Open-source, 3D-printed, high-pressure (50 bar) liquid-nitrogen- cooled para-hydrogen generator

The signal of magnetic resonance imaging (MRI) can be enhanced by several orders of magnitude using hyperpolarization. In comparison to a broadly used Dynamic Nuclear Polarization (DNP) technique that is already used in 10 clinical trials, the para-hydrogen (pH2) based hyperpolarization approaches are less cost-intensive, scalable and offer high throughput. However, a pH2 generator is necessary. Available commercial pH2 generators are relatively expensive (10,000 – 150,000 €). To facilitate the spread of pH2 hyperpolarization studies, here, we provide the blueprints and 3D-models as open-source for a low-cost (<3,000 €) 50 bar liquid nitrogen pH2 generator.


Introduction 15
Nuclear Magnetic Resonance (NMR), as well as Magnetic Resonance Imaging (MRI), are widely used in medical imaging and chemical analysis. Despite the great success of these techniques (Feyter et al., 2018;Lange et al., 2008;Watson et al., 2020), the low signal-to-noise ratio of NMR limits promising applications such as in vivo spectroscopy or imaging nuclei other than 1 H (Wilferth et al., 2020;Xu et al., 2008). The hyperpolarization of nuclear spins boosts the signal of selected molecules by orders of magnitude. This way, imaging of the lung or metabolism has become feasible (Beek et al., 2004;Kurhanewicz et 20 al., 2011).
The production of para-hydrogen (pH2) is relatively easy: H2 gas is flowing through a catalyst at cold temperatures; maximum 25 para-enrichment of almost 100 % is achieved at about 25 K (Gamliel et al., 2010;Jeong et al., 2018;Kiryutin et al., 2017). To reach low temperatures, hence enrich pH2, liquid cryogens Jeong et al., 2018) or electric cryopumps (Feng et al., 2012) are used. Electronic setups were reported, e.g. for pressures up to 50 bar of ≈ 100 % pH2 (Hövener et al., https://doi.org/10.5194/mr-2020-27 Discussions Open Access Preprint. Discussion started: 5 November 2020 c Author(s) 2020. CC BY 4.0 License. 2013). Liquid nitrogen (lN2)-based systems were described, however, often with limited description, low production rate and pressure. 30 Thus, in this contribution, we report a para-hydrogen generator (PHG) based on lN2 that operates at a pressure of up to 50 bar at a cost of less than 3000 €. The setup is easy to replicate as it is fully open-sourced (Ellermann, 2020b) and all parts are either off-the-shelf, 3D-printed or can be constructed easily. Besides, we introduce an automated pH2 quantification method using a 1 T benchtop NMR and Arduino-based process control.
Background In 1933 Werner Heisenberg received his Nobel Prize "for the creation of quantum mechanics, the application of 35 which has, inter alia, led to the discovery of the allotropic forms of hydrogen" (NobelPrize.org, 2020). Allotropy is a property of substances to exist in several forms, in the same physical state. Two forms of hydrogen usually are referred to as spin isomers; they are para-hydrogen (pH2) and ortho-hydrogen (oH2). Hydrogen is not the only compound that has stable or longlived spin isomers at room temperature (rt) there are many examples: deuterium (Knopp et al., 2003), water (Mammoli et al., 2015;Meier et al., 2015), ethylene (Zhivonitko et al., 2013), and even naphthalene derivative (Stevanato et al., 2015). 40 The spin of hydrogen nuclei (proton) is the origin of the two allotropic forms or two spin isomers of hydrogen. Protons have spin-½, hence they are fermions. Fermions are particles that follow Fermi-Dirac statistics, therefore the sign of the total wave function of H2 has to change when two nuclei are exchanged. The spin space of two spin-½ consists of (2 • 1 2 + 1) 2 = 4 states.
