Higher Dimensional NMR Spectroscopy (2D And 3D)

Nuclear magnetic resonance (NMR) spectroscopy has emerged as a key tool in pharmacological investigation, enabling researchers to uncover the structures of complex organic molecules with previously unheard-of precision (Simpson et al., 2018); (Drevet Mulard et al., 2025). 2D NMR spectroscopy is a particularly useful technique among the various NMR techniques that examines the connections between a molecule's nuclei to uncover intricate structural details (Kuballa et al., 2023); (Dasset al., 2017). Two-dimensional (2D) NMR spectroscopy has developed into a versatile technique for drug development, chemical characterization, and structural elucidation in the pharmaceutical industry (Shaikhah et al., 2024; Seger & Sturm, 2022; LeBlanc & Mesleh, 2020). An essential tool in analytical chemistry is nuclear magnetic resonance (NMR) spectroscopy (Letertre et al., 2021). Because it provides vital insights into the molecular structures and dynamics of substances, it is particularly helpful in many scientific domains, including pharmaceuticals (Wang et al., 2024). One subfield of NMR, two-dimensional (2D) NMR spectroscopy, has become essential to pharmaceutical analysis and drug development (Emwas et al., 2020).
This article aims to demonstrate the vital role that 2D NMR plays in drug development, including drug characterization, formulation development, and quality control. The study also intends to assess the impact of technological advancements on 2D NMR sensitivity and resolution, such as cryogenic probes and high-field spectrometers. By investigating 2D NMR's adaptability. The research also aims to discover the technique's limitations and constraints, including its cost and complexity, particularly in the areas of pharmaceutical innovation and intellectual property protection.
The method has been used in research to create components and applications in NMR spectroscopy1, separation science, and microfluidics, as well as lab apparatus and specially designed reaction containers for chemical synthesis and structures with integrated active chemistry.  The structure of molecules Chemistry schools have used 3D printing to teach a variety of topics, including point groups, crystal symmetry, orbital theory, and VSEPR theory. Furthermore, some innovative work using 3D printing has been done to display reaction progress surfaces19. More recently, several companies have used the technique to print mathematical functions and additional representations of complex data sets, such as chemical spectra20–21. The work on printing chemical data by Higman et al. (17) and Bakker et al. (19) is also interesting.

To make the process easier to understand, a thorough explanation is included on how to create the stereolithography (stl) and/or object (obj) files needed to print 2D spectroscopic or chromatographic data. Two methods of file preparation are discussed. The first uses MATLAB for NMR data, while the second uses Mathematical for 2D HPLC data. An exported data matrix from a standard HPLC or NMR instrument is the starting point for both methods, after which it is converted into an ASTL file. To assess the processes, two NMR spectra—one from correlation spectroscopy (COSY) and one from heteronuclear single quantum coherence (HSQC)—and a 2D HPLC chromatogram (of biro ink) were 3D printed23–24. The data used was either generated internally (COSY and HPLC) or obtained from an online source (HSQC). Our results show that the file preparation methods discussed in this article are suitable for producing digital files of chromatograms and spectra that may be printed in three dimensions.

This paper offers a technique for using 3D printing to create physical models of 2D nuclear magnetic resonance (NMR) and 2D high-performance liquid chromatography (HPLC) data using standard software. The created models are robust and flexible, and they provide a way to grab students' attention. The models also assist students develop representational competency, which enhances their understanding of difficult subjects, by facilitating participatory learning in the classroom and providing an alternative method of data visualization.Students who learn best visually or tactilely can find these models to be quite beneficial. Thanks to 3D printing, a blind or visually challenged student can learn about structure-function relationships with minimal assistance. The 3D print data is easily portable between countries, and the created models can be utilized again and again. This lowers the cost of materials and resources and facilitates large-scale distribution, which helps many educational institutions reach their sustainability goals.

Principle: According to the NMR principle, many nuclei have spin8 and all nuclei are electrically charged. An energy transfer from the base energy to a higher energy level is achievable when an external magnetic field is applied. When there is an external magnetic field, energy can transfer from lower to higher energy levels. The radio frequency1 is the wavelength at which energy is transferred. The NMR principle usually consists of three consecutive steps. Polarization (or alignment) of magnetic nuclear spins in the presence of a strong, constant magnetic field, B0.A radio-frequency (RF) pulse, which is a weakly fluctuating magnetic field, altered this nuclear spin alignment. Identification and analysis of the electromagnetic waves released by the atomic nuclei of the sample due to this nucleus rupture.

