Colorimetry, spectrophotometry and nephelometry.pptx

Colorimetry,
spectrophotometry and
nephelometry
Dr Sole padmasree
2nd year post graduate
MD Biochemistry

colorimeter
It is an analytical instrument used for measuring the absorbance of light
passing through a coloured solution based on the intensity of colour
developed when a specific reagent is added to it.

colorimetry
• It is a scientific technique that is used to determine the concentration of the
coloured compounds in solutions by the application of Beers - Lamberts
law

INTRODUCTION
The body fluids such as blood , urine , CSF etc contains several organic
and inorganic substances.
These substances will be biochemically equilibrium to their rates of
production and degradation.
This equilibrium is disturbed with many of the diseased conditions . The
detection of biochemical abnormality through tissue sample is not
possible

Cont. ….
And thus it is easy to access the concentration of biochemical
substances through body fluids colorimetrically.
In colorimetric determinations , a specific reagent reacts with a specific
component to form a coloured substance.
The concentration of this coloured substance is measured by colorimeter
based on BEER’S – LAMBERT’S law

Relationship between transmittance and absorbance:
When an incident light beam with intensity I0 passes through a square cell
containing a solution of a compound that absorbs light of specific
wavelength given that the intensity of the transmitted beam is Is , the
transmittance is defined as
 T =
𝑰𝑺
𝑰𝑶

Some of the incident light may be reflected by the surface of the cell or
absorbed by the cell wall.
These factors are eliminated by using a reference cell identical to the
sample cell , expect the compound of interest is omitted from the solvent
in the reference cell.
The transmittance through the reference cell is IR divided by Io .

The transmittance for the compound in the solution is then defined as
𝐼𝑆
𝐼𝑅
The reference sample which is actually the blank is inserted in colorimetry
and the instrument is adjusted to an arbitrary scale reading of 100 i.e 100%
transmittance.
Now the amount of light absorbed as the incident light passes through the
sample is equivalent to
 A = - log
𝑰𝑺
𝑰𝑹
= - Log T

1. What is absorbance?
The amount of light absorbed as incident light passes through a sample
, which is equal to log 1/T , where T is the transmittance.
A = Log
𝟏
𝑻
or - Log T

2. What is absorptivity?
A proportionality constant for a compound that is the measure of the
absorption of radiant energy at a given wave length as it passes
through a solution of that compound at a concentration of 1g/L.
Expressed mathematically as absorbance divides by the product of the
concentration of a substance in g/L and the sample path length in cm.
a =
𝑨
𝒃𝒄

BEER’S – LAMBERT’S law
BEER’S LAW : A mathematical equation stating that the concentration
of substance is α to the amount of light absorbed by the coloured
solution expressed mathematically as
A= abc
A is the absorbance , a=proportionality constant or absorptivity b= light
path in cm and c= concentration of absorbing compound.

Because of the inverse relationship between absorbance and
transmittance , the concentration is also inversely proportional to the
log of transmitted light.
Absorbance values have no units as absorbance is calculated from the
ratio of the intensity of the light transmitted through sample to intensity
of light transmitted through blank.

‘A’ have no units hence , the units for a are reciprocal to b and c , b is 1
cm and c is moles per lit , the symbol ε is substituted for a.
The value of ε is constant for a given compound at a given wavelength
under conditions of temp , pH etc.
Molar absorptivity is a proportionality constant for a compound that is
the measure of the absorption of radiant energy at a given wave length
as it passes through a solution of that compound at a given
concentration of 1 mol / lit. ε=
𝐴
𝑏𝑐

Deviation from BEER’S law
The graph of absorbance versus concentration shows
the intercept at zero and a linear plot.
 Deviations from Beer’s law may be caused by
changes in instrument functions or chemical reactions.

 Deviations become apparent at higher concentration (i.e.,
higher absorbance).
changes in chemical or physical equilibrium involving the
absorbing species. Changes in solution pH, ionic strength,
and temperature can result in such deviations

LAMBERT’S law states that the amount of light absorbed by the
coloured substance depends on the length of the column or the depth of
liquid in which light is passed.
 The length of the column through which light is passing will be always
kept constant by using test tubes / cuvettes of same diameter for both
test and standard.

The concentration of the coloured complex is directly proportional to the
concentration of component in the specimen.
The depth of the coloured complex is measured by the photometer or
spectrophotometer
The photometric readings are compared with a known primary standard.