They are three symmetric spin states: | + ⟩ = | ⟩, | 0 ⟩ = (| ⟩ + | ⟩)/√2, | − ⟩ = | ⟩ and one asymmetric nuclear spin state | ⟩ = (| ⟩ − | ⟩)/√2 (Fig. 1). Here conventionally | ⟩ and | ⟩ states are nuclei spin states with the projection of spin 45 on 0Z axis ½ and -½, | + ⟩, | 0 ⟩ and | − ⟩ are triplet spin states of two spin-½ with a total spin 1 and the projection on 0Z axis +1, 0 and -1, and | ⟩ is a singlet spin state of two spin ½ with the total spin 0. The angular distribution of the two lowest rotational states ( 00 corresponds to = 0 and 1+1 ± 1−1 and 10 corresponds to = 1) and spin states of ortho-and para-hydrogen are indicated. The numbers in 50 parenthesizes are the degeneracies of the state 2 + 1. The energy of rotation spin states in units of K is equal to = ( + 1) R with R = https://doi. org/10.5194/mr-2020-27 Discussions Open Access Preprint. Discussion started: 5 November 2020 c Author(s) 2020. CC BY 4.0 License. 87.6 K (Atkins and De Paula, 2006). The distance between two adjacent energy levels is +1 − = 2( + 1) R . The figure was inspired by an illustration of I. F. Silvera (1980). The rotational wave function after nuclei permutation does not change, because of the molecular symmetry, and is only 55 multiplied by (−1) , with being the rotational quantum number of the state. Hence, H2 with a symmetric nuclear spin state (triplet states) can have only an asymmetric rotational state ( is odd); such H2 is called oH2. And vice versa, H2 with an asymmetric nuclear spin state (singlet state) can have only symmetric rotation states ( is even); such H2 is called pH2.
The difference in the energy levels of two ground states of ortho ( = 1) and para ( = 0) hydrogen is =1 − =0 = 2 R ≅ 175 K ( Fig. 1) (Atkins and De Paula, 2006). Such a big energy gap allows a relatively simple way of spin-isomer enrichment: 60 for H2 the ground state is pH2 and its population can be increased by cooling down the gas (Fig. 2) (M. Richardson et al., 2018). The ratio of the number of molecules of pH2, H 2 , to oH2, H 2 , in thermal equilibrium is given by Boltzmann distribution of rotational energy levels:  (Haynes, 2011). All these PHGs were specifically designed with PHIP (Para-Hydrogen Induced Polarization) in mind; meaning for a relatively low scale of production and in-lab use (not for the industry). These setups required some on-site assembling and were realized in different 80 designs, e.g. with pulsed injection (Feng et al., 2012) or continuous flow (Hövener et al., 2013). The continuous flow setup was reported to operate at a conversion temperature of 25 K, 4 SLM flow rate, 50 bar maximum delivery pressure, and experimentally obtained H 2 ≅ 98 ± 2 % (Hövener et al., 2013).
These setups work reliably and don't require much in terms of service (e.g. no liquid cryogens). Disadvantages, however, include high initial investments (40.000 -150.000 €), some maintenance of the He-compressor and cryostat (≈ 10.000 € every 85 25.000 h operational time), some site requirements (~4 kW cooling water, appropriate safety precautions) and operational cost in form of electricity (>4 kW electrical power) ( Table 1).
A 100 % pH2 enrichment, however, may not always be needed. 50 % pH2 fraction provides already 1/3 of the maximum polarization at 1/10 of the costs (or less) (M. Richardson et al., 2018). To achieve 2 of 50 % the temperature of lN2, 77 K, is sufficient. Indeed, much of the initial studies were performed with lN2-based PHGs -and still are (Kiryutin et al., 2017;90 Meier et al., 2019). The design of such PHGs is generally simple -a catalyst chamber or tube immersed in lN2. But just like cryostat-based PHGs, lN2-based PHGs are continuously improving. As such, recent advances included remarkable work, where 20 l lN2 were sufficient to provide pH2 continuously for 20 days (Jeong et al., 2018).

