This document describes the components and operation of a spectrophotometer. The major components are a light source, monochromator, sample chamber, detector, and recorder/meter. The monochromator selects a specific wavelength of light from the polychromatic light source. Common types of monochromators include filters, prisms, and diffraction gratings. Samples are placed in cuvettes in the sample chamber. Double-beam spectrophotometers compare the light transmitted through a sample cuvette to a reference cuvette containing only solvent. Absorbance is then measured to analyze samples.

1. 1
Spectrophotometric Instruments
◘ The major components of a spectrophotometer is :-
• light source .
• Monochromator .
• Sample chamber.
• Detector .
• a meter or recorder .
- All of these components are
usually under the control of a
computer .
◘ Is used to measure absorbance experimentally .
◘ Operation :
- This instrument produce light of preselected wavelength , direct it
through the sample ( usually dissolved in a solvent and placed in a
cuvette )
- Measure the intensity of light transmitted by the sample .

2. 2
◘ components of a spectrophotometer:
1-Light source :
♦ The Incandescent Tungsten
( Tungsten-iodide lamp )
• The most common source of light for
work in the visible and near-infrared
region .
• Only about 15% of radiant energy
emitted falls in the visible region ,
with most emitted as near-infrared .
• Often , a heat-absorbing filter is inserted between
the lamp and the sample to absorb the infrared
radiation .
♦ The Deuterium-discharge lamp & The Mercury-arc
lamp :
• Most commonly used for ultraviolet UV work .
♣ The Deuterium-discharge lamp
- Provides continuous emission down to 165 nm .
♣ The Mercury-arc lamp
• low-pressure mercury lamps :
- Emit a sharp-line spectrum , with both UV and visible lines .
• Medium and high-pressure mercury lamps :
- Emit a continuum from UV to the mid visible region .

3. 3
♦ The most important factors for a light source are :
• Range ( spectral distribution within the range ) .
• The source of radiant production .
• Stability of the radiant energy .
• Temperature .
2-Monochromator :-
• Lamps produce continuous emission of all wavelengths within their
energy .
• Therefore , a spectrophotometer must have an optical system to select
monochromatic light ( light of a specific wavelength ) which is the
function of a monochromator .
• Monochromator select monochromatic light from polychromatic light
- monochromatic light : one wavelength ( narrow band ) , laterally (
one color )
- polychromatic light : many wavelengths ( wide band ) , laterally (
many color )
• The reflected light rays in the monochromator constructively and
destructively interfere depending upon whether they are in phase or
out of phase .

4. 4
• The degree of wavelength isolation is a function of :-
- the type of device used .
- the width of entrance and exit slits .
• The band pass of a monochromator defines the range of wavelengths
transmitted and is calculated as width at more than half the maximum
transmittance .

5. 5
◘ Monochromator types :
1- Colored-glass Filters :
• The least expensive .
• Usually pass a relatively band of radiant energy .
• have a low transmittance of the selected wavelength .
• Although not precise , they are simple , inexpensive , and useful .
2- Interference Filters :
♦ The principle:
• produce monochromatic light based on the principle of
constructive interference of waves .
♦ Component :
• Two pieces of glass , each mirrored on one side , are
separated by a transparent spacer that is precisely one-half the
desired wavelength .
♦ Operation :
• light waves enter one side of the filter and are reflected at the
second surface .
• wavelengths that are twice the space between the two glass surfaces will reflect
back and forth , reinforcing others of the same wavelengths , and finally passing on
through .
• Other wavelengths will cancel out because of phase differences (destructive
interference ) .
• Because interference filters also transmit multiples of the desired wavelengths , they
require accessory filters to eliminate these harmonic wavelengths .
♦ Interference filters can be constructed to pass a very narrow range of wavelengths
with good efficiency .

6. 6
3- The Prism :
• A narrow beam of light focused on a prism is refracted as it enters the
more dense glass .
• Dispersion by prism depends on refraction of light which is wavelength
dependent .
• Short wavelengths ,higher energy ( ex: violet color ) are refracted more
than long wavelengths , lower energy (ex: red color ) , result in dispersion
of white light into a continuous spectrum .
• The poly-chromatic white light is dispersed to its individual colors .
• The prism can be rotated , allowing only the desired wavelength to pass
through an exit slit .

7. 7
4- Diffraction Gratings :
• Are most commonly used as
monochromators .
• consist of many parallel grooves (
15,000 or 30,000 per inch ) etched
onto a polish surface .
• The principle :
- Diffraction , the separation of
light into component wavelengths , is based on the
principle that wavelengths bend as they pass a sharp
corner .
- The degree of bending depends on the wavelength .
• The Operation :
- The wavelengths move past the corners , wave fronts are formed .
- Those that are ( in phase ) reinforce one another .
- Those that are not in phase ( out of phase ) cancel out and disappear .
- This results in complete spectra .
• Gratings with very fine line rulings produce a widely dispersed spectrum ,
• They produce linear spectra , called orders , in both directions from the
entrance slits .
• Because the multiple spectra have a tendency to cause stray light
problems , accessory filters are used .

8. 8
5- Sample Chamber & ( sample cell ) :
♦ sample cell or cuvette may be rounded or square
• Square cuvettes :
- have plane-parallel optical surfaces and a
constant light path .
- They have an advantage over round cuvettes in
that there is less error from the lens effect ,
orientation in the spectrophotometer , and
refraction .
• cuvettes with scratched optical surfaces scatter light and should be
discarded .
• Inexpensive glass cuvettes can be used for applications in the visible
range , but they absorb light in the UV region .
• Quartz cuvettes must , therefore ,be used for applications requiring UV
radiation , they may be used throughout the UV and visible regions (200-
800 nm) .
• Disposable plastic cuvettes are now commercially available in
polymethacrylate ( 280-800 nm) .

9. 9
♦ Sample Chamber :
1-Single-beam spectrophotometer :
• Those Holding only one cuvette at time .
2-Double-beam spectrophotometer :
• Those holding two cuvettes , one for a reference , usually ( solvent ) , and one
for a sample .
♣ The operation :
- In double-beam optics , the light beam is split into two paths by directing it
through a monochromator .
- the two beams , which are of identical wavelength and intensity , pass through
the sample cell ( Analyte + Solvent ) and reference chamber ( Solvent only ) .