SPECTROSCOPIC TECHNIQUES

MUHAMMAD AWAIS
QUAID-E-AZAM COLLEGE OF PHARMACY SAHIWAL
SESSION 2021-2026

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 Dedicated to all my friends and class-fellows of Pharm-D 3 rd prof. session
2021-2026 who enabled me to make this humble contribution
Muhammad Awais

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 ATOMIC ABSORPTION SPECTROSCOPY
Introduction:
 Atomic spectroscopy is an analytical technique for detecting and quantifying metals and
metalloids.
 The technique was introduced in 1955 by Walsh in Australia. The first commercial
atomic absorption spectrometer was introduced in 1959.
Atomic absorption spectroscopy (AAS) is a spectro analytical procedure for the
quantitative determination of chemical elements using the absorption of optical radiation
(light) by free atoms in the gaseous state.
Atomic absorption spectroscopy is based on absorption of light by free metallic ions. •
Only elemental analysis can be done. • Atomic absorption is a very common technique for
detecting metals and metalloids in environmental samples

Principle:
When a beam of electromagnetic radiation of a particular wavelength is passed through
atoms in vaporized form, the ground state atoms are excited due to the absorption of
radiation. The decrease in the intensity of radiation is causally related to the atoms in the
ground state present in the sample. Atomic-absorption spectroscopy quantifies or measures
the absorption of energy radiation by ground state atoms in the gaseous state.

The absorption of the visible light energy that has the right wavelength causes the electrons
of the sample to be promoted from a lower energy level to a higher energy level. The
analyte concentration is determined from the amount of absorption.

In AAS, two processes and/or phenomenon are involved that are as follows:
 When light energy is absorbed by the metal sample, free atoms are produced from
the sample metal but are not ionized.
 Once the free atoms are produced, they further absorb radiation from an external
source.

The amount of light absorbed by specific element present in the sample is directly
proportional to the density of atoms present in the flame or in the sample (beer lambart
law)
According to the Beer-Lambert law, the absorbance of a solu on is directly propor onal to the
concentra on of the absorbing material present in the solu on and path length.

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Instrumentation:
Atomic absorption spectrometer has four principal components;
 A light source (usually a hollow cathode lamp)
 Beam chopper
 Atomizer
 Monochromator
 A detector, and read out device

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Hollow Cathode Lamp:
The light source is usually a hollow cathode lamp of the element that is being measured. 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.

Working:
 Applying a potential difference between the anode and the cathode leads to the ionization
of some gas atoms.
 These gaseous ions bombard the cathode and eject metal atoms from the cathode in a
process called sputtering. Some sputtered atoms are in excited states and emit radiation, as
they fall back to the ground state.

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  The shape of the cathode which is hollow cylindrical concentrates the emitted radiation
into a beam which passes through a quartz window all the way to the vaporized sample.
 Since atoms of different elements absorb characteristic wavelengths of light.
 Analyzing a sample to see if it contains a particular element means using light from that
element.
Example:
A lamp containing lead emits light from excited lead atoms that produce the right mix of
wavelengths to be absorbed by any lead atoms from the sample.
A beam of the electromagnetic radiation emitted from excited lead atoms is passed through
the vaporized sample. Some of the radiation is absorbed by the lead atoms in the sample.
The greater the number of atoms there is in the vapor, the more radiation is absorbed.

Beam Chopper:
It is present between the hollow cathode lamp and flame. It rotates and breaks the steady
light into intermittent light. This gives a pulsating current in photo cell.

Atomizer:
 Elements to be analyzed needs to be in atomic sate.  Atomization refers to the separation
of particles into individual molecules and breaking molecules into atoms. This is done by
exposing the analyte to high temperatures in a flame or graphite furnace.  The role of the
atom is to primarily dissolvate a liquid sample and then the solid particles are vaporized
into their free gaseous ground state form. In this form atoms will be available to absorb
radiation emitted from the light source and thus generate a measurable signal proportional
to concentration.
There are two types of atomization;
 Flame atomization
 Graphite furnace atomization
Atomization method or energy source is selected according to the sensitivity and selectivity
of the sample.

Flame Atomization:
 Flame Atomic absorption can only analyze liquids and solution samples, where it
uses a burner to increase the path length, and therefore to increase the total absorbance.
 Sample solutions are usually introduced into a nebulizer by being sucked up by a
capillary tube. In the nebulizer the sample is dispersed into tiny droplets, which can be
readily broken down in the flame.
 The fine mist of droplets is mixed with fuel (acetylene) and oxidant (nitrous oxide)
and burned. The flame temperature is important because it influences the distribution
of atoms.

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Process Taking Place in Flame:
Following is the process that occurs in the flame;
 Nebulization: Conversion of liquid sample into a fine spray.
 Desolvation: Solid atoms are mixed with the gaseous fuel.
 Volatilization: Solid atoms are converted into to a vapor in a flame.

Graphite Furnace Atomization:

Graphite furnace is used for the atomization of the sample. The sample is dried then
burned to ash and finally atomized. The technique is thus named as Graphite atomic
absorption spectroscopy. This technique should be used only when the sample size is
small and/ or when a greater sensitivity is needed. Graphite atomic absorption can
analyze liquid, solid, semi-solid and solution samples. It should not be used when
ordinary flame AA would do as well, since there are disadvantages relating to sample
size and precision.

Monochromator:
This is a very important part in an Atomic Absorption spectrometer. It is used to
separate out all of the thousands of lines. Without a good monochromator, detection
limits are severely compromised.
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.

Detector and Read Out Device:

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.

Amplifier
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.

Recorder
After passing through the amplifier the signals are recorded.

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Double Beam AAS
In this type, before it reaches the sample, the light source is split into two separate
beams. From these one passes through the sample and second one is used for reference.
This gives an advantage because the reference reading and sample reading can take
place at the same time.

Calibration Curve:
A calibration curve is used to determine the unknown concentration of an element in a
solution. The instrument is calibrated using several solutions of known concentrations.
The absorbance of each known solution is measured and then a calibration curve of
concentration vs. absorbance is plotted.
The sample solution is fed into the instrument, and the absorbance of the element in
this solution is measured. The unknown concentration of the element is then calculated
from the calibration curve.

Applications
 Atomic absorption spectroscopy has become one of the most frequently used tools in
analytical chemistry.
 Atomic absorption spectrometry has many uses in different areas of chemistry such as
clinical analysis of metals in biological fluids and tissues such as whole blood, plasma,
urine, saliva, brain tissue, liver, hair, muscle tissue.

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  Atomic absorption spectrometry can use in qualitative and quantitative analysis.
 Atomic absorption is used for the determination of ppm levels of metals.
 Pharmaceuticals: In some pharmaceutical manufacturing processes, minute quantities of a
catalyst used in the process (usually a metal) are sometimes present in the final product.
Therefore, by using AAS the amount of catalyst present can be determined. For example,
in vitamin preparations.

Advantages:
 Precise and accurate results can be obtained by the usage of this technique.
 It is a very sensitive. It can detect concentrations as small as a few parts to μg / Liter
(parts per million)
 It is generally very specific as the set wavelength is strongly absorbed by the
particular metal ion being analyzed (and not by other components).
 Only a little quantity of the sample is required about 1ml – 2ml.
 It can analyze specific elements because of the unique light-absorbent qualities of
their atoms
 AAS can determine concentrations of over 65 elements.
 AAS supports a broad range of industries and sectors, including environmental,
chemical, petrochemical, food and drink and pharmaceutical

Disadvantages:
 It is a cost-effective technique.
 Flame atomic absorption spectroscopy, can analyze only liquid sample.
 Graphite atomic absorption spectroscopy should not be used when ordinary
flame AA would do as well, since there are disadvantages relating to sample
size and precision.

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PREPARED BY: MUHAMMAD AWAIS

SPONSORSHIP & COMPANY BY: MUHAMMAD IMRAN
BLOOM-JASMINE
QUAID-E-AZAM COLLEGE OF PHARMACY SAHIWAL
SESSION 2021-2026

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 ATOMIC EMISSION SPECTROSCOPY
Emission Spectrum:
The collection of spectral lines produced by an excited atom is called emission spectrum
and will be characteristic of that atom.

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:
Atom are surrounded by orbits (energy levels) containing electrons. The energy gap (between
excited and ground state) for all atoms is different due to the ratio of protons and electrons in the
structure. Hence, upon excitation, when the electrons come back to the ground state, emit
different wavelength radiation (produce characteristic spectra).
When the element is heated in the flame, the absorption of energy by the ground state electron in
an atom results in excitation of some on these electrons to higher energy resulting in excitation,
when these excited atom come to ground state they emit radiations that are the area of interest we
study in AES.
A solution of sample to be analyzed is sprayed into flame possessing the thermal energy require
to excite the element at which it will radiate its characteristic bright line emission. Atomic
emission spectroscopy uses quantitative measurement of the optical emission from excited atoms
to determine analyte concentration Sample in liquid Heating converts sample into vapor form
containing atoms In vapor state, some atoms are in excited state and some in ground state If
excited atoms are measured (Atomic emission spectroscopy) If ground state atoms that get
excited due to the incident radiation are measured (atomic absorption spectroscopy)
Types of Emission Spectrum:
There are three types of emission spectrum:
 Line emission spectrum
 Band emission spectrum
 Continuous emission spectrum

Line Emission Spectrum: It consists of sharply defined and often widely and irregularly
spaced individual lines of a single wavelength. These spectra are characteristic of element. They
are also called atomic spectrum.

