A spectrophotometer is a laboratory equipment that can measure the number of photons (the intensity of light) absorbed after passing through the solution of the sample. It can also detect the concentration of the solution by measuring the intensity of detected light. “Spectrophotometry” is the method used by the spectrophotometer for measuring the amount of light absorbed by any chemical substance when a light beam passes through it.
The spectrophotometer generally consists of two different devices; spectrometer and photometer. The spectrometer emits light of the desired wavelength, and the photometer detects the amount of light absorbed by the solution.
Table of Contents
Principle of Spectrophotometer
A spectrophotometer is based on the Beer-Lambert law, which states that absorbance (amount of light absorbed) of the solution has a linear relationship with the length of light and the concentration of a sample.
That is; A∝ CL
Where; A= Absorbance
C= concentration of the sample
L= path length
OR, A= εCL where ε= molar extinction coefficient, the value of which is constant for a specific molecule.
Here, A= -log (T)
Where T= Transmittance; the fraction of light passing through the sample, expressed as; T= It/Io
Here, It= transmitted light, and Io= incident light.
Therefore, A= -log (It/Io)
Now, we know that a standard spectrophotometer uses a cuvette of the length of 1 cm, which is the path length. Since path length (L), absorbance (A), and molar extinction coefficient (ε) are known, the concentration of the solution (C) can now be calculated.
Parts of Spectrophotometer
A spectrophotometer consists of four general parts; light source, an optical system (monochromator), sample holder, and detector (photometer).
Any spectrophotometer requires light of various wavelengths. Commonly tungsten lamp provides a visible spectrum of light in a spectrophotometer. Likewise, hydrogen and deuterium lamps provide ultraviolet radiation, and Nernst filament or globar provide IR (infrared) radiation.
Optical system (monochromator)
An optical system of spectrophotometer consists of the following parts:
- Lenses: It collects the radiation from the source and directs it into the slit.
- Entrance slit: It provides a narrow image of the radiation.
- Collimator lens: It depicts the light from the entrance slit parallel.
- Exit slit: It selects the desired spectrum of the light-emitting from the exit slit.
- Dispersive device: A prism and grating function as dispersive devices. These help in dispersing the incident ray of light. The action of the prism depends on the refraction of light. Quartz or fused silica prism is a must for ultra-violet spectrum below 350 nm. A diffraction grating consists of many parallel lines ruled at highly close intervals, likely 15000/30000 lines per inch on a highly polished surface like aluminum. The recommended ruling number is from 20 grooves/mm in infrared to as many as 3600 or more grooves/mm for the visible and ultraviolet rays. The grating is better than a prism.
The sample solution is placed in the cuvette ( glass tubes) directly before the detector and after the dispersive device. Cuvette varies from test tubes because it has uniform thickness and optical path length. Usually, the cuvette is made up of glass or quartz.
After the desired light passes through the sample solution in the cuvette, the photometer detects the photons and gives the signals to the galvanometer for digital display.
Working Mechanism of Spectrophotometer
- Using the spectrophotometer requires calibration of the machine using standard solutions of known concentration.
- Depending on the solution, a specific wavelength of light is selected for the test. The ray of light passes through the diffraction grating, prism and mirror before reaching the solution.
- The mirror is for light navigation in the spectrophotometer, and the prism splits the light into different wavelengths.
- The light diffraction grating allows only the desired wavelength of light to pass through it.
- Then the light reaches the cuvette with solution (test/standard). When monochromatic light reaches the cuvette, some are absorbed, some are reflected, and the remaining parts are transmitted.
- After that, the photodetector system detects the transmitted light, which measures its intensity and converts it into the electrical signals sent to the galvanometer.
- Now the galvanometer measures the received signal and displays it in digital form; it is the absorbance of the solution.
- The concentration of the solution is calculated after determining the absorbance by using the formula; C= A/εL, where A is absorbance, C is the concentration of the solution, L is path length, and ε is the molar extinction coefficient.
- A simple exception in a double beam spectrophotometer is that the monochromatic light splits into two beams, one for the standard solution and another for the test solution. In this type of spectrophotometer, the processing of the test and the standard solution occurs simultaneously.
Calibration of spectrophotometer requires standard/known solution. Comparison of the calculated value with previous value will help determine the condition of the equipment.
Types of Spectrophotometer
Depending on the range of wavelength of the light spectrophotometer is of two types, that are:
It uses light over the ultraviolet range (185-400 nm) and visible range (400-700 nm) of the electromagnetic radiation spectrum.
It uses the wavelength of light over the infrared range (700-1500 nm) of the electromagnetic radiation spectrum.
Similarly, based on the number of beams of light, it is of two types, which are:
Single beam spectrophotometer
It operates between 325-1000 nm wavelength using only a light beam. The light travels in a single direction, and the test solution and the blank solution are read in the same cuvette one after another.
Double beam spectrophotometer
It works between 185-1000 nm wavelength. The two photocells split the light from the monochromator into two beams. A beam is used for reference, and another is used for the reading samples.
Uses of Spectrophotometer
The uses of a spectrophotometer are as follows:
- A spectrophotometer is used for the quantitation of nucleic acid (DNA or RNA).
- It determines the concentration of color and colorless compounds by measuring the absorbance of the solution. It can also determine the concentration of biological materials like nucleic acid and proteins.
- Similarly, it determines the phase of reaction by measuring the formation and disappearance rate of the light-absorbing compounds in the range of the visible and UV region of the electromagnetic spectrum.
- It also identifies compounds by determining the absorption spectrum in the visible region of the light and the UV region of the electromagnetic spectrum.
- The spectrophotometer should be turned on 10 to 15 minutes before use.
- The device should be calibrated each time.
- The selected wavelength should be the maximum wavelength that the solution can absorb.
- The sample used should not contain any substance which can dissociate, react or change during the measurement.
- Since the absorbance depends on the concentration, sample preparation should be within the acceptable concentration range.
- BATRA, S. (2022). SPECTROPHOTOMETER – Principle, Components, Working & Application | BIOCHEMISTRY PRACTICALS. Paramedics World. Retrieved 6 June 2022, from https://paramedicsworld.com/biochemistry-practicals/demonstration-of-spectrophotometer-principle-components-working-applications/
- Manandhar, S., & Sharma, S. (2017). Practical Approach to Microbiology (3rd ed., pp. 18-20). National Book Centre.
- Phillips, K. (2022). What Is a Spectrophotometer and How Does It Work? – HunterLab Horizons Blog. HunterLab Horizons Blog. Retrieved 6 June 2022, from https://blog.hunterlab.com/blog/color-measurement/what-is-spectrophotometer/