# Hyperpolarised \({}^{1}\)H-\({}^{13}\)C Benchtop NMR Spectroscopy
## 3 Results  
### Optimal Detection of SABRE-Enhanced \({}^{13}\)C Benchtop NMR Spectra  
To explore the optimal detection approach for SABRE-hyperpolarised benchtop \({}^{13}\)C NMR spectroscopy, we compared two methods for hyperpolarised signal acquisition. In the first pulse-and-acquire (PA) approach, the \({}^{13}\)C NMR signal was detected directly following a single 90\({}^{\circ}\) excitation pulse (Figure 2a). SABRE-enhanced benchtop \({}^{13}\)C NMR spectra acquired using PA have been reported previously [49]. Here we compared this approach with a second method where the \({}^{13}\)C NMR signal was detected indirectly following a \(J\)-based INEPT transfer of hyperpolarisation from \({}^{1}\)H to \({}^{13}\)C (Figure 2b). A comparison of the SABRE-hyperpolarised \({}^{13}\)C benchtop NMR spectra of 4-methylpyridine (4-MP) using these two approaches is presented in Figure 3a. In both cases, SABRE hyperpolarisation was achieved using the manual sample shaking method. Figure 2: NMR pulse sequences where \(\tau_{1}=1/2J_{CH}\) and \(\tau_{2}=1/3J_{CH}\). (**a**) Pulse and acquire (PA) for direct \({}^{13}\)C detection, (**b**) INEPT for transfer from \({}^{1}\)H to \({}^{13}\)C, (**c**) PA with refocusing delay and \({}^{1}\)H decoupling, (**d**) INEPT with refocusing delay and \({}^{1}\)H decoupling, (**e**) single-shot variable flip angle hyperpolarisation lifetime measurement (see Table S1 for values of \(\theta_{n}\)), and (**f**) 2D HETCOR, where \(t_{1}\) is incremented to encode \({}^{1}\)H chemical shift into the indirect dimension. For hyperpolarisation experiments, the pulse sequences are applied immediately following the SABRE-hyperpolarisation step and sample transfer into the benchtop NMR spectrometer. Inspection of the fully-coupled, SABRE-enhanced \({}^{13}\)C NMR spectra in Figure 3a reveals that a higher overall \({}^{13}\)C NMR signal enhancement was observed for the PA detection scheme (Figure 3a, top) when compared to the INEPT approach where polarisation was transferred from \({}^{1}\)H to \({}^{13}\)C (Figure 3b bottom). This suggests that the efficiency of the spontaneous indirect transfer from \({}^{1}\)H to \({}^{13}\)C during SABRE in the PTF is higher than the efficiency of the RF-driven transfer achieved by the INEPT sequence following transfer of the sample to the NMR detector. One potential explanation for the higher \({}^{13}\)C signal observed in the PA experiment is relaxation. If the lifetime of the \({}^{1}\)H polarisation is shorter than for the \({}^{13}\)C hyperpolarisation, a higher proportion of available signal will decay during sample transport in the INEPT case when compared to the PA case. Figure 4 presents a comparison of the \({}^{13}\)C and \({}^{1}\)H hyperpolarisation lifetimes as a function of concentration of 4-methylpyridine, where the concentration of the catalyst is 5.2 mM in all cases. These single-shot lifetime measurements were acquired on the benchtop NMR spectrometer using the variable flip angle sequence in Figure 2e immediately following hyperpolarisation using the manual shaking SABRE procedure. Contrary to the hypothesis, we found that the lifetimes for the \({}^{1}\)H hyperpolarisation are longer than for the directly detected \({}^{13}\)C polarisation. Therefore, these results do not support the proposition that relaxation effects lead to higher observed \({}^{13}\)C signals in the PA case. However, it should be noted that these experiments do not distinguish between \({}^{1}\)H hyperpolarisation in molecules with and without \({}^{13}\)C.  
Figure 3: 1D SABRE-hyperpolarised \({}^{13}\)C NMR spectra of 260 mM 4-methylpyridine at natural abundance in MeOD with 5.2 mM active SABRE catalyst. Each spectrum was acquired in a single scan on a benchtop (43 MHz) NMR spectrometer following SABRE hyperpolarisation in a PTF of 6.1 mT. (**a**) Fully-coupled \({}^{13}\)C NMR spectra acquired immediately following a 90\({}^{\circ}\)\({}^{13}\)C pulse (top) and following INEPT transfer from \({}^{1}\)H with \(J_{CH}\) = 10 Hz.