3D design of PHG 105
The principal scheme of lN2 base of a complete PHG consists of H2 gas supply, generator and pH2 storage ( Aldrich, St. Louis, U.S.A.) was filled into the coil. In both ends of the coil, the cotton wool was pressed to keep the catalyst in place and protect the rest of the system from contaminations. All fittings, T-pieces, ball-valves, overpressure-valve, flow regulators, the pressure gauge, and fast connectors (Swagelok, Solon, U.S.A.) were connected with the same copper tube. For 115 storage of pH2, a 1 L cylinder made from aluminium was used (C1, A6341Q, Luxfer, Nottingham, UK). All parts were chosen to be rated for 100 bar or more to allow for a 100 % safety margin. A list of all parts is given in Appendix A. The models of PHG, 3D-printing parts and experimental macros (experimental protocols) are available (Ellermann, 2020b). The para-hydrogen-enriched gas exits ortho-para-conversion unit, warms up and passes another particle filter. The filters reduce the contamination of the setup with catalyst. A ball-valve (A4) is used to start or stop gas flow. Two needle valves (A5) are used to control the flow rate. A 3-way-valve (A6) allows to fill or drain the storage cylinder. A 100 bar safety valve (A1) is connected to the system to relief 125 potential excess of pressure.

Safety concept
A crucial part of a PHG is the development of a safety concept which includes a detailed risk assessment and comprehensive operating manual. The handling of pressurized H2 gas entails the risk of pressured gases, forming a potentially explosive mixture with air as well as hydrogen embrittlement (Beeson and Woods, 2003;National Aeronautics and Space 130 Administration, 1997). To reduce these risks, the following safety requirements were set:

Flow quantification
We refrained from including a flow meter in the setup to keep it simple and robust. Instead, we used the time t p,V needed to fill a cylinder of a given volume V0 to a given pressure pout to measure the average flow rate fr of pH2 production. To obtain 180 Standard Liters per Minute (SLM) we used the following equation: where rt is the temperature of the quantification experiment (here: 22 °C) and "norm" stands for standard pressure and temperature values (pnorm = 10 5 pascals ≜ 1.0 bar, Tnorm = 273.15 K) (Nič et al., 2009). The measurement r is performed in a regime where Pout in still linear as a function of time ( , ), hence it coincides with an initial flow rate that is usually reported. 185

Gas system
A medium-pressure 5 mm NMR tube (522-QPV-8, Wilmad-LabGlass, Vineland, U.S.A.) was used for pH2 quantification and heavy wall 5 mm NMR tube (Wilmad-LabGlass, 522-PV-9) for experiments with Magnetic Field Cycling (MFC). Each of these NMR tubes was equipped with input and output gas lines (1/16" PolyTetraFluoroEthylene capillary (PTFE) with 0.023" inner diameter) by glueing to the cap. The other end of these tubes was connected to a custom made valve system. The 190 pressure in the system was set by changing the reducers of respective gases and back pressure valve in the gas system (P-785, P-787, Postnova). Inlet gas pressure was regulated to achieve a steady bubbling for the given backpressures of 2.8 bar or 6.9 bar. The valve system is controlled with an Arduino which was linked to the spectrometers software synchronizing the gas supply, venting of the NMR tube, and data acquisition. Using this gas system we supplied to NMR tube N2 (99.999 %, Air Liquide), H2 (99.999 %, Air Liquide) or pH2. 195 pH2 quantification protocol The pH2 quantification was performed according to a quantification protocol (schematically shown in Fig. 4). kept open during the NMR measurement. Because the NMR signal was not locked during the experiment, the H2 resonance was moved to 0 ppm during post-processing for convenience.
All NMR spectra of gases were acquired with a standard excitation and acquisition of free induction decay pulse sequence (12.6 µs excitation pulse that corresponds to 90° flipping angle, 20 ms acquisition time, 50 kHz spectral width, 0.5 s repetition 205 time, 100 transients for averaging, SpinSolve Expert v3.54, Magritek, Aachen, Germany). Spectra were subjected to 20 Hz exponential apodization and phase-correction. To remove background signals, a spectrum of N2 was acquired also and subtracted from the rtH2 (H2 in thermal equilibrium at room temperature) and pH2 spectra. After that, an automatic baseline correction (MNova v14.1.2, Santiago de Compostela, Spain) was applied to the phased spectrum. The spectral lines of rtH2 and pH2 were integrated within the borders of -15 ppm and +15 ppm giving S(rtH2) and S(pH2). And finally the fraction of pH2 210 H 2 was calculated: Here it is taken into account, that only oH2 contributes to MR signal and H 2 = 1 4 at room temperature (Green et al., 2012).
215 Figure 4: Scheme of pH2 quantification protocol. The NMR tube is flushed with N2, pH2 or rtH2 gas for 180 s before the exhaust is closed.
A rest time of 30 s is allowed for the system to settle down. Finally, the NMR spectra are acquired, before the gas is released.
Magnetic field cycling experiment. The NMR spectrometer was equipped with an in-house built MFC setup that will be A PHG fulfilling the initial design requirements was successfully constructed (Fig. 6). Most parts were either commercially available, 3D printed or simple to construct on-site. The holders for the bottles and a bottom plate were the sole part prepared by a mechanical workshop. All parts were rated for more than 100 bars and no H2 leaks were detected at 50 bars of H2 using 245 an H2 detector (GasBadge Pro H2, Industrial Scientific, Pittsburgh, U.S.A.). Inspection and operation were facilitated by easy access and open construction design. The total cost was below 3,000 € (Appendix A).
We deliberately abstained from including a flow meter into the setup to keep the cost low and increase the robustness. Instead, we monitored the pressure Pout in the storage cylinder and calculated the flow rate (Fig. 7a). The expected increase in pressure 250 and decrease in the flow rate of pH2 was observed. The flow rate is an important parameter since it affects the collisions of H2 with the catalyst in the ortho-para conversion unit (Fig. 3, A3). At optimal parameters, lN2 based PHG can provide H 2 ≅ 52 % (Fig. 2,7b). These collisions are responsible for the fast ortho-para conversion. If the flow rate is too fast, the gas will not have enough time to reach ortho-para thermal equilibrium while passing through the unit. Hence the pH2 fraction will be reduced. 255 Thus, to find optimal performance conditions of the PHG we quantified H 2 as a function of the flow rate (Fig. 7c) set by the needle valves (Fig.3, A5). At the given settings of Pin = 20 bar and Pout = 10 bar, H 2 ≈ 51.7 % was found for a flow up to fr = 2 SLM. For larger flow rates, the enrichment dropped significantly. Given this data, and to allow for some variation, we chose a standard operating flow of ≈ 0.9 SLM. This flow rate was fast enough for convenient ad-hoc pH2 production. For example, l L of 49 bar pH2 with H 2 = (51.7 ± 0.76) % were produced in 29 min (Pin = 49 bar, initial flow rate of 2.9 SLM, 260  Preprint. Discussion started: 5 November 2020 c Author(s) 2020. CC BY 4.0 License.

