Instruments

Types of Spectroscopy with Definition, Principle, Steps, Uses

It is the science of studying the interactions between light and matter , where the emission and absorption of the light radiations...

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Sourav Bio
This article writter by Sourav Bio on February 02, 2022

Writer and Founder of Microbiologynote.com. I am from India and my main purpose is to provide you a strong understanding of Microbiology.

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Types of Spectroscopy with Definition, Principle, Steps, Uses
Types of Spectroscopy with Definition, Principle, Steps, Uses

Definition of spectroscopy

It is the science of studying the interactions between light and matter , where the emission and absorption of the light radiations by the material are investigated and assessed. The focus of spectroscopy is the dispersion of light as well as other radiations caused by the object, which allows the examination of the various characteristics that the objects.

The measurement of the field of spectroscopy is dependent on the frequency of the radiation observed. The spectroscopy technique has been extensively utilized because it permits the analysis of physical, composition and electronic structure of different particles at molecular or atomic scales.

Definition of spectrometer

The spectrometer is a science instrument that can be used to determine the variations or difference in different properties of an object within a certain distance. The characteristic that is measured by a spectrometer differs depending on the kind of spectrometer employed. NMR spectrometer measures variations in frequencies of nuclear resonance. mass spectrometers measure the variation in charge to mass ratio, while an optical spectrometer analyzes the changes in electromagnetic radiation. By measuring these fluctuations various characteristics of particles can be assessed and observed.

Definition of spectrophotometer

The spectrophotometer is one particular type of spectrometer that studies the interaction (absorption reflection, scattering) in electromagnetic waves emitted by a sample and the emission (fluorescence electroluminescence) from electromagnetic waves from various samples. It is also referred to as electromagnetic spectrometer since it measures the measurement of various characteristics of light and its interactions with matter. These are often used in laboratories to test the quantity of various substances based on the amount of light absorbed by a specimen.

Definition of spectroscope

A spectroscope also known as optical spectrometer, is a device which measures the various characteristics of light across certain areas of the spectrum that is used in the examination of various objects. The characteristic measured is typically its intensity but polarization of light is also measured under certain conditions.

Spectroscopes are often employed in research on Chemistry and astronomy to aid in the examination of diverse types of. The past, prisms were employed as spectroscopes. However today, diffraction gratings and mobile slits, and photodetectors are utilized. They are typically utilized to determine what the composition and chemical makeup of an object is from the amount of radiation produced by different objects.

Definition of spectrograph

The spectrograph is an scientific instrument that detects various wavelengths of light and then separates them by their wavelengths or frequencies that are recorded by multiple detectors. These instruments are employed to capture and record the spectrum of astronomy. The spectrograph is used for studying astronomy, as telescopes.

In a spectrograph light rays enter the spectrograph via the telescope, which is equipped with a mirror which functions to make all light rays of light parallel to one another. The rays are then directed to the diffraction grating which disperses the light into various wavelengths that are then sent onto the detectors to allow the analysis of each wavelength. These detectors are very useful in the analysis of the incoming light coming from various celestial objects to determine the nature on the compositional chemical of these objects.

Definition of spectra

Spectrums, also known as singular spectrum in optics, refers to the colours that are observed when light beams are scattered through the prism. Spectrum refers to the spectrum of variables that are related to light and other waves. In the case of light it is the spectrum of electromagnetic radiation that’s most frequently employed. The electromagnetic spectrum encompasses the frequency range of electromagnetic radiation. These frequencies are used to define the spectrum of electromagnetic radiation that is absorbed or released by objects.

In addition the mass spectrum, it’s utilized in spectroscopy, using the ion abundance as a function of weight to the charge ratio. Electron spectrum is yet another spectrum in physics which is the intensity or number of a particle beam based on the energy of the particle.

1. Absorption spectroscopy

Absorption spectroscopy is founded on the premise that materials possess an absorption spectrum that comprises a variety of radiation that the material absorbs at various frequency. The spectrum of absorption for materials is influenced by the molecular and atomic composition of the material. The frequency of light absorption by a substance is influenced by the energy difference between two states of energy in the molecules.

The absorption causes the creation lines of absorption which, when combined alongside other lines creates the absorption spectrum. So, when a photon that is sufficiently powerful reaches an object, it is absorbed by electrons, causing them to move into a higher energy level. The amount of light (radiation) that is absorbed creates an absorption spectrum, which can be measured in terms of the absorbance. The absorbance of a specimen is influenced by the quantity of electrons excited that in turn are dependent on the quantity of the molecules present within the sample.

The steps of absorption

  1. Solvent liquid as well as the sample solution are positioned in two vessels for transport known as cuvettes.
  2. The solvent-filled vessel is then placed into the spectrometer for analysis of the amount of light lost due to scattering and absorption from the solvent. Any absorption observed during this procedure is subtract from the sample’s absorbance.
  3. The cuvette that contains samples is placed into the spectrometer.
  4. Absorbances of the sample can be recorded at different frequencies, which typically range from 200-800 nm.
  5. A similar spectrum emerges by a different amount of the sample.
  6. The graph of the absorbance determined by the level of concentration in the sample can be plotted which is then used to determine the unidentified concentration of the sample.

Applications

  • Absorption spectroscopy can be used to identify whether there is a specific substance in the sample and then to measure the present substance.
  • This method has been used to remote sensing that permits the measurement of the amount of harmful substances without direct interaction between the instrument with the sample.
  • Absorption spectroscopy can be utilized to determine the molecular and atomic structure of different substances.

