Low-cost High-Pressure Clinical-Scale 50% Parahydrogen Generator Using Liquid Nitrogen at 77 K

Abstract

We report on a robust and low-cost parahydrogen generator design employing liquid nitrogen as coolant. The core of the generator consists of catalyst-filled spiral copper tubing, which can be pressurized to 35 atm. Parahydrogen fraction >48% was obtained at 77 K with three nearly identical generators using paramagnetic hydrated iron oxide catalyst. Parahydrogen quantification was performed on the fly via bench-top NMR spectroscopy to monitor the signal from residual orthohydrogen—parahydrogen is NMR silent. This real-time quantification approach was also used to evaluate catalyst activation at up to 1.0 standard liter per minute flow rate. The reported inexpensive device can be employed for a wide range of studies employing parahydrogen as a source of nuclear spin hyperpolarization. To this end, we demonstrate the utility of this parahydrogen generator for hyperpolarization of concentrated sodium [1-¹³C]pyruvate, a metabolic contrast agent under investigation in numerous clinical trials. The reported pilot optimization of SABRE-SHEATH hyperpolarization yielded ¹³C signal enhancement of over 14,000-fold at clinical relevant magnetic field of 1 T corresponding to approximately 1.2% ¹³C polarization – if near 100% parahydrogen would have been employed, the reported value would be tripled to ¹³C polarization of 3.5%.

Graphical Abstract

INTRODUCTION

NMR hyperpolarization techniques enhance the detection sensitivity of NMR spectroscopy and imaging by several orders of magnitude.^1–4 These tremendous gains in detection sensitivity enable new applications, including molecular imaging of exogenous contrast agents.^5–7 The nuclear spins of these new contrast agents are hyperpolarized (HP) using a wide range of techniques.^{1, 8–10} Some hyperpolarization techniques have been successfully employed in clinical trials.^11–14 Despite the major successes in clinical research, none of these methods have enjoyed widespread or routine clinical use so far, in part because of high instrumentation cost and low hyperpolarization throughput.¹⁴

Parahydrogen-Induced Polarization (PHIP) is a simple, fast, and low-cost hyperpolarization approach^15–16 that has the potential to revolutionize production of HP contrast agents for clinical use. Canonical PHIP requires pairwise parahydrogen (p-H₂) addition to an unsaturated molecular substrate.^17–18 More recently, the non-hydrogenative variant called Signal Amplification by Reversible Exchange (SABRE) has emerged^19–20: The latter method employs chemical exchange of p-H₂ and to-be-hyperpolarized substrates on metal complexes.^21–22 Both PHIP and SABRE approaches have produced a range of HP contrast agents with some validation success in cellular and pre-clinical models^23–29 as also described in recent reviews.^{10, 30–31}

Parahydrogen, employed as the source of nuclear spin order in PHIP,^{15, 32–33} is produced by transient exposure of normal dihydrogen gas (with its ambient 1:3 para-to-ortho-state distribution) to a low-temperature.^{26, 34–38} Because p-H₂ is a lower energy state, the equilibrium shifts to the para- state at sufficiently low temperatures³⁹; nearly 100% p-H₂ can be obtained at ≤20 K.^{2, 40} When pure p-H₂ is employed for PHIP, near-unity proton polarization can be unlocked after the magnetic equivalence of the nascent p-H₂-derived protons is broken.^{15, 17, 41} Moreover, in both hydrogenative PHIP and its non-hydrogenative variant SABRE, it has been demonstrated that the polarization of nascent p-H₂-devrived protons can be transferred via the network of spin-spin couplings to other spin-1/2 nuclei including ¹³C,^{24, 42–45 15}N,^{46–48 1}H,^{21 31}P,^{49 19}F,^50–51 and others.⁵² Nuclear spin polarization (P) values in excess of 50% have been demonstrated^53–55 when polarization transfer is optimized using pure p-H₂ gas.

