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Abstract
NMR spectroscopy is the analytical instruction significant used for quantification of the metabolites in mammalian cells. Characterization of metabolomics is prevailing with the effective identification of the small molecules, which tends to change its chemical structure and conformation. Spectrum used in the NMR is 13C NMR and 1H NMR for its prominent use in the visual graph. This lies the significance of the utility of the NMR technique. The method used during the experiment is the simple NMR technique with the use of the MestReNova software for the computational graphical output. 5-hydroxy-5-methylhexan-2-one and 6-hydroxy- 6-methyl heptan-3-one are the sample compound used for the experiment. The results obtained after running the samples of heptanoic and octanoic acid has been interpreted in the results and discussion section. The structural nomenclature at 500MHX and the processed data obtained in JeolFID have been analysed. The data obtained in both H NMR and C NMR has been interpreted along with the evaluation of the purification percentage.
The study has the major aim and objectives associated with NMR spectroscopy of mammalian metabolites. The main aim of this study started with the use of NMR spectroscopy for analyzing mammalian metabolites with chemical shift aspects. Therefore, two major objectives of this study are mentioned below-
Objectives
“Nuclear magnetic resonance” (NMR) spectroscopy refers to the study of different molecules. The interactions between different molecules are usually differed with radiofrequency. Electromagnetic radiation is used with a molecule’s nuclei in a very strong magnetic field. According to Emwas et al. (2019), the application of this spectroscopy is usually recognized from chemical, physical and biological elements testing. The important application of this process is highly significant to provide molecular structure and identity. Owing to the interior and exterior chemical potential of the polymer network, it forces water to go through between the
networks by diffusion. As stated by Alderson and Kay (2021), the function of bimolecular has been used in this process to analyze and collect different molecules based on information. The molecule's physical and chemical properties seem to have different dimensions for interacting with other elements. The broader description and application process of NMR is reflected in two major types in testing the structure and properties of molecules, such as Continuous Waves (C.W.) and “Fourier-Transform (FT-NMR)” (Li et al. 2021). However, the highest reach has been explored in the case of testing and examining the organic compounds.
This process of chemical and physical analysis of compounds is simple. The magnetic fields of different atoms have been identified to have tiny magnetic associations and properties. The measures of magnetic fields within the elements are significant to analyze with spectroscopy to calculate the structural and physical properties. As stated by Kubicki et al. (2021), the main principle of NMR is that all the nuclei have electrical charges and many others have spin. Gathering information and knowledge about the structure and vibration rates of molecules in their natural environment is the key advantage of using this process. According to Dervisevic et al. (2019), NMR spectroscopy is the simple and speedy process of analyzing and acquiring information. The information and sources refer to the e-library and other sources of information collected with NMR spectroscopy of compounds. Moreover, the chemical shift in NMR spectroscopy has been identified as the resonating frequency analysis by atomic nucleus magnetic field charges. Therefore, the testing process of chemical shift with NMR has helped to diagnose the structure of molecules. The usage of a large magnet to probe the spin properties in an atom is recognized to use the radio frequency waves in promoting the transitions.
The metabolites of mammals refer to the compounds involved with metabolism development. The metabolism in mammals seems to have different mono and polycyclic compounds. The commonly identified elements and compounds linked with metabolites are ranged from ethanol, aspartic acid, glutamic acid, acetic acid and lactic acid. However, the broader division of elements is identified as primary and secondary. The compounds mentioned earlier are mainly recognized as the primary laments, while the secondary elements are antibiotics, peptides, alkaloids, resins, terrenes, and naphthalene. According to Kuang et al. (2021), metabolism in a living organism's body is reflected in the breakdown process of foods into energy substances. Therefore, the metabolites are the compounds that are involved in the process of breaking down elements. The complex structures and compounds are basically breakdown into mono and simple structures.