Photometry involves the measurement of light transmitting power of a solution in
order to determine the concentration of light absorbing material present .
According to beer’s lambert’s law , the absorbance of a solution containing light
absorbing material depends on the following factors:
• The nature of the substance
• The wavelength of the light
• Path of light
• The amount of coloured material in the light path

Colorimetric procedures are limited to visible portion of spectrum
(380-760 nm)
Spectrophotometric procedures involves use of ultraviolet ( >380
nm) visible and IR portion of spectrum.
 Analytical procedures based upon the direct measure of colour
intensity in terms of light absorption at specific wavelengths are
known as photometric procedures and instrument used is the
photometer.

Transmittance : the intensity of a light beam that passes through a cell
containing a solution of a compound that absorbs light at a
particular wave length, divided by the intensity of an incident light beam.
T =
𝐼
𝐼𝑂

Nominal wavelength represents the wavelength in nanometers at peak
transmittance.
The maximum percent emission at nominal wavelength is peak
transmittance.
Spectral bandwidth is the range of wavelengths above one-half peak
transmittance.

According to beer’s law A α concentration
According to lambert’s law A α path length of light ( L)
A=KCL
Absorbance = -Log T
-Log T = KCL
So OD = KCL since – log T = OD
Since K and L is constant OD α C

OD T α CT and OD std α C std
So OD T / OD Std = CT /C Std
Hence CT=
𝒐𝒅𝒕
𝒐𝒅𝒔
× 𝒄𝒔𝒕𝒅
This formula is used to determine the concentration of the analyte
present in the solution.
C std is the standard which is known concentration .

Since – Log T = OD
OD = log 1/T since T = I/Io
OD = Log Io/I
Since Io or incident light is 100 % Io is 100%
% Transmitted is X
And lets say I = some X

A =log ( 100/X)
A = log 100 – log X
A = log 102 – log X 2log10 – log X since log10=1
A = 2- log X or 2- log %T

Basic components of colorimeter
1. Light sources ( radiant energy sources )
2. Monochromator
3. Cuvette
4. Photo detectors
5. Readout devices.

1. What is light ?
• Light : the energy transmitted via electromagnetic waves that are
characterised by frequency and wave length.
• Light is composed of photons whose energy is inversely proportional to
the wavelength.
• So UV has higher energy than the IR

Light sources:
The function of the light source is to provide incident light of sufficient
intensity for photometric measurements.
A tungsten lamp is used in case of colorimeters where a visible range
of light is required.
It emits continuous spectrum of visible light.

Monochromator
The important characteristics of monochromator are its bandpass width ,
nominal wavelength and peak transmittance.
The usual filters used are a coloured glass or a gelatin containing
suitable dyes. But these offer wide band width and low peak emission.
Now semi transparent silver films and cut off filters are placed on each
side of these films to control incident light , this narrowing the band width
to 8 -17 nm.

Selection of wavelength.
The type of monochromator chosen depends upon the analytical
purpose for which it is to be used.
Narrow band spectrum is required in colorimeter and
spectrophotometer for identifying sharp absorbing peaks.

Lack of agreement to Beer’s law when a part of spectral energy
transmitted by the monochromator is not absorbed by the substance
being measured , most commonly observed with wide band pass
instruments.
An increase in absorbance and improved linearity with concentration are
usually observed with instruments that operate a narrow bandwidths of
light

Cont…
The narrow bandwidth of an absorbing material is defined as “the
bandwidth of the spectral absorbance curve at a point equal to one half of
the maximum absorbance”.
In precise form the spectral bandwidth passing through a solution should
not exceed 10 % of its natural bandwidth.

cuvette
It is a small vessel used to hold a liquid sample to be
Analysed in the light path of a colorimeter.
It may be 1. round 2. square or 3. rectangle.
It is made of 1. glass 2. silica 3. plastic.

Square and rectangle has a plain parallel faces and a light path if
constant length 1.0cm
Ordinary borosilicate glass cuvettes are suitable for measurements in
visible portion of spectrum
Satisfactory cuvette size suitable for the reading of 1ml to 3ml of reaction
mixture.

photodetectors
These devices convert light energy into electrical energy when light
transmitted by a solution falls on them which can be measured by a
galvanometer.
Most common of these devices are the barrier layer calls or photocells.
These cells are composed of an iron back plate coated with a layer of
crystalline selenium or cadmium on the surface. ( front active layer)
The light sensitive material in turn coated with thin transparent film of
gold or copper.

