Raman spectroscopy is named after its discoverer ‘Sir C.V. Raman’, who discovered it in 1928. It is a Chemical analysis technique that provides detailed information about the molecular structure without causing any physical and chemical changes. It studies the vibrational modes along with translational and rotational modes of the molecule. In 1930, ‘Sir C.V. Raman’ received the Nobel prize in Physics for his work on the scattering of light and the discovery of the Raman effect.
Principle of Raman Spectroscopy
Raman spectroscopy works on the principle of Raman scattering. When a monochromatic radiation incident on the sample, the radiation gets reflected, absorbed, or scattered. The scattered light photons have a different frequency from the incident photon because of the change in the vibrational and rotational properties of the molecules, which results in the change of wavelength of the incident and the scattered light. This change in the frequencies of the incident photon and the scattered photon is known as the Raman shift. When the scattered photon has less energy, hence a longer wavelength than the incident photon, it is called Stokes scattering. When the scattered photon has more energy, hence a shorter wavelength than the incident photon, it is called anti-stokes scattering.
How Does Raman Spectroscopy Work?
The phenomenon of Raman Spectroscopy includes the interaction of light with the chemical bonds inside the molecule. When a high-intensity beam of light falls on the molecule, then it gets scattered in different directions. Most of the scattered light is of the same wavelength as that of the incident light, this is called Rayleigh scattering. Some amount of light is scattered at different wavelengths, this is called Raman scattering. Rayleigh scattering does not provide any information, but Raman scattering provides the Raman spectrum that consists of a number of peaks that represent the intensity and wavelength position of the Raman scattered light. Each peak shows the specific vibrational energy associated with the chemical bonds in a molecule. In this way, Raman spectroscopy helps to interpret the chemical structure of a molecule.
Uses of Raman Spectroscopy
Raman Spectroscopy is used in many various fields for both qualitative and quantitative analyses of the molecule because it provides information very easily and rapidly. Let us discuss some of the major fields where Raman spectroscopy is widely used.
Identification and Classification of Cells
Raman Spectroscopy plays a significant role in the identification and classification of cells as it uses laser beams that generate unique signals at various wavelengths that can be further used to identify specific molecules like nucleic acid, protein, sugar, etc. This process is accomplished by bombarding a laser beam on a single cell and depending on its nature, the cell may absorb, reflect, or scatter the light waves that eventually generate a pattern known as Raman spectra; this is often termed as the signature or fingerprints of the cells.
The changes in the behavior of a cell that occurs upon its interaction with any toxic agent can be observed with the help of Raman Spectroscopy. Different toxic agents have different effects on the living cells, which results in the different Raman spectra. The more advanced versions of Raman spectroscopy like surface-enhanced Raman Spectroscopy (SERS) and coherent anti-stokes Raman spectroscopy (CARS) are also used nowadays for analyzing living cells. These techniques enhance the Raman signal of the chemical bonds present in cells and are widely used in the development of sensors and biosensors.
Stem Cell Analysis
Stem Cell therapy is a widely used medical technology that can be used to cure many chronic diseases like diabetes, Parkinson’s disease, and cardiovascular diseases. This therapy process involves the replacement of the damaged cells and tissues with healthy ones. Hematopoietic stem cell transplantation is the most extensively used stem cell therapy; the stem cells in this transplantation are usually obtained from the bone marrow or umbilical cord. This technology uses Raman spectroscopy to get the fingerprints of the cells because different cells are specialized to perform specific functions, which, with the help of Raman micro-spectroscopy, can be detected easily.
To Diagnose Diseases
With the help of Raman spectroscopy, diseases can be easily diagnosed as the molecular composition of healthy cells is different from that of diseased ones. Raman spectroscopy is capable of diagnosing both the tissue samples, i.e., healthy and diseased tissue samples. When a laser beam passes through the sample that needs to be diagnosed, the scattered light that is of a different frequency than the incident light is observed. The scattered light consists of both the Rayleigh and Raman scattered light. The frequencies of incident light and the Raman scattered light are different. The difference between the Rayleigh scattered light’s intensity and Raman scattered light’s intensity is called Raman shift, and this shift represents the vibrational frequencies of the molecular sample that is under study. Each chemical bond of the sample molecule has different vibrational frequencies, hence their fingerprints are also unique, and this fingerprint help in diagnosing the ailment in living cells. Nowadays, various clinical diagnostic tools based on this principle of Raman spectroscopy are used to detect diseases. Latest techniques like tip-enhanced Raman Spectroscopy (TERS) and Surface-enhanced Raman spectroscopy (SERS) are able to investigate Protein-ligand binding interactions.
