SPECTROSCOPY

Spectroscopy is the study of the interaction between matter and electromagnetic radiation.
Historically, spectroscopy originated through the study of visible light dispersed according to its
wavelength by a prism. The electromagnetic spectrum is the range of frequencies of electromagnetic
radiation and their respective wavelengths and photon energies.

Principle of spectroscopy

The term "spectroscopy" defines a large number of techniques that use radiation to obtain
information on the structure and properties of matter. The basic principle shared by all spectroscopic
techniques is to shine a beam of electromagnetic radiation onto a sample, and observe how it responds
to such a stimulus.

The history of spectroscopy began with Isaac Newton's optics experiments (1666–1672). Newton
applied the word "spectrum" to describe the rainbow of colors that combine to form white light and that
are revealed when the white light is passed through a prism. During the early 1800s, Joseph von
Fraunhofer made experimental advances with dispersive spectrometers that enabled spectroscopy to
become a more precise and quantitative scientific technique.

Application of spectroscopy

Spectroscopy is used as a tool for studying the structures of atoms and molecules. The large
number of wavelengths emitted by these systems makes it possible to investigate their structures in detail.

Spectroscopy also provides a precise analytical method for finding the constituents in material
having unknown chemical composition. • In a typical spectroscopic analysis, a concentration of a few parts
per million of a trace element in a material can be detected through its emission spectrum.

ATOMIC SPECTROSCOPY

Atomic spectroscopy is the study of the electromagnetic radiation absorbed and emitted by atoms.
It includes a number of analytical techniques used to determine the elemental composition of a sample
(it can be gas, liquid, or solid) by observing its electromagnetic spectrum or its mass spectrum.

Technique for determining the elemental composition of an analyte by its electromagnetic or mass
spectrum. It helps in understanding of the atom and the atomic process involved.

Atomic spectroscopy uses the electromagnetic radiation or mass spectrum of a sample to

determine elemental composition. The wavelength of energy absorbed or emitted by atoms is
characteristic to each element and can be used for element identification and quantification.

Principle of Atomic Spectroscopy

Atomic spectroscopy is the determination of elemental composition by its electromagnetic or

mass spectrum. The study of the electromagnetic spectrum of elements is called Optical Atomic
Spectroscopy. Electrons exist in energy levels within an atom. These levels have well defined energies and
electrons moving between them must absorb or emit energy equal to the difference between them.
 In optical spectroscopy, the energy absorbed to move an electron to a more energetic level and/or
the energy emitted as the electron moves to a less energetic energy level is in the form of a photon. The
wavelength of the emitted radiant energy is directly related to the electronic transition which has
occurred. Since every element has a unique electronic structure, the wavelength of light emitted is a
unique property of each individual element.

Types of Atomic Spectroscopy

1. Atomic absorption spectroscopy

2. Atomic emission spectroscopy
3. Atomic Fluorescence spectroscopy
4. Atomic Mass spectroscopy

Working Principle

The following can be used to express the basic principles of AAS.

All atoms or ions can absorb the energy (light) when it is at specific, unique wavelengths. The
electrons within an atom exist at various energy levels. When the atom is exposed to its own unique
wavelength, it can absorb the energy (photons) and electrons move from a ground state to excited states.
It is possible to determine the concentration of the element in the sample by measuring the amount of
light that is absorbed.

An atomic absorption spectrometer uses these basic principles and applies them in practical
quantitative analysis. A typical atomic absorption spectrometer consists of four main components: the
light source, the atomization system, the monochromator and the detection system (Figure 1).

In AAS, an analyte-containing solution is infused into a flame. Samples are changed by the flame
into excitable, free ground state atoms. When a lamp with a wavelength tailored to the atoms is passed
over the flame, the absorbed light energy causes the atoms' electrons to be activated.

Example:
When a sample containing copper (Cu) and nickel (Ni), for example, is exposed to light at the
characteristic wavelength of Cu, then only the Cu atoms or ions will absorb this light. The concentration of
the absorbing ions or atoms directly correlates with the amount of light that is absorbed at this
wavelength.

Parameters of Atomic Absorption Spectroscopy

• Due to the peculiar arrangement of their electrons, atoms absorb energy in their own distinctive
pattern of wavelengths. Based on these distinct arrangements, atomic absorption spectroscopy
can quantify the presence of known elements in a substance.
 • Atomic absorption spectroscopy involves processes that ultimately result in the sample's
destruction. The material must first be converted into an atomic gas before being subjected to an
AAS analysis.
• High levels of precision are offered by AAS. Results typically have an accuracy range of 0.5% to 5%,
however depending on the situation, this could increase even more.
• When analyzing certain elements, precautions must be taken to avoid readings being tainted by
background light source absorption by other atoms or molecules.

Instruments Used

● Radiation/Light Source
The most used type of light source in atomic absorption spectroscopy is a hollow cathode lamp. It
consists of a hollow cylindrical cathode made up of the same element under consideration. The cylindrical
metal cathode of such a lamp is filled with gas such as Ar or Ne, which contains the necessary element as
well as an anode, under low pressure. High voltage is applied to the cathode and anode, which ionizes the
filled gas. As the gas ions are directed towards the cathode during the glow discharge, the cathode material
is energized to release the radiation of the target element. Deuterium lamps (DL) and electrodeless
discharge lamps (EDL) are two more popular radiation sources.

● Atomizer
Atomizers are used to break analytes into free atoms. This is in order to analyze an element for its
atomic constituents. The most commonly used atomizers in atomic absorption spectroscopy are flames
and graphite tube atomizers.
Flame Atomic Absorption Spectroscopy (FAAS) - In a flame atomizer, a solution of the sample is
nebulized (sprayed) by a gaseous mixture of an oxidant and a fuel and carried into a flame where
atomization occurs.
Graphite Furnace Atomic Absorption Spectroscopy (GFAAS) - Atomization takes place in an open-
ended graphite tube in the graphite-furnace AAS process. The material is atomized as the tube’s
temperature rises. Radiation enters the tube from one end and excites the analytes. The detector at the
other end determines the absorbed fraction.

