Nitrogen fixation using reactions in water containing microdroplets

Abstract

The invention generally relates to nitrogen fixation using reactions in water containing microdroplets. In certain aspects, the invention provides methods of producing ammonia that involve generating a water containing microdroplet in presence of gaseous nitrogen from a gas source, wherein the water containing microdroplet comprises a metal catalyst and the metal catalyst reacts with the gaseous nitrogen to produce ammonia, optionally including in situ derivatization.

Description

[0001] Attorney Docket No.: PURD-149 / 01WO 28593 / 721

[0002] PATENT APPLICATION NITROGEN FIXATION USING REACTIONS IN WATER CONTAINING MICRODROPLETS

[0003] Related Application

[0004] The present application claims the benefit of and priority to U.S. provisional patent application serial number 63 / 555,979, filed February 21, 2024, the content of which is incorporated by reference herein in its entirety.

[0005] Government Support

[0006] This invention was made with government support under DGE- 1842166, awarded by the National Science Foundation and under FA9550-21-1-0170, awarded by Air Force Office of Scientific Research. The government has certain rights in the invention.

[0007] Field of the Invention

[0008] The invention generally relates to nitrogen fixation using reactions in water containing microdroplets.

[0009] Background

[0010] Ammonia is among the most important of all commodity chemicals due to its use as a fertilizer. It is synthesized in the energy intensive Haber-Bosch process at high temperature and pressure, using methodology that has changed little in over a century. Many alternatives have been explored, including methods based on ions generated by mass spectrometry. For example, hydrocarbons undergo nitrogen atom insertion in N2 discharges a result that may be due to reaction with N3-a key intermediate in N2 discharge chemistry. It has been shown that Ta2N+gives Ta N3+with a key step involving N-N cleavage. An alternative is that the key intermediate is anionic, e.g.2dinitrogen dianion, which is known to facilitate reduction of N2. It is also well-known that N2 is cleaved by solid lithium under mild conditions.

[0011] Summary

[0012] The invention generally relates to nitrogen fixation using reactions in water containing microdroplets. The work herein builds upon and goes beyond certain different prior work doneAttorney Docket No.: PURD-149 / 01WO 28593 / 721

[0013] PATENT APPLICATION

[0014] related to ammonia formation in droplets. In certain aspects, Nitrogen (N2) is converted into ammonia in a spray of water containing microdroplets containing a lithium salt as precatalyst and the product is captured in situ using, for example, formaldehyde as reagent. Direct analysis of the sprayed solution by positive ion MS shows hexamethylenetetramine (1), also known as hexamine, the product of NH3 reaction with formaldehyde. This product is also observed when the spray is directed at a conducting or non-conducting surface and the collected material is analyzed offline by nESLMS performed on extracted material or by direct desorption electrospray ionization (DESI) MS from the surface. The chemical identification of product 1 was confirmed by MS / MS using data for the authentic compound as a reference. By adding authentic product to the extract, the amount of ammonia formed in a typical 3 -hour deposition experiment, measured as the derivative 1, was 7 pg. Multiplexing the sprayers using 12 nanoESI emitters increased yields. The method of nitrogen fixation should be applicable as a new route to the synthesis of nitrogen-containing organic compounds, distinguished by the low cost and environmentally friendly nature of the starting materials. An example is the generation of 1-imino-l,2,4-triphenyl-5-hydroxy-2,4-pentadiene using 1,3,5-triphenylpyrylium tetrafluoroborate as reagent.

[0015] In Certain aspects, the invention provides, methods of producing ammonia that involve generating a water containing microdroplet in presence of gaseous nitrogen from a gas source, wherein the water containing microdroplet comprises a metal salt catalyst and the metal salt catalyst reacts with the gaseous nitrogen to produce ammonia, optionally including in situ derivatization.

[0016] In certain embodiments, the reaction occurs at an air / liquid interface of the water containing microdroplet. In certain embodiments, the metal catalyst is lithium. In certain embodiments, the water containing microdroplet further comprises a capture reagent that reacts with the produced ammonia. In certain embodiments, the capture reagent is formaldehyde or a pyrylium salt. In certain embodiments, the gas source that produces the gaseous nitrogen is compressed nitrogen gas or compressed air comprising gaseous nitrogen.

[0017] In certain embodiments, generating the water containing microdroplets comprises producing a spray discharge from a spray source. In certain embodiments, the spray source is at least one spray source selected from the group consisting of electrosonic spray, electrospray, nano-electrospray (nESI), and sonic spray (without an applied high voltage). In certainAttorney Docket No.: PURD-149 / 01WO 28593 / 721

[0018] PATENT APPLICATION

[0019] embodiments, the method is conducted using more than one spray source. In certain embodiments, the water containing microdroplet is directed into a mass spectrometer for analysis.

