Difference Between UV, IR, and NMR Spectroscopy

In the complex and rapidly advancing field of structural biology, one of the key challenges remains the elucidation of molecular structures and a detailed understanding of how these structures determine biological function. Techniques such as X-ray crystallography, cryo-electron microscopy (cryo-EM), and nuclear magnetic resonance (NMR) spectroscopy have become cornerstones of structural biology, providing unparalleled insight into the spatial organization of proteins, nucleic acids, and other biomolecules. Among the many complementary methods available, ultraviolet (UV) and infrared (IR) spectroscopy also play an important role, particularly in the context of molecular characterization, conformational changes, and functional analysis. Despite their common goal of providing detailed molecular information, UV, IR, and NMR spectroscopy differ fundamentally in their principles, techniques, and the types of information they provide.

Each of these spectroscopic methods sheds light on different aspects of molecular structure and behavior. UV spectroscopy is essential for studying electronic transitions and chromophore interactions, IR spectroscopy reveals the vibrational motions of functional groups, and NMR spectroscopy provides high-resolution insights into atomic-level structures and dynamics. Understanding the differences between these techniques is critical for researchers who want to use the most effective tool for their investigations.

At Creative Biostructure, we provide in-depth insights and comparisons of three advanced spectral technologies. Serving the academic, biotech and pharmaceutical sectors, we specialize in structural biology and membrane protein technology. Our services include X-ray Crystallography, Cryo-Electron Microscopy (Cryo-EM), NMR Spectroscopy, Molecular Dynamics Simulations, and more, all designed to support your research needs.

UV Spectroscopy: Exploring Electronic Transitions

Ultraviolet (UV) spectroscopy is a technique that measures the absorption of ultraviolet light by a molecule, providing insight into the electronic transitions occurring within the molecule. The basis of UV spectroscopy is the excitation of electrons in the molecule from a lower energy state (ground state) to a higher energy state (excited state) upon absorption of UV light. This technique is particularly sensitive to the electronic properties of molecules containing conjugated π-bond systems or chromophores that absorb light in the UV region (typically 200-400 nm).

Three Typical Diagrams in UV Spectroscopy

In UV-Vis spectroscopy, the three typical diagrams that illustrate electronic transitions under UV-Vis light illumination are:

• Absorption Spectrum: This diagram shows the absorbance (y-axis) versus wavelength (x-axis) for a sample. It indicates the wavelengths at which the sample absorbs UV or visible light. Absorption peaks correspond to the energies required for electronic transitions. In the UV-Vis region, the spectrum typically shows absorption bands related to the transitions between molecular orbitals, such as π → π*, n → π*, and σ → σ* transitions.
• Electronic Energy Level Diagram: This diagram represents the energy levels of electrons within a molecule. It typically shows the ground state (lowest energy) and excited states (higher energy levels). Upon absorbing UV-Vis light, an electron in the molecule transitions from a lower energy molecular orbital (such as the HOMO, Highest Occupied Molecular Orbital) to a higher energy molecular orbital (such as the LUMO, Lowest Unoccupied Molecular Orbital). This type of diagram visually illustrates the types of electronic transitions (e.g., π → π*, n → π*, or σ → σ*) that occur in response to UV-Vis light.
• Jablonski Diagram: Although primarily used in fluorescence and phosphorescence studies, the Jablonski diagram also shows the electronic energy states of a molecule. In the context of UV-Vis absorption, it represents the excited states following photon absorption. The diagram depicts the transitions between the ground state and excited states and shows potential relaxation pathways such as non-radiative decay, fluorescence, and phosphorescence. It helps to illustrate how absorbed UV-Vis light promotes electronic transitions and how energy can be released from the excited states.