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Band Emission Spectrum: It consists of group of lines each of which has single
wavelength that becomes closely spaced as they approach the end of band. They are also called
molecular spectrum.

Continuous Spectrum: They are obtained when solids are heated to incandescence. They
are characterized by absence of any sharp lines as a function of wavelength. On the other hand
when gases and vapors are heated to high temp they yield a series of bands or lines.

When we study the emission of energy by the atoms in the flame, we call this technique as
Atomic Emission Spectroscopy AES.
Basic principle of AES
In atomic emission the sample is atomized and the analyte atoms are excited to higher
energy levels. The analyte concentration is determined from the amount of emission. The
analyte atoms are promoted to a higher energy level by the sufficient energy that is
provided by the high temperature of the atomization sources. The excited atoms decay
back to lower levels by emitting light. Emissions are passed through monochromators or
filters prior to detection by photomultiplier tubes.

Instrumentation:
The instrumentation of atomic emission spectroscopy is the same as that of atomic absorption,
but without the presence of a radiation source.

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Atomic absorption spectrometer has three principal components;

 Atomizer (Flame, Graphite furnace, ICP)

 A Monochromator
 A detector, and read out device

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 Sample Cell
• The sample cell contains the test solution of the element or metal, the quantity of which
is to be determined.
Nebulizer
• Draw up liquid samples at controlled rate. • Creates a fine aerosol spray for
introduction into flame.
• Mix the aerosol, fuel and oxidant thoroughly for introduction into flame
Atomizer:
Atomizer converts the test solution into fine spray with the help of compressed air or gas
under pressure. The atomizer may be automated or manually operated. • The most
common methods are flames and plasmas, both of which are useful for liquid or solution
samples. • Solid samples may be analyzed by dissolving in a solvent and using a flame or
plasma atomizer
The major energy sources in Atomic Emission spectroscopy are:
 Graphite furnace: This technique should be used only when the sample size is small
and / or when a greater sensitivity is needed. The substance sample is dried at 200oC for
60 sec. Then it is burned at 1200oC for 30 sec. to burn all organic samples. And then
finally the atomization is brought about by heating at 2700oC for 60 sec.
 A flame: The flame (1700oC – 3150oC) is most useful for elements with relatively low
excitation energies like sodium potassium and calcium.
 Inductively coupled plasma: The ICP (6000oC – 8000oC) has a very high
temperature and is useful for elements of high excitation energies.
 Electric arc: Sample is heated by an electric arc.
 Electric spark: Sample is excited in high voltage spark.
Monochromator:
• It is used to select the specific wave length of light which is emitted by the samples and
exclude all the other wave lengths.
• It consists of an entrance and exit slits and mirror or lenses to deflect or focus the
radiation.
A monochromator, in atomic emission spectroscopy, is simply a wavelength selector. It is
used to select the specific wavelength of light which is emitted by the sample, and to
exclude other wavelengths.
Detector:
The selected wavelength 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.

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 Applications of AES:
 Atomic emission is widely used for the analysis of trace metals in a variety of sample
matrices.
 Well suited for Multi-elemental analysis.
 Determination of zinc, sodium and potassium in urine.
 Quantification of Fe in green vegetables.
 To determine water hardness.
 Also used in agriculture, industrial, food and forensics.

Comparison between AAS & AES

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 FLAME PHOTOMETRY
History:
During 1980s Bowling Barnes, David Richardson, John Berry and Robert Hood developed an
instrument to measure the low concentrations of sodium and potassium in a solution. They named
this instrument as Flame photometer.

Introduction:
 Flame photometry is a process where in the emission of radiation by neutral atoms is measured.
 The neutral atoms are obtained by introduction of the sample into flame. Hence the name flame
photometry.  Since radiation is emitted, it is also called as flame emission spectroscopy.

Definition:
Flame photometry (more accurately called Flame Atomic Emission Spectrometry) is a branch of
spectroscopy in which the species examined in the spectrometer are in the form of atoms.
A photoelectric flame photometer is an instrument used in inorganic chemical analysis to
determine the concentration of certain metal ions among them sodium, potassium, calcium and
lithium.

Basics:
Flame Photometry is based on measurement of intensity of the light emitted when a metal is
introduced into flame.
o The wavelength of colour tells what the element is (qualitative)
o The color's intensity tells us how much of the element present (quantitative)

Principle:
 The basic principle upon which Atomic Spectroscopy works is based on the fact that
"Matter absorbs light at the same wavelength at which it emits light".
 Atoms of elements are subjected to hot flame and specific quantum of thermal energy
absorbed by orbital electrons so they become unstable at high energy level and release
energy as photons of particular wavelength and change back to ground state.
 When a metal salt solution is burned, the metal provides a colored flame and each metal
ion gives a different colored flame.
 Flame tests, therefore, can be used to test for the absence or presence of a metal ion.

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Overview:
The solvent is first aspirated to obtain fine solid particles.
 These molecules in the solid particles are moved towards the flame to produce gaseous atoms
and ions.
 These ions absorb the energy from the flame get excited to high energy levels from the ground
state.
 But as these ions are unstable, they return back to ground state. While returning they emit
characteristic radiation.
 The intensity of emitted light is proportional to the concentration of the element.

Basic Concept:
 Liquid sample containing metal salt solution is introduced into a flame.  Solvent is first
vaporized, leaving particles of solid salt which is then vaporized into gaseous state  Gaseous
molecule dissociates to give neutral atoms which can be excited (made unstable) by thermal energy
of flame.  The unstable excited atoms emit photons while returning to lower energy state.  The
measurement of emitted photons forms the basis of flame photometry.  Under constant and
controlled conditions, the light intensity of the characteristic wavelength produced by each of the
atoms is directly proportional to the number of atoms that are emitting energy, which in turn is
directly proportional to the concentration of the substance of interest in the sample.  Various
metals emit a characteristic colour of light when heated.

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Instrumentation:
 Burner
 Monochromators
 Detectors
 Recorder and display
Structure of Flame
The flame may be divided into the following regions or zones:
 Preheating zones
 Primary reaction zone or inner zone
 Internal zone
 Secondary reaction zone
1. Preheating Zone: In this, combustion mixture is heated to the ignition temperature by
thermal conduction from the primary reaction zone.

2. Primary Reaction Zone: This zone is about 0.1 mm thick at atmospheric pressure. There
is no thermodynamic equilibrium in this zone and the concentration of ions and free radicals is
very high. This region is not used for flame photometry.

3. Interconal Zone: It can extend up to considerable height. The maximum temperature is

achieved just above the tip of the inner zone. This zone is used for flame photometry.

4. Secondary Reaction Zone: In this zone, the products of the combustion processes are
burnt to stable molecular species by the surrounding air.

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Interferences:
In determining the amount of a particular element present, other elements can also affect the result.
Such interference may be of 3 kinds:

 Spectral Interferences: Occurs when the emission lines of two elements cannot be resolved
or arises from the background of flame itself. They are either too close, or overlap, or occur due to
high concentration of salts in the sample

Ionic Interferences: high temperature flame may cause ionization of some of the metal atoms,
e.g. sodium. o The Na+ ion possesses an emission spectrum of its own with frequencies, which are
different from those of atomic spectrum of the Na atom .

 Chemical Interferences: The chemical interferences arise out of the reaction between
different inter-ferents and the analyte. Includes:

 Cation-anion interference: The presence of certain anions, such as oxalate,

phosphate, sulfate, in a solution may affect the intensity of radiation emitted by an element.
E.g., calcium + phosphate ion forms a stable substance, as Ca3(PO4)2 which does not
decompose easily, resulting in the production of lesser atoms.
 Cation-cation interference: These interferences are neither spectral nor ionic in
nature E.g. aluminum interferes with calcium and magnesium.

Detail of instrumentation of flame photometry

Sample Delivery System:

There are three components for introducing liquid sample:
 Nebulizer – it breaks up the liquid into small droplets.

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 o Nebulization the is conversion of a sample to a mist of finely divided droplets using a jet of
compressed gas. o The flow carries the sample into the atomization region.
o Pneumatic Nebulizers: (most common use)
 Aerosol modifier: It removes large droplets from the stream and allow only smaller droplets
than a certain size to pass
 Flame or Atomizer – it converts the analyte into free atoms.

FUEL AND OXIDANTS:

 Several burners and fuel + oxidant combinations have been used to produce analytical flame
including Premixed, Mecker, Total consumption, Lundergarh, Shielded burner, and Nitrous
oxideacetylene flames
 Fuel and oxidant are required to produce the flame such that the sample converts to neutral
atoms and get excited by heat energy. The temperature of flame should be stable and also ideal. If
the temperature is high, the elements in sample convert into ions instead of neutral atoms. If it is
too low, atoms may not go to excited state. So a combination of fuel and oxidants is used such that
there is desired temperature.