Once one has a supply of p-H₂ in hand, the remaining hardware required to accomplish polarization transfer in PHIP and SABRE is relatively straightforward and low-cost (e.g., approximately $10k for a setup employing a mass-flow controller and mu-metal shields for SABRE⁵⁶ or PHIP field cycling studies⁵⁷ at micro-tesla fields), because no cryogenic or high-field hardware is required. However, the ostensible need for pure p-H₂ would require the use of cryogenic equipment in the range of $50,000–125,000 (e.g., Bruker or ARS generators),^{26, 29, 34, 36, 58} representing a substantial investment and a barrier for those working in (or desiring to enter) the field of p-H₂-based hyperpolarization. Moreover, the quantification of the p-H₂ fraction is often required to ensure reproducible results in PHIP and SABRE. In the NMR hyperpolarization community, the measurement is typically performed using high-field NMR spectroscopic quantification of the residual orthohydrogen fraction—because p-H₂ is NMR silent⁵⁹—although other methods have been demonstrated.^60–62 Once created, the p-H₂ gas can then be stored in pressurized aluminum cylinders for weeks.^{34, 36, 63} The requirement of a high-field NMR spectrometer adds additional complexity and cost to the infrastructure for robust and reproducible operation of a p-H₂-based hyperpolarization facility. As an alternative, we have recently demonstrated that the residual orthohydrogen fraction in near-100% p-H₂ gas can be monitored in real time using low-field bench-top NMR spectroscopy.⁶⁴ Bench-top NMR spectrometers have substantially lower cost than high-field NMR devices; they are also portable, have a small footprint, require no cryogens to operate, and are increasingly becoming a standard “workhorse” in routine hyperpolarization studies.^65–67

To mitigate the cost and complexity of cryogenic hardware, p-H₂ production can be conducted at liquid N₂ temperature (ca. 77 K at 1 atm) resulting in approximately 50% p-H₂ fraction.³⁸ Moreover, liquid He can also be employed as a chilling source resulting in 97.5% p-H₂ fraction.⁶³ The key disadvantage of using 50% (versus near 100%) p-H₂ is the reduction of the resulting hyperpolarization effect by a factor of ~3. Such substantial polarization decrease can be unforgiving for many signal-to-noise ratio (SNR)-challenged applications, e.g., most notably in vivo studies.^{27, 68} However, many other applications—including the development phase of PHIP and SABRE-based contrast agents—can be accomplished with this ‘lower’ p-H₂ grade, which is much easier and cheaper to achieve in practice.^69–71

Several parahydrogen converter/generator designs employing a wide range of ortho-to-para conversion catalysts have been reported for operation at liquid N₂ temperature.^{38, 62, 71–73} Moreover, very recently liquid-He-based system has been employed in the production of nearly pure p-H₂ using an inexpensive design ($1,200 in parts),⁶³ although the design relies on liquid He (which may impose a substantial additional running cost and infrastructure), requires a ~90-min. cool-down time, and has limited production capacity at maximum specs (200 standard cubic centimeters per minute, sccm).

Here, we report a robust and inexpensive design of a p-H₂ generator for operation with liquid N₂ at a tested pressure of up to 35 atm. The reported design is based on more than ten years of experience in our laboratories. The produced compressed H₂ gas is quantified by ‘real-time’ NMR spectroscopy of exiting p-H₂ using a bench-top 1.4 T NMR spectrometer. The design reproducibility has been evaluated with 3 separately constructed devices. Moreover, we have also investigated ortho-para catalyst activation by catalyst exposure to >100 °C to achieve a production rate of 1,000 sccm with ~49% p-H₂ fraction. The utility of the reported device has been tested in the feasibility demonstration of [1-¹³C]pyruvate hyperpolarization via SABRE, following the work of Duckett and co-workers.⁷⁴ HP [1-¹³C]pyruvate is a leading HP contrast agent employed for tracking of metabolism in vivo^{7, 11–12, 14} and is currently being evaluated in many clinical trials and preclinical models of numerous human diseases.^{13–14, 75} Taken together, the reported design augmented by real-time p-H₂ quantification using benchtop NMR spectroscopy will hopefully be of interest not only to those already working in the field of NMR hyperpolarization in general (and p-H₂-based hyperpolarization in particular), but also to those seeking a low-barrier entryway into NMR hyperpolarization techniques.