Cont…..
The back side of the metal plate is connected to galvanometer.
Light passing through the transparent films sets up a flow of electrons in
one direction
This develops potential difference between two poles of the cell and
causes the current to flow which is proportional to conc of solution and
detected by galvanometer.

Read out devices:
Electrical energy from a detector is dispayed on a digital read out device.
This provides a numerical display of absorbance or converted values of
concentrations.

Basic steps involved in photometric procedure:
Pipetting of a reagent in three test tubes blank , standard and test
Addition of sample and standard in respective tubes
Incubate them at room temperature
Read the intensities of test and standard at a specific wave length
Calculating the unknown concentration by using beers-lamberts law.

Basic steps in working of photometer :
Light emitted by a light source is focused on a filter through a condenser lens.
Filter transmits a narrow band of light.
When a narrow band of light is passed through a coloured solution the light
absorbed α concentration of coloured solution
The light falls on the photo cells, which converts radiant energy into electrical
energy.
Galvanometer readout device records %T or OD

spectrophotometer
• Spectrophotometer is an analytical instruments used to quantitatively
measure the transmittance of including UV range.
• This instrument uses prism or diffraction grating with a slit is used as
monochromator to transmit single wavelength.
• Spectrophotometers are classified as single beam or double beam.

Single beam spectrophotometer:
With this instrument a beam of light is passed through a monochromator
that isolates a desired range of UV spectrum.
Slits are used to isolate a narrow beam of light and improves its
chromatic purity.
The light next passes through a cuvette where portion of radiant energy
is absorbed depending on nature and concentration of substance
present.

Cont….
Any light not absorbed is transmitted to a detector which converts light energy
into electrical energy that is registered digitally on galvanometer.
A reagent blank or water blank is used to adjust the device to 100% T or zero
absorbance.
In the standard solution with known concentration is taken and reading is
recorded
Finally a reading is noted for the unknown solution and now its concentration is
determined.

Double beam spectrophotometer:
This instrument uses a light beam chopper inserted after the exit slit.
A system of mirrors passes portions of light reflected off the chopper
alternatively through the sample and the reference cuvette onto a
common detector.
The chopped beam approach and the use of one detector compensates
for light source variation and for sensitivity changes in the detector.

comparision
Single beam spectrophotometer Double beam spectrophotometer
In single beam it is not possible to compare sample
and blank together
In double beam its possible to do direct one step
comparison of sample in one path with a standard in
the other path.
In single beam radiant energy wavelength has to be
adjusted every time
In this scanning can be done over a wide wavelength
region.
Working on single beam is tedious and time
consuming
Working in double beam is fast and nontedious

Components in spectrophotometer which are different from
colorimeter:
• LIGHT SOURCE:
Types of light sources used in spectrophotometers include
incandescent lamps and lasers.
Incandescent Lamps:
 The light source for measurements in the visible portion of the
spectrum is usually a tungsten light bulb.
The lifetime of a tungsten filament is greatly increased by the presence
of a low pressure of iodine or bromine vapor within the lamp.

An example is the quartz-halogen lamp, which has a fused-silica
envelope and which provides high-intensity light over a wide spectrum
and for extended operating periods
 A tungsten light source does not supply sufficient radiant energy for
measurements below 320 nm.

 In the UV region of the spectrum, a low-pressure mercury-vapor lamp that
emits a discontinuous or line spectrum is useful for calibration purposes, but is
not practical for absorbance measurements because it can be used only at
certain wavelengths.
 Hydrogen and deuterium lamps provide sources of continuous spectra in the
UV region with some sharp emission lines, as do high-pressure mercury and
xenon arc lamps.
These sources are more commonly used in UV absorption measurements. A
deuterium lamp is more stable and has a longer life than a hydrogen lamp

Cont….
Laser sources from argon , fluoride , nitrogen ,nd YAG (neodymium-doped
yttrium aluminium garnet) , aluminium garnet.
Laser Sources A laser (light amplification by stimulated emission of radiation)
is a device that also is used as a light source in spectrophotometers.
These devices transform light of various frequencies into an extremely
intense, focused, and nearly nondivergent beam of monochromatic light.
 Through selection of different materials, different wavelengths of light emitted
by the laser are obtained

Various types of lasers and wavelengths for which they operate

Spectral isolation:
A system for isolating radiant energy of a desired wave length and
excluding that of other wave lengths is called a monochromator.
Devices used for spectral isolation includes 1.filters and 2. prisms.
A combination of slits and lenses are often inserted before or after the
monochromatic device. To render light rays parallel or to isolate narrow
portion of light beam.

