Nuclear Magnetic Resonance of a Nitrogen and Sulfur Containing Ligand
Introduction
1.1 NMR Spectrometer
Nuclear Magnetic Resonance (NMR) spectroscopy is an analytical technique extensively used by chemists to determine chemical structures. This potent method depends on the NMR spectrometer, which acquires the necessary data for structural interpretation. Figure 1 depicts the basic mechanism of an NMR spectrometer. To begin with, a thin glass tube placed between a strong magnetic field usually contains an organic sample dissolved in deuterochloroform (CDCl3) a common solvent used in NMR spectroscopy. The sample is then exposed to a short pulse of radiation emitted by the rf (radiofrequency) generator. As the sample absorbs the rf energy, a detector measures the intensity of the energy absorption. Ultimately, the detector’s electrical signal is amplified and plotted on an NMR spectrum.
Figure 1: NMR Spectrometer
1.2 Nuclear Magnetic Resonance Spectroscopy
NMR spectroscopy specifically identifies atomic nuclei and the chemical environments they occupy within a molecule. To accomplish this, NMR spectroscopy utilizes the magnetic properties of specific nuclei. Nuclei have a quantum property called nuclear spin, which determines how sensitive they are to an external magnetic field. The nuclear spin value is determined by the number and composition of protons and neutrons within the nucleus as can be seen in Table 1.
Table 1: Nuclear spins and their common features.
All nuclei with an odd number of protons (1H, 2H, 14N, 19F, 31P) or neutrons (13C), are NMR active. Not all nuclei have this property; namely the ones with even numbers of both protons and neutrons (12C) and thus they do not exhibit the NMR phenomenon. To some extent, though this is an oversimplification, nuclei with a non-zero spin value behave like tiny magnets and can interact with the external magnetic field, denoted B0. Particularly, due to their spin, nuclei can create their own magnetic field. These nuclei are referred to as magnetic nuclei and devoid of an external magnetic field their spins are positioned randomly. In contrast, when an external field is applied the nuclei take on specific orientations as can be seen in figure 2. Certain spins align with the external field (parallel), whereas others align in the opposite direction (antiparallel). The parallel orientation is in lower energy than antiparallel orientation and is therefore favoured. When nuclei are exposed to electromagnetic radiation with adequate frequency, the nuclei absorb the energy which flips them from a low-energy state to high-energy. When this “flip” occurs, it indicates that nuclei are in resonance with the applied radiation.
Figure 2: Orientations of nuclei a) without an external magnetic field b) parallel (pink arrows) and antiparallel (blue arrows) orientations due to applied magnetic field.
Three factors are prominent in determining the exact radio frequency needed for resonance: strength of the external magnetic field, characteristics of the nucleus and the electronic environment within the nucleus. Among these factors, the strength of the external magnetic field directly influences the energy difference, denoted ΔE, between the two spin states. As illustrated in figure 3, two spin states are equal in energy in the absence of an external field. However, the energy gap between the two spin states grows as the applied magnetic field gets stronger. Therefore, a higher-frequency radiation is essential to convert the nucleus from its ground state to a higher-energy spin state.
Figure 3: When a stronger external field is applied, the energy difference between the two spin states simultaneously gets greater.
1.3 Shielding and Chemical Shifts
All nuclei within a molecule are surrounded by electrons which are in different electronic (magnetic) environments. The electrons surrounding the nuclei can create their own local magnetic fields that partially cancel out the strength of the applied field. More specifically, the actual field strength that the nuclei feel is slightly weaker than the applied field. Nuclei are said to be shielded by electrons from the external magnetic field. The shielding effect can be summarized with the following expression:
Beffective = Bapplied – Blocal2
While shielding is affected by several factors, including electronegativity, magnetic anisotropy of π systems and hydrogen bonding, this discussion will concentrate on electronegativity, as it plays a prominent role in determining the chemical shifts. Electronegativity is the ability of an atom to attract electrons toward itself, this can be compared with the data in Table 2. For instance, fluorine (F) is the most electronegative atom, and it decreases the electron density surrounding an adjacent nucleus. This results in the nucleus being deshielded and experiencing a stronger external field compared to a highly shielded nucleus.
The applied field strength increases from left to right on an NMR chart, implying that nuclei that absorb energy on the at a higher ppm are less shielded and result in a higher chemical shift.