The metabolic pathway for drugs in the body is mainly found to be limited to the intestines or livers. The number of metabolites can be easily identified with NMR spectroscopy. However, the 1D-NMR was used to analyze metabolomics. As stated by Jang et al. (2019), the exchanges of metabolites in mammals have been identified to be exploited with major inhibitory refluxing. The profiling of metabolites and different biofluids in tissues is disclosed with the NMR process. The advantages of using this process in metabolites have been assumed to refer to the whole composition extraction. According to Soliman et al. (2020), this process of compound structure analysis has influenced the diagnosis of different diseases and treatments. Therefore, cancer treatment and diagnosis have experienced significant impacts from this process. Therefore, the NMR spectroscopy has the capability to detect less than 200 metabolites. On the other hand, the M.S. analysis technique can identify and detect more than a thousand metabolites. Transportation and energy transfer have been principled with the direct flow from the base to higher energy (Alderson and Kay, 2021). Moreover, the application of NMR spectroscopy in analyzing mammalian metabolites has been limited, with insufficient concentration detection for samples.
The NMR spectroscopy with the technique of H NMR is involved in studying the metabolic pathway. The metabolic pathway in living with metabolites in the case of applying the hydrogen 1 NMP procedure is highly relevant. According to O'Callaghan et al. (2021), the NMR profiling with food samples is allowed to identify the significant components present in the samples. Moreover, difficulties are usually developed during the profiling process of samples with NMR spectroscopy. However, the chemical shift in the NMR process renders the samples errors without proper application of resonance frequency application. The radiation frequency and magnetic fields are the two commonly used approaches in NMR analysis. The mammalian metabolites are highly diversified in the nature of atomic construction. The bipolar nature of several elements has been stated as the key risk factor for analyzing NRM spectroscopy. As stated by Mateo-Otero et al. (2020), the metabolites examination with NMR spectroscopy is highly popular. However, the major interest of chemists, as well as others in the laboratory, as this process has been required lower time. This is the speediest process of obtaining results from samples. The compounds with mono and polybases are being run through this analysis process. Moreover, this 1H NMR procedure is effective in analyzing the structure and properties of compounds.
The property of some carbon atoms to possess signals of different intensities makes it difficult for a 13C NMR signal to detect the number of atoms. 13C NMR uses peak integration to investigate molecules that have been enriched with isotopes of 13C. The resonance frequency of the nucleus of this 13C is comparatively lower than the protons and this advantage is utilised by 13C NMR to view the 13C signals in a completely different window having a separate radio frequency (Chem. 2021). Similar to H NMR, experiments involving C-NMR can also be used to view only the carbon atoms and the standard used in 13C NMR is also tetramethylsilane (TMS). However, unlike H NMR, the four equivalent carbon atoms of TMS are used as the reference in the case of 13C NMR. The chemical shifts in the nuclei of 13C are more widely spread than the proteins present and the typical difference between the values is almost 188 ppm (Chem. 2021). This property helps in visualizing each carbon atom distinctly with the help of the peaks formed by their signals.
The factors influencing the chemical shifts in the nuclei of the 13C atoms are the same as the protons. These factors include the bonds of the electronegative atoms and “diamagnetic anisotropy effects” that tend to move the produced signal downfield (Rashid et al. 2021). The sp2 hybridisation also adds to the effect of this large downshift and the combining effects of this hybridisation and the double bond in oxygen cause the carbonyl carbons to raech the farthest downfield. Generally, two 13C atoms cannot be found near each other due to the low abundance of 13C and due to this, the spin-spin coupling cannot be observed in 13C NMR. However, the heteronuclear coupling can be observed between the neighbouring carbon atoms and the hydrogen atoms. Chemists for turning off the coupling between C and H use a technique called broadband decoupling because the large coupling constant between these can alter the clarity of obtained results (Lankhorst et al. 2018). Hence, the spectrum obtained in a 13C NMR is in the form of singlet carbon atoms.
