In terms of chemistry, the spectrophotometer is the measurement in the qualitative term of the different properties of a material specifically the reflection or transmission in from of wavelength. Spectrophotometer is all the more specific than the general term like the “electromagnetic spectroscopy in the spectrophotometry which deals with the visible light, near-ultraviolet, and near-infrared, but does not cover time-resolved spectroscopic techniques.”
Spectrophotometry is the instrument that depends on the qualitative analysis of the various molecules of a compound based on the amount of light that is being absorbed by the “coloured compound”. “Spectrophotometry uses photometers, which are also known as spectrophotometers. It can measure a light beam's intensity as a function of its color wavelength”. Some of the significant features of the spectrophotometers are the “spectral bandwidth which is the range of colors spectrophotometers can transmit through the test sample, the amount of sample-transmission, the logarithmic range of sample-absorption, and sometimes a percentage of reflectance measurement”.
A spectrophotometer is mainly used to measure the “transmittance or reflectance” of the different surface like the solutions, transparent or opaque in terms of solid which may include polished glass or gasses. Since many “Biochemical” are in colour form, as a result, they absorb many visible lights and so they can only be calculated in the “colorimetric procedures”. Many a times a colourless biochemical is converted into different coloured compounds which are more suitable for “chromogenic color-forming reactions to yield compounds suitable for colorimetric analysis”. In addition, a spectrophotometer is also made in such a specific way that it can also calculate the “diffusivity on any of the listed light ranges that usually cover around 200 nm - 2500 nm using different controls and calibrations”. For the above mentioned range of 200nm to 2500nm of light, specific calibrations are required in the spectrophotometer which uses the basic standards that may differ in the type depending on the “wavelength of the photometric determination”.
For example, Spectrophotometer was use to calculate the “equilibrium constant” of a particular chemical solution. The chemical reaction that occurs within the solution may happen in the forward or in the backwards direction. It happens when the “reactants form products and products break down into reactants”. At a point of time, the following chemical reaction will reach a balance point which is also known as the equilibrium point. If the scientist or the researcher would want to find out the level of concentrations of the different reactants and the products at that particular point of time, spectrophotometry could be used in order to check the light transmittance of the chemical solution. The amount of light passing through the chemical solution determines the level of concentration of the solution. The more concentrated the solution is, the less amount of light that will be passed through the solution. The lesser the concentration of the solution, the more amount of light that will pass through that chemical solution. The happens because not all chemical that were present in the solution have same concentration level. Some chemical have high level of concentration than the other. Some chemical may have a lower level of concentration than the other. “The absorption of light is due to the interaction of light with the electronic and vibrational modes of molecules. Each type of molecule has an individual set of energy levels associated with the makeup of its chemical bonds and nuclei, and thus will absorb light of specific wavelengths, or energies, resulting in unique spectral properties. This is based upon its specific and distinct makeup”.
Spectrophotometer is used in a wide range of different scientific field including physics, chemistry, materials science, biochemistry, molecular biology and chemical engineering. Spectrophotometer are also be extensively used in a wide range of industries such as semiconductors, laser and optical manufacturing, printing and forensic examination. In addition, a spectrophotometer is also used in the laboratories for the study of various chemical substances. A Spectrophotometer is used many a time for the study of the different “enzyme activities, determinations of protein concentrations, determinations of enzymatic kinetic constants, and measurements of ligand binding reactions. Ultimately, a spectrophotometer is able to determine, depending on the control or calibration, what substances are present in a target and exactly how much through calculations of observed wavelengths”.
In the field of Astronomy, the word Spectrophotometer indicates the calculation of the spectrum of a celestial objects or bodies. There the flux state “of the spectrum is calibrated as a function of wavelength, usually by comparison with an observation of a spectrophotometric standard star, and corrected for the absorption of light by the Earth's atmosphere”.
There are two different types of Spectrophotometer. One of which is a transits a single beam other one is a double beam. A double beam spectrometer compares the intensity of light between two light paths. One path has the reference sample while the other light beam has the test sample. A single beam Spectrophotometer is used to measure the relative intensity of the light before and after the insertion of the test sample. In spite of the fact that all the required calibrations and calculation can be done easily through a double beam instrument, which is all the more easier and stable, a single beam spectrophotometer has a much larger wider and more dynamic range. In addition, single beam spectrophotometers are also much more optically simpler and are more compact.
A Spectrophotometer usually uses a monochromator which has a diffraction grating in order to create the “analytical spectrum”. These diffraction grafting is either movable or may be fixed as per the requirements. “If a single detector, such as the photomultiplier tube or photodiode is used, the grating can be scanned stepwise like scanning the spectrophotometer” so as to make sure that the detector is able to measure the intensity of the light at almost all the “wavelength which was synchronise with each step”. A different array of spectrophotometer like the charged couple devices (CCD) which is also used in the DSLR cameras in recent times. In addition photodiode arrays (PDA) is also a good option. “In such systems, the grating is fixed and the intensity of each wavelength of light is measured by a different detector in the array. Additionally, most modern mid-infrared spectrophotometers use a Fourier transform technique to acquire the spectral information. This technique is called Fourier transform infrared spectroscopy”.