JEOL USA Blog

What Is Surface Analysis?

Surface analysis is an analytical technique to elucidate elemental composition, chemical state, and micro structure from material surface layer (several nm to several µｍ). As phenomena such as corrosion, wear, adhesion, and reactions that impact performance and reliability occur primarily on the surface, surface analysis is vital for material evaluation, quality control, and failure analysis.

For analysis, it is necessary to select the most suitable method, according to the sample state (target point, size, material, etc.) and analysis purpose.

What information we want to know? What is the material of the sample?
What is the range of information that we want to know? How deep?
Is it water-soluble? Does it react with solvents? Pre-treatment needed?

It is important to select the analytical method suitable for the purpose.

Types and features of surface analysis instruments

The figure below shows the comparison of typical surface analysis methods from various points of view, such as excitation source, detection signal, quantitativeness, whether the chemical state can be analyzed or not, sensitivity, handling of an insulator, and analysis capability of depth direction. It is important to understand the feature of each method and properly select the analysis method according to the purpose.

Analytical methods	EPMA (WDS)/SXES/EDS	AES	XPS	XRF	SIMS
Excitation source	Electron beam	Electron beam	X-ray	X-ray	Ion
Signal	Characteristic X-ray	Auger electron	Photoelectron	Fluorescence X-ray	Secondary ion
Detectable element	Be ~ (WDS, EDS) Li (SXES, Windowless EDS)	Li ~	Li ~	C ~	H~
Quantitative analysis	○	○	○	○	×
Chemical state	△	○	○	×	Organic compound
Detection depth	Several µm	Several nm	Several nm	Several mm	Several nm
Sensitivity	Several ten ppm （Mass concentration）	Several thousand ppm （Atomic concentration）	Several thousand ppm （Atomic concentration）	Several ten ppm （Mass concentration）	Several ppm （Atomic concentration）
Insulator	○ (Conductive coating)	△	○	○	○
Depth analysis	△	○	○	×	○
Strength	Qualitative analysis Quantitative analysis Wide area ~ micro area analysis	Micro area analysis Chemical bonding state analysis Depth profile analysis	Insulator analysis Chemical bonding state analysis Depth profile analysis	Qualitative analysis Thin film analysis Trace element analysis	Organic substance analysis Trace element analysis
Challenge	Chemical bonding state analysis (Strong at SXES) Organic substance analysis	Wide area analysis Insulator analysis Organic substance analysis	Micro area analysis Trace element analysis	Micro area analysis	Qualitative analysis Quantitative analysis

In this column, we explain surface analysis instruments that JEOL offers, such as XPS (photoelectron spectrometer), AES (Auger microprobe) , XRF (X-ray Fluorescence Spectrometer) , EPMA (Electron Probe Microanalyzer) with *standard wavelength-dispersive X-ray spectrometer, SEM+EDS (Scanning Electron Microscope＋Energy Dispersive X-ray Spectrometer), and SXES (soft X-ray emission spectrometer) that can be installed to EPMA (WDS) and SEM.
We clearly explain each mechanism, its strengths and weaknesses in analysis, and key points for selecting the instrument.

Difference of analysis area/depth according to surface analysis instrument

XRF enables elemental analysis in the deepest and widest region. It is suitable for understanding the average composition of the entire bulk material and is utilized in qualitative/quantitative analysis in a wide field of view.

On the other hand, SEM + EDS and EPMA (WDS) can investigate the local elemental distribution by detecting x-rays that are generated in a local area of about several micrometers. SEM+EDS enables simultaneous evaluation of morphology and elemental analysis, while EPMA provides superior capabilities in more precise quantitative analysis and area analysis.

Moreover, AES and XPS makes it possible to obtain signals from the very shallow surface layer of about several nanometers deep. They are optimal for evaluating chemical state of the surface layer such as surface processing, contamination, and oxidization state.

Thus, to investigate the extreme surface at the nanometer scale, AES or XPS is suitable. For local analysis at the micrometer level, SEM combined with EDS or EPMA is appropriate. If the target is a wide area on the millimeter scale, XRF is the best choice. The appropriate instrument varies depending on the required analysis depth and the field of view size.

Difference in principles and detection signals of surface analysis instruments

As shown below, each instrument has a different excitation source (incident probe) and detection signal, and the information obtainable is different according to their features.

Points for selecting surface analysis method

In surface analysis, it is important to select the appropriate technique based on the properties of the specimen and the purpose of the analysis. For specimens that are susceptible to vacuum, such as biological or liquid specimens, methods like XRF, which can be performed under atmospheric pressure, or SEM equipped with a low-vacuum mode are effective. If the specimen can withstand a vacuum environment, more sensitive and higher-resolution techniques such as XPS, AES, or EPMA can also be considered. This section introduces the optimal analytical method for each purpose, along with the figures.

Qualitative/Quantitative Analysis

Area Analysis

State Analysis

Summary

Surface analysis is a technique to obtain key information that directs to performance and reliability of the instrument. This article explains the tips of instrument selection through the features of typical analysis methods and points of selection, strength by instrument, and concrete application examples.

JEOL Ltd. has product line-ups that can satisfy a wide range of needs from beginners to researchers, and that can be utilized with support from introduction to operation.

COSY/TOCSY Analysis│Interpreting spin correlations using 2D NMR

In this article, we explain the basic principles and analysis methods of COSY and TOCSY, which are representative techniques of 2D NMR. Starting from confirming correlations between adjacent protons using COSY, we introduce spin network analysis with TOCSY and analysis of carbohydrate using 1D and 2D TOCSY, with concrete examples.

What is COSY?

COSY（COrrelation SpectroscopY）is a basic method for visualizing couplings between adjacent ¹Hs by using 2D NMR. Traditionally, homodecoupling of ¹Hs was used to locate neighboring ¹H atoms one by one. However, with the appearance of COSY, it became possible to analyze ¹H-¹H coupling correlations simultaneously over a broad range, dramatically improving the efficiency of structural analysis. Currently, COSY is widely used as an introductory 2D NMR method for confirming ¹H-¹H correlations.

COSY shows correlations between adjacent ¹Hs - in other words, ¹Hs that are 3 bonds apart. The spin coupling of 3 bonds apart is expressed as ³J_HH. If the adjacent ¹H is known, the connection of ¹Hs in a molecule can be understood.

Fig.1 COSY spectrum of cinnamic acid cis-3-hexenyl ester

Fig.1 shows a COSY spectrum for the region corresponding to ¹Hs at 1 to 6 in "cinnamic acid cis-3-hexenyl ester". As indicated above, when the correlation signals and ¹H spectrum of 1D on both axes are connected, 5 correlations can be observed: 1-2, 2-3, 3-4, 4-5, and 5-6. The result allows us to infer that ¹Hs from 1 to 6 have a neighboring structure each. In addition, the COSY allows us to understand the connection between carbon atoms indirectly from the coupling information of neighboring ¹Hs. The information is extremely important for determining the substructure.