Monitoring the contents of Proteins, Lipids, and Nucleic acid
Raman spectra provide more detailed information about various biomolecules like proteins, lipids, and nucleic acid than the infrared spectra. Raman spectra bands are sharper while the infrared spectra bands are broader, so we get better results by using Raman spectroscopy. Hence, Raman spectroscopy is more beneficial for the detailed analysis of complex biomolecules.
Single-cell analysis is of great importance in cellular biology because most diseases initially start from a single cell, so it becomes crucial to analyze the detailed molecular contents like proteomic, genomic, and even the epigenome content of a single cell. Raman spectroscopy provides the detailed imaging of a single cell without harming and modifying it. This single-cell analysis is very useful in the early detection of harmful diseases like cancer.
Art and Archeology
Pigments characterization plays a crucial role in detecting the age and quality of artifacts found at various archeological sites. The quantitative and qualitative analysis of inorganic and organic compounds can be done by using Raman spectroscopy. It is a widely used analytical tool because of its highly sensitive nature that enables us to detects pigments like chlorophyll, carotenoids, and many other micro-biological pigments. Nowadays, mini portable instruments based on the principle of Raman spectroscopy are also available to detect different microbial pigments.
Ceramics and Gemstones
The structure of ceramics is very complex, but with the help of various Raman spectroscopy techniques, we can study them easily. Traditionally, various methods like UV light test, scratch test, conductivity test, fog test, and heat test were used in the analysis of gemstones that, sometimes, resulted in the distortion of the gemstones, whereas Raman spectroscopy is a fast and low-cost method that can analyze the gemstones without disturbing their physical and chemical properties. The Raman spectra of gems help gemologists to determine their origin and chemical properties by comparing them with the library spectra of gems obtained from various sources. It is possible to identify all gemstones using a special Raman spectroscopy technique involving three light sources.
Paintings, Dyes, and Binders
Raman spectroscopy is used to analyze various inorganic pigments present in paints and dyes. It is also used to identify individual pigments used in paintings to get an insight into the artist’s work. Some advanced techniques like Surface-Enhanced resonance Raman spectroscopy (SERRS) are used for samples that have very small diameters (even less than 25 micrometers) that help in the identification of organic colorants present in sculptures, dyes, and paintings.
The prominent reason for Raman spectroscopy being used in the analysis of archeological materials is that it does not contaminate the article as it does not come in direct contact with it, unlike the age-old traditional methods while investigating the article. In this way, we can find out various contents present in age-old items without interfering and destroying them and protect our cultural heritage.
Chemistry, Geology, and Mineralogy
Raman spectroscopy can provide real-time data of chemical reactions. It provides detailed information of chemical processes without distracting the reaction. Also, the process of sampling and quenching is not needed. In the reaction monitoring process, Raman spectra of the reactants, products, and solvents are observed.
Identifying Molecular Structure
Every molecule has its own unique spectra, Raman spectra provide all the information of vibrational and rotational modes of the molecule that helps to identify the structure of different compounds and also analyze their purity. It also helps to understand the mechanism of crystal formation.
Raman Spectroscopy helps in the identification of the minerals that are obtained from natural sources. It also helps in analyzing the complex man-made microstructures. Raman spectra provide detailed information like the presence of foreign ions, crystals orientation, compositions, stress, and strain, which gives the comprehensive physicochemical description of the minerals.
Natural rocks obtained from the earth usually consist of one or more minerals; these are also analyzed using the technique of Raman spectroscopy. Thus, Raman Spectroscopy is also used in geological studies because it is able to detect very small phase changes that a classical optical microscope can not detect. It gives details like polymorphic changes and amorphization.
Semiconductors are the most crucial components in microelectronics. They are used in device fabrications, microelectronic processing, and thin film deposition. Due to its wide applications, it is very much important to have complete information about the structure of semiconductors. Raman spectroscopy gives a detailed analysis of the geometry and orientation of their structure. It provides precise information about the intrinsic properties of semiconductors, which is most crucial in designing and manufacturing good quality high-performance integrated circuits and devices. Raman spectroscopy also helps in analyzing complex superlattice structures.
Intrinsic Stress or Strain of the Molecular Structure
Any kind of stress and strain in the molecules of semiconductors can be detected by observing the shift in the position of the Raman band. Due to this stress and strain, the band shape changes; broadening and deformation in peaks might also occur. The effects on Raman spectra depends on the characteristics of materials and the geometry of stress and strain.
