In this article we will discuss about:- 1. Meaning of Spectrophotometer 2. Parts of Spectrophotometer 3. Operation.
Meaning of Spectrophotometer:
Spectrophotometer measures light absorption as a function of wavelength in UV as well as visible regions and follows the Beer Lambert’s law of light absorption.
Beer Lambert’s Law:
When a monochromatic light passes through an absorbing medium, the amount of light that is absorbed is directly proportional to the number of light absorbing molecules in that medium or the concentration of substance in that medium.
Unlike calorimeters, in spectrophotometers the compounds can be measured at precise wavelengths.
Parts of Spectrophotometer:
There are six parts in a spectrophotometer:
(1) Light sources,
(2) Condensing lens,
(3) A monochromator,
(4) Sample holders,
(5) Sample detectors, and
There are two light sources i.e. a tungsten lamp which generates visible light and a deuterium or hydrogen lamp which generates UV light. Deuterium lamp gives wider and more intense light in UV region than a hydrogen lamp.
The light from the light source is known as polychromatic or heterochromatic, since it is composed of wide range of wave lengths. The polychromatic light is reflected back using a plane mirror which passes through an entrance slit, condensing lens and falls on to the monochromator. Monochromator disperses the light and the desired wavelength is focussed on the exit slit using the wavelength selector.
These monochromators produce radiations of single wavelength. They are based either upon refraction by a prism or by diffraction by a grating. For visible region, prisms are made up of glass and for UV region, of quartz or silica.
A grating consists of ruled lines (almost 2000 lines per mm) on a transparent or reflecting base. Resolving power of a grating is directly proportional to the closeness of these lines. Compared to prisms gratings are superior since they yield resolutions of the spectrum for the entire range of wavelengths. Use of a double monochromator enhances efficiency of monochromation where a selected part of the spectrum from the first grating is further resolved by a second grating which results in a band width as low as 0.1 mm.
Cuvettes are optically transparent cells made of glass / silica / plastic / quartz.
Plastic and glass cannot be used for light measurements in UV region since they absorb UV light below 310 nm. Silica and quartz can be used for both UV and visible light measurements since they do not absorb UV light. Since quartz absorbs light below 190 nm, cuvettes of lithium fluoride can be used which transmits radiations down to 110 nm.
Oxygen also absorbs light at wavelengths less than 200 nm. Therefore, if spectra are required in this region, the apparatus must be evacuated. Standard cuvettes are made up of quartz and have an optical path of 1 cm and hold one to three ml of solution. Minicuvettes have a capacity of 0.3-0.5 ml.
Photocell or photomultiplier tube:
A photocell (Fig. 1.3) is a photoelectric device which converts light energy into electrical energy. This is then amplified, detected and recorded. The photons strike on a photoelectric cathode in vacuum, causing emission of electrons which is proportional to the intensity of radiation, in photocells. These electrons are attracted by a positive electrode and a current flows, causing potential difference across a resister incorporated in the system. This current is amplified electronically and measured.
A photomultiplier tube (Fig. 1.4) is similar to a photo cell since it has a cathode with photoemissive surface and a wire anode. Besides the photocathode, it has a circular array of nine additional cathodes known as dynodes. Dynode-1 is maintained at a potential of 90 V more positive than that of the photoemissive cathode and because of this, the electrons are accelerated towards it.
Each photoelectron causes emission of several additional electrons when it strikes dynode-1. This in turn is accelerated towards dynode-2 which is 90 V more positive than dynode-1. Several electrons are again emitted for each electron and the process is repeated nine times and for each photon, 106-107 electrons are produced. These amplified electrons flow to the anode and a much larger photoelectric current is generated than that in a photocell.
Operation of Spectrophotometer:
Absorption spectrum and absorption maxima (λ max) of protein.
λ max is the particular wavelength at which light absorbing molecules absorb light maximally for a particular compound. X max of each compound is characteristic of that compound.
λ max of proteins:
1. Make 1 mg/ml and 0.1 mg/ml bovine serum albumin (BSA) in distilled water.
2. Switch on the spectrophotometer and UV lamp and wait for five minutes, for the instrument to warm up.
3. Select the wavelength, say 200 nm.
4. Adjust the instrument to 100% transmittance or 0% absorbency for blank (distilled water) using quartz cuvettes.
5. Replace distilled water with 1 ml BSA and record absorbance.
6. Change the wavelength to 205 nm and repeat steps 4 and 5.
7. Record readings for other wavelengths upto 320 nm and plot absorbance against wavelength (Fig. 1.5).
The wavelength at which protein absorbs the light maximally is the λ max of protein, i.e. 280 nm. 280 nm λ max is due to tryptophan and tyrosine residues in protein. The other λ max between 215-225 is due to the peptide bonds in protein.
X Max of Nucleic Acids
Prepare nucleic acid (DNA at 20-30 µ g/ml) in Tris EDTA. Repeat steps 2-7 of the above experiment and plot a graph (Fig. 1.6).
One A260 unit of double stranded DNA = 50 µg/ml.
One A260 unit of single stranded DNA = 40 µg/ml.
Yeast and Bacterial Cells:
The optical density (OD) of bacterial or yeast cell suspension with a spectrophotometer is a measure of light scattering and not light absorption, since light is scattered by cells. All the transmitted light does not reach the photomultiplier tube and the amount reaching the photomultiplier tube is a function of cell density as well as the distance between the cuvette and the photomultiplier tube.
An absorbance of 1 at 600 nm
= 8 X 108 bacterial cells/ml
For yeasts 1A600 = 1X107 cells/ml.