Synthesis of Hyponitrite Complexes for the Reduction of NO

Introduction

Climate change has progressively gotten worse since the industrial evolution, to a point where the United Nations has declared we have 10 years to make changes to prevent irreversible 1 damage to Earth. Nitrous oxide (N2O) makes up 6% of the U.S. greenhouse gas emissions, however, it can stay in the atmosphere for around 114 years and has a global warming potential 2 300 times that of 1 pound of carbon dioxide. N2O is naturally reduced though chemical reactions done by N2O reductase, a copper centered enzyme found in a denitrifying bacteria, however, the 3 mechanism is not known and replication is difficult as N2O is a poor ligand for transition metals. The bacteria is unable to keep up with the amount of nitrous oxide produced by humans. Studies regarding the denitrification process began by looking at the heme proteins in humans that are responsible for the regulation of the signal transduction by utilizing NO.4 This has led to the understanding of the intermediate steps for the process, such as the coordination and reduction.5 My research focuses on the speculations that are made about the denitrification process done by nitrous oxide reductase with the goal of creating a complex that can replicate the reduction, leading to a man-made way of reducing the nitrous oxide in the air. Complexes that reduce NO to N2O have signifying factors that are clear indications that the reduction is either complete or there is an unexpected side reaction. Few mechanistic studies provide insight to the transformation between NO to N2O, although the insertion of the coordinated NO is the key to the mechanistic step. Not thermodynamically favorable, the reaction uses a metal catalyst to proceed the reaction, ideally a first-row transition metal, due to the price and abundancy of the metals. Previous studies have also ruled out rate limiting steps as excess NO converts to N2O in a copper catalyst with a single NO attached. The copper gains another NO forming a 6 dinitrosyl, or hyponitrite (N2O2), species that quickly reacts with NO to produce the N2O. The copper catalyst is one of the more stable and effective metals to use and is the center of the study as previous studies using nickel catalyst were unsuccessful due to reactivity. In this study, instrumentation available on campus will be used to identify the product of each step of the synthesis alongside higher methods of analysis for verification of each experimentally determine product. Complexes would be synthesized using common bench workspace and the glovebox in the Stieber Lab to ensure air sensitive are not decomposed. Infrared absorption spectrometry (IR) and X-ray diffraction (XRD) instruments on campus complement each other and will be utilized for the identification of the complexes synthesized for understanding and characterizing hyponitrite intermediates for the reduction process of NO. Nitrogen rich fertilizer used in agriculture is the main contributor for the amount of N2O emission.2 Previous publications indicate that a NO ligand can also bond in either a linear, or a side-on configuration which have an effect of the bonding patterns with a second NO. The linear configuration experiences higher yields (77%), however, it is unable to accept the second NO for reduction. The side on configuration (23% yield) is thought to configure the second NO to promote N-N coupling because of the presence of spin density between the nitrogen attached to the nitrosyl.7 With this configuration, the nitrogen is exposed for binding with a second NO to produce a hyponitrite complex.8 Synthesizing a complex that is able to only side on orientated will be studied. The IR spectroscopy instrument on campus is a Fourier transform infrared spectrometer (FT-IR) which uses a single optical path to collect a spectrum of the complex while also collecting a spectrum of atmospheric molecules for the background to be subtracting by the ratio.9 The data is composed of the sum of sine and cosine terms which produce the constructive and destructive inference of waves.10 An IR beam is shot at the complex which produces energy differences in different molecular species. IR radiation produces small differences between vibrational and rotational states that are amplified in the complex as it exhibits changes in the dipole moment. The dipole moment, determined by the magnitude of the charge difference between the two atom centers, produces a regular fluctuation which can then interact with the electric field from the IR. When the fluctuations produced by the vibrational frequencies and the rotation around asymmetric center matches the frequency of the radiation, the radiation is absorbed and calculated to produce a stretch on the spectrum.11 The data is rapidly