Fig. 2 Complex COSY spectrum

However, what happens when the COSY spectrum is as complex as the one in Fig. 2? Looking at the region highlighted in green, you can see that the signals overlap, making it hard to identify correlations. In this situation, moving forward with structure determination seems almost impossible.

For compounds like polysaccharides or cyclic molecules, where ¹H signals densely appear in a narrow range as in Figure 2, relying on COSY alone can lead to a dead end in structural analysis.

One solution for the situation when the COSY does not adequately analyze is TOCSY. We will explain TOCSY in the next section.

What is TOCSY?

TOCSY（TOtal Correlation SpectroscopY）is a 2D NMR technique that allows you to visualize all ¹Hs belonging to the same spin network at once. Because spin couplings propagate through the network, TOCSY reveals not only adjacent positions but also correlations across the entire spin system, making it highly effective for analyzing complex organic molecules, sugars, and amino acids. TOCSY is also known as HOHAHA（HOmonuclear HArtmann-HAhn spectroscopy), which essentially refers to the same experiment.

Fig. 3 Network of continuous ¹H spin couplings

For example, suppose that there are atomic connections as shown in Figure 3. H_A・H_B・H_C・H_D are connected through spin couplings between neighboring ¹Hs, and this connection is called spin system (spin network). The spin system does not propagate through quaternary carbons without attached H or ¹H coupled via oxygen, so the network is interrupted at these points. TOCSY allows us to observe correlations between nuclei within the same spin system even if they are not directly coupled--for instance, between ¹H_A and ¹H_D. In TOCSY, the magnetization is transferred step by step: from H_A to H_B, then from H_B to H_C, and finally from H_C to H_D. In other words, magnetization moves through the spin system, connecting everything along the way. This magnetization transfer is called "relay". TOCSY observes the correlation signals based on this relay.

Fig. 4 Pulse sequences of COSY and TOCSY

Fig. 4 shows the pulse sequences for COSY and TOCSY. COSY uses two 90° pulses, while with the TOCSY, the second pulse is a spin lock pulse. The duration of this spin lock pulse is called "mixing time," which is a key parameter with TOCSY. By increasing the mixing time, you can observe correlation signals that have been relayed over longer distances within the spin system.

Mixing time and correlation signal of TOCSY

Fig. 5 Schematic diagram of TOCSY spectrum

Fig. 5 shows a schematic diagram of a TOCSY spectrum of a compound having two independent spin networks. Each spin network is expressed in yellow and in green. As shown, TOCSY observes correlation of all ¹Hs belonging to each spin network. At that time, if signals like "A" and "a" within the primary spectrum on both axes appear away from the region where signals appear densely, the signals are easily classified separately in the networks of yellow and green, enabling confirmation of each spin network.

Fig.6 Schematic diagram of TOCSY (Correlation of A)

Next, let's look at how the correlation signal of A changes when the mixing time is varied. Figure 6 is an enlarged view of the correlation of A from the schematic TOCSY diagram in Figure 5. With a short mixing time, the magnetization of ¹H_A moves only to the neighboring ¹H_B, and the correlation signal B with ¹H_B appears. When the mixing time is further increased, the magnetization moves to ¹H_C, and correlation signal C appears. When the mixing time is increased even more, the magnetization moves to ¹H_D, and correlation signal D appeared. As a result, it becomes clear that there is a connection of ¹H_A - ¹H_B - ¹H_C - ¹H_D.

The longer the mixing time, the farther the magnetization can move. Therefore, if a sufficiently long mixing time is used, the magnetization can be transferred through all 1Hs within the same spin system, allowing the detection of correlation signals for all 1Hs in that spin system. Furthermore, by comparing spectra obtained with different mixing times, the sequential order of the 1H connections can also be known.

Example of analysis for sucrose using 2D TOCSY

20 mg / 0.6 mL D2O solution (400 MHz)

Here, we present an example of analysis for sucrose using 2D TOCSY. Sucrose is a disaccharide composed of glucose and fructose linked by a glycosidic bond. Sucrose contains a total of 22 ¹Hs, but since the sample is dissolved in heavy water, the ¹Hs of the hydroxyl groups (-OH) are not observed due to deuterium exchange. Therefore, in this case, 14 ¹Hs are observed, excluding the 8 ¹Hs of hydroxyl group.

Fig.7 COSY spectrum of sucrose

Let us show you the COSY spectrum of sucrose in Fig. 7. The part highlighted in the red frame is enlarged and shown in Fig. 8.

Fig. 8 COSY spectrum of sucrose

In Figure 7, six correlation signals can be found relatively easily. However, it is difficult to interpret the correlations of signals appearing in areas where chemical shifts are close to each other, such as those indicated by the red circles in Figure 8. Many people may get stuck at this point in the analysis. Therefore, we will try using the TOCSY method.

TOCSY spectrum with mixing time of 20ms

TOCSY spectrum with mixing time of 150ms

Fig. 9 TOCSY spectra measured with mixing times set to 20 ms and 150 ms

Figure 9 shows the TOCSY spectra measured with mixing times set to 20 ms and 150 ms, respectively. First, we focus on the correlation signal of the ¹H at the anomeric position of glucose, which appears at a chemical shift well away from other signals. As shown earlier in Figure 6, we will examine how the correlation signals change with different mixing times, to confirm the relay information from the ¹H at the anomeric position. It can also be observed that more correlation signals appear as the mixing time increases. Furthermore, for regions where signals overlap, it is easier to compare by using sliced data rather than the 2D spectrum. Therefore, we will compare each 1D spectra obtained by extracting slices along the X-axis (in the area indicated by the green frame) for the confirmation of correlation signal of the ¹H.

Figure 10. One-dimensional spectra sliced along the X-axis for the correlation signal of the 1H at the anomeric position of sucrose

The top spectrum in Figure 10 shows a conventional ¹H spectrum. Below it are sliced data obtained with mixing times varied from 20 ms to 200 ms. The signal on the left corresponds to the Glu H-1, which we consider as the starting point of the relay. At a mixing time of 20 ms, only the correlation with the neighboring Glu H-2 appears, revealing only the connection between positions 1 and 2. As the mixing time increases, correlation signals appear sequentially, and eventually, the connections from position 1 to position 6 in the glucose moiety can be confirmed. While it was difficult to clearly identify the correlation partner of the Glu H-5 using COSY, TOCSY provided this information.

Next, let us examine the relay starting from the Fru H-3′. As with the sucrose moiety, Figure 11 shows the TOCSY spectra obtained with different mixing times.

TOCSY spectrum measured with mixing time of 20ms

TOCSY spectrum measured with mixing time of 150ms

Figure 11. TOCSY spectra measured with mixing times set to 20 ms and 150 ms

As before, we compare sliced data along the X-axis for a signal that appears in a region without significant overlap--this time focusing on the Fru H-3′.

