Session 2: Spectroscopy Techniques in Practice - Methods and Motivations

This article corresponds to the second session of the "Fundamentals of Spectroscopy Webinar" series presented by Shayne Harrel, a Spectroscopy Application Specialist at Andor. Harrel has a Ph.D. in physical chemistry, focusing on time-resolved terahertz spectroscopy, and has experience developing spectroscopy solutions and applications. After Adam’s first presentation covering the basics of spectroscopy, the present session will deepen the understanding of spectroscopy in practice.

Atomic and molecular spectroscopy are the main types to explore. In atomic spectroscopy, the focus is on the interaction of atoms with light, providing information about the atomic or elemental identity of the sample. On the other hand, molecular spectroscopy involves the interaction of molecules with light, offering insights into molecular identity, molecular structure, and the local environment. In atomic spectroscopy, electronic transitions (electrons moving from ground state to excited states) are primarily studied, while molecular spectroscopy explores electronic transitions, molecular vibrations, and rotations.

For instance, UV-Vis spectroscopy probes valence electron transitions and reports measurements in units of wavelength (nanometers or angstroms) while infrared absorption spectroscopy, focuses on molecular vibrations, reporting measurements in wave numbers (inverse centimeters). Considering absorption spectroscopy, this technique involves the absorption of electromagnetic radiation by a sample, with the absorption varying based on the frequency or wavelength of the light and its interaction with the matter. Examples of different types of light used in absorption spectroscopy and the corresponding transitions they probe include X-rays and transmission with core electrons, UV-Vis light and valence electrons, IR and molecular vibrations, microwave and molecular rotations, and RF and electrons in nuclear spins.

In this technique, transitions occur between molecular orbitals, specifically the Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO). UV-Vis absorption experiments excite electrons from the HOMO to the LUMO, and within these electronic molecular orbitals, there are vibrational and rotational levels that contribute to the transitions observed in experiments. Examples of UV-Vis absorption spectrometers include single-beam and dual-beam configurations that consist of components such as a broadband light source, a dispersion element for separating light, a sample chamber, a detector, and a recorder.

In a UV-Vis experiment, the absorbance of a sample is calculated as the logarithm of the ratio of the intensity of light before and after passing through the sample. This is represented by the equation Absorbance = log I₀ / I_T, where I₀ is the intensity of light before passing through the sample (for the reference sample) and I_T is the intensity after passing through the sample. The absorbance can be used to determine the concentration of a species in the sample using Beer's law. Beer's law states that absorbance (A) is directly proportional to the concentration (C) of the species, the molar absorptivity (ɛ), and the path length (D) through the sample, expressed as A= ɛ x C x D.

An example of how Beer's law works can be seen in how the concentration of proteins in a sample is measured. Proteins contain aromatic amino acids like phenylalanine, tryptophan, and tyrosine, which absorb strongly in the UV region. By measuring the absorbance at a characteristic wavelength (e.g., 280 nanometers) where these amino acids absorb, the protein concentration can be determined. However, this method may not be entirely accurate due to interference from nucleic acids absorbing around 260 nanometers and variations in the composition of proteins. Despite these limitations, this technique is commonly used for monitoring processes like protein purification, often integrated into commercial instruments like HPLC (High-Performance Liquid Chromatography).

The analysis of HOMO and LUMO transitions provides insight into the redistribution of electron density upon electronic excitation. In the case of 4-dimethylamino-4'-nitrostilbene (DMANS), a dye molecule, electronic excitation involves transferring an electron from the HOMO to the LUMO. The electron density diagrams show regions of relative electron richness (green) and electron deficiency (red) for both orbitals. Further examination involves comparing the square of the wave functions for the HOMO and LUMO orbitals to understand the changes in electron density. By subtracting the square of the wave function for the HOMO from that of the LUMO and multiplying the result by the charge of an electron, one can visualize the changing electron density. In the resulting image, red indicates areas of electron loss, while blue indicates areas of electron gain. Experimental data obtained for DMANS in different solvents (benzene, toluene, and 1,3-dichlorobenzene) reveals a shift in the UV-Vis absorption spectrum. This shift indicates changes in the electronic environment or interactions between the solvent molecules and DMANS, influencing its absorption properties.

Photoluminescence, the emission of light from matter following the absorption of photons, occurs in both atoms and molecules. In the context of molecules, there are two primary types: fluorescence and phosphorescence. Fluorescence is characterized by relatively intense and fast emission, typically occurring on picosecond to nanosecond time scales. On the other hand, phosphorescence exhibits weaker emission and longer time scales, often on the order of microseconds or even longer. Both fluorescence and phosphorescence emissions are redshifted from their excitation wavelength. This means that the emitted light has a longer wavelength (lower energy) compared to the absorbed photons. The distinction between fluorescence and phosphorescence lies in the mechanisms governing the return of excited electrons to lower energy states within the molecule.

Aleksander Jablonski, known as one of the pioneers of modern photophysics, extensively studied the processes underlying fluorescence and phosphorescence. His research culminated in the development of the Jablonski Diagram, a schematic representation of the photophysical processes in molecules, where the ground state of a molecule is depicted at the bottom, while excited states, including both singlet and triplet spin states, are shown above. Fluorescence occurs when a molecule absorbs a photon, transitioning from the ground state to an excited singlet state, and subsequently emits a photon as it returns to the ground state. Alternatively, a singlet state can undergo intersystem crossing to a triplet state, facilitated by spin-orbit coupling. Phosphorescence occurs when a molecule in a triplet state emits a photon upon returning to a singlet state, despite the quantum mechanical prohibition against this transition. Phosphorescence is generally less intense and slower than fluorescence, with emission occurring on the order of microseconds or longer. Additionally, molecules may undergo internal conversion, where they relax non-radiatively to the ground state without emitting a photon with no emission is observed. It's important to note that not all molecules exhibit fluorescence or phosphorescence, as these processes depend on the specific molecular properties and electronic transitions involved.