acquired, however, there is an increase to the signal to noise ratio.9 IR spectrums can be divided into two regions, the fingerprint region and the functional group region.12 The functional group, or near-IR, region consist of the earlier portion of the spectrum, or 4000 to 15,000 cm-1 displays common functional groups in the complex, such as oxygen, nitrogen, sulfur and some hydrocarbons and can be used for qualitative analysis of the complexes synthesized as able to differentiate between NO and N2O. The fingerprint region is used to identify between different organic and inorganic compounds and consists of the higher region of the IR spectrum.11 IR is a powerful method that can differentiate between many different possibilities for the types of outcomes. To begin, IR can provide information of the metal binding with the NO or even 13 if it is bound to multiple NO molecules. NO reduces to N2O and IR is useful to provide insight qualitative properties of the complexes as it can differentiate between the two. This allows periodic check to ensure the synthesis is indeed successful. Other information provided can be the symmetry of the complex, the coordination, and isomerization of the complexes. IR produces specific stretches for asymmetric N-O (970 cm-1) and can signify the possibility of a trans- hyponitrite complex. A symmetric complex would show a N-O stretch (around 900-1,000 cm-1) due to the nature around the complex.14 A linear configuration would exhibit a longer M-N bond and a peak around 1800 cm-1 while the side on configuration would consist of values similar to other three-coordinate NO species, around 17,000 cm-1.15 IR spectrums also have the capability to distinguish if the NO is reduced as a stretch at 1555 cm-1 would be consistent with a highly reduced linear NO ligand.6,8 IR spectroscopy would be a key source of analysis for the complexes synthesized because of the vast of information provided about the NO chemistry in the complex. IR spectroscopy is unable to characterize all electronic transitions or homonuclear species, making it difficult to understand some of the binding interactions in the complex. This method is useful for ensuring the synthesis is continuing in the right direction. The use of home source XRD would allow characterization of the complete complex that would ideally be built of the hints given from the IR spectrum. X-rays have the same wavelength as 1 Å allowing the determination of bonds to be possible. An X-ray beam is emitted to hit the crystalized complex which diffracts on the lattice planes of the complex. The waves interact constructively and destructively before hitting the detector to produce spots that are reflections of the electrons of the atom. The reflections are then correlated and solved to give a crystalized complex. This method requires crystals, unlike IR spectroscopy which involves a screening process for the crystallization conditions for the complex as well as longer acquisition times, are more complex molecules require longer run periods. The crystallization process is based on trial and error so finding appropriate solvents and time for crystallization further increases the time for this experiment. Some complexes are difficult, and sometimes too reactive to crystallze16 making this an inefficient method for some complexes. Copper is the transition metal of choice because it is less reactive than nickel allowing the XRD to collect a spectrum, but it is anticipated that the X- ray would excite some of the complexes making IR spectroscopy a more efficient method. The XRD would be a beneficial method of analysis for my project, as it could provide clarity on the binding and chemical structure of the complex. Using the XRD would allow the binding of the NO to be analyzed for insight on the linear or side on interaction which could be dependent on the properties of the ligands attached to the complex. Further experimentation of ligand properties, such as electron donating, withdrawing, and steric hinderance, could be further analyzed for future complexes. It would also provide further information on bond lengths and bond angles between NO and the metal of choice. Currently there are limited resources on the relationship between the bond angle, NO length and the IR peak. Creating a table with expected IR stretches for the given NO configuration would provide useful for the future steps of the 17 mechanism. The reduction of NO to N2O is one step of a longer mechanism which still unknown. Understand one portion of the reaction and having a complete table of the expected trends is beneficial for the future steps of the analysis as well. Instruments utilized for this experiment would provide a useful insight on the steps of the process as well ensure the synthesis process is producing the expected outcome.