Complex biological samples are most analysed with the help of “Nuclear Magnetic Resonance Spectroscopy” due to its ability to provide straightforward quantification of samples. H NMR mostly performs quantification of metabolites without the involvement of individual standards for each metabolite. The chemical nature possessed by the analyte does not alter the results in NMR but a proper extraction of the metabolite is mandatory (Saborano et al. 2019). It is also crucial to maintain appropriate NMR pulses and the quantification strategy for the metabolite needs to be appropriate too. The property of NMR to characterise complex mixtures and their chemical compositions is the basic reason behind using NMR in metabolomics. Although the sensitivity of NMR procedures might not be similar to other techniques, its versatility in the elucidation of structures and the property to allow molecule selection based on isotopes makes it reliable.
Spectrum analysis by NMR spectroscopy is an analytical technique based on physiochemical analysis under the magnetic field. Spectrum in the NMR involves identifying the molecular structure by determining the configuration of the atomic structure, and the difference in the potential chemical shift. According to Garcia-Perez et al. (2002), NMR spectroscopy is considered a useful technique for correlation. 1H-1H COSY in the NMR spectra with the utility of the correlation spectroscopy is determined. Coupling protons reaction is primarily determined by the cross peak and the identification of the COSY spectrum. Interpretation of the spectrum such as 1H NMR and 13C NMR is the promptly used compound for determine the structure of the exciting compound.
Metabolism is the downstream response that occurs within the cells, proliferation of the cell and alteration of the structure chemically leads to change in the epigenetics environment. As stated by Alshamleh et al. (2020), mammalian metabolites trigger the balancing of the cell cycle and external stimuli; therefore, determination of the level of the metabolite within cells is significant. NRM spectroscopy in the identification and determination of the sudden alteration of the level of the metabolite within the mammalian cells is incorporated at the specific site. Labelling of the NMR spectra is profound with the use of isotopes 15N and 13C are effective for the examination and analysis process. Synthesis of the metabolites in the NMR develops an extreme sensitivity during the analysis, which renders the maximum sensitivity during the experiment in order to obtain validated results.
Synthesis of the mammalian metabolites during the sample preparation needed to be quick due to the metabolic nature of changing metabolic concentration. As referred by Emwas et al. (2019), metabolomics in the NMR is exclusively used for the protein-bound metabolites, which are comparatively impossible in other analytical techniques. NMR's ability in determining the properties, characteristics and structural compound in the chemical shift of the compound develops a complexity that leads to the 1H spectrum binding with the proton coupling with the coupled constants. Metabolites data profiling in NMR spectroscopy with the resonance visual in the database enables to provide the determination of the metabolic process.
Purification of the metabolites is the systematic process that involves the use of the spectrum to determine the structure elucidation. Determination of the computational database and analysis of the visual graph reflects the correlation between the structural compound. As opined by Crooks et al. (2019), the 1H-1H COSY spectrum is effective as it determines the off-diagnoal peak of the hydrogen compound in the coupling proton. Heteronuclear single quantum coherence spectroscopy (HSQC) reflects the signalling of the hydrogen attached to heteronuclear. Heteronuclear Multiple Bond Correlation Spectroscopy (HMBC) explains the long ranges of correlation between carbon and the proton. Therefore purification of the metabolites involves in the lysis of the cells leads to the overspill of the internal constituents. It is followed by the binding effect of the protein to a matrix. Removal of the extra continue and elution of the metabolites at the centrifugation, purification process can be attained. NMR analytic tool used in the metabolic reflects the NMR analysis with quenching of the cells.
