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

Mon, 19 Jan 2026 23:40:34 GMT

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.

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

Mon, 19 Jan 2026 23:03:21 GMT

Structural Analysis of Organic Compound Using 2D - NMR Spectrum

Mon, 19 Jan 2026 19:48:38 GMT

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)

Mon, 19 Jan 2026 19:48:04 GMT

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 (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

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

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).

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.

Structural biology 101: Principles, techniques and applications

Tue, 29 Apr 2025 20:40:00 GMT

Structural biology 101: Principles, techniques and applications

The molecular machinery of life operates with astonishing precision. Proteins fold into intricate geometries, nucleic acids assemble into double helices and tertiary structures, and multi-subunit complexes carry out processes fundamental to cellular survival. Understanding these structures, and how they determine biological function, is the domain of structural biology.

It is not enough to know what molecules are made of. We must comprehend how they are built.

The Central Premise: Structure Determines Function

Structural biology rests on a fundamental axiom of molecular life sciences: the three-dimensional shape of a biomolecule dictates its biological role. From enzymatic catalysis and signal transduction to genome maintenance and immune recognition, structural conformation governs interaction, specificity, and activity.

Even minor perturbations in structure, such as point mutations, deletions, or misfolding, can compromise function, drive pathogenicity, or confer drug resistance. In this context, elucidating structure is not merely descriptive; it is diagnostic, predictive, and, increasingly, design-oriented — making structural biology a critical tool in modern molecular research.

Molecular Architecture: Hierarchical and Informative

Biological macromolecules exhibit a multi-tiered architecture:

Primary structure: Linear sequences of amino acids (proteins) or nucleotides (DNA/RNA).
Secondary structure: Localized folding motifs, including α-helices, β-sheets, and loops, stabilized by hydrogen bonds.
Tertiary structure: The complete three-dimensional conformation of a single polypeptide or nucleic acid strand, shaped by hydrophobic interactions, ionic bridges, disulfide bonds, and van der Waals forces.
Quaternary structure: The spatial organization of multiple subunits into higher-order complexes, often essential for cooperative function.

This structural framework enables researchers to interpret molecular behavior, predict function based on form, and design biological systems with enhanced or novel capabilities.

Techniques in Focus: Methods for Determining Molecular Structure

Structural biology relies on a suite of experimental and computational techniques to determine the architecture of macromolecules with precision and clarity. Each method offers distinct advantages, and together, they provide a comprehensive view of biological structure and function.

X-ray Crystallography

A cornerstone of structural biology, X-ray crystallography involves directing X-rays through a crystallized biomolecule and analyzing the resulting diffraction pattern. Using a variety of techniques to modify the original diffraction amplitudes so as to extract phases for each of the Bragg reflections enables the generation of high-resolution electron density maps and the construction of detailed atomic models. It is widely used to study enzymes, nucleic acids, and protein-ligand interactions, particularly where static, well-defined structures are needed for applications such as drug design and mechanistic elucidation.

Cryo-Electron Microscopy (Cryo-EM)

Cryo-EM allows scientists to visualize macromolecular complexes in near-native states by imaging specimens frozen in vitreous ice. Thousands of two-dimensional images are computationally aligned to reconstruct a three-dimensional structure. Its strength lies in resolving large and flexible biological assemblies, making it essential for studying membrane proteins, viral particles, and ribonucleoprotein complexes. Also, typically much smaller quantities of the biomolecule of interest can be used for a structural study using cryo-EM and image processing, colloquially known as SPA. Although not always reaching down to atomic resolution, SPA nonetheless has proven itself to be the promising technique over x-ray diffraction and NMR for resolving many important biomolecules. Thus, this method has become pivotal in comprehending structural heterogeneity and conformational dynamics in structural biology.

Nuclear Magnetic Resonance (NMR) Spectroscopy

NMR provides atomic-level insights into biomolecules in solution by measuring the magnetic properties of specific atomic nuclei. It is particularly effective for probing protein dynamics, conformational changes, and transient interactions that occur in a physiological environment. In structural biology, NMR is indispensable for characterizing smaller proteins and intrinsically disordered regions. The technique provides a complementary view to the models provided by crystallography or Cryo-EM.

MicroED

This technique is an equivalent of xray diffraction in that electrons are used to diffract off a crystalline sample. Since electrons interact so much more with matter than xray photons, much smaller crystals can be used in this technique. Applied to small molecules this technique has proven to be incredibly successful in solving structures with great precision and speed often in a matter of minutes.