● Monochromators
The hollow cathode lamp emits many narrow emission lines. A monochromator is used to isolate
a single resonance emission line. This happens after the light passes through the sample within the
atomization source, e.g. the flame.
A monochromator is an optical device with the sole purpose of collecting light containing many
wavelengths and isolating (selecting) a narrow wavelength band. A simple monochromator consists of the:
1. Entrance slit: This is at the entry of the monochromator immediately after the light beam passes
through the flame. The entrance slit effectively confines the source radiation to a narrow beam.
2. Internal mirrors: These direct the light through a monochromator.
3. Dispersing element: At the heart of the monochromator, the dispersing element takes the light
and disperses the radiation into its component wavelengths (you may be familiar with a prism that
separates white light into a rainbow).
4. Exit slit: This is the exit from the monochromator. The wavelength selected for analytical
measurement is focused onto the exit slit where it will then pass to the optical detector for measurement.
All other wavelengths will remain inside the monochromator.
The AA spectrometer’s monochromator is at the heart of the system. Its role is to select the
wavelength of interest from the beam passing through the flame containing the analyte of interest.

● Detectors
A detector converts the transmitted light coming from a monochromator to a simplified electrical
signal proportional to the light intensity. The most commonly used detectors in atomic absorption
spectroscopy are photomultiplier tubes. The PMT measures the photon intensity of the analytical line
 coming out of the monochromator. In order to be read out, signal from the PMT is converted to digital
format by a transducer.

● Amplifier
The output from detectors is amplified by the amplifiers several times.

● Readout devices
Readout devices receive and re-code electrical signals coming from the detector to convert them
into a readable response through the use of a computer system with suitable software.

Atomic Emission Spectroscopy

Atomic Emission Spectroscopy is one of the four main types of Atomic Spectroscopy that is used
for the determination of the elemental composition of different substances. Atomic emission occurs when
a valence electron in a higher energy atomic orbital return to a lower energy atomic orbital.

I.Concept and Working Principle of Atomic Emission Spectroscopy (AES)

A phenomenon that works by exciting an atom to a higher energy level and then studying the
energy emitted by the electron when it falls back to its initial energy level. So basically, it focuses on the
emissions given out by atoms. By studying the emissions, the user can identify the type of element as well
as its quantity. This means that one can perform both qualitative and quantitative analysis on the sample
under study.

Figure 2.1 Atomic Emission Spectroscopy Concept

Applications of Atomic Emission Spectroscopy:

Throughout the years of usage, Atomic Emission Spectroscopy (AES) is proven to be effective in a
variety of applications. Some are as follows:

1. Metallic deficiency in living organisms can be diagnosed.

2. Clinical diagnosis of different metals i.e. Na, K, Ca, Mg, Fe, Zn, etc in body fluids like blood,
urine, etc.
3. Trace metal and rare earth elemental detections.
4. The analysis of agricultural substances like fertilizers, composts, etc
5. Different metals like Fe, Ni, Mn, etc can be analyzed in motor lubricants.
6. Pharmaceutical tablets can be analyzed for hazardous substances.
7. Determination of alkali and alkaline earth metals
8. Igneous and metamorphic rocks can be analyzed for mineral compositions which can be
used for the dating process.
 9. Detection of metallic ions and minerals in water i.e. sea and freshwater have such
differences.
10. The analysis of trace and other elements, including multi-elemental analysis in stainless
steel, frozen lava, emerging islands, etc.

Figure 2.2 Fireworks metal swords color representation

To have a partial grasp about Atomic Emission Spectroscopy (AES), consider fireworks as an example.
Long to know, the different colors in fireworks is due to the presence of different metal swords. The yellow
light in fireworks is the atomic emissions of sodium so as other colors correspond to its other metal
content. AES involves three major steps:

1. Atomization where atomization of the sample occurs converting it into gas phase atoms and ions.
2. Excitation where we provide sufficient energy to promote the valence electrons of an atoms from
the ground state to an excited state.
3. Relaxation where the excited atoms relax back to the ground state giving up their energy as
photons this is when the atomic emission occurs.

• Atomic Emission Spectra are produced when excited electrons return to the ground
state. When electrons return to a lower energy level, they emit energy in the form of
light.

Figure 2.3 Sodium Atom Atomization, Excitation and Relaxation State

Figure 2.3 shows sodium atoms as an example. If enough heat or electrical energy is supplied, we
can promote the electrons from the ground state 3s orbitals to 3p 4p or 5p excited state orbitals. After a
few nanoseconds the excited atoms relax back to the ground state giving up the energy as atomic
emissions. In the previous example, the wavelength of 589 nanometers is responsible for the yellow light
we see in fireworks that contain sodium salts.
 Figure 2.4 Atomic Emission Spectra

In figure 2.4, some samples of atomic spectra are shown. One key principle of AES is that the
wavelength of the emitted light depends on the energy gap between the atomic orbitals which is unique
for every element. As seen in the example, one element can have a set of characteristic emission
wavelengths, AES takes advantage of the unique line spectrum of elements to identify and to quantify
them.

WORKING PRINCIPLE

This section contains the process particularly utilized in Atomic Emission Spectroscopy (AES) and
all other underlying principles.

Emission Process

Figure 2.5 Atomic Emission Process

The atomic emission process is the starting point of the Atomic Emission Spectroscopy. Before
proceeding to the next step, the following must be properly attained:

1. Solvent evaporates leaving behind the solid residue.

2. The solid residue gets vaporized due to the high temperature of the flame and the molecules
dissociate to atoms.
3. The metal atoms are vaporized by the flame and are thermally excited to higher energy levels.
4. The excited metal atom returns to ground state by emitting photon or light of its own
characteristic wavelength.

Atomic Emission Spectroscopy Process

Before proceeding to the actual process, it is important to understand all the components and
their corresponding function in the process.
 1. The holder which in most cases are the beakers. These are used to hold the analytes.

Figure 2.6 Sample holder

2. The atomiser with flow controls for oxygen and fuel that converts the sample to aerosol and
passes this aerosolized sample to the next unit (e.g nebulizer and mixing chamber)

Figure 2.7 Atomiser with flow controls for oxygen and fuel

3. The flame/burner where air and fuel combine in the burner to produce the flame. This is where
the emission process takes place.

Figure 2.8 Flame/ Burner

4. The monochromator/ filter which allows the chosen and absorbs all the other wavelengths

Figure 2.9 Monochromator/ Filter

5. The detector that detects the intensity of the emitted light coming out of the cell and generates
current proportional to it. E.g photocell or photodiode. Display usually in the form of computer

Figure 2.10 Detector and Display

After specifying all the important components used in AES, the following is the standard working
principle of instruments performing AES:

Figure 2.11 Atomic Emission Spectrometer

1. The sample is converted into aerosol/mist form through the atomiser.

2. Then this aerosol is made to pass into the flame. The aerosol contains the atoms from the sample.
 3. Once in the flame, these atoms absorb energy. The energy differences between the atomic states
varies for different atoms. As a result, different atoms will absorb different amounts of energy as
required by them.