Fig. 12 One-dimensional spectra sliced along the X-axis for the correlation signal of the H-3′ proton of fructose

The top spectrum in Figure 12 shows a conventional ¹H spectrum, and below it are sliced data obtained with different mixing times. As before, we take the Fru H-3′ as the starting point of the relay. As the mixing time increases, correlations with the Fru H-4′, Fru H-5′, and Fru H-6′ appear, allowing us to confirm the connections within the fructose moiety.

In this way, TOCSY enables the separation of spin networks within a compound that contains multiple spin networks. By varying the mixing time, it is also possible to assess the distance from the starting ¹H. Even when the COSY spectrum becomes complicated, using TOCSY provides significant assistance in determining the molecular structure.

Example of analysis for glucose using 1D TOCSY

1D TOCSY is the one-dimensional version of 2D TOCSY, and the basic principle is the same. While 2D TOCSY is used to analyze the spin network of the entire molecule, 1D TOCSY is more effective when you want to examine only the spin system to which a specific ¹H belongs. 1D TOCSY observes the magnetization transfer--i.e., the relay--from a selectively excited ¹H. By gradually increasing the mixing time, the propagation of the magnetization relay along the spin network can be tracked. For selective excitation, it is best to choose a 1H whose chemical shift is sufficiently separated from other signals. Another advantage of 1D measurement is that, compared to 2D measurement, it offers higher digital resolution and makes it easier to avoid signal overlap.

Now, let us present an example of glucose analysis.

Fig 13. ¹H spectrum of glucose

Glucose exists in aqueous solution as α- and β-anomers. Therefore, the ¹H NMR spectrum is a mixed spectrum of α-glucose and β-glucose, as shown in Figure 13. For selective excitation of a ¹H signal, it is typically to choose ¹H appearing at a lower field than others (in the case of sugars, ¹H at the anomeric position). The result of a 1D TOCSY experiment by selectively exciting the anomeric ¹H at position 1 of α-glucose is shown in Figure 14.

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Fig.14 1D TOCSY spectrum of glucose

By varying the mixing time from 20 ms to 200 ms, it was possible to trace the spin network starting from the anomeric ¹H at position 1 of α-glucose as the relay point. The bottom spectrum with a mixing time of 200 ms represents the extraction of only the spin network of α-glucose from the mixed spectrum of α-glucose and β-glucose. Furthermore, as explained earlier, 1D TOCSY provides better digital resolution than 2D sliced data. Thus, 1D TOCSY is more effective for extracting and confirming spin systems connected through spin couplings from crowded spectra. It is recommended to use 1D TOCSY and 2D TOCSY appropriately depending on the purpose.

Basics of NOE/NOESY: Causes and Solutions When NOE Is Not Detected

NOE (Nuclear Overhauser Effect) is an important NMR measurement technique that can reveal spatial proximity relationships within molecules. In this column, we discuss the difference between difference NOE, 1D NOESY, and 2D NOESY, and how to select each method, the relaxation time that is key for analysis. The causes and solutions for cases when NOE is not detected are also covered. Moreover, we answer the frequently asked questions and explain the key points for effective use of NOE measurement.

What is NOE measurement?

NOE measurement (Nuclear Overhauser Effect) is a method to observe nuclei in spatially close distance. The NOE measurement is utilized to obtain stereochemistry of molecules, and distinguish isomers and confirm location of substituent group, for application of small molecular compound.

Principle of NOE

The NOE is a phenomenon where spatially close nuclei magnetically affect each other and cause an NMR signal intensity change.

How to observe NOE

Different from the splitting by spin-coupling, the NOE is not observed with the NMR spectrum (1D, 2D NMR) that we have introduced so far.
Therefore, the following steps are required for the NOE measurement.

Irradiating radio wave to specific proton (excitation/saturation)
Observing signal intensity change of other protons
Judging if this change is coming from the NOE

Types of NOE Measurement

There are three main types of NOE measurements.

Difference NOE: observe a steady state NOE by taking the difference in spectra with and without radio wave irradiation.
2D NOESY: observe transient NOE by using a 2D spectrum.
1D NOESY: selectively excite protons and observe transient NOE in one dimension.

Steady state NOE is to irradiate a radio wave for a long time, make a specific proton saturated, and observe the signal intensity change of other protons once the saturation state becomes steady.

Transient NOEs are observed during the mixing time after the spin state is temporarily changed by a short period of radio wave irradiation. Since the mechanisms by which NOEs are generated are different, the measurement conditions for each method must be determined by considering the characteristics of each.

Difference NOE Measurement

Difference NOE measurement is a method of checking the change in signal intensity of nearby protons by taking the difference between the spectra of a specific proton when irradiated with radio waves and when not irradiated with radio waves.

As shown above, two protons, H_I and H_S are going to be considered. HI and HS are in spatially close distance and having a dipole interaction. (B) is 1H spectrum when HS was not irradiated.

Here, when a radio wave is irradiated to H_S to saturate H_S, the signal of H_S becomes invisible. At this time, the signal intensity of H_I becomes large due to the dipole interaction between H_I and H_S. This is the "Change due to NOE". Since the change of signal intensity by NOE is very small, the difference between (A) and (B) is calculated to make the difference clear. The NOE observed by difference NOE is called "steady state NOE". In general, for a small molecular compounds having high speed molecular motion in solution, when the signal change of irradiated proton is expressed downward, the NOE signal is observed as an upward signal. This is called "Positive NOE".

Actual example of difference NOE measurement is introduced, by using ethyl crotonate.

We would like to determine whether the conformation of a double bond is cis or trans by using the difference NOE measurement. The proton of methyl group expressed as A is irradiated, and the related NOE is observed.

In the upper part of the above figure, the ¹H spectrum of ethyl crotonate is shown. In the lower part, difference NOE spectrum when a proton of methyl group at A is saturated, is shown. This data is measured by using 400 MHz instrument and the NOE signals are observed upward at B and C.

Based on the obtained results, irradiation of the proton of the methyl group at A led to observable NOEs at B and C, indicating that ethyl crotonate has a trans conformation.

1D NOESY

1D NOESY is a method to selectively excite a specific proton and observe the transient NOE between the specific proton and the other spatially nearby protons as a one-dimensional spectrum. In this measurement, soft pulse (narrow bandwidth pulse) and pulsed field gradient are used to excite only the proton of interest, and the change in NOE over the mixing time is recorded. Since there is no need to subtract spectra as in difference NOE, spectra that are easy to analyze are obtained. 1D NOESY is suitable when you have a specific proton of interest or when you want to check the NOE in a simple way.

Here, we present an example of a 1D NOESY measurement using ethyl crotonate. The (B) in the above figure shows 1D NOESY spectrum obtained by selective excitation of proton A, while (C) shows the spectrum obtained by selective excitation of proton C. Unlike difference NOE spectra, 1D NOESY does not show the residual signals caused by subtraction of two spectra obtained before and after radio wave irradiation, making the spectrum easier to analyze.

