ESR and X-Ray spectroscopy

Submitted to Submitted by

Patel Shrushti Anilkumar

Dr. N.J. Patel
Associate professor
Dept. of Biochemistry,
BACA, AAU
Reg no.- 2080120008
M.sc. (Horti) vegetable
science
 Introduction

• ESR (Electron spin resonance spectroscopy) also known as Electron Paramagnetic Resonance (EPR) or
Electron Magnetic Resonance (EMR)

• It is discovered by E.K. Zavoisky, in 1944.

• It is spectroscopy of unpaired electrons.

• It is a branch of absorption spectroscopy in which radiation having frequency in microwave region.

Electron spin resonance (ESR) is also known as Electron Paramagnetic Resonance(EPR).

• This is a technique for detecting paramagnetism. the technique may be used for detecting transitional
metal ion and their complexes, free radicals and their excited states.

• ESR spectroscopy are similar to those of nuclear magnetic resonance (NMR), except that an electron spin,
rather than a nuclear spin, is the focus Unpaired electrons in biological systems are in much lower
abundance than nuclei, so ESR is a technique that focuses on local sites while NMR is more global.
 Principle of ESR

• The unpaired electrons are excited to a high energy state under the magnetic field by the absorption of
microwave radiations.

• The excited electron changes its direction of spin and relaxes in to the ground state by emitting its
energy.

• The transition between two different energy levels takes place by absorbing a quantum of radiation of
frequency in the microwave region.

• Microwave absorption is measured as a function of the magnetic field by ESR Spectroscopy.

 ESR Phenomenon is applied to….

• Atoms having odd number of electrons.

• Ions having partly filled inner electron shells.

• Free radicals having unpaired electrons.( Free radicals are reactive molecules with
unpaired electrons. They cause the oxidative breakdown of oils, food products, and play
a role in the biochemisty of disease.)
 How does the spectrometer work?

• The radiation source usually used is called a klystron

• They are high power microwave sources which have low-noise

characteristics and thus give high sensitivity

• A majority of EPR spectrometers operate at approximately 9.5

GHz, which corresponds to about 32 mm ( Q-band)

• The radiation may be incident on the sample continuously or

pulsed

• The sample is placed in a resonant cavity which admits

microwaves through an iris.

• The cavity is located in the middle of an electromagnet and

helps to amplify the weak signals from the sample.
 •Numerous types of solid-state diodes are sensitive to microwave energy.

• Absorption lines are detected when the separation of the energy levels is equal to the energy of the
incident microwave.

• In practice, most of the external components, such as the source and detector, are contained within a
microwave bridge control.

•Additionally, other components, such as an attenuator, field modulator, and amplifier, are also included
to enhance the performance of the instrument.

•The diode detector is mounted along the E-vector of the plane=polarized microwave and produced a
current proportional to the microwave power reflected from the cavity.

•The generated microwaves are sent through the attenuator, through the circulator, and onto the sample. 
Microwaves reflected back from the sample cavity are routed in the circulator to the detector diode. The
signal comes out as a decrease in current at the detector analogous to absorption of microwaves by the
sample.
• If an external magnetic field is no present, the two electron
spin states (spin up and spin down) are degenerate. The
degeneracy of the electron spin states characterized by the
quantum number, ms =+/- 1/2, is lifted by the use of a
magnetic field.

• Transitions between the electron spin levels are induced by

radiation at the matching frequency. When a magnetic field is
induced, atoms with unpaired electrons spin either in the same
direction (spin up) or in the opposite direction (spin down) of
the applied field. These two possible alignments have different
energies are no longer degenerate.

• The alignments are directly proportional to the applied

magnetic field strength. This is called the Zeeman Effect. An
un-paired electron interacts with its environment, and the
details of ESR spectra depend on the nature of those
interactions. The readings can provide information on
structural and dynamic information, even from the chemical or
physical process, without influencing the process itself.
 (ESR) Applications of Electron Spin Resonance

• ESR spectroscopy is one of the main methods used to study metalloproteins, particularly those containing
molybdenum, copper, iron, etc.

• Both copper and non-haem iron do not absorb radiation in visible and ultra violet range, posses ESR
absorbance peak in one of their oxidation state.
• Hence their appearance and disappearance of their ESR signal are used to monitor their activity in multi
enzyme system.

• In metalloproteins, the metal atom has characteristic number of ligands coordinated to it in a definite
geometrical arrangement. Studies using ESR have shown that their geometry is frequently distorted.
• The technique ESR has been extended by spin labelling . Thus spin labelling glycerophosphatides with a
suitable nitrox free radical the laterate diffusion of the labelled molecules can be studied.
• Electron spin resonance is also extensively used to study the free radicals caused by irradiation
of biological material.

• The concentration of free radicals in samples can also be determined.

• Study of naturally occurring substances such as minerals with transition elements, minerals
with defects (e.g; quartz), Haemoglobin (Fe), Petroleum, Coal, Rubber etc.
 X ray spectroscopy

• X-ray spectroscopy is a form of optical spectroscopy

that utilizes emission, absorption, scattering,
fluorescence, and diffraction of X-ray radiation.
• X-Rays are a form of electromagnetic radiation.

• X-rays have a wavelength ranging from 0.01 to 10 nano meters.

• X-ray wavelengths are shorter than those of UV rays and typically longer than those of gamma rays.

• X-Rays were discovered by Wilhelm Roentgen in 1895.

