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 the 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
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
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

12. 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

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

14. 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.

15. 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.

16. 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

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

19. 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

20.  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.

21.  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.

22.  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.

24.  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.

25. 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.

27.  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.

28. 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).

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

30.  • 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.

31. • 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.

32.  • 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.

33. 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.

35. 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

36. 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.

37. 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.

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

40. 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.

41. 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.

42. 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

43. 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.

44. 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.

45. 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.

46. 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.