INTRODUCTION:

• Atomic Spectroscopy is assumed to be the oldest instrumental method for

the determination of elements. These techniques are introduced in the mid of
19th Century, 2

• Bunsen and Kirchhoff showed that the radiation emitted from the flames
depends on the characteristic element present in the flame.
The potential of atomic spectroscopy in both the qualitative as well as
quantitative analysis were then well established.

It is divided into three types which are

• Absorption Spectroscopy
• Emission Spectroscopy
• Luminescence Spectroscopy

Another branch of Atomic Absorption Spectroscopy is

• Flame Photometry or Flame Atomic Emission Spectrometry (in which the
species is examined in the form of atoms)
• Atomic Absorption Spectrophotometry (AAS)
• Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP- AES)

INTRODUCTION:
• 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.
• Flame Photometry is based on measurement of intensity of the light
emitted when a metal is introduced into flame.
– The wavelength of colour tells what the element is (qualitative)
– The colour's intensity tells us how much of the element present
(quantitative)
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  subjected to hot flame  specific quantum of thermal
energy absorbed by orbital electrons  become unstable at high energy level
 release energy as photons of particular wavelength  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.
BASIC CONCEPT:
• Liquid sample contaning metal salt solution is introduced into a flame
• Solvent is first vaporized, leaving particles of solid salt which is then
vaporised into gaseous state
• Gaseous molecule dissociate 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 • 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.

Principle
• When a solution is aspirated into a low temperature flame, in an aerosol,
each droplet of water evaporates leaving a solid core of the residue of
evaporation.
• The core further breaks down to the molecular level, and provided the
molecules are not too refractory, progress to form atomic species.

. • The atom then is excited by the flame and its electron temporarily moves to
a higher energy state.
• When the electrons return to the ground state, they lose the excitation
energy and a discrete wavelength of visible light is emitted. 14
. • The emitted light can be isolated from other light wavelengths by an
optical filter.
• The amount of light being emitted is proportional to the number of
atoms in the flame, and it follows, the conc. of that atom in the original
solution.
• The amount of light emitted can be measured by a suitable photo detector.
• A photo detector generates an electrical signal which is amplified and
displayed on a digital readout.
• Various metals emit a characteristic colour of light when heated:
Element Emission wavelength Flame colour
(nm)
Sodium (Na) 589 Yellow
Potassium (K) 766 Violet
Barium(Ba) 554 Lime green
Calcium (Ca) 662 Orange
Lithium (Li) 670 Red
Structure of Flame: As seen in the figure, the flame may be divided into the
following regions or zones.
– Preheating zones
– Primary reaction zone or inner zone
– Internal zone
– Secondary reaction zone
Requirements of flame
It should have proper temperature
• Temperature should remain constant throughout the operation
• There should not be any fluctuation during burning 19
• Preheating zone- In this, combustion mixture is heated to the ignition
temperature by thermal conduction from the primary reaction zone.

• 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

• 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.
• Secondary reaction zone - In this zone, the products of the combustion
processes are burnt to stable molecular species by the surrounding air.

Functions of flame
 To convert the analyte of the liquid sample into vapour state
 To decompose the analyte into atoms and simple molecules
 To excite the formed atoms/free atoms/simple molecules to emit radiant
energy

sample Delivery System:

There are three components for introducing liquid sample:
• Nebulizer – it breaks up the liquid into small droplets.
– Nebulization the is conversion of a sample to a mist of finely divided
droplets using a jet of compressed gas.
– The flow carries the sample into the atomization region.
– Pneumatic Nebulizers: (most common) •
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

1)BURNERS
BURNERS used in the flame photometry should have following properties :
 The flame should have ability to evaporate the solvent to give a residue.
 The flame should convert this residue into gases state atoms and finally to
individual atoms.
The effect of flame is depends upon the temperature of flame and this
temperature can be monitored by following methods :
 Fuel to air ratio
 Type of solvent for preparing sample solution.
  Amount of solvent which is entering into the flame.
 Type of burner used in flame photometer.

1)MECKER BURNER
This burner was used earlier and employed natural gas and oxygen.
Produces relatively low temp. and low excitation energies.
This are best used for ALKALI metals only.
Now-a-days it is not used.

2)TOTAL CONSUMPTION BURNER

In this the air & fuel are mixed in a chamber which is delivered through nozzle,
where the sample is aspirated.
The main advantage of this burner is the total usage of sample solution.