Metabolic labelling in the NMR spectroscopy develops a stable isotope in metabolomics. The extraction process for the analytical process is the development of polarity in the hydrophilic compound. As stated by Lutz and Bernard, (2022), it involves 1H NMR spectroscopy. Higher proportion extraction with the use of the methanol-water in the intracellular metabolite through the process of the NMR analysis of mammalian cells. Biological variability within the metabolites is the primary cause of the change in the metabolite. This renders the changes in the normal structure and development of the different structures. It is a utility in determining the cancerous cells; the purification process involves measuring the frequency signaling from the metabolites molecules under the magnetic field (Saborano et al. 2019). Therefore, NMR characterisation in the mammalian metabolic cells is highly significant for the specific nuclei measurement, which reflects the hydrogen correlation with the adjacent molecule.
Metabolite identification using NMR is an effective and valuable analytic tool in streaming the chemical structure of this molecule and its conformation. According to Fernández-García et al. (2002), NMR has efficiency in developing multinuclear structural information, which enables this technique to achieve structural purification. Metabolomic implementation involves the extraction of the mammalian cells to determine the dynamic of the spectra in overlapping. Quantification in metabolomics determines the metabolic pathways which develop a metabolic network, therefore mapping the involvement of the protein, which acts as the intermediate and the quantified level of the metabolic concentration. The chemical shift within the cells leads to the issue linked with the inherent, genetic which tend to develop a change in the different functional groups. Application of the NMR in metabolomics in human health, determination of the cancerous cell lines which cause due to alteration of the concentration level of the metabolic. Uses of the functional group within the cells are effective in peptide bonding.
Metabolite quantification is essential for the determination of the chemical shift, which might change, and lead to the development of the biofluid. Enumeration of the signal intensity signifies the alteration level of the concentration. As referred to by Smith et al. (2020), identification of the liver function can be significantly determined through metabolomics. This provides an accurate measurement of the metabolism contained in the specific pathway analysis (Siegal and Selenko, 2019). Therefore, the application of NMR spectroscopy in metabolomics has multiple uses in the analysis of human health and prevailing diseases. The various environment friendly methods were applied to synthesize nanogels. When comparing between the synthesis methods of nanogels and micro gels, the synthesis methods nanogels are almost as similar as the methods for producing microgels.
Materials required for the completion of the experiment
Parameters of 1H NMR
Relaxation delay 2 sec
Number of scans 64
Sweep 10
Offset 4
Parameter of 13C NMR
100-200 ppm C13
Relaxation delay 2 sec
Number of scans 256
Sweep 250
Offset
COSY 20: proton correlation
Parameter of DEPT135
CH3, CH comes as +ve peak
CH2 comes as –ve peak
Relaxation delay 2 sec
Number of scans 128
Sweep 10
Parameter of HMBC
Number of scans 325
Interaction 256
Delay 2 sec
Offset 3 ppm
Sweep 7
Parameter of HSQC
Number of scans 16
Interaction 256, each has 16 scans
Relaxation 2 sec
Offset 3ppm
Sweep 1
Synthesis of 1H NMR Spectrum develops a structure of the chemical equivalent that can be achieved by determining the number of signals. As stated by Ma et al. (2019), three H bind C=O with the same chemical in the structural environment. The resonance frequency in the external magnetic fields leads to the development of the signalling of the 1H NMR spectrum. Hb protons bind with the methyl group, which is chemically equivalent to the other methyl bond. Distinguishing between the chemical equivalent and the proton of the non-equivalent proton is of tremendous importance for determining the NMR spectrum. The principle of this technique is measuring the fluctuations of cattered light from a sample and detecting a scattering angle and interpreting as an average light intensity by a photomultiplier or photon detector. The chemical shift in the synthesis of the 1H NMR Spectrum is significant for the determination of the spectrum analysis enabling to accumulation of information about the chemical, biological and physical properties of the compound structure. The high-energy electron beam exposes on the sample surface and reflects the scattered light into the detector to generate the numerous signals. The high-resolution texture images of the analyzed sample surface, including sample structure rearrangement, are provided
Chemical shifting is significant for the development of the structural chemical shows signal in the significance of the scale in an appropriate environment. The smaller shift of the chemical molecule develops a lower resonance with a large shift of the chemical developing a high resonance frequency. Integration within the NMR spectroscopy involves reflecting the signal by interacting with the spectrum to produce a signal in the magnetic field, which develop the resonance at the magnetic field. According to Abdullabass et al. (2020), signal spilling in the NMR is the splitting of the proton on bombarding into the system. It reflects the frequency of the small molecule and helps in identifying the structure, and size of the molecule.
