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Photometric Calibration Procedure

This document provides an overview of photometric calibration procedures. It discusses various methods of determining compound concentrations including colorimetry, which uses a solution's light absorption properties. Colorimeters objectively measure absorbance, unlike earlier methods relying on human vision. Components of colorimeters and spectrophotometers are described, including light sources, monochromators, sample cells, and photodetectors. The document also covers calibration curves, filter selection, Beer's and Lambert's law, atomic absorption spectrophotometry, densitometry, turbidimetry, nephelometry, flame photometry, fluorometry, and chemiluminescence.

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Mustafa Khandgawi
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128 views29 pages

Photometric Calibration Procedure

This document provides an overview of photometric calibration procedures. It discusses various methods of determining compound concentrations including colorimetry, which uses a solution's light absorption properties. Colorimeters objectively measure absorbance, unlike earlier methods relying on human vision. Components of colorimeters and spectrophotometers are described, including light sources, monochromators, sample cells, and photodetectors. The document also covers calibration curves, filter selection, Beer's and Lambert's law, atomic absorption spectrophotometry, densitometry, turbidimetry, nephelometry, flame photometry, fluorometry, and chemiluminescence.

Uploaded by

Mustafa Khandgawi
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
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PHOTOMETRIC

CALIBRATION
PROCEDURE

1. COLORIMETRY &

SPECTROPHOTOMETR

Many biochemical experiments involve the measurements of


compound or group of compounds present in a complex
mixture

The most widely used method for determining the concentration


of biochemical compounds is colorimetry, which makes use of
the property that when white light passes through a colored
solution, some wavelength are absorbed more than others
Many compounds are not themselves colored but can be made to
absorb light in visible region by reaction with suitable reagents
The earliest colorimeters relied on the human eye to match the
color of a solution with that of one of a series of colored discs.
The results obtained were too subjective and not particularly
accurate

THE COLORIMETER

Colorimeter is generally any tool that characterizes


colour samples to provide an objective measure of
colour characteristics
In chemistry, the colorimeter is an apparatus that
allows the absorbance of a solution at a particular
frequency (colour) of visual light to be determined.
Colorimeters hence make it possible to determine the
concentration of a known solute, since it is
proportional to the absorbance

THE COLORIMETER

Different chemical substances absorb varying


frequencies of the visible spectrum
Colorimeters rely on the principle that the
absorbance of a substance is proportional to its
concentration i.e., a more concentrated solution
gives a higher absorbance reading. And this what
Beers states.

LIGHT AFFECTED BY SAMPLE


CONCENTRATION

BEERS AND LAMBERTS LAW

Beers law states that the concentration of a substance is


directly proportional to the amount of light absorbed or
inversely proportional to the logarithm of the transmitted light
A = 2- log %T
A ~C
Lamberts law states that the rate of decrease in intensity with
the thickness of the medium is proportional to the intensity of
light
Beer-Lambert law gives
T= e -kct

Finally gives
Concentration of Test = (absorb of Test/absorb of STD)x
concentration of STD
This law represents the basic for colorimetric and
spectrophotometric measurements

COMPONENTS
Components:
Light source : The most important factors for a
light source are range, spectral distribution
within the range, the source of radiant
production, stability of the radiant energy, and
temperature
Monochromators : Filter in the colorimeter is used
to select the color of light which the solute absorbs
the most, in order to maximize the accuracy of the
experiment. Note that the colour of the absorbed
light is the 'opposite' of the colour of the specimen,
so a blue filter would be appropriate for an orange
substance

COMPONENTS

Sample cell
Photodetectors : could be barrier-layer cell,
phototube, photomultiplier tube, or
photodiode. The light then falls on to a
photocell which generates an electrical
current in direct proportion to the intensity
of light falling on it. This small electrical
signal is increased by the amplifier which
passes to a galvanometer of digital readout
to give absorbance reading directly

CALIBRATION CURVES

Calibration curves used to help in determining


concentrations when for instance analyzing
many samples. This is done by plotting
absorbance at a specific wavelength versus
concentration for standards of known
concentration
Each method reads within a specific range,
below and above this range the reading will
not be convenient ,and sample then must be
treated before measurement. Deviations from
linearity are typically observed at high
absorbances