Computational Modeling and AI-Based Prediction

Computational methods have become an essential component of structural biology, offering powerful tools for predicting molecular structures directly from sequence information. These approaches leverage biophysical principles and evolutionary relationships to generate models of three-dimensional conformation with growing precision. When integrated with experimental techniques, computational modeling helps interpret structural data, identify functionally relevant regions, and explore the effects of sequence variation. This synergy extends the reach of structure-based research, enabling a deeper mechanistic understanding and more informed molecular design.

Applications: Translational Insights and Technological Innovation

Structural biology informs a broad spectrum of scientific and industrial domains:

Structure-Based Drug Design (SBDD): Precise knowledge of target geometry enables rational ligand design, optimizing binding affinity and selectivity while reducing off-target effects.
Mechanistic Studies of Disease: Structural analysis elucidates how mutations destabilize protein folding, promote aggregation (e.g., in neurodegenerative disorders), or alter enzymatic activity.
Immunology and Vaccine Development: Structural insights into antigen presentation and epitope accessibility have accelerated monoclonal antibody engineering and the rational design of immunogens.
Synthetic and Systems Biology: De novo protein design, pathway reengineering, and the development of synthetic molecular machines all rely on structural characterization.
Environmental and Agricultural Biotechnology: Enzyme engineering for pollutant degradation or enhanced crop resilience often begins with structural templates.

Pioneering Structural Biology with JEOL USA

Structural biology offers a powerful framework for exploring the architecture of life at the molecular scale. Techniques such as Cryo-EM, X-ray crystallography, and NMR spectroscopy reveal the structures that govern biological activity, offering insights essential to fields ranging from drug discovery to synthetic biology.

As a global leader in scientific instrumentation, we, JEOL, support this work by providing high-performance tools tailored to the complex demands of structural biology. Our CRYO ARM™ series and advanced NMR spectrometers are designed to deliver the resolution, stability, and analytical precision required to decode molecular structures with confidence.

By equipping researchers with the means to visualize and interpret molecular form, we enable discovery at the atomic level— advancing not only scientific understanding but also broadening the impact of structural biology across research and innovation.

NMR Analysis of Lithium Ion Batteries

Mon, 01 Apr 2024 20:07:24 GMT

LiB: Next Generation Energy Storage

Lithium-ion batteries (LIBs) are used to power portable electronics, electric vehicles, and grid storage solutions; they play a crucial role in driving sustainability and are an essential energy storage device. With the demand for electric vehicles and renewable energy sources continuing to rise, there is an increasing need to improve electrochemical storage. The search for new battery materials, alongside the drive to improve performance, and lower the cost of existing and new batteries, comes with its challenges.

Lithium has one of the highest electrochemical potentials compared to other metals, making it very active. Therefore, it releases the electron from the outer shell much faster than other metals, which makes it a good choice for battery research. 6/7Li are called ‘spin spies’ because they detect changes in structure, state of deterioration, Li-ion mobility, and quantitation during charging and discharging of the battery and have guided the synthesis of new anode, cathode and electrolyte materials.

A primary concern in finding new forms of electrolytes in secondary batteries is safety because electrolytes can leak in a battery and are very sensitive to temperature change, especially high temperatures.

Leveraging NMR for LiB Analysis

One method to observe lithium ions is nuclear magnetic resonance (NMR). NMR is one of the few analytical methods to characterize the local structure and ion dynamics of LIB materials. NMR spectroscopy is crucial in studying the electrochemical and physical properties of the LIB components. NMR applications are used for three of the components of LIBs: cathode, anode, and electrolyte. The material that is being analyzed will determine the appropriate NMR technique, such as solid-state NMR, in-situ NMR, and diffusion NMR.

Characterizing Li-ion cells and batteries can involve a galvanostatic cycle which can study the behavior of batteries being cycled. A current is applied to cause an electrochemical reaction, followed by a reverse reaction, and this is repeated until the battery degrades, usually because of temperature. NMR is used to determine the time this process will take.

NMR: Non-Destructive Analysis

The main benefits of NMR spectroscopy over alternative approaches are its non-destructive nature and ability to study a range of operating storage devices in situ. Further research will provide key observations that can lead to the development of more efficient, safer batteries in the future. Magic-angle spinning improves spectral resolution for solid-state samples by physically spinning the sample.