2. Astronomical spectroscopy

Astronomical spectroscopy studies the study of astronomical patterns applying the concept of spectroscopy in the analysis of electromagnetic radiation that is the radiation emitted by stars and other celestial bodies.

Principles

The spectrum created by light that comes from celestial objects may not be as clear as that from white light. Both the emission and absorption spectrum of light are created by passing light through a diffraction-grating. When light is dispersed into emission and absorption lines, which depend on the energies of electrons in the molecules and atoms of the object at the source.

On the basis of absorption by radiation of a certain wavelength the graph is drawn with respect to the frequency and wavelength of emission and absorption lines. The majority of these lines develop because of being surrounded by metals. So, based on the length and the width of such lines, the amount of these metals is determined.

Steps

  1. In astronomical spectroscopy, visible light from different stars is reflected by a telescope and into the spectroscope.
  2. The moment the light reaches the diffraction grating on the spectroscope is scattered into various wavelengths.
  3. The wavelengths dispersed by dispersion land on the photodetectors that analyze the wavelengths.
  4. The detectors create the spectrum’s flux scale dependent on the wavelength, in comparison to observations of the standard stars.

Applications

  • Light absorption by the stars shows various properties of stars such as chemical composition temperatures as well as density, mass as well as relative movement.
  • Studying the spectrum of stars aids to understand galaxies as well as the structure of galaxies.
  • Astronomical spectroscopy may also be employed to determine the motion of galaxies and stars by using it’s Doppler effects and the redshift.

3. Atomic absorption spectroscopy

Atomic absorption is an analytical method that utilizes the principle of spectroscopy in the identification of the chemical components.

Principle

Absorption spectroscopy employs the idea that free electrons created by an atomizer absorb light with different wavelengths. Free electrons absorption UV and visible light causing electrons to shift onto higher energy orbits. In this process the absorption spectrum gets released, and is observed by photodetectors.

The absorption spectrum produced allows the quantification of electrons free that are in gaseous states of matter. The amount of light (radiation) absorption results in an absorption spectrum, which can be determined by measuring the absorbance. The absorption of a sample depends on the quantity of the molecules present in the sample.

The steps of Atomic absorption spectroscopy

  1. A liquid is mixed into a certain volume of spirit , which is then added to a flask that will then be vaporized to the form of a gas through an acetylene-nitrous-oxyde flame that is fuel-rich.
  2. An LED is selected to the right wavelength to function as an illumination source.
  3. The gas that is formed by the sample of liquid is taken through a detector that determines the absorbance of gas’s atoms.
  4. Similar procedures are used to determine the absorbance of solvent banks and conventional solution.
  5. A graph is drawn to show the absorbance in relation to the concentration of molecules present in the sample.

Applications of Atomic absorption spectroscopy

  • The spectroscopy of absorption can be utilized for the quantitative and qualitative identification of elements made of metals in biological systems.
  • This is also helpful in the detection of the presence of certain metals as impurities in mixtures of alloys and other compounds.
  • The spectroscopy of absorption has been used to aid in the purification of samples from environmental sources like soil and water.
  • The detection of the presence of metals in oil and pharmaceutical products is also possible using this method.

4. Circular dichroism spectroscopy

Circular Dichroism Spectroscopy is a kind of spectroscopy of light absorption which measures the difference in the absorption of left and right polarized light.

Principle of Circular Dichroism Spectroscopy

Right-handed and left-handed parts of the light incident are taken in in different ways from the specimen, leading to different absorption coefficients. This difference is referred to as circular dichroism. Light rays that are circularly polarized traverse the optically-active medium that has different speeds due to different indices of refractive for circularly polarized left and right light.

The chiral molecules that are optically active will more often absorb one side of circle-polarized beam of light. Protein peptide bonds function as chromophores. The peptide bonds are optically active chiral proteins of proteins, and the amount of chromophores is proportional the absorption capacity. This means that the degree of absorption can be used to verify the secondary structure that is adopted by proteins.

Steps of Circular Dichroism spectroscopy

  1. The sample is put in a vessel for transport with buffers , and then placed into the spectrometer.
  2. In the spectrometer circularly polarized light pass through samples in an alternate manner.
  3. The detector for the photomultiplier within the spectrometer generates an electric charge in proportion to the circle dichroism (the different between the absorption of left and right-polarized radiation) of the beam that emerges out of the specimen.
  4. It is examined against normal proteins to identify the differences in second structure of proteins.

Applications of Circular Dichroism Spectroscopy

  • The most important use of CD spectrum is the confirmation of the secondary structure that is assumed to be that the proteins have. This method permits the determination of the proportions of a-helix and the b-sheet of proteins based upon their circular dichroism.
  • Circular Dichroism spectroscopy can be used to observe changes in secondary structure within the sample in time.
  • This technique may be utilized to compare two macromolecules and to identify variations in the structure and structure of molecules.
  • It can also be utilized to study pharmaceutical products to verify that they’re still present in their fold-up active conformations.

5. Electrochemical impedance spectrum (EIS)

Electrochemical impedance spectroscopy is a sophisticated electrochemical method that determines the impedance of an electrical system by applying various AC frequency potentials.

Principles of Electrochemical impedance spectrum (EIS)

Electrochemical Impedance Spectroscopy (EIS) is the method used to determine how a brand new material or device hinders the movement of electricity. This is achieved using the AC signal through the electrodes that are connected with the specimen. AC voltage of various frequencies is applied to the sample, and then the electrical current is recorded.