The interpretation method is similar to that of difference NOE: we check how other signals appear when the selectively excited proton signal is shown downward.
In example of ethyl crotonate, when proton A is selectively excited, positive NOEs are observed at B and C. Conversely, when proton C is excited, a positive NOE is observed only at A. These results also support the estimation that ethyl crotonate has a trans type stereochemistry. Thus, when determining stereochemistry with ambiguous spatial arrangements, it is important to measure 1D NOESY of the selective excitation of each part.

2D NOESY

2D NOESY is a method to observe interaction (NOE) between spatially close protons in a molecule as a two-dimensional spectrum. This experiment observes the appearance of how NOE changes (transient NOE) during the mixing time after temporarily changing the proton spin state. The obtained spectrum expressed the self-signals on a diagonal and correlated signals between spatially close protons. Since the 2D NOESY allows for comprehensive NOE correlation in the entire molecule, it is suitable for analysis of complicated structures and confirmation of entire signal assignment.

From here, we will explain how to read 2D NOESY spectra. As shown in the above figure, when A and C, B and D are in close proximity to each other, such correlation signals appear. The data sliced at positions A and B (in blue dotted line) in X-axis direction, are equivalent to 1D NOESY spectra by selective excitation of A and B, respectively.

This figure shows 2D NOESY of ethyl crotonate. Looking at each sliced data, the signals at the selective excited part (in red) appear downward, while signals with NOE correlation (in black) appear upward (positive NOE).

When looking at the correlation signals in black, the following NOE correlations can be found:

A：B and C
B：A
C：A
D：E
E：D

In addition, focusing on the NOE correlations among A, B, and C, the results support the presence of the trans type conformation, consistent with the previously mentioned difference NOE and 1D NOESY results.

Easy guideline in selecting NOE measurement method

As we have explained, for an NOE measurement, it is possible to choose the method according to the purpose. We recommend that you begin with 1D NOESY which allows for easy condition setting and clear spectra to be obtained.

Target proton has been specified: 1D measurement
Want to see NOE across the molecule, or signal assignment is uncertain: 2D NOESY

Important factor for NOE measurement: Relaxation time

One of the most frustrating problems in NOE measurements is, without a doubt, "when no NOE signal is detected!" In such a case, please measure the longitudinal relaxation time (T₁) of the target sample, first. Then, for difference NOE measurement, please set the irradiation time of the excitation pulse to be more than 5 times that of T₁ of the target sample. Also, please set the mixing time with 1D/2D-NOESY, to be about 0.8 times that of T₁ of the target sample.

Now, why do we need to consider the relaxation time of the target sample, for an NOE measurement? This is because NOE has a deep relationship with relaxation time, as NOE is a relaxation phenomenon based on the dipole interaction.

Here, we would like to provide a simple explanation about relaxation in NMR.

The above is the schematic diagram of energy levels of nuclear spin of spin number= 1/2 when it is placed in a static magnetic field. Nuclear spin takes up two energy states after undergoing Zeeman splitting by being affected by magnetic field. Stable state with low energy is called α state, while the one with high energy is called β state. When a radio wave having a frequency equivalent to this energy gap is irradiated, the spin in the α state absorbs the energy of radio wave and excited into the β state. When the spin number of the α state and the β state becomes the same by irradiation of a radio wave, energy absorption stops, which is called the saturation state. In the saturation state, the spin in the β state emits energy and returns to the α state, finally, it even returns to the thermal balanced state. The whole process is called relaxation. Then, a series of processes from excitation to relaxation, are called the NMR phenomenon that we are observing.

The interaction works between two neighboring nuclear spins, which is the driving force for the relaxation explained earlier. In solution NMR, the dipole interaction is one of the major factors in promoting relaxation. Therefore, setting the parameter of relaxation time which has deep relationship with dipole interaction is an important key for successful NOE observation.

Cause and solution for undetectable NOE signals

The likely cause and action when NOE is not detected, are explained in the following 4 points.

Optimize sample adjustment/measurement conditions
Change molecular motion
Change resonance frequency
Change measurement method

First, check if 1. is optimized, and if the NOE is not observed, try 2 to 4.

1. Optimize sample adjustment/measurement conditions

Since the NOE signal is very weak, the result is greatly affected by the sample concentration and purity, and measurement condition setting.

Concentration: if the concentration is too high, relaxation tends to occur between molecules, weakening the NOE.
Caution against contamination of paramagnetic substances: dissolved oxygen and metal ions (Fe, Cu, etc.) promote relaxation, thus preventing NOE.
Setting of irradiation/mixing times: Based on T1(longitudinal relaxation time), irradiation time of more than 5 times, mixing time of about 0.8 times are guidelines.

2. Change molecular motion

The intensity of NOE also depends on molecular motion. The following graph shows the relationship between NOE intensity and molecular motion.

This curve was theoretically brought from the relational expression of NOE intensity and dipole interaction.

The vertical axis expresses the NOE intensity, while the horizontal axis expresses the product of observation frequency ω multiplied by correlation time of the molecule t_C. t_C is the time required for a molecule to make a full rotation in solution and is a parameter expressing the motion of the molecule. Which means, the smaller the molecule is, the shorter the t_C becomes. The larger the molecule is, the longer the t_C becomes.

The left shows the region of low molecule where t_C is short and the motion of molecule is fast. Here, a positive NOE is observed. On the other hand, the right shows the region of polymer where t_C is long and the motion of the molecule is slow. Here, the negative NOE is observed. Which means, the graph indicates that NOE intensity greatly changes depending on the motion of the molecule.

What matters in performing measurement is that there is a region where NOE becomes zero. When the molecule is close to the region where the product of multiplication of observation frequency ω and correlation time of the molecule t_C is 1, the NOE becomes very weak or the NOE is not observed.

Therefore, if NOE is not observable even if measurement conditions are reviewed with your instrument, it is possible that the product of multiplication of observation frequency ω and correlation time of the molecule t_C is in this region.

In such a case, changing ω or t_C is a possible choice. However, changing ω means changing the measurement magnetic field, and it may be difficult to try it at once if you hold only one instrument. Therefore, we suggest that you consider changing t_C , which is, changing the factor that can affect tC(measurement temperature and viscosity of a solvent). We recommend that you change the measurement temperature, first.

3. Change resonance frequency

Next, I will explain the case where the observation frequency is changed, which is, where the measurement magnetic field is changed.

The above figure shows the magnetic field dependence of NOE intensity and correlation time t_C .The vertical axis represents NOE intensity, while the horizontal axis represents correlation time t_C. Each curve corresponds to a magnetic field from 90 MHz to 920 MHz. The curves shift to the left as the measurement magnetic field increases. If we focus on the tC at which the NOE becomes zero, we can see that the point at which the signal intensity becomes zero shifts to the region of low molecules (where the molecules are in fast motion) as the magnetic field is increased. This means that under the same measurement conditions, the signal intensity of positive NOE becomes weaker at higher magnetic fields. For example, it can be said that a molecule with zero NOE in a 600 MHz instrument may have a positive NOE observed in a 300 MHz instrument. In addition, for the case of a molecule with about 1000 molecular weight, the possibility is high that it can be in the region in a circle where NOEs are extremely small, so attention is needed.