4. These atoms cannot remain in the higher energy state for too long. As a result, they fall back to
their original atomic state by releasing energy. The energy is released in the form of photons. They are
atomic emissions.
5. The emitted light passes through a lens. Then passes through a monochromator. Monochromator
is important because we cannot analyze more than one element at a time. Monochromator is set to a
specific wavelength depending on the element being analyzed. For instance Sodium (Na), the
monochromator, is then set to 589 nm. It absorbs all the other wavelengths but allows one wavelength
to pass through to the detector.
6. The detector depends on the intensity of the emitted light.
7. Intensity of the light is proportional to the concentration. The higher the concentration, the more
electrons get excited, the more electrons return back to its ground state by emitting light. The intensity
of the emitted light will be higher in the meter reading.

III. Samples & Signal Sources

Samples
• Sodium (Na)
• Has a wavelength of 589 nanometers
• The flame test for sodium displays a brilliantly bright yellow emission

• Lithium (Li)
• Has a wavelength of 670 nanometers
• The atomic spectrum of lithium has a strong red line

• Calcium (Ca)
• Has a wavelength of 622 nanometers, the first being an arc line and the others being
molecular bands.
• The flame spectrum of calcium has emissions at 422.7, 554, and 622 nm, the first being
an arc line and the others being molecular bands.
• The flame test for sodium displays an orange emission

• Barium (Ba)
• Has a wavelength of 554 nanometers
• barium emits a yellow-green color.

Signal Sources
• Flames
• It consists of a complete total consumption burner in which the sample is drawn through
a capillary tube and fed directly into the flame, and the flame is a high temperature source
used to dissolve and evaporate the sample and create free atoms for spectroscopic
examination.
• It is utilized for molecules that do not require extremely high temperatures for atom
excitation during quantitative analysis.
• The same nebulization and spray chamber assembly used in atomic absorption is
employed for atomization and excitation in flame atomic emission. The burner head is
made up of a single or many slots, as well as a Meker-style burner. Older atomic emission
 devices frequently employed a complete consumption burner, which draws the sample
through a capillary tube and injects it straight into the flame.

• Plasmas
• Plasma is a gaseous mixture that is electrically conductive and contains high quantities of
cations and electrons.
• by ionizing a flowing stream of argon gas produces argon ions and electrons, which are
then utilized in atomic emission. The high temperature of a plasma is caused by resistive
heating as electrons and argon ions flow through the gas.
• Since a plasma works at a much higher temperature than a flame, it has a higher
atomization efficiency and a larger population of excited states.

Three Types of High-temperature plasma

o Inductively Coupled Plasma (ICP)
• Plasma generated in a device called torch
• Tesla coil produces initiation spark
o Direct Current Plasma (DCP)
• created by the electronic release of the two electrodes.
o Microwave Induced Plasma (MIP)
“ The most important of these plasmas is the inductively coupled plasma (ICP)”

• Arc and Spark

• Flowing between two electrodes
• Usually performed on solids
Arc - used in quantitative analysis

Types of Arc
• AC Arc - it employs potential difference of 1000 volts or more
• Is drawn at a distance 0.5 - 3mm.
• DC Arc - is generated with a potential gradient of 50 - 300 volts
• The temperature in the arc stream ranges from 2273 - 5273 K.
• Used for identification and determination of elements present in very small
concentration
Spark
• High voltage transfer 10 - 50kV across two electrodes gives a spark
• The use of condenser increases the current
• Brilliant spark is obtained at 0.005 mF capacity to the capacitor.

IV. Instruments and Instrumentation

Instrument that Uses AES

1. Inductively coupled Plasma (ICP-AES)
2. Direct Current Plasma (DCP-AES)

Inductively Coupled Plasma (ICP)

• Plasma induced by radio frequency.
• It is a method of emission spectroscopy that excites atoms and ions with a plasma, causing
it to emit electromagnetic radiation at wavelengths characteristic of a particular element.
• It is one of the popular instruments in environmental labs in which this method is capable
of running and analyzing a bunch of metals and samples per day.
• Its main purpose is to detect trace elements and determine very precisely the elemental
composition of samples; however there is an exception to that which is the Argon
Element.
 TYPES OF INSTRUMENTS FOR ICP-AES
1. Sequential spectrometers
2. Simultaneous spectrometers

Sequential Spectrometers
These instruments use a moveable grating monochromator to select wavelengths in
sequential order and use a single photomultiplier tube to detect them. The grating of the
monochromator is of holographic type having a large number of grooves.

Fig. 4.1. A schematic diagram showing the optical diagram of a sequential ICP optical emission
spectrometer

Simultaneous Spectrometers
These instruments are designed to simultaneously measure the response at different
wavelengths so as to overcome the drawbacks of the sequential spectrometers.

There are two types of spectrometers belonging to this category:

• Polychromators
These simultaneous spectrometers are designed with a fixed diffraction grating
monochromator and multiple ‘exit slits’ and associated photomultipliers.
• Solid state array based spectrometers
These spectrometers are of recent origin and as the name suggests these make
use of solid state detectors. These have allowed the analysts to get over the problems of
conventional sequential and simultaneous spectrometers.

Direct Current Plasma (DCP)

• Developed by William Schenk over the last 1960s and early 1970s
• Simpler, use less Ar, and less expensive than ICP
• It is very tolerant of complex samples (solids and slurries) which make it a good technique.

Fig. 4.2. Three Electrodes DCP or Inverted Y-shaped Plasma

Figure shows the schematic diagram of the positioning of the electrodes and the sample
insertion in a typical DCP. It has two anodes set at an included angle of about 60o and a cathode.
The anodes are made up of graphite and are surrounded by ceramic sleeves.
Instrumentation

Fig. 4.3. Schematic Layout of Different Components of an AES

Different Components of an AES

1. Sample
2. Nebulizer
3. Monochromator
4. Detector
5. Signal Processing
6. Computer System / Readout Devices

Sample
A cup-shaped graphite electrode acts as a sample cell in atomic emission spectroscopy. The
sample is introduced into plasma with the help of nebulizers.

There are two steps in the sample introduction

1. Sample preparation
The solution preparation for the analysis using AES depends on the nature of the sample
and the concentration of elements to be determined. There are two main types of sample
preparation methods used:
• Acid Digestion Method
• Dry Attack Method
2. Nebulization
It is used to convert the liquid stream into an aerosol consisting of particles that are 1–10
mm in diameter.