• Roentgen noticed crystals near a high-voltage cathode-ray tube exhibiting a fluorescent glow, even when he
shielded them with dark paper. Some form of energy was being produced by the tube, and it was
penetrating the paper and causing the crystals to glow. Roentgen called the unknown energy “X-radiation”

• The use of X-ray spectroscopy actually began a bit earlier, in 1912, starting with a father-and-son team of
British physicists, William Henry Bragg and William Lawrence Bragg.

• X-rays are short-wavelength (hence, high frequency, and hence, relatively high energy) electromagnetic
radiation. Two ways to produce X-rays: 1) Deceleration of high-energy electrons 2) Electronic transitions
involving inner-orbital (e.g. - d or f) electrons The wavelength range of X-Rays is from about 10-5 Å to
100ÅConventional X-Ray spectroscopy is largely confined to the region of about 0.1 Å to 25 Å
 How X-ray spectroscopy works?

• When an atom is unstable or is bombarded with high-energy particles, its electrons transition
from one energy level to another. As the electrons adjust, the element absorbs and releases high-
energy X-ray photons in a way that's characteristic of atoms that make up that particular chemical
element. X-ray spectroscopy measures those changes in energy, which allows scientists to identify
elements and understand how the atoms within various materials interact.
Three types of X-ray spectroscopy

1) X-ray absorption

2) X-ray diffraction

3) X-ray fluorescence
 X –Ray Absorption

• This is similar to absorption methods in other regions of

electromagnetic spectra like uv- visible spectroscopy etc. It gives
information about the absorbing material.

• In this method a beam of x-ray is pass through the sample and the
fraction of x-ray photons absorbed is considered to be a measure of
concentration of sample.
• It is only used to detect the internal structures, elemental analysis,
thickness measurements etc.

• An x-ray is absorbed by an atom when the energy of the x-ray is

transferred to a core-level electron (K, L, or M shell) which is ejected
from the atom.
• The atom is left in an excited state with an empty electronic level (a
core hole). Any excess energy from the x-ray is given to the ejected
photoelectron.
• Principle of XAS is The Beer-Lambert law states that: for a given material sample path length and
concentration of the sample are directly proportional to the absorbance of the light.

• Thus The absorption is measured directly by measuring what is transmitted through the sample .
 Applications Of XAS

• XAS is a technique used in different scientific fields including molecular and condensed matter
physics, chemistry, earth science, and biology.

• Determining the true oxidation state of atoms in minerals, oxides, and mixed-metal systems.

• Determining the types of metals present in enzyme active sites and their local structures.

• Determining structural changes of metals during alloying processes.

• Determining the chemical composition of impurities in common material.

 For Analytical Purposes, X-rays are Generated in Three Ways:

1) Bombardment of metal target with high-energy electron beam

2) Exposure of target material to primary X-ray beam to create a secondary beam of X-ray fluorescence

3) Use of radioactive materials whose decay patterns include X-ray emission

 X-ray Diffraction

• These methods are based on the scattering of x-rays by

crystals.

• By using these method, analyst can easily identify the crystal

structure of any solid sample with high degree of specificity
and accuracy.

• XRD is most important method as compared to other x-ray

spectroscopy.

• The periodic lattice found in crystalline structure may act as

diffraction grating for wave particles of electromagnetic
radiation with wavelength of a similar order of magnitude
(1Aº).
• The atomic planes of a crystal causes an incident beam of X-
rays to interfere with one another as they come out from the
crystal. This phenomenon is called X-ray diffraction.
 Applications of XRD

• XRD Applications is in most widely used for the identification of unknown crystalline materials (e.g.
minerals, inorganic compounds). Determination of unknown solids is critical to studies in geology,
environmental science, material science, engineering and biology. Other applications include
• characterization of crystalline materials

• Identification of fine-grained minerals such as clays and mixed layer clays that are difficult to determine
optically.
• determination of unit cell dimensions measurement of sample purity.
 X-Ray Fluorescence Spectroscopy

• X-Ray Fluorescence (XRF) can be considered in a simple three step process occurring at the atomic
level:

• An incoming X-Ray knocks out an electron from one of the orbitals surrounding the nucleus within an
atom of the material.

• A hole is produced in the orbital, resulting in a high energy, unstable configuration for the atom.

• To restore equilibrium, an electron from a higher energy, outer orbital falls into the hole. Since this is a
lower energy position, the excess energy is emitted in the form of a fluorescent X-Ray. 
• The wavelength of fluorescence is characteristic of the element being
excited, measurement of this wavelength enable us to identify the
fluorescing element. The intensity of the fluorescence depends on how
much of that element is in x-ray beam. Hence measurement of the
fluorescence intensity makes possible the quantitative determination of
an element.
• Almost any solid or liquid sample can be measured. As this is not a
diffraction technique, the sample materials need not be crystalline or
ordered, although for elemental analysis purposes, the sample must be
completely homogeneous.
 Instrumentation of X-Ray Spectroscopy

• X-ray generating equipment (X-ray tube)

• Collimator
• Monochromators
• Detectors

A. 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.
B. Collimators

• A collimator is a device that narrows a beam of particles or waves.

• Narrow mean to cause the directions of motion to become more
aligned in a specific direction (i.e., collimated or parallel).
• Collimation is achieved by using a series of closely spaced ,parallel
metal plates or by a bundle of tubes ,0.5 or less in diameter.

C. 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
D. X-ray Detectors

The most commonly employed detectors include:

1. 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.

2. 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.