3)PREMIX OF LAMINAR FLOW BURNER :

In this type of the burner, aspirated sample, fuel and oxidant are thoroughly
mixed before reaching the burner opening and then entering the flame.
There is high loss of sample(95%) as large droplets which are drained out.

4)LUNDERGARTH BURNER :
In this sample & air mixed in chamber,this mixed composition send to fuel
nozzle where it burn.

MIRRORS :
The radiation from the flame is emitted in all the directions in space. Much of
the radiation is lost and loss of signal results.
A mirror is located behind the burner to reflect the radiation back to the
entrance slit of the monochromator .
The reflecting surface of the mirror is front-faced. These mirrors are very easily
scratched when subjected to chemical attack e.g. acid vapors. Great care
should be taken to protect them from corrosive atmosphere.

MONOCHROMATORS:
-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:
– 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.
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.

APPLICATION
• Flame photometer has both quantitative and qualitative applications.
• Flame photometer with mono-chromators emits radiations of characteristic
wavelengths which help to detect the presence of a particular metal in the
sample.
• To estimate sodium, potassium, calcium, lithium etc. level in sample of serum,
urine, CSF and other body fluids.
• Na+ and K+ ions in, muscles and heart can be determined by diluting the
blood serum and aspiration into the flame.

INTERFERENCES:
• In determining the amount of a particular element present, other elements
can also affect the result.
Such interference may be:
• 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 ionisation of some of
the metal atoms, e.g. sodium.
– 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 interferon's and the analyte. Includes:
i. 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.
ii. Cation-cation interference: – These interferences are neither spectral
nor ionic in nature – Eg. aluminum interferes with calcium and
magnesium.

ADVANTAGES
• Simple quantitative analytical test based on the flame analysis.
• Inexpensive.
• The determination of elements such as alkali and alkaline earth metals is
performed easily with most reliable and convenient methods.
• Quite quick, convenient, and selective and sensitive to even parts per million
(ppm) to parts per billion (ppb) range.
Disadvantages
• The concentration of the metal ion in the solution cannot be measured
accurately.
• A standard solution with known molarities is required for determining the
concentration of the ions which will corresponds to the emission spectra.
• It is difficult to obtain the accurate results of ions with higher concentration.
• The information about the molecular structure of the compound present in
the sample solution cannot be determined.
• The elements such as carbon, hydrogen and halides cannot be detected due
to its non- radiating nature.

APPLICATIONS OF FLAME PHOTOMETRY:

QUALITATIVE APPLICATIONS:
 Some of these elements can be detected visually by the color in the flame,
e.g. sodium produces yellow flame.
 The best method is to use flame photometer and when a radiation of the
characteristic wavelength is detected, it will indicate the presence of the
metal in the sample. This is done by peak matching technique.
 Flame photometry is used to determine the concentration of various soil
components.
 For agricultural purposes, an analysis of a proper mixture of surface soil and
subsoil is carried out to determine the fertilizer requirement of the soil.
 In clinical chemistry, it is very important to determine the concentration of
sodium and potassium ions in body fluids since their ratio controls the
action of muscles including the heart.
 This is achieved by diluting the blood serum and aspiration into the flame.
 Food and drinks are analyzed for alkali metals.
QUANTITATIVE APPLICATIONS:
 Fruit juices and alcoholic beverages can be analyzed.
 Flame photometry is used in quantitative estimation of alkali and alkaline
earth metals in Mineral Extraction and Pharmaceuticals.
 It is also used in cement, petroleum industries.

FLUORIMETRY
When the electro magnetic radiation passed through a sample, the molecule
absorb radiation energy and moves from ground state to excited state.
When the excited molecules come back to ground state by they emit a
radiation of longer wavelength (λ) then the incident radiation. This entire
process is called “ LUMINESCENCE ”.
It is of 3 types.
Fluorescence spectroscopy.
Phosphorescence spectroscopy.
Chemiluminescence spectroscopy

Fluorescence:
In this excited molecules comes back to the ground state by emission radiation
from 10 -8 to 10 -2 sec. after the incident radiation is cut off.
Or
When a beam of light is incident on certain substances they emit visible light
or radiations. This is known as fluorescence.
Fluorescence starts immediately after the absorption of light and stops as soon
as the incident light is cut off. The substances showing this phenomenon are
known as flourescent substances.

Phosphorescence: In this excited molecules comes back to the ground state

by emission of radiation from 10 -2 to 100sec. even after cut off incident
radiation.
Or
When light radiation is incident on certain substances they emit light
continuously even after the incident light is cut off.
This type of delayed fluorescence is called phosphorescence. Substances
showing phosphorescence are phosphorescent substances.