MATERIALS AND METHODS

Generator design.

The core component employs copper tubing (0.25 in. outer diameter, OD; 0.03 in. wall; 0.19 in. inner diameter, ID, McMasterCarr, P/N 5174K21; ~115 cm length) that was filled with ~21 g of hydrated iron(III) oxide (Fe₂O₃·H₂O, 371254, Sigma-Aldrich, St. Louis, MO)—this material is produced as Ionex Type OP Catalyst (https://www.molecularproducts.com/products/ionex-type-op-catalyst Molecular Products, Louisville, Colorado, USA). Prior to loading, the catalyst material was purged of microparticles by mechanical filtration via ABN strainer cone funnels with disposable 190-micron mesh https://www.amazon.com/gp/product/B01H7PEHEK/. Each funnel was filled to ~1/5 of its capacity and the catalyst was washed with ethanol or isopropanol until washing liquid passing through it became practically colorless. The alcohol-washed catalyst was further washed by hexane until the washing liquid became colorless as well. The washed catalyst was placed in a glass beaker and dried overnight in the oven at ~60 ℃. If not removed, microparticles can degrade p-H₂ generator performance if they escape downstream of the cryogenic region. The catalyst-filled copper tube was wound into a spiral with ~2.36 in. (6 cm) OD consisting of approximately 6 turns [~2.75 in. (7 cm) height], Figure 1. The ends of the copper tubing were filled with glass wool to ensure the catalyst stays in the 0.25 in. copper tubing segment. Next, each end of the catalyst-filled 0.25 in. spiral tubing segment was adapted to a heat-exchange 0.125 in. OD copper tubing spiral (0.03 in. wall; 0.065 in. OD, McMasterCarr, P/N 5174K1;) using brass Yor-lok reducers (McMasterCarr, P/N 5272K214). The two hollow spirals (~20 turns of similar diameter) made of 0.125 in. copper tubing are designed to serve two purposes. The 0.125 in. copper tubing spiral is reinforced by aluminum brackets (Figure 1) to enhance structural rigidity. In case of the liquid N₂ level is above these heat exchangers, the inlet heat exchanger allows for pre-cooling of the incoming H₂ gas. Alternatively, if the liquid N₂ level is below the heat exchangers, heat exchange between incoming and exiting hydrogen gas flows allows pre-cooling of the incoming H₂ gas while warming the exiting para-enriched H₂, Figure 1.

Figure 1.

Photographs of the p-H₂ generator device core, outlining the orientations and interfaces of key components.

Experimental setup for ‘real-time’ bench-top NMR spectroscopy of hydrogen gas.

To monitor the p-H₂ enrichment on the fly, we have employed the setup described previously,⁶⁴ which was adapted for operation with the present generator, Figure 2. A high-pressure tank equipped with a dual-stage pressure regulator and containing ultra-high purity (>99.999%) hydrogen was connected to the input port of the generator using a Yor-lok brass coupling. The other end of the generator was connected directly to the input of a mass-flow controller (MFC; Sierra Instruments Inc., Monterey, California, USA, P/N C100L-DD-1-OV1-SV1-PV2-V1-S0, 1000 sccm model). The hydrogen tank pressure was set to ~125 PSI. The flexible 0.125 in. copper lines allow for easy maneuvering of the generator core to insert into/remove from the liquid N₂ bath (in a Styrofoam container) or exposing the catalyst-filled section to a heat gun for catalyst activation studies (see below). Parahydrogen quantification was performed using a 1.4 T NMR spectrometer operating at 61 MHz proton resonance frequency with gas samples at 8 atm (100 PSI overpressure) employing the following acquisition parameters: 1024 scans, 5 kHz spectral width, 52 ms acquisition time, 0.1 s repetition time, ~102 s total acquisition time, 90° excitation pulse of ~10 μs duration.

Figure 2.

Experimental setup schematic employed for p-H₂ quantification studies using real-time bench-top 1.4 T NMR spectroscopy. The safety valve allows for 100 PSI overpressure, and the normal hydrogen (n-H₂) pressure of the main hydrogen tank was set to 125 PSI. Switching the valve to the “OFF” position directs hydrogen gas to an NMR tube via a 0.065 in. OD Teflon catheter.

