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INT. Ed. 2017, 5G, 9174 –9177 T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim xxx.xxxxxxxxxx.xxx 9175 between the diffusion of analytes in their free and catalyst- bound forms to be small. As previously reported,[22] this is due to the 1-methyl-1,2,3-triazole cosubstrate that dramatically slows down analyte dissociation in NHC-iridium SABRE complexes; dissociation rates lower than 1 s@1 have been determined for complexes analogous to the one used herein. The combination of the slow analyte exchange and a short diffusion delay (50 ms) cause systematic deviations on the determined diffusion coefficients to be below detection.[23] Note that the regular DOSY displays a much smaller linewidth in the indirect (diffusion) dimension due to the higher signal-to-noise ratio achieved at high concentrations. A similar resolution might be achieved by SABRE-DOSY at low concentrations without any penalty in experimental time by simply employing fully enriched pH2 instead of the circa 50 % enrichment used herein, which would provide a nearly three-fold additional signal increase. All analytes in the low-micromolar mixture are resolved in the SABRE-DOSY plot of Figure 2. That enables assess- ment of the number of analytes and correlation of distinct peaks from a given analyte. Two of the signals can be dismissed from as not being from the analyte pool, since they also appear in the SABRE spectrum of the neat catalyst-mtz solution (Supporting Information, Figure S4). Among the analytes, signals of the slowest diffusing member 1 were easily correlated, while the lightest analyte 6 is clearly resolved as the fastest diffusing component. Furthermore, the two pairs of isomeric alkyl pyridines 2–3 and pyrazines 4–5 are clearly resolved. This is an impressive result, considering that diffusion coefficients are expected to correlate to the size of analytes and this set varies by no more than one atomic mass unit and comprises the same basic molecular shape. However, we have recently demonstrated that [D4]MeOH enhances resolution in the diffusion of nitrogenous aromatics, including the differentiation of isomers.[24] Finally,a remaining problem is that one of the peaks from 5 (asterisk in Figure 2) overlaps with a catalyst-mtz signal (Figure S4) and does not properly correlate with other resonances from 5. Resolving such overlap is a well-recognized issue in DOSY that could potentially be solved by more advanced data processing techniques.[25] In summary, we demonstrate a method to greatly lower the concentration floor for DOSY. This was achieved in the face of flow and mixing steps necessitated for SABRE hyperpolarization. We acknowledge that the approach is limited to analytes that can reversibly bind the iridium-based SABRE catalyst, however, key analytical applications within this realm have been demonstrated[9a–b] and are poised to grow. Without SABRE and its corresponding (100 +)-fold sensitivity gains, DOSY analysis of low-micromolar mixtures would not be feasible. Furthermore, additional gains will be straightforward to achieve in this framework by moving to > 90 % pH2. Combining the present approach with recent advances in single-scan DOSY[16] could allow < 1 min mea- surement times, but the spatial encoding employed in such experiments would reduce sensitivity with a corresponding tradeoff on the detection limit of SABRE-DOSY. The resolution and sensitivity of the method herein provide an important new piece in the toolbox for mixture analysis by NMR spectroscopy and take steps forward in the ever- expanding fields of DOSY and nuclear hyperpolarization. Experimental Section Managing convection: Experiments were carried out in a SABRE polarizer (Figure 3). Mixing of the sample while the flow cell is filled helps to eliminate temperature gradients across sample volume if the

INT. Ed. 2017, 5G, 9174 –9177 T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim xxx.xxxxxxxxxx.xxx 9175 between the diffusion of analytes in their free and catalyst- bound forms to be small. As previously reported,[22] this is due to the 1-methyl-1,2,3-triazole cosubstrate that dramatically slows down analyte dissociation in NHC-iridium SABRE complexes; dissociation rates lower than 1 s@1 have been determined for complexes analogous to the one used herein. The combination of the slow analyte exchange and a short diffusion delay (50 ms) cause systematic deviations on the determined diffusion coefficients to be below detection.[23] Note that the regular DOSY displays a much smaller linewidth in the indirect (diffusion) dimension due to the higher signal-to-noise ratio achieved at high concentrations. A similar resolution might be achieved by SABRE-DOSY at low concentrations without any penalty in experimental time by simply employing fully enriched pH2 instead of the circa 50 % enrichment used herein, which would provide a nearly three-fold additional signal increase. All analytes in the low-micromolar mixture are resolved in the SABRE-DOSY plot of Figure 2. That enables assess- ment of the number of analytes and correlation of distinct peaks from a given analyte. Two of the signals can be dismissed from as not being from the analyte pool, since they also appear in the SABRE spectrum of the neat catalyst-mtz solution (Supporting Information, Figure S4). Among the analytes, signals of the slowest diffusing member 1 were easily correlated, while the lightest analyte 6 is clearly resolved as the fastest diffusing component. Furthermore, the two pairs of isomeric alkyl pyridines 2–3 and pyrazines 4–5 are clearly resolved. This is an impressive result, considering that diffusion coefficients are expected to correlate to the size of analytes and this set varies by no more than one atomic mass unit and comprises the same basic molecular shape. However, we have recently demonstrated that [D4]MeOH enhances resolution in the diffusion of nitrogenous aromatics, including the differentiation of isomers.[24] Finally,a remaining problem is that one of the peaks from 5 (asterisk in Figure 2) overlaps with a catalyst-mtz signal (Figure S4) and does not properly correlate with other resonances from 5. Resolving such overlap is a well-recognized issue in DOSY that could potentially be solved by more advanced data processing techniques.[25] In summary, we demonstrate a method to greatly lower the concentration floor for DOSY. This was achieved in the face of flow and mixing steps necessitated for SABRE hyperpolarization. We acknowledge that the approach is limited to analytes that can reversibly bind the iridium-based SABRE catalyst, however, key analytical applications within this realm have been demonstrated[9a–b] and are poised to grow. Without SABRE and its corresponding (100 +)-fold sensitivity gains, DOSY analysis of low-micromolar mixtures would not be feasible. Furthermore, additional gains will be straightforward to achieve in this framework by moving to > 90 % pH2. Combining the present approach with recent advances in single-scan DOSY[16] could allow < 1 min mea- surement times, but the spatial encoding employed in such experiments would reduce sensitivity with a corresponding tradeoff on the detection limit of SABRE-DOSY. The resolution and sensitivity of the method herein provide an important new piece in the toolbox for mixture analysis by NMR spectroscopy and take steps forward in the ever- expanding fields of DOSY and nuclear hyperpolarization. Experimental Section Managing convection: Experiments were carried out in a SABRE polarizer (Figure 3). Mixing of the sample while the flow cell is filled helps to eliminate temperature gradients across sample volume if thethe Figure 3. Schematic of SABRE polarizer. The system provides auto- mated flow control and handles mixing with pH2 in a controlled low- field environment appropriate to SABRE (details in the Supporting Information). sample and flow probe temperatures are matched (Supporting Information, Figure S5). Residual convection was evaluated with the asymmetric double stimulated echo sequence[26] that separates diffusion from convection (Figure S6). Convection dampens to below detection in 5 s, suggesting that DOSY is possible after a 5 s delay. This is in accordance with earlier reports on stop flow experiments in flow probes, in which convection dampened quickly to levels that had little effect on gradient-encoded experiments.[27] Considering that the effective T1 relaxation time[28] of protons in analytes 1–6 is 10–12 s, the relaxational loss of signal during the 5s delay is 30–40 %. SABRE-DOSY experiment: The sample consisted of 6 low- micromolar analytes, 0.5 mm of catalyst, and 15 mm of cosubstrate in