Fig. 4.4. Pneumatic Nebulizers used in AES (a) Concentric Tube Type (b) Cross Flow Type
 Fig. 4.5. (a) Frit Nebulizer and (b) Ultrasonic Nebulizer used in AES

Monochromators
These are used to choose the specific radiation type, emitted by the analyte and the removal of
all other unwanted radiations. For this reason, it is sometimes called ‘the wavelength selector’.

Fig. 4.6. Principle of Monochromator

Detectors
Photomultiplier tubes, photoemissive cells, or array detectors act as detectors in atomic emission
spectroscopy. They are used to convert optical signals into electrical current which is then amplified by
the amplifier.

Computer System / Readout Devices

Computers are used as readout devices in atomic emission spectroscopy. Computers analyze the
data in the form of spectra and plot the calibration curve using the atomic emission ranges library.
 ATOMIC FLUORESCENCE SPECTROSCOPY

What is Fluorescence?
● Fluorescence is an emission phenomenon where the energy transition from a higher to a lower state
within the molecule concerned is measured by the detection of the emitted radiation rather than the
absorption.
● Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic
radiation.
● A molecule absorbs light at one wavelength and reemits light at a longer wavelength.
● An atom or molecule that fluoresces is termed a fluorophore.
● At room temperature, most molecules occupy the lowest vibrational level of the ground electronic state,
and on the absorption of light, they are elevated to produce excited states.
• Excitation can result in the molecule reaching any of the vibrational sub-levels associated with each
electronic state.
● Having absorbed energy and reached one of the higher vibrational levels of an excited state, the
molecule rapidly loses its excess of vibrational energy by collision and falls to the lowest vibrational level
of the excited state.
● From this level, the molecule can return to any of the vibrational levels of the ground state, emitting its
energy in the form of fluorescence at a lower wavelength than the absorbed light.
● This shift to a longer wavelength is called the Stokes shift

When the atom absorbs the energy, it gets excited and mostly excited state from the ground state. And
during relaxation, it comes back to the ground state. It emits radiation which is called fluorescence.

What Is Fluorescence Spectroscopy?

Fluorescence spectroscopy (fluorimetry or spectrofluorometry) is a type of electromagnetic spectroscopy
which analyzes fluorescence from a sample.
● Fluorometry is defined as the measurement of emitted fluorescent light.
● It involves the use of a beam of light, usually ultraviolet light, that excites the electrons in molecules of
certain compounds and causes them to emit light of lower energy; typically, visible light.
● In fluorescence spectroscopy, the signal being measured is the electromagnetic radiation that is emitted
from a sample as it relaxes from an excited electronic energy level to its corresponding ground state.
● The analyte is originally activated to a higher energy level by the absorption of radiation in the UV. The
processes of activation and deactivation occur simultaneously during a fluorescence measurement.
● Devices that measure fluorescence are called fluorometers or fluorimeters.
● In simple words, when a sample is introduced into flame or other radiation sources, it absorbs photons
and gets excited to a higher level. The atoms at an excited state are unstable and hence return to the
ground state by emitting a photon. This emitted photon is used to determine the concentration of
elements present in a sample. The relationship of concentration to the intensity of fluorescence is
calculated from the Beer-Lambert law.

TYPES OF ATOMIC FLUORESCENCE TRANSITIONS

Fluorescence emission occurs through different pathways based on the wavelength of emitted radiation
when compared to absorbed radiation. The six types of atomic fluorescence transitions are given below.
1. Resonance fluorescence
2. Stokes direct line fluorescence
3. Stepwise line fluorescence
4. Two-step excitation or double resonance fluorescence
5. Thermal fluorescence
6. Sensitized fluorescence

1. Resonance Fluorescence
● Most useful type because it generates the most intense fluorescence.
● Used for most analytical determinations.
● Occurs when the atom re-emits a spectral line having the same wavelength as that used for excitation.
● The wavelength of emitted radiation is equal to the wavelength of absorbed radiation
2. Stokes Direct Line Fluorescence
● Also called Stokes fluorescence.
● It is observed when an electron in an atom is excited to a higher energy state by absorption of radiation
and then goes to a lower energy level (intermediate level) which is above ground state by emission of
radiation.
● From this intermediate level, the electron returns to the ground state by a radiationless process.
● So, the wavelength of absorption is shorter than the wavelength of emission or the wavelength of the
emitted radiation is longer than the wavelength of the absorbed radiation.
● Direct line fluorescence will always occur at a higher wavelength than that of the resonance line which
excites it.
Note: Radiationless deactivation is the loss of electronic excitation energy without photon emission or
chemical change.

3. Stepwise Line Fluorescence

● In this type of fluorescence an atom initially excited to a higher energy state by absorption of radiation
undergoes a partial deactivation by a collision or a radiationless process to a lower excited state
(intermediate level) which is above the ground state. Then from this (intermediate level), a transition
occurs and the atom emits radiation (fluorescence) to return to the ground state.
● It is also a type of Stokes fluorescence

4. Two-Step Excitation or Double Resonance

● Two dye lasers are used in a two-step excitation procedure for the double resonance fluorescence.
● The analyte is excited by the first laser from its ground state to an excited state, from which it is excited
by the second laser to an even higher excited state.
● Fluorescence emission occurs along with the de-excitation of this more highly excited state to a lower
energy state

5. Thermally Assisted Fluorescence

● Also called anti-Stokes fluorescence.
● Thermally assisted fluorescence is the opposite of stepwise line fluorescence.
● It occurs when the electron from the ground state is excited to a higher energy level (intermediate level)
by absorption of radiation (radiative excitation).
● Then electron from the intermediate level gets further excited to a higher energy level (above the
intermediate level) with the help of thermal energy in a radiationless process.
● The fluorescence emission occurs from both the excited energy levels.
● In this case, part of the emitted radiation has a shorter wavelength (higher energy) as compared to the
exciting radiation.
● This type of fluorescence is weak, tough, and has found few analytical applications

6. Sensitized Fluorescence
● In sensitized fluorescence, the energy of the excited atom (called the donor) is transferred to another
atom (called the acceptor) which gets excited and then relaxes back to the ground state accompanied by
fluorescence emission.
● However, thermally assisted fluorescence and sensitized fluorescence are not generally employed for
analytical purposes