Source:
 A Burner used to spray the sample solution into fine droplets.

 Pre-mixed Burner: widely used because uniformity in flame intensity. In this energy type
of burner, aspirated sample , fuel and oxidant are thoroughly mixed before reaching the burner
opening.
 Total Consumption Burner: in this fuel and oxidant are hydrogen and oxygen gases. Sample
solution is aspirated through a capillary by high pressure of fuel and Oxidant and burnt at the tip
of burner. Entire sample is consumed.

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Monochromator:
Filters and monochromators are needed to isolate the light of specific wavelength from remaining
light of the flame. For this simple filters are sufficient as we study only few elements like Ca, Na,
K and Li. So a filter wheel with filter for each element is taken. When a particular element is
analyzed, the particular filter is used so that it filters all other wavelengths.

 Prism: Quartz material is used for making prism, as quartz is transparent over entire region
 Grating: it employs a grating which is essentially a series of parallel straight lines cut into a
plane surface

Detectors:
Flame photometric detector is similar to that used in spectrophotometry. The emitted radiation is
in the visible region, i.e., 400nm to 700nm. Further, the radiation is specific for each element, so
simple detectors are sufficient for the purpose of photovoltaic cells, phototubes, etc.
 Photomultiplier tubes
 Photo emissive cell
 Photo voltaic cell

Photovoltaic Cell:
 It has a thin metallic layer coated with silver or gold which act as electrode, also has metal base
plate which act as another electrode
 Two layers are separated by semiconductor layer of selenium, when light radiation falls on
selenium layer.
 This creates potential diff. between the two electrode and cause flow of current.

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Read-Out Device:
 It is capable of displaying the absorption spectrum as well absorbance at specific wavelength

 Nowadays the instruments have microprocessor controlled electronics that provides outputs
compatible with the printers and computers  Thereby minimizing the possibility of operator error
in transferring data.

Applications:
Flame photometry is used in various industries like chemicals, soil, agriculture, pharmaceuticals,
glass and ceramics, in plant materials and water, oceanography, and in biological and
microbiological laboratories.
 It is used in determination of potassium, sodium, magnesium and calcium in biological
fluids like serum, plasma, urine etc, is routinely carried out by flame photometer
 To estimate sodium, potassium, calcium, lithium etc. level in sample of serum, urine, CSF
and other body fluids.
 Determining the concentration of sodium and potassium ions in infusion solutions, such as
NaCl solution, Ringer solution or others. Product control and indirect quality testing of
various substances over sodium, potassium or lithium.
 Concentration determination in pharmaceutical reagents. In the production of blood
collection tubes, the finished products are checked for correct chemical composition by
means of flame photometry.
 Determination of certain metals like lead, manganese, in petroleum products like gasoline,
lubricating oils and organic solvents
 Flame photometry is useful for the determination of alkali and alkaline earth metals.(imp
point)
 Used in determination of lead in petrol.
 Used in the study of equilibrium constants involving in ion exchange resins.
 Used in determination of calcium and magnesium in cement.

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Advantages:
 The method of analysis is very simple and economical.
 It is quick, convenient, selective and sensitive analysis.
 It is both and qualitative and quantitative in nature.
 Even very low concentrations (parts per million/ppm to parts per billion/ppb range) of
metals in the sample can be determined.
 This method compensates for any unexpected interfering material present in the sample
solution.
 This method can be used to estimate elements which are rarely analyzed.

Disadvantages:
In spite of many advantages, this analysis technique has quite a few disadvantages:
 The accurate concentration of the metal ion in the solution cannot be measured.
 It cannot directly detect and determine the presence of inert gases.
 Though this technique measures the total metal content present in the sample, it does not
provide the information about the molecular structure of the metal present in the sample.
 Only liquid samples may be used. Also sample preparation becomes lengthy in some cases.
 Flame photometry cannot be used for the direct determination of each and every metal
atom. A number of metal atoms cannot be analyzed by this method. The elements such as
carbon, hydrogen and halides cannot be detected due to their non-radiating nature.

***********************************************************************

PREPARED BY: MUHAMMAD AWAIS

SPONSORSHIP & COMPANY BY: MUHAMMAD IMRAN& TEHSEEN
GHAFOOR
QUAID-E-AZAM COLLEGE OF PHARMACY SAHIWAL
SESSION 2021-2026

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 Mass Spectrometry
Mass Spectrometry is the generation, separation and characterization of gas phase ions according
to their relative mass as a function of charge
Mass Spectrometry is an analytical technique which involves the production of gaseous ions from
the substance under investigation, their separation according to their mass -to-charge ratio (m/z)
and the measurement of relative abundance of these ions
Spectrometry instead of Spectroscopy→ no absorption of light is involved
Spectroscopy:
OLD CONCEPT: It is the combination of two words spectrum & scope. Spectrum means, “a set
of specific no. or set of specific values or a set of digits,” while the word Scope means “Look or
Watch.”
LATEST DEFINITION: A type of physics in which we study the interaction of matter with
radiant energy (radiation) – Electromagnetic radiations (EMR)
Spectrometry:
It is the combination of two words spectrum & meter. Spectrum means, “a set of specific no. or
set of values or a set of digits,” while the word Meter means “measurement.”
So, Spectrometry means measurement of spectrum. When there is no absorption or emission of
electromagnetic radiations, then this study is known as spectrometry, but here the spectrum is not
made up by the electromagnetic radiations. The spectrum is Mass spectrum. If it has the concept
of mass then it is referred to as Mass spectrometer.
Basic Theory:
Mass Spectrometry is the most sensitive technique for structural determination (elucidation). In
MS the molecule is broken down into fragments. These fragments are separated according to their
mass to charge ratio (m/e). Each fragment gives its peak (response) on reaching to detector. These
fragments are recorded as Mass spectrum.
Principle:
Charged molecules or molecular fragments are generated in a high-vacuum region, or immediately
prior to a sample entering a high-vacuum region, using a variety of methods for ion production.
The ions are generated in the gas phase so that they can then be manipulated by the application of
either electric or magnetic fields to enable the determination of their molecular weights.

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Mass Spectrum:
Mass spectrum is the graphical representation and comparison in between mass to charge ratio vs.
relative intensity. The highest peak is recorded as 100% which is also called as base peak. The
other peaks are recorded with reference to the base peak (100% peak).
The total energy is 70 eV. Some amount of energy (10 eV) is utilized to ionize the compound and
the remaining energy (70 eV) is utilized to break the bonds. MS break all bonds of different
compounds and fragments are formed (those compounds which have too much similarity).
 Highest peak (base peak) represents the highest quality.
 Every fragment shows its specific response.
Detector provides a peak of every fragment. Detector detects the fragments on mass to charge
ratio (m/e) and their relative intensity use. Retention time is not checked over here as we use mass.
Recorder made comparison and it shows results. It is then attached with digital library. After
fragmentation the fragments are again combined to check the structure of the sample.
Exception: There may be two 100% peaks, but this is rare.

Instrumentation and Data analysis

There are five major parts of Mass spectrometer, which are as follows:
1. Ion source
2. Mass analyzer (include electric field which work as accelerated the ions and
magnetic field which separate the ions on basis of m/e ratio)
3. Detector
4. Recorder
Schematic Diagram:

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Ion source: It is also called ionization chamber or ionic chamber or ionization source. Ionization
of the sample occurs in ionization chamber.
Ionization Potential: The minimum energy required to ionize an atom or molecules is called
Ionization potential. The energy may be supplied by different ways depending upon the physical
or chemical nature of sample.
Methods of Ionization:
Several methods are employed for the ionization of the sample depending upon the physical state
and chemical nature of the sample.
Electron impact / Electron Ionization (EI):
 The first and the common method of ionization is Electron impact or electron ionization.  In
this method electron beam is used for the purpose.  The sample is first volatilized in a separate
chamber and vapors at pressure 10-4 – 10-6 torr are allowed to enter in the ionization chamber
(ion source / ionic chamber / ionic compartment).  In the ionization chamber there is a
bombardment of electron beam which can remove or eliminate electron from sample creating
Radical cations.  The source of electronic beam is electrically heated filament of tungsten. On the
opposite side of filament there is an anode plate which acts as “Electron trap. Ions which are
produced move towards mass analyzer.  Detector signals are directly proportional to the no. of
ions hitting to the detector.  By adjusting the magnates, ions of all the masses are collected and
countered.

Chemical Ionization (CI):

 In chemical ionization a reagent (Methane, Ethane, Butane or isobutene) is introduced into high
pressure source (0.1 – 1.0 torr) and ionized by electrons bombardment.  Primary ions are
produced. These ions undergo collision resulting in the production of secondary stable ions e.g.
methane is bombarded by electron beam and the equation will be:
CH4 + e- → CH4+• + 2 e-
Due to the high pressure there is a possibility or colliding a primary ion with the other molecules
and the reaction will be like;
CH4+• + CH4 → CH3 • + CH5+

25 | P a g e
 Remember that only positively charged species reach the collector.