[0020] In other aspects, the invention provides methods of producing ammonia and collecting a reaction product that involve generating a water containing microdroplet in presence of gaseous nitrogen from a gas source, wherein the water containing microdroplet comprises a metal catalyst and the metal catalyst reacts with the gaseous nitrogen to produce ammonia; and collecting the ammonia on a surface as a reaction product.

[0021] In certain embodiments, the reaction occurs at an air / liquid interface of the water containing microdroplet. In certain embodiments, the metal catalyst is lithium. In certain embodiments, the water containing microdroplet further comprises a capture reagent that reacts with the produced ammonia to produce the reaction product that is collected on the surface. In certain embodiments, the capture reagent is formaldehyde or a pyrylium salt. In certain embodiments, the gas source that produces the gaseous nitrogen is compressed nitrogen gas or compressed air comprising gaseous nitrogen. In certain embodiments, generating the water containing microdroplet comprises producing a spray discharge from a spray source.

[0022] In certain embodiments, the surface is a conductor or an insulator. In certain embodiments, the method further comprises desorbing the reaction product from the surface for analysis.

[0023] In other aspects, the invention provides methods of producing ammonia that involve generating an water containing microdroplet in presence of gaseous nitrogen from a gas source such that an amount of nitrogen becomes associated with the water containing microdroplet; and directing the water containing microdroplet onto a surface comprised of a metal catalyst to thereby cause the metal catalyst to react with the gaseous nitrogen that has become associated with the water containing microdroplet to produce ammonia.

[0024] Brief Description of the Drawings

[0025] FIG. 1 panels A-D show four reaction formats (other than direct electrosonic spray used with online MS analysis) used to convert N2 into NH3 and condense the ammonia with formaldehyde. (Panel A) Electrosonic spray deposition onto various surfaces, the principal format in this work. (Panel B) Multiplexing achieved using 12 nano-electrospray ionizationAttorney Docket No.: PURD-149 / 01WO 28593 / 721

[0026] PATENT APPLICATION

[0027] (nESI) emitters. (Panel C) Sonic spray of water droplets using a sheath gas stream of N2 with the spray being passed through a corona discharge for direct droplet charging. (Panel D) Sprayed water droplets with N2 pneumatic assistance impacting Li metal surface (form of DESI). Both nESI and DESI were used for offline analysis of the various surfaces after deposition to show the presence of (1), the ammonia reaction product with formaldehyde.

[0028] FIG. 2 shows mass spectra corresponding to various experimental conditions as described in the inset of each spectrum. The surface in all SSI deposition experiments was washed with 10 pL of water at the center of the deposition spot and sprayed via SSI to the mass spectrometer. The identity of the high intensity peak at m / z 141 in the case of SSI deposition was verified with MS / MS.

[0029] FIG. 3 shows DESLMS images collected from two graphene deposition surfaces. (Top left) Trace amounts around of hexamine were homogenously distribution around the periphery of the graphene sheet when a spray voltage was not applied. (Top right) A similar distribution but with greater product intensity was found when ESSI was performed. (Bottom left, bottom right) In both cases paraformaldehyde was primarily found around the center of the deposited spray plume, though again with greater intensity in the case of ESSL The yellow circle indicates the impact area of the incident spray.

[0030] FIG. 4 shows possible mechanism for the reduction of Li+in at the air- water interface of microdroplets followed by subsequent reaction with N2 and H2O leading to NH3 formation and Li+regeneration.

[0031] FIG. 5 shows MS / MS spectra of (top) hexamine produced from the bulk reaction of formaldehyde and ammonium hydroxide, (middle) hexamine formed from sonic spray ionization deposition of water containing LiBr and formaldehyde, and (bottom) a hexamine standard purchased from a commercial vendor. All spectra were collected in the positive ion mode and identical trapping and dissociation parameters were used.

[0032] FIG. 6 panels A-D show MS / MS of m / z 141 when performing ESSI of a 3% water containing formaldehyde solution (v / v) and 100 mM LiBr solution with (Panel A) On-axis spray with a 250-pm ID fused silica capillary, (Panel B) On-axis spray with a 50-pm ID fused silica capillary, and (Panel C) Off-axis spray and 10-cm spray distance using 250-pm ID ESSI capillary. (Panel D) Off-axis (approximately 30 degrees) and increased 10-cm spray distance gives traces of m / z 141 with MS / MS that provide the highest relative intensity peaks thatAttorney Docket No.: PURD-149 / 01WO 28593 / 721

[0033] PATENT APPLICATION

[0034] correspond to the hexamine standard (red) compared to those which arise from trace amounts of an isobaric m'z contaminant ion (black). This experiment reinforces the role of small droplets in facilitating the interfacial redox chemistry necessary to form NH3. Note that the intensity of the m 'z 141 peak in Panel A-C was so low that it could not be observed in the full scan mass spectra and the resulting MS / MS spectra required 100+ averages acquire enough signal to produce the above spectra. The signal in the MS / MS spectra for Panel D was over an order of magnitude greater intensity than Panel A-C.