A Nyquist plot is created by comparing the frequency response to the electrical impedance drawing the impedance on the y-axis, and the speed along the x-axis. The instrument is able to apply an oscillating field to the test sample and then measures the response of the current. The imaginary and real components of impedance are determined by calculating the phase shift and the change in amplitude with various frequency.

The steps of electrochemical impedance spectroscopy (EIS)

  1. The test module is then connected to the EIS to confirm that the wires that connect to the system are connected properly, and that all components are functioning properly.
  2. To begin the flow of current through the system to begin the flow of current, it is necessary to start the flow of current. Zplot software is installed by the computer. The necessary parameters are set and the AC amplified value is set to 10mV. Initial frequency set at 1×106 and the final frequency is set to 100Hz.
  3. The Zview software can then be used to display the results.
  4. The electrodes are taken off of the testing module.
  5. The sample is made and then put into the test furnace, which is located inside the fumes from the hood.
  6. The electrodes are connected to the assembly and the EIS is operated using the same way as before.

The use of electrochemical impedance spectroscopy (EIS)

  • The impedance of a specimen could be used as a method to identify the number of microorganisms that grow in the sample.
  • EIC can also be used to test for cancerous tissues, where the electrical current’s impedance changes as the structure of the cell and size alters.
  • It is also used to study the effects of chemical layers, polymers, or coatings on electrodes, which can be beneficial in the transfer of electrons and sensitiveness.

6. Electron spin resonance (ESR) the study of spectroscopy

Electron Spin Resonance (ESR) often referred to by the name of Electron Magnetic Resonance (EMR) also known as Electron Paramagnetic Resonance (EPR) can be described as an absorption spectroscopy whereby radiations with a frequency within the microwave range (0.04 25 cm – 0.04 cm) are absorption by paramagnetic materials which cause a transition between the magnetic energies of electrons that have unpaired spins.

Principles of ESR spectroscopy

The spectroscopy of electron spin resonance is founded on the notion that molecules, atoms or ions with unpaired electrons have magnetic properties due to the electron spin. If a compound or molecule is placed within a magnetic field the spin of electrons that are not paired can be aligned in two distinct states. In the event that the electrons’ spin is aligned with that direction as the field of magnetic energy it will result in the state of lower energy. Contrarily, the opposite direction to the field leads to an energy state that is higher.

In the end, an electron that is not paired can shift between the two states of energy through either absorption or emission of electrons, which creates the resonance condition. The spectrum generated during the absorption process is sent to detectors for study of the electronic structure within the samples.

The steps of ESR spectroscopy

  1. It is the ESR instrument is switched on and then warmed up over a period of 30 mins. The required parameters are set by the ESR.
  2. Calibration is done by using to an unfilled ESR tube to make sure there are there aren’t any background signals coming emanating from the tube or instrument are present.
  3. The sample is placed inside the ESR tube after which its ESR spectrum is then measured.

Applications of ESR spectroscopy

  • Electronic spin resonance (ESRS) (SRS) is one of the primary methods for studying the metalloproteins that contain transition metals.
  • This technique also permits to study the denaturation of proteins as well as the folding of proteins.
  • ESR helps to determine the catalytic rate of various metals.
  • It is also utilized in the field of biological research , which includes qualitative and quantitative analyses of the reactive nitrogen species (RNS) and reactive oxygen species (ROS).

7. Emission spectroscopy

Emission spectrum is a technique that analyzes the wavelengths of light particles that are emitted by molecules or atoms as they transition from a high energy state to a lower energy level.

The Principle of Emission Spectroscopy

When molecules or electrons are heated, either by an open flame or with the use of an electrical heater, they release energy by emitting light. The light produced by the compound is then absorbed by an spectrometer, which then splits the light into distinct wavelengths.

Every element has its own spectrum of atoms that indicate that an atom emits only a limited quantity of electricity. Every element emits a specific set of distinct wavelengths distinctive to it due to its electronic structure. using this spectrum, the structure of the specimen is identified.

The steps of emission spectroscopy

  1. The sample’s solution is heated, either by the form of a flame or by the aid of an electrical heater.
  2. The solvent first evaporates and the finely divided solid particles remain in the middle of the flame , along together with the other molecules as well as Ions.
  3. This results in the stimulation of electrons, which results in the emission of a particular wavelength.
  4. The light is then passed through the spectrometer and the monochromator scatters light into various wavelengths.
  5. Detectors are able to detect wavelengths within the spectroscope.
  6. An image of the wavelength and the amount of concentration can be plotted to calculate how much concentration is present in the specimen.

Utilization of Emission spectroscopy

  • Emission spectroscopy can be used in environmental and agricultural analysis as well as in industrial analysis to detect of alloys and metals.
  • It is also used to determine lead in petrol.
  • This method has been utilized to determine the equilibrium constant for Ion Exchange resins.

8. Energy dispersive spectrum

Energy dispersive spectroscopy is also known as Electron dispersive analysis, is a method to determine the chemical or elemental characterization of the sample using the interaction of X-rays and the sample.

The principle of Energy dispersive spectroscopy

The basic principle behind energy dispersive spectroscopy is comparable to emission spectroscopy, except that the stimulation of electrons is caused through the X-ray beams. The sample contains electrons present in its ground state that are bound in the nucleus. When X-rays focus on an element, electrons at the ground level are excited and travel to inside the shell.

The result is an electron-hole within the outer shell, which is then filled by an electron from the outer high-energy shell. The energy difference between the high-energy shell and low-energy shell is released into the form of beams of X-rays. Energy dispersive spectroscopy is used to determine the amount of energy as well as the number of beams of X-rays that are released in the samples. The energy difference between two shells is a sign in the structural atomic makeup the sample. This aids in determining the chemical composition of the specimen.