Photoluminescence (PL) is frequently employed to analyze various materials, particularly emerging nanostructures such as gallium nitride. Gallium nitride is a significant wide bandgap semiconductor known for emitting blue light, making it valuable for applications like blue LEDs and diode-based lasers. In a study of gallium nitride nanostructures, researchers utilized PL to investigate defects within the material, mapping the spatial distribution of phosphorescence intensity across the sample surface using an XY translation stage. By examining the photoluminescence properties of these materials, researchers gained insights into their electronic structure and performance, facilitating the development of more efficient solar cell technologies. This process leads to a shift in the energy of the scattered photon, resulting in either higher or lower energy compared to the incident photon. When the scattered photon has lower energy than the incident photon, it's called Stokes scattering, and when it has higher energy, it's called anti-Stokes scattering. These energy shifts correspond to vibrational transitions in the molecule.

Fluorescence spectroscopy involves the study of emitted light from a sample following excitation by photons. In a typical fluorescent spectrometer setup, a light source, which can be broadband or laser, is used to excite the sample. The fluorescence emitted from the sample is usually detected at a 90-degree angle from the excitation source to minimize background interference. The emitted light is then passed through a monochromator for wavelength resolution and detected by a detector. The resulting data, fluorescence intensity as a function of wavelength, is recorded for analysis.

The fluorescence quantum yield, represented by Φ, is an important parameter in fluorescence measurements. It quantifies the efficiency of fluorescence by indicating the ratio of emitted photons to absorbed photons. A perfect emitter would have a quantum yield of 1, meaning it emits one photon for every photon absorbed. Time-resolved fluorescence measurements can be performed using a pulsed laser and a time-resolved detection scheme. This allows researchers to study the fluorescence dynamics of molecules over time. An example shown here is the time-resolved fluorescence of Near-Infrared Fluorescent Proteins (iRFPs), genetically engineered fluorescent probes used in biological imaging, particularly for deep tissue imaging due to their absorption towards the infrared region. By plotting the intensity of fluorescence as a function of time, they can determine the fluorescence lifetime, denoted as tau (τ). The fluorescence lifetime is defined as the time it takes for the fluorescence intensity to decrease to one over the base of the natural logarithm (1/e) of its initial value. This demonstrates how the solvent or local environment can influence photo-physical processes.

Like the solvatochromic shift observed in absorption spectra, fluorescence can also exhibit sensitivity to changes in solvent polarity. Solvatochromic shifts in fluorescence spectra can be significant and serve as sensitive probes for alterations in the local environment surrounding fluorophores or fluorescent molecules. The process involves electronic excitation from the ground state to the excited state, leading to a redistribution of electron density or charge density within the molecule, as observed in the case of DMANS. This redistribution can be influenced by the polarity of the solvent or the nature of the surrounding environment.

FRET, or Förster Resonance Energy Transfer, occurs when two molecules capable of fluorescence are near each other, typically referred to as a donor-acceptor FRET pair. In FRET, the donor molecule is excited from its ground state to the excited state. Instead of emitting fluorescence as usual, the donor transfers its energy to the acceptor molecule, causing the acceptor to become photoexcited and subsequently fluoresce. The efficiency of FRET, denoted by E, depends on the distance between the donor and acceptor molecules. This distance dependence is described by the Förster equation, where the FRET efficiency decreases as the sixth power of the distance (1 / R⁶), making it highly sensitive to spatial proximity. FRET has numerous applications in studying molecular interactions and dynamics. It can be used to investigate protein-protein interactions, monitor conformational changes in biomolecules, probe molecular binding events, and even map molecular distances within complex systems.

Raman spectroscopy offers several advantages over other spectroscopic techniques. It does not require sample preparation, making it suitable for analyzing samples in various states. Additionally, it provides detailed information about molecular structure and chemical composition, making it valuable for a wide range of applications, including materials science, pharmaceuticals, and forensic analysis. The wavenumber in Raman spectroscopy is defined as the number of waves per unit distance, typically measured in inverse centimeters (cm^-1). It represents the energy difference between the scattered photon and the incident photon, normalized to the energy of the excitation source. This normalization allows for consistent comparison across different Raman spectra obtained using different excitation sources.

One of the advantages of using wavenumbers in Raman spectroscopy is that it provides a direct measure of the vibrational energy levels in the sample, allowing researchers to identify specific molecular vibrations and analyze chemical composition based on characteristic Raman peaks. As the excitation wavelength increases, the Raman shift for a given wave number also increases. Conversely, when the excitation wavelength decreases, the corresponding Raman shift decreases. This phenomenon highlights how the choice of excitation wavelength affects the distribution and location of Raman modes in the spectrum. Shorter excitation wavelengths result in Raman modes being concentrated within a smaller spectral range, while longer excitation wavelengths lead to broader dispersion of Raman modes across the spectrum. Raman spectroscopy's specificity enables various applications, such as chemical composition analysis, structural characterization, and multilayer analysis of nanomaterials.