Materials and Methods

A mechanism like the one developed by Kogut et. al will be followed with the same backbone but a Cu center to investigate the binding patterns of the NO in this model.18 Using Cu would investigate the low coordination between the M(NO) pairs that would ideally accommodate both linear and side of coordination of NO. A copper center enzyme is used by bacteria to reduce NO to N2O and the proven success using nickel, the one element to the left of copper, should ideally be possible. Copper is readily used by nature and is a low-cost transition metal with low coordination so success with this as a metal center would be highly beneficial. The synthesis will begin with Cu(I) lutidine and β-diketiminato potassium salt i [ Pr2NNF6]K(THF) in equimolar amounts. This could form a blue colored solution and the excess solvent would be evaporated using the rotovap. This would allow the solvent to dry and form crystals that ideally should also be blue colored. Both the solution and crystals would be analyzed using IR spectroscopy and the crystals would be analyzed using the XRD to ensure the binding of the metal center to the ligand. The following steps would involve a recrystallization of the complex to create pure crystals for future use. The recrystallization process involves finding appropriate solvents were the crystals dissolve in. Tests will be completed to test solvents with different densities to find a solvent were the crystals are both soluble and insoluble. Crystals will be then grown using solvent layering as the technique. The soluble solvent with a higher density would be purified first by using glass wool in a glass pipette and running the solution through there. It would then be placed in a vial and then topped with a lighter insoluble solvent using a glass pipette to avoid extreme aggregation of the solution. Multiple trials should be set up with different solvents and at different temperatures since crystals may not form. Crystals may take weeks to grow, so letting the solutions sit for around a week before checking for growth is a crucial step. If crystals due grow, another analysis using IR spectroscopy and the XRD would be beneficial as these crystals would be higher purity than the last. At this point, the copper center should be attached to the ligand and it would be possible to proceed to attach the NO and focus on the binding patterns and possibilities of a hyponitrite. Attaching the NO would be done by adding 1 equivalent of NO at 1atm with diethyl ether. This step is fundamental for understanding the binding of the NO and notice if it can also provide linear and side on attachment. Once this complex is mixed, it will again be crystalized and analyzed for binding coordination with will be notice with the disorder of the crystal structure. The complex will be kept at low temperatures to ensure there is a lack of thermal motion in the unit cell. ORTEP diagrams can be generated to observe the uncertainty of each atom in the complex as well as measure the bond distances and angles with the NO and metal. All crystals will be analyzed using Olex, a software used with Shelx, and solved to obtain an R value less than 5. If this is unobtainable, then further crystallization methods, such as different crystallization techniques and methods of analyzing the product, would be used to produce crystals with better diffraction patterns for solving and that we indeed have the complex we should have. Methods like IR could be utilized in this step to notice any peaks that should be present were the NO would be expected to be seen. N2O is also visible in IR so ensuring that we are just analyzing one NO bounded to our metal is also important. Following that analysis, a reaction with the initial starting material will be ran again using three equivalents of NO to observe any hyponitrite binding if possible. Ideally, if there is side on bonding in the complex, we should notice some hyponitrites forming. A similar reaction with nickel was able to accomplish a 46% yield of the hyponitrite which should be a slightly lower percentage for a copper center as copper is less reactive than nickel is.15 IR should be taken of the complex and compare with the previous step to identify if the reaction went to completion. There should be two visible NO peaks in the IR spectra for the complex in comparison to the first. At this point, steps should be taken to crystalize the product in a similar manner, to evaluate the complex with the XRD. Although copper is less reactive than nickel so it would have a lower yield, it would be more likely to be analyzed using the XRD. Nickels reactivity prevents it from being analyzed using the XRD as the X-ray can excite it even at low temperature. Copper should be able to withstand the X-ray beam during the analysis. This analysis would provide insight of the hyponitrite nature. Future steps would include use of other methods to determine the redox nature of the complex and to have mechanistic studies using UV/vis to determine the rate and binding and release of NO as well as using computational methods to check the accuracy of all the findings.