The steps in Energy dispersive spectroscopy

  1. Two samples with known and undiscovered concentrations are collected in an instrument for transport, often called cuvette.
  2. These vessels then are next placed one after another inside the spectrophotometer, which is equipped with an excitation source, which could be either one of electrons or x-ray beam, and detectors.
  3. The spectrophotometer is able to transmit an x-ray across the sample.
  4. The photosensitive detectors within the spectrophotometer can detect radiation that passes through the sample. It is later converted to voltage signal.
  5. The voltage signals enter the pulse processor, which measures the signals before passing the signals to an analyzer to display data and then for further analysis.

Applications for Energy dispersive spectroscopy

  • Energy-dispersive X-ray spectrum (EDS) as part of electron microscopy is extensively utilized in various research fields because it gives precise information about the chemical substance and subcellular/subcellular structures.
  • EDS has been utilized in medical diagnostics, as the X-ray microanalysis could be helpful in determining the guidelines for malignant tissue growth.
  • The use of electron dispersive spectrum in environmental analysis is an effective instrument to determine the danger of air pollution regarding the health of humans.
  • Pesticide droplets (0.5 ul to 1 ul) in varying amounts on the plant’s surface can be analysed using this method.

9. The use of fluorescence spectroscopy

The fluorescence spectroscopy technique is a form of electromagnetic spectroscopy that makes use of the fluorescence generated by the objects within an area that is not necessarily visible in the range within the spectrum.

Principle of Fluorescence Scroscopics

The fundamental principle of fluorescence spectroscopy resembles emission spectroscopy. In both, the change of electrons from one state to the next creates emissions in the spectrum. It is an emission process that occurs when a transition from a higher state to less energy-rich state can be preceded by radiation. Only molecules that are in their excited forms are able to emit fluorescence and consequently, they need to be brought to an energy state that is higher in order to trigger the phenomena.

The radiation emitted appears as bands because there are numerous closely related wavelengths dependent on the rotational and vibrational energy levels achieved. The spectrum of the fluorescence of the molecule is independent of what wavelength is used by the exuberant radiation and also has an image mirror relationship and the absorption spectrum.

The probability of transition from the electronic excited state to that of the grounded state will be dependent on what intensity is light. The properties of fluorescence of molecules are determined by the properties of the molecule and aid in the identification of the composition of molecules.

The steps of fluorescence spectroscopy

  1. Two samples of unknown and known concentrations are collected from the transport vessel, which is also called cuvette.
  2. These vessels then are placed one after another into the spectrofluorimeter, which comes with a detectors and a light source.
  3. The spectrofluorimeter transmits light of a certain length through the material.
  4. The photosensitive detectors in the spectrophotometer are able to detect sunlight that is passing through the sample. The light transforms into digital data.
  5. The graph shows the intensity of fluorescence measurement by the level of concentration in the sample drawn, which is then used to determine the unidentified concentration of the sample.

Applications of fluorescence spectroscopy

  • The use of fluorescence spectroscopy can be found in medical, biomedical, and chemical research to aid in the study and analysis of organic chemicals.
  • It is also used to differentiate benign tumors from malignant ones.
  • The spectroscopy of atoms can be utilized to determine the presence of various metals in different environmental samples , including water, air, and soil.
  • In the field of analytical chemistry, fluorescence detectors are employed in conjunction with HPLC.

10. Fourier-transform infrared (FTIR) spectroscopy

Fourier-transform infrared spectroscopy can be described as an instrument used to make identification of the spectrum infrared that shows absorption as well as emission molecules in liquid, solid or gaseous phase. The term Fourier-transform was applied because the procedure requires an algorithmic process known as Fourier transform to transform the initial data to the spectrum.

Principle of FTIR spectroscopy

Fourier-transform spectroscopy employs the same dispersive principle as dispersive spectroscopy, but instead of focusing the beam on only one frequency, this method makes use of the beam to have several frequencies. This technique will then determine the extent to which the beam is absorbed into the sample. A second beam of different frequencies is utilized to process samples and data is taken.

The process is repeated several times over a brief period of time. All of the information gathered is later passed on to a computer which is able to work backwards to find the wavelength of absorption. The spectrometer features a specific design known as Michelson interferometer, which is comprised of a number of mirrors, with one which is moved toward the source of light. When the mirror, the wavelength of light is blocked and then transmitted as a result of waves of interference. Because of this the light that comes out from the interferometer displays an entirely different spectrum every time.

Steps of FTIR spectroscopy

  1. The sample is then placed into the FTIR the spectrometer. The sample must be thin enough for IR light to travel through.
  2. The device directs IR to the sample and determines the extent and the frequencies of beam are taken up from the specimen.
  3. Utilizing an online reference database the item can be recognized.

Uses of FTIR spectroscopy

  • FTIR spectrum is widely used to study organic, polymeric and even organic compounds.
  • This method permits the measurement about the chemical content of different samples.
  • FTIR is used in conjunction in conjunction with gas chromatography to aid in the identification of substances that are that are not chromatographically separated.
  • FTIR can be used to determine the polarity of a given location while studying the hydrophobic membranes.

11. Gamma-ray spectroscopy

Gamma-ray spectroscopy can be described as an analytical method used to studying the spectrum of energy of gamma rays generated from radioactive substances in the form of a sample.

