Fluorometer

The Fluorescence Phenomenon

• Let us consider further how photons are emitted by fluorescence. As we have
seen, some of the excess excitation energy that is lost by an electronic relaxation
process is lost by heat, and the remaining energy is lost by the emission of a
photon. It follows that the energy of the emitted photons will be of a lower energy
than the absorbed photons and, since energy is proportional to frequency, the
frequency of the emitted radiation will be lower than that of absorbed radiation. If
the frequency of the emitted radiation is lower than that of the absorbed
radiation, it follows that the fluorescent radiation must be of a longer wavelength
than that of the radiation which was originally absorbed.
 The Block diagram
• The block diagram of a generalized luminescence instrument
is illustrated in next Fig. It consists of
• (1) A source of radiation,
• (2) A collimator or baffle on the absorption side,
• (3) A primary filter or excitation monochromator,
• (4) A sample cell,
• (5) A secondary filter or emission monochromator,
• (6) A baffle on the emission side,
• (7) A photo detector, and
• (8) A data readout device.
 Function of :
1. Baffle: Make the needed geometric confined to
the light beam which is going through the sample.
2. Primary filter: Wavelength selection of the light
that will be absorbed by the atom in the solution.
3. Secondary filter: Wavelength selection of the
largest emitted wavelength.
There are two advantages of flourometery

• A special advantage of fluorometry is its great

specificity. High specificity results from dependence
on two spectra, the excitation and emission spectra.
• Fluorometry offers much greater sensitivity, which
may exceed that of spectrophotometric methods by
as much as four orders of magnitude or even more.
 Disadvantages of flourometery

• The principal disadvantage of fluorometry is

the sensitivity of its determinations to
temperature and pH of the sample
(fluorescence in general is pH-sensitive).
Flourometer categories
 Applications of Fluorescence
• Fluorescence plays a major role in analysis,
particularly in the determination of trace
contaminants in our environment, industries and
bodies because of the high sensitivity and specificity
for applicable compounds, as was mentioned before.
• And also:
– Enzyme assays
– Nucleic acid measurement and detection (gels)
Flame photometer
 Flame photometers differ in three important
ways from conventional spectrophotometers

• First, the power source and the sample-holder are combined in a certain
flame.

• Second, in most applications of flame photometry (flame emission

spectroscopy), the objective is measurement of the sample's emission of
light rather than its absorption of light, as shown in next block diagram of
flame photometer fig(1).

• Third, flame photometers can determine only the concentrations of pure

metals.
 Basic Theory of Operation
• When metal is dissolved in solutions it exists as ions. When
aspirated into a combustion flame the solvent in the aerosol
quickly evaporates leaving a solid residue, which is then
broken down to, form an atomic species.

• The energy of the flame is able to excite the atoms and move
their electrons to a higher energy state by one of two
methods: absorption of additional thermal energy from the
flame (atomic emission) or absorption of radiant energy from
an external source of radiation (atomic absorption).
• When these electrons return to the ground state, they lose
the excitation energy and a discrete wavelength of visible
light is emitted.

• This light wavelength can be isolated from other light

wavelengths by an optical filter and the amount of light
emitted can be detected with a suitable photo detector.

• The amount of light emitted is proportional to the number

of atoms in the flame and hence the number of ions in
solution.

Electrical signal from the photo detector is amplified and

displayed on a digital readout.
 Atomic emission instruments
• Are based on the principle that when an atom in an excited
state returns to the ground state, it emits power at a
characteristic wavelength. The power required to raise the
atoms to an excited state is supplied by the flame. The
amount of radiation emitted at a particular wavelength is
proportional to the concentration of the corresponding atom.
 Atomic absorption instruments
• A source of radiant power at the characteristic wavelength of
the atom to be analyzed is used to produce a beam that is
passed through a flame. The atoms in the flame selectively
absorb power at their characteristic wavelengths as they are
raised to an excited state
 Note that ……..
• One additional feature of atomic absorption flame
photometer is that because the atoms in the flame emit as
well as absorb power at the characteristic wavelength, it is
necessary to be able to differentiate between the two sources
of power reaching the detector.

• A rotating sector disk between the source and the flame is

used to produce pulses of power rather than a steady output.
 Examples of metals
Element Color Wavelength
Li Red 670.8 nm
K Violet 404.7 nm
Na Yellow 589.0 nm
Table(1)-Typical Characteristic Color for Certain Metals Undergoing Flame Photometry
 Steps of flame instrumentation
(1)Pretreatment of the sample
(2)Delivering the analyte to the flame.
(3)Inducing the spectral transitions (absorption or emission) necessary
for the determination of the analyte.
(4)Isolating the spectral lines required for the analysis.
(5)Detecting the increase or decrease in intensity of radiation at the
isolated line(s).
(6)Recording these intensity data.
 1. Pretreatment of Sample
• Atomic absorption spectroscopy (AAS) and flame
emission spectroscopy (FES) require that the analyte
be dissolved in a solution in order to undergo
nebulization (see the next section). The analyst must
be aware of substances that interfere with the
absorption or emission measurement. When these
substances are in the sample, they must be removed
or masked (complexed). Reagents used to dissolve
samples must not contain substances that lead to
interference problems.
 2. Sample Delivery (Nebulization)
• The device that introduces the sample into the flame plays a
major role in determining the accuracy of the analysis. The
most popular sampling method is nebulization of a liquid
sample to provide a steady flow of aerosol into a flame. An
introduction system for liquid samples consists of three
components:
(1) A nebulizer that breaks up the liquid into small droplets,
(2) An aerosol modifier that removes large droplets from the
stream, allowing only droplet smaller than certain size to pass,
and
(3) The flame or atomizer that converts the analyte into free
atoms.
 3. Nebulization
• Nebulization is the technique in the sample solution is introduced through
an orifice into a high velocity gas jet usually the oxidant. The sample
stream may intersect the gas stream in either a parallel or perpendicular
manner. Liquid is drawn through the sample capillary by the pressure
differential generated by the high-velocity gas stream passing over the
sample orifice. The liquid stream begins to oscillate, producing filaments.
Finally, these filaments collapse to form a cloud of droplets in the aerosol
modifier or spray chamber. In the spray chamber the large droplets are
removed from the sample stream or broken up into smaller droplets by
impact beads. The final aerosol, now a fine mist, is carried into the burner.
 4. Atomization

• The atomization step must convert the analyte

within the aerosol into free analyte atoms in
the ground state. Very small sample volumes
(5-100 μL) or solid samples can be handled by
flameless electrothermal methods.
Thank you