WORKING PRINCIPLE OF ATOMIC FLUORESCENCE SPECTROSCOPY

● AFS is a method of elemental analysis that involves using a light source to excite gaseous atoms
radiatively to a higher energy level, followed by a deactivation process involving a photon's emission. This
emission process provides the measured fluorescence signal. AFS can be distinguished from the related
atomic spectrometric techniques of atomic absorption spectrometry (AAS) and atomic emission
spectrometry (AES) because it involves both radiative excitation and deexcitation.
● Atomic Fluorescence occurs when neutral atomic Species emit radiation after being excited by a line or
continuous source.
● Photoexcitation is the same as in AAS but we measure the emitted (rather than absorbed) radiation in
AFS.
How do analytes react to it?
● The use of AFS has been boosted by the production of specialist equipment that is capable of
determining individual analytes at very low concentrations (at the ng l1 level). The analytes have tended
to be introduced in a gaseous form and hence sample transport efficiency to the atom cell is very high.
● In atomic fluorescence spectrometry, the analyte is brought into an atom reservoir which could be a
flame, plasma, glow discharge, or a furnace, and is excited by focusing a beam of monochromatic
electromagnetic radiation emitted by a suitable primary source.
● The location of the fluorescence emission signal indicates the identity of the analyte whereas the
intensity is a measure of its concentration. The intensity of the ‘fluorescence’ increases with increasing
atom concentration in the flame, providing the basis for its quantitative determination.

SOURCES OF ATOMIC FLUORESCNECE SPECTROSCOPY

Light Sources
The light source is employed to excite atoms radiatively in the atom cell. It is advantageous to use an
intense light source to obtain the maximum AFS signal. Light sources for AFS are placed at right angles to
the detector. This is to ensure that incidental radiation from the source is not detected. Since a more
intense source will lead to greater sensitivity.
○ Hollow Cathode Lamp
■ Standard Hollow Cathode Lamp (HCL) - often insufficient for the majority of applications. A range of
alternatives has therefore been developed.
■ Boosted-Discharge Hollow Cathode Lamps (BDHCLs) - similar to standard HCLs but can be operated at
greatly increased current. The light output is consequently several times more intense. A similar effect
may be obtained by pulsing a standard HCL to 100–1000 mA, although this will reduce the lifetime of the
lamp.
○ Electrodeless Discharge Lamp (EDL) - suitable because it has an intensity 200–2000 times greater than
that of an HCL.
○ Laser - the greatest output intensity and would therefore be expected to yield the highest sensitivity.

Continuum Source
○ Xenon-Arc Lamp - 50–500W lamp, relatively poor intensity offered at each individual wavelength by the
blackbody irradiator. Particularly bad in the ultraviolet (UV) region of the spectrum. Scatter has also been
reported as being problematic.

Inductively coupled plasma (ICP) - initiate fluorescence is not yet a routine technique.

As with AAS, modulation of the light source enables an optimal signal-to-noise ratio to be obtained. The
frequency of modulation depends on the light source, but may be up to 10 kHz for an EDL.

INSTRUMENTATION OF ATOMIC FLUORESCENCE SPECTROSCOPY

1. Light Source
2. Excitation Monochromator
3. Sample Holder / Cuvette
4. Emission Monochromator
5. Detector
6. Readout Device

1. Light Source
● This may be a xenon lamp, high-pressure mercury vapor lamp, xenon-mercury arc lamp, laser, LED,
hollow cathode lamp, or another type.
● The critical feature is that the electromagnetic source should be of constant high intensity, and
fluorescence depends on the intensity of the light and the concentration of the sample.
● The light source is employed to excite atoms radiatively in the atom cell.
● Light sources for AFS can be classified into two primary types: conventional (nonlaser) sources and laser
sources.

Xenon Lamp
● Xenon produces a continuous spectrum.
● Provides relatively high-intensity radiant energy over the spectral region of 250 to 800 nm.
● Widely used for certain fluorescence applications because of o its high energy output, o stability of lamp
flashes, o higher ultraviolet and visible spectral output.

2. Monochromator / Filter
a. Excitation monochromator
▪ Used for tuning the wavelength of the exciting beam.
▪ It allows only the radiation which excites molecules/atoms in the sample.
b. Emission monochromator
▪ Used for analysis of the fluorescence emission.
▪ It passes radiation only emitted by the sample molecules to the detector.

• Due to the emitted light always having a lower energy than the exciting light, the wavelength of the
excitation monochromator is set at a lower wavelength than the emission monochromator.
• Monochromators have different types of filters:
o Interference Filters
o Colored Glass Filters
⮚ used for both excitation and emission wavelength selection, but they are more susceptible to
transmitting stray light and unwanted fluorescence.
o Grating Monochromator
⮚ Isolate regions of the spectrum
⮚ An advantage of the grating monochromator provides selectivity of the excitation and emission
wavelengths required when working with new fluorophores with absorbance
o Prisms

Note: Always remember that the emission monochromator is always placed at 90 degrees from the
incident light.

3. Sample Holder / Cuvette

• Often made up of quartz or glass.
• It could be square or cylinder in shape.
• With spectrofluorometers, placement of the cuvette and excitation beam relative to the photodetector
is critical in establishing the optical geometry for fluorescence measurements.
• Because fluorescence light is emitted in all directions from a molecule, several excitation/emission
geometries are used to measure fluorescence.
• In practice, most commercial spectrofluorometers use the right angle-detector approach, because it
minimizes the background signal that limits analytical sensitivity
•The front surface approach provides the greatest linearity over a broad range of concentrations because
it minimizes the inner filter effect.
● The front surface approach shows similar sensitivity to the right-angle detectors but is more susceptible
to background light scatter.
● Widely applied to heterogeneous solid-phase fluorescence immunoassay systems

4. Detectors
• It will convert light energy into an electrical signal that is displayed on readout devices.
• Types of detectors:
o Barrier Layer Cell
o Phototube
o Photomultiplier tube (PMT) is commonly used as a detector in fluorescence spectroscopy.
o Thermocouple
•The detection system is at ninety degrees to the radiation source to ensure that only emitted light is
measured.
• The detector is usually solar-blind to not pick up any ambient light.
• All commercial fluorescence instruments use photomultiplier tubes as detectors and a wide variety of
types are available.
• The material from which the photocathode is made determines the spectral range of the photomultiplier
and generally two tubes are required to cover the complete UV-visible range.
5. Readout Device
• An amplifier amplifies the signal coming from the detector and a recorder records them which is
displayed on the readout device.
• The output current from the detector is fed into a measuring device that indicates the extent of
fluorescence produced by the sample.