13C NMR spectrum in the NMR spectroscopy provides a solution to the overlapping with prominently occurs due to the low sensitivity. Magnetic file d strength of the the13C NMR spectrum, this help in reducing the acquisition time during the process of the metabolite. As opined by Granda et al. (2018), this led to the determination of the fast exchange of the molecules with the cells. The fractional distribution of the isotope can be eventually determined by the cross peaks in the computational database. The carbon nucleus in the NMR spectroscopy is less sensitive than the carbon nuclei, which reflect the comparatively lower frequency of the carbon than the hydrogen spectroscopy. 13C NMR spectrum synthesis in metabolomics leads to the accumulation of rich information regarding the metabolites.
Interaction between the H and the 13C is removed due to the second involvement of the radio frequency. This phenomenon cancels the coupling of the proton. As referred by Rosenau et al. (2018), this method is done to gather significant information about the structure and the conformation of the hydrogen bond between the adjacent sides. Therefore, the synthesis of the 13C NMR spectrum in the NMR spectroscopy is effective in developing the image in the graph format by displaying it in a single line. This process is significantly important for developing the cross peak and reducing the coupling, thereby displaying the least overlapping peak.
1H NMR Spectrum is characterized by the determination of the molecular structure, which is significant for the identification the development of human disease identification. Alteration of the molecule in the cell is the causative agent for the prevailing chance of the disease. As stated by Pham et al. (2019), this is highly reflected through the single band of four hydrogens, primarily at a single chemical shift. Therefore, 1H NMR Spectrum in the NMR represents the characteristics of the development of the bond, which influence the probable change of the metabolite in the tissue. According to Field et al. (2020), 1H NMR Spectrum is developed by the bond formation by the methyl group, which reflects the equivalent distribution of the chemical bond. Therefore, 1H NMR Spectrum characteristics determination in the experiment using the mammalian metabolite is the symmetry that encompasses the two signals in total. This effectively leads to the determination of the significant utility of the 1H NMR Spectrum in the identification of the changes in the metabolites.
13C NMR spectrum in the NMR spectroscopy has a significant advantage in comparing the 13C NMR and the 1H NMR in the standard of the TMS. It is interpreted by determining the aldehyde which reflects the acid and the ester. As opined by Sarker et al. (2018), characteristics of the 13C NMR spectrum determination in the mammalian metabolites are significant for analysing the chemical shift and the structure of the molecule. 13C NMR spectrum in NMR unable to split due to spin-spin coupling, this leads to the interaction with the 1H NMR. 13C NMR spectrum in the NMR spectra reveals the importance of the chemical shift and the separate performance of the chemical compound, which is highly required for the analysis of the metabolites. Tetramethylsilane (TMS) enables to development of an equivalent carbon. This reflects the ongoing process in the identification and enumeration of the metabolite concentration. According to Lane et al. (2019), changes in the carbon nuclei are the reflection of the spin changes; this change has a range of 0-240 ppm. Therefore, this reflects the characteristics of the 13C NMR spectrum in NMR spectroscopy. It enables to determine the revealed peak in the computational database for its structure change due to chemical shifting.