Parahydrogen quantification

Parahydrogen quantification was performed using the previously described method,⁶⁴ which was adapted for operation with the described generator. Briefly, on the day of the operation, the setup (Figure 2) was first operated at room temperature, i.e., without a liquid N₂ bath. The MFC flow rate was set to 150 sccm, and the safety valve was set to 100 PSI overpressure (as confirmed by the pressure gauge, Figure 2). The valve was placed in the “OFF” position, and normal hydrogen was allowed to pass through the catheter and run through a standard 5 mm NMR tube equipped with a “Y” connector for 10 minutes. This “purge” stage was required to remove any residual air and moisture from the setup, and to fill the NMR tube to 100 PSI overpressure with normal H₂ gas (containing 75% o-H₂).

Next, the valve is switched to the “ON” position and the gas flow is directed via bypass rather than through the NMR tube. As a result, normal hydrogen (25% para-H₂ and 75% ortho-H₂) in the tube was not flowing during NMR acquisition (instead, the flow was directed via bypass). Next, an NMR spectrum of normal H₂ gas was acquired using the acquisition parameters listed above. The signal (integrated area under the curve, AUC) was computed using SpinSolveExpert software supplied by the vendor (Magritek, New Zealand). The corresponding signal from an empty NMR tube was also acquired and subtracted from each NMR measurement to account for any background signal using the same spectral processing parameters.

The generator’s catalyst-filled spiral was then submerged into a liquid N₂ bath and allowed to equilibrate at cryogenic temperature for 10 min. with a continuous H₂ flow at 150 sccm. The valve was switched to the “OFF” position to direct the gas flow through the NMR tube for ~2 min. Next, the valve is switched “ON”. As a result, the para-enriched hydrogen in the tube was not flowing during NMR acquisition (instead, the flow was directed via bypass). Next, an NMR spectrum of para-enriched H₂ gas was acquired using the acquisition parameters listed in the Figure 3 caption. The NMR signal was processed in the same fashion as for normal H₂ as described above. All measurements for p-H₂-enriched and normal H₂ gas were repeated three times and averaged. The p-H₂ fraction (f) was computed using Eq. 1:

$$f=1-\frac{3*\mathit{\text{Senriched}}}{4*\mathit{\text{Snormal}}},$$ (1)

where S_enriched and S_normal are the corresponding NMR signals for p-H₂ enriched and normal (i.e., non-enriched) hydrogen gas samples. Note the multipliers 3 and 4 are used to reflect the 75% o-H₂ in normal (unenriched) H₂ gas.³⁰ Three p-H₂ generators were tested for test-retest reproducibility.

Figure 3.

a) Parahydrogen quantification using a 1.4 T NMR spectrometer operating at 61 MHz proton resonance frequency using gas samples at 8 atm (100 PSI overpressure). Acquisition parameters: 1024 scans, 5 kHz spectral width, 52 ms acquisition time, 0.1 s repetition time, ~102 s total acquisition time, 90° excitation pulse (~10 μs long). b) Dependence of p-H₂ fraction on the flow rate for activated and non-activated catalyst.

Catalyst activation.

Catalyst activation was performed by heating the catalyst-containing spiral using a heat gun to >100 °C for ~15 minutes under continuous 150 sccm flow of H₂ gas.