ATOMIC MASS SPECTROSCOPY

Mass Spectrometry is an analytical chemistry technique that helps identify the amount and type
of chemicals present in a sample by measuring the mass-to-charge ratio and abundance of gas-phase ions.
In this technique, the molecular bombardment with a beam of energetic electrons. The molecule gets
ionized and broken up into many fragments, some of which are positive ions. And each kind ion has a
particular ratio of mass to charge, i.e. m/e ratio. This technique is useful for solid, liquid and gas.
Mass spectrometry, also called mass spectroscopy, an analytic technique by which chemical
substances are identified by the sorting of gaseous ions in electric and magnetic fields according to their
mass-to-charge ratios. The instruments used in such studies are called mass spectrometers and mass
spectrographs, and they operate on the principle that moving ions may be deflected by electric and
magnetic fields. The two instruments differ only in the way in which the sorted charged particles are
detected. In the mass spectrometer they are detected electrically, in the mass spectrograph by
photographic or other non-electrical means; the term mass spectroscope is used to include both kinds of
devices. Since electrical detectors are now most commonly used, the field is typically referred to as mass
spectrometry. A mass spectrum is a graph obtained by performing mass spectrometry. It is a relation
between the mass to charge ratio and ion signal.

General Mass Spectrometer

Mass Spectrometers measure the weights of molecules. This is typically done by electric and magnetic
fields in a vacuum. In order to measure the masses of the molecules, the ions must be ionized and either
formed in the vacuum or transferred to the vacuum.

The ionization usually takes place in the mass spectrometer source. The source is designed for the
ionization method that is going to be used. On some instruments the ionization source can be changed for
the ionization method. The choice of the ionization method is determined by the nature of the molecule.
Some molecules may be ionized by multiple types of ionization methods; however, with some molecules,
the mass spectroscopist will have to try many ionization methods to ionize the molecule. Knowledge of
the ionization methods and the molecules could help reduce the time the mass spectroscopist needs to
find the correct ionization method.

The ions formed in the source are transferred to the analyzer, which is the heart of the spectrometer. The
analyzer uses magnetic and electric fields to measure the mass to charge ratio of the ion. The compatibility
of the analyzer to specific ionization sources vary significantly depending on the type of analyzer. This
compatibility issue comes from the fact that some analyzers prefer to form ions in pulses while other
analyzers prefer ions to be formed continuously. In addition, each analyzer has its own characteristics for
such parameters as resolution, sensitivity, and speed of acquisition. Another feature of analyzers is that
some may be operated in such a way to increase the sensitivity by increasing the selectivity. Oftentimes,
a compromise is needed in the choice of analyzer to use.

Finally, the ions are detected. This is usually done by sending the ions into an electron multiplier or a micro
channel plate, which multiplies the current and sends it onto a computer that plots the current vs. the
mass to charge ratio. One other method of detection is by image current, which is used on FTMS analyzers
(both ion cyclotron resonance and orbitrap based instruments). Image current detection, detects the ions
going around the instrument in pattern by the induced current on electrodes, this current measures the
frequency of the pattern, which the computer uses to come up with the mass spectrum, which is the mass
to charge ratio vs. intensity graph.
Working Principle of Mass Spectrometry

The evolution of mass spectrometry has been marked by an ever-increasing number of applications in
science and technology. New applications and new developments have gone hand in hand to create a
complex array of instruments, but all may be understood by tracing the ions through three basic elements:
an ion source, a method of analyzing the ion beams according to their mass-to-charge ratio, and detectors
capable of measuring or recording the currents of the beams. These elements exist in many forms and are
combined to produce spectrometers with specialized characteristics. The needs of users vary, as do the
chemical form and the amount of sample available for analysis, which may be in sub microgram quantities.
The result is a great variety of design.

The basic principle of mass spectrometry (MS) is to generate ions from either inorganic or organic
compounds by any suitable method, to separate these ions by their massto-charge ratio (m/z) and to
detect them qualitatively and quantitatively by their respective m/z and abundance. The analyte may be
ionized thermally, by electric fields or by impacting energetic electrons, ions or photons. The ions can be
single ionized atoms, clusters, molecules or their fragments or associates. Ion separation is affected by
static or dynamic electric or magnetic fields.

Based on Newton’s second law of motion and momentum, a mass spectrometer uses this property of
matter to plot ions of varying masses on a mass spectrum. From the law, we infer how much mass is
relevant to the inertia and acceleration of a body. This principle is applied to the aspect where ions with
different mass to charge ratios are deflected by different angles in an electric or magnetic field.

Components

The instrument consists of three major components:

1. Ionization Source

Molecules are converted to gas-phase ions so that they can be moved about and manipulated by external
electric and magnetic fields. In our laboratory we use a technique called nanoelectrospray ionization,
which is somewhat similar to how cars are industrially painted. This method allows for creating positively
or negatively charged ions, depending on the experimental requirements.
Nanoelectrospray ionization can directly couple the outlet of a small-scale chromatography column
directly to the inlet of a mass spectrometer. The flow from the column is passed through a needle that is
10- 15 um at its tip.
Ionization occurs when the collision removes an electron from the sample molecule, creating
predominantly singly charged positive ions. Because EI is a high-energy process, it cleaves covalent bonds,
producing repeatable fragmentation that can be used to identify compounds using mass spectrallibraries.
2. Mass Analyzer

Once ionized, the ions are sorted and separated according to mass-to-charge (m/z) ratios. There are a
number of mass analyzers currently available, each of which has trade-offs relating to speed of operation,
resolution of separation, and other operational requirements. The specific types in use at the Broad
Institute are discussed in the next section. The mass analyzer often works in concert with the ion detection
system.
Once ionized, the ions are sorted and separated according to mass-to-charge (m/z) ratios. There are a
number of mass analyzers currently available, each of which has trade-offs relating to speed of operation,
resolution of separation, and other operational requirements. The specific types in use at the Broad
Institute are discussed in the next section. The mass analyzer often works in concert with the ion detection
system.

3. Ion Detection System

The separated ions are then measured and sent to a data system where the m/z ratios are stored together
along with their relative abundance. A mass spectrum is simply the m/z ratios of the ions present in a
sample plotted against their intensities. Each peak in a mass spectrum shows a component of unique m/z
in the sample, and heights of the peaks connote the relative abundance of the various components in the
sample.
The objective of ion current detectors used with mass spectrometers is to provide a measure of the
intensity of ions with different m/z values that have been separated in a mass analyzer.

