Spectroscopy

Spectroscopy using spectrophotometers of different types like: U.V, Mass Spectrophotometer, absorption , Emission, Nuclear magnetic resonance and X-rays Spectrophotometer

1. SpectroScopy

2. Spectroscopy “ Spectroscopy is t he interaction between material and electromagnetic radiation. It is a technique used for the analysis of pharmaceutical and biomedical materials”. Previously it was believed light travels in straight line but some important phenomenon like Interference, Refraction, Diffraction etc , could not be explained. Later it was proposed that light travels in wave form. Light is a form of electromagnetic radiation some other forms of electromagnetic radiations are UV, Visible, Infra-red, X-rays , Radiowaves , cosmic rays etc. The study of spectroscopy relates to emission and absorption spectra of the matter.

3. Emission Spectra : It is produced by the excitation of atom of the element. Excitation may be due to heat or electrical energy. Electrons moving to lower energy level releases radiations which are analyzed with help of spectroscope as emission spectrum. Absorption Spectra : It is produced when an atom of an element absorbs energy from surrounding. Electromagnetic Radiation: These can be produced by changing electric and magnetic field simultaneously which act at right angle to each other and also perpendicular to the direction of electromagnetic wave . Properties: • These are transverse waves. • Can propagate in vacuum also. • May produce optical effects. • Can be polarized.

4. Electromagnetic Spectrum:

5. Electronic Excitation Electron excitation is the transfer of a bound electron to a more energetic, but still bound state. This can be done by photoexcitation, where the electron absorbs a photon and gains all its energy or by electrical excitation, where the electron receives energy from another, energetic electron. E = hv = hc / λ Where h is plank’s constant , c is velocity of light, E is energy released or absorbed.

6. Beer-Lambert law: The Beer-Lambert law states that the quantity of light absorbed by a substance dissolved in a fully transmitting solvent is directly proportional to the concentration of the substance and the path length of the light through the solution. Beer's Law may be written simply as: A= log Iₒ/ It or A = ε b c . Where : Iₒ is incidhent radiation It is transmitted radiation A = absorptivity b = Length of absorption pathₒ c = concentration of absorbing where A is absorbance (no units) ε is the molar absorptivity with units of L mol-1 cm-1 (formerly called the extinction coefficient) b is the path length of the sample, usually expressed in cm.

7. Transmittance: The quantity of radiation transmitted by the molecules of sample under consideration. T = It / Iₒ Where It is the intensity of transmittance radiation Iₒ is the intensity of incident radiation If the transmittance is 100 it means that no absorption occurred. If transmittance is 0 then it means that no radiation occurred.

8. Spectrophotometer: The spectrophotometer is an optical instrument for measuring the intensity of light relative to wavelength. Electromagnetic energy, collected from the sample, enters the device through the aperture (yellow line) and is separated into its component wavelengths by the holographic grating. Types These are as follow: 1. UV – Visible Spectrophotometer 2. Atomic Absorption Spectroscopy 3. Atomic Emission Spectroscopy 4. Mass Spectroscopy 5. Nuclear Magnetic Resonance (NRM) 6. X- Ray Spectroscopy

9. 1. UV – Visible Spectrophotometer It includes excitation of electrons from lower energy level to higher energy level. It involves the measurement of UV (190-380nm) or visible (380-800nm) radiation absorbed by the electrons of solution. PRINCIPLE UV spectrophotometer principle follows the Beer-Lambert Law. This law states that whenever a beam of monochromatic light is passed through a solution with an absorbing substance, the decreasing rate of the radiation intensity along with the thickness of the absorbing solution is actually proportional to the concentration of the solution and the incident radiation. This law is expressed through this equation : A = log (Iₒ /I) = ECI

10. A stands for the absorbance, I0 refers to the intensity of light upon a sample cell. l refers to the intensity of light departing the sample cell, C stands for the concentration of the solute, L stands for the length of the sample cell and E refers to the molar absorptivity. Instrumentation : It consist of following parts:  Radiation source  Monochromator  Sample cell  Detector  Recorder

11. Radiation source Various UV radiation sources are as follows : a. Deuterium lamp b. Hydrogen lamp c. Tungsten lamp d. Xenon discharge lamp e. Mercury arc lamp Various Visible radiation sources are as follow: a. Tungsten lamp b. Mercury vapour lamp c. Carbonone lamp

12. Monochromators • Wavelength selector that can continuously scan a broad range of wavelengths. Used in most scanning spectrometers including UV, visible, and IR instruments.

13. SAMPLE COMPARTMENT Spectroscopy requires all materials in the beam path other than the analyte should be as transparent to the radiation as possible . The geometries of all components in the system should be such as to maximize the signal and minimize the scattered light. Some typical materials are: – Optical Glass - 335 - 2500 nm – Special Optical Glass – 320 - 2500 nm Detectors After the light has passed through the sample, we want to be able to detect and measure the resulting light. These types of detectors come in the form of transducers that are able to take energy from light and convert it into an electrical signal that can be recorded, and if necessary, amplified.