Ex-situ NMR can uncover the charging and discharging cycle during lithium-ion battery operation. It explored the new cathode material with a multi-layer structure with domains where lithium-ion is contained.

NMR is a valuable tool for researchers because of its high flexibility and chemical sensitivity whilst remaining non-invasive. NMR spectroscopy is a vital tool for investigating the chemical and physical properties and electrochemical performance of LIBs. It will help advance current research into finding more sustainable and efficient solutions to support the future of our planet. Further applications of NMR in battery research will support battery manufacturing and prevention of battery failures; furthermore, it will improve technologies to meet the demand for high efficiency, longer lifetime and lower costs.

To learn more about JEOL USA’s air-isolated microscopy workflow proving its value in advanced battery research and production, check our our press release on our Science of Energy theme at Pittcon 2023!

Optimizing NMR Processing: Techniques and Best Practices

Fri, 01 Dec 2023 09:51:53 GMT

How Does a Nuclear Magnetic Resonance Spectrometer Unveil Molecular Structures?

Fri, 01 Dec 2023 09:35:32 GMT

Deciphering Complex Chemical Structures with COSY NMR

Fri, 01 Dec 2023 09:22:23 GMT

Unveiling the Future of NMR: Empowering Research with the JEOL ROYALPROBE HFX

Fri, 27 Oct 2023 04:01:00 GMT

As research continues to push the boundaries of what's possible, the tools at our disposal must keep up with the demands of innovation. Enter the JEOL ROYALPROBE HFX, a revolutionary technology that's redefining how multi-nuclei experiments are done.

The JEOL ROYALPROBE HFX represents the culmination of cutting-edge engineering and innovation, merging the capabilities of the ROYALPROBE and TFH probes to create a versatile and high-performance solution. This innovative merge empowers researchers with the flexibility to perform routine experiments seamlessly, including ¹H{¹⁹F,X}, ¹⁹F{¹H,X}, ¹³C{¹H,¹⁹F}, and X{¹H,¹⁹F} experiments.

To provide further insight into this technology, we’ve crafted the ROYALPROBE HFX Digital Guide. In addition to application notes and example data, with this guide you’ll discover:

Comprehend the Mechanics of ROYALPROBE HFX: The fusion of ROYAL and TFH probes gives birth to the ROYALPROBE HFX. Gain a deep insight into the intricacies of the ROYALPROBE HFX, including its integration of dual tune modes and AutoTune extension.
Recognize the Versatility of the Probe: Discover how the ROYALPROBE HFX's exceptional performance metrics and adaptability transcend traditional boundaries, enabling a wide range of multi-nuclei experiments.
Explore Real-world Applications: Witness the probe's capabilities in real-world scenarios, from spectral assignments to complex hybrid decoupling effects, and understand how it enhances research across various domains.

This technology is the result of innovative engineering that empowers researchers with the flexibility to perform routine experiments seamlessly, opening doors to a myriad of possibilities. Download the guide and explore a new realm of possibilities with the JEOL ROYALPROBE HFX: [Link to HFX Guide].

Unlock the Power of Multi Frequency Drive System (MFDS): Your Gateway to Advanced NMR Experiments

Wed, 13 Sep 2023 00:38:39 GMT

Structure Elucidation Challenges: How Can Advanced Spectroscopy Overcome Them?

Thu, 31 Aug 2023 11:27:55 GMT

Upgrade or Replace? Making the Right Choice for Your NMR Instrument.

Thu, 13 Jul 2023 13:01:10 GMT

ENC 2023 Conference Notes & Mini Symposium

Mon, 01 May 2023 13:58:44 GMT

The Experimental Nuclear Magnetic Conference (ENC) celebrated its 64^th conference April 16-21, 2023 at the Asilomar Conference Center in Pacific Grove, CA. Beginning in 1959 and established as a 501(c)(3) non-profit in 1987, the conference was organized with the following stated goals:

Promote the interest of molecular spectroscopy in general and NMR spectroscopy, in particular;
Organize an international meeting for the exchange of state-of-the-art information with special attention on experimental aspects of NMR spectroscopy.