With all the above components, a mass spectrometer should always perform the following processes:

1. Produce ions from the sample in the ionization source.

2. Separate these ions according to their mass-to-charge ratio in the mass analyzer.
3. Eventually, fragment the selected ions and analyze the fragments in a second analyzer.
4. Detect the ions emerging from the last analyzer and measure their abundance with the detector that
converts the ions into electrical signals.
5. Process the signals from the detector that are transmitted to the computer and control the instrument
using feedback.

PROCESS OF ATOMIC MASS SPECTROSCOPY

Mass spectrometry is used to accurately measure the mass of the various molecules within a sample. The
four stages/ process of mass spectrometry are – ionization, acceleration, deflection, and detection

1. Ionization
The sample is vaporized before being passed into an ionization chamber where it is bombarded by a stream
of electrons emitted by an electrically heated metal coil. The forceful collisions knock off one or more
electrons from the particle, resulting in a positively charged ion. Most of these have a +1 charge because
of the inherent difficulty in removing a second electron from an ion that is already positive.

2. Acceleration
The positively charged ionization chamber repels the positively charged ions, which accelerate towards
three negatively charged slits with progressively decreasing voltage. The speed at which they accelerate
depends on their mass so the lighter ions move faster than the heavier molecules.

3. Deflection
In this stage, the stream of positively charged ions are deflected by a magnetic field. The extent of the
deflection depends on the mass and charge of the ion. The lighter the mass of the ion, the more the
deflection. Ions with a charge greater than +1 will also be deflected more.

4. Detection
In this final stage, the beam of ions passing through the mass analyzer is detected by a detector on the
basis of the mass-to-charge ratio (m/z). When an ion hits the detector, the charge is neutralized by an
electron jumping from the metal onto the ion. This generates an electrical current which is proportional
to the abundance of the ion. The mass spectrum generated on completion of these four stages is sent to
a computer for analyses, where it shows the different m/z values of the ions present and their relative
abundance.

Mass Spectrometry Ionization Methods

There are many types of ionization methods used in mass spectrometry methods. The classic methods
that most chemists are familiar with are electron impact (EI) and Fast Atom Bombardment (FAB). These
techniques are not used much with modern mass spectrometry except EI for environmental work using
GC-MS. More modern techniques of atmospheric pressure chemical Ionization (APCI) , electrospray
ionization (ESI), matrix assisted laser desorption ionization (MALDI) and other derivative methods have
taken their place in the mass spectrometry laboratory. The reason for APCI over EI is that APCI forms a
protonated molecule and is completely compatible with Liquid Chromatography (LC), while EI is more likely
to fragment the ion leading to a possible more ambiguous identification of the molecule weight and is
incompatible with LC. ESI along with Matrix assisted laser desorption ionization (MALDI) has basically
eliminated the use of FAB, because it produces the protonated molecules, which made FAB so popular,
but is much more sensitive. In addition, MALDI along with ESI allowed for ionization and measurement of
large molecular weights that could not be done before, even by FAB. ESI has an advantage in its easy
compatibility with LC. MALDI has advantages for imaging mass spectrometry.

Electron Impact ionization (EI)- Electron ionization (EI), also known as electron impact ionization and even
electron bombardment ionization, is an ionization method where high energy electrons interact with other
atoms or molecules in solid or gas phases to produce ions. It is considered a hard ionization method
because it uses highenergy electrons which causes high fragmentation. In other words, the ionized
molecules often break into their smaller constituent parts. EI is done by volatilizing a sample directly in
the source that is contained in a vacuum system directly attached to the analyzer. The gas phase molecules
are bombarded by a beam of electrons formed by heating a filament bias at a negative voltage compared
to the source. The bias voltage is most commonly at -70 volts. The electron beam ejects an ion from the
gas phase molecule producing a radical ion. This technique is considered a hard ionization technique,
because it causes the ion to fragment. EI is also the method that is most commonly used for GC-MS.

Fast Atom Bombardment (FAB) - FAB is a technique that was popular in the 80's to early 90's because it
was the first technique that allowed ionization of non-volatile compounds that could be done simply. It
was done by bombarding a sample in a vacuum with a beam of atoms, typically Ar or Xe, accelerated to
Kilovolt energies. The sample was typically mixed in a matrix. The two most common matrices were
glycerol and 3 Nitro-benzoic acid. The matrix allowed the sample to refresh itself. The ions formed by FAB
were adducts to the molecule, where the adducts could be protons, sodium ions, potassium ions or
ammonium ions. A variation of FAB was replacement of the atom beam with a beam of ions, typically
cesium ions, which was called secondary ion mass spectrometry (SIMS). SIMS spectra were typically
identical to FAB spectra and the terms became interchangeable.

Electrospray ionization (ESI) - ESI is the ionization technique that has become the most popular ionization
technique. The electrospray is created by putting a high voltage on a flow of liquid at atmospheric pressure,
sometimes this is assisted by a concurrent flow of gas. The created spray is directed to an opening in the
vacuum system of the mass spectrometer, where the droplets are de-solvated by a combination of heat,
vacuum and acceleration into gas by voltages. Eventually the ions are ejected from the droplets and
accelerated into the mass analyzer by voltages. For larger molecules, the ions may contain multiple
charges, allowing the detection of very large molecules on analyzers that have limited mass to charge
(m/Z)) ratio ranges. Because of the natural use of a flowing liquid, it is easily adapted to liquid
chromatography (LC).

There are many other techniques that are variations of electrospray, for example, nanospray is a derivative
of ESI that basically is a low flow version and is highly sensitive because ESI sensitivity is dependent on
concentration not the amount of sample used. Static nanospray or picospray is a similar derivative, but
flow is created by only the electrostatic spray or a small gas pressure, this allows a few microliters to last
over one hour. In addition, static nanospray has the advantage of less carryover, because the needle is
disposed of after each sample. Another similar ionization technique is to spray a surface with the ESI spray
and have the spray extract ions from the surface into the mass spectrometer. This technique is called
desorption electrospray ionization (DESI) and produces spectra similar to electrospray, but is analyzing the
sample on a surface.

Atmospheric Pressure Chemical Ionization (APCI)- APCI is a method that is typically done using a similar
source as ESI, but instead of putting a voltage on the spray itself, the voltage is placed on a needle that
creates a corona discharge at atmospheric pressures. This discharge creates ions, in theory mostly H3O+
or water clusters. The sample is injected into the discharge by a spray created by a flow of liquid combined
with a heated gas that volatilizes the sample. The ions are formed by proton transfer from the H3O+ or
the water clusters to the sample. These ions are then extracted into the same opening vacuum that is used
for electrospray. Another variation of this technique is atmospheric pressure photoionization (APPI),
where the initial ionization is performed by photo ionization, usually of a dopant that absorbs the light and
is added to the sample flow.

