# Hyperpolarised \({}^{1}\)H-\({}^{13}\)C Benchtop NMR Spectroscopy
## 4 Discussion and Conclusions  
In this work we have demonstrated the acquisition of high-resolution 1D and 2D \({}^{13}\)C[\({}^{1}\)H] benchtop NMR spectra of relatively low concentrations of the target analyte at natural abundance in a single scan. The SABRE-enhanced \({}^{13}\)C[\({}^{1}\)H] spectra are easily interpreted at 1 T, including the separation of peaks with chemical shift differences of less than 0.5 ppm. Using SABRE hyperpolarisation with a PTF of 6.1 mT, the optimal SNR was achieved by the PA approach in the fully-coupled spectra but the refocused INEPT approach provided optimal SNR in the decoupled spectra. The superior performance of the INEPT sequence is likely due to the wide range of anti-phase \({}^{13}\)C-\({}^{1}\)H terms that are excited in the PA approach and that are not easily refocused using a single delay. While the INEPT approach  
Figure 5: 2D \({}^{13}\)C–\({}^{1}\)H HETCOR benchtop NMR spectra of 4-methylpyridine (4-MP). **(a)** Thermally polarised spectrum of neat 4-MP (10.3 M) acquired with 64 steps each with 16 scans in a total experiment time of 307 min (5.1 h). **(b)** 1D slices through the 2D spectrum in **(a)** at the chemical shift of the _methyl_ proton resonance (top), _meta_ proton resonance (middle), and _ortho_ proton resonance (bottom). **(c)** SABRE-hyperpolarised 2D spectrum of 260 mM 4-MP with 5.2 mM active SABRE catalyst in methanol-\(d_{4}\) acquired with 90 steps each with a single scan in a total experiment time of 69 min. **(d)** 1D slices through the 2D spectrum in **(c)** as in **(b)**. Note, the differences in chemical shifts between the two spectra are due to the presence of the solvent (methanol-\(d_{4}\)) in the SABRE case. provided the highest SNR for the \({}^{13}\)C(\({}^{1}\)H) spectra, our results highlight that \({}^{1}\)H nuclei directly bonded to \({}^{13}\)C are not hyperpolarised efficiently under these experimental conditions. In future work, adopting the approach of [PERSON] and coworkers [52] to perform SABRE in a much higher PTF within the bore of the benchtop NMR spectrometer could lead to much more efficient \({}^{1}\)H-\({}^{13}\)C hyperpolarisation via a one-bond INEPT transfer. One of the key advantages of the SABRE approach over other hyperpolarisation methods for analytical applications is that it is reversible and so a single sample can be re-polarised multiple times. We have exploited this feature to acquire 2D \({}^{13}\)C-\({}^{1}\)H benchtop NMR spectra, with re-polarisation achieved outside of the spectrometer between each step of the 2D experiment. However, in our flow-based approach to SABRE-enhanced 2D NMR, evaporation of the solvent during sample transfer and \(p\)-H\({}_{2}\) bubbling ultimately limits the maximum number of transients that can be achieved for a single sample. In addition, the transfer of the sample between the spectrometer and the mixing chamber is time-consuming relative to the other steps in the experiment. These limitations could potentially be overcome by using more efficient sampling methods, such as the single-shot 2D methods [67], which have previously been demonstrated using SABRE and high-field detection [68]. Alternatively, hyperpolarisation within the bore of the benchtop spectrometer, as suggested above, would significantly limit the transfer time and distance between the SABRE-polarisation transfer step and the signal detection step. This approach has the potential to make SABRE-enhanced 2D \({}^{13}\)C benchtop NMR viable for more routine applications by significantly decreasing experiment times, increasing the maximum number of transients, and improving sensitivity by increasing the efficiency of the INEPT transfer. SABRE hyperpolarisation was achieved here using a model analyte, 4-methylpyridine. In order for an analyte to be strongly enhanced by SABRE, it needs to reversibly bind to the catalyst on an appropriate timescale. The residence time on the catalyst must be long enough for significant polarisation transfer to occur but not too long such that NMR relaxation dominates.