Although ENC is not hosted in a single location, the conference has a long history with the Asilomar Hotel and Conference Grounds. The conference grounds are situated along Asilomar State Beach, which is part of the Asilomar Marine Reserve and boasts a rich and biodiverse ecosystem, and close to the Asilomar Dunes Natural Preserve, which features 25 acres of pedestrian boardwalk through the sand dune ecosystem.

As a conference, ENC is excellent for networking with the thought leaders of today’s NMR community in company-hosted hospitality suites. JEOL hosted their events in the Fred Farr Forum, including the JEOL Announcement Night, NMR Mini Symposium, iPad Raffle, and more.

JEOL Announcement Night

JEOL’s Announcement Night kicked off Tuesday, 4/18. Highlights of the presentation included the following:

New ECZL System – Our new NMR ECZL G series is a flagship model for cutting-edge NMR methods. The footprint of the spectrometer has been reduced to less than 60% of ECZR, while maintaining the expandability needed to support a wide range of applications. It is flexible in terms of expansion, with support for three or more channels, high-power amplifiers, and high-output magnetic field gradients, allowing for future functional expansion even when installed in the minimum configuration
ROYALPROBE™ HFX – The ROYALPROBE™ HFX is the world's first liquid NMR probe with the capability to switch between single tune and dual tune modes on the high frequency coil. This expands the capability of a standard workhorse NMR, making it an excellent choice for scientists looking to achieve the maximum impact of their instrument.
Cryogenic Probes for NMR – JEOL offers two ultrahigh sensitivity autotune probes using cryogenic probe technology: The SuperCOOL Cryogenic Probe and the UltraCOOL Cryogenic Probe.

The SuperCOOL Probe features significantly improved sensitivity as its thermal noise is reduced by cooling of both the detection coil and preamplifier. The SuperCOOL probe reduces measurement times to up to 75% to enable many more samples to be measured in a single day.
The UltraCOOL Probe achieves more than 4 times the sensitivity of conventional probes while thermal noise is reduced by cooling of both the detection coil and preamplifier. Measurement times using the UltraCOOL probe are only 1/16 that of a conventional probe.

Cryogen Reclamation System – JEOL’s new Cryogen Reclamation System offers a convenient, reliable, and highly effective solution to managing and maintaining your NMR instrument’s cryogen levels. This system maximizes NMR instrument uptime and reduces the risk of shutdown due to cryogen supply issues by substantially reduces the evaporation of liquid helium and liquid nitrogen from the superconducting magnet.

NMR Mini Symposium

The JEOL NMR Mini Symposium included six scientific presentations covering a range of NMR topics. Speakers included:

Paul Ellis, Daniel Arcos, and F. David Doty, Doty Scientific - Spin Echoes, Sensitivity, Wurst (Adiabatic) Pulses, and Artifact Suppression Utilizing Modern ssProbes
Professor Federico Del Rio, UNAM, Mexico - Structure of Arachnid Toxin by NMR
Ronald Crouch, JEOL USA - Balancing the Robust and Convenient with the Challenging
Yusuke Nishiyama, JEOL LTD - 14N Solid State NMR at Fast MAS
Peter Kirali, JEOL UK - The Hidden Gem of Data Processing in JASON
Manuel Perez, JEOL UK - JASON: Advanced Functionality, Enabling Automaton

In case you missed it, you can view our NMR Mini Symposium on-demand.

NMR Technical Presentations

In addition to our Mini Symposium, JEOL scientists contributed to two technical talks. The first took place on Monday 4/17 and was presented by Genevieve Seabrook:

“Small GTPases are regulators mediating important cellular functions. These sGTPases are often mutated in human cancers. We have developed a real-time multiplex NMR assay allowing the following of several sGTPases nucleotide exchange in a single experiment. Along with sGTPase proteins strategically selectively labeled, time-shared NMR methodology was used to reduce acquisition time. Analysis of sGTPases amides chemical shift changes, allowed us to identify residues that have been perturbed during the nucleotide exchange and the resulting structural changes within the sGTPases. A mixture of six sGTPases was used to assay GEF activities present in cells lysates and in organoids lysates. A combination of selective isotopic labeling and real-time, time-shared NMR experiments can be extended to other biological processes”

The second, 1H CSA: Friend or Foe?, was presented by Frederic A. Perras:

“Despite the high sensitivity, and recent resurgence, of 1H solid-state NMR, measurements of 1H chemical shift anisotropy (CSA) have remained rather niche. In many instances, we would even consider it a nuisance that leads to decoherence and t1 noise in 1H dipolar recoupling. This presentation will cover the development of highly stable dipolar recoupling methods that decouple the 1H CSA in addition to new 1H CSA recoupling schemes that enable the measurement of tensor skew, and small anisotropies. Lastly, the utility and limitations of 1H CSA for the measurement of dynamic information in low sensitivity samples, such as heterogeneous catalysts, will be discussed.”

