What is spectroscopy?
Spectroscopy is the study of interactions of light (electromagnetic radiation) with matter.
There are different types of spectroscopic techniques, the basic principle shared by all of them is: irradiating a specific electromagnetic beam to the sample and checking the response of the sample to this stimulus. The spectroscopic technique allows scientists to obtain the information they need regarding the structure and properties of the material from the examined sample.
What is light?
Light carries energy in the form of small packets called photons. Each photon has a discrete and certain amount of energy called a quantum. Light also has wave properties with wavelength and frequency characteristics. The photon energy is related to its frequency and wavelength through the following relationship.
E=hν and ν=c/λ
where h is Planck’s constant and c is the speed of light. Therefore, high energy radiations have high frequency and low wavelength and vice versa.
The range of frequencies and wavelengths of light is known as the electromagnetic spectrum. This spectrum is divided into different regions from short wavelengths with very high energy (gamma rays and X-rays) to high wavelengths with low energy microwave and radio waves. (Visible region) White light forms only a small part of the electromagnetic spectrum, which covers 380-770 nm. When a material absorbs electromagnetic radiation, the change produced depends on the type of radiation and its energy amount. Absorption of energy leads the electron or molecule to go from the basic state of energy (initial energy) to the excited state (higher energy), and this excited energy can be in the form of rotation, vibration or electron excitation.
UV-Visible Spectroscopy:
Absorption of UV radiation is associated with the excitation of electrons of atoms and molecules from the lowest energy level to the highest level. Since the energy levels of the material are quantized, only radiation with a certain amount of energy, which is equal to the energy difference between different levels, is absorbed. The possible electron transfers are as follows: When the specified amount of energy is exactly equal to the energy required for each of these transfers, then energy will be absorbed.
The greater the gap between energy levels, the more energy is needed to transfer electrons to higher levels, which indicates radiation with smaller wavelengths and higher frequency.
Absorption of light in the visible ultraviolet region occurs only due to the following transitions:
Therefore, to absorb light in the region of 200-800 nm, molecules must contain π bonds or atoms with non-bonding orbitals. A non-bonding orbital is a pair of electrons only on atoms such as oxygen, nitrogen or halogens.
It is possible to predict the wavelength that is most likely to be absorbed by the pigment. When white light passes through a colored sample or is reflected by it, a certain part of the wavelengths are absorbed. The color of the sample solution indicates that the wavelength corresponding to its complementary color is absorbed.
This relationship is shown by the color wheel:
UV-Visible Spectrophotometer:
The spectrophotometer device can be used to measure the absorbance of samples in the visible ultraviolet region either in a single wavelength or by scanning in a range of wavelengths. They include the UV range from 400-190 nm and the visible range from 400-800 nm. This technique can be used for both quantitative and qualitative measurements.
In the figure below, you can see the components of a spectrophotometer.
The light source, which includes a tungsten halogen lamp with a deuterium lamp, covers the visible and near-ultraviolet regions – 200-800 nm. The output of the light source is focused on the selected wavelength (grid or prism) so that the light is separated into its constituent colors with different wavelengths.
Liquid samples should be poured into cuvettes or transparent cells with flat walls. The reference cell, control or blank contains the solvent in which the sample is dissolved, in fact, the control has all the texture of the sample except for the parameter to be measured. For each wavelength, the intensity of light passing through the sample (I) and the blank (I0) is measured. If I is less than I0, it indicates that the sample has absorbed light.
The absorption of the sample is also calculated according to the following formula:
?=???10?0?
The detector converts the intensity of the incoming light into a current, and the higher the amount of this current, the higher the intensity. In the visible ultraviolet region, the absorption graph is drawn in terms of wavelength (nm) (note: absorption has no unit).
Visible UV spectrum:
The diagram below shows the absorption spectrum of 1 and 3 Buta DN. It is easy to identify the wavelengths that matter absorbs (peaks) and the wavelengths that transmit light (valleys) from this diagram.
As you can see in the diagram above, the maximum absorption occurs at the wavelength of 217 nm, which is located in the ultraviolet region and there is no color absorption, so 1 and 3 Buta DN is colorless.
The spectrum of the blue copper complex shows that its complementary color, i.e. yellow, is absorbed.
Beer–Lambert law:
According to Beer-Lambert’s law, absorption is proportional to the concentration of the desired parameter in the sample. So, by using visible ultraviolet spectroscopic technique, it is possible to determine the concentration of the sample. Beer-Lambert’s law is as follows:
A=εcl
A = absorption
ε = molar absorption coefficient which is constant for a specific parameter at a specific wavelength
c = concentration of the solution
l = the length of the optical path, dimensions of the cuvette or cell
If the absorption of a series of sample solutions with a certain concentration is drawn in relation to their concentration, while following Beer-Lambert’s law, this graph will be linear and is known as a calibration curve. The concentration of unknown samples can be determined by measuring their absorbance and using calibration curves.
Since the absorption of solutions is directly related to their concentration. Spectroscopic data can also be used for kinetic studies. The rate of change in the concentration of reactant or product can be measured by changing the amount of absorption. By drawing the absorption curve against time, the order and also the rate equation can be determined according to the reactants.
New applications of spectroscopy:
✓ In medicinal chemistry, spectroscopy is widely used in studying the kinetics of enzymes, investigating tissue damage, and diagnosing diseases of organs such as the liver and pancreas.
✓ It is used in the pharmaceutical industry to perform analytical tests in order to design the formula and quality control. Also, in-vivo and in-vitro tests can be done by checking drug degradability.
✓ In the field of biochemistry and genetics, he used spectroscopy to determine the amount of DNA and the ratio of protein/enzyme activity.
✓ It is used in paint industries for quality control and product development.
✓ It is used in environmental and agricultural fields to quantitatively determine organic substances and heavy metals in water.
Artin Azma Mehr is the representative of HACH in Iran, a supplier of analytical equipment for oil and gas and petrochemical laboratories and specialized spectrophotometers such as HACH DR-6000. Contact us to supply the devices and consumables you need.
source:
The Royal Society of Chemistry (RSC), 2009, Spectroscopy in a suitcase