Table 2: Chemical Shifts and how electronegativity can affect them.
The delta (δ) scale is an arbitrary scale used to calibrate NMR spectra. It is expressed in parts per million (ppm), where 1 δ equals 1 ppm of the spectrometer's operating frequency. Because the NMR chart has an arbitrary value, a reference point is necessary to define the position of energy absorption. TMS (tetramethylsilane; ([CH3)4Si]) is assigned a chemical shift of zero and is used as the reference point for both 1H and 13C NMR. Chemical shift can be calculated with the following equation:
𝛿 = 𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑 𝑐ℎ𝑒𝑚𝑖𝑐𝑎𝑙 𝑠ℎ𝑖𝑓𝑡 (𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐻𝑧 𝑎𝑤𝑎𝑦 𝑓𝑟𝑜𝑚 𝑇𝑀𝑆) / 𝑆𝑝𝑒𝑐𝑡𝑟𝑜𝑚𝑒𝑡𝑒𝑟 𝑓𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦 𝑖𝑛 𝑀𝐻𝑧
Regardless of the spectrometer's operating frequency, the chemical shift expressed in δ (ppm) remains constant. This consistency allows NMR spectra to be compared even when recorded on different instruments. For example, a ¹H nucleus that absorbs at 2.0 ppm on a 200 MHz spectrometer will also appear at 2.0 ppm on a 500 MHz instrument.
1.4 Types of NMR
As stated above, all nuclei with an odd number of protons or neutrons are NMR active. Thus, 1H NMR and 13C NMR are not unique in their ability to produce signals. They are, however, the most common and can give useful information about L1 as most of the atoms in L1 are C or H atoms. This discussion will therefore concentrate on 1H, 13C NMR and their correlation NMRs: COSY (Correlated Spectroscopy) and HSQC (Heteronuclear Single Quantum Coherence).
1.4.1 1H NMR
Hydrogen NMR, also referred to as proton NMR, is one of the most important techniques in organic chemistry for the characterization of every molecule. One use of proton NMR is to determine how many chemical environments are present within a molecule. Accordingly, different absorption peaks in the proton NMR spectrum correspond to chemically non-equivalent 1H nuclei in the molecule. On the other hand, a chemically equivalent proton shows identical absorption peaks. Most 1H NMR absorption peaks appear within a quite narrow range downfield, about 0 to 10 ppm.
It is possible to count the number of protons by looking at the relative integration of each peak in a ¹H NMR spectrum. Integration refers to the area under each peak, which is proportional to the number of protons producing that peak. Modern NMR instruments can measure the area which is represented by the integral line or/and by integration values under each peak. When integration values are displayed on the spectrum, dividing each value by the smallest one gives the relative ratio of the protons. This ratio can then be scaled to match the total number of protons in the molecule. In contrast to this, an older NMR instrument, which can be seen on figure 4, integrates the peaks using the stair-step method; the height of each step indicates the area under the peaks and therefore corresponds to the relative number of protons.
Figure 4: NMR spectrum produced by an older instrument.
Upon analyzing a proton NMR spectrum, an absorption signal can split into multiple peaks (multiplet). These splitting patterns are caused by tiny magnetic fields created by neighbouring nuclei, which affect the magnetic field felt by a nucleus of interest. This phenomenon is called spin-spin splitting, where the interactions are referred to as coupling. Note that chemically equivalent protons do not split each other’s signal, as they show identical absorption peaks.
Table 3: Names of peaks they have a certain number of adjacent protons.
As mentioned above, a nucleus can have different spin states depending on whether it aligns with or against the applied magnetic field. When adjacent protons are parallel to the applied field, their own magnetic field can amplify the strength of the applied field felt by a proton of interest. Otherwise, the antiparallel orientation can slightly diminish the strength. Though this is an oversimplification, the splitting generally tells us how many adjacent protons are present within the range of three bonds or less. The n + 1 rule, where n is the number of the neighboring protons, states exactly how many peaks a single signal would split into. The coupling constant, denoted J, is expressed in Hz typically ranging from 0 to 18 Hz; is the distance between split peaks. When two proton groups are related to each other, meaning that their magnetic fields affect each other, they have the same coupling constant.