[0035] FIG. 7 is an illustration showing an exemplary data analysis module for implementing the systems and methods of the invention in certain embodiments.

[0036] Detailed Description

[0037] The starting point for the present study was the evidence accumulated since 2011 that the rates of organic reactions in microdroplets can be accelerated by very large factors. This topic has been reviewed and ascribed to partial solvation of reagents at the solution / air interface as well as the high electric field that develops at the interface provided that the solution contains at least some water. Amongst the many reactions that have been found to be accelerated is the reduction of N2 by water microdroplets in the presence of FeaCh. Although only traces of ammonia were formed, this is extremely important chemistry which we sought to extend by providing a derivatizing reagent to capture the ammonia in situ. Dehydration in microdroplets is a facile reaction so we elected to use formaldehyde reagent which readily reacts with ammonia to form hydrazine (1). This chemistry is fast in bulk and the product has been observed in a meteorite sample.

[0038] The experimental methods used to generate products and analyze them in situ or after deposition on a surface are described herein. The main method used was electrosonic spray (ESSI) along with nESI while analysis in situ of deposited samples used desorption electrospray ionization (DESI). The effect of charge was investigated by pneumatic sprays without applying a voltage, the method known as sonic spray ionization (SSI). The scale up experiments using multiple sprayers are based on methods described. FIG. 1 panels A-D show four reaction formats (other than direct electrosonic spray used with online MS analysis) used to convert N2 into NH3 and condense the ammonia with formaldehyde.Attorney Docket No.: PURD-149 / 01WO 28593 / 721

[0039] PATENT APPLICATION

[0040] The data herein shown confirm formation of ammonia by MS and MS / MS for the formaldehyde reaction product, m / z 141, across various conditions. Specifically, as shown in FIG. 2, experiments were done using pure water, water containing Li salts, water containing formaldehyde, and mixtures of these reagents by dropcasting onto a surface to provide control data. MS / MS confirms the formation of the ammonia derivative (1) in protonated form at m / z 141. This result is consistent with a theory that ammonia is formed in very small amounts from nitrogen in sprayed water droplets when using a heterogeneous catalyst (FIG. 2). Full scan mass spectra corresponding to various experimental conditions as described in the inset of each spectrum. The surface in all SSI deposition experiments was washed with 10 pL of water at the center of the deposition spot and sprayed via SSI to the mass spectrometer. The identity of the high intensity peak at m,'z 141 in the case of SSI deposition was verified with MS / MS (FIGS. 5-6).

[0041] Results are further summarized in Table 1.

[0042] Table 1

[0043] rMS / MS of m / z 141 Experiment Ionization Method Gas Presence of m / z 141 .x, rnstcnes stciDdsrd Deposition - Li' + Formaldehyde SSI N, ¥ Y Deposition - Li* + Formaldehyde SSI Ar N* Deposition - Li1+ Formaldehyde (+) ESSI N2¥ Y Deposition - id* + Formaldehyde (+) ESSI Ar N* Deposition - Formaldehyde SSI N2N Deposition - Formaldehyde (+) ESSI N2N Deposition - Li* SSI N* Deposition - Li* (+) ESSI N, N* Drop Cast - H-0 {+) nESl N2N Drop Cast - Lt* (+) nESi Y N Drop Cast - Formaldehyde (+) nESl N Drop Cast - Li* + Formaldehyde (+) nESl N, Y N 250 pm ID - Li* + Formaldehyde On-Axis (+) ESSI N2N 50 pm ID - Li" + Formaldehyde On-Axis (+} ESSI N2N 250 pm ID - Li* + Formaldehyde On-Axis (+) ESSI N2N

[0044]

[0045] 50 pm ID - Li* + Formaldehyde On-Axi$ (+) ESSi N, Y Y # Dependence of the nature of gas (N2 vs. Ar), ESSI (+ 4 kV potential) vs. SSI (zero voltage), initial droplet size (capillary diameter), and composition of drop cast deposited solutions on theAttorney Docket No.: PURD-149 / 01WO 28593 / 721

[0046] PATENT APPLICATION

[0047] formation of the desired product (1). Asterisks denote cases where, if nESI is used for the ionization post-deposition, a Li+adduct of propylene glycol is observed at m z 141.