Mass Analyzer:
Ions are produced in the ionic source and then these ions leave the ionic source and enter into the
mass analyzer. In mass analyzer, these ions are separated according to their m/e ratio. It is also
called as ion separator.
Following are the commonly used mass analyzers in Mass spectrometer;
 Magnetic sector mass analyzer
 Time of flight analyzer
 Ion trap analyzer
 Quadrupole mass Analyzer
Magnetic Analyzer

➢ The analyzer tube is surrounded by a magnet whose magnetic field deflects the positively
charged fragments in a curved path. At a given magnetic field strength, the degree to which the
path is curved depends on the mass-to-charge ratio (m/z) of the fragment. The path of a fragment
with a smaller m/z value will bend more than that of a heavier fragment. In this way, the particles
with the same m/z values can be separated from all the others. If a fragments path matches the
curvature of the analyzer tube, the fragment will pass through the tube and out the ion exit slit
When an ion of charge z is being repelled by electrostatic field of voltage V, ion is accelerated

➢ Its potential energy is zV→ is converted to kinetic energy = 1/2mv2

26 | P a g e
Where, m = mass of ion
V = velocity of ion
Under the influence of the magnetic field, the ion assumes a curved path with the radius r and
experience a centrifugal force given by mv2/r.
The magnetic field also exerts centripetal force on the ions which is equal to zHv, where H is the
magnetic field strength

The Radius of curvature of an ion in the magnetic field depends on the m/z value of ion. The ion
of larger m/z value follow a path of lager radius. The ions of smaller m/z value follow a path of
smaller radius. Consequently, the ions emerging from the magnetic field are separated in space
according to their m/z value.
The efficiency of a mass spectrometer is measured from the ability of its analyzer to resolve
(separate) the ions having close m/z values. The resolving power of a mass spectrometer is
acceptably defined by measuring the depth of the valley between the two adjacent peaks produced
by the ions of close m/z values. While deriving the Eq. 3, it was assumed that all ions enter the
magnetic field with the same kinetic energy. This is not strictly true. the ions enter the analyzer
with a slightly different kinetic energy ➢This energy-spread makes the ions to follow a slightly
different path in the analyzer, resulting in a rather broadened peak.

27 | P a g e
Ion Collector/ Detector:
➢Once the ions are separated by the mass analyzer, they reach the ion detector, which generates
a current signal from the incident ions.
Three types of commonly used detectors are discussed below;
1. Faraday cup detector: It is a metallic cup which is maintained at potential which allows
the ions to be captured by the cup.
2. Photographic plate detector: It is cupped at right angle to path of ions. So that, there is a
linear formation of images and abundance of ions is determined by the intensity of each
image.
3. Electron multiplier detector: It is a series of electrodes (dynodes) which are connected
to each other. When ions hit the first dynode, there is a release of large no. of electrons.
These electrons hit the second dynode and then it will release large no. of electrons. This
process goes on throughout the series of electrodes, which are normally 10.
As a result of this sequence there is a production of electric current which is amplified and
presented in the form of graph. The graph is called as Mass spectrum.

Vacuum system:
All mass spectrometers operate at low pressure, just to avoid the ion collision. Any collision of
ions may result ionic reactions; neutralization and scattering. To minimize the collision whole
procedure is operated at low pressure or high vacuum (10-4 – 10-8 torr). The vacuum system also
attracts the ions towards the detector.

Recording of Mass Spectrum:

The most important method of recording the mass spectrum is the use of online computer. The
whole system is known as data system. A printer is also attached to the data system to take out the
print of recorded mass spectrum.

Mass Spectrum
Mass spectrum is a plot of the relative intensity of the ions (corresponding to their abundance)
against their m/z value. The molecular ion and fragment ions produced in a mass spectrometer and
recorded by it are unique for each compound. A mass spectrum, therefore, is like a fingerprint of
the compound

28 | P a g e
Mass spectrum is produced in the form of bar graph which is interpreted by using the following
peaks.
 Molecular ion peak: It has highest mass-to-charge ratio (m/z) in mass spectrum created
due to loss of one electron from molecule, and its m/e value roughly represents the
molecular weight of compound.
 Base peak: It is the most intense peak of the mass spectrum. It has 100% relative
abundance. • the relative abundance of each of the other peaks is reported as a percentage
of the base peak.
 Fragment ion peak: Peak of fragment ion (peaks with smaller m/z value) in mass spectrum
is called fragment ion peak. Fragmentation pattern is characteristic for a particular organic
compound. So we can confirm the compound by comparing with the library of
fragmentation pattern of reference compounds. The way by which the molecular ion to be
fragmented depends on the strength of its bonds and stability of the fragments

Limitations of MS:
 Mass spectrometry is not currently used in routine quality control (QC) but is placed in a research
and development (R&D) environment, where it is used to solve specific problems arising from
routine processes or in process development.  The instrumentation is expensive and requires
support by highly trained personnel and regular maintenance. However, these limitations are
gradually being removed with open access instruments becoming more common.

29 | P a g e
Applications of MS:
1. Preparation of isotopes: MS is very useful for preparations of pure isotopes, high polymers
and natural products can be analyzed by this.
2. Cis-trans isomers: It is also used to distinguish between Cis & Trans isomers, since stability
of ions produced may differ for Cis & Trans ions significantly.
3. Study of free radicals: It is useful to study free radicals, determination of bond strength,
evaluation of heat of sublimation etc.
4. Study of closely related compounds: It is useful in the analysis of closely related compounds.
For example; hydrocarbons, petroleum, products, lubricating oils etc.
5. Identification of Molecular formula: It provides important information for identification by
help of molecular weight, molecular formula and by fermentation pattern.
6. Determination of molecular weight: It is best tool for the determination of molecular weight
of the substance (where substance is bombarded with moving electrons and its mass spectra is
recorded, the mass of peak at highest m/e reveals molecular mass accurately also the molecular
formula.
7. Trace analysis: The inorganic trace analysis MS can used for trace analysis of elements in
alloys and minerals and in super conductors.
8. In proteomics: Characterization of proteins and protein complexes, sequencing of peptides
and identification of post-translational modifications
9. Metabolomics: Cancer screening and diagnosis, global metabolic fingerprinting analysis,
biomarker discovery and profiling, bio-fuel use and generation, lipidomics studies and
metabolic disorder profiling
10. Pharmaceutical Analysis: Drug discovery and absorption, ADME studies, pre-clinic
development, pharmaco-economics and dynamics analysis
11. Forensic Analysis: Analysis of trace evidence, arson investigation, conformation of drug
abuse, bomb investigation
12. Clinical Applications: Clinical drug development, phase 0 studies, clinical tests, disease
screening, drug therapy monitoring, investigation of infectious agents for targeted therapies.

*****************************************************************************
PREPARED BY: MUHAMMAD AWAIS
SPONSORSHIP & COMPANY BY: MUHAMMAD IMRAN
QUAID-E-AZAM COLLEGE OF PHARMACY SAHIWAL
SESSION 2021-2026

30 | P a g e
 MOLECULAR FLUORESCENCE
SPECTROSCOPY
Luminescence: The optical emission from a molecule at a wavelength longer than the one used
to excite it from G0 to Es is called luminescence. It is defined as, the emission of photons from
electronically excited state.
Luminescence is divided into two types, depending upon the nature of the ground and the excited
states.
There are two states:
1. Singlet state
2. Triplet state

SINGLET STATE: In singlet excited state, the electron in the higher energy orbital has the
opposite spin orientation as the second electron in the lower orbital. These two electrons are said
to be paired. Return to the ground state from an excited singlet state does not require an electron
to change its spin orientation.
TRIPLET STATE: In a triplet state these electrons are unpaired, that is, their spins have the same
orientation. A change in spin orientation is needed for a triplet state to return to the singlet ground
state e.g. in case of free radicals.
Types of Luminescence

Fluorescence: When a substance absorbs radiation and immediately emits the radiation after
the radiation absorption, this phenomenon is called as fluorescence. The substances which exhibit
phenomenon of fluorescence are called as fluorescent. The phenomena of fluorescence happens
instantaneously and begins immediately after the absorption of radiation of light and terminates
quickly as the incident light radiations are cut off.

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Phosphorescence: When a substance absorbs the radiation and subsequently emits the
radiation contineuously after the absorption of radiation is cut off. This type of phenomenon is
called as phosphorescence. The substances exhibit such characteristic are called as phosphorescent
substances.

Chemiluminescence:During the process of chemiluminescence, light is produced from

chemical reaction. During chemical reaction, the light is emitted because this reaction produces
electrically excited molecules. When these excited molecules return to their ground state, the emit
light.
In many biological systems, the chemiluminescence reactions occur where this phenomenon is
known as bioluminescence.
One of the distinguished characters of chemiluminescence is that it does not need any sophisticated
instrumentation. Additionally, no external radiation source is also required for excitation purpose.
The instrument may contain only two things, one is reaction vessel and the other one is a
photomultiplier tube. The devices that are used for the selection of wavelength are not needed
because the source of radiation is only chemical reaction. Moreover, there is no need of excitation
source.