Figure 1. Three typical diagrams of UV-Vis and electronic transitions under UV-Vis light illuminations. (Khan et al., 2018)

Instrumentation: Components and Advancements

Modern UV-Vis spectrophotometers are composed of several key components that contribute to their precision, accuracy, and versatility in various applications. These include:

• Light Source: The spectrophotometer requires a stable and continuous light source for UV and visible wavelengths. Deuterium lamps provide a consistent output in the ultraviolet (UV) range (typically 190–400 nm), while tungsten or halogen lamps cover the visible (Vis) spectrum (400–1100 nm). Some advanced instruments incorporate xenon flash lamps, which offer a broad spectrum and longer lifespan.
• Monochromator: To isolate specific wavelengths, the monochromator uses either a diffraction grating or a prism. Diffraction gratings are more commonly used in modern instruments due to their higher resolution and wavelength precision, ensuring minimal stray light interference.
• Sample Holder: The sample chamber contains cuvettes that hold the liquid samples. Quartz cuvettes are preferred for UV measurements due to their transparency in the UV range, while glass or plastic cuvettes are sufficient for visible light applications. Some advanced spectrophotometers support fiber optic probes for direct in situ sample measurements.
• Detector: The detector converts transmitted or absorbed light into an electrical signal. Photomultiplier tubes (PMTs) offer high sensitivity and rapid response, making them ideal for detecting low-intensity light. Photodiode arrays (PDAs) enable simultaneous multi-wavelength measurements, improving speed and efficiency.

UV Spectroscopy in Structural Biology

In structural biology, UV spectroscopy is widely used to study aromatic amino acids such as tryptophan, tyrosine, and phenylalanine, which are abundant in protein structures and serve as intrinsic chromophores. The absorption spectra of these amino acids provide critical information about protein folding, conformational changes, and interactions with other molecules. For example, shifts in UV absorption maxima can indicate changes in the environment surrounding aromatic residues that may occur during ligand binding, protein folding, or protein-protein interactions.

Figure 2. Spectral Density Curves for Aromatic Amino Acids: Trypotophan (Trp), tyrosine (Tyr), Phenylalanine (Phe) and albumin derived from bovine serum (BSA). (Hammond et al., 2014)

UV spectroscopy is also valuable in quantifying the concentration of nucleic acids and proteins by measuring absorbance at specific wavelengths—typically 260 nm for nucleic acids and 280 nm for proteins. This application is essential in structural biology for assessing the quality of samples prior to further analysis with more advanced techniques. Additionally, UV spectroscopy can provide data on the secondary structure of proteins by analyzing the absorption of light in the far-UV region (190-250 nm), where peptide bonds absorb.

Figure 3. A simple UV/vis absorption spectrum displaying the peak absorption wavelength, λ_max, whose intensity is given by the molar extinction coefficient, ϵ, whose computational analog is the oscillation strength, f. (Beard et al., 2018)

Despite its versatility, UV spectroscopy is limited in its ability to provide detailed three-dimensional structural information about biomolecules. While it can indicate changes in conformation and the presence of specific chromophores, it does not offer atomic-level resolution or the ability to resolve complex structural features like bond angles or distances. Furthermore, UV spectroscopy is not suitable for studying biomolecules that lack chromophores or those that do not absorb in the UV range.

IR Spectroscopy: Probing Molecular Vibrations

Unlike UV spectroscopy, infrared (IR) spectroscopy focuses on the vibrational motions of atoms within a molecule. When infrared radiation is passed through a sample, the molecules in the sample absorb energy at specific frequencies that correspond to the vibrational modes of the chemical bonds within the molecule. These vibrations occur at characteristic frequencies depending on the mass of the atoms involved and the strength of the bonds. IR spectroscopy therefore provides information about the functional groups present in a molecule and the molecular environment that surrounds them.

Figure 4. Symmetric stretch-compress mode of the C–H bonds of bromomethane.

Fourier Transform Infrared (FTIR) Spectroscopy

Fourier Transform Infrared (FTIR) spectroscopy is a powerful analytical technique that enhances traditional dispersive infrared (IR) spectroscopy by using interferometry instead of a monochromator to measure the absorption of infrared light by a sample. This method provides faster, more sensitive, and higher resolution spectral analysis of various materials, making it essential in fields such as chemistry, pharmaceuticals, materials science, and environmental analysis.

Key Components and Working Principle

• Michelson Interferometer: At the core of an FTIR spectrometer is the Michelson interferometer, which consists of a beam splitter, a fixed mirror, and a moving mirror. The beam splitter divides the infrared light into two paths: one directed to a fixed mirror and the other to a moving mirror. When these beams recombine, they produce an interferogram, a complex signal that contains information about all the wavelengths in the infrared spectrum.
• Fourier Transformation: The resulting interferogram is a function of time (or mirror position). A Fourier Transform (FT) algorithm is used to convert this time-domain data into the more familiar frequency-domain spectrum, which plots absorption intensity versus wave number (cm⁻¹). This transformation allows all infrared frequencies to be detected simultaneously, greatly improving data acquisition speed and spectral resolution.

