The spectrophotometer measures absorption and spectral bandwidth of the given sample. Absorption is the logarithm of transmittance, i.e. Log (T), whereas transmittance is the portion of light moved through the sample. Transmittance can define as the ratio of light incidents on the test sample to the light transmits through the solution, i.e. IO/I. Spectral bandwidth also refers to as “Bandpass”, and define as a range of colours it can reflect through the test sample.
The spectrophotometer must be calibrated before taking the readings of the test sample, by putting a cuvette holding a control solution. The process of calibrating the device commonly refers to as “Zeroing of spectrophotometer”. The amount of light transmitted or absorbed can be calculated by the readings obtained in a digital meter of a spectrophotometer.
Definition of Spectrophotometer
The spectrophotometer can define as an instrument, which measures the absorbance of the test sample at a specific wavelength, by measuring the amount of light transmitted by the sample. This device contains several components like a light source, collimator, monochromator, cuvette, light detector, and digital meter.
Spectrophotometry is a science which deals with the study of light intensity absorbed or transmitted by the different solutions when a ray of light moves through the test sample. Its basic theory concludes that each chemical compound transmits or absorbs certain light according to their relative wavelength.
Working of Spectrophotometer
The principle of spectrophotometer depends upon the Beer-Lambert law, which states that when a beam of light incidents on the homogenous solution, a fraction of incident light reflects, some of it absorbs and the remaining transmits through the solution. Both Beer and Lambert have given their own theories on the absorption of radiation.
According to Beer-Lambert law, the absorbance is equal to the given mathematical expression:
A= log I0/I
A= ε C l
Where, A= Absorbance of light
I0= Intensity of incident light
I= Intensity of transmitted light
ε= Absorption coefficient
C= Concentration of the absorbing material
l= Path length (cm)
The amount of light transmitted from the solution is inversely proportional to the absorption of light.
A= log10 1/T
As the absorbance of the medium increases, the transmittance of light through the solution decreases and vice versa. Absorbance is a non-dimensional quantity whose value ranges between 0-1.
A spectrophotometer includes the following sequential events:
- First, a light source falls onto the monochromator (Dispersion device).
- Then the monochromator will produce a single source of light that falls onto the focusing wavelength selector.
- The focusing convex lens will pass a fraction of the monochromatic source of light from the sample solution to the photocell detector.
- A photocell detector converts the light energy into the electrical energy, and an amplifier transmits this electrical signal to the internal circuit.
- Finally, an internal circuit inside a spectrophotometer gives out a final output on a digital meter.
Based on Light Source
Single beam spectrophotometer
In this, a fraction of light from the diverging devices is wholly passed from the sample solution. A beam of light from the light source falls onto the collimator convex lens and moves to the diaphragm. The diaphragm ensures the 100% transmittance and allows the light to fall onto the monochromator device.
A dispersion medium or monochromator device allows the transmittance of a single source of light onto the focusing convex lens. The focusing convex lens transmits light at a particular wavelength from the sample to the photocell detector. A photocell detects the portion of light transmitted or absorbed and gives the reading on the display meter.
Double beam spectrophotometer
In this, a fraction of light coming from the monochromator device divides into two beams; one falls onto the reference sample and the other onto the test sample. Its mechanism is more or less similar to single beam spectrophotometer but differs when the dual mirrors divide a single beam of light into two.
One beam of light passes from the test sample to the photocell and the other passes from the reference sample to another photocell. A photocell detects the amount of light transmitted or absorbed and gives the reading on the display meter.
Based on Light Wavelength
It makes the use of cuvettes made of quartz, and hydrogen or deuterium lamps as a light source. The hydrogen lamp emits continuous or discontinuous spectral UV- light ranging between 200-450 nm. This device determines the absorbance or transmittance for the fluids and even solutions.
It makes the use of plastic and glass cuvettes and tungsten halogen light source. The tungsten lamp consists of a tungsten filament, which emits a visible spectral range between 330-900nm. The tungsten lamp has a long life of 1200h. This device can measure the change in colour intensity according to the change in concentration of moderately diluted solutions.