[0048] Subsequently, a surface analysis was performed. As shown in FIG. 3, DESI-MS images were collected from two graphene deposition surfaces. (Top left) Trace amounts around of hexamine were homogenously distribution around the periphery of the graphene sheet when a spray voltage was not applied. (Top right) A similar distribution but with greater product intensity was found when ESSI was performed. (Bottom left, bottom right) In both cases paraformaldehyde was primarily found around the center of the deposited spray plume, though again with greater intensity in the case of ESSI. The yellow circle indicates the impact area of the incident spray.

[0049] Reactions were performed by spraying the LiBr and 3% (v / v) formaldehyde solution or a similar mixture (formaldehyde replaced for 10 mM water containing 2,4,6-triphenylpyrylium tetrafluoroborate (2) i.e. a Katritzky salt) onto a graphene surface using ESSI. The product of these reactions are hexamine and a non-aromatic hydroxypyridinium salt, respectively - both a result of reactions with the produced NH3. This allows macroscopic volumes of solution to be processed, e.g. 4500 pL in a 3 h period. The water containing solutions were sprayed and the presence of (7) or (2) on the surface was confirmed by MS / MS on eluted material while their distribution on the surface was established by DESI-MS. After the deposition, each surface was washed repeatedly with 1 mL of water to remove all product(s). The yield for hexamine was determined through standard addition of authentic hexamine to an equal volume of water used to wash surfaces for which the preparative experiment was performed again but with Ar gas. The hexamine yield was found to be 2.4 pg / hour using a single sprayer. The yield of the Katritzky product was approximately an order of magnitude greater.

[0050] At the simplest level, the experimental evidence suggests that the reduction of Li cations to the atomic metal which is a strong reducing agent enabling reduction of N2 to NH3. The driving force is the very low reduction potential of Li+. One can speculate that the well-known superacidity / superbasicity of the droplet interface [9] can then drive the reaction sequence N2' + H+— > N2H — > N2H’ -> NXH2‘ --> — > NH3 in the microdroplets at the air / solution interface where the ultimate source of the hydrogen is water but the proximate source is not known. Evidence forAttorney Docket No.: PURD-149 / 01WO 28593 / 721

[0051] PATENT APPLICATION

[0052] these simple mechanistic concepts is found in the fact that no product is generated in the absence of lithium, even in experiments with other metals.

[0053] The catalytic effect of lithium is assumed to be due to its reduction to the metal, as there is much evidence in the literature that lithium metal catalyzes the corresponding bulk phase reaction and no evidence that lithium salts do so. It is therefore reasonable to propose that the reduction of lithium occurs in the microdroplets allowing catalysis of N2 fixation to give ammonia and that this step is followed by a reaction that is well-known in bulk phase, viz. condensation of ammonia and formaldehyde to give hexamine (1). Note that the bulk reaction does not occur in water but its occurrence in water microdroplets is entirely consistent with the fact that condensation reactions in microdroplets are well known, and that the proposed occurrence, in water containing microdroplets, follows the reaction sequence: (i) lithium reduction (ii) Li catalyzed N2 fixation perhaps to give LisN (iii) protonation of LisN by H2O or HaO formed at the droplet interface to produce NH3 and LiOH (iv) NH3 condensation with formaldehyde to give 1.

[0054] We have no direct evidence for the first reaction in this sequence (i), reduction of Li+in microdroplets note that the reduction potential of lithium is extremely low (- 3.04 V) meaning that a super base is required to generate the metal atom. The evidence for strong electric fields and an electrical double layer at the water containing air interface means that difficult reductions (and oxidations) are possible in microdroplets. [9]

[0055] Indirect evidence for the third reaction in the sequence, (iii), condensation of NH3 condensation with formaldehyde to give the azaadamantane 1 comes from the fact that condensation in microdroplets is extremely common, with examples including peptide formation from amino acids amongst many others.

[0016] Direct evidence was obtained by an experiment in which a spray of formaldehyde solution was intercepted by ammonia vapor yielding an intense azaadamantane peak observed in the MS. Evidence for the second reaction in the sequence, (ii), comes from the observed overall reaction and the evidence for steps (i) and (iii) just presented. Without limitation, FIG. 4 shows potential mechanisms of action.

[0056] Desorption Electrospray Ionization and Sonic Spray Ionization (SSI)

[0057] Desorption electrospray ionization (DESI) is described for example in Takats et al. (U.S. patent number 7,335,897), the content of which is incorporated by reference herein in its entirety.Attorney Docket No.: PURD-149 / 01WO 28593 / 721

[0058] PATENT APPLICATION

[0059] Sonic Spray Ionization (SSI) is DESI without the applied voltage. DESI or SSI each allows ionizing and desorbing a material (analyte) at atmospheric or reduced pressure under ambient conditions.