The precision of pH2 production, quantification and lifetime
To test the reproducibility of the quantification method, H 2 of a single batch was quantified 5 times in a row (including venting, flushing, and filling of the tube). The average H 2 was found to be (51.5 ± 0.36) %, corresponding to a coefficient of variation (CV) of 0.7 % (Fig. 8). 270 To access the reproducibility of the entire production process, four pH2 batches were produced on different days. An average H 2 of (51.6 ± 0.88) % was observed (CV = 1.7 %) (Fig. 8).
For evaluating the lifetime of pH2 in the 2 L cylinder, a 10 bar pH2 batch was produced (Pin = 20 bar, fr = 0.9 SLM). Over 22 days, five samples were taken from the batch and H 2 was quantified. An exponential decay function was fitted to the data and yielded a constant of 35.5 ± 1.48 days (Fig. 9). 275

Application: 1 H-low-field SABRE at different pH2 pressures
The presented setup was designed to allow for pressures up to 50 bar. High pressures are beneficial for hyperpolarization because the concentration of pH2 in solution increases with pressure. Low concentration of pH2 is often the limiting factor of 290 the hyperpolarization yield (polarization level × concentration of polarized species). To demonstrate the effect, we polarized nicotine amide by SABRE and magnetic field-cycling (scheme at Fig. 5) at two different pH2 pressures: 2.8 and 6.9 bar (Fig.   10). Strong polarization was observed on 1 H resonances of nicotine amide and hydrogen in solution and increased at higher pressure. A 2.5 fold increase in pressure yielded a 2.3 fold increase of nicotine amide polarization.