4. Change the measurement method

If NOE is not observed after trying methods 1 through 3 introduced so far, a measurement method called ROESY is used. ROESY is a NOE measurement in a rotational coordinate system.

The above figure is the 1D NOESY and 1D ROESY spectra of gramicidin S measured at 90°C. At this temperature, the NOE of gramicidin S becomes nearly zero, but using ROESY allows the observation of correlation signals. The NOE observed in ROESY is referred to as ROE. The greatest advantage of ROESY is that, as illustrated in the schematic diagram, ROE is always positive and does not have a zero region. You might wonder, "Then why not just use ROESY from the beginning?" However, ROESY requires more careful parameter settings and tends to produce unwanted signals. Therefore, ROESY is better used when NOESY does not work well.

Frequently Asked Question about NOE Measurement

The shape of the difference NOE signal looks strange!!

In difference NOE measurements, if the irradiation position is not set at the peak maximum, spin coupling effects can occur, causing changes in the signal shape. Therefore, the irradiation position should be set precisely at the peak maximum.

In difference NOE measurements, when multiplets more than doublets are selectively irradiated and if the irradiation position is shifted from the peak maximum, the signal shapes are changed. The two spectra above show difference NOE results when the methyl group protons of ethyl crotonate are irradiated. Positive NOE signals are observed at B and C. If the irradiation position is shifted to around the center ppm of signal A instead of the peak maximum, a large, asymmetric, residual-like signal appears, as shown in the dotted area of the top spectrum. This is due to spin coupling interactions with proton B. Always select the irradiation position at the peak maximum in difference NOE measurements.

Why does a small molecule show a negative NOE?

This phenomenon is known as indirect NOE (three-spin effect), and in small molecules, it can be observed when protons are arranged nearly linearly.

Normally, small molecules undergoing rapid motion in solution exhibit positive NOE signals. However, signals may sometimes appear in the same direction as a negative NOE. For example, consider the spatial arrangement shown in the figure on the left, where protons A-B, and B-C are spatially close, but A-C are far apart. The figure on the right shows a 1D NOESY spectrum when proton A is selectively excited. Since A and B are close, a positive NOE is observed at B. However, an indirect NOE may also appear at C via B. This indirect NOE appears in the opposite direction to the positive NOE. It's important to note that this signal does not indicate that A-C are spatially close. This phenomenon is most likely to occur when the three protons are arranged nearly linearly in space, and it is known as the "three-spin effect."

Why NOE is not available, even though the distance should be close?

When, three protons are arranged spatially special, NOE may not be observed even if the distances are close.

As a special case of the three spin effect, let me explain the triangle problem. As shown in the above figure, when three protons are arranged spatially special, this problem can occur.

When A is selectively excited, not only the distance between A and B, but also A and C are close. Thus, positive NOE is supposed to be seen between A and C, the forecasted spectra is the right in the above figure.

However, the positive NOE from A to C shown in orange and the indirect NOE via B shown in green can cancel each other out, resulting in no observable NOE. Theoretically, this cancellation occurs when the ratio of distances between each pair of protons is 1:1:1.26. In other words, even if the protons are spatially close, NOE may not be observed.

Reference

Claridge, D.W.T.(2004), *Yūki kagaku no tame no kōbun Kainou NMR tekunikku* [High-resolution NMR techniques for organic chemistry] (T.Takeuchi & M. Nishikawa (Trans.) Kodansha Scientific.
Fukushi, E. & Sohmiya H. (2007), *Korenara wakaru nijigen NMR* [Understanding 2D NMR made easy]. Kagaku Dojin.

Structural Analysis of Organic Compound Using 2D - NMR Spectrum

In this column, we will explain the structural analysis of organic compound by using 2D-NMR spectrum.

Structural Analysis of one-dimensional NMR (1D-NMR)

One-dimensional NMR (1D-NMR) is the most basic measurement of NMR spectroscopy, in which the spectrum is displayed along a horizontal axis (chemical shift). As introduced in the previous column, information on chemical shift, integration ratio, and splitting pattern (coupling) is used to analyze the structure of an object. However, it can be difficult to perform structural analysis using only 1D-NMR data when the number of detected signals is large, when signals appear in overlapping positions, or when couplings are complex.

The two-dimensional (2D-NMR) spectral analysis method introduced in this column can be used extensively regardless of the object to be analyzed, since the basic sequence of steps is followed. In addition, by following the basic procedures, even beginners can mechanically perform structural analysis.

What is 2D-NMR?

2D-NMR is a method used to check a more detailed molecular structure than 1D-NMR. Since there are many 2D-NMR measurement methods, here, we introduce representative measurement methods.

Measurement Name	What you can find out
COSY	A method to observe coupling between neighboring same nuclides (frequent:¹H/¹H)
INADEQUATE	A method to observe coupling between neighboring ¹³Cs
HMQC/HSQC	A method to observe coupling of a directly coupled heterogeneous nuclei (frequent: ¹H/¹³C)
HMBC/H2BC	A method to observe the correlation signals of heterogeneous nuclei(frequent: ¹H/¹³C) via 2-3 bonds
ADEQUATE	A method to detect ¹³C-¹³C coupling by ¹H observation
NOESY/ROESY	A method to observe the correlation signals between ¹Hs that are closely located(with NOE interaction)

Structural Analysis Using J Coupling Correlation

This time we introduce an example of structural analysis using "J Coupling Correlation" which is an interaction via chemical bonding. Structural analysis of an object is conducted by following the 4 steps below.

In Step 1, we use a 1D-NMR spectrum of ¹³C and DEPT to determine and estimate the atomic group and functional group.

In Step 2, we use HMQC and 1D spectrum of ¹H to find ¹³C and ¹H that are directly bonded.

In Step 3, we use COSY to examine and locate which is the neighboring ¹Hs.

By using information obtained in Step 2 and 3, substructures can be determined to some extent.

In Step 4, we use HMBC to determine the structure of the remaining parts, by considering the connection of ¹³C and ¹H which are further apart.

Structural analysis example of C₆H₁₀O₂

Now we will explain the structural analysis of a specific sample.

This time, we use a substance whose molecular formula C₆H₁₀O₂ is only known and dissolve in heavy chloroform.

Step 1 ¹³C (1D-NMR) & DEPT

In Step 1, we use a 1D-NMR spectrum of ¹³C and DEPT to determine and estimate atomic group and functional group. First, as shown in Fig. 1, we put a symbol on each signal in 1D-NMR spectrum of ¹³C. Starting with the signal on the right side of the spectrum, from the high-field side, add symbols A, B, C... and so on. The chemical shift of each signal is read to one decimal place. The read information is summarized in Table 1.