Chemiluminescence has wide range of applications. It can be used for the detection of arsenic in
water and can also be applied to fabricated microarrays on a flow chip, allowing for patterned
biosensor applications.

Jablonski Diagram:
An electron was present in its ground state S0, it absorbs light or electromagnetic radiation and
gets excited to a high intermediate energy level.
According to Kasha’s rule, the electron will require to attain a stable energy level near to that
intermediate level, so it moves to S1. After attaining a stable energy state it jumps back to its ground
state SO either in non-radiative fashion, or in radiative fashion thus, producing fluorescence.

32 | P a g e
The non-radiative fashion is due to the loss of energy during interconversion. In interconversion
only vibrational and rotational changes takes place within the molecules.

Properties of fluorescence:
Fluorescence is a specific property of each molecule and it depends upon the auxochrome of
molecule. The emitted light has two important characteristics;  It is usually of longer wavelength
(lower energy) than the excited light. This is because part of the energy associated with S state is
lost as heat energy.  The emitted light is composed of many wavelengths which results in
fluorescence spectrum.

Schematic representation of electronic levels and transitions in a fluorescence and phosphorescence

33 | P a g e
Theory
Internal conversion: When the lower and upper electronic states of the excited singlets have same
multiplicity, this phenomenon is known as internal conversion
Photon emission: The molecules in the singlet excited state come back to the ground state and
cause the emission of the photon that is called as fluorescence.
Energy transfer: The energy is transferred from singlet state to the triplet state when the molecule
come back to the ground state which is also called as intersystem crossing.

Fluorescence Spectroscopy:
Fluorescence spectroscopy FS is an analytical technique that measures the concentration of
fluorescent substance present in the sample. It makes the use of the fluorescence in order to
measure their concentration while, fluorescence is the emission of the light by a substance which
absorbs light or Electromagnetic radiation.

Principle:
Molecular fluorescence spectrometry is based on the emission of light by molecules that have
become electronically excited subsequent to the absorption of electromagnetic radiation. It uses
the UV visible spectra along with some part of IR region of electromagnetic radiation. It is a
sensitive technique by which we can detect the sample raging from ppm – ppt.
Molecular fluorimetry: It is a technique that measures the fluorescence from molecules that have
been excited from Go to Es by absorption of EMR of a particular wavelength.

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Instrumentation:

Schematic representation

 Light Source:
Xenon-Arc Lamp: Xenon gas is used as a source of light. It also gives steady supply. The
wavelength of light produce is about 300 – 1300 nm.
 Excitation Monochromator:
Various types of monochromators can be used to select the specific type of wavelength to
fall on the sample so that the sample excites that is why it is also called as excitation
monochromator. It is also called wavelength selector.
Commonly used monochromators are:
 Prisms: Various salt or glass prisms are used for the process.
 Grating: In general, gratings are used in the design of the instrument and offer
better resolution at high frequency than the prisms. They offer much better
resolution at low frequency.
 Sample Holder:
Cuvette or quarts cell is used as a sample holder. The cell is transparent from all sides so
that the fluorescence produced can be observed by the detector easily.
 Emission Monochromator:
A second monochromator is used to observe the produced fluorescence without difficulty.
It acts as wavelength filter. It is placed right angle (90 degree) to the first monochromator.
It directs the fluorescence towards the detector.
 Detector:
The detector observes the fluorescence produce by the substance and generates the
electrical signal. An amplifier is attached that amplifies the intensity of the signal. The
commonly used detectors are phototube & photomultiplier tubes.
 Read Out Devices:
The computer reads the detected signal and plots a graph.

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Applications of Fluorescence Spectroscopy:
Though the Fluorescence is a specific property of substances, but this type of spectroscopy
has limited applications as not all substances show fluorescence.
Some applications are as follows:
 Quinine was the first medicine that showed fluorescence.
 The technique is used for the quantitative analysis as well as for qualitative analysis.
 The technique is also useful for protein identification. This is done by tagging a fluorescent
molecule within the protein and fluorescence is observed.
 This technique is used for the assay of different medications that shows fluorescence
 It is utilized in medical diagnostics, such as detecting biomarkers in patient samples.
 It is applied in drug discovery and quality control, especially for compounds that exhibit
fluorescence.
 Analyzes fluorescent materials and polymers.
Qualitative Analysis of Crude Drugs: The powdered crude drugs (alone or after treatment
with different reagents) can be analyzed for fluorescent properties under visible light and UV
light (short and long wavelength). The data generated is used for quality control of the plant
material.

Quenching :
The decrease in fluorescence intensity is known as quenching.
Reasons of Quenching:
 Concentration of the molecule
 pH
 Presence of chemical substances
 Temperature
 Viscosity

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 Types of Quenching:
 Self-quenching
 Collisional quenching
 Static quenching
 Chemical quenching

*********************************************************************

PREPARED BY: MUHAMMAD AWAIS

SPONSORSHIP & COMPANY BY: MUHAMMAD IMRAN & TEHSEEN
GHAFOOR
QUAID-E-AZAM COLLEGE OF PHARMACY SAHIWAL
SESSION 2021-2026

37 | P a g e
 UV – VISIBLE SPECTROSCOPY
It refers to measurement of absorbance of EMR in UV/VI region of spectrum.
UV-VIS spectroscopy is considered as the most important spectrophotometric technique that is
most widely used for the analysis of variety of compounds. This technique works on the basis of
the measurement of interaction of electromagnetic radiations (EMR) with matter at particular
wavelength. For proper working of UV-VIS spectroscopy and to get accurate results, it is very
important to understand the components of UV-VIS spectroscopy and their individual role in the
proper functioning of UV-VIS spectrophotometer.

Introduction
UV-VIS spectroscopy is considered as the oldest analytical technique that can be defined as the
spectrophotometric technique which is used to measure the intensity of light in UV (10–400 nm)
and VIS (400–800 nm) regions as a function of wavelength. The wavelengths of UV and VIS
radiations are usually expressed in nanometers (nm). The analyte absorbs the light of specific
wavelength (UV and VIS only) and the amount of radiation absorbed by the analyte is measured.
The spectrum produced after the absorption of UV-VIS light results from the interaction of EMR
in UV-VIS region with the analyte. It forms the basis to analyze a variety of substances like
organic, inorganic, biochemical, and pharmaceutical compounds. In UV-VIS spectroscopy,
absorption of radiations occurs at electronic energy levels (one of the three basic energy levels,
i.e., electronic, vibrational, and rotational energy levels) of molecules; therefore, this technique is
also known as electronic spectroscopy.

Principle:
UV-VIS spectroscopy works on the basis of the absorption phenomenon of light and the amount
of absorbed light is directly proportional to the amount of analyte present in a sample solution. As
the concentration of analyte is increased, absorption Fig. 3.1 Schematic representation of wave
phenomenon 30 3 Ultraviolet-Visible (UV-VIS) Spectroscopy of light is also increased linearly,
whereas the transmission of light is decreased exponentially.
An electronic energy level consists of various vibrational energy levels, whereas a single
vibrational energy level consists of various rotational energy levels. When a photon interacts with
a molecule, it may induce a transition in electronic energy levels if energy provided by the photon
matches with energy difference in these levels. The amount of radiations absorbed by the analyte
is measured and plotted against the wavelength of EMR in order to obtain the spectrum. Thus, a
typical UV-VIS spectrum is a plot of wavelength or frequency versus the intensity of absorption.

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Instrumentation and Working:
The UV-visible spectrometer consists of following parts:
 Light source
 Monochromator (Prism type, Grating type)
 Quarts cell (sample holder)
 Detector (phototubes, photomultiplier tubes)
 Recorder

Components of UV-VIS Spectrophotometer

Following are the main components of UV-VIS spectrophotometer:

Light Source: It provides a sufficient light in the form of polychromatic light that is appropriate
for marking the measurements. Usually, light source provides polychromatic light over a wide
range of spectrum . In UV-VIS spectrophotometer, two types of light sources are used. For UV
sources, deuterium, hydrogen, tungsten, mercury, and xenon lamps are used, whereas for VIS
sources, tungsten, mercury vapor, and carbon lamps are used.

Monochromator: It is a device that receives the polychromatic light as input from a lamp and
provides output in the form of monochromatic light. This device is used to disperse the radiations
of polychromatic light according to the wavelength.

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Components of Monochromator: Monochromator consists of three essential components
that are collectively involved to disperse polychromatic light into monochromatic light. These are
entrance slit, dispersing device, and exit slit.
 Entrance Slit It sharply defines the incoming polychromatic beam of light and imparts it
on dispersing element.
 Dispersing Device It is a special plate that contains hundreds of parallel grooved lines.
These lines are used to separate the polychromatic light (white light) into visible light
spectrum. It disperses the polychromatic light that is coming from the entrance slit into its
component wavelengths with the help of focusing lens. Dispersing element, such as prism
or grating is usually made up of quartz, fused silica, or glass.
 Exit Slit allows the minimal wavelength together with the band of wavelengths on either
side. The position of exit slit is not fixed and it is mostly needed to adjust it by rotation to
vary the minimal wavelength passing through the exit slit.