The sample compound for the experiment has been processed with the isolation of the metabolite compound from the cells. This involves the cell lysis, followed by the centrifugation and the elution for the NMR spectroscopy. Sample prepared by extracting the compound synthetically to get the pure form of the compound. NMR spectra determination is highly sensitive for which compound synthesis is prominently required for the extraction of accurate data. As referred by Bartma et al. (2021), the Sample compound is a chemical compound that needs to be isolated with an intact chemical structure. This enables to gather of information from the metabolite for its normal or alter the structure, configuration and confirmation, which promotes the human health issue. The compounds used for the experiment have been soluble in the methanol with the standard of TMS. This signifies the right structure implementation into the experiment. Therefore, the preparation of the sample compound is the important phase for the accuracy and validation of the interpretation of the result. The preparation of the sample in the experiment reflects the importance of the prevailing disease the human health. It is highly significant for the development of accurate and validates results.
NMR spectroscopy involves measuring the molecule resonance under the magnetic field. It primarily involves determining the structural analysis to determine the alteration of chemical structure. As stated by Crook and Powers (2020), metabolomics analysis provides aq detailing for the metabolic with quantification. This promotes the evaluation of the concentration and the level of the metabolites within the cells. Validation of the mammalian metabolic compound reflects the clinical significance in determining the environmental factor, which is prevailing and leading to the development of the disease. Evaluation of the compound by incorporating the right compound into the instrument is preferred. This influences the interaction of the metabolites and determines the pathway of the biological process. According to Abdul-Hamid et al. (2019), computational software is required for visual analysis of the graph, which reflects, by the radio frequency. The wavelength is gathered in the spectrum, which is followed by the measurement of the frequency length. This reflects the interpretation of the change in the structure and its conformation. Evaluation of the analytical technique is promptly required for the analysis of metabolite identification.
Heptanoic acid can exist in its equilibrium in the forms of “keto and hemi ketal tautomer”.
The NMR spectra of 1H, 13C, and DEPT were obtained in the 500MHz instrument Jeol400.
Spectra obtained this H NMR for the OH atoms. 4 major peaks are obtained at 2.091, 1.288, 1.287 and 1.121 respectively with the largest peak being the third one. 4 obtained peaks are singlet, one doublet and more than 3 triplets in high resolution. The hydrogen in the three major peaks is in the ratio 2:2:2:2 using the n+1 rule. Hence, there are three major environments surrounding the H atoms implying that the total number of hydrogen atoms is 14. The data obtained from the chemical shift is used to know w about the environment surrounding the hydrogen atoms (Balcerczyk et al. 2020). Therefore, the result demonstrated that the formulation provided low stability, and there was a possibility for aggregation or flocculation-taking place in the formulations. The first major peak is a doublet hence the carbon next to it has one hydrogen attached to it, i.e. a CH3 group. The next two major peaks are singlets and hence their surrounding carbon atoms are not paired with any hydrogen atoms. The highest peak is a double and hence the surrounding carbon atom has two hydrogen atoms paired with it. Followed by this, the C NMR spectrum was also conducted and the results obtained were as follows:
This is the C NMR spectra which obtained for the OH atoms. 6 major peaks are obtained at -209.08, -117.352, 29.111, 28.601, and 28.575 and the last peak is a merge of 4 peaks in the range of 0.055 to 0.058. The peak obtained at 28.575 is a singlet and it is the highest obtained peak. The first peak is a singlet and hence there are no hydrogen atoms attached to the surrounding carbon atom. The same applies to the second and third major peaks, which are also singlets. The particles were isolated brought on the narrow distribution of the particles in the system. The fourth major peak however is a doublet and hence the number of hydrogen atoms attached to the neighbor carbon atom is one. The hydrogen in the six major peaks is in the ratio 2:2:2:3:2:3 using the n+1 rule. Hence, there are six major environments surrounding the H atoms and the total number of hydrogen atoms is 14.
