COLORIMETRY

is an instrument used to measures the absorbance of a particular wavelength of

light by a specific solution.
Used to determine the concentration of colored samples with the help of human
eyes.
used for measuring absorption in the visible region of spectroscopy.

Visible region 380-760 nm or 3800 7600 A
o

400-800 nm or 4000 8000 A
o
.
Used to determine the concentration of an unknown solute in a given solution
by the application of the absorption laws like Beer-Lambart Law.
SPECTROPHOTOMETRY is an instruments used to make the same
measurements.
THEORY OF COLORIMETRY AND SPECTROPHOTOMETRY

When light is incident on an object or sample a part of it is absorbed, a part is
reflected and a part may be transmitted.
Reflected Light



Transmitted light
Absorbed light
Incident Light
Sample
It is clear from the figure that the intensity of the incident light decreases as they
passes through the medium or samples.
If, Io = intensity of incident light, Ia = absorbed light, Ir = reflected light and It =
intensity of transmitted light
Then,
Io = Ia + It + Ir
However,
Ir = reflected light is very very small only about 1-2% and can be eliminated then,
Io = Ia + It
NOTE
Colored samples absorb radiation from the visible region while colorless
samples absorb radiation from the UV region of the EM radiation.
The amount of radiation absorbed by the sample is directly proportional to the
intensity of the colored sample as well as thickness or concentration of the
medium.
ABSORPTION LAWS
There are the laws which govern the absorption of light by the molecules.
These are
Lambert's Law
Beer' s Law
Beer Lambert's Law (combination of Lambert's Law and Beer' s Law)
First of all the absorption of light with the thickness of the medium was
investigated by Bouguer.
But this credit was enjoyed by Lambert, who simply extends the concepts
developed by Bouguer.

LAMBERT'S LAW
When a beam of light or monochromatic radiation is allowed to pass through an
absorbing medium, then the intensity of radiation decreases with thickness of the
medium.
Mathematically, may be stated as
-

------------------- (1)
Or -

= ------------------ (2)
Where,
I = Intensity of the incident light or radiation
t = denotes the thickness or path length of the medium
K = is a constant and is called proportionality constant or absorption coefficient

The term dI/dt denotes the rate of change of intensity with the thickness of the
medium
And ve sign indicates the decrease of intensity with increase the thickness of
the medium.

On integrating the equation (2) then,
It becomes
I = Io e
kt
------------- (3)
BEERS LAW
When a beam of light or monochromatic radiation is allowed to pass through an
absorbing medium, then the intensity of radiation decreases with concentration of
the absorbing medium.
Mathematically, may be stated as

-------------------- (1')
Or

= -------------------- (2')
Where,
I = Intensity of the incident light or radiation,
c = molar concentration of the solution
K = is a constant and is called proportionality constant or absorption coefficient
On integrating the equation (2') then,
It becomes
I = Io e
kc
------------------- (3')
Where, c = conc. of the soln. in moles/liter
BEER LAMBERT'S LAW
Combination of two laws i.e. combination of Beers and Lamberts Law
or we can say that combination of equations (3) and (3),
Then,
I = Io e
kct
-------------------- (4)
This is the fundamental equation of Colorimetry and Spectrophotometry.
Now taking the log of equation (4) then, we get the following equation
Log Io /It = k / 2.303 ct -------------------- (5)
We know log Io / It = Absorbance (A) and k / 2.303 = then,
A = ct -------------------- (6)
Where, A = absorbance
= molar absorptivity or molar extinction coefficient
c = concentration of the medium, t = thickness of the medium
Question
Let's suppose that a sample has molar absorptivity 10000 and absorbance 2 with a 1 cm cell
then calculate the concentration of that solution.
Since, A = ct
c = A / t
= 2 / 10000 1
= 2x10
-4
mol per liter
INSTRUMENTATION
All photometers, colorimeters and spectrophotometers have the following
basic component
Light source is the continuous source of radiant energy. It emits all colors of
light.
Ex- Tungsten filament lamp and hydrogen deuterium lamp.

Filter or Monochromator it allows the light of the required wavelength but
absorb the light of other wavelength.
It selects one wavelength and that wavelength is sent through the sample.

Sample cells all instruments must contain a container or cuvettes for the
sample, which are round or square shape made up of glass or plastic and have
thickness of 1 centimeter.
Detector used for measuring the wavelength of transmitted light that has
passed through the sample.
In some systems it is called as read out system.
It measures the intensity of the incident light and generates an electrical
signal which is proportional to the intensity.
Ex- photovoltaic cell, phototubes, photomultiplier tubes.

Amplifier it increases the signal so that it is easier to read out.

Computer-
Recorder -
Summary-
Colorimetry is a light sensitive instrument that measure how much light is
absorbed by an object or substance or sample.
It determines color based on the red, blue and green components of light
absorbed by the object or substance or sample.

Example consider the bananas
When white light hits the bananas, all colors are absorbed except for yellow.
The yellow light reflect back and hits our eyes, so we perceive the banana to
be yellow.
The working start from the light source which produce white light
(which is a combination of VIBGYOR).

The white light passes through a lens or prism then it breaks into VIBGYOR

Then the light hit on monochromator, that select required wavelength but
absorb the light of other wavelength

When the white light hits the color filter then it filter the light.

Working
Now the filtered light hits the sample then some of the light is absorbed, some of the
light is reflected and some are transmitted through the sample.

Darker sample will absorb more of the light while pale samples will absorb less of the
light.

The detectors measure the amount of the light which is transmitted through the
sample.

Finally read out by computer.
can be used to monitor the growth of a bacterial or yeast culture. (if the culture
grows then the medium becomes cloudy and absorbs more light).
to determine the concentration of a known solute in a given solution.
used to test for water quality, by screening for chemicals such as chlorine,
fluoride, cyanide, dissolved oxygen, iron, molybdenum, zinc and hydrazine.
also used to determine the concentrations of plant nutrients such as
phosphorus, nitrate and ammonia etc. in the soil.
also used to determine hemoglobin in the blood.
also used in industries like color printing, textile manufacturing, paint
manufacturing, in food industries etc.
APPLICATION
it can detect the smallest color difference that the human eye cannot pick up.
used to find out the concentration of any colored subsistent in food & beverage
for quality control.
also used for clinical and medical applications like routine analysis of
Albumin, Cholesterol, Glucose, Creatinine, Total Protein, Haemoglobin etc.