FILTER SELECTION

The filter chosen is usually complementary


to the solution colour to be measured
Solution colour

Usual filter

Blue
Bluish-green
Purple
Red
Yellow
Yellowish-green

Yellow
Red
Green
Bluish-green
Blue
Violet

FILTER SELECTION

We should measure the absorbance of the


coloured reaction throughout the visible
spectrum and then deciding the wavelength
of filter which gives the highest absorbance
Once the correct wavelength has been
chosen to give maximum absorption, the
selectivity is determined by taking further
readings at various wavelengths with two
different concentrations of the same solution

SCIENCE IS AMAZING

SPECTROPHOTOMETERS

SPECTROPHOTOMETERS

Is a sophisticated type of
colorimeter
where
monochromatic light is provided
by prism or diffraction grating
The band width of the light
passed by a filter is quite board,
so that it may be difficult to
distinguish
between
two
components of closely related
absorption with a colorimeter. A
spectrophotometer
is
then
needed

How to calibrate
colorimeters/spectrophotometers

Think about
(Source of errors in
spectrophotometric
measurements)

ATOMIC ABSORPTION
SPECTROPHOTOMERS

The analyzed sample must contain the reduced metal in the


atomic vaporized state. Commonly, this is done by using the heat
of a flame to break the chemical bonds and form free, unexcited
atoms
The sample, in solution, is aspirated as a spray into a chamber,
where it is mixed with air and fuel. This mixture passes through
baffles, where large drops fall and are drained off. Only fine
droplets reach the flame
Light from the hollow-cathode lamp passes through the sample of
ground-state atoms in the flame. The amount of light absorbed is
proportional to the concentration
When a ground-state atom absorbs light energy, an excited atom is
produced. The excited atom then returns to the ground state,
emitting light of the same energy as it absorbed
Flameless atomic absorption requires an instrument modification
that uses an electric furnace to break chemical bonds
(electrothermal atomization)

Atomic absorption spectrophotometry is


sensitive, accurate, specific and Precise
It is routinely used to measure concentration of
trace metals that are not easily excited
It is generally more sensitive than flame
emission because the vast majority of atoms
produced in the usual propane or airacetylene
flame remain in the ground state available for
light absorption

2. DENSITOMETRY

Densitometry is the quantitative measurement


of optical density in light-sensitive materials,
such as photographic paper or photographic
film, due to exposure to light. Optical density
is a result of the darkness of a developed
picture and can be expressed absolutely as the
number of dark spots (i.e., silver grains in
developed films) in a given area, but usually it
is a relative value, expressed in a scale
The densitometer is used to detect and
differentiate electrophoresis patterns

TURBIDIMETRY

Turbidimetric measurements are made with a


spectrophotometer to determine
concentration of particulate matter in a
sample. The amount of light blocked by a
suspension of particles depends not only on
concentration but also on size

NEPHELOMETRY

Nephelometry is similar, except that light


scattered by the small particles is measured
at an angle to the beam incident on the
sample holder

FLAME PHOTOMETRY

A sample aspirated into a flame, producing


atoms in an excited state, which are capable
of emitting light of a specific wavelength
depending on the element of interest
The intensity of the emitted light can be
correlated to the quantity of ion present in
the sample

FLUOROMETRY

The source emits short-wavelength high-energy


excitation light. A mechanical attenuator controls
light intensity
The primary filter, placed between the radiation
source and the sample, selects the wavelength that
is best absorbed by the solution to be measured
The fluorescing sample in the cuvet emits radiant
energy in all directions
The photodetector must be placed at a 90 angle
from the initial light source. This eliminates any
interference from the initial light source and detects
only fluorescent light
The electrical output of the photodetector is
proportional to the intensity of fluorescent energy

FLUOROMETRY

FLUOROMETRY

Definition of fluorescence :
Certain molecules absorb light and a given
frequency, and then re-emit that light at a
different and longer frequency
Advantages of fluorescence:Very specific and
sensitive
Disadvantages of fluorescence: Few
molecules fluoresce and very susceptible
to pH and
temperature changes

CHEMILUMINESCENCE

A chemical reaction that emits energy in the


form of light. When used in combination with
immunoassay technology, the light produced
by the reaction indicates the amount of
analyte in a sample

THANKS FOR
ATTENTION

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