Fig. 1 ¹³C Spectra of C₆H₁₀O₂

Signal	Chemical Shift
A	14.1 ppm
B	17.6 ppm
C	59.8 ppm
D	122.8 ppm
E	144.0 ppm
F	166.2 ppm

Table 1 ¹³C Spectra Information

Next, we will confirm the results of the DEPT spectra. Since DEPT observes the carbon to which hydrogen is directly bonded, the series of the carbon atom of interest (the number of hydrogen directly bonded to the carbon of interest) is known. In other words, it is possible to identify atomic groups such as CH₃, CH₂, and CH. (DEPT cannot detect spectra of quaternary carbons.) DEPT provides three types of spectra, depending on the measurement parameters (DEPT135, DEPT90, and DEPT45).

Table 2 lists the signal appearance patterns in the three types of DEPT spectra. In DEPT135, the signals of CH₃ and CH are detected upward, while the signal of CH₂ appears downward (opposite phase). It is often possible to discriminate CH₃ or CH from the chemical shift. In many cases, the DEPT135 measurement alone is sufficient. If discrimination by DEPT135 alone is difficult, measure DEPT90, in which only the signal of CH is detected.

Table 2 DEPT Signal Appearance Patterns

We will use the DEPT spectra to determine the atomic groups of the sample. As shown in Figure 2, we will discriminate the atomic groups of each signal by comparing the signal appearance patterns of the 1D-NMR spectrum of ¹³C and the DEPT spectrum.

-----
DEPT135・・・Upward signal: CH₃ or CH, Downward signal: CH₂
DEPT90・・・ Detectable signal : CH
Signal detected only in the 1D-NMR spectrum of ¹³C and not detected in the DEPT spectrum: quaternary carbon
-----

Using the information so far, we were able to determine that the atomic groups of each signal are, from right to left, A (CH₃), B (CH₃), C (CH₂), D (CH), E (CH), and F (quaternary carbon).

Fig. 2 ¹³C Spectra and DEPT Spectra of C₆H₁₀O₂

The information so far is summarized in Table 3. This information is used to estimate the atomic groups and functional groups.

Signal	Chemical Shift	Atomic Group
A	14.1 ppm	CH ₃
B	17.6 ppm	CH ₃
C	59.8 ppm	CH ₂
D	122.8 ppm	CH
E	144.0 ppm	CH
F	166.2 ppm	C

Table 3 ¹³C Spectra Information (2)

Figure 3 shows the chemical shift table for ¹³C. At an approximate border of 100 ppm, the saturated carbon signals are appeared on the right and the unsaturated carbon signals on the left. We will compare the information summarized in Table 3 with the chemical shift table. First, if we look at D (122.8 ppm) and E (144.0 ppm), we see that they are in the region of unsaturated carbon, indicating that this CH is derived from unsaturated carbon. Next, if we look at F, the chemical shift value is 166.2 ppm, which is in the ester region. The molecular formula of this substance is C₆H₁₀O₂, and since there are two Os, the quaternary carbon of F is presumed to be derived from the COO group.

Figure 3 ¹³C Chemical Shift Table

The information so far is summarized in Table 4.

Signal	Chemical Shift	Atomic Group
A	14.1 ppm	CH₃
B	17.6 ppm	CH₃
C	59.8 ppm	CH₂
D	122.8 ppm	CH=
E	144.0 ppm	CH=
F	166.2 ppm	COO

Table 4 ¹³C Spectra Information (3)

Step 2 HMQC & ¹H

In Step 2, we will use HMQC and 1D-NMR spectra of ¹H to find directly bonded ¹³C and ¹H combinations. HMQC will tell you the combination of ¹H and ¹³C that are directly bonded. Spin couplings are written with a symbol, such as ¹J_CH. The number of bonds is written in the upper left corner of the J representing the spin coupling, and the nucleus to which it is bound is written in the lower right corner of the J.

Figure 4 shows an HMQC spectrum. In a 2D-NMR spectra, a high-resolution 1D-NMR spectrum is displayed at each axis; for HMQC, the ¹H spectrum is displayed on the X-axis and the ¹³C spectrum on the Y-axis. In Step 2, the same symbols that were attached to each signal in the ¹³C spectrum in Step 1 are attached to the signals in the ¹³C spectrum on the Y-axis, with the upper side of the Y-axis representing the high-field side (small chemical shift) and the lower side representing the low-field side (large chemical shift).

Next, draw a line from the ¹³C signal on the Y-axis to the HMQC correlation signal and confirm which ¹H is directly bonded to ¹³C. For example, if we focus on the ¹³C signal of C, we draw a line toward the side, and when the line hits the correlation signal, we draw a line up from there. The ¹H signal that we have reached here is the counterpart directly bonded to signal C of ¹³C. When the corresponding ¹H is found in this way, the ¹H is marked with the same symbol as the counterpart ¹³C. Since it is the counterpart of the signal C of ¹³C, we will also put the symbol C on this ¹H. All correlated combinations will be given a similar symbolization.

Fig.4 HMQC Spectra

Step 3 ¹H-¹H COSY

In Step 3, we will use ¹H-¹H COSY to see which are the neighboring ¹Hs. COSY shows the spin-coupled ¹H connections. What can be observed mainly is the correlation between ¹Hs via three couplings, as shown in Figure 5. In symbols, we denote this as ³J_HH. As shown in Fig. 5, if ¹Hs in a ³J_HH relationship are lined up, we can follow the ¹H connections one after another.

In COSY, in addition to the ³J_HH, a ²J_HH through two couplings and a remote ⁴J_HH can also be observed.

However, the most important and necessary information is the correlation between ¹Hs at the neighboring ¹³C, ³J_HH.

Fig. 5 Correlation of ¹Hs via Three Bonds

Fig. 6 shows the actual COSY spectra. COSY shows ¹H spectra both on the X-axis and Y-axis.

In Step 1, we assigned a symbol to each ¹³C signal. In Step 2, we assigned a symbol to each ¹H signal that is directly coupled to ¹³C using HMQC. In Step 3, we first assigned the same symbols to the signals in the ¹H spectrum on the X and Y axes as we did in Step 2. We looked at the HMQC spectra we used earlier (Figure 4) and copied the symbols attached to the signals in the ¹H spectra on the X-axis as they were.

Note that in Figure 6, the symbols are alphabetically ordered from the high-field side, but this just happened to be the case for this sample. The symbols on the ¹H signal are not always in alphabetical order.

Also, in the COSY spectra, signals line up on the diagonal line (called diagonal signals) when the diagonal line is drawn. But these are not used as information for structural analysis. The off-diagonal signals are the COSY correlation signals.

We draw a line from the correlated signal toward the X- and Y-axes spectra and find the ¹Hs that are spin-coupled to each other. For example, if we focus on the correlation signal circled in green, we find the ¹H signal C on the X-axis and the ¹H signal A on the Y-axis. Therefore, we can see that signals C and A are spin-coupled (neighboring) to each other. Once the counterpart is found, we add a symbol to the correlated signal. For example, C/A, to specify that they are ¹H signals in a ³J_HH relationship. Since the correlated signals of COSY appear in a line symmetric position with respect to the diagonal, we find the ³J_HH counterpart in the same way for all correlated signals.