Prisms and diffraction gratings are widely
used as monochromatic in
spectrophotometers.
A prism separates white light into a
continuous spectrum by refraction with
shorter wavelength bents.
The slit is adjusted to pass the desired
wavelength of light into the solution.

Cuvettes:
It is a small vessel used to hold a liquid sample to be analysed in the
light path of a spectrophotometer.
It may be round or square or rectangle made up or glass or plastic or
slilica (quartz)
As spectrophotometers are mainly designed for the UV spectrum of
light below 340 nm Quartz cells are usually preferred.

Cont…
As the UV light is partially absorbed by the glass and hence quartz
cuvettes are used.
Same plastic cells may be used but often present with problems like 1.
tolerance , 2. cleaning , 3. etching by solvents , 5. temperature
deformation.
So plastic cuvettes may be used for disposable single time use
applications.

The advantage of spectrophotometer over the colorimeter is that it is
1000 times more sensitive.
Therefore even minute quantities of the substance in a diluted solution
can be assessed.

Nephelometry is used to measure the
concentrations of large particles (such as
antigen–antibody complexes, prealbumin, and
other serum proteins) .
It detects light that is scattered at various
angles, scattered light yields a small signal that
must be amplified.

Nephelometry is based on the scattering of radiation by particles in
suspension.
When a collimated light beam strikes a particle in suspension, portions
of the light are absorbed, reflected, scattered, and transmitted.
Nephelometry is the measurement of the light scattered by a
particulate solution.

Three types of light scatters occur according to the relative size of the
light wavelength .
If the wavelength (λ) of light is much larger than the diameter (d) of the
particle (d < 0.1λ), the light scatter is symmetrical around the particle.
Minimum light scatter occurs at 90 degrees to the incident beam .

 If the wavelength of light is much smaller than the particle diameter (d > 0.1λ),
then the light scatters forward owing to destructive out of phase back scatter..
If the wavelength of light is approximately the same as the particle size, more
light scatters in the forward direction than in other directions.
A common application of nephelometry is the measurement of antigen–antibody
reactions. Because most antigen– antibody complexes have a diameter of 250–
1500 nm, and the wavelengths used are 320–650 nm, light is scattered forward

A typical nephelometer consists of a light source, a collimator, a
monochromator, a sample cuvette, a stray light trap, and a
photodetector.
 Light scattered by particles is measured at an angle, typically 15–90
degrees to the beam incident on the cuvette.

 Light scattering depends on the wavelength of incident light and
particle size. For macromolecules with size close to or larger than the
light wavelength, measurement of forward light scatter increases the
sensitivity of nephelometry.
Light sources include a mercury-arc lamp, a tungsten–filament lamp, a
light emitting diode, and a laser.

Lasers produce stable, nearly ideal monochromatic light of narrow bandwidth
and emit radiant energy that is coherent, parallel, and polarized.
A laser beam can be maintained as a very slim cylinder only a few micrometers
in cross-section.
 A typical helium–neon laser lamp consists of a helium-pumping electrode
(cathode) and a hollow glass laser core surrounded by a laser plasma tube
(anode).
Both the plasma tube and the core are filled with free helium and neon gases.

The electrical discharge between the cathode and the anode is
confined to the hollow glass core to keep it concentrated for maximum
energy transfer.
Two mirrors are positioned at the ends of the laser tube. One of them is
fully reflective, and the other partially transparent.

When the electrode is charged, the helium atoms are excited to a
higher energy state and then transfer this energy to the neon atoms by
collision
 In turn, the excited neon atoms emit photons.
Photons bounce back and forth between the two end mirrors,
stimulating other atoms to emit additional photons, resulting in an
amplification process.

The amplified light eventually emerges as a laser beam through the
partially transparent mirror.
 With the high intensity monochromatic beam, a substantial increase
in sensitivity has been seen with lasers over conventional light
sources.
 Disadvantages of laser sources include cost, safety and cooling
requirements, and limited availability of wavelengths.

Colorimetry, spectrophotometry and nephelometry.pptx

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Colorimetry, spectrophotometry and nephelometry.pptx