Working of Monochromator: Polychromatic light is collimated and hits the dispersing

element at an angle and splits into its component wavelength by prism or grating after entering
monochromator through the entrance slit. By rotating the dispersing element and exit slit, radiation
of required particular wavelength is obtained from monochromator via exit slit.

Sample Device/Cuvette Cuvettes are available in various forms for UV-VIS region. They
are specifically designed to hold the sample for spectrophotometric analysis. They vary with
respect to shape, size, path length, and transmission characteristics of required wavelength.
Cuvettes are made up of plastic, glass, or optical grade quartz that does not absorb the λ range of
interest. Before to do the analysis, cuvette should be cleaned thoroughly and they should be free
from all types of impurities that may alter the spectroscopic reading.

Detector: It is a device that can be used for the measurement of the amount of light passing
through the sample and convert the light signals (coming from the sample) into the electrical
signals. In order to detect the radiation in UV-VIS spectroscopy, three kinds of photosensitive
devices are used which are as follows:

1. Photovoltaic Cells or Barrier Layer Cell: It contains metallic base plate such as
iron or aluminum that acts as an electrode. Semiconductor, like silicon or germanium, is
deposited on its surface. Over it, thin layer made of gold or silver is present that acts as
second collector tube. When light falls over Se, electrons are generated at Se-Ag surface
and these electrons are collected by the silver. Excess of electrons on silver surface
produces the voltage difference between the silver and base of cell.
2. Phototubes or Photoemissive Tubes:It contains an evacuated glass tube. A light
sensitive cathode is present inside it. Cathode is coated from inner side with light sensitive
material like silver oxide and potassium oxide. When cathode is exposed to radiation, there
is the emission of photoelectrons and they are collected by anode and are returned through
the external circuit. Current is amplified and recorded by this process.

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 3. Photomultiplier Tubes: It is the most commonly used type of detector. It contains a
photoemissive cathode. It contains numerous dynodes (emit numerous electrons in
response to each electron hitting them) and an anode. A photon strikes to the cathode after
entering the tube and it causes emission of numerous electrons that accelerate to the first
dynode. First dynode is 90 V more positive as compared to that of cathode, several
electrons emitted for each incident electron are further accelerated and more electrons are
produced by this process, eventually electrons are collected at anode. By this way, each
original photon produces 106–107 electrons, resulting current is amplified and recorded.

Recorder: Spectrometer provides the signals to the recorder and intensity of signals depends
upon light absorption by the analyte at a particular wavelength. These signals are amplified by
amplifier and elaborated by the personal computer. Digital read-out 3.8 Components of UV-VIS
Spectrophotometer 37 devices like light emitting diode or liquid crystal display (LCD) are used
for clarity purpose that removes the ambiguity.

Working:
 The solvent used to dissolve the sample are the ethanol, methanol, n-hexane and distilled
water. For recording the UV-visible spectrum the sample is dissolved in a solvent, which
itself don’t absorb light in that’s range.
 A quartz cell made up of special glass of 1cm path length is used as a container for the
sample solution (sample holder).
 The solution is exposed to UV-Visible light by the prism selector. The prism selector is
rotating continuously to emit lights of different wavelength.
a. Hydrogen lamp is used for emitting UV light.
b. Tungsten lamp is used for emitting visible light.
The instrument provides a graph between wavelengths of radiations absorbed and the intensity of
absorption. The wavelength corresponding to top of peak shows the maximum absorption. The
wavelength is denoted by λ and maximum absorption is denoted by λmax . The intensity of
absorption corresponding to the wavelength is called ‘Molar extinction constant,’ and it is denoted
by epsilon or Ɛ.

Types of UV-VIS Spectrophotometer:

Design of spectrophotometer is based on the fundamental principle of light absorption by
absorbing species. Based on the number of cuvettes and beams used, UV-VIS spectrophotometer
is classified into the following two types:
Single-Beam UV-VIS Spectrophotometer:
In this type of spectrophotometer, single beam and/or cuvette is used for the analysis (Fig. 3.8). In
single-beam UV-VIS spectrophotometer, a single light beam passes across the cuvette. The
spectrophotometer is calibrated by putting the reference and/or standard solution in the cuvette and
then the resulting value of absorbance is subtracted from sample measurements in order to remove

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the effects produced by the solvent and cell. This type of spectrophotometer is appropriate for
applications in the wavelength range of 190–1100 nm. Various types of samples including nucleic
acid, proteins, and a number of organic molecules are often analyzed in this region. Whereas, the
visible region of the electromagnetic spectrum is from 340 to 750 nm, and this region is appropriate
for the analysis of colored samples.
Double-Beam UV-VIS Spectrophotometer:
In this type of spectrophotometer, two beams and/or cuvettes are used for the analysis (Fig. 3.9).
The light beam coming from the source of light is split into the sample and reference beam with
the help of mechanical chopper. The role of reference beam is to monitor the intensity of light
energy while the sample beam shows absorption of light from the sample. The value of observed
absorbance which is the ratio of the sample and reference beam is recombined before it enters into
the monochromator. This arrangement compensates the effects of light reference and sample beam
due to drift in lamp intensity, electronic and mechanical fluctuations which affect the reference
and sample beams equally.
Although, double-beam UV-VIS spectrophotometers are bit complicated as compared to that
of single beam, still they have some advantages which are as follows:
 The sample and reference run simultaneously. There is no need to adjust zero at each
wavelength or replace the blank with sample.
 The ratio of the powers of reference and sample is constantly obtained.
 The scanning speed is rapid over a wide range of wavelength and digital read-out device is
used to record the electrical signals.

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 Basic Concepts about UV-Visible spectrophotometer
TRANSMITTANCE: Proportion of the radiant power “I” transmitted by a sample to the radiant
power “I o” incident upon the sample OR The fraction of incident EMR transmitted through a
sample.
T= I / IO
ABSORBANCE: The fraction of incident EMR absorbed by a sample. Negative logarithm to the
base-10, of transmittance.
A= log(IO / I) or A= log(1 / T)
ABSORPTIVITY (a): Absorbance of sample of concentration (g/L) in 1 cm cell is called
absorptivity or extinction coefficient. The values of absorptivity vary with the wavelength of
incident energy but at specified wavelength, its value is constant.
MOLAR ABSORPTIVITY (ε): Absorbance of sample of molar concentration (moles of solute
per liter of the solution) in 1 cm cell is called molar absorptivity or extinction coefficient.

The Intensity of absorption:

Beer’s Lambert’s Law:

History:
 In 1760, Johann Heinrich Lambert cited Bouguer's work and stated that the loss of light
intensity when it propagates in a medium is directly proportional to path length.
 In 1852, August Beer discovered another attenuation relation according to which the
loss of light intensity when it propagates in a medium is directly proportional to
concentration.
The modern derivation of the Beer–Lambert law combines the two laws and correlates the
absorbance to both the concentrations of the attenuating species and the thickness of the
material sample.

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 Beer’s Law:
When a beam of monochromatic light is passed through a substance dissolved in a non-
absorbing medium the absorption of light is directly proportional to molar concentration of the
substances.
Mathematically;
𝑙𝑜𝑔10 𝐼𝑜/ 𝐼 ∝ C

Lambart’s Law:
When a beam of light is passed through substance the absorption of light is directly
proportional to the path length of the substance.
𝑙𝑜𝑔10 𝐼𝑜/𝐼 ∝ I
The two laws are combined to obtain the absorption of light by a substance therefore;
𝑙𝑜𝑔10 𝐼𝑜/ 𝐼 ∝ C . I
𝑙𝑜𝑔10 𝐼𝑜 /𝐼 = Ɛ𝐶𝐼
Where;
 Io stands for intensity of incident light
 I stands for transmitted light
 C stands for ‘concentration of substance in centimeter
 Ɛ stands for proportionality constant known as molar extinction constant.
Expression of absorbance is;
𝑙𝑜𝑔10 𝐼𝑜/ 𝐼 = A (Absorbance)
So, final equation can be written as;
𝐴 = Ɛ𝐶𝐼

Deviations from Beer-Lambert Law:

The relationship between concentration and absorbance is not always linear (as observed
during experimentation) and this is because of following factors that cause deviations from
Beer-Lambert law;
 High concentration of absorbing species
 Light scattering due to particulate matter
 Fluorescence or phosphorescence of sample
 Change in refractive index

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  Non-monochromatic radiation
 Stray light

Witt Theory:
German scientist O.M. Witt in 1876 suggested the chromophore auxochrome theory for colored
organic compounds. The various terms used in this are as follows:

Chromophore:
Chromophore is derived from the Greek word “Chromophorus” which mean the color carrier and
it can be defined as “the part of a molecule that is covalently bonded with unsaturated group
responsible for imparting the color after absorbing the light in UV or VIS region” is called as
chromosphere, or it can also be defined as “the functional group containing multiple bonds capable
of absorbing radiations above 200 nm.”
For example, in nitro compounds, the yellow color is produced due to presence of NO 2 group and
hence NO2 is considered as chromophore. Typical examples of chromophore are

Auxochrome:
There are certain functional groups, such as –SH, –NH2, – OH, and halogens, which are not
chromophore themselves (do not show absorption at wavelength higher than 200 nm), once they
are attached to a chromophore, they enhance the absorption of chromophore to shift towards a
longer wavelength, accompanied by an increased intensity. Such functional groups are known as
auxochromes. Auxochromes when attached to a particular chromophore, they alter the ability of a
chromophore to absorb light and alter the intensity of light absorption.