¹³C SABRE hyperpolarization of [1-¹³C]pyruvate

¹³C SABRE hyperpolarization of [1-¹³C]pyruvate was performed using SABRE in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH)^47–48 tailored for the ¹³C nucleus^{45, 76} using the DMSO-co-ligand approach developed by Duckett and co-workers.⁷⁴ Sodium [1-¹³C]-pyruvate and deuterated methanol-d₄ solvent were purchased from Sigma-Aldrich and used without any further purification. The [IrCl(COD)(IMes)] SABRE catalyst precursor was synthesized according to a literature procedure.²¹ The sample was prepared with a fixed ratio of substrate to Ir-IMes SABRE pre-catalyst and DMSO in 0.6 mL of methanol-d₄ in a 5 mm NMR tube with a typical ratio of substrate, catalyst (12 mM) and DMSO as 7:1:10. Ultra-high-purity H₂ gas (Airgas) was fed into a p-H₂ generator and enriched to about 50% para- fraction using liquid N₂ as described above. The p-H₂ flow is directed via PTFE tubing using MFC (Sierra Instruments SmartTrak 100 series) set at 80 sccm flow rate and directed to a conventional 5 mm NMR tube (Norell) to allow p-H₂ bubbling through the sample. The entire p-H₂ line was pressurized to 40 PSI overpressure. SABRE-SHEATH requires the use of micro- o sub-microtesla magnetic fields to enable efficient polarization transfer from p-H₂-derived hydrides to heteronucleus (e.g., ¹³C targeted here). In practice, these fields are achieved by attenuating the Earth’s magnetic field, and creating a minute magnetic field inside the shield using electromagnet. Here, magnetic fields near or below ~1 μT were achieved with a home-built apparatus consisting of a solenoid coil placed inside a mu-metal shield (Magnetic Shield Corporation, model No. ZG-206). This solenoid is 41 mm in diameter: 40 mm core, 20 cm long windings with 220 turns AWG20 (0.9 mm) Cu wire and with 220 Ω resistor in series. The solenoid coil was driven by commercial 1.5 V batteries with a variable-resistance decade box in series to provide finer control of the internal magnetic field of the shield, which is monitored using a Lakeshore Cryotronics Gaussmeter (Model No. 475 DSP with HMMA-2512-VR Hall Probe). NMR experiments were performed using a 1 T Magritek Spinsolve benchtop NMR spectrometer. All ¹³C NMR spectra were taken without ¹H decoupling throughout the duration of the experiment. The time required to manually transfer the sample from the shield region to the magnet for low-field NMR acquisition was usually < 5 s. The ¹³C signal enhancement was computed by comparing HP signal AUC to external ¹³C signal thermal signal reference (4M sodium [1-¹³C]acetate) using Eq. 2:

$$\epsilon \left({}^{13}C\right)=\frac{S_{HP}}{S_{REF}}\cdot \frac{C_{REF}}{C_{HP}}\cdot \frac{A_{REF}}{S_{HP}},$$ (2)

where S_HP and S_REF are ¹³C signals from HP [1-¹³C]pyruvate and thermal signal reference [1-¹³C]acetate, C_REF and C_HP are concentrations of thermal signal reference [1-¹³C]acetate (4 M) and of HP [1-¹³C]pyruvate, respectively, and A_REF and A_HP are effective cross-sections of the NMR tubes for the thermal signal reference [1-¹³C]acetate and HP [1-¹³C]pyruvate samples.

RESULTS AND DISCUSSION

Parahydrogen enrichment.

Three identical copies of the generator were employed for quality assurance prior to catalyst activation. Under conditions of liquid N₂ and 150 sccm p-H₂ flow rate, the bench-top NMR quantification yielded the following p-H₂ enrichment fractions: 48.4±0.5%, 48.1±0.5%, and 48.2±0.5% respectively, demonstrating the robustness of the design in the context of reproducible generator construction. A representative NMR quantification of p-H₂ fraction at 150 sccm flow rate is shown in Figure 3a. The remaining p-H₂ quantification studies were performed with one of the three devices. The flow rate was then varied from 150 sccm to 1000 sccm (Figure 3b, blue bars) clearly demonstrating the reduction of p-H₂ fraction with increased flow rate. This finding is rationalized as follows: the non-activated catalyst has some potency for ortho↔para conversion, which is sufficient for slow-flowing H₂ gas. When the flow rate is fast (i.e., 1000 sccm), the slow ortho↔para conversion rate is no longer sufficient to allow the system to reach an equilibrium conversion while the gas moves along the catalyst-filled copper spiral, thus yielding a lower than expected p-H₂ fraction.

Catalyst activation by heating under H₂ atmosphere.

