Introduction:
It is an instrument used to measure absorbance at various wavelengths which is composed of two units: a spectrometer and a photometer. Moreover, this is used to measures the number of photons (the intensity of light) absorbed after it passes through sample solution.
Basically, the spectrometer is to use to produce light of any required wavelength, while the photometer is to measure the intensity of light. Moreover, it is designed in a way that the sample is placed between spectrometer and photometer. Eventually, the amount of light transmitted or absorbed through the sample is measured by photometer which will convert a voltage signally to display for the results and interpretation accordingly. The voltage signal is linked to the changes in the intensity of light absorbed.
All spectrophotometers contain several elements:
- A source of light
- An optical system, or monochromator, to select a narrow band of wavelengths from the whole spectrum emitted by the source of light.
- A sample compartment that holds a sample tube or cuvette with fixed path length
- A photomultiplier tube (PMT) that magnifies emergent light because its intensity is too diminished to be accurately read
- This cell is connected to a galvanometer which translates the electrical output of the activated into a specific transmittance value
- A processor and a detector of radiation and its auxiliary equipment.
Principle:
This instrument is based on photometric techniques that is operated in the UV region, Visible spectrum and IR spectrum
According to photometric technique, when a beam of incident light of intensity I0, passes through a solution, a part of the incident light is reflected, a part is absorbed and the rest of the light is transmitted. If the part of incident light which got reflected is Ir, the part which got absorbed is Ia and the part which got transmitted is It
If I0 denotes the incident light, Ir is the reflected light, Ia is the absorbed light and it the transmitted light then
I0 = Ia + It + Ir
Instrumentation:
Light source
Tungsten filament lamps are the best source of light for the visible spectrum whereas hydrogen and deuterium lamps are the most widely used light sources for Ultraviolet radiation. In addition, Nernst filament or globar is the most satisfactory sources of IR (Infrared) radiation.
Prisms and Gratings
A prism is an optical component that is used to disperse light into its constituent colours, forming a spectrum of light. The process of using a prism to separate light into its component wavelengths is called dispersion. Light is refracted when it travels through a prism. The speed of light changes as it passes through different media, and this change in speed causes the light to bend. The extent of bending depends on the wavelength of light, with shorter wavelengths (like blue and violet) being bent more than longer wavelengths (like red). As a result of refraction, different colours of light are spread out, or dispersed, into a spectrum. This spectrum is a continuum of colours arranged according to their wavelengths. The order of colours in a typical prism spectrum is red, orange, yellow, green, blue, indigo, and violet (ROYGBIV).
Monochromator/Filters
To select the particular wavelength after the prism or diffraction grating is used to split the light from the light source. It selects the required wavelengths of light from a given light source that has been diffracted through prism or graft.
Slits
Entrance slit: Â This is placed to provide narrow source of light to avoid overlapping of monochromatic image.
Exit slit: This is placed in order to provide rout for the transmitted light form the sample tube. Moreover, both the entrance and exit slits have equal width.
Sample and reference cells holder
It hold the sample /test solution which is known as cuvettes. They can be of any size such as square, round, rectangular that are made up of glass, pyrex, silica, plastic, and quartz.
Beam splitter
It can only be found in a double beam spectrophotometer that is used to divide the single beam of light originating from the light source into two beams.
Detector system
When light falls after transmission through the sample to the detector system, an electric current is generated that reflects the galvanometer reading Phototubes, photomultiplier tube (PMT) and photovoltaic cell are generally used as a detector.
Types:
Single beam spectrophotometer
Generally, this spectrophotometer uses single beam of light and operates between 325 nm to 1000 nm wavelength in which the light will travel in one direction and the test solution and blank are read in the same.
Double beam spectrophotometer
Double beam spectrophotometer operates between 185 nm to 1000 nm wavelength that possess two photocells. This instrument splits the light from the monochromator into two beams. One of the beams is used for reference and the other for reading of the sample in tube holder.
Fig: Single and Double Beam spectrophotometer
Applications:
- Pharmaceutical analysis– It is used to purify and concentration of individual components of complex drug
- Nucleic Acid (DNA/RNA) analysis–Â In the molecular biology it is generally used in order to identify the purity and concentration of RNA and DNA. In general, the 260/280 absorbance ratio (260/280) is effective in identifying possible DNA contamination in isolated nucleic acid samples. Pure DNA often has a 260/280 ratio of 1.8, whereas pure RNA typically has a ratio of 2.0. Protein contamination reduces the 260/280 ratio due to greater absorbance at 280 nm.
- Clinical Chemistry– It is used to analyse blood samples for various components, including glucose, cholesterol, and electrolytes.
- Haematology– It is employed to measure the concentration of hemoglobin in blood samples.
- Urinalysis– It can be used to detect and quantify proteins and glucose in urine samples. In which abnormal levels may indicate kidney or metabolic problem.
- Therapeutic Drug Monitoring– It is used to quantity concentration of drug or other chemical or compounds in blood or other biological fluids.