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Auxochrome in fact is a color enhancing group that contains non-bonding (n) electrons that do not
absorb EMR themselves in near UV region, but when they attached to a chromophore, alter the
wavelength and intensity of absorption of that chromophore.

Absorption Maximum: The wavelength at which a substance shows maximum absorbance

is called absorption maximum or lambda max.

Absorption Shifts:
 Bathochromic shift (Red shift): When λmax of a compound shifts to longer
wavelength, it is known as bathochromic shift or red shift.
Reason:
 Presence of an auxochrome group (–OH, -OCH)
 Increased conjugation of system
 Change of solvent
 Complexation

 Hypsochromic shift (Blue shift): When λmax of a compound shifts to shorter

wavelength, it is known as hypsochromic shift or blue shift.
Reasons:
 Adding a group in molecule that causes removal of conjugation
 Change of solvent

Intensity Shifts:
 Hypochromic effect: When absorption intensity (ε) of a compound is decreased, it is
known as hypochromism.
 Hyperchromic effect: When absorption intensity (ε) of a compound is increased, it is
known as Hyperchromism

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Electronic Transitions:
Electrons in an organic molecule are involved in bonding as strong (σ bond), weak (π bond) or
present as non-bonding lone pair. Based on the nature of the bond present in organic molecule,
absorption of electronic transitions that are associated with the absorption of UV-VIS radiations
are of following four types

 σ to σ* transition:
σ electron from outermost orbital is excited to corresponding anti-bonding orbital σ*. For
such transitions, a huge amount of energy is required.
Examples:
Hydrocarbons e.g., Methane (CH4) has C-H bond only and can undergo σ→ σ* transition
and shows absorbance maxima at 125 nm. Measurement in vacuum UV region
 π to π* transition:
π electron in a bonding orbital is excited to corresponding anti-bonding orbital π*.
Compounds containing multiple bonds like alkenes, alkynes, carbonyl, nitriles, aromatic
compounds etc. undergo π → π* transitions.
Examples:
Unsaturated hydrocarbons e.g., butadiene generally absorb in the region 217nm.

 n to σ* transition:
Saturated compounds containing atoms with lone pair of electrons like O, N, S and
halogens are capable of n → σ* transition. These transitions usually require less energy
than σ → σ* transitions.
Examples:
Alkyl halides, ethers, amines, alcohols etc. Measurement in vacuum UV region
 n to π* transition:
An electron from non-bonding orbital is promoted to anti-bonding π*=orbital.
Compounds containing double bond involving hetero atoms (C=O, C≡N, N=O) undergo such
transitions.

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 Examples: Aldehydes and ketones n → π* transitions require minimum energy and show
absorption at longer wavelength around 300 nm

Spectrophotometric Determination of Absorbance of Known Solution:

Suppose we want to determine the amount of substances in the solution by spectrophotometric
method than the procedure will be as fallows;
1. Place the solution in a quartz cell.
2. Now put the cell in a cell holder and move it into a position so that the solution comes in a light
path.
3. Go on changing the wave length or the incident radiation and note the absorbance for each value
of the wave length.
4. Now plot a graph between absorbance and wavelength and find out the value of λmax
5. This wavelength is used in the measurement of absorbance throughout the experiment.
6. Prepare a series of different concentration of solution that is for known concentration measure
the absorbance of each known concentration at that wavelength as mentioned in step 2.
7. Plot a graph between different values of absorbance and concentration. This is called a
calibration graph or standard curve.

Applications of UV-Visible Spectroscopy:

1. Detection of conjugation: With the help of UV-visible spectroscopy figures out the presence
of double and triple bond in a compound. The conjugation can be C=O, C=C, C≡C. the absorption
of that compound and by the observation of λmax values, we can also predict the location of
substituents.
2. Detection of functional group: It is possible to detect certain functional group with the help of
UVVisible spectroscopy. Absence of absorption above 200nm is indication of the absence of the
conjugation.

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3. Detection of geometrical isomerism (Cis/Trans): When compounds shows geometrical
isomerism. The Trans isomer shows the absorption at higher wavelength with larger values of
extinction co-efficient. That is absorption as compared to Cis-isomers.
4. Qualitative analysis: (Identification of unknown compounds) The detection of unknown
compounds is carried out by making the comparison of unknown spectrum with the known
spectrum.
5. By using this technique the structures of some vitamins and organic compounds can be
identified.
6. By the use of this technique the no. of chromophores such as aldehyde, isolated double bonds
can be identified.
7. By this technique we can also perform conformational analysis and we can also determine the
rate of reaction.
8. By this technique we can also determine the quantitative analysis of drug having even low
concentration in the sample. The examples of the drugs are paracetamol, vitamin B2 (riboflavin),
Vitamin C (ascorbic acid, Vitamin B12 cyanocobalamine.
9. We can also determine the concentration of the known compound if it follows Beer’s Lambart’s
law.
10. We can also measure the kinetic measurement.

Advantages:
1. Analysis is quick.
2. Sample analysis is easy.
3. Absorption spectrum provides valuable information regarding the presence of analyte in sample.

Disadvantages
1. Lack of sensitivity and selectivity.
2. Limited to UV/VIS absorbing compounds.
3. Need spectrophotometer capable of reading in the UV-VIS region.
4. Samples should be in solution form.
5. Mixture of substances poses difficulty to analyze and requires prior separation.
6. Interferences from sample mixture make the measurement difficult.

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7. Samples should be in solution. Mixture of substances poses difficulty to analyze and requires
prior separation.
8. Interference from the sample’s matrix makes the measurement difficult.

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PREPARED BY: MUHAMMAD AWAIS

SPONSORSHIP & COMPANY BY: MUHAMMAD IMRAN
QUAID-E-AZAM COLLEGE OF PHARMACY SAHIWAL
SESSION 2021-2026

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 IR SPECTROSCOPY
Introduction:
 It is the measurement of different IR frequencies absorbed by a sample positioned in the path
of an IR beam  This technique is based upon the fact that the molecule of a chemical substance
shows selective absorption in IR region. After absorption of IR radiation, a molecule of a
chemical substance vibrates at various rates and those vibrations result in absorption bands
which are called as absorption spectrum.

Specifications:
 Complete identification of drug is not possible.  It determines the presence of groups and
functional groups.  This technique is also called as fingerprinting of molecule.  It deals with
the region of electromagnetic spectrum that lies in the infrared region (frequency range 500cm-
1 – 670cm-1 ).  It doesn’t tell about no. of groups.

Principle:
Electromagnetic radiation ranging between 400 cm-1 and 4000 cm-1 (2500 and 20000 nm) is
passed through a sample and is absorbed by the bonds of the molecules in the sample causing
them to stretch or bend. The wavelength of the radiation absorbed is characteristic of the bond
absorbing it. IR Radiations: Electromagnetic radiations that are lower in energy than visible
radiations are called infrared radiations. Absorption in IR region is due to the rotational and
vibrational levels. When radiations of frequency range less than 100 cm-1 are absorbed,
molecular rotation takes place in the substance. Therefore, on the bases of molecular rotation,
the vibrational spectrum appears as vibrational-rotational bands.

Regions of IR:
There are three main regions of IR. Absorption in IR region is mainly due to the changes in the
rotational and vibrational levels of molecules.

Modes of Vibration:
There are two types of vibrational modes.
i. Stretching vibration
ii. Bending vibration

1. Stretching Vibration:
In this type of vibration, that occurs along the bond axis where the distance between the two
atoms increases or decreases but the atoms remain at the same bond axis.

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 There are two types of stretching vibration.
 Symmetric stretching: in this type of the moment of atoms with respect to the particular
atoms with respect to particular atom in the same molecule is in the same direction.
 Asymmetric stretching: in case of asymmetric stretching one atom approaches the central
atom while the other departs from it.

2. Bending Vibration: In this type of vibration, the position of atoms changes with
respect to the original bond axis. We can say that stretching absorption of the bond appears
at higher frequencies, (high energy) as compared to the bending absorption of the same
bond.
There are four types of bending vibrations:
 Scissoring: o When to atoms joined to a central atom move towards and away from each
other like a scissor the deformation is known as Scissoring o In this type two atom
approaches each other.
 Rocking: o Two atoms joining a central atom move back and forth in the plane of molecule
o In this type of the movement of atoms takes place in the same direction.
 Wagging: In this deformation the structural unit moves back and forth out of the plane of
molecule o In this type two atom moves up and below the plane with respect to the central
atom.
 Twisting: o When the structural unit rotates about the bond which joins to the rest of the
molecule the deformation produced known as twisting o In this type one of the atoms
moves up the plane while the other moves down the plane with respect to central atom.

