Spectrometry:

Most of the optical techniques are described as Photometry.  In short, photometry is the detection of light radiation and changes in radiation energy, usually within the visible spectrum.

In most practical applications, light originated from a light source and it passes through a chemical compound with the sample placed in its path.  The resultant light is then detected and analyzed.  This form of measurement is called absorptiometry.  Alternatively, the sample itself can act as a light source as is the case of bioluminesence.

Absorptiometry:

Absorbance- is the most common optical assay technique used in clinical chemistry.

If a beam of light from a light source passes through a cuvette containing a chemical compound in solution, part of the beam can be absorbed.  In formal terms one can say that some photons collide with ions or molecules in the solution and thus impart their energy to these.  These applies only to those photons with precisely the correct energy level, i.e. only light of a certain wavelength is absorbed.

The intensity of light leaving the sample will thus be less than its intensity when it entered.  The loss of intensity is a function of the concentration of the compound.

Intensity of light leaving the sample will thus be less than its intensity is a function of the concentration of the compound.

Intensity is generally understood to mean a stream of radiation which is power and can thus be measured in units such as Watts.  Since measurements always relate to a ratio between two intensities, the actual units of measurement are, however, not important.  Instead, convenient units such as readings on an arbitrary scale are normally used.

Calorimetry is the term applied to measurement of light in the visible portion of the spectral range.  The extent to which light is absorbed by a compound in solution depends on the wavelength of the incident light and the colour of the solution.  For clinical chemistry applications the compound usually absorbs light in the visible range of the electromagnetic spectrum (400 to 700).

For certain applications, this range is extended into the ultraviolet (UV) region.  Even though the human eye cannot detect UV radiation, it is still termed light.

Each absorbing compound has a typical absorption spectrum as detailed in the above table.  If the absorbance peak of a compound is within the blue wavelength region, the eye will see the solution as a yellow colour since yellow is the complementary colour to blue.  The more blue light that is absorbed, the more yellow the solution will appear.

If the resultant yellow colour is compared with a given scale of yellow standards, the human eye can estimate the concentration of the compound in the solution.

When an electronic detector is substituted for the human eye, the decrease in light energy caused by an absorbing substance is easily registered and therefore be measured.  In practice, monochromatic light (light  is of single colour) used as the light source rather than polychromatic light (light of several colours).            
Monochromatic light can be produced from white light light by the use of such wavelength selection devices as filters, prisms or diffraction gratings.  These wavelength selection devices are described later in this chapter.

Absorption Photometer:

Simple photometers often use a simple meter or a chart recorder as a registering device while automated photometers use a microprocessor or computer on-line with the photometer.

The path length of each individual light ray through a cuvette must be identical.  Hence a cuvette with parallel sides must receive a parallel light beam.  If the cuvette is cylindrical, every light ray must pass through an imaginary point at its center, which is achieved by placing the cuvette between two cylindrical lenses.

Depending on the particular application, photometers can be divided into several groups.

Filter Photometers - where the wavelengths are selected by coloured filters or interference filters.

Spectral Line Photometers - where the light is supplied at discrete wavelengths using a spectral line source.

Spectrophotometers - where wavelengths are selected by a dispersing device, e.g. a prism or diffraction grating.

Light sources:

Two different types of light sources are used to produce light.  One uses blackbody radiation such as the tungsten filament lamp and the other which uses radiation produced by specific energy, such as a gas discharge lamp or LED.

Tungsten halogen lamps may also be used.  These consist of a tungsten filament in a quartz envelope which also contains traces of a halogen such as iodine.  These types of lamp give intense light in the visible and near UV range, but, Tungsten lamps can only be usefully used for the production of light with wavelengths down to 340 nm.  In order to work satisfactorily in the UV region, different light sources are therefore required.  A gas discharge lamp is the most satisfactory.


It should be noted that for UV radiation below 340 nm the bulb/tube shall have atleast a window area made of quartz, since glass appears to be opaque to UV radiations.

Wavelength selection:

In absorbance chemistry it is often necessary to utilise narrow wavelength bands.  In some instances a line source provides the narrow bands required, but more often it is desirable to select a band of wavelengths from a continous source of radiation, such as the halogen lamp.  This gives greater flexibility in choosing the wavelength bands.

There are two different methods of wavelength slection:
1. by using a filter
or
2. by the use of a dispersing device such as a prism or a diffraction grating in a monochromator.

Filters:

Glass filter:

The simplest and economical way to isolate a definite range of wavelengths is to use a filter of coloured glass or other coloured material which transmits some wavelengths and absorbs others.