A variation of the APCI technique used in our laboratory is atmospheric solids analysis probe (ASAP), which
basically uses the APCI source. The sample is not injected into the flow of liquid, but placed on a probe
placed directly into the corona discharged. In this technique the liquid flow may be turned off, but heated
gas is needed to volatilize the sample. A flow of liquid can be used to change the chemistry in the discharge
to reduce the fragmentation. The advantage of this technique over the traditional method is ease of use
and less carryover of sample.

Matrix Assisted Laser Desorption Ionization (MALDI)- MALDI is a method of ionization in which the
sample is bombarded with a laser. The sample is typically mixed with a matrix that absorbs the laser
radiation and transfers a proton to the sample. Some small mass samples can be ionized without a matrix,
but this is typically called laser desorption. The laser is always pulsed, and typically in a vacuum. In
addition, MALDI mostly forms singularly charged ions. This means MALDI is mostly performed on specially
built time-of-flight instruments. One major application of MALDI besides simple analysis is imaging mass
spectrometry.

Analyzer

There are several types of analyzers used for analyzing the mass to charge (m/z) ratio of ions created in
the source; these analyzers can also be combined to form tandem mass spectrometers. When multiple
types of analyzers are combined, they are called hybrid instruments. Below are simplistic explanations of
the types of analyzers with their advantages and disadvantages.

Magnetic Sector Instruments- Sector type instruments are made of electric and magnetic fields that bend
a beam of ions traveling at relatively high energies. They were once the most popular analyzer for high
resolution analysis, but have gone out of favor because of a lack of sensitivity when scanning, the space
required and the complexity of actually running them. One area that it is still being used is dioxin analysis,
because of its high sensitivity in a high resolution selected ion monitoring (SIM) mode. SIM is setting the
instrument to a specific mass and monitoring that one mass as a function of time to create a
chromatogram of the chemical.

Quadrupole mass spectrometers - Quadrupole mass spectrometers are probably the most common mass
spectrometers, because of the simplicity to use, sensitivity, and quick scan speeds. These properties make
them ideal for such applications as GC-MS and LC-MS. It is basically 4 rods that have a combination of
Radio-frequency (RF) and direct current (DC) voltages applied to them. The combination is chosen to allow
only ions with a specific m/z or a range of m/z to transmit through the analyzer. The voltages are all
controlled by a computer. The quadrupole is a low resolution analyzer. They are often used as the first two
parts of a tandem mass spectrometer with the first part acting as a mass selector, while the second is used
as a collision cell allowing all the products to be transmitted to a third analyzer. When the third analyzer is
a quadrupole, the instrument becomes ideal for quantitation by using selected reaction monitoring (SRM).
SRM is similar to SIM mode, but uses two analyzers, one set to select out a precursor for the molecule,
while the second set for a fragment of the precursor ion.

Ion Trap mass spectrometer - Ion trap mass spectrometers are basically a three dimensional quadruple.
This type of mass spectrometer can store and manipulate ions. It is more sensitive than a simple
quadrupole for full scan mode. The ability to store and manipulate ions is used not only to acquire tandem
mass spectra, but multiple stage tandem mass spectra. Two limitations on the tandem mass spectrometer
is that the lower m/z in the tandem mass spectra is 1/3 m/z of the precursor, and the fragmentation is not
as extensive as instruments that use two quadrupole as the front end.

Time-of-Flight (TOF) mass spectrometer - TOF mass spectrometers are relatively simple mass
spectrometers, because they basically just measure the time an ion takes to travel a specific distance with
a specific kinetic energy. Larger m/z takes longer time to travel than the smaller m/z. These instruments
are typically quite sensitive and fast. Improvements in technology have increased significantly the
resolution of these instruments by use of reflectrons, increasing path length, and the use of pulsed
voltages in the source. They do not have a natural m/z range, but are only limited by the detector and an
ability to ionize the molecule. A variation of TOF is orthogonal acceleration TOF instruments, where a beam
is pulsed into the instrument at a right angle. These orthogonal TOF instruments are commonly used for
continuous ionization sources and as the third analyzer in hybrid instruments

Fourier Transform mass spectrometry (Fourier Transform ion cyclotron resonance spectrometry)(FTMS)
- While other mass analyzers have used Fourier Transformation in their analysis (for example, there was a
FT-TOF instrument developed), FTMS usually refers to Fourier transform ion cyclotron resonance mass
spectrometers. These instruments measure the frequency of the cyclotron motion caused by the ions in a
magnetic field. The frequency of that motion is dependent on the m/z of the ions. These instruments can
have extremely high resolution and mass accuracy, with the resolution dependent on time of observation
(scan time) and the magnetic field. For a 7 Tesla magnet, a m/z ion would have 100,000 resolution at m/z
400 with a scan rate of 1 second per spectra (at m/z 800, it would be 50,000). A higher magnetic field
increases the resolution. Two disadvantages of FTMS is the relatively slow scan speed and the maintenance
of the superconducting magnets.

Orbitrap - Orbitraps are the newest mass analyzer, introduced in 2005 by Thermoelectrics. They are very
similar in their advantages to FTMS, but do not require the superconducting magnets. Basically the
analyzer measures the frequency of ions injected simultaneously into an electrostatic trap. The motion is
harmonic in the electrostatic trap.

Types of Ions and Peaks in Mass Spectroscopy

Molecular Ion
● Ion formed by the loss of a single electron at lowest ionization potential from a molecule.

Fragment Ion
● Generated by the fragmentation of molecular ions in the ionization chamber.

Metastable ion
● Some fragmentation may occur during their flight down the ion tube field free region instead of the
ionization chamber which is known as Metastable ions.
● They reach the detector at masses lower than the actual mass and give broader peaks.

Quasi Molecular ion

● A protonated molecular ion.
● An ion formed by removal of one H-atom from a molecular ion is known as Quasi molecular ion. Multiple
charge ions Some double/triple charged ions are observed.
Mainly occurs in the ESI spectrum. Different m/z ratio.

Base Peak
● The most intense/tallest peak in the mass spectrum.
● It is due to the greatest relative abundance.

Isotope Peaks
● It is due to the presence of heavier isotope element
● It gives less intense peaks.