Fig. 6 COSY Spectra

Once the information on the correlated signals of COSY is available, a correlation table for each signal is created.

Table 5 is the correlation table for COSY. Write the symbols for the ¹H signals in vertical and horizontal order, as per the spectrum of the data on the ¹H-¹HCOSY axis. (In the case of this sample, they happen to be arranged alphabetically, but you should look at the COSY spectra and list them in chemical shift order.) For example, if the correlated signals are C and A, mark the intersection of C and A. In this way, all correlated signals are entered in the COSY correlation table.

Table 5 COSY Correlation Table

Fig.7 shows the diagram of correlation signal of COSY.

In this sample, COSY correlation signal was observed between ¹H of C and ¹H of A.

The HMQC spectra also indicated that ¹H of C and ¹³C of C are directly coupled. In the same way, ¹H of A and ¹³C of A are directly coupled.

Therefore, COSY correlation signals between C and A can induce that ¹³C of C and ¹³C of A are neighboring. In other words, using the COSY information, we can indirectly find the neighboring ¹³C connections from the neighboring ¹H connections.

Fig. 7 COSY Correlation Signals

The information that we have so far is the ¹³C spectrum information (Table 4) and the COSY correlation table (Table 5). From the COSY correlation table, we can see that the carbon atoms derived from the ¹³C signals A-C, B-E, and D-E are neighboring to each other. Combined with the information from the ¹³C spectrum, when we rewrite the symbols as atomic groups, we have A-C : "CH₃-CH₂-", B-E-D : "CH₃-CH=CH-", and F : "COO" in Step 1. Therefore, we know that this compound is composed of the following three substructures.

----
Substructure 1：CH₃-CH₂-　・・・ A-C
Substructure 2： CH₃-CH=CH-　・・・ B-E-D
Substructure 3： COO　・・・ F
----

Next, we will consider what kind of molecule these will make by connecting each substructure (Figure 8). We will list all possible molecular structures from the combination of the three substructures. When considering molecular structures, it is easier to focus on the substructure containing CH₃ because CH₃ is located at the end of the structure. In this case, there are two possible structures. Firstly, Substructures 1 and 2 are found to be the ends of the molecular structure because they contain CH₃.And since 1 and 2 are located at the ends, we can predict that substructure 3 is sandwiched between 1 and 2. The different orientations of substructure 3 allowed us to create two different inferred structures (I, II). The orientation of the COO can be used to determine which inferred structure is more reasonable. Therefore, HMBC measurements are performed to confirm the long-range spin coupling with respect to the COO.

Fig. 8 Combination of Three Substructures

Step 4 HMBC

In Step 4, we will consider long-range spin coupling between ¹³C and ¹H by using HMBC.

In HMBC, we can observe the correlations of ²J_CH through two bonds or ³J_CH through three bonds, as shown in Figure 9.

Fig. 9 CH Correlation through 2 or 3 Bonds

Fig. 10 is the actual spectra of HMBC. The HMBC axes are the same as HMQC's case, ¹H spectra on X-axis and ¹³C spectra on Y-axis. In Step 4, we will put the same symbol that we assigned for HMQC spectra in Step 2, for each signal of a 1D-NMR spectrum on the X and Y axes.

Next, we draw lines from the correlation signals to the X- and Y-axis spectra to find the spin-coupled ¹³C and ¹H combinations. Here, the two horizontally-lined up signals circled in orange are the signals of ¹J_CH, a direct coupling of ¹H and ¹³C. Since these are remnant signals, they are not observed in all direct couplings. As the direct couplings have already been observed by HMQC, it is not necessary to focus on them here. In the HMBC spectrum, only the long-range information is read, ignoring the direct coupling signal. For example, if we focus on the correlation signal circled in green, we can see that ¹³C of C and ¹H of A are long-range spin coupled to each other, since tracing to Y-axis results in C and to X-axis results in A.

Once the counterpart is known, add a symbol to the correlation signal, e.g., A/C, to indicate that it is a long-range spin-coupled ¹H and ¹³C of ²J_CH or ³J_CH. For all correlation signals, do the same to find a long-range spin-coupling counterpart and mark the correlation signal with a symbol. Once you have written out the information for the HMBC correlation signals, create a correlation table.

Fig. 10 HMBC Spectra

Table 6 is the correlation table for HMBC. We write the symbol for the ¹H signal horizontally and the symbol for the ¹³C signal vertically, as per the spectrum. (Note that the symbols for the ¹H signals are not necessarily in alphabetical order.) Correlation signals for the HMBC spectra will be filled in. For example, if the correlation signal is ¹³C for C and ¹H for A, mark the intersection of C for ¹³C and A for ¹H in the correlation table. In this way, all correlation signals are entered in the HMBC correlation table.

Table 6 HMBC Correlation Table

We use the information in the HMBC to connect the substructures.

Look at the HMBC correlation table (Table 7) that you filled out earlier. In the correlation signal marks, bonds that have already been analyzed by COSY in Step 3 are marked with ○, and bonds that were found for the first time in HMBC are marked with ●. The bond that was found for the first time in HMBC is the COO correlation for F. This indicates that F is a long-range spin couplings with C, D, and E.

Table 7 HMBC Correlation Table (2)

We will look at the long-range correlations of F-C, F-D, and F-E to consider which direction is correct for the COO to be attached, among the inferred structures I and II. (Figure 11).

First, in inferred structure I, F's ¹³C and C's ¹H have three bonds "³J_CH", ; F's ¹³C and D's ¹H have two bonds "²J_CH", ; and F's ¹³C and E's ¹H have three bonds "³J_CH".

Next, let us look at the inferred structure II. The ¹H of ¹³C and C in F is "²J_CH" with two bonds, the ¹H of ¹³C and D in F is "³J_CH" with three bonds, and the ¹H of ¹³C and E in F is "⁴J_CH" with four bonds.

Since ⁴J_CH is not likely to be observed, we can expect that the inferred structure I is a reasonable structure.

Fig. 11 Comparison between Inferred Structures I and II

Finally, the inferred structure I is checked against the information in the 1D-NMR spectrum of ¹³C to confirm if it is really correct. In the inferred structure I, CH₂ of the symbol C is bonded to oxygen. And the ¹³C chemical shift of this C was 59.8 ppm (Table 1).

The ¹³C chemical shift table for CH₂ (Table 8) shows that normal CH₂ is observed at 20-45 ppm, while CH₂ bonded to oxygen is observed at 40-70 ppm with a lower field shift.

From this fact, we can conclude that "Structure I is correct".

	Chemical Shift
- CH₂ -	20 -45 ppm
- CH₂O -	40 - 70 ppm

Table 8 ¹³C Chemical Shift for CH₂

How to read NMR spectra from the basics (chemical shift, integration ratio, coupling)

This column provides easy-to-understand explanations about what we can learn from the NMR spectra (chemical shift, integration ratio, and coupling)

What we can learn from the NMR spectra

There are three main things that we can learn from the NMR spectra.

