The visible light absorption spectra can be taken on any material that is visibly clear. Polystyrene, quartz, and borosilicate cells (Pyrex) are the most commonly used materials. Ultraviolet light is absorbed by most crystals and plastics, which is why quartz cells are used. The Si-O present in the glass and quartz and the CC in the plastic absorb the infrared light. The infrared absorption spectra are usually performed with a thin film of the sample held between plates of sodium chloride. Other methods involve suspending the compound in a substance that does not absorb in the study region. Mineral oil emulsions (Nujol) and potassium bromide crystals are usually the most common. The NaCl and KBr, being ionic, do not absorb the infrared significantly,
Spectrometry as an analytical instrument
It is often of interest to know not only the chemical composition of a given sample but also the relative concentrations of several compounds in a mixture. To do this, a scale, or calibration curve, must be constructed using several known concentrations for each compound of interest. The graph that results from the concentration with respect to the absorbance is made by hand or using an appropriate curve fitting software, which uses a mathematical formula to determine the concentration in the sample. The repetition of this process for each compound in a sample gives a model of several absorption spectra that together reproduce the absorption observed. In this way it is possible, for example, to measure the chemical composition of comets without having samples of them on Earth.
A simple example: standard cyanide at 200 parts per million gives an absorbance with an arbitrary value of 1540. An unknown sample gives a value of 834. Since this is a linear relationship and passes through the origin, the unknown value is easily calculated like 108 parts per million. With this proportion method, it is not necessary to know the values of the governing coefficients, or chromophores, or the experimental cell length.
In practice, the use of a calibration curve, rather than a single comparison point, reduces the uncertainty in the final measurement by excluding random interference (noise) in the preparation of the standards.
How to determine the concentration
A spectrophotometer analyzes the reflected or transmitted light energy of a molecule.
A spectrophotometer has a system that separates the different wavelengths of a light beam: the monochromator. There are two types of monochromator:
- light scattering through a prism
- diffraction of light by a network or by a crystal
The spectrum is reconstructed by software from measurements made at fixed wavelength intervals (example: measurements every 1, 3, 5 nm, …).
- deuterium discharge lamp: range 190 to 400 nm (maximum emission at 652 nm)
- tungsten filament lamp: area 350 to 800 nm
- Xenon discharge lamp (high energy) used in the UV and visible range. It works as a flash, at the moment of measurement
A. A densitometer measures the opacity of a film (transmission densitometer) or the absorption of light by a reflective object (reflection densitometer).
b. A colorimeter contains a detection system consisting of a sensor associated with at least three interference filters. These filters have properties close to those of the peaks of the spectral curve of the human eye to simulate the trichromatic response of the eye.
c. A spectrophotometer has more detectors than a standard colorimeter. It measures the spectral reflectance of an object for each wavelength.