[0060] A DESI system generally includes a device for generating a DESI-active spray by delivering droplets of a liquid into a nebulizing gas. The system also includes a means for directing the DESI-active spray onto a surface. It is understood that the DESI-active spray may, at the point of contact with the surface, include both or either charged and uncharged liquid droplets, gaseous ions, molecules of the nebulizing gas and of the atmosphere in the vicinity. The pneumatically assisted spray is directed onto the surface of a sample material where it interacts with one or more analytes, if present in the sample, and generates desorbed ions of the analyte or analytes. The desorbed ions can be directed to a mass analyzer for mass analysis, to an IMS device for separation by size and measurement of resulting voltage variations, to a flame spectrometer for spectral analysis, or the like.

[0061] In an exemplary DESI system, a spray is generated by a conventional electrospray device. The device includes a spray capillary through which the liquid solvent is fed. A surrounding nebulizer capillary forms an annular space through which a nebulizing gas such as nitrogen (N2) is fed at high velocity. In one example, the liquid was a water / methanol mixture and the gas was nitrogen. A high voltage is applied to the liquid solvent by a power supply via a metal connecting element. The result of the fast-flowing nebulizing gas interacting with the liquid leaving the capillary to form the DESI-active spray comprising liquid droplets. DESI-active spray also may include neutral atmospheric molecules, nebulizing gas, and gaseous ions. Although an electrospray device has been described, any device capable of generating a stream of liquid droplets carried by a nebulizing gas jet may be used to form the DESI-active spray.

[0062] The spray is directed onto the sample material which in this example is supported on a surface. The desorbed ions leaving the sample are collected and introduced into the atmospheric inlet or interface of a mass spectrometer for analysis, optionally by an ion transfer line which is positioned in sufficiently close proximity to the sample to collect the desorbed ions. The surface may be a moveable platform or may be mounted on a moveable platform that can be moved in the x, y or z directions by well known drive means to desorb and ionize a sample at different areas, sometimes to create a map or image of the distribution of constituents of a sample. Electric potential and temperature of the platform may also be controlled by known means. AnyAttorney Docket No.: PURD-149 / 01WO 28593 / 721

[0063] PATENT APPLICATION

[0064] atmospheric interface that is normally found in mass spectrometers will be suitable for use in the invention. Good results have been obtained using a typical heated capillary atmospheric interface. Good results also have been obtained using an atmospheric interface that samples via an extended flexible ion transfer line made either of metal or an insulator.

[0065] Nano ESI (nESI)

[0066] Inductive nESI can be implemented for various kinds of nESI arrays due to the lack of physical contact, and is described for example in US 9,184,036, the content of which is incorporated by reference herein in its entirety. Examples of circular and linear modes are illustrated in US 9,184,036. In the rotating array, an electrode placed ~2 mm from each of the spray emitters in turn was supplied with a 2-4 kV positive pulse (10-3000Hz Hz) giving a sequence of ion signals. Simultaneous ions signals were generated in the linear array using pulsed voltages generated inductively in the adjacent nESI emitters. Nanoelectrospray spray plumes were observed and analytes are detected in the mass spectrum, in both positive and negative detection modes.

[0067] Electrosonic Spray Ionization

[0068] Electrosonic spray ionization is described for example in Takas et al. (Analytical Chemistry, Vol 76 / Issue 14). Electrosonic spray ionization (ESSI), a variant on electrospray ionization (ESI), employs a traditional micro-ESI source with supersonic nebulizing gas. The high linear velocity of the nebulizing gas provides efficient pneumatic spraying of the charged liquid sample. The variable electrostatic potential can be tuned to allow efficient and gentle ionization. This ionization method is successfully applied to aqueous solutions of various proteins at neutral pH, and its performance is compared to that of the nanospray and micro-ESI techniques. Evidence for efficient desolvation during ESSI is provided by the fact that the peak widths for various multiply charged protein ions are an order of magnitude narrower than those for nanospray. Narrow charge-state distributions compared to other ESI techniques are observed.

[0069] System Architecture

[0070] In certain embodiments, the systems and methods of the invention can be carried out using automated systems and computing devices. Specifically, aspects of the inventionAttorney Docket No.: PURD-149 / 01WO 28593 / 721

[0071] PATENT APPLICATION

[0072] described herein can be performed using any type of computing device, such as a computer, that includes a processor, e.g., a central processing unit, or any combination of computing devices where each device performs at least part of the process or method. In some embodiments, systems and methods described herein may be controlled using a handheld device, e.g., a smart tablet, or a smart phone, or a specialty device produced for the system.

[0073] Systems and methods of the invention can be performed using software, hardware, firmware, hardwiring, or combinations of any of these. Features implementing functions can also be physically located at various positions, including being distributed such that portions of functions are implemented at different physical locations (e.g., imaging apparatus in one room and host workstation in another, or in separate buildings, for example, with wireless or wired connections).