TYPES OF NMR 

1.H NMR (proton NMR) spectroscopy

2. Carbon-13 NMR (¹³C NMR) Spectroscopy

3. Fluorine-19 NMR (¹?F NMR) Spectroscopy

4. Phosphorus-31 NMR (³¹P NMR) Spectroscopy

5. Two-Dimensional (2D) NMR Spectroscopy

6. Solid-State NMR

7. Dynamic NMR (DNMR)⁶

8. High-Resolution Magic Angle Spinning (HR-MAS) NMR¹

9. Quantitative NMR (q NMR)

10. Multinuclear NMR

Two-Dimensional (2D) NMR Spectroscopy

Two-dimensional NMR (2D NMR) extends the capabilities of one-dimensional NMR by introducing a second frequency axis, providing a powerful method for probing molecular connectivity, spatial relationships, and dynamics. 2D NMR experiments are broadly classified into homonuclear and heteronuclear types and can also be differentiated based on through-bond or through-space interactions. Here is a structured overview of the main types:

1. Homonuclear Correlation Experiments (Same Nuclei, e.g., ¹H–¹H)

a. COSY (Correlated Spectroscopy)

• Purpose: Identify scalar (J) coupling between nuclei, mapping which protons are coupled
• Mechanism: Detects through-bond interactions (usually 2-3 bonds apart) using two 90° RF pulses with a variable evolution period (t?) and detection period (t?).
• Applications: Elucidating proton connectivity in organic molecules, peptides, and small biomolecules.

b. TOCSY (Total Correlation Spectroscopy)

• Purpose: Extends COSY by connecting all protons in a given spin system, including longer-range networks.
• Mechanism: Uses isotropic mixing to transfer magnetization throughout the spin system.
• Applications: Assigning complete spin systems, mapping amino acids or sugar residues in polymers or carbohydrates.

c. NOESY (Nuclear Overhauser Effect Spectroscopy)

• Purpose: Detect spatial proximity (<5 Å) between protons, independent of covalent bond
• Mechanism: Through-space dipole-dipole cross-relaxation generates cross-peaks, enabling distance measurements.
• Applications: Determining 3D structure of biomolecules, stereochemistry, ligand binding.

d. ROESY (Rotating-frame Overhauser Effect Spectroscopy)

• Purpose: Similar to NOESY, but suitable for molecules with intermediate correlation times where NOE intensities are weak or zero.
• Mechanism: Spin-locking along the x-axis, allowing cross-relaxation in a rotating frame.
• Applications: Small to medium-sized molecules with moderate rotational correlation times (~1 kDa).

e. J-Resolved Spectroscopy

• Purpose: Separate chemical shifts from J-couplings; useful when overlapping multiplets occur in 1D spectra.
• Mechanism: Spreads each multiplet in the indirect dimension while keeping chemical shift along the direct dimension.
• Applications: Determining coupling constants and simplifying complex spectra.

2. Heteronuclear Correlation Experiments (Different Nuclei, e.g., ¹H–¹³C)

a. HSQC (Heteronuclear Single Quantum Coherence)

• Purpose: Correlates protons directly bonded to heteronuclei (¹³C, ¹?N, etc.).
• Mechanism: Transfers polarization from ¹H to heteronucleus and records one cross-peak per bonded pair.
• Applications: Assigning heteronuclear shifts, protein backbone analysis, characterizing isotopically labeled molecules.

b. HMBC (Heteronuclear Multiple Bond Correlation)

• Purpose: Detects indirect (long-range) proton–heteronucleus couplings (2–4 bonds apart).
• Mechanism: Cross-peaks reveal long-range connectivity; one-bond correlations are suppressed.
• Applications: Determining molecular skeletons, locating quaternary carbons, elucidating complex organic structures.

c. HMQC (Heteronuclear Multiple Quantum Coherence)

• Purpose: Alternative to HSQC; correlates protons and heteronuclei via multiple quantum coherence.
• Applications: Structural elucidation similar to HSQC but traditionally less sensitive

d. INADEQUATE (Incredible Natural Abundance Double Quantum Transfer Experiment)

• Purpose: Maps ¹³C–¹³C couplings for carbon skeleton determination.
• Mechanism: Requires detecting rare ¹³C–¹³C pairs (1% natural abundance gives ~0.01% of molecules).
• Applications: Precise carbon skeleton analysis of organic molecules

3. Pulse Sequence Phases in 2D NMR

All 2D NMR experiments generally involve four stages:

1. Preparation: Magnetization is created using RF pulses.
2. Evolution (t?): Spins freely precess, encoding frequency information along the indirect dimension.
3. Mixing: Magnetization is transferred between nuclei (through-bond for COSY/TOCSY or through-space for NOESY/ROESY).
4. Detection (t?): Free induction decay (FID) is observed; data are converted to frequency dimensions via 2D Fourier transform.

Three-Dimensional (3D) NMR Spectroscopy

Using a set of computer programs, the location of atoms in a crystal structure is translated into a three-dimensional (3D) representation of a solid form that can subsequently be 3D printed. Then, using 3D printing, these files are used to create to-scale physical models that bring crystal structures to life. An increasingly popular method of manufacturing is three-dimensional (3D) printing, which is the creation of a physical three-dimensional model via an additive process from a corresponding digital model file.