NMR Technical Posters

JEOL presented four technical posters at ENC 2023. The first, titled “Simplifying triple resonance experiment for high quality NMR spectra with Multi Frequency Drive System” and presented by Hiroaki Sasakawa, explored our new Multi Frequency Drive System, which is available on our ECZ Luminous NMR console:

“Organic compounds with phosphorus and boron nuclei often exhibit spectral complexity and reduced sensitivity in NMR analysis due to J couplings between hydrogen and carbon with these nuclei. We developed a triple resonance system called Multi Frequency Drive System (MFDS) to address this issue, enabling triple resonance experiments with a standard 2-channel NMR system. Using a JEOL JNM-ECZL600G spectrometer equipped with the ROYALPROBE™ P+, we conducted various solution NMR measurements. We present examples of signal enhancement and spectrum simplification achieved by triple resonance measurements of 1H, 31P, and 11B collected with a 2-channel NMR instrument.”

The second poster focused on our new 1.01 GHz NMR system. Titled “Development and applications of a 1.01 GHz (23.7 T) NMR system,” this poster was presented by Yoshitaka Ishii:

“We discuss development of an ultra-compact 1.01 GHz NMR magnet, and its preliminary NMR applications. The new ultra-compact 1GHz NMR magnet utilizes high-temperature superconducting (HTS) coils made of bismuth-based cuprates besides conventional low temperature superconducting coils. Because of the high current density of the HTS coil, the magnet weighs only 1.6 tons and its footprint is the smallest among the existing 1 GHz NMR systems. The cryogenic refrigerator mounted on the magnet eliminates needs of regular liquid-helium refilling. We have successfully collected multi-dimensional solution NMR and solid-state NMR data for proteins at a 1H frequency of 1.01 GHz. The quality of the NMR data and other research progress from the ongoing project to develop 1.3 GHz NMR will be also discussed.”

The third technical poster, presented by Takuya Matsumoto and title “Cryogen Reclamation System for NMR Magnets” offers a solution for evaporation of cryogens:

“NMR magnets are usually cooled by two kinds of cryogens, ie.liquid helium and liquid nitrogen. The boil-off rates of the cryogen in general NMR magnets are typically around 20 cc/h for liquid helium and 200 cc/h for liquid nitrogen. We have developed new cryogen reclamation system that can greatly suppress the evaporation both of liquid helium and liquid nitrogen. The system was tested with an NMR magnet, and it was confirmed that the noise generated by system vibration was at a level that would not interfere with NMR measurements. It has also confirmed that the magnet maintained stable zero boil-off status for more than 6 months.”

The fourth technical poster was presented by Yutaro Ogaeri and titled “Internuclear Distance Measurements between 1H and 14N in Multi-Component Rigid Solids at Fast MAS”:

“1H-14N internuclear distances are readily and accurately measured using the symmetry-based phase modulated resonance-echo saturation-pulse double-resonance (PM-S-RESPDOR) method in rigid solids. Analytical equation of the fraction curve easily provides 1H-14N couplings. However, this treatment is only applicable when NH proton resonance is well separated from the other proton peaks, which is not necessarily satisfied even at fast MAS >60kHz, especially in multi-component systems. To overcome this problem, THMQC filtering is applied to suppress the 1H signals other than NH proton prior to the PM-S-RESPDOR experiments. The method is well demonstrated on two components acetaminophen-oxalic acid (APAP-OXA) systems.”

New ECZ Luminous NMR Console

Our new NMR ECZL G series is a flagship model for cutting-edge NMR methods. The footprint of the spectrometer has been reduced to less than 60% of ECZR, while maintaining the expandability needed to support a wide range of applications. It is flexible in terms of expansion, with support for three or more channels, high-power amplifiers, and high-output magnetic field gradients, allowing for future functional expansion even when installed in the minimum configuration. Learn more about the ECZL.