Discussion
Design. The design of the presented PHG is simple and compact without compromising on performance and safety. The PHG 300 is small and portable (although a heavy bottom plate was added to add stability). Since there are no electrical components, it can be placed indoor as well as outdoor and does not require any electrical power supply. Note, electric components can be an ignition source which may lead to an explosion in case of a hydrogen leak.
For the framework, mostly, off-the-shelf parts were used. More complex geometries as e.g. holders for valves or gauges were 3D-printed. They have individual shapes and dimensions and manufacturing in a workshop might lead to high costs and long 305 production lead times. 3D-printing turned out to be a versatile manufacturing method enabling fast prototyping, complex shapes, and low-cost for one-off productions. The design of the PHG, all 3D-models (STL-files, Standard Triangulation Language, and CAD-files, Computer-Aided Design) are provided enabling other groups to adjust the parts to their individual needs (Ellermann, 2020b).
Choosing a small 2 L dewar keeps the design compact and the running costs low since less than 2 L of liquid-nitrogen were 310 required to prepare 1 L of pH2 at 50 bar. In combination with a short cooling time, the setup is perfectly suited for on-site pH2 production in a hyperpolarization lab.
Costs. The final cost of the PHG incl. the hydrogen sensor is 2,988 € incl. VAT (19 %). If a hydrogen sensor is already available in the lab, the overall costs for the PHG can be pushed down to less than 2,500 € incl. VAT (19 %). A complete set including the PHG, a hydrogen sensor, hydrogen/nitrogen gas as well as a variety of essential tools costs about 3,700 € incl. 315 VAT (19 %).
Safety. All parts which are in contact with pressurized gas are rated to at least 100 bar. However, we fixed the operation pressure to 50 bar to get a generous safety margin of 100 %. Additionally, the design of the PHG incorporates a gas path which also enables safe ventilation of storage bottle. The design and the choice of parts also consider potential handling errors. For example, the output connectors are closed for pressures up to 17 bar when they are disconnected, i.e. the storage bottle is 320 disconnected. Thus, no contact between the air in the room and hydrogen in the PHG occurred. Furthermore, we included a handheld hydrogen sensor that can measure hydrogen concentrations in the parts per million (ppm) regime. The sensor should be always turned on during operation and will indicate potential leakages of H2 gas.
The setup includes low-temperature cryogens as liquid nitrogen. To prevent the spilling of liquid nitrogen, the dewar is restrained by the copper tubing inside and the surrounding metal frame. Moreover, a lid covers the liquid nitrogen bath that 325 also reduces the evaporation rate of the cryogen. Since the flask only holds around 2 L of cryogen, the amount of liquid nitrogen that has to be handled is greatly reduced.
Note that PHG should be placed in a non-public lockable enclosure or room with sufficient ventilation and only instructed personnel should operate it.
Our presented safety concept is economical and practical without sacrificing any safety measures. Nevertheless, after setting up the PHG, it should be tested for leakage with inert gas or nitrogen. The part-list also contains a leak detection spray and sensor.
Performance The enrichment achieved here, e.g. H 2 = 51.6 ± 0.88 % for Pin = 20 bar, fr = 0.9 SLM, was close to the maximum of 51.8 % conditioned by the boiling point of lN2, and somewhat higher than reported elsewhere H 2 = 50 % 335 (Barskiy et al., 2016a(Barskiy et al., , 2016bShchepin et al., 2016). Determining the enrichment as a function of flow allowed us to choose an optimal flow of 0.9 SLM for Pin = 20 bar: this rate is sufficient e.g. to fill 1 L bottles to 10 bar in 10 min. The central design criterium of high pressure was successfully met as 1 L of 49 bar pH2 were produced in 28 min (Pin = 50 bar). We demonstrated that an increase of pH2 pressure can give a proportional increase in polarization (Fig. 10). Obviously, this approach is limited as soon as the hyperpolarization yield is no longer determined by the availability of pH2 and cannot provide polarization above 340 33% (Korchak et al., 2018).
pH2 quantification and production reliability The automatic quantification process features a CV of 0.7 %; the pH2 production and quantification together feature a CV of 1.7 % (Fig. 8). Both values are suited for routine pH2 quality control without a need for an expensive high-field NMR system. The automatization certainly helps to make the process more reliable but is not necessary. Feng et al. used the same quantification approach and reported precision of 1-3 % for quantification 345 (2012). NMR is a convenient method for pH2 quantification, but optical methods may be used, too (Parrott et al., 2019). Feng et al. reported a lifetime in aluminium tanks of (63.7 ± 8.3) days and about 2 % points loss of H 2 per week (2012). With ~120 days of lifetime, Hövener et al. reported even longer values (2013). We found here a shorter lifetime of (35.5 ± 1.48) days in our 2 L aluminium storage bottle. Note, that we did not vacuum our cylinder that can increase the lifetime. Still, the lifetime is sufficiently long to produce pH2 once a week; the lifetime of 350 (35.5 ± 1.48) days corresponds to about 5.5 % points loss of H 2 per week.

Conclusion
The presented PHG provides H 2 ≈ 52 % at a high pressure of 50 bar reliably (CV = 1.7 %) that provides about 1/3 of the polarization achieved with H 2 ≈ 100 %. Because the device provides high-pressure pH2, however, this effect can be partially compensated in the PHIP/SABRE experiment. A new, automated quantification routine at 1 T benchtop NMR proved to be 355 reliable and simple (CV = 0.7 %). The design of PHG is straightforward, easy to manufacture with openly available blueprints and at a cost of less than 3,000 €. The device may facilitate further research on the promising method of parahydrogen-based hyperpolarization.  (Ellermann, 2020a) and via git (Ellermann, 2020b). 370

Author contribution
Data curation, investigation, formal analysis, software development (here programming of macros), validation, visualization 380 and writing of the original draft was done by FE. ANP and JBH contributed equally to conceptualization, supervision and reviewing the manuscript.

Competing interests
There are no competing interests to declare.

Financial Support
We acknowledge support by the Emmy Noether Program "metabolic and molecular MR" (HO 4604/2-2), the research training circle "materials for brain" (GRK 2154/1-2019), DFG -RFBR grant (HO 4604/3-1, № 19-53-12013), the German Federal Ministry of Education and Research (BMBF) within the framework of the e:Med research and funding concept (01ZX1915C), Cluster of Excellence "precision medicine in inflammation" (PMI 1267). Kiel University and the Medical Faculty are 390 acknowledged for supporting the Molecular Imaging North Competence Center (MOIN CC) as a core facility for imaging in