Ultraviolet and visible spectrometers have been in general use for the last thirty five years and over this period have become the most important analytical instrument in the modern day laboratory.
In many applications other techniques could be employed but none rival UV-Visible spectrometry for its simplicity, versatility, speed, accuracy and cost-effectiveness.
Spectroscopy involves the analysis of electromagnetic radiation absorb emitted or scattered by molecules or atoms as they undergo transitions between energy levels.
• There are two types of spectroscopy:
1. Atomic spectroscopy
2. Molecular spectroscopy
1.Atomic Spectroscopy: Atomic spectroscopy is the interaction of electromagnetic radiation with atoms.
For example Atomic absorption spectroscopy and Atomic emission spectroscopy
2. Molecular Spectroscopy: Molecular spectroscopy is interaction of electromagnetic radiation with molecules.
For example Nuclear magnetic resonance and Infrared spectroscopy.
Beer Lambert Law
The Beer-Lambert Law states that the concentration of a substance in solution is directly proportional to the ‘absorbance ‘, A, of the solution.
When monochromatic radiation passes through a homogeneous solution in a cell, the intensity of the emitted radiation depends upon the thickness (l) and the concentration (C) of the solution.
There are two types of UV visible spectroscopy:
• Single beam
This instrument represents a fully functional single beam UV visible. It is called single beam because all measurements are based on the intensity of a single beam of light in a single optical beam.
• Double beam
There are two beam. First is sample beam which directed through the sample cell and another is reference beam which is directed around the sample.
The law is only true for monochromatic light, that is light of a single wavelength or narrow band of wavelengths, and provided the physical or chemical state of the substance does not change with concentration.
For UV H2 discharge lamp and deuterium lamp are used. Deuterium lamp provide high intensity emission and adequate continuity in the region of 190-380nm.
For visible light tungsten filament lamp is used, Xenon discharge lamp and mercury arch lamp. Tungsten filament provides a higher output in a cross over region of 320-380nm and longer range up to 900nm.
A Monochromator is an optical device that transmits a mechanically selectable narrow band of wavelength of light or other radiation chosen from a wider range of wavelength available at the input.
It allows passing of radiation of particular wavelength.
It consists of two measures parts:
• Entrance silts
• Exit silts
• Differential grating focusing mirror.
Sample cell is called cuvette.
• For visible region glass cuvette are used.
• For UV silica or quartz cuvette are used.
Light may or may not be absorbed by passage through the sample. This is determined by quantitative measurement of light passing through the instrument by means of detector such as photo multiplier tube, photo voltaic cell, photo conductive cell and barrier layer cell.
Photo Multiplier Tube
Photo multiplier tube is generally used in spectroscopy. It consists of an evacuated tube containing one photocathode and 9 to 16 electrodes known as dynodes. Photocathode is negatively charged and then a photon hits the photocathode. Emission of electrodes takes place due to photovoltaic effect. When incident radiation falls on the metal surface of cathode and get emitted which are attracted towards the first dynode. These with are attracted by the second dynode and with are emitted by second dynode. Hence, the process is repeated at all the dynodes. At the end of dynode chain and anode is present which acts as collector of electrons. The current flowing from anode is directly proportional to photo electron flux generated by photo cathode.
It consists of semi-conductor like selenium which is deposited on a strong metal base such as iron. A thin layer of gold or silver is sprayed over the semiconductor which then acts as second collective electrodes. When radiations fall on the surface, it produces electrons at the selenium silver interface. Since, barriers exist between selenium and iron. The flow of electron is prevented which results in accumulation of electron on the silver surface and produces electrical voltage.
Forensic drug analysis deals with the identification and quantification of illegal drugs. The testing of drugs is generally carried out in two steps, namely, screening and confirmation. UV visible spectroscopy is widely used as a very significant tool for both qualitative and quantitative drug analysis.
Some of the applications of UV/visible spectroscopy in forensic science as discussed below:
• Analysis of dyes and pigments from hair and fibers may appear identical in color with human eye, the fibers may have been dyed with different pigments.
• Analysis of dyes form pen. A UV visible provides a mean a discriminating a similarly colored inks.
• Analysis of drugs- various drug molecule produce characteristics UV-visible spectra. UV Visible molecular absorption is routinely used for the analysis of narcotics and for drug testing. The presence of these drugs can be confirmed from their absorption maxima i.e. λ max or by comparing the UV/visible spectra of these drugs with spectra of authentic sample.
• Analysis of explosives- the organic explosives like nitro aromatic and nitro amines show typical absorption in UV visible region.
• Analysis of paint-dyes and pigments.
• UV visible spectroscopy provide an important method for both qualitative and quantitative analysis in forensic investigations.
• It also found application for determination of alcohol in blood.
• UV visible spectroscopy can also used for study of carbon monoxide poising by analysis of carboxyhemoglobin in forensic Blood Samples.
• Determination of metals in forensic samples can also done with UV visible spectroscopy. Lead, arsenic are sometimes encountered at industrial work places and many time used as poisons. Arsenic salts are commonly used as insecticides in houses.
In addition to these metal ions are also found in paints, dyes, explosives such as gunpowder and in glass. The presence and concentration of these metal ions can be easily measured by UV visible spectroscopy
• UV visible spectroscopy can also used to find the age and for analysis of blood stains commonly present at crime scenes.
• UV visible spectroscopy can be used for quantitative determination of hemoglobin in tooth and bone samples.