Instrumentation: �Spectrophotometer, Colorimeter

By Dr J Onubi

MBBS, MSc, FMC Path

Table of contents

• Definition
• Types
• Components
• Principles
• Conclusion

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Definition

• Spectrophotometer-

an instrument for making photometric measurements

• It is used to measure the light transmitted by a solution to determine the concentration of the light absorbing substance in the solution.

Spectrophotometer

Colorimeter

Types

• 1. single-beam spectrophotometer
• 2. double-beam spectrophotometer

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• Single-beam spectrophotometer

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light source Monochromator Cuvet Detector Read out/meter

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• Double-beam spectrophotometer

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Components

• Light source
• Monochromator
• Cuvet
• Photodetectors
• Read out devices

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Light Source

• . Incandescent lambs. Tungsten light bulbs – has low pressure of iodine or bromine vapor within the lamp which increases the life time. E.g the quartz–halogen lamp.
• A tungsten light source does not supply sufficient radiant energy for measurements below 320nm.
• In the uv = use of low pressure mercury vapor- lamp. No continuous spectrum.

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• Hydrogen lamb:- provides sources of continuous spectra in the uv region as do high-pressure mercury and xenon arc lamps.
• A deuterium lamp is more stable and has a longer life than a hydrogen lamp. The lamps operate for 2000-5000hrs before replacement is necessary.

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• 2. Laser sources- light amplification by stimulated emission of radiation (laser). Certain materials have the capability of absorbing energy and storing all or part of it. If the loss of absorbed energy is forbidden by quantum mechanical rules, the material will remain in an excited state.

Monochromator- used for isolation of radiant energy.

• Filters –thin layer of coloured glass.
• Prisms- a prism separates white light into a continuous spectrum by refraction.
• Diffraction gratings – prepared by depositing a thin layer of aluminum- copper alloy on the surface of a flat glass plate, then ruling many small parallel grooves into the metal coating. Better gratings contain 1000 to 2000 lines/mm and must be made with great care. Each line ruled on the grating when illuminated, giving rise to a tiny spectrum. Waves fronts are formed that reinforce those wavelengths in phase and cancel those not in phase. The net result is a uniform linear spectrum.

• . Cuvets –round, square or rectangular constructed from

-Glass- visible portion of spectrum

- Silica(quartz)- mostly reading <340nm

- Plastic- disposable, single line applications.

• optical surfaces must have constant light path, Clean and clear.

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• Photodetectors.- devices that convert light into an electric signal that is proportional to the no of phatons striking its photosensitive surface.

-Photomultiplier-uv and visible regions of the spectrum

-Solid state devices – photodiodes

-photodiodes arrays

-Charge–coupled detectors

-Barrier layer cells used in older instruments.

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• A photomultiplier is an electron tube that is capable of significantly amplifying a current.
• Photo multiplier tubes have extremely rapid response times.
• They are very sensitive and do not show on much fatigue as other detectors.
• F. Readouts devices.
• G. Recorders.

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