14. Applications  This is used to detect a functional group. It can be used to detect the absence or the presence of chromophore in a complex compound.  This can also be used to detect the extent of conjugation in polyenes.  UV spectroscopy can also help determine the configurations of a geometrical isomer.

15. 2 ) Atomic Absorption Spectroscopy Atomic absorption spectrometry (AAS) is a technique in which free gaseous atoms absorb electromagnetic radiation at a specific wavelength to produce a measurable signal. It is used to detect metals in a sample. Principle The technique uses basically the principle that free atoms (gas) generated in an atomizer can absorb radiation at specific frequency . Atomic-absorption spectroscopy quantifies the absorption of ground state atoms in the gaseous state .The atoms absorb ultraviolet or visible light and make transitions to higher electronic energy levels. The analyte concentration is determined from the amount of absorption. Concentration measurements are usually determined from a working curve after calibrating the instrument with standards of known concentration

16. Instrument It consist of following parts :  LIGHT SOURCE  Atomizer  MONOCHROMATOR  DETECTOR

17. LIGHT SOURCE: Hollow Cathode Lamp are the most common radiation source in AAS. It contains a tungsten anode and a hollow cylindrical cathode made of the element to be determined. These are sealed in a glass tube filled with an inert gas(neon or argon ) . Each element has its own unique lamp which must be used for that analysis

18. Atomizer Elements to be analyzed needs to be in atomic sate. Atomization is separation of particles into individual molecules and breaking molecules into atoms. This isdone by exposing the analyte to high temperatures in aflame or graphite furnace . MONOCHROMATOR : This is a very important part in an AA spectrometer. It is used to separate out all of the thousands of lines. A monochromator is used to select the specific wavelength of light which is absorbed by the sample, and to exclude other wavelengths.The selection of the specific light allows the determination of the selected element in the presence of others.

19. DETECTOR: The light selected by the monochromator is directed onto a detector that is typically a photomultiplier tube , whose function is to convert the light signal into an electrical signal proportional to the light intensity. The processing of electrical signal is fulfilled by a signal amplifier . The signal could be displayed for readout , or further fed into a data station for printout by the requested format. APPLICATIONS: Determination of even small amounts of metals (lead, mercury, calcium, magnesium, etc ) as follows:  Environmental studies: drinking water, ocean water, soil.  Food industry.  Pharmaceutical industry.

20. 3 ) Atomic Emission Spectroscopy Atomic emission spectroscopy (AES) is a method of chemical analysis that uses the intensity of light emitted from a flame, plasma, arc, or spark at a particular wavelength to determine the quantity of an element in a sample . Principle The electrons of an atom moves from higher energy level to lower energy level, they emit extra amount of energy in the form of light which is consist of photons. This emitted energy is detected by detector.

21. 4 ) Mass Spectroscopy It is an instrumental method for identifying the chemical constitution of a substance by means of the separation of gaseous ions according to their differing mass and charge. Principle A mass spectrometer generates multiple ions from the sample under investigation, it then separates them according to their specific mass-to-charge ratio (m/z), and then records the relative abundance of each ion type.

22. Principle The first step in the mass spectrometric analysis of compounds is the production of gas phase ions of the compound, basically by electron ionization. This molecular ion undergoes fragmentation. Each primary product ion derived from the molecular ion, in turn, undergoes fragmentation, and so on. The ions are separated in the mass spectrometer according to their mass-to-charge ratio, and are detected in proportion to their abundance. A mass spectrum of the molecule is thus produced. Ions provide information concerning the nature and the structure of their precursor molecule.

23. Applications  It is used for the analysis of Oligonucleotides.  It is used for the analysis of Proteins and Peptides.  It is used for the analysis of Lipids.  It is used for the analysis of Glycans .

24. 5) Nuclear Magnetic Resosnance Nuclear magnetic resonance (NMR) is a phenomenon where nuclei in a magnetic field absorb and re-emit electromagnetic radiation. This resonance energy (E) or frequency (ν) is determined by the strength of the magnetic field (B), the magnetic properties of the isotope of the atom. Principle The principle behind NMR is that many nuclei have spin and all nuclei are electrically charged. If an external magnetic field is applied, an energy transfer is possible between the base energy to a higher energy level (generally a single energy gap).

25. Instrumentation  Sample tube/sample holder  Permanent magnet  Magnet coil  Sweep generator  Radio frequency transmitter  Radio frequency reviever  Read out system

26. • Sample tube / sample holder It should be chemically inert, durable & transparent to NMR radiation. Generally about 8.5 cm long & approximately 0.3 cm in diameter is employed . Sample probe It’s the device that hold sample tube in position & is provided with an air driven turbine for rotating the sample tube almost 100 revolutions per min.

27. • Permanent Magnet It provide homogenous magnetic field at 60-100MHz. • Magnetic coil It induce magnetic field when current flow through them. • Sweep generator To produce equal amount of magnetic field pass through the sample.