Principle of Gamma-ray spectroscopy

In the course of radioactive decay, gamma decay can also be observed within radioactive compounds. Gamma decay is when nucleons that are found in the nucleus in different levels shift toward a lower energy state and the energy gap is released as the Gamma-ray. Gamma rays then transmitted through detectors which transform the radiation into electrical signals that are used by computers.

The identification and quantification of gamma radiations can help identify the type of source. By comparison of the measured energy with the energy that is known for radioisotopes that emit gamma-rays, the source of the emission can be identified.

Steps of Gamma-ray spectroscopy

  1. The chamber that counts is opened by using the handle. The inside of the chamber is usually surrounded by lead, and then covered with copper in order to limit the radiation background caused by xrays that are emitted by the lead.
  2. The sample container is set delicately on top of the detector. Then the chamber for counting is sealed.
  3. Background counting is used to determine whether radiation or contamination is present in quantities that could interfere with the sampling data.
  4. Additional analysis is performed using computer systems.

Uses of Gamma-ray spectroscopy

  • Gamma-ray spectrum is only used to study the nuclear structures, transitions as well as nuclear reaction.
  • This is also used in astronomical research, like the detection of forts in other stars.
  • Gamma-ray spectroscopy is used for analysis of the elemental and isotopic composition of space-less bodies within the solar system particularly mars and the moon.

12. Infrared (IR) spectrum/ vibrational analysis

Infrared spectroscopy is also known as vibrational spectrum, is a method which makes use of the interaction between the infrared rays and the sample.

The principle of IR spectrum/ Vibrational analysis

The wavelength that is used in the study of organic compounds varies from between 2,500 and 16,000 nm and the frequency range is between 1.9×1013 to 1.2×1014 in Hz. The rays aren’t able to generate enough energy to trigger electrons, however they do trigger vibrational excitation in the covalently bonded atoms and groups.

The sound that is observed in atoms is typical of these atoms, and aids in the identification of molecules. The spectrum of infrared is the most fundamental measurement made by Infrared spectroscopy.

It is the diagram of the measured intensity infrared versus frequency (or frequency) of light. IR Spectroscopy analyzes the frequency and vibrations of atoms and on the basis of this, you can identify what functional groups.

The steps of IR spectrum/ vibrational analysis of spectroscopy

  1. The IR spectrumrometer is switched on and left to warm up for about 30 minutes.
  2. The unidentified sample is collected, and its appearance recorded.
  3. The background spectrum is gathered to eliminate the spectrum derived from natural causes.
  4. A small portion of the sample is placed in the probe with the metal spatula.
  5. The probe is positioned in its place with a twist.
  6. It is then the IR spectrum from the unidentified sample is gathered. This process is repeated in case necessary, to obtain an acceptable quality spectrum.
  7. The frequencies of absorption that show the functional groups in the present are recorded.
  8. The spectrum obtained is then analyzed to identify the most likely identity of the sample that is not known.

Applications of IR vibrational spectroscopy and spectroscopy

  • Infrared spectroscopy is widely employed to characterize proteins and for the examination of various solids liquid, gaseous, and solid samples.
  • IR The spectroscopy of IR can be utilized to detect functional groups, which aids in the determination of the molecules and their components.
  • Applications of IR methods of spectroscopy allow for the detection of molecular changes triggered by changes in the body, understanding the molecular mechanisms behind various illnesses, and identifying particular biomarkers using spectral spectroscopy that can be utilized in diagnosing.

13. Magnetic resonance spectroscopy

Magnetic resonance spectroscopy (RSS) is a diagnostic method that is used in conjunction with the magnetic resonance imaging (MRI) that measures the changes in biochemistry that occur throughout the body.

The basic principle of Magnetic resonance spectroscopy

Magnetic resonance spectroscopy analyses the hydrogen atoms that are present within the specimen. The nucleus of an atom is activated with a magnetic force which results in the nucleus to spin in a specific direction. The variation in intensity of the various directions of spin creates an array of spectrums that are specific to the nucleus of an atom.

The frequency of the spectrum are determined, which allows to determine the amount of different compounds. By measuring the concentration of compounds, and then comparably it with the typical concentration of various cells, the kind of tissue that is found in the body is identified.

Steps to Magnetic resonance spectroscopy

  1. It is the MR instrument is switched on and then warmed up for about 30 minutes. The parameters that are required are set by the magnetic resonance instrument.
  2. The calibration process is carried out by using the empty MR tube to make sure there are no background signals either from the tube or instrument are present.
  3. The sample is then put inside the MR tube and the spectrum of magnetic resonance is then measured.

Applications for Magnetic resonance spectroscopy

  • Magnetic resonance spectroscopy allows doctors and chemists to get biochemical information about the tissues in the body, which aids in the process of diagnosing.
  • MR spectrum is utilized in conjunction with the MRI procedure that is typically used to detect problems in hospitals.
  • It is now increasingly used in medical research projects.
  • This method has also been used to create profiling serum samples that are metabolically analysed from patients who have been diagnosed with hepatocellular cancer (HCC).

14. Mass spectroscopy

Mass spectroscopy is one type of spectroscopic method that allows to determine the quantity and types of chemicals found in the sample through analysing the mass to charge percentage of the charged ions.

The principle behind Mass spectroscopy

Mass spectroscopy is founded on the premise that when a substance is bombarded with electrons molecules present in the substances are transformed into ions. The separation of ions depends on their charge to mass ratio. In the majority of ions, they have a charge of one, which indicates that the ratio is the molecular mass of an Ion. The ions are then exposed to magnetic and electric fields that cause deflection of the particles. Ions that have a similar mass to charge ratio exhibit similar deflection.