1.4.2 13C NMR
While ¹³C NMR is based on principles like those of proton NMR, there are subtle differences in their operation. Absorption peaks in ¹³C NMR spectra appear over a wide range from 1 to 220 ppm. 6 Carbon-13 has a natural abundance of 1.1% making it rather tricky for NMR instruments to detect. However, signal averaging performed by Fourier-transform NMR (FT-NMR) has smoothed the path for detecting 13C. Due to the low natural abundance, 13C is relatively weak compared to background electronic environments. The noisy background seen in figure 5, is removed by running hundreds or thousands of trials and subsequently averaged by a computer; hence the term signal averaging. On an ordinary NMR instrument 1H and 13C spectra can’t be observed at once, as they require different rf energy to flip. FT-NMR, on the other hand, allows all 1H and 13C nuclei to resonate simultaneously; which results in complex signals that are mathematically manipulated by Fourier transforms and plotted on an NMR spectrum as usual.
In contrast to proton NMR, 13C NMR spectra do not exhibit splitting patterns since it is unlikely that another 13C nucleus would be adjacent due to its low natural abundance. Likewise, protons’ magnetic fields will not affect 13C nuclei as they are usually recorded with broadband decoupling. This technique performs a second rf energy irradiation that causes the protons spin-flip so quickly that their local fields become equal to zero. 13C nuclei do not uniformly generate the same peak intensity, therefore their spectra are not integrated in the same way as proton NMR spectra. This significant difference in peak size is also a result of broadband decoupling.
Figure 5: Carbon-13 NMR spectra of 1-pentanol, CH3CH2CH2CH2CH2OH.
1.4.3 Two-dimensional NMRs: COSY and HSQC NMR
When looking only at a one-dimensional proton NMR spectrum, it can be challenging to determine which protons are coupling. This is where COSY (Homonuclear Correlation Spectroscopy), seen in figure 6, comes in handy. It is a two-dimensional NMR technique that shows exactly which protons are coupled to each other. COSY NMR takes advantage of the fact that coupled peaks share the same coupling constant. The one-dimensional NMR is plotted against itself, as shown in figure 6. It may be useful to draw a diagonal line directly through a COSY spectrum since any signal that appears on this line corresponds to the signal coupling to itself. Additionally, the COSY spectrum is in fact symmetrical around the diagonal line because coupling is a reciprocal interaction between protons. The points of interest are therefore the off-diagonal peaks or cross peaks which indicate the protons that couple to each other.
Figure 6: COSY spectrum with a diagonal line.
Similar to COSY, HSQC (Heteronuclear Single Quantum Coherence) is also a two-dimensional NMR method. However, while COSY shows coupling between protons, HSQC on figure 7 focuses on identifying which protons are directly bonded to each 13C. In other words, the cross peaks in an HSQC spectrum indicate the relation between protons and 13C. In the HSQC spectrum, the proton NMR signals are plotted along the x-axis, while the 13C NMR appear along the y-axis. Whereas HSQC NMR solely determines a single bond connectivity between protons and 13C, HMBC (Heteronuclear Multiple Bond Correlation) can give the correlation between these nuclei despite them being separated by multiple bonds away. Nevertheless, to narrow the scope, HMBC will not be considered further in this study.
Figure 7: HSQC NMR spectrum.
In this paper, I will be analyzing the 1H, 13C, COSY and HSQC NMRs of the following ligand. L1 is a nitrogen and sulfur containing ligand with the molecular formula C19H30N2S4 and the molecular weight 414.703 g/mol. The goal of this study is to confirm the identity of the ligand and to provide a deeper insight into the application of NMR spectroscopy techniques.
Figure 8: L1 structure.
2. Experimental Section
2.1 Materials and methods
All chemicals and solvents were purchased from Sigma-Aldrich, Merck, Fluorochem and TCI-Europe, and were utilized as supplied unless stated otherwise.
A Bruker Advance 600 spectrometer was used to record solution state 1H-NMR spectra, 13C NMR spectra, COSY spectra and HSQC spectra (600 MHz).
2.2 Synthesis
Figure 9: Reaction scheme of L1.