Advantages of FTIR Over Dispersive IR Spectroscopy

• Higher Signal-to-Noise Ratio (SNR): The multiplexing (Fellgett's) advantage allows multiple data points to be collected simultaneously, improving sensitivity.
• Rapid Scanning and Real-Time Analysis: The ability to measure all wavelengths simultaneously allows for much faster spectral acquisition, making FTIR ideal for high-throughput applications.
• Improved Resolution: FTIR provides higher spectral resolution than traditional dispersive IR spectrometers, allowing differentiation of closely spaced absorption bands, which is critical when analyzing complex mixtures.
• Improved Sensitivity for Weak Absorptions: Because of its higher energy throughput, FTIR is more effective at detecting weakly absorbing functional groups, even in low concentration samples.

IR Spectroscopy in Structural Biology

In structural biology, IR spectroscopy is particularly useful for studying the secondary structure of proteins and the conformational dynamics of biomolecules. For example, the amide I band (approximately 1600-1700 cm⁻¹), which corresponds to the C=O stretching vibration of the peptide backbone, is commonly used to determine the content of α-helices and β-sheets in proteins. The amide II band (approximately 1500 cm⁻¹) provides additional information about protein structure, as it is associated with N-H bending and C-N stretching vibrations. These features are invaluable for characterizing protein folding, conformational changes, and protein-ligand interactions.

IR spectroscopy can also be used to study nucleic acids, although it is less commonly applied to nucleic acids than to proteins. The vibrational modes of nucleic acids, particularly in the 1200-1300 cm⁻¹ range, provide insight into conformational changes of RNA and DNA, including transitions between different double helix structures and the binding of small molecules.

Figure 5. Typical biological spectrum showing biomolecular peak assignments from 3,000–800 cm^{− 1}, where ν = stretching vibrations, δ = bending vibrations, s = symmetric vibrations and as = asymmetric vibrations. The spectrum is a transmission-type micro-spectrum from a human breast carcinoma (ductal carcinoma in situ). The sample was cryosectioned (8 μm thick) and mounted on BaF₂ slides (1 mm thick) before IR microspectroscopy. Equipment: Bruker IR scope II, circular diameter of aperture ~60 μm; a.u., arbitrary units. (Baker et al., 2014)

A major advantage of IR spectroscopy is its ability to study molecules in solution without requiring the high concentrations or crystalline forms that may be necessary for techniques such as X-ray crystallography. However, IR spectroscopy has its limitations. The spectral resolution is often insufficient to provide detailed information at the atomic level, and the technique can be affected by sample heterogeneity. In addition, while IR is excellent for observing functional group vibrations, it does not provide direct information about atomic interactions or three-dimensional structure in the same way as other techniques.

NMR Spectroscopy: Atomic-Resolution Structural Information

Nuclear magnetic resonance (NMR) spectroscopy differs from UV and IR spectroscopy in its ability to provide high-resolution, atomic-level structural information about biomolecules in solution. NMR spectroscopy is based on the interaction of atomic nuclei with an external magnetic field and radiofrequency radiation. Certain nuclei, most notably hydrogen (¹H) and carbon (¹³C), exhibit magnetic properties and can be observed in a magnetic field. By measuring the energy transitions of these nuclei as they interact with the magnetic field, NMR can provide detailed information about the chemical environment, connectivity, and spatial arrangement of atoms in a molecule.

Figure 6. Schematic diagram of NMR principle. (Cao et al., 2021)

NMR Spectroscopy in Structural Biology

In structural biology, NMR is a gold standard for studying the three-dimensional structures of small to medium-sized proteins, nucleic acids, and other biomolecules in solution. Unlike X-ray crystallography, which requires crystals of the biomolecule, NMR allows for the study of molecules in a near-native, dynamic state, making it ideal for studying proteins and nucleic acids that are flexible or difficult to crystallize. NMR data can provide detailed information on atomic spacing, torsion angles, and the overall fold of the molecule.