After catalyst activation in the copper spiral as described above, the performance of the same generator was evaluated at various flow rates (Figure 3b, red bars). The results clearly indicate that catalyst activation is indeed important in order to maximize the ortho↔para conversion, allowing the system to achieve full conversion at high flow rates up to 1,000 sccm. Although higher hydrogen flow rates were not tested due to limitations of the mass flow controller, we expect the generator to perform well at substantially higher flow rates of at least 4000 sccm. Our expectation is based on the performance of a recently published cryogenic design, which employs catalyst-filled copper tubing filled with half the quantity of the catalyst (10 g vs. 21 g employed here) in smaller ID/OD copper tubing.⁶⁴ This recently published design performed well at flow rates of up to 4000 sccm.⁶⁴

The utility of the parahydrogen generator for ¹³C SABRE-SHEATH hyperpolarization.

Hyperpolarization of [1-¹³C]pyruvate was evaluated using another copy of the generator at a different site. It was employed for SABRE hyperpolarization studies of [1-¹³C]pyruvate using SABRE-SHEATH. The simultaneous exchange of p-H₂ and [1-¹³C]pyruvate on activated Ir-IMes catalyst leads to buildup of ¹³C hyperpolarization, Figure 4a. Figure 4b shows a representative spectrum of ¹³C-hyperpolarized [1-¹³C]-pyruvate with signal enhancement ε of over 14,000-fold, corresponding to P_13C of 1.2% obtained via comparison of the NMR signal intensity with a reference sample, Figure 4c.

Figure 4.

a) Schematic of the catalytic system for SABRE-SHEATH hyperpolarization. Activated Ir complex catalyst, [Ir(H₂)(η²-pyruvate)(DMSO)(IMes)], transfers magnetization from p-H₂ to [1-¹³C]pyruvate through a J-coupled spin network. Both p-H₂ and pyruvate have weak, transient binding to the iridium complex. b) Single-scan HP ¹³C spectrum selected from SABRE-SHEATH experiments; enhancement ɛ~14,000. Sample: 30 mM sodium [1-¹³C]pyruvate, 20 mM DMSO, 7.8 mM Ir-IMes catalyst in methanol-d₄; spectrum acquired immediately following manual sample transfer to 1 T after 55 s p-H₂ bubbling at B_T=−0.7 μT. The inset of (b) shows a close-up ¹³C spectrum. c) Single-scan thermally polarized ¹³C signal from 4 M sodium [1-¹³C]acetate using similar acquisition parameters.

If near-100% p-H₂ would have been employed, P_13C would be tripled to P_13C = 3.5%.³⁰ We note that P_13C strongly depends on the experimental conditions. To the best of our knowledge, the extrapolated P_13C value reported here exceeds the highest reported value (P_13C of ~1%)^{74, 77} by more than threefold representing a substantial advancement for HP [1-¹³C]pyruvate production via SABRE-SHEATH technique.

The pilot optimization of ¹³C SABRE-SHEATH conditions reveal ¹³C signal dependence on the microtesla magnetic field (Figure 5a), temperature (Figure 5b), polarization buildup time (i.e., the duration of p-H₂ bubbling, Figure 5c) and catalyst concentration (Figure 5e). The ¹³C T₁ in-shield relaxation value of 31±4 seconds at [catalyst]=7.8 mM are substantially longer than ¹⁵N T₁ of ca. 12–15 s of [¹⁵N₃]metronidazole at [catalyst]~2 mM⁷⁸ despite that fact that ¹³C gyromagnetic ratio is 2.5 times greater than ¹⁵N one and therefore ¹³C spin would be more prone to the catalyst-induced relaxation. We rationalize this observation by greater distance of ¹³C1 nucleus from Ir due to the presences of bridging oxygen (i.e., Ir-O=¹³C) versus direct Ir interaction with ¹⁵N nucleus (i.e., Ir-¹⁵N). This observation is important because longer in-shield ¹³C T₁ at microtesla magnetic field effectively results in greater P_13C.⁷⁸

Figure 5.

