Visible - Ultraviolet SpectroscopyWhen a compound absorbs certain amount of light against certain wavelengths, it gives its characteristic absorption spectrum. The colored compound will have one or more absorption maximum in the visible region of the spectrum i.e. between 400-700nm. One of the main reasons for absorption spectra in visible and ultraviolet region (190-390nm) is due to the energy transition of the electron in the molecule. Normally (-bonding electron of carbon-carbon double bonds and lone pairs of oxygen and nitrogen atoms are involved.

Principle

UV and visible spectrophotometry works on the principle of Lambert and Beers law in which when light of monochromatic radiation or heterogeneous in nature impinges upon a homogeneous medium , a portion of the incident light is reflected, a portion is absorbed within the medium and the remaining light is transmitted. If the intensity of the incident light is expressed as I0, that of the absorbed light by Ia, that of the transmitted light by It and that of the reflected light by Ir then,I= Ia + It + IrDescription of Instrument

The optical system of simple UV-Visble spectrophotometer consists of monochromators, cuvettes, photocells, slits and recorder.

Light Source The light source is usually a tungsten lamp for the visible spectrophotometer and either a hydrogen or deuterium lamp is used in UV spectrophotometer.

Monochromators- Monochromators are optical systems which can produce parallel beam of radiation of single wavelength from a multi wavelength source of radiation. The monochromatic wavelength is possible due to refraction by a prism or diffraction by a grating. In visible spectrophotometer the prism is made up of glass. However, it is made up of quartz or silica in UV spectrophotometer since glass absorbs at wavelengths below 400nm. Diffraction grating monochromators consists of a series of ruled lines on a transparent base. A series of overlapping spectra is formed by diffraction of white light. In a monochromator prism can pre-select a portion of spectrum of light which is then diffracted to obtain monochromatic light.Cuvettes- Cuvette is an optically transparent cell in which the material under study is dissolved in a suitable solvent and placed to check absorbency. The glass cuvette and quartz cuvette is used in visible and UV spectrophotometer respectively. Usually in a spectrophotometer cuvette holder can hold between two to four cuvettes. The cuvette is used as reference cuvette and test cuvette. The test cuvette is used to set spectrophotometer to zero absorbency. Absorption cuvette is always positioned in the light path of an instrument. The optical path length of the normally used cuvettes is about 1cm and requires 2.5-3.0 cm3 of sample for taking readings.

Photocells- Photocells transform light radiation into electrical energy which is then amplified, detected and recorded. When photons fall on a metal surface in vacuum condition, emission of electrons takes place in proportion to the intensity of radiation. The ejected electrons are then attracted by the positive electrode causing a flow of current, in turn generating a potential difference across a resistor in the system. The absorption of light is accurately measured after electronically amplifying this potential. Typical photocells that are sensitive to different wavelengths are used accordingly.Slits- The variation of the slit width ensures adequate radiation reaching the photocell and the absorbency depends on the slit width. Zero absorbency can be achieved in most spectrophotometers by adjusting the slit width. It is due to its effect on the bandwidth and the variation in the sensitivity of the photo cell with Wavelength. A narrow slit width is highly preferred to obtain reliable data in spectrophotometer.

Recorder (Read Out) Recorder is able to scan the spectrum as well as measure variation in the absorption at predetermined wavelength with time. The better recorder can scan the spectrum at varying speeds.

Operation

In spectrophotometer light from the tungsten lamp is focused on to the entrance slit by condensing lens. The light from the slit is collimated and directed on to the grating which disperses the beam and the spectrum obtained, as a result is focused on to the exit slit. Various wavelengths are scanned by rotating the grating over its axis by rotating the wavelength disc which is coupled to the grating mounting. The monochromatic light is isolated by the exit slit passes through a blank, standard or sample placed in cuvette and the transmitted light falls on a photo diode. The output signal from the photo diode is amplified, processed read to transmittance (T), absorbance (A) and concentration(C) as selected.

Atomic Absorption Spectroscopy

PrincipleAtomic absorption spectroscopy (AAS) is based on volatilization of atoms in a flame causing them to absorb light of specific wavelength. When the molecules come in contact with the flame their atoms gets dissociated due to vaporization and the gaseous atoms in the ground state absorb light of specific wavelength. Absorption lines are characteristic to atoms present in the compound. Atomic absorption spectrophotometers measure the absorption of a beam of monochromatic light by atoms in the flame. The number of atoms present in the light path can determine the absorption of energy.In a AAS the wavelength of radiation emitted on volatilization of element in a flame may be readily resolved into line spectra. The amount of radiation absorbed is proportional to the number of excited atoms present.

Instrumentation

The main components of an atomic absorption spectrometer are the following.

Radiation source- The most commonly used radiation source to produce a beam of radiation with a narrow bandwidth are hollow cathode tube , discharge lamps, or white light with double monochromators. The hollow cathode tube consists of a tungsten anode and a cylindrical cathode in a sealed inert gas tube. Utility of the discharge lamps are specific to the element.

Nebulisers- Nebulisers are otherwise known as atomizers. These are spray type in which a stream of air passes over a capillary tube dipping into the test solution. A very tiny drop of test solution is produced which is then passed with the help of direct injection system along with the air stream into the burner for volatilization of atom.

Various gases are mixed with air to produce flame. These mixtures are air natural gas, air propane gas and air acetylene gas, which can generate temperatures from 1500-25000C.Monochromators and detectors- A simple filter, prism or grating monochromators are commonly used in AAS. Instruments with both single and double beam optics are used. Photocells, photodiodes etc are used as detectors. The read out for AAS is in the visible region. i.e., the extinction values range from zero to infinity. The desirable wavelength falls between 190-800nm.

Application of AAS

1. Extensively used in biochemistry to study the body fluid composition.

2. Elements like sodium, potassium, calcium, magnesium levels can be measured directly.

3. In order to study copper, lead and mercury in the biological fluids and in the food industry.

4. AAS is extensively used to study elements in plant after extraction from the plant sample.

5. Both quantitative and qualitative analyses are possible using AAS.

Photo

cell

Ref.

Cuvette

Sample

Cuvette

Read out device

Light Source

Mirror

Mirror

Chopper

Light Source

Monochromator

Flame/

Nebulizer

Sample

Monochromator

Detector

Amplifier

Read Out Device