Figure 7. Solution NMR structures of cytochrome c (A) and calmodulin (B) in the confined environment of reverse micelles. (Hu et al., 2021)

Figure 8. Model DNA (secondary) structures by NMR: (a) B-DNA (PDB ID: 1ZF7); (b) G-quadruplex (PDB ID: 139D); (c) i-motif (PDB ID: 1A83); (d) AGCGA-quadruplex (PDB ID: 5M1L); and (e) G-hairpin (PDB ID: 5M1W). (Plavec, 2022)

NMR spectroscopy relies on the analysis of chemical shifts, scalar couplings, and NOE (nuclear Overhauser effect) interactions to determine the three-dimensional structure of a molecule. Chemical shifts arise from the electron density around a nucleus, and they are highly sensitive to the local environment, such as the presence of nearby functional groups or the spatial arrangement of atoms. Scalar couplings reflect the interactions between coupled nuclei, while NOE interactions provide spatial distance constraints, which can be used to calculate the relative positions of atoms within the molecule.

Figure 9. (A) Energy diagram showing the effects of J-coupling for the molecule hydrogen fluoride. (B) Example of the chemical shift: NMR spectrum of hexaborane B₆H₁₀ showing peaks shifted in frequency, which give clues as to the molecular structure (click to read interpretation details). (C) Nuclear Overhauser effect.

The ability of NMR to provide atomic-level structural information is unparalleled, especially when combined with multidimensional NMR techniques such as 2D, 3D, and 4D NMR. These techniques allow researchers to obtain high-dimensional data that can be used to generate detailed models of protein-ligand interactions, conformational changes, and protein folding.

One of the great strengths of NMR is its ability to study biomolecular dynamics. By tracking changes in chemical shifts and relaxation times, NMR provides insight into the flexibility and motion of biomolecules, such as proteins that undergo conformational changes during enzymatic catalysis or ligand binding. In addition, NMR is invaluable in the study of protein-protein and protein-nucleic acid interactions, providing detailed information on the binding sites, affinities, and kinetics of these interactions.

However, NMR spectroscopy has its challenges. While capable of high-resolution structural determination, NMR requires relatively high sample concentrations and can become difficult for large biomolecules (e.g., >40 kDa) due to spectral overlap and signal attenuation. In addition, interpretation of NMR data can be computationally intensive, requiring sophisticated algorithms to extract structural and dynamic information from the spectra.

Key Differences Between UV, IR, and NMR Spectroscopy

At Creative Biostructure, we have developed all-in-one gene-to-structure pipelines for the structure determination of macromolecules of your interest. We also offer a comprehensive list of products and other related services to facilitate your structural biology research. Explore our comprehensive range of services and products and contact us with any questions or inquiries.

References

1. Baker MJ, Trevisan J, Bassan P, et al. Using Fourier transform IR spectroscopy to analyze biological materials. Nat Protoc. 2014;9(8):1771-1791.
2. Beard EJ, Sivaraman G, Vázquez-Mayagoitia Á, Vishwanath V, Cole JM. Comparative dataset of experimental and computational attributes of UV/vis absorption spectra. Sci Data. 2019;6(1):307.
3. Cao R, Liu X, Liu Y, et al. Applications of nuclear magnetic resonance spectroscopy to the evaluation of complex food constituents. Food Chemistry. 2021;342:128258.
4. Khan SA, Khan SB, Khan LU, Farooq A, Akhtar K, Asiri AM. Fourier transform infrared spectroscopy: fundamentals and application in functional groups and nanomaterials characterization. In: Sharma SK, ed. Handbook of Materials Characterization. Springer International Publishing; 2018:317-344.
5. Hammond BR, Johnson BA, George ER. Oxidative photodegradation of ocular tissues: Beneficial effects of filtering and exogenous antioxidants. Experimental Eye Research. 2014;129:135-150.
6. Hu Y, Cheng K, He L, et al. NMR-based methods for protein analysis. Anal Chem. 2021;93(4):1866-1879.
7. Plavec J. NMR study on nucleic acids. In: Sugimoto N, ed. Handbook of Chemical Biology of Nucleic Acids. Springer Nature Singapore; 2022:1-44.