The amount of each of these ions is later determined using detectors. Mass spectrums are created by charting the relative abundance of ions in relation to the proportion of charge to mass. The spectrum can be used to determine the basic structure for the samples, mass of the particles or molecules as well as how the samples chemical structures are formed.

The steps of Mass spectroscopy

  1. 200 milliliters of the sample are mixed with 1.8 milliliters in 65% nitric acids.
  2. The mixture is then added to the bath overnight, at 50°C.
  3. They are chilled down to room temperature and the sample is then diluted by adding 8ml of distillate water in order to achieve nitric Acid concentrations below 20%..
  4. It is added back to the spectroscope, and the experiment is then carried out.
  5. The results are recorded by the software that runs on computers and are displayed in the form of the mass spectrum.

Applications of Mass spectroscopy

  • Mass spectrum is an effective instrument to measure the properties of known materials.
  • It also allows identification of compounds that are not known and the determination of the chemical and structural composition of different substances.

15. Molecular spectroscopy

Molecular spectroscopy is one type of spectroscopy which utilizes the interaction of molecules and electromagnetic radiation to identify the chemical composition of samples.

The principle of Molecular spectrum

Molecular spectroscopy is the process of interfacing of molecules with electromagnetic radiation to create Absorption patterns (i.e. the spectrum) that structural or compositional data can be derived. The molecular spectrum develops by the movement of molecules from one state of energy to another through interactions with various electromagnetic radiation.

The processes involved are identical to those of atoms, but the process is more complex. The interaction between electrons and nuclei occurs in molecules that cannot be seen in the atoms. These interactions may be electrical, rotational or vibrational. Based on the nature of interaction, various techniques for spectrometric analysis are available to analyze the substances. Similar to the absorption or emission spectrum could be used in accordance with the type of interactions to be used.

The steps of Molecular Spectrum

  1. Different types of spectrometers such as Fourier-transform and emission spectrometers can be utilized to perform molecular spectroscopy.
  2. All of these systems comprise the radiation source along with a sample, as well as the system to detect and analyzing the samples.
  3. The sample is placed in the spectrometer and the radiation source is focused on the sample.
  4. Two samples with known and undiscovered concentrations are collected from the transport vessel, which is also known as cuvette.
  5. These vessels then are placed one after another into the spectrophotometer, which comes with a the light source and detectors.
  6. The spectrophotometer operates by transmits light of a certain length through the specimen.
  7. The photosensitive detectors in the spectrophotometer are able to detect light that passes through the sample. The light is converted into digital data.
  8. An absorption graph calculated in relation to the amount of sample present is drawn that can be later used to determine the concentration that is unknown for the sample.

The applications of Molecular spectrum

  • Molecular spectroscopy permits the analysis of the molecular and atomic structure of many substances.
  • It is also helpful in determining gas composition, as well as the composition of other compounds.

16. Mossbauer spectroscopy

Mossbauer spectroscopy is a method that is based in the Mossbauer effect, which was discovered through Rudolf Mossbauer which utilizes the spectrum produced by emission or absorption of nuclear gamma rays within solid particles.

The principle behind Mossbauer spectroscopy

Mossbauer effect says that the recoil energy related to emission or absorption of a photon could be explained by the conservation of momentum. In solids the nuclei of atoms aren’t free, and are bonded to a specific lattice. When a nucleus inside solid particles emits or absorbs gamma-rays, the energy is always lost in discrete packets known as phonons which are quantized oscillations of the crystal’s lattice.

In certain instances there are instances where there is a possibility that no phonons are released in some cases, and momentum conservation in these cases is sustained through the force of the entire lattice. Therefore, gamma rays released by a single nucleus could be completely absorbent by samples containing nuclei that share the same isotope. the absorption of this absorbed energy can be determined. The observed Mossbauer spectrum is used to determine any radioactive sample.

The steps of Mossbauer spectroscopy

  1. The sample is then placed in the spectrometer.
  2. Gamma radiation is then absorbed by the sample. The atoms that are part of the source of radiation emitting radiation must be of the same isotopes as the atoms of the sample that absorb them.
  3. The detector measures then the strength of the beam which is transmitted by the specimen.
  4. The spectrum is then examined for various reasons.

The uses of Mossbauer spectroscopy

  • Mossbauer spectroscopy can be a sensitive method that is able to detect even subtle changes to your nuclear surroundings.
  • It gives precise information on the magnetic, chemical and structural properties of various objects.
  • It is also utilized in geology to determine the chemical composition of meteors as well moon rock.
  • This technique is widely utilized in bioorganic Chemistry to study enzymes and iron-containing proteins.

17. NMR spectroscopy (NMR) analysis of spectroscopy

The spectroscopy of nuclear magnetic resonance a method that makes use of nuclear magnetic resonance in order to identify the structure of different substances.

The principle behind NMR spectroscopy

The spectroscopy of nuclear magnetic resonance founded on the notion that the nuclei of molecules, atoms, or ions are charged and possess spin. If a molecule is placed within a magnetic field the nucleus’s spin can be aligned in two different states. When the spin aligns with an axis of the magnetic field it leads to an energy state that is lower and the reverse aligning of the field leads to an energy state that is higher.

In the end, the nucleus may change between these two states of energy by either absorption or emission of electrons, which creates the resonance condition. The spectrum that is produced by the absorption process is sent to detectors to allow the study of the nuclear structures that are present in the specimen.