To identify the Contamination
There is a high possibility of contamination of semiconductors due to various reasons. Raman spectroscopy provides the information-rich Raman spectra that can be used to locate the contaminant’s locations, and it also helps in identifying the source of contamination.
The semiconductor structures, whose chemical compositions change with positions, are called heterostructure. They are used for making high-speed digital and analog devices. Surface-Enhanced Raman Spectroscopy (SERS) provides useful information on Heterostructure characterization and gives quantitative microanalysis, which enables us to understand their chemical properties. It also analyzes the effect caused by the implementation of foreign ions in heterostructures.
Photoluminescence Imaging of Semiconductors
Photoluminescence is the process that involves the emission of light by the material after the absorption of photons. This phenomenon occurs in many bulk materials such as semiconductors having a direct bandgap. Raman spectrometer that has a high spectral resolution is used in performing photoluminescence spectroscopy to get information about the conformation, and solid-state structure of semiconductors. So, Raman spectrometer can be called a two-in-one instrument when used to perform photoluminescence spectroscopy.
Raman spectroscopy is an ideal tool for examining the geometric structure and determining the bonding type within the carbon molecules. Because of its highly sensitive nature, it gives an insight into carbon allotropes like fullerenes and graphene structures. Raman spectra can also reveal the doping and stress in these allotropes that might be caused by temperature or pressure changes.
To Collect Spectra of Carbon Nanotubes (CNT)
Carbon nanotubes are used in various fields that include electronics, microelectronics, solar cells, super-capacitors, and organic light-emitting diodes. The purity of nanotubes is very much important for industrial purposes. Since Raman spectroscopy is a non-contact and non-destructive technique that can be operated at room temperature as well as under controlled environmental conditions without causing any changes in intrinsic properties of nanotubes; it is widely used to obtain the Raman spectra of Nanotubes and determining their purity. Surface-enhanced Raman spectroscopy is also widely used these days as it amplifies the Raman signal and provides better Raman spectra.
Detect Structural Morphology of Carbon Nanomaterials
All the carbon nanomaterials have nearly the same structure, but Raman spectra can detect even the small structural morphologies. Every peak that we observe in Raman spectra corresponds to the particular vibrational energy of bonds in a carbon compound. The sensitivity of Raman spectroscopy in determining structural morphologies makes it a prominent tool used by most material scientists who study carbon nanomaterials.
Pharmaceuticals and Cosmetics
To Determine the Distribution of API
Active pharmaceutical ingredient (API) is the active component in medicines that are used to cure various ailments. Raman spectroscopy helps to distinguish API and raw materials present in a tablet. Nowadays, combination tablets that contain multiple APIs in a single tablet are also manufactured, which makes it easy for the consumer to avoid the stress of consuming multiple tablets. Most of these combination tablets have bisection lines for the equal distributions of the API. Raman Spectroscopy imaging can help in distributing the APIs equally.
Early Drug Development
In biomedical science, Raman spectroscopy has emerged as the most widely used tool. It helps pharmacists design innovative drugs by doing preformulation studies that include the physical, chemical, and analytical analysis of candidate drugs and develop required drugs in the early stages.
Content uniformity involves testing the final product; the concentration of API in the dosage should be under the prescribed limit for consumer safety. Raman spectroscopy is very helpful in doing the content uniformity testing as it does not affect the quality of the product and easily analyzes the concentration of API.
Nowadays, cosmetologists use the technology of Raman spectra to analyze their cosmetics product’s permeability that how easily do the particular products absorb through the skin. The better the permeability, the better is the product. It is also used to check any changes in the skin caused by the cosmetic products by analyzing the dominant biomolecule present in the skin, i.e., Keratin.
Drug Supply Surveillance
The technique of Raman spectroscopy is effectively used by police and various surveillance agency to check the illegal moment of banned substances as Raman spectra can easily distinguish between two similar-looking white powders, which might look exactly the same to the human eye.
Process Analytical Technology (PAT)
Raman Spectroscopy is a very important PAT tool in pharma-Industries. PAT is a mechanism that includes designing, analyzing, and controlling pharmaceutical manufacturing processes by measuring the critical parameters that affect the quality of an active pharmaceutical ingredient (API).
Quality control for the Final Product
After the production of pharmaceutical products, quality control is the next crucial step. The final product includes the dosage as well as excipients; excipients are those substances that directly do not involve in the medicinal action but are added to support the manufacturing process. The Raman spectra of APIs show sharp peaks, while excipients do not show sharp peaks, which enables us to find the actual API concentration and analyze the performance of the final product.