L1 (C19H30N2S4) was synthesized according to the following procedure: 1.213 g of (1) was dissolved in methanol and refluxed for 15 minutes. 0.311 g of 2,2-dimethylpropane-1,3-diamine was then added to the solution and stirred for 48 hours. The resulting mixture was filtered, and the precipitate was washed with cold methanol. The residue was subsequently dried in a vacuum. The yield was 0.713 g and 56.6%.
3. Results and Discussion
3.1 1H NMR of L1
Figure 10: `H-NMR of L1
Figure 11: 1H-NMR of L1 in a narrow range.
A line of symmetry is drawn on the molecular structure of L1 to help identify the number of signals or chemical environments. The line shows that the two sides of the molecule are identical, meaning the protons on the left and right sides are chemically equivalent.
The integration values under each signal are used to determine the number of protons each peak represents. Dividing all values by the smallest (2.00) results in the approximate ratio of the protons: 3 : 2 : 3 : 2 : 2 : 2 : 1. Since L1 contains a total of 30 protons, the ratio is multiplied by 2 to obtain the actual number of protons shown in the spectrum above.
To determine the chemical shifts of each signal, electronegativity was considered. Signal (g) is seen at about 12.5 ppm, which means the proton it belongs to us highly deshielded. The NH proton is expected to be the most deshielded, as N is very electronegative, and would decrease the electron density on the proton it’s bonded to. Hence signal (g) most likely belongs to the NH proton.
Taking a closer look at the splitting patterns, it is evident that signals (a) and (c) are singlets, as these proton groups do not couple with any others. The remaining signals are multiplets because they couple with adjacent proton groups. Namely, signal (b) appears as a quintet, indicating that this proton group has four adjacent protons. Signals (d) and (e) appear as triplets, as each has two neighboring protons. Lastly, signal (f) appears solely as a doublet, since it only has one neighbouring proton. It is quite difficult to determine which signals are coupling simply by analyzing a one-dimensional proton NMR, which is why COSY NMR becomes very helpful.
Figure 12: 1H-NMR spectrum of L1, a closer look at peaks b, d, e and f.
3.2 COSY NMR of L1
Figure 13: COSY NMR spectrum of L1.
Figure 14: COSY NMR spectrum of L1 at a narrow range.
As mentioned above, COSY NMR is extremely useful for determining which protons are coupled to each other. Based on the spectrum above, protons giving signal (f) are coupled to those giving signal (g) and they are only separated by two bonds. Although signal (g) appears as a singlet, it represents the NH group which typically display as a broad singlet on NMR spectra. The OH proton, which does not appear on the spectrum behaves similarly because they are labile – meaning that they can suddenly disappear off the molecule and return. OH and NH proton groups are not entirely present to interact with adjacent protons. Moreover, they are moving way to rapidly for the NMR instrument to analyze energy absorption from them. Finally, signal (b) has four neighbouring protons and couples to signals (d) and (e).
1.3 13C NMR of L1
Figure 15: 13C-NMR of L1
Electronegativity was also utilized to assign each chemical shift to 13C signals. Signal (a) is expected to appear at 16.68 ppm as the 13C atom is multiple bonds away from the NH proton. Whereas signal (j) at 197.93 ppm is likely the most deshielded since it is directly double bonded to an adjacent sulfur. Note that there are only 19 carbons in total, as there is one atom on the line on the line of symmetry. As mentioned earlier, 13C NMRs do not exhibit splitting patterns due to its low natural abundance. They also do not generate peak intensity uniformly, so integration cannot be used in this context.
3.4 HSQC NMR of L1
Figure 16: HSQC of L1
The HSQC spectrum above helps determining which protons are directly bonded to 13C in L1. It is quite difficult to assign peaks for 13C just by considering electronegativity. Besides indicating the connectivity between protons and carbons, HSQC was very useful in assigning 13C peaks. This spectrum was observed with the following manner: signal 1Ha is bonded to 13Cc , which confirms that peak c) at 24.14 ppm on 13C-NMR belongs to carbon C on figure 17 (blue). The following figure further confirms the results from the HSQC spectrum above.
Figure 17: L1 structure with 1H atoms and 13C atoms highlighted.