Pilot optimization of SABRE-SHEATH hyperpolarization of [1-¹³C]pyruvate: a) magnetic field sweep of a sample of [Ir(COD)(IMes)] (13 mM) with sodium [1-¹³C]pyruvate (90 mM) and DMSO (120 mM) in 0.6 mL methanol-d₄ at room temperature; b) temperature sweep of a sample of [Ir(COD)(IMes)] (7.8 mM) sodium [1-¹³C]pyruvate (30 mM) and DMSO (20 mM) in 0.6 mL methanol-d₄ at B_T=−0.7 μT; c) p-H₂ bubbling duration sweep using a sample of [Ir(COD)(IMes)] (7.8 mM) with sodium [1-¹³C]pyruvate (30 mM) and DMSO (20 mM) in 0.6 mL methanol-d₄ at B_T=−0.7 μT; d) In-shield ¹³C T₁ signal decay using a sample of [Ir(COD)(IMes)] (7.8 mM) with sodium [1-¹³C]pyruvate (30 mM) and DMSO (20 mM) in 0.6 mL methanol-d₄ at B_T=−0.7 μT; e) SABRE catalyst concentration sweep using samples of 30 mM of sodium [1-¹³C]pyruvate and 20 mM DMSO in 0.6 mL methanol-d₄ at B_T=−0.7 μT. All experiments were performed using with 100 PSI p-H₂ (~50% para-) overpressure at ~100 sccm flow rate.

We envision that additional future additional improvements for ¹³C pyruvate polarization can be made through increase of p-H₂ pressure and flow rate⁷⁹ and the use of recently reported hardware for more precise calibration of in-shield nanotelsa magnetic field.⁸⁰

The reported results clearly demonstrate the utility of our generator to produce a HP state that can be easily detectable, even when using a bench-top NMR spectrometer operating at 1 T. We note that although ¹³C1-labeled pyruvate was employed, the resonance at 205 ppm corresponds to natural ¹³C abundance signal from ¹³C2 locked in a singlet state with ¹³C1.⁷⁷ Thus, we anticipate that our generator can enable a wide range of p-H₂ based hyperpolarization studies in the context of development, optimization and quality assurance of HP ¹³C compounds and biocompatible contrast agents even at natural abundance ¹³C level. We also anticipate that other nuclei (¹⁵N, ¹⁹F, ¹H, etc.) can also be readily studied using our low-cost and easy-to-maintain p-H₂ generator in combination with a bench-top NMR spectrometer. Such combination should provide a straightforward gateway to HP studies with p-H₂ for a wide range of laboratories.

CONCLUSIONS

In summary, we report a robust design of a p-H₂ generator developed for operation at liquid N₂ temperature based on many years of experience in our laboratories. We employed near real-time bench-top NMR spectroscopy for quantification of p-H₂ fraction, indicating p-H₂ enrichment of ~48% (3 separately constructed devices) at flow rates of up to 1000 sccm; moreover, it is expected that flow rates of up to 4000 sccm should be attainable without performance loss. Catalyst activation by heat under H₂ atmosphere was shown to be important for efficient operation at high flow rates. The utility of the generator has been investigated for SABRE-SHEATH ¹³C-hyperpolarization of [1-¹³C]pyruvate, the leading metabolic ¹³C contrast agent under investigation in clinical trials. Despite low p-H₂ fraction resulting in ~3-fold signal reduction (vs. near-100% p-H₂), it was possible to successfully hyperpolarize [1-¹³C]pyruvate for detection using a 1 T bench-top NMR spectrometer (ε~14,000, P_13C~1.2%). We anticipate that the reported generator design will be useful for those working on development of p-H₂-based hyperpolarization technologies (e.g., PHIP and SABRE), and particularly those working on developing new biocompatible compounds that can be employed as exogenous HP contrast agents. Taken together, the combination of the described p-H₂ generator and a bench-top NMR spectrometer embodies a low-cost and robust gateway to the field to p-H₂ hyperpolarization without substantial investment in complex infrastructure. Although on-demand p-H₂ production for utility in SABRE hyperpolarization was demonstrated here, the produced p-H₂ gas can also be stored in an aluminum tank for weeks, because p-H₂ back conversion to normal hydrogen is slow.^{36, 64}