Theory of florescence and Phosphorescence:

1. Single/triplet state:
A molecular electronic state in which all of the electrons are paired are called
singlet state.
In a singlet state molecules are diamagnetic.
Most of the molecules in their ground state are paired.
When such a molecule absorbs uv/visible radiation, one or more of the
paired electron raised to an excited singlet state /excited triplet state.
 2. Electron transition state:
when a molecule in ground singlet state absorb energy it moves from
different vibrational level of ground singlet state to excited singlet state.
In each electronic excited state there are no. of vibrational levels. This
means in excited singlet state the electrons are at different vibrational
levels.
When the electrons move from excited state to ground state following
possible changes occur.
- The electrons in excited state comes back ground state by without
emission of radiation by release of heat. The electrons in the excited
singlet state posses different vibrational level and reach the ground
state.
- The electrons in the higher vibrational level should come back to the
lower vibrational level and this processes takes 10 -8 to 10 -2 sec.
Once it reaches lower vibrational level, immediately comes to the
ground state this process is said to be “ fluorescence ”
INETR SYSTEM CROSING:
When the electrons in the excited state, the S 1 vibrational levels are coupled
with vibrational levels of T 1 .
The electrons in the triplet state take some more time to cross the vibrational
level with in the triplet state to reach the ground state and this process takes
about 10 -2 to 100sec.This phenomena is said to be ”phosphorescence”
FLUORESCENCE AND CHEMICAL STRUCTURE
Fluorescence is most commonly observed in compounds containing aromatic
functional groups with low energy.
Most unsubstituted aromatic hydrocarbons show fluorescence - quantum
efficiency increases with the no: of rings and degree of condensation.
Simple heterocyclic do not exhibit fluorescence.
1. pyridine
2. Furan
3. Thiopene
4. Pyrole

Fusion of heterocyclic nucleus to benzene ring increases fluorescence.

- Quinoline
- Isoquinoline
- Indole
STRUCTURAL RIGIDITY

Fluorescence is favored in molecules with structural rigidity.

 organic chelating agents complexed with metal ion increases
fluorescence.(A chelating agent is a substance whose molecules can form
several bonds to a single metal ion).

Factors affecting fluorescence intensity

Nature of molecule
Nature of substituent
Effect of concentration
Adsorption
Light
Oxygen
pH
Temp . &viscosity
Intensity of incident light
Path length

1.Temperature: it is inversely proportional to luminescence.

Increase in temp. causes increase in collision of molecules and finally increase
in F & P. Decrease in temp. Causes decrease in collision of molecules and
finally decrease in F & P

2.Viscosity : It is directly proportional to luminescence. I

ncrease in viscosity causes decrease in molecule collision and finally increase in
F & P. Decrease in viscosity causes increase in molecules collisions and finally
decrease in F& P.

3. Oxygen: It decrease the fluorescence in 2 ways; Oxidises the fluorescent

substance to non fluorescent substance.  Decrease fluorescence because of
paramagnetic property as it has triplet ground state.

4. pH: its effects depends on chemical structure of molecules.

E.g- Aniline in neutral & alkaline medium gives visible flourescence & in acidic
medium gives fluorescence in uv region only.
Phenols in acidic condition do not give f but in alkaline medium they give
fluorescence.

5. Rigidity in structure: Fluorine has rigid structure and it shows more

fluorescence. Biphenyl has flexible structure and shows less fluorescence
 6. Nature of group: Electron donating groups like amino, hydroxyl etc causes
increase in fluorescence. Electron withdrawing groups like NO2, COOH etc
causes decrease in fluorescence.

QUENCHING:
Decrease in fluorescence intensity due to specific effects of constituents of
the solution.
Due to concentration, pH, pressure of chemical substances, temperature,
viscosity, etc.
Types of quenching
 Self quenching
 Chemical quenching
 Static quenching
 Collision quenching

CHEMICAL QUENCHING
Here decrease in fluorescence intensity due to the factors like change in
pH,presence of oxygen, halides &heavy metals.
pH- aniline at pH 5-13 gives fluorescence but at pH <5 &>13 it does not
exhibit fluorescence.
Halides- like chloride,bromide,iodide & electron withdrawing groups like
NO2,COOH etc. leads to quenching.
Heavy metals -leads to quenching, because of collisions of triplet ground
state.
 STATIC QUENCHING: This occurs due to complex formation. e.g- caffeine
reduces flourescence of riboflavin by complex formation.