28. • Radio frequency transmitter Transmitter is fed on to a pair of coils mounted on right angles to the path of field. 60 MHz capacity is normally used. • Radio frequency receiver Detect radio frequencies emitted as nuclei relax at lower energy level. • Signal detector & recording system The electrical signal generated is amplified by means of amplifier & then recorded.

29. Chemical Shift The position of peaks in spectrum indicates the type of group present (i.e. aliphatic , aromatic or acetylenic ) while the number of peaks tells number of sets of H-atom (proton) present in the molecule. Each type of proton has different electronic environment hence they absorb specific amount of energy. This difference in the absorption position (energy) of the H-atom with respect to standard TMS peak is known as Chemical shift .

30. Application • NMR used for structural elucidation of organic and inorganic solids • determines the physical and chemical properties of atoms • Application in medicine • Anatomical imaging • Measuring physiological function • Flow measurement and angiography • Tissue perfusion studies • Tumours detection

31. 6) X – Ray Spectroscopy X-ray spectroscopy is a technique that detects and measures photons, or particles of light, that have wavelengths in the X-ray portion of the electromagnetic spectrum. It's used to help scientists understand the chemical and elemental properties of an object. Principle XRF works on methods involving interactions between electron beams and x-rays with samples. It is made possible by the behavior of atoms when they interact with radiation. When materials are excited with high-energy, short wavelength radiation (e.g., X-rays), they can become ionized. When an electron from the inner shell of an atom is excited by the energy of a photon, it moves to a higher energy level.

32. When it returns to the low energy level, the energy which it previously gained by the excitation is emitted as a photon which has a wavelength that is characteristic for the element (there could be several characteristic wavelengths per element). These X-rays since have characteristic energies related to the atomic number, and each element therefore has a characteristic X-ray spectrum which can be used to identify the element.

33. Instrumentation Components for X-ray spectroscopy are:  X-ray generating equipment (X-ray tube)  Collimator  Monochromators  Detectors

34. X-ray generating equipment (X-ray tube) X-rays can be generated by an X-ray tube. X-rays tube is a vacuum tube that uses a high voltage to accelerate the electrons released by a hot cathode to a high velocity.The high velocity electrons collide with a metal target, the anode, creating the X-rays.

35. Collimator A collimator is a device which narrows a beam of particles or waves. To narrow can mean either to cause the directions of motion to become more aligned in a specific direction, or to cause the spatial cross section of the beam to become smaller.

36. Monochromator Monochromator crystals partially polarize an unpolarized X-ray beam. The main goal of a monochromator is to separate and transmit a narrow portion of the optical signal chosen from a wider range of wavelengths available at the input . Types of Monochromator  Metallic Filter Type  Diffraction grating type

37. X-ray Detectors The most commonly employed detectors include:  Solid State Detectors  Scintillation Detectors Solid State Detectors The charge carriers in semiconductor are electrons and holes. Radiation incident upon the semiconducting junction produces electron-hole pairs as it passes through it . Electrons and holes are swept away under the influence of the electric field, and the proper electronics can collect the charge in a pulse.

38. Scintillation detectors Scintillation detectors consist of a scintillator and a device, such as a PMT (Photomultiplier tubes), that converts the light into an electrical signal. It consists of an evacuated glass tube containing a photocathode, typically 10 to 12 electrodes called dynodes, and an anode. Electrons emitted by the photocathode are attracted to the first dynode and are accelerated to kinetic energies equal to the potential difference between the photocathode and the first dynode. When these electrons strike the first dynode, about 5 electrons are ejected from the dynode for each electron hitting it. These electrons are attracted to the second dynode, and so on, finally reaching the anode. Total amplification of the PMT is the product of the individual amplifications at each dynode. Amplification can be adjusted by changing the voltage applied to the PMT.

39. APPLICATIONS It is used to study Structure of crystals. It is used to study Polymer characterization. It is used to study Particle size determination. It is used to study Applications of diffraction methods to complexes. It is used to study Low-angle scattering.

40. References Allen, DW; Cooksey, C; Tsai, BK (Nov 13, 2009). "Spectrophotometry". NIST. Retrieved Dec 23, 2018. Ishani , G (2006). "The first commercial UV-vis spectrophotometer". The Scientist. p. 100. Retrieved Dec 23, 2018 – via Science In Context. Simoni , RD; Hill, RL; Vaughan, M; Tabor, H (Dec 5, 2003). "A Classic Instrument: The Beckman DU Spectrophotometer and Its Inventor, Arnold O. Beckman". J. Biol. Chem. 278 (49): e1. ISSN 1083-351X. Beckman, A. O.; Gallaway , W. S.; Kaye, W.; Ulrich, W. F. (March 1977). "History of spectrophotometry at Beckman Instruments, Inc ". Analytical Chemistry. 49 (3): 280A–300A. doi:10.1021/ac50011a001.

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