Based on the H NMR spectra, the number of hydrogen atoms can be presumed and these have been presented in the table below. Using TMS as the internal standard, NMR was conducted at 500 MHz and based on the chemical shift, the following assumptions can be made:
|
No. of protons |
Heptanoic acid |
Tautomer |
Designation |
|
1 |
--- |
3.340 (s, 1H, OH), |
g |
|
2 |
2.632 (s, 1H, OH), |
--- |
f |
|
3 |
2.520-2.475 (m, 2H) |
2.520-2.475 (m, 2H) |
e |
|
4 |
--- |
--- |
2.09 for water |
|
5 |
--- |
-- |
1.93 CD3CN |
|
6 |
1.646-1.615 (m, 2H) |
1.646-1.615 (m, 2H) |
d |
|
7 |
1.361 (s, 3H) |
--- |
c |
|
8 |
-- |
1.288 (s, 3H) |
b |
|
9 |
1.122 (s, 6H) |
1.122 (s, 6H) |
a |
Table 1: Proton assignment with a chemical shift of heptanoic acid and its tautomer
Based on the C NMR spectra, the number of carbon atoms can be presumed and these have been presented in the table below.
|
No. of C atoms |
Heptanoic acid |
Tautomer |
Designation |
|
1 |
209.1 |
-- |
k |
|
2 |
-- |
117.3 |
j |
|
3 |
69.1 |
i |
|
|
4 |
-- |
38.4 |
h |
|
5 |
38.3 |
-- |
g |
|
6 |
-- |
37.3 |
f |
|
7 |
36.9 |
-- |
e |
|
8 |
29.5 |
-- |
d |
|
9 |
29.1 |
-- |
c |
|
10 |
-- |
28.5 |
b |
|
11 |
-- |
27.6 |
a |
Table 2: Carbon assignment with a chemical shift of heptanoic acid and its tautomer
Table 3: Integration area of hydrogen signal of heptanoic acid and its tautomer
The values obtained at the 1st, 2nd, 7th and 8th entries are different in the case of heptanoic acid and its tautomer. However, the hydroxyl peak cannot be relied upon due to the acidity it possesses. The strength of the OH bond is variable due to the H bond and the absorbed wavelength is partially dependent on this (Lombó et al. 2021). Hydrogen b and c of the 7th and 8th entry are being considered and these represent the methyl groups of both the compounds.
3.1.3.1 Peak Picking
The primary, secondary and tertiary peaks of the hydrogen-containing carbon atoms of heptanoic acid were obtained. The peaks observed for primary and tertiary carbons were observed as positive peaks and the peak obtained for secondary carbon was negative. No peaks were observed in the case of quarternary carbon. Hence, two negative peaks were obtained in the case of each tautomer resulting in 4 negative peaks representing CH2.
The peaks were obtained at the delta values of 38.4, 36.89, 29.11 and 28.59 respectively.
Table 4: Hydrogen signal correlation in heptanoic acid and its tautomer
3.1.3.2 Peak Integration
The integration of the peaks of hydrogen in each case is observed to be 3.1. The percentage of purity is measured by multiplying the area surrounding characteristic hydrogen by 100. In this case, the total area of the two characteristic hydrogens b and c are being considered, hence the total area is 3.1+3.1=6.2.
Hence, the purity percentage = 3.1/(6.2)*100
=0.5*100
=50% purity
Octanoic acid can also exist in its equilibrium in the forms of “keto and hemi ketal tautomer”.
The NMR spectra of 1H, 13C, and DEPT were obtained in the 500MHz instrument Jeol400.
This is the H NMR spectra which obtained for the OH atoms. 9 major peaks are obtained at 4.892, 2.514, 1.938, 1.359, 1.169, 1.160, 1.035, 1.016 and 0.000 respectively with the largest peak being the sixth one. In high resolution, 6 obtained peaks are singlets, 2 doublets and the remaining peaks are triplets and quartets. 6 peaks can be considered as significant among the 9 peaks and the ratio of carbon is 2:2:2:2:2:2. The number of parks obtained in NMR spectroscopy (Emwas et al. 2019) detects the number of environments surrounding the carbon atoms. Hence, there are 9 major environments surrounding the H atoms and 6 significant ones. The total number of hydrogen atoms in octanoic acid is 16. The first major peak is a singlet; hence there are no hydrogen atoms attached to the carbon group next to it. The distortion signal expressed by the metabolites compound reflects the interaction of the adjacent compound. Analysis of the dietary compound within the human serum is eventually developed. It has identified using the metabolites concentration and the change of the chemical shift. Most of the major peaks are singlets and their surrounding carbon atoms are not paired with any hydrogen atoms. The highest obtained peak is also a singlet and hence there are no CH3 groups nearby any atom.
