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 (Ir), a part is absorbed (Ia) and rest of the light is transmitted (It)
I0 = Ir + Ia + It
⇒ In photometers (colorimeter & spectrophotometer), (Ir) is eliminated because the measurement of (I0) and It is sufficient to determine the (Ia). For this purpose, the amount of light reflected (Ir) is kept constant by using cells that have identical properties. (I0) & (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.
⇒ 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
as = Absorbency index
c = Concentration of Solution
⇒ 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
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
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
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.
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
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