The main dsadvantage with these types of filters is that they allow a considerable amount of the radiation outside their pass band to be transmitted.

Interference Filters:
The most common types of filters used in photometers based on electronics are interference filters.  This type of filters is composed of a very thin, transperent plate with a semi-transplant mettalic film on each side.  The films are applied through evaporation in a vacuum.

The thickness of the plate determines the wavelength at which radiation can pass through the filter.  When white light strikes the filter, at right angles to the surface, part is reflected off the first metallic film while the rest passes through.  The light which passes through is similarly split when it strikes the second metallic mirror.  If this second reflected part has the correct wavelength then it will be reflected again, off the inside of the first metallic mirror, in phase with the incoming light of the same wavelength.  Therefore, light at those special wavelength is reinforced.  Light at other wavelengths interfere destructively, so that essentially no energy passes through the filter.
Reinforcement occurs according to the formula
lamda = 2d/m
where lamda is the wavelength of light, d is the effective thickness of the layer and m is an integer.

Prisms:

To obtain better definition a prism can be used as an alternative to the filter.  A prism seperates white light into its components.  The entrance slit acts as a monochromatic light source and is located at the focul point of the first lens, because the light beam must be parallel when it passes through the prism.  On leaving the prisms all light rays with the same wavelengths are still parallel, but red rays have been deflected less than violet, etc.  The lens following the prism concentrates these rays into one plane, the focul plane, where the exit slit is placed.

The part of the spectrum required can be selected by rotation of the prism.  This techniqe produces monochromatic light, and the combind prism and mounting is called a monochromator.

Gratings:

Diffraction grating provide an alternative means of producing monochromatic light.  A grating comprises a large number of parallel lines or grooves etched closely together on a highly polished surface such as steel or glass.

In practice several spectra of different orders are produced and by using higher order spectra the dispersion can be increased.

The dispersion capability of a grating is determined by the total number of grating grooves and by the order number of the spectrum, but is not dependent on the light wavelength or the grating space.

The advantages of using a grating is that the dispersion of the different wavelengths is linear over the whole spectrum making it easier to calibrate a linear wavelength scale.  By rotating the grating, different parts of the spectrum can be made to pass through the exit slit.

Grating Mounting:

There are many ways of mounting a grating in a mono-chromator.  the most common method is the Ebert mounting.  The required wavelength is selected by rotating the grating around its central axis.  The position of the grating determines which wavelength reaches the exit slit/  

Having obtained a narrow band of energy, a means of measuring the energy is required.  This accomplished by converting light energy into an electric current by means of a transducer.  A transducer is a device which converts one from of energy into another.  Two common transducers, so called photo-emissive devices, are the phototube and the photo-multiplier, described below.

Phototube: The simplest type consists of two electrodes in a glass vacuum tube.  The cathode is coated with an alkali metal oxide such as caesium oxide.  When light strikes the electrode, some negatively charged electrons are released.  These electrons are attracted to the positively charged anode, causing an electric current, propotional to the incident light, to flow in the anode circuit.  The anode current of the phototube causes an output voltage to be developed across the anode load resistor.  This voltage requires amplification prior to measurement.

Photo-multiplier:

The second photo-emissive device is the photo-multiplier.  The photo-multiplier contains a series of collecting dynodes (anodes) set at progressively increasing potentials.  Light strikes the cathode and releases a few electrons, these accelerate towards a few electrons, these accelerate towards the first dynode and on impact, release a secondary cloud of electrons.  The electrons released by the secondary emission are then accelerated towards the next dynode liberating even more electrons as they strike the dynode. 

Measuring Cells or Cuvettes:

In automated clinical analyser systems the measuring cell is usually either a stopflow cell or a cuvette.  The name stop-flow indicates that a flow of samples passes a cell but stops each time an absorbance measurement is taken.  The cuvette may be discarded or alternatively washed after each measurement.  In continous flow systems, the absorption is continuously monitored.

In manual use a cuvette is often a plain parallel container of glass or crystal with a volume ranging from a few micro-liters to several mL.  Cylindrical cuvettes of polystyrene or acrylate are conveniently used in many photometers.

Some instruments use cuvettes arranged in circular trays allowing serial measurements to be interpreted by the analysers.
        
Slit Width:

To obtain the best results, the absorbance should be measured using the narrowest possible slit, otherwise significant spectrum details can be lost.  This applies particualrity to qualitative analyses.

There are, however, a number of other factors which limit minimum bandwidth, principally the "radiant power" of the light source, and the sensitivity of the detection system.      


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