Horizontal axis (chemical shift):
The horizontal axis contains information about the type of functional group and molecule conformation. From the position where the spectrum appears (numerical value on the horizontal axis), it is possible to predict what kind of functional group and molecule conformation are contained in the molecule to be measured.
Integration ratio (signal area ratio):
By comparing the integral values of each signal, it is possible to compare the number of functional groups contained in a molecule and to obtain information on the mixing ratio of a mixed sample consisting of multiple molecules.
Splitting pattern (coupling):
The signal is split due to the influence of another nuclear spin existing near the nuclear spin of interest. Figure 1 shows the 1H NMR spectrum of ethanol. The methyl and methylene group signals show that the signal is not a single signal, but is split into multiple signals. Since the splitting pattern of the signal depends on the number and type of other nuclear spins existing nearby, it is possible to predict the substituents contained in the system from the splitting pattern.

Fig.1 1H NMR spectrum of ethanol (CH3CH2OH)

Fig.1 ¹H NMR spectrum of ethanol (CH ₃CH ₂OH)

Reasons for causing differences in horizontal axis (chemical shift)

The difference of chemical shift is due to the strength of the magnetic field received (felt) by the nuclear spin we are focusing on.

As shown in Fig 2, depending on the height of the electron density existing near the nuclear spin, the strength of shielding of the magnetic field (the strength of the magnetic field that the nuclear spin receives) varies.

The electron density existing near the nuclear spin depends on the magnitude of the electronegativity of the atoms existing near the nuclear spin of interest.

When an O (oxygen) atom with high electronegativity exists nearby, electrons are attracted by the O atom, the electron density near the nuclear spin of interest decreases, and the magnitude of the magnetic field that the nuclear spin receives becomes greater.

As the electron density near the nuclear spin decreases(shielding becomes lower), the corresponding signal shifts to the left.

Fig. 2 Difference of strength of shielding the magnetic field

Example of chemical shift table of ¹H

Fig. 3 Correlation diagram of typical functional groups and 1HNMR signal positions

Fig. 3 Correlation diagram of typical functional groups and ¹HNMR signal positions

Figure 3 shows a correlation diagram of typical functional groups and ¹HNMR signal positions. In the NMR spectra, the right side is generally called as the high-field side and the left side as the low-field side. The signal appearing at 0 ppm is the signal of the reference material TMS (tetramethylsilane). The chemical shift value is a numerical value that represents the shift from other signals) So, it is necessary to calibrate the reference point with a reference material such as TMS etc. ¹H signals from alkyl chains, such as methyl, methylene, and methine, often appear near 1 ppm. And as mentioned above, ¹H signals near alcohol and ether groups with neighboring oxygen atoms and 1H signals derived from amino groups with neighboring nitrogen atoms are detected near 3ppm to 4ppm. The signal appearing near 5 ppm is an alkene-derived ¹H signal. Furthermore, ¹H signals derived from aromatic rings are observed around 7 ppm, and a signal derived from formyl groups such as aldehydes appears around 9 ppm. Signals derived from carboxyl and phenol groups appear around 11 ppm. The position at which the signal appears allows a rough prediction of the type of functional group.

When performing a structural analysis using NMR, please be careful of heavy water exchange in the case where OH or COH groups are included. In solution NMR, the sample is dissolved in a heavy solvent for measurement. If the solvent to be used is heavy water or heavy methanol, heavy water exchange occurs between the D(²H) in the solvent molecule and the ¹H in the OH or COH groups, and the ¹H signal from the OH or COH groups may not be observed.

Integration ratio

Fig.4 ¹HNMR spectrum of benzyl acetate

The following is a brief introduction to the use of integration ratios. Figure 4 shows the structural formula of benzyl acetate and the ¹H spectrum. Looking at the molecular structure of benzyl acetate, we can guess that ¹H signals would be observed in three areas related with the CH₃ group, the CH₂ group, and the aromatic group.

Furthermore, a closer look reveals that benzyl acetate has three ¹Hs derived from CH₃, two 1Hs derived from CH₂, and five ¹Hs derived from one substituted aromatic CH. The integration ratio of each signal is calculated to be CH₃:CH₂:CH = 3:2:5, which indicates that the values predicted from the structure and the actual measured values coincide.

It can also be seen that CH₃ is shifted to the left from the area where ¹H signal derived from CH₃ is often observed (around 1 ppm) due to the influence of the neighboring O atoms.

Examples of the use of integration ratios in mixed samples include the following:

Relative quantitative evaluation by comparison of integration values of each component
Absolute quantitative evaluation using a standard substance of known purity (q-NMR)
Calculation of reaction rate by comparison of integration values before and after the reaction

In both examples, it is important to find the signal that is specific to each component and that can be integrated correctly (i.e., not overlapping with other signals).

Coupling and Spin-Spin Coupling Constant "J"

Fig.5 1H NMR spectrum of 2,4 dimethyl pyrimidine

Fig.5 ¹H NMR spectrum of 2,4 dimethyl pyrimidine

Finally, we introduce couplings. Coupling refers to the interaction between the nuclear spin of interest and another neighboring nuclear spin. In 1D measurements of ¹H NMR, the interaction, "coupling" occurs when nuclear spins are in proximity to each other and induces the NMR signal splits. The unit of the splitting width of spin coupling is expressed in Hz. This number is called the spin coupling constant or J-coupling constant (j-value).

Formula for calculating spin coupling constant (J value)

It is also known that the splitting widths have the same j-value when coupled to each other. In the compound in Fig. 5, Ha and Hx are coupled, so the splitting widths of both Ha and Hx have the same value, 6hz. Thus, when a split peak is observed, the j-value information can be used to determine which signals are coupled.

Splitting pattern due to coupling

Let us explain a little more about the splitting pattern caused by coupling. An unsplit signal is called a singlet, denoted by the symbol "s"; a two-divided signal is a doublet, denoted by the symbol "d"; a three-divided signal is a triplet, denoted by the symbol "t"; a triplet has a signal strength ratio of 1:2:1, : A signal that splits into four is a quartet, denoted by the symbol "q." The signal strength ratio for a quartet is 1:3:3:1. Signals with five or more segments are multiplets, indicated by the symbol "m".

Using the ¹H NMR spectrum of ethanol as an example, we will explain the splitting of the ¹H signal. Focusing on the signal derived from the CH₃ group around 1ppm, the number of ¹H near by CH₃ group is 2 (coupled with CH₂ group), so it splits into 2+1=3.

Looking at the signal derived from the CH₂ group around 3.5 ppm, the number of ¹H near by CH₂ group is 3 (coupled with CH₃ group), which splits into 3+1=4.

Because the OH signal around 5ppm is not coupled to the near by ¹H, it does not split and is in the singlet state. Basically, we can see that the signal splits into the "n+1", "n" means the number of nuclei spins positioning around the nuclear spin of interest.