4. Conclusion
L1 (C19H30N2S4), a nitrogen and sulfur containing ligand, was analysed using 1H, 13C, COSY and HSQC NMR spectroscopy techniques. These methods further confirmed the presence and connectivity of 1H and 13C in L1. Taking everything into account, NMR spectroscopy is a paramount technique in determining the chemical structure of any molecule, as each chemical shift, signal and splitting pattern directly informs about the chemical environment of the nuclei of interest. Notably, COSY and HSQC were very valuable in determining which protons were coupled to one another and which were directly bonded to ¹³C.
5. References
1. McMurry, J. Why This Chapter? In Organic Chemistry: A Tenth Edition [Online]; 10th ed.; OpenStax, 2023. https://openstax.org/books/organic-chemistry/pages/13-why-this-chapter
2. McMurry, J. The Nature of NMR Absorptions. In Organic Chemistry: A Tenth Edition[Online]; 10th ed.; OpenStax, 2023. https://openstax.org/books/organic-chemistry/pages/13-2-the-nature-of-nmr-absorptions
3. Smirnov, S. L.; McCarty, J. Nuclear Spin and Magnetic Field. In Biophysical Chemistry[Online]; LibreTexts Chemistry, n.d.https://chem.libretexts.org/Courses/Western_Washington_University/Biophysical_Chemistry_(Smirnov_and_McCarty)/05%3A_Nuclear_Magnetic_Resonance_(NMR)_Spectroscopy_-_Introduction/5.01%3A_Nuclear_Spin_and_Magnetic_Field
4. Kennepohl, D.; Farmer, S.; Spinney, R. Chemical Shifts and Shielding [Online];LibreTexts Chemistry, n.d.
5. Wong, F. NMR Made Easy! Part 1 - Electronegativity and Shielding - Organic Chemistry.Youtube, Oct 31, 2012. https://youtu.be/9orcRVTKcS0?si=nZzeWcOHWvx_EEr_
6. McMurry, J. Chemical Shifts. In Organic Chemistry: A Tenth Edition [Online]; 10th ed.;OpenStax, 2023. https://openstax.org/books/organic-chemistry/pages/13-why-this-chapter
7. McMurry, J. 1H NMR Spectroscopy and Proton Equivalence. In Organic Chemistry: A Tenth Edition [Online]; 10th ed.; OpenStax, 2023. https://openstax.org/books/organic-chemistry/pages/13-7-1h-nmr-spectroscopy-and-proton-equivalence
8. McMurry, J. Integration of 1H NMR Absorptions: Proton Counting. In Organic Chemistry: A Tenth Edition [Online]; 10th ed.; OpenStax, 2023.https://openstax.org/books/organic-chemistry/pages/13-5-integration-of-1h-nmr-absorptions-proton-counting
9. McMurry, J. Spin–Spin Splitting in 1H NMR Spectra. In Organic Chemistry: A Tenth Edition [Online]; 10th ed.; OpenStax, 2023. https://openstax.org/books/organic-chemistry/pages/13-7-1h-nmr-spectroscopy-and-proton-equivalence
10. McMurry, J. 13C NMR Spectroscopy: Signal Averaging and FT–NMR. In Organic Chemistry: A Tenth Edition [Online]; 10th ed.; OpenStax, 2023. https://openstax.org/books/organic-chemistry/pages/13-10-13c-nmr-spectroscopy-signal-averaging-and-ft-nmr
11. McMurry, J. Characteristics of 13C NMR Spectroscopy. In Organic Chemistry: A Tenth Edition [Online]; 10th ed.; OpenStax, 2023. https://openstax.org/books/organic-chemistry/pages/13-11-characteristics-of-13c-nmr-spectroscopy
12. Silverstein, R.M; Webster, F.X; and Kiemle D.J. Two Dimensional Homonuclear NMR Spectroscopy [Online]; LibreTexts Chemistry, n.d. https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Introduction_to_Organic_Spectroscopy/07%3A_Two-Dimensional_NMR_Spectroscopy/7.03%3A_Two_Dimensional_Homonuclear_Spectroscopy
13. Silverstein, R.M; Webster, F.X; and Kiemle D.J. Two Dimensional Heteronuclear NMR Spectroscopy [Online]; LibreTexts Chemistry, n.d. https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Introduction_to_Organic_Spectroscopy/07%3A_Two-Dimensional_NMR_Spectroscopy/7.04%3A_Two_Dimensional_Heteronuclear_NMR_Spectroscopy