SPECTROPHOTOMETER

A spectrophotometer is an instrument used to measure absorbance at various wavelengths. It is

similar to calorimeter except that it uses prism or diffraction grating to produce monochromatic
light. It can be operated in UV (Ultraviolet) region, Visible spectrum as well as IR (Infrared)
region of the electromagnetic spectrum.

⇒ Absorption of light – Light falling on a colored solution is either absorbed or transmitted. A

colored solution absorbs all the colors of white light and selectively transmits only one
color. This is its own color.

PRINCIPLE OF SPECTROPHOTOMETER
Spectrophotometer is based on the photometric technique which states that When a beam of
incident light of intensity I0 passes through a solution, a part of the incident light is reflected (I r),
a part is absorbed (Ia) and rest of the light is transmitted (It)

Thus,

I0 = Ir + Ia + It

⇒ In photometers (colorimeter & spectrophotometer), (Ir) is eliminated because the

measurement of (I0) and It is sufficient to determine the (I a). For this purpose, the amount of light
reflected (Ir) is kept constant by using cells that have identical properties. (I 0) & (It) is then
measured.

⇒ The mathematical relationship between the amount of light absorbed and the concentration of
the substance can be shown by the two fundamental laws of photometry on which the
Spectrophotometer is based.

COLORIMETER – PRINCIPLE, COMPONENTS, WORKING & APPLICATIONS

Beer’s Law

⇒ This law states that the amount of light absorbed is directly proportional to the concentration
of the solute in the solution.

Log10 I0/It = asc

where,

as = Absorbency index

c = Concentration of Solution
Lambert’s Law

⇒ The Lambert’s law states that the amount of light absorbed is directly proportional to the
length and thickness of the solution under analysis.

A = log10 I0/It = asb

Where,

A = Absorbance of test

as = Absorbance of standard

b = length / thickness of the solution

The mathematical representation of the combined form of Beer-Lambert’s law is as follows:

Log10 I0 / It = asbc

If b is kept constant by applying Cuvette or standard cell then,

Log10 I0/It = asc

The absorbency index as is defined as

as = A/cl

Where,

c = concentration of the absorbing material (in gm/liter).

l = distance traveled by the light in solution (in cm).

In simplified form,

The working principle of the Spectrophotometer is based on Beer-Lambert’s law which states
that the amount of light absorbed by a color solution is directly proportional to the concentration
of the solution and the length of a light path through the solution.

A ∝ cl

Where,
 A = Absorbance / Optical density of solution

c = Concentration of solution

l = Path length

or, A = ∈cl

∈ = Absorption coefficient

TYPES OF SPECTROPHOTOMETER

Spectrophotometer is of 2 types –

 Single beam spectrophotometer

 Double beam spectrophotometer
Single beam spectrophotometer operates between 325 nm to 1000 nm wavelength using the
single beam of light. The light travels in one direction and the test solution and blank are read in
the same.

SINGLE BEAM SPECTROPHOTOMETER

Double beam spectrophotometer operates between 185 nm to 1000 nm wavelength. It has two
photocells. This instrument splits the light from the Monochromator into two beams. One beam
is used for reference and the other for sample reading. It eliminates the error which occurs due to
fluctuations in the light output and the sensitivity of the detector.

PARTS OF SPECTROPHOTOMETER

There are 7 essential parts of a spectrophotometer

Light source – In spectrophotometer three different sources of light are commonly used to
produce light of different wavelength. The most common source of light used in the
spectrophotometer for the visible spectrum is a tungsten lamp. For Ultraviolet
radiation, commonly used sources of are the hydrogen lamp and the deuterium lamp. Nernst
filament or globar is the most satisfactory sources of IR (Infrared) radiation.

Monochromator – To select the particular wavelength, prism or diffraction grating is used to

split the light from the light source.

Sample holder – Test tube or Cuvettes are used to hold the colored solutions. They are made up
of glass at a visible wavelength.

Beam splitter – It is present only in double beam spectrophotometer. It is used to split the single
beam of light coming from the light source into two beams.

Mirror – It is also present only and double beam spectrophotometer. It is used to the right
direction to the splitted light from the beam splitter.

Photodetector system – When light falls on the detector system, an electric current is generated
that reflects the galvanometer reading.

Measuring device – The current from the detector is fed to the measuring device – the
galvanometer. The meter reading is directly proportional to the intensity of light.

WORKING OF THE SPECTROPHOTOMETER

⇒ When using a Spectrophotometer, it requires being calibrated first which is done by using the
standard solutions of the known concentration of the solute that has to be determined in the test
solution. For this, the standard solutions are filled in the Cuvettes and placed in the Cuvette
holder in the spectrophotometer that is similar to the colorimeter.

⇒ There is a ray of light with a certain wavelength that is specific for the assay is directed
towards the solution. Before reaching the solution the ray of light passes through a series of the
diffraction grating, prism, and mirrors. These mirrors are used for navigation of the light in the
spectrophotometer and the prism splits the beam of light into different wavelength and the
diffraction grating allows the required wavelength to pass through it and reaches the Cuvette
containing the standard or Test solutions. It analyzes the reflected light and compares with a
predetermined standard solution.

⇒ When the monochromatic light (light of one wavelength) reaches the Cuvette some of the
light is reflected, some part of the light is absorbed by the solution and the remaining part is
transmitted through the solution which falls on the photodetector system. The photodetector
system measures the intensity of transmitted light and converts it into the electrical signals that
are sent to the galvanometer.
⇒ The galvanometer measures the electrical signals and displays it in the digital form. That
digital representation of the electrical signals is the absorbance or optical density of the solution
analyzed.

⇒ If the absorption of the solution is higher than there will be more light absorbed by the
solution and if the absorption of the solution is low then more lights will be transmitted through
the solution which affects the galvanometer reading and corresponds to the concentration of the
solute in the solution. By putting all the values in the formula given in the below section one can
easily determine the concentration of the solution.

⇒ In double beam spectrophotometers, the beam splitters are present which splits the
monochromatic light into two beams one for the standard solution and the other for test solution.
In this, the absorbance of Standard and the Test solution can be measured at the same time and
any no. of test solutions can be analyzed against one standard. It gives more accurate and precise
results, eliminates the errors which occur due to the fluctuations in the light output and the
sensitivity of the detector.

APPLICATIONS OF THE SPECTROPHOTOMETER

⇒ The spectrophotometer is commonly used for the determination of the concentration of

colored as well as colorless compounds by measuring the optical density or its absorbance.

⇒ It can also be used for the determination of the course of the reaction by measuring the rate of
formation and disappearance of the light absorbing compound in the range of the visible & UV
region of electromagnetic spectrum.

⇒ By spectrophotometer, a compound can be identified by determining the absorption spectrum

in the visible region of the light spectrum as well as the UV region of the electromagnetic
spectrum.

⇒Here is the formula used for determining the Concentration of a substance in the Test
solution using the absorbance values measured by the spectrophotometer….

A = ∈cl

⇒ For two solutions i.e. Test and standard,

∈ = Constant

l = Constant (using the same Cuvette or Standard cell)

AT = CT ….. (i)

AS = CS ….. (ii)
⇒ From (i) & (ii),

AT × CS = AS × CT

CT = (AT/AS) × CS

Where,

CT = Concentration of the Test solution

AT = Absorbance/ Optical density of the test solution

CS = Concentration of the standard

AS = Absorbance / Optical density of the standard solution