The steps of NMR spectroscopy

  1. It is the NMR instrument is switched on and then warmed up over 30 mins. The required parameters are set by the NMR.
  2. The calibration process is carried out by using the empty NMR tube in order to check for background signals either from the tube or instrument are present.
  3. The sample is placed inside the NMR tube after which its NMR spectrum is then measured.

Utilization of NMR spectroscopy

  • NMR spectrum analysis is utilized to control quality.
  • It also aids in the analysis of the amount and purity of the sample as well as looking for the structure of the substances.
  • Solid-state NMR spectrum is employed to study the molecular structure of solids.
  • It is also employed to determine the protein’s structure.

18. Photoelectron spectroscopy

Photoelectron spectroscopy is also known as photoemission spectroscopy is the spectrometric method used to measure the release of electrons from substances as a result of the photoelectric effects to determine the energies of binding for the electrons.

Principles of Photoelectron spectroscopy

Photoelectron spectroscopy is based on principles of photoelectric effects. The sample is exposed UV rays which cause photoionization in the materials. The photoelectrons emitted have energy levels that correspond to their initial energy states as well as the rotational and vibrational levels of electrons. Their energy can be utilized to calculate the energy of binding for the electrons within the atoms using the following formula:

BE= hn-KE where hn represents the radiation that is incident and KE is the energy produced by the photoelectrons that are emitted. The BE-KE graph can then be shown. The peaks of the spectrum are the electrons that reside in the different subshells within an atom. The lower peaks are the electrons in the valence region, while the top peaks are the electrons in the core.

The steps of Photoelectron spectroscopy

  1. The sample is then placed inside the spectrometer. It is exposed to the ionizing radiation of the source of radiation.
  2. The electrons that are released hit the detectors. This transforms their energy in an electrical signal.
  3. The signal is then transferred to an analyzer in order to extract the analog information out of the signal.
  4. A graph is then plotted that shows the energetic energy of emitting electrons and the energy that is bound to them.
  5. On the basis of the peak that are formed by the graphs, mystery element can be identified.

The applications of Photoelectron spectroscopy

  • Electron spectroscopy is sensitive all elements, with the exception of hydrogen and helium. Therefore, it plays a significant function in all aspects of chemistry.
  • Because the energetic levels in atoms can be influenced to their surroundings This method is the most effective method for determining the oxidation levels of molecules’ atoms.
  • By analyzing the spectra of the photoelectron One can determine the ratios between different elements and atoms of the same element.
  • This method is sensitive to a variety of important areas of study, such as catalysis studies, corrosion studies study, semiconductor research as well as adsorbed gas works and electrode research.

19. Raman spectrum

Raman spectrum is an analytical method that gives precise information on its chemical composition, the polymorphism crystallinity and molecular interactions in a compound.

The principle behind Raman spectrum

A Raman spectrum is derived by analyzing scattering light from the specimen. The bulk of the incident light beam traverses it (transmission). A small portion of the beam is scattered isotropically i.e. uniformly across all directions and has the same wavelength that the beam that was incident. The Raman spectrum is derived due to the fact that only a small percentage of the light incident by the test has distinct frequencies from that of the light incident.

As a result of the incident photon, the molecules move to an exciting rotational-vibrational-electronic state, and the photons are emitted from the sample. To maintain its energy following the molecule has moved to a higher level that is, the scattered photons shift into an entirely new state. The difference in energy between the new and original condition of the photon scattered is equivalent to the energy difference between the two states of vibration for the molecules.

The primary requirement for a peak to show up on the Raman spectrum is a change in the polarizability of the molecule in the transition. The depolarization can be calculated by comparing two intensities that are perpendicular and parallel polarization relative to incident light.

The steps of Raman spectrum

  1. Aqueous solutions are the most common choice. to turn the laser on following the selection of the suitable wavelength for the.
  2. The spectrometer then gets calibrated using a sample test sample using the correct timing and exposure energy.
  3. The sample is then put under the microscope, focusing on the area to be studied.
  4. A variety of wavenumbers are processed by the monochromator which produces what is known as the Raman spectrum.
  5. This data then gets examined using appropriate software, and the analysis is by analyzing the peaks in the spectrum.

The uses of Raman spectrum

  • Raman spectroscopy can be used in conjunction with infrared spectroscopy because it permits the study of the sample under water conditions.
  • This method is also employed for determining the composition of chemicals and the structure of different samples.

20. UV spectroscopy

UV spectrum is a kind of absorption spectroscopy in which UV light sources are attracted by electrons, which cause them to become excited and reach the state of high energy.

The principle behind UV spectrum

In UV spectroscopy, UV radiations that are passed through the sample are absorbed by electrons, which increase intensity of the process. This results in the stimulation of electrons that move from the lower energy state to an energy level higher. The excitation creates an absorption spectrum, which can be observed by detectors inside the spectrometer.

The amount of light (radiation) absorption results in an absorption spectrum that can be evaluated by measuring the absorbance. The absorption of a sample depends on the amount of excited electrons , which in turn are dependent on the amount of the molecules present in the sample.

Steps of UV spectrum

  1. Two samples of unknown and known concentrations are collected from the transport vessel, which is also known as cuvette.
  2. They are placed one after the next into the spectrophotometer, which has a detectors and a light source.
  3. The spectrophotometer transmits light of a certain length through the specimen.
  4. The photosensitive detectors that are present in the spectrophotometer are able to detect light that passes through the sample. The light is converted into digital data.

An absorption graph calculated in relation to the amount of sample present is drawn which is then used to determine the concentration that is unknown for the sample.