[0074] Processors suitable for the execution of computer program include, by way of example, both general and special purpose microprocessors, and any one or more processor of any kind of digital computer. Generally, a processor will receive instructions and data from a read-only memory or a random access memory or both. The essential elements of computer are a processor for executing instructions and one or more memory devices for storing instructions and data. Generally, a computer will also include, or be operatively coupled to receive data from or transfer data to, or both, one or more mass storage devices for storing data, e.g., magnetic, magneto-optical disks, or optical disks. Information carriers suitable for embodying computer program instructions and data include all forms of non-volatile memory, including by way of example semiconductor memory devices, (e.g., EPROM, EEPROM, solid state drive (SSD), and flash memory devices); magnetic disks, (e.g., internal hard disks or removable disks); magnetooptical disks; and optical disks (e.g., CD and DVD disks). The processor and the memory can be supplemented by, or incorporated in, special purpose logic circuitry.

[0075] To provide for interaction with a user, the subject matter described herein can be implemented on a computer having an I / O device, e.g., a CRT, LCD, LED, or projection device for displaying information to the user and an input or output device such as a keyboard and a pointing device, (e.g., a mouse or a trackball), by which the user can provide input to the computer. Other kinds of devices can be used to provide for interaction with a user as well. For example, feedback provided to the user can be any form of sensory feedback, (e.g., visualAttorney Docket No.: PURD-149 / 01WO 28593 / 721

[0076] PATENT APPLICATION

[0077] feedback, auditory feedback, or tactile feedback), and input from the user can be received in any form, including acoustic, speech, or tactile input.

[0078] The subject matter described herein can be implemented in a computing system that includes a back-end component (e.g., a data server), a middleware component (e.g., an application server), or a front-end component (e.g., a client computer having a graphical user interface or a web browser through which a user can interact with an implementation of the subject matter described herein), or any combination of such back-end, middleware, and frontend components. The components of the system can be interconnected through network by any form or medium of digital data communication, e.g., a communication network. For example, the reference set of data may be stored at a remote location and the computer communicates across a network to access the reference set to compare data derived from the female subject to the reference set. In other embodiments, however, the reference set is stored locally within the computer and the computer accesses the reference set within the CPU to compare subject data to the reference set. Examples of communication networks include cell network (e.g., 3G or 4G), a local area network (LAN), and a wide area network (WAN), e.g., the Internet.

[0079] The subject matter described herein can be implemented as one or more computer program products, such as one or more computer programs tangibly embodied in an information carrier (e.g., in a non-transitory computer-readable medium) for execution by, or to control the operation of, data processing apparatus (e.g., a programmable processor, a computer, or multiple computers). A computer program (also known as a program, software, software application, app, macro, or code) can be written in any form of programming language, including compiled or interpreted languages (e.g., C, C++, Perl), and it can be deployed in any form, including as a stand-alone program or as a module, component, subroutine, or other unit suitable for use in a computing environment. Systems and methods of the invention can include instructions written in any suitable programming language known in the art, including, without limitation, C, C++, Perl, Java, ActiveX, HTML5, Visual Basic, or JavaScript.

[0080] A computer program does not necessarily correspond to a fde. A program can be stored in a file or a portion of file that holds other programs or data, in a single file dedicated to the program in question, or in multiple coordinated files (e.g., files that store one or more modules, sub-programs, or portions of code). A computer program can be deployed to be executed on oneAttorney Docket No.: PURD-149 / 01WO 28593 / 721

[0081] PATENT APPLICATION

[0082] computer or on multiple computers at one site or distributed across multiple sites and interconnected by a communication network.

[0083] A fde can be a digital file, for example, stored on a hard drive, SSD, CD, or other tangible, non-transitory medium. A file can be sent from one device to another over a network (e.g., as packets being sent from a server to a client, for example, through a Network Interface Card, modem, wireless card, or similar).

[0084] Writing a file according to the invention involves transforming a tangible, non-transitory computer-readable medium, for example, by adding, removing, or rearranging particles (e.g., with a net charge or dipole moment into patterns of magnetization by read / write heads), the patterns then representing new collocations of information about objective physical phenomena desired by, and useful to, the user. In some embodiments, writing involves a physical transformation of material in tangible, non-transitory computer readable media (e.g., with certain optical properties so that optical read / write devices can then read the new and useful collocation of information, e.g., burning a CD-ROM). In some embodiments, writing a file includes transforming a physical flash memory apparatus such as NAND flash memory device and storing information by transforming physical elements in an array of memory cells made from floatinggate transistors. Methods of writing a file are well-known in the art and, for example, can be invoked manually or automatically by a program or by a save command from software or a write command from a programming language.