Introduction to IR Spectrometer:
The following figure gives the schematic representation or block diagram for the use of
spectrophotometer.

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 In this spectrometer the source of radiant energy is “Nernst glower.” It is consisting of
Zirconium and Yttrium oxides in the shape of tube which is electrically heated to 1500oC to
2000oC because IR rays are not transmitted by glass.
A prism made of salt (NaCl) is used as a monochromator. The radiations from the Nernst
glower are polychromatic. When these are passed through the salt prism, the different
wavelength got separated a slit is placed in the path of radiation emerging from the prism so
that only radiant energy of the desired wavelength passed through and falls onto the solution
under examination.
The radiant energy transmitted by the solution is then allowed to fall on a detector for
measuring the intensity of transmitted infrared radiations.
Thus, IR spectrometer can give the absorption spectrum of a substance. Therefore, by
analysis the spectrum and the information regarding the structure of the substance can be
obtained.
Determination of IR Spectrum of a Substance:
KBr method:
1. Take the 100mg of the dried KBr in a clean pestle and mortar and grind it thoroughly with
1mg of sample.
2. Now carefully place the sample mixture into processing chamber of the mold (dye). In such
a manner that it is held between the polished surface of the bottom and top processing dyes.
3. Subsequently, attach the chamber to the vacuum line and switch on the vacuum pump.
4. Initially applied a slight negative pressure to as to compact the powder and then gradually
increasing the pressure less than 15mm Hg for 30 seconds. Finally enhance the pressing force
to 10,000 pound/ inch2 pressure for 1 to 2 minutes.
5. Now release the pressure and remove the dyes and take out the disc from the mold and kept
it into a position onto the sample holder to study the various spectrum of organic compounds.

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 Sampling of Liquid:
 Liquid sample cells can be sandwiched using liquid sample cells of highly purified alkali
halides, normally NaCl. & KBr can also be used.  Aqueous solvents cannot be used because
they cannot dissolve alkali halides. Organic solvents like chloroform can be used.  The For
most liquids, the sample cell thickness is 0.01-0.05 mm. sample thickness should be selected
so that the transmittance lies between 15- 20%.  For most liquids, the sample cell thickness is
0.01-0.05 mm.  Some salt plates are highly soluble in water, so the sample and washing
reagents must be anhydrous

Sampling of Gases:
 The sample cell is made up of NaCl, KBr etc.  It is similar to the liquid sample cell.  A
sample cell with a long path length (5 – 10 cm) is needed because the gases show relatively
weak absorbance.  Gas analysis are not commonly used because of lack of sensitivity.

Instrumentation of IR Spectrophotometer:
The IR spectrophotometer is based on either single monochromator or double monochromator.
The important features of an IR spectrometer are as follows.
1. IR source
2. Monochromator
3. Detector
4. Mode of operation
1. IR SOURCE:
Instruments for measuring infrared absorption all require a source of continuous infrared radiation
and a sensitive infrared transducer, or detector. Infrared sources consist of an inert solid that is
electrically heated to a temperature between 1,500 and 2,200 K. The heated material will then emit
infra-red radiation.
i. The Nernst Glower: It is a cylinder (1-3 mm diameter approximately 20mm long).
The Nernst glower is constructed of rare earth oxides (ZrO2, Y2O3) in the form of a

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 hollow cylinder. Platinum leads at the ends of the cylinder permit the passage of
electricity. Nernst glowers are fragile. They have a large negative temperature
coefficient of electrical resistance and must be preheated to be conductive. Its lifetime
depends on the operating temperature and the care taken in handling it.
ii. The Globar Source: A globar is a rod of silicon carbide (5 mm diameter, 50 mm long)
which is electrically heated to about 1,500 K. Water cooling of the electrical contacts
is needed to prevent arcing. The spectral output is comparable with the Nernst glower,
except at short wavelengths (less than 5 mm) where its output becomes larger. It is less
convenient to use, more expensive & less intense than Nernst Glower.
3. Monochromator:
Two types of substances are normally used as monochromator:
i. Prism
ii. Grating
 Used as dispersive element  Constructed of various metal halide salts  Sodium chloride
is most commonly prism salt used  These salts are subjected to mechanical & thermal
instability or water solubility  Protection against damage must be continuously exercised
 Metal Halide prisms: various metal halide prisms such as Pot. Bromide KBr and Lithium
Fluoride LiF have been used.
 NaCl prism: Sod. Chloride can be used for the whole of the region from 4000cm -1 –
650cm-1 .
Grating:
 Gratings are nothing but rulings made on some materials like glass, quartz or alkyl halides
depending upon the instrument. The mechanism is that diffraction produces reinforcement.

3. Detectors:
Thermal Detectors: Includes thermocouples, thermistors and pneumatic devices (Golay detectors).
They measure the heating effect produced by the infrared radiation.
A variety of physical property changes are quantitatively determined.
 Expansion of non-absorbed gas (Golay detector)
 Electrical resistance (Bolometer)
 Voltage at junction of dissimilar metals (Thermocouples)

 Bolometer: is an excellent detector for measuring IR radiation. It gives electrical signal

as a result of the change in resistance of metallic conductor with temperature.
 Thermocouples: consist of a pair of junctions of different metals; for example, two
pieces of bismuth fuse to either end of a piece of antimony. The potential difference

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 (voltage) between the junctions changes according to the difference in temperature between
the junctions.
 Golay cells: This detector utilizes the expansion of the gas as the basis for detection. It
is mostly used in commercial spectrophotometers. It consists of a small metal cylinder
closed by a rigid blackened metal plate at one end and a flexible silver diaphragm at the
other end. The whole chamber is filled with xenon gas. IR radiation passes through a
radiation transmitting window. Heat conducted causes the gas to expand and causes the
flexible metal diaphragm to deform. The light from lamp is made to fall on the metallized
diaphragm which reflects the light on the photocell. Motion of diaphragm changes the
output of cell. The signal seen by the phototube is modulated in accordance with the power
of the radiant beam incident on the gas cell. Thermal detectors provide a linear response
over a wide range of frequencies but exhibits slower response times and lower sensitivities
than photon detectors.
 Pyroelectric detectors: are made from a single crystalline wafer or a pyroelectric
material, such as triglyceride sulphate

Schema c diagram:

Interpretation of IR:
IR spectrum is divided into two parts;
 Functional group region: In case of functional group region the range is 4000 cm -1 – 1600
cm-1 .

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 Fingerprint region: In case of fingerprint region the range is 1600 cm-1– 625 cm-1. As, the
recent approach is to examine the functional group region because most the compounds have
only few strong bonds due to characteristic stretching vibrations of their functional group.
Example:
 The functional group such as C-H, O-H, and C-N absorbs in the region of 3700 cm-1 – 2500
cm-1 due to their stretching vibrations.  The absorption due to triple bond C≡C, C≡N occurs in
the region of 2300 cm-1 – 2100 cm-1 .  The absorption due to double bond that is C=C, C=O
occurs in the region of 1900 cm-1 – 1600 cm-1 . The confirmation of functional group should also
be checked in the other region of spectrums as well.
Limitations:
 Rarely used as a quantitative technique because of relative difficulty in sample preparation and
the complexity of spectra.
 Usually, can only detect gross impurities in samples.
 Sample preparation requires a degree of skill, particularly when potassium bromide (KBr) discs
are being prepared.
 The technique is lacking in robustness since sample handling can have an effect on the
spectrum obtained and thus care has to be taken in sample processing.

Applications of IR Spectrophotometer
The important applications of IR spectroscopy are discussed below;
Qualitative analysis and quantitative analysis of products
Quantitative analysis: By using IR spectrum of the sample of drug and by making the
comparison of IR spectrum of standard drug we can also determine the percentage purity of the
drugs.
Hydrogen bonding: By using IR spectroscopy, we can determine the H-bonding of the organic
molecules highly electronegative atoms such as Nitrogen, Oxygen and Fluorine are involved in
strong H-bond formation.
Purity of sample: Generally, the pure sample shows fairly sharp spectrum whereas the material
of high molecular weight generally shows poor spectrum because of the presence of several
kinds of functional groups.
Chromatographic separation studies: The process of chromatographic separation can readily
be monitored by taking the spetra of selected fractions.
Determination of aromaticity: By taking the IR spectrum of different organic aromatic drugs
we can study the relative proportion of saturated and unsaturated rings present in hydrocarbons.

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 Tautomerism: Tautomeric equilibria can be studied with the help of IR spectroscopy. Most
common system such as keto-enol and Lactum to Lactum contain a group such as C=O, OH,
NH2, C=S group which shows characteristic frequency which make it possible for identification
of particular drug.
Identification of different groups and bonds: By taking the IR spectrum of different functional
groups we can identify on the bases of spectrum having their specific frequency range.

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