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Colour is everywhere. Every chemical compound absorbs, transmits, or reflects light over an electromagnetic spectrum in wavelengths. When light passes through any solution a section of it is absorbed. Spectrophotometry allows both qualitative and quantitative analysis. As the concentration of a substance increases light absorption increases, and light transmission decreases.
Spectrophotometry is used in chemistry, biochemistry (for enzyme-catalysed reactions), physics, biology, and clinical studies (examining haematology or tissues). It allows scientists to analyse different samples without having any skin contact as the samples are contained in a small tube called a cuvette or in case of the Photopette, measurements are done directly in the sample container without having to transfer it.
Spectrophotometry is a standard and inexpensive technique to measure light absorption or the amount of chemicals in a solution. It uses a light beam which passes through the sample, and each compound in the solution absorbs or transmits light over a certain wavelength. The instrument used is called a spectrophotometer. It measures the number of photons absorbed and is made up of two instruments: a spectrometer and a photometer. The spectrometer produces the light of the wavelength and the photometer measures the intensity of light by measuring the amount of light that passes through the sample.
There are two types: a single beam, and double beam. Single beam spectrophotometers are generally more compact and have a higher dynamic range but the optics in a double beam can permit higher levels of automation, better precision and can correct for background absorption of the solvent. With the double beam spectrophotometer, one beam passes through the sample, and the other through a reference solution or the solvent.
Spectrophotometers consist of a light source, a monochromator (which separates the polychromatic radiation of the light source into all its wavelength), a sample chamber containing a cuvette, a detector (such as a photomultiplier tube or photodiode) to detect the transmitted light, a digital display and a data analysis software package.
The monochromator (such as a prism or grating) inside the machine refracts the light into a single spectrum and disperses polychromatic light into the essential wavelengths. A grating divides the light available into different segments. Gratings are common in spectrophotometers that use UV, visible and infrared regions.
In the spectrophotometer, the number of photons absorbed by a solution is called the absorbance readout. The longer the path-length that the light must travel through a solution prior to it reaching the detector, the greater the chance of a photon being absorbed.
Different compounds absorb best at different wavelengths. A UV-visible spectrophotometer uses light over the ultraviolet range (185 – 400 nm) and visible range (400 – 700 nm) of the electromagnetic radiation spectrum. Whereas an IR spectrophotometer uses light over the infrared range (700 – 15000 nm).
Ultraviolet (UV) and visible (VIS) spectroscopy show electronic transitions in atoms and molecules, to measure this a spectrophotometer is used. Compounds that absorb in the visible region are coloured, whereas ones that absorb only in the UV region are colourless.
UV-VIS spectrophotometer usually use two light sources. . A deuterium lamp is used for the UV region and a tungsten lamp for the VIS region. These lights reach the monochromator via a mirror. The wavelength for red light is between 700 and 750 nm and blue between 400 and 450 nm. If the wavelength is shorter than 350 nm it is UV and has more energy.
Spectrophotometers measure absorbance (A) and transmittance (T). The intensity of light (I0) measures photons per second. When light passes through a blank sample, it does not absorb light so is symbolised as (I). Scientists use blank samples without chemical compounds as a reference. They contain everything that is in the sample cuvette, except the one material which absorbance is being measured.
To calculate the transmittance the following equation is used:
Transmittance (T) = It/I0
It = Light intensity after passing the cuvette (transmitted light)
I0 = Light intensity before passing the cuvette (incident light)
Absorbance (A) = – log10 T = – log IS/IR
The Beer-Lambert law indicates that the amount of light that is absorbed by a substance is proportional to the amount of the sample concentration. It is also determined by the amount of solute that is present.
The sample molecules or ions in a solution can be detected and quantified using a spectrophotometer and The Beer-Lambert Law with this equation: A = ƐCL
A = absorbance of light at a specific wavelength
Ɛ = molar extinction coefficient (the absorbance of 1 mole of a substance dissolved in 1 litre solvent)
C = the molar concentration of a sample
L = the optical path length of a sample