[0085] Suitable computing devices typically include mass memory, at least one graphical user interface, at least one display device, and typically include communication between devices. The mass memory illustrates a type of computer-readable media, namely computer storage media. Computer storage media may include volatile, nonvolatile, removable, and non-removable media implemented in any method or technology for storage of information, such as computer readable instructions, data structures, program modules, or other data. Examples of computer storage media include RAM, ROM, EEPROM, flash memory, or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, Radiofrequency Identification tags or chips, or any other medium which can be used to store the desired information and which can be accessed by a computing device.Attorney Docket No.: PURD-149 / 01WO 28593 / 721

[0086] PATENT APPLICATION

[0087] As one skilled in the art would recognize as necessary or best-suited for performance of the methods of the invention, a computer system or machines of the invention include one or more processors (e.g., a central processing unit (CPU) a graphics processing unit (GPU) or both), a main memory and a static memory, which communicate with each other via a bus.

[0088] In an exemplary embodiment shown in FIG. 7, system 200 can include a computer 249 (e.g., laptop, desktop, or tablet). The computer 249 may be configured to communicate across a network 209. Computer 249 includes one or more processor 259 and memory 263 as well as an input / output mechanism 254. Where methods of the invention employ a client / server architecture, steps of methods of the invention may be performed using server 213, which includes one or more of processor 221 and memory 229, capable of obtaining data, instructions, etc., or providing results via interface module 225 or providing results as a file 217. Server 213 may be engaged over network 209 through computer 249 or terminal 267, or server 213 may be directly connected to terminal 267, including one or more processor 275 and memory 279, as well as input / output mechanism 271.

[0089] System 200 or machines according to the invention may further include, for any of I / O 249, 237, or 271 a video display unit (e.g., a liquid crystal display (LCD) or a cathode ray tube (CRT)). Computer systems or machines according to the invention can also include an alphanumeric input device (e.g., a keyboard), a cursor control device (e.g., a mouse), a disk drive unit, a signal generation device (e.g., a speaker), a touchscreen, an accelerometer, a microphone, a cellular radio frequency antenna, and a network interface device, which can be, for example, a network interface card (NIC), Wi-Fi card, or cellular modem.

[0090] Memory 263, 279, or 229 according to the invention can include a machine-readable medium on which is stored one or more sets of instructions (e.g., software) embodying any one or more of the methodologies or functions described herein. The software may also reside, completely or at least partially, within the main memory and / or within the processor during execution thereof by the computer system, the main memory and the processor also constituting machine-readable media. The software may further be transmitted or received over a network via the network interface device.Attorney Docket No.: PURD-149 / 01WO 28593 / 721

[0091] PATENT APPLICATION

[0092] EXAMPLES

[0093] Example 1 : A sustainable method for ambient nitrogen and CO2 fixation at water microdroplet interfaces to yield industrially valuable products

[0094] The rates of many solution-phase organic and redox reactions have been reported to be accelerated by up to 106times their corresponding bulk reaction rate when at the interface of aqueous microdroplets. These observations have historically been attributed to the large interfacial electric field, partial solvation of reagents, and increased reagent concentration during de-solvation. However, gaseous reagents such as carbon dioxide and methane have also demonstrated unique reactivity at the droplet surface. Here we take advantage of this phenomenon to efficiently capture CO2 and N2 from ambient air and rapidly transform the corresponding carbamic acids and ammonia to industrially relevant compounds. Successful reactions were transferred to a solvent recycling microdroplet reactor to produce up to 100+ milligrams of product per hour.

[0095] All solutions were prepared at 10 mM with ultrapure water except for any lithium salts utilized which were prepared at 100 mM. Microdroplets were generated via nebulization with CO2, N2, or compressed ambient air sheath gas using sonic spray ionization (SSI). CO2 capture was performed by spraying amine solutions into a custom 4-neck flask attached to a Soxhlet apparatus and condenser. Initial solutions and condensed solvents were recycled through the flow system via polytetrafluoroethylene tubing and a peristaltic pump. Nitrogen fixation experiments were performed in a similar manner using lithium salts, ammonia trapping reagents, and depositing products on a grounded graphene surface. When necessary, products were isolated via column chromatography and analyzed by MS, MS / MS, FT-IR, and both1H- and13C-NMR

[0096] Water containing icrodroplets containing aliphatic amines were found to react with pure carbon dioxide nebulization gas to form the corresponding carbamic acid at conversions between 18% and 73% during the microsecond-to-millisecond flight to the inlet of the mass spectrometer. Additionally, several normally unreactive electron-deficient amines were found to form smallAttorney Docket No.: PURD-149 / 01WO 28593 / 721