 Once limited to industrial settings where certain components would be quickly prototyped before large-scale manufacturing, this technique has rapidly expanded into previously unexplored areas over the past 20 years due to the growing availability and affordability of 3D printing technologies. As a result, the scientific community is using 3D printing and 3D-printing-inspired techniques more frequently to support research on topics like biomimetic microvascular systems, tissue growth scaffolds, electronic and pneumatic devices, and the creation of functional devices in lab settings .The Cronin group has been working on the concept of "reaction ware," which uses 3D printing techniques to create custom reactors where the geometry and orientation of the printed devices can control the outcome of reactions or sequences of reactions, enabling the explicit production of complex reactors for particular experimental needs. In tandem with the advancement of 3D printing techniques in the physical and biological sciences, 3D printing is being utilized more and more in education as well as for medical or forensic imaging data. This allows for the intuitive physical representation of complex data while maintaining important characteristics (like scale and connectivity) of the original structures to provide insights into the creation and use of such data. Since the earliest attempts to comprehend the structural structure of chemistry, physical models of chemical systems have been used. Hofmann is credited with using these models in public lectures for the first time in the 1860s.

However, the application of molecular models goes beyond simple visual representation and instruction; they have been essential to the development of several structural theories, most notably Watson and Crick's use of a skeleton-type model to explain the double helical structure of the DNA molecule. These conventionally tangible items have been transformed into (typically) two-dimensional (2D) rotatable screen representations of chemical structures in recent decades due to the exponentially growing processing capacity of computers. A. Similar to how computer technology has been used to solve and refine single crystal and powder X-ray diffraction experiments, these 2D computer representations are based on the physical data of a particular system rather than averaged or ideal bond lengths. While this is an improvement over the physical models that were previously used, it loses the natural ability to recognize 3D ordering that can be obtained from real-world physical models. Here, we describe how to convert crystallographically derived structural information on inorganic metal-oxide clusters into to-scale physical models using a suite of software and several 3D printing platforms.

 Uses

• Verification and identification of small molecules.
• Clarification of 3D structures (small and macromolecules).
• Keeping an eye on changes in the environment or disruptions caused by ligand binding.
• Hydrogen bond measurement.
• Keeping an eye on pKa values.
• Investigating molecular movements throughout a broad range of temporal scales.
• After enzymatic processes.
• Control and assurance of quality.

Methodology: The essential code for exporting, displaying, and creating a final model must be developed in order to handle spectrum data and develop methods for 3D printing chromatograms. Figure 1 summarizes the procedure developed in this paper. NMR There were two printed NMR spectra. These included an HSQC (1H, 13C) of taurocholic acid that was obtained from the human metabolome database (HMDB0000036) and a COSY (1H, 1H) spectrum of an extract of Daphnia magna from a prior metabolomics work by the lead author, which was the first on that species.For additional processing, the datasets were first opened in TOPSPIN Version 4.0 (Bruker Biospin, Preston, Victoria, Australia), exported as a text file, and then opened in MATLAB (matrix laboratory) software (Version 9.4, Natick, MA).

Chromatograms To keep with the "printing" idea, a 2D HPLC chromatogram of ink from a standard biro was used for the 3D printed chromatogram. Using two columns instead of one to separate out a sample mixture results in two-dimensional HPLC chromatograms. Before being reinjected onto the second column and run once again before detection, the eluent from column 1 is gathered and concentrated. For every data point, this yields two retention times and an intensity value. As with the NMR data, the 2D data are thus a set of Cartesian coordinates. Retention durations on columns 1 and 2 are represented by the x and y axes, and intensity (or detector response) is represented by the z axis.

Similar to the NMR spectra, a 3D print of an unprocessed raw HPLC trace was only millimeters long and meters high due to the disparities in dynamic range on each axis of the HPLC data set. Such a model would be quite delicate and impractical for most instructional purposes, since no 3D printer has a printer bed that size. Therefore, before any further processing, the raw data were normalized to 1 by dividing all values by the greatest value. Additionally, the larger peaks were clipped and the data were rescaled to 6 × 4 × 4 in.3 (the same dimensions as a photo). After that, the information was exported as an STL file that could be used with a 3D printer.

NMR Spectroscopy – Instrumentation: An NMR spectrometer consists of nine major parts.
holder of the sample: The sample is held in glass tubes that are 8.5 cm long and 0.3 cm in diameter.
Magnetic coils: A magnetic field is created when current passes through a magnetic coil.
Permanent magnet: It helps create a consistent magnetic field between 60 and 100 MHz.
Sweep generator: Modifies the strength of the magnetic field that has already been applied.
A short, powerful radio wave pulse is emitted by a radiofrequency transmitter. It facilitates radiofrequency-based receiver radio wave detection. Unabsorbed radio frequencies can be found with the help of a radio frequency detector.
Recorder: It records the NMR signals detected by the RF detector.
Readout System: A readout system is a computer that logs the data.

Figure 3: Instrumentations