined by themagnetic field inhomogeneity, solvent suppression methods do not work properly.

This is because the selective excitation used by the method only saturates the nuclei with a resonancefrequency close to the center of the peak. As the tails are simply the signal of nuclei located where themagnetic field has a different strength, such as close to the wall of the tube, they will not be efficiently
saturated because they have a different resonance frequency to the one of the nuclei in the homogeneous
center of the tube. Depending on the frequency spreading caused by the magnetic field inhomogeneity, theselective excitation part of the sequence will saturate the center of the peak, burning a hole in the center,leaving, in extreme cases, the tails unaffected. So, although the center of the peak can be attenuated,the overlapping of the tails would remain and the solvent suppression would be of no advantage for ourmeasurements.
As the homogeneity of the magnetic field defines the line shape of the peaks in the spectrum, theaccepted way to specify the homogeneity of a magnet is by providing the line width (LW) at different heightsof the NMR peak of a chloroform standard sample. While it is common to use the LW at 50% of the peakas main indicator, it is actually the LW at its base which determines the performance that can be expected
for the solvent suppression method. For high field systems, the width of the line is typically specified
at 50%, 0.55%, and at 0.11% of the peak height. To achieve the highest performance in applications
where solvent suppression is required, Magritek introduced in 2017 the Spinsolve ULTRA model. Whilethe classic version of the Spinsolve delivers the highest homogeneity available in the benchtop market
today (with a LW <0.5 Hz at 50% and <20 Hz at 0.55% of the peak height), the ULTRA model specifies aneven superior linewidth <0.2 Hz at 50%, <6 Hz at 0.55%, and <12 Hz at 0.11% of the peak height, whichis comparable to the homogeneity delivered by high field magnets. An example of such a line shape isshown in Fig. 4 where a standard chloroform sample was used for the measurement. At this point it isimportant to mention that the Spinsolve ULTRA is the only model on the market that specifies the LW
at 0.11%. This ULTRA high homogeneity makes it possible to resolve the smallest signals close to thesolvent signals like no other benchtop system.

Figure 4: Spectrum of a reference chloroform sample dissolved in deuterated acetone at a concentration of 20%.
The inset shows the full line shape where the linewidth at 50% of the peak amplitude is specified. The zoomed
spectrum shows the carbon satellites of chloroform as a reference to identify the 0.55% of the peak height. The line
width at the base of the peak is shown also at 0.11%.

C decoupling

13

In the previous examples water was used as solvent. However organic solvents, with multiple lines in thespectrum, are frequently used depending on the solubility of the sample, or the reaction under investigation.
Figure 5a shows the full scale spectrum of a paracetamol sample dissolved in ethanol at a concentrationof 170 mM. As ethanol has a concentration of about 17 molar only the signals of the solvent are visible
at full scale. A zoom x100 allows us to identify the signals of paracetamol in the base of the large solvent
peaks (Fig. 5b). This example confirms again that the Spinsolve receiver can acquire the signal of a neatsolvent without showing effects of saturation or compromising the sensitivity. The result after applying
solvent suppression at the center of each of the three solvent signals can be observed in Fig. 5c. The
selective excitation pulse applied before the acquisition of the spectrum achieves a saturation of about ahundred fold and the solvent peaks are now comparable in amplitude to the paracetamol signals.

a)b)zoom x100c)
zoom x100

Figure 5: a) Spectrum of paracetamol dissolved in Ethanol at a concentration of 170 millimolar. b) Zoom x100 of
the spectrum in a). The signal of paracetamol gets visible and appears with a similar intensity as the signal of thecarbon satellites of the ethanol peaks. c) Spectrum of the paracetamol sample acquired using multi peak solvent
suppression method that saturates the three main signals of ethanol.

While the solvent suppression sequence does the job of removing the tails of the solvent peaks, we canquickly identify a new problem introduced by the organic solvent. Because the solvent has C-H groups,
the carbon satellites of these groups are visible in the spectrum. The natural abundance of 13C is about
1%, so with a neat concentration of 17 Molar, there are 170 mM of ethanol molecules with a 13C nuclei.
In this example, it is the same concentration as the paracetamol in the sample. The concentrations
used here are similar to the concentrations you can expect in a typical reaction, confirming that carbon
satellites are a common problem to expect when using organic solvents. A powerful NMR method toremove the satellites from the spectrum is known as carbon decoupling. It requires continuous excitationof the 13C nuclei either during the solvent suppression period or during signal acquisition. Figure 6 shows
the results obtained with the second option implemented on a Spinsolve Carbon ULTRA system, whichallows simultaneous acquisition of the 1H signals and excitation on the carbon channel. With this we canconclude that an instrument with a carbon channel can also be of advantage for Reaction Monitoring
applications, although 13C is not a nucleus that one would typically try to detect in flow applications (thisis because of the longer measurement times required for 13C measurements).

a)zoom x100b)zoom x100

Figure 6: Comparison of the spectra acquired with (a) and without (b) carbon decoupling. By acquiring the signal
in the presence of carbon decoupling the 13C satellites are completely eliminated, removing the overlap with thesignals of the product that need to be integrated.

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