Applications of UV spectrum

  • UV spectrum is a technique that is used to detect organic compounds that contain impurities.
  • This is also employed to determine the quantity of compounds that are able to absorption of UV radiation.
  • It is also used for studying the kinetics of reactions in which UV rays are absorbed by the cell of the reaction and the changes in absorbance are examined.

21. Ultraviolet and visible (UV/Vis) spectroscopy

Ultraviolet and visible spectroscopy an absorption spectroscopy method that utilizes radiation from the UV range, as well as the visible range of electromagnetic radiation.

Principle of UV/Vis spectroscopy

UV/Vis spectroscopy operates on the premise that materials create an absorption spectrum that is an array of absorption that results from radiation absorbed by the materials at various frequency. The spectrum of absorption for materials is determined by the molecular and atomic composition of the material. The intensity of light absorption by a substance is influenced by the energy difference between two states of energy in the molecules.

The absorption causes the formation of an absorption line. This along and other lines creates the absorption spectrum. The light that is incident in this spectrometer falls within the spectrum between the UV and visible spectrums in the electromagnetic spectrum.

So, when a light with enough energy hits an object, its energy is absorbed by electrons and causes them to shift into a higher-energy state. The amount of light (radiation) that is absorbed creates an absorption spectrum, which can be later measured by the absorption. The absorption of a substance is influenced by the quantity of electrons that are excited, which depends on the amount of the molecules present within the sample.

Steps of UV/Vis spectroscopy

  1. Solvent liquid and sample solution are positioned into two transport vessels called cuvettes.
  2. The solvent-filled vessel is then placed into the spectrometer to measure the loss of light due to scattering and absorption from the solvent. Any absorption observed during this procedure is subtract from the absorbance in the sample.
  3. The cuvette containing liquid sample is put in the spectrometer.
  4. Absorbances of the sample can be measured at various frequencies that generally ranges between 200 and 800 nm.
  5. A similar spectrum emerges by the various levels of the substances.
  6. The graph of the absorbance calculated in relation to the amount of sample present is drawn which is then used to determine the unidentified concentration of the sample.

Uses of UV/Vis spectroscopy

  • Qualitative analysis can be conducted in the UV/Vis areas to determine certain categories (proteins as well as nucleic acid) of compounds, both in their pure form and within biological mix-ups.
  • This kind of spectroscopy can be employed to quantify biological samples, either directly or by using colorimetric assays.

22. X-ray photoelectron spectroscopy

The X-ray photoelectron spectrum is a precise, accurate method of spectroscopy based on the photoelectric effect that is used to aid in the detection of electrons within the compound, on its surface, as well as the chemical composition and electronic configuration of the substance.

Principle of X-ray photoelectron spectroscopy

Photoelectron spectroscopy is based on an idea of the effect of light. The sample is exposed the X-rays at specific wavelengths to cause photoionization of the substance. Photoelectrons that are emitted emit energy levels that are a reflection of their energy states at the time of their creation and the level of rotation and vibration of electrons. The electrons’ energy is utilized to calculate the energy of binding for the electrons inside the atoms using the formula:

BE= hn-KE where hn represents the radiation that is incident as is KE the energy produced by photoelectrons that are emitted. The BE-KE graph can then be shown. The peaks on the spectrum are the electrons within the various subshells within an atom. The lower peaks are the electrons in the valence region, while the top peaks are the electrons in the core.

Steps of X-ray photoelectron spectroscopy

  1. The sample is put inside the spectrometer. It is exposed to the ionizing radiation of the source of radiation.
  2. The electrons emitted hit the detectors, which transforms their energy in an electrical signal.
  3. The signal is then transferred to the analyzer , which extracts the analog information out of the signal.
  4. A graph is then drawn on the energy kinetic of released electrons as well as the energy of binding.
  5. By analyzing the high points by the graphs, unidentified element can be identified.

Uses of X-ray photoelectron spectroscopy

  • X-ray photoelectron spectrum is widely employed to measure chemical composition, chemical state and electronic structure of various organic and inorganic compounds.
  • This can also aid in the analysis of surface areas of different compounds.

References

  • https://www.platypustech.com/5-different-types-of-spectroscopy
  • https://www.biologydiscussion.com/biochemistry/spectroscopic-techniques/top-14-types-of-spectroscopic-techniques-explained-biochemistry/12900
  • https://www.slideshare.net/guest824336/introduction-to-spectroscopy
  • https://www.news-medical.net/health/Spectroscopy-Types.aspx
  • https://microbenotes.com/types-of-spectroscopy/
  • https://www.britannica.com/science/spectroscopy
  • https://en.wikipedia.org/wiki/Spectroscopy
  • https://www.azooptics.com/Article.aspx?ArticleID=1382
  • https://chem.libretexts.org/Courses/Northeastern_University/10%3A_Spectroscopic_Methods/10.1%3A_Overview_of_Spectroscopy
  • https://microbiologynotes.org/spectroscopy-and-its-types/
  • https://www.vedantu.com/physics/spectroscopy
  • https://www.toppr.com/guides/physics/electromagnetism/spectroscopy/
  • https://www.slideshare.net/BivekTimalsina/introduction-to-spectroscopic-methods-65218657
  • https://www.slideshare.net/mariomS7/uvvis-spectroscopy
  • https://www.slideshare.net/Santachem/uv-visible-spectroscopy
  • https://www.biologydiscussion.com/biochemistry/spectroscopic-techniques/top-14-types-of-spectroscopic-techniques-explained-biochemistry/12900
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