PANIC 2023: Conference Notes & Recorded Symposium

Tue, 28 Mar 2023 16:49:46 GMT

PANIC 2023 Conference Notes

PANIC (Practical Applications of NMR industry Conference) started out in 2012 as a scientific conference seeking to promote real-world, practical solutions for modern NMR problems. PANIC seeks to address the daily problems that scientists encounter when they apply NMR to wide range of analytical problems.

This year, for their 11^th conference to date, scientists from across the world traveled to Nashville, TN, to learn, network, and share their latest discoveries in the field of NMR. Here at JEOL, we were excited to share our new ECZ Luminous series NMR spectrometer and some recent developments in the application of qNMR during our Mini Symposium on Sunday morning.

NMR Mini Symposium

Three speakers presented at the meeting. The first talk, given by Ronald Crouch of JEOL USA, was titled "Strategies to Evaluate Low-Sensitivity Experiments" and explored the use of older but underutilized experiments that are still very valuable, as well as using data that seems to be wrong at first glance.

Our second speaker, Iain Day from JEOL UK, gave an overview of current tools in JASON as well as some of the projects under development in his presentation "qNMR with JASON: The power of SMILEQ".

Lastly, Jessie Ochoa of Genentech summarized some methods and techniques developed and used in a real life industrial setting for quantitative NMR work. in her discussion of “Quantitative NMR of Small Molecule Pharmaceuticals”.

In case you missed it, you can view our NMR Mini Symposium on-demand:

Delta College Adapts To New Routine With Online Microscopy Training And New Technology

Mon, 11 May 2020 09:50:00 GMT

CRAFT For NMR: Challenging Conventions To Achieve Faster, More Accurate Analysis

Mon, 04 Nov 2019 21:15:00 GMT

JEOL USA blog

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

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

Structural Analysis of Organic Compound Using 2D - NMR Spectrum

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

What is 2D-NMR?

Structural Analysis Using J Coupling Correlation

Structural analysis example of C6H10O2

Step 1 13C (1D-NMR) & DEPT

Step 2 HMQC & 1H

Step 3 1H-1H COSY

Step 4 HMBC

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

What we can learn from the NMR spectra

Reasons for causing differences in horizontal axis (chemical shift)

Example of chemical shift table of 1H

Integration ratio

Coupling and Spin-Spin Coupling Constant "J"

Splitting pattern due to coupling

Structural biology 101: Principles, techniques and applications

Structural biology 101: Principles, techniques and applications

The Central Premise: Structure Determines Function

Molecular Architecture: Hierarchical and Informative

Techniques in Focus: Methods for Determining Molecular Structure

X-ray Crystallography

Cryo-Electron Microscopy (Cryo-EM)

Nuclear Magnetic Resonance (NMR) Spectroscopy

MicroED

Computational Modeling and AI-Based Prediction

Applications: Translational Insights and Technological Innovation

Pioneering Structural Biology with JEOL USA

NMR Analysis of Lithium Ion Batteries

LiB: Next Generation Energy Storage

Leveraging NMR for LiB Analysis

NMR: Non-Destructive Analysis

Optimizing NMR Processing: Techniques and Best Practices

How Does a Nuclear Magnetic Resonance Spectrometer Unveil Molecular Structures?

Deciphering Complex Chemical Structures with COSY NMR

Unveiling the Future of NMR: Empowering Research with the JEOL ROYALPROBE HFX

Unlock the Power of Multi Frequency Drive System (MFDS): Your Gateway to Advanced NMR Experiments

Structure Elucidation Challenges: How Can Advanced Spectroscopy Overcome Them?

Upgrade or Replace? Making the Right Choice for Your NMR Instrument.

ENC 2023 Conference Notes & Mini Symposium

JEOL Announcement Night

NMR Mini Symposium

NMR Technical Presentations

NMR Technical Posters

New ECZ Luminous NMR Console

PANIC 2023: Conference Notes & Recorded Symposium

PANIC 2023 Conference Notes

NMR Mini Symposium

Delta College Adapts To New Routine With Online Microscopy Training And New Technology

CRAFT For NMR: Challenging Conventions To Achieve Faster, More Accurate Analysis

Structural analysis example of C₆H₁₀O₂

Step 1 ¹³C (1D-NMR) & DEPT

Step 2 HMQC & ¹H

Step 3 ¹H-¹H COSY

Example of chemical shift table of ¹H