Spectra obtained this C NMR for the OH atoms. 7 major peaks are obtained at -214.440, -83.853, -70.726, 49.067, 48.641, 38.043 and 9.293. The peak obtained at 49.067 is the highest and it is a singlet. All the 7 major peaks obtained are singlets and hence there are no carbon atoms surrounding them that have hydrogens attached to them. The highest major peak at 49.067, however, can be said as a merge of several peaks and the exact number cannot be detected even at the highest resolution. The ratio of hydrogen atoms in the seven major peaks can be said as 2:2:2:2:2:2 using the n+1 rule. There are seven environments surrounding the H atoms and the total number of hydrogen atoms is 16.
TMS has been used as the internal standard and the NMR was conducted at 500 MHz. The strong and sharp resonance line of TMS and its chemical shift at a low frequency makes it appropriate as a standard (Alderson and Kay, 2020). Based on the data obtained from the H NMR spectra, the probable number of hydrogen atoms can be presented in the table below.
Based on the C NMR spectra, the number of carbon atoms can be presumed and these have been presented in the table below.
The values obtained at the 7th and 8th entries are different in the case of octanoic acid and its tautomer. The hydroxyl atom is prone to lose its hydrogen due to the fact that it is an OH group attached to a single molecule and hence it is not reliable (Balthazar et al. 2021). The OH bond’s strength is variable due to this and the absorbed wavelength is partially dependent on the OH group. The a and b hydrogen of the 7th and 8th entry are being considered and these represent the methyl groups of both the compounds. Hence, the peaks obtained at the 7th and 8th entries were chosen to measure the purity percentage. It is significant for the determination at the early stage in order to develop drugs for human health benefits. The physiological stage of the metabolite determination in the NMR involves analysing the stability of the metabolite in the complex mixture.
3.2.3.1 Peak Picking
The primary, secondary and tertiary peaks of the hydrogen-containing carbon atoms of octanoic acid were obtained. No negative peaks were obtained in this case as expected by the CH2 group. Hence, no proper correlation could be made. No peaks were observed in the case of quarternary carbon as well. This ambiguity in the obtained data suggests the use of homonuclear correlation. This is a 2D spectroscopy used for detecting signals that are coupled to one another by more than one chemical bond (Drabi?ska et al. 2020). In this case, the spectroscopy hydrogens are coupled to the neighbouring hydrogen atoms and others with the help of off-diagonal peaks.
No negative peaks have been obtained for which an extended analysis was conducted on the 1H-1H COSY spectrums. The signals obtained from the results of “Heteronuclear single quantum coherence spectroscopy” (HSQC) of octanoic acid displays directly attached hydrogen to 13C heteronuclei.
The 2D spectroscopy helped in obtaining the structure elucidation and helped uncover the signals that were overlapping in the standard 2D spectroscopy. The elucidation, quantification and identification were performed for both the compounds using NMR spectroscopy.
3.2.3.2 Peak Integration
The integration of the peaks of hydrogen in each case is observed to be 0.9. The percentage of purity is measured by multiplying the area surrounding characteristic hydrogen by 100. In this case, the total area of the two characteristic hydrogens a and b are being considered, hence the total area is 0.9+0.9=1.8.
Hence, the purity percentage = 0.9/(1.8)*100
=0.5*100
=50% purity
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