Specific Aims

Completion of this project would be an accomplishment for inorganic and synthetic chemist. If this project can work the way intended, humans would begin to reduce the amount of N2O in the atmosphere leading to a planet with a longer life. The initial steps are straight forward and should work without much issues. Starting complex will be synthesized just by adding the 1,3-Diketimines(nacnac) as the backbone and copper, allowing it to bind at the nacnac center. The nacnac should be an efficient choice for the backbone as it uses sterics to allow only a small binding site for anything else, without drastically changing the oxidation state of the complex. Next would be to add one equivalence of NO to study the binding with the copper center and understand the capabilities of the side on and end on interactions. Crystallographic information would provide bond angles and bond lengths for the complexes. It should be visible using the XRD and it would be portrayed as disorder as both configurations, as both should be crystalized in the same crystal. When solving the crystal structure, it should be possible to identify the two and separate out the structure as it would be portrayed as disorder. Mechanistic studies will accomplished to understand potential alternative methods as well as the yields of the complexes. This would lead into the next step, understanding the formation and isolating a hyponitrite complex without an added cation. Inorganic chemists have been working on ways to synthesize and isolate hyponitrite complexes. So far there has been very limited success, since most first row transition metals tend to be reactive and quickly reduce when the complex is thought to have synthesized. There has been some success with heavier metals in the f block, as they are more stable and can be isolated but that would not be a feasible method for our problem at hand. One of the few isolated published copper hyponitrites has replicated in current work and similar work has been done with a nickel metal center. Being able to synthesize and isolate a copper hyponitrite complex would be a beneficial contribution to the other chemist that are working in a similar project. Issues with the synthesis procedure include the delicacy of the amount of NO that must be added into the system. Most papers with successful work have only added a specific amount of NO to the system for the formation of the hyponitrites, however, the reactivity is unpredictable to testing multiple equivalences of NO is a crucial step. Finding the amount of NO needed for the system might be able to contribute to a common range to begin to see the amount of hyponitrite formation. An isolated hyponitrite copper complex has very few examples in literature and there has been even fewer complexes without an added counter cation. Counter cations have been necessary to aid in the crystallization of the complex since isolated complexes have not been successfully crystalized. This project would include the first few trials to have counter cations to aid in the crystallization process. These counter cations would also aid in the mechanistic studies as the increase stability would allow studies throughout the steps. Using IR analysis can provide signals to show the progress in the synthesis and start a proposed mechanism. Mechanistic studies can then provide insight to the general reaction which can test other metals in the future. Understanding each step of the mechanism as the experiment is running would add further insight to how the complexes could potentially be formed in current biological systems. As these complexes are more readily crystallizable, it is possible to study the result using all methods, such as IR and XRD, to set a reference for future runs without the counter cation in place. Future studies involving probing the oxidation state, as it is important to have a low enough oxidation state to hold the hyponitrite configuration, but be able to reduce in the presence of excess NO. This is a study that can be done in the future using cyclic voltammetry, another instrument that is provided by the labs in the university. Cyclic voltammetry uses two sequential electron transfers to oxidize and reduce the complex as it predicts the intramolecular decomposition. After the trials with the counter cations, analysis of the complexes without the stabilizing force would be implemented and the previous runs would act as references for the complexes. Since mechanistic studies will be completed, is should be possible to repeat the synthesis without the counter cation, but the reaction would probably be too reactive to complete mechanistic studies. Instrumental verification should be completed whenever possible to understand if the reaction is proceeding the way it is expected to. This is when IR will be able to identify the state of the reaction as previously mentioned in other literature sources. Crystallization of the complex is expected to be difficult and the conditions previously used in the counter cation complex are not guaranteed to work for this complex as well. Several test should be running to find an ideal condition by varying the temperature, solvent, and crystallization method and giving it time to crystalize. During this period, methods such as cyclic voltammetry could be used to understand more about the oxidation state since different binding modes would change the electron contributions to the oxidation state. Ideally, the complex would crystalize which would then allow XRD studies to be conducted. If the complex is unable to crystalize, other outside sources, such as a synchrotron source, may need to be utilized to determine the structural geometry of the complex. A synchrotron has the capability to work on any complex regardless of physical state and examination of the spectra would provide verification of an isolated hyponitrite complex as well as provide the oxidation state. The reduction on NO is what the project is about, so the following steps would include adding additional NO to understand the reductions process as well as the products. By varying the amount of NO that is added to different trials, potential products can be studied and understood. Ideally a complex that can reduce the large amount of NO in the atmosphere while regenerating itself and lasting long periods of time would be preferred. The complex should act as a catalyst, which acts in low quantities, is affordable, and regenerates itself at the end of the reaction. Both complexes, with and without the counter cation, should be studied to understand the NO reduction process. The last step would be to understand what happens to the complex when it is exposed to an excess amount of NO, as it would ideally be because of the current atmospheric conditions, and the decay process from prolonged exposure to NO. These studies would provide information about what the complex decomposed to and if there are any observable signs when it is decomposing. Once both complexes are verified, the next step would be to use computational methods to understand the differences in the binding patterns for both complexes. Since the linear method is produced in a higher yield in previous studies, it would be expected to do the same. Computational methods can probe the electron states and provide additional information about the binding patterns and areas with the highest electron density, providing insight on potential ways to make the complexes stable and more likely to reduce the NO, done by using a computational method of X-ray emission spectroscopy. The different binding patterns could be analyzed computationally to understand the different effects in an ideal environment. Computational methods at the synchrotron could act as a structural source for the complexes that are unable to be analyzed using the XRD. An unstable complex is not what biological systems use to naturally reduce the NO, so making a stable complex that is not air sensitive would be a future project. The reduction of NO is a multi-step reduction process that needs to be understood for the future of the planet. Understanding the reduction of NO to N2O is the first step to understand the biological systems that naturally do this in the soil and even in the human body. Forming a complex that can reduce NO to N2O would be the first step for the problem. Mechanistic studies would provide answers to the different binding patterns of NO and might provide some insight on how to create a mechanism that probes for side on only interactions. Allowing the complex to be exposed to constant NO would provide insight on the possibility of the linear NO being able to bend under the pressure. Using the instruments on campus and outside sources should allow a complete study of the complex and mechanism. Future uses of the project include being able reduce NO as a catalyst while being regenerated and stable at STP. The complexes will be verified using the IR and XRD which are able to differentiate between the different binding patterns and determine progress on the synthesis that we expect to see. These methods complement each other as when the IR is unable to provide enough information, the XRD can get the details that are necessary. The XRD has limitations that can be analyzed using the IR or outside methods. Together, these methods would provide a detailed mechanistic study of the reduction of NO to N2O.