[0097] PATENT APPLICATION

[0098] amounts of carbamic acids when nebulized into microdroplets. Coupling reagents were added to the amine solutions to facilitate trapping of the carbamic acids as symmetrical ureas. The reactions were then moved off-line to a solvent recycling apparatus that enables the continuous spray and collection of re-condensed solvent for microdroplet reaction scale-up. Hundreds of milligrams of various industrially valuable symmetrical ureas, asymmetrical ureas, and carbonate esters were formed in approximately 1 hour using pure CO2. Ambient laboratory air (470 ppm CO2, 28% RH) was then compressed and used as a nebulization gas for the above reaction in the solvent recycling system. By doing so, direct air capture of CO2 and fixation into urea compounds was achieved in microdroplets with total yields between 10 and 105 mg in 1 hour. A similar experimental setup was used to achieve nitrogen fixation from ambient air and subsequent ammonia trapping in microdroplets. Aqueous solutions containing a lithium salt and an ammonia trapping reagent were nebulized with pure N2 gas or compressed ambient air and deposited onto various surfaces lining the reaction chamber. The product of the ammonia trapping, hexamethylenetetramine, was detected on the milligram scale after 2 hours of deposition. Applying a positive potential to the solution syringe needle increased the yield of the deposited product significantly but was not necessary. Furthermore, the product is observed in trace amounts when sprayed directly toward the mass spectrometer. Based on desorption electrospray ionization (DESI) imaging and control studies, the mechanism appears indicate the spontaneous reduction of Li+at the air- water interface of nm-size droplets to Li° which is known react rapidly with ambient N2 and H2O to yield ammonia and regenerate Li+.

[0099] Incorporation by Reference

[0100] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

[0101] Equivalents

[0102] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein.

Claims

Attorney Docket No.: PURD-149 / 01WO 28593 / 721PATENT APPLICATIONWhat is claimed is:

1. A method of producing ammonia, the method comprising: generating a water containing microdroplet in presence of gaseous nitrogen from a gas source, wherein the water containing microdroplet comprises a metal catalyst and the metal catalyst reacts with the gaseous nitrogen to produce ammonia.

2. The method of claim 1, wherein the reaction occurs at an air / liquid interface of the water containing microdroplet.

3. The method of claim 1, wherein the metal catalyst is lithium.

4. The method of claim 1, wherein the water containing microdroplet further comprises a capture reagent that reacts with the produced ammonia.

5. The method of claim 4, wherein the capture reagent is formaldehyde or a pyrylium salt.

6. The method of claim 5, wherein the gas source that produces the gaseous nitrogen is compressed nitrogen gas or compressed air comprising gaseous nitrogen.

7. The method of claim 1, wherein generating the water containing microdroplet comprises producing a spray discharge from a spray source.

8. The method of claim 7, wherein the spray source is at least one spray source selected from the group consisting of electrosonic spray, electrospray, nano-electrospray (nESI), and sonic spray (without an applied high voltage).

9. The method of claim 7, wherein the method is conducted using more than one spray source.

10. The method of claim 1, wherein the water containing microdroplet is directed into a mass spectrometer for analysis.Attorney Docket No.: PURD-149 / 01WO 28593 / 721PATENT APPLICATION11. A method of producing ammonia and collecting a reaction product, the method comprising:generating a water containing microdroplet in presence of gaseous nitrogen from a gas source, wherein the water containing microdroplet comprises a metal catalyst and the metal catalyst reacts with the gaseous nitrogen to produce ammonia; andcollecting the ammonia on a surface as a reaction product.

12. The method of claim 11, wherein the reaction occurs at an air / liquid interface of the water containing microdroplet.

13. The method of claim 12, wherein the metal catalyst is lithium.

14. The method of claim 11, wherein the water containing microdroplet further comprises a capture reagent that reacts with the produced ammonia to produce the reaction product that is collected on the surface.

15. The method of claim 14, wherein the capture reagent is formaldehyde or a pyrylium salt.

16. The method of claim 5, wherein the gas source that produces the gaseous nitrogen is compressed nitrogen gas or compressed air comprising gaseous nitrogen.

17. The method of claim 11, wherein generating the water containing microdroplet comprises producing a spray discharge from a spray source.

18. The method of claim 11, wherein the surface is a conductor or an insulator.

19. The method of claim 11, wherein the method further comprises desorbing the reaction product from the surface for analysis.

20. A method of producing ammonia, the method comprising:Attorney Docket No.: PURD-149 / 01WO 28593 / 721PATENT APPLICATIONgenerating a water containing microdroplet in presence of gaseous nitrogen from a gas source such that an amount of nitrogen becomes associated with the water containing microdroplet; anddirecting the water containing microdroplet onto a surface comprised of a metal catalyst to thereby cause the metal catalyst to react with the gaseous nitrogen that has become associated with the water containing microdroplet to produce ammonia.

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