COLLISIONAL QUENCHING
It reduces fluorescence by collision. where no. of collisions increased hence
quenching takes place.

Types of flourescence
1. Chemiluminescence (when excited by chemicals)
2. Electrochemiluminescence (when excited by electrochemical reaction)
3. Photoluminescence (when excited by electromagnetic radiation)

Based on the wavelength of emitted radiation

1. Stroke’s flourescence (wavelength of the emitted radiation is longer than the
absorbed radiation)
2. Anti-stroke’s Flourescence (wavelength of emitted radiation is shorter than
absorbed radiation)
3. Resonance Flourescence (wavelength of emitted radiation = absorbed
radiation)

Based on Phenomena:
1. Sensitised flourescence (when elements like Th, Zn, Cd or an alkali metal are
added to mercury vapour these elements are sensitised & thus gives
fluorescence.
2. Thermally assisted Flourescence (the excitation is partly by electromagnetic
radiation and partly by thermal energy)
COMPONENTS OF FLUORIMETERS AND SPECTROFLUORIMETERS
filters and monochromators
sample cells
detectors

SOURCE OF LIGHT
mercury arc lamp.
xenon arc lamp.
tungsten lamp.
tunable dye lasers.

MERCURY ARC LAMP

Produce intense line spectrum above 350nm.
High pressure lamps give lines at 366,405, 436, 546,577,691,734nm.
Low pressure lamps give additional radiation at 254nm.

XENON ARC LAMP

Intense radiation by passage of current through an atmosphere of xenon.
Spectrum is continuous over the range between over 250- 600nm,peak
intensity about 470nm.

TUNGSTEN LAMP
Intensity of the lamp is low.
If excitation is done in the visible region this lamp is used.
It does not offer UV radiation.

TUNABLE DYE LASERS

Pulsed nitrogen laser as the primary source.
Radiation in the range between 360 and 650 nm is produced.

FILTERS AND MONOCHROMATORS

FILTERS:- Primary filter-absorbs visible light & transmits uv light. Secondary
filter-absorbs uv radiations & transmits visible light.

MONOCHROMATORS : Exitation monochromaters-isolates only the radiation

which is absorbed by the molecule.
Emission monochromaters-isolates only the radiation emitted by the molecule.
SAMPLE AND SAMPLE HOOLDER:
The majority of fluorescence assays are carried out in solution.
Cylindrical or rectangular cells fabricated of silica or glass used.
Path length is usually 10mm or 1cm.
All the surfaces of the sample holder are polished in fluorimetry.

DETECTORS
photovoltaic cell
photo tube
photomultiplier tubes – Best and accurate.

INSTRUMENT DESIGNS
single beam fluorimeter double beam fluorimeter
spectrofluorimeter(double beam)

SINGLE BEAM FLUORIMETER

Tungsten lamp as source of light.
The primary filter absorbs visible radiation and transmits uv radiation.
Emitted radiation measured at 90o by secondary filter.
Secondary filter absorbs uv radiation and transmits visible radiation.

DOUBLE BEAM FLUORIMETER

Similar to single beam instrument.
Two incident beams from light source pass through primary filters separately
and fall on either sample or reference solution.
The emitted radiation from sample or reference pass separately through
secondary filter.

SPECTROFLUORIMETER
The primary filter in double beam fluorimeter is replaced by excitation
monochromaters.
The secondary filter is replaced by emission monochromaters.
The incident beam is split into sample and reference beam using a beam
splitter.
The detector is photomultiplier tube.

APPLICATIONS OF FLUORIMETRY
1] Determination of inorganic substances
 Determination of ruthenium ions in presence of other platinum metals.
Determination of aluminum (III) in alloys.
Determination of boron in steel by complex formed with benzoin.

2]Nuclear research
Field determination of uranium salts.
3]Fluorescent indicators
Mainly used in acid-base titration.
e.g.: eosin- colorless-green.
Fluorescein: colourless-green.
Quinine sulphate: blue-violet.
Acridine: green-violet

5] organic analysis
Qualitative and quantitative analysis of organic aromatic compounds present
in cigarette smoke, air pollutants, automobile exhausts etc.

6] pharmaceutical analysis
7] Liquid chromatography
Fluorescence is an imp method of determining compounds as they appear at
the end of chromatogram or capillary electrophoresis column.

8]determination of vitamin B1 &B2.