Budget

Chemicals and reagents $1,000 Flame resistant lab coat $100 Computer software for calculations $100 Synchrotron visit $500

Work Cited

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Reflection Essay My research question was about forming a research proposal using the chemistry instrumentation in the College of Science. The college of science has analytic instruments that were used throughout the semester for lab work in my current course, CHM 5510L, however, I planned on creating a lab proposal that would be closes related to my graduate research work, which is finding ways of reducing nitrous oxide in the atmosphere. The research paper was assigned to us during this quarantine, so I have been unable to access physical sources of information from the actual library. I knew that I had to investigate the biological systems that normally do this kind of reductions, so I began with a simple search on the CPP library website of simply “nitrous oxide” to get an overview of the matter. I found a useful digital encyclopedia and ebooks that were available to read online with information on the matter, but I wanted to get more details on the reduction process and what is known about the nitrogen cycle that normally does this reduction. This led me to change the search result to be slightly more refined. I searched “nitrous oxide reduction” and began reading peer reviewed articles and reading some of the sources they were citing. There was a lot of useful information and I search for these articles using the online database. I utilized the scientific search engines such as the Web of Science and the American Chemical Society (ACS) Journals to find the reference material. These are two that I learned about in class that provide many chemistry related journals and mostly primary sources. The great thing about both resources is that they have a small panel on the side with other side articles based on the citations. The CPP library search engine also searched though other resources and journal publishing areas I would have not thought to look at mainly because I did not know there was such a thing. The key beneficial part of the library search page though, is the peer review articles that show up to use as a starting point for any research paper. Peer review articles are filled with references and primary sources that can be used to understand more about the issue. During my research, I did also use google scholar as a search engine, however, some of the articles were inaccessible though the CPP database. I have learned that we can request access to these articles, I have previously requested the delivery of a book, which would be something I would try in the future. I have also noticed the emails that have been sent during the quarantine of the library research assistants that want to help with research questions and that will be something else that I will utilize in the future. For past research papers I have wanted to access an article that is printed and stored in the filing cabinets in the back of the second floor but I haven’t been sure on how to look at those, so asking someone for help would be beneficial. Most importantly, I learned the library’s search feature is very strong and can pull information from multiple types of articles and references. I can find articles from places that are not directly tied to my department but are relevant. I found new scholarly journals such as the Journal of Bioscience and Bioengineering which has additional details from a biological perspective of the reaction I am trying to replicate. These resources would aid in forming a stronger research paper in the future.