It makes the use of Nernst glowers as a conductive device having a long life. This kind of spectrophotometer helps in the study the vibrations of different molecules at a specific wavelength. Near and mid-IR-rays cause rotational and harmonic vibrations.
Components of Spectrophotometer
A spectrophotometer includes the following elements:
The source of illumination usually includes hydrogen, tungsten, xenon flash lamps and Nernst glower for the UV, Visible, UV-visible and infrared spectrophotometers respectively. The light source can emit continuous or discontinuous spectral bands at varying wavelength. An illuminator provides a polychromatic source of light to the collimating device.
It is an optical device which contains a tube having a convex lens on one side and an aperture on the other. Collimator primary function is to convert the radiating polychromatic source of light into a parallel beam by the adjustable aperture. In the focal plane of a collimator convex lens, an opening or aperture passes the parallel beam onto the dispersion device via the diaphragm.
A dispersion device is positioned between the diaphragm and wavelength selector. The diaphragm functions as an entry slit of the polychromatic source of light that ensures 100 % transmittance from the collimator to the dispersion medium. The dispersion medium reflects the light of the selected wavelength through the exit slit that allows the monochromatic source of light to escape. Prisms, diffraction gratings and filter system are the most common dispersion medium.
It disperses the polychromatic source of light coming from the diaphragm into the constituent monochromatic source of light. A prism disperses the light of variable wavelength to a different extent, depending on:
- Its optical angle (60 degrees)
- And, the material from which it is composed
For the commercial use generally, 60 degrees cornu quartz and 30 degrees littrow prism are used.
These are the optical components, commonly used in the visible, UV and IR spectrophotometers. Its degree of dispersion depends upon:
- Spacing between the gratings
- Wavelength of light
Diffraction grating comprises of few ridges or rulings on the surface, coated by aluminium. It can cause transmissive and reflective kind of diffraction.
The filters are first layered with gelatin, then coloured with organic dyes and finally sealed within the glass plates. Now, these are the most common dispersion medium used for the biological and biochemical assays. The filters can split the different parts of electromagnetic radiation by absorbing and transmitting a particular wavelength. Spectrophotometers which consist of a filter system as a dispersion media refers to as “Filter photometers”.
The optical filter provides a monochromatic source of light and contains absorption or interference filter. An absorption filter can transmit the desired range of wavelength or spectral elements by blocking some of the spectral components from a beam of light through an illuminator.
Filter wheel system is a commercial device incorporated with several filters having different wavelength responses, and positioned nearly the circumference of the rotating wheel. Each filter will show various spectral bands at a particular wavelength. This kind of system only selects discrete bands.
A cuvette is a sample holding tube that can be made of plastic, glass, fibre etc. A cuvette with a blank solution helps in the calibration of the spectrophotometer by giving zero reference number. The calibration of the spectrophotometer is necessary to check the accuracy of the light source.
It also refers as “Photoresistor” which detects the range of light transmitted from the test sample and transforms the light into an electrical signal. A photocell detector shows the following properties:
- High sensitivity
- Short response time
- Long-term stability
- An electrical signal which can be easily amplified
Amplifier and digital meter
A photocell produces an electrical signal, directly proportional to the light reflected from the solution. An amplifier boosts the electrical signal and transfers it to the internal circuit, which can finally convert this signal into a readable form. The readings in a digital meter can be noted down, and a standard graph between absorption and transmittance can be plotted accordingly.
A spectrophotometer helps in the qualitative analysis like the type, molecular weight and structure of different compounds, as the different compounds absorb light at different wavelengths.
Example: Aliphatic or acyclic hydrocarbons or their derivatives absorb light at a wavelength ranging between 220-280nm.
It also helps in quantitative analysis of protein, enzymes, the amino acid (tyrosine), and blood glucose level by the use of UV-Visible spectrophotometer.