Understanding Light and Colour (With Diagram)| Microbiology

Let us learn about understanding light and colour.

It is important to understand and to differentiate this technique and others which involve measuring electromagnetic radiation from various parts of the spectrum.

The following table 22.1 should clarify the types of radiation that constitute the electromagnetic spectrum: 

For those who are unfamiliar with the definition of wavelength and its units here is a brief guide.

Radiation may be considered as a wave Fig. 22.1. The wavelength is the distance between two successive peaks of that wave.

The wavelengths in the table are expressed in nanometers (nm) these are related to metres thus:

1 nanometer = 10 ^{− 9} metre

Most of us are more familiar with the visible UV and IR segments of the spectrum so lets look at these regions more closely and label them according to the kind of measurements made in the respectie regions.

Colorimetry is just one of the types of photometric analysis techniques, i.e., it is a light measuring analytical procedure Fig. 22.2.

Colorimetric measurements are made using a white light source which is passed through a colour filter or alternative wavelength selection device. This incident light then passes through a cuvette containing a chemical compound in solution. The intensity of the light leaving the sample will be less than the light entering the cutette. The loss of light or absorption is proportional to the concentration of the compound.

Colorimetry however only applies to measurements made in the visible region of the electromagnetic spectrum, e.g., (380 -780 nm) The extent to which light is absorbed by a sample is dependant upon many factors. The main general contributors are the wavelength of the incident light and the color of the solution.

Each compound in solution has a typical (and usually unique) absorption spectrum, an example is shown in fig. 22.3

The spectrum is a pattern of the amount of light absorbed by the substance in the solution plotted against the wavelength of the light.

In most cases the spectrum will have a peak i.e., a wavelength at which absorption is at a maximum. This is often referred to as the λ_max for the compound in question.

If the absorption is being quantified it is essential that it is measured as close as possible to the λ_max. Sensitivity is reduced at any other wavelength.

Different objects absorb some wavelengths and reflect others. If white light passes through a yellow solution, it absorbs all colours except yellow. Similarly, a book cover appears red since it absorbs all colours except red. If solution is clear and colourless it has not absorbed any visible radiation and therefore all the white light is transmitted, i.e., it is transparent.

The solution absorbs blue light strong a λ_max at460nm and therefore appears yellow.

If the concentration of the yellow solution is reduced by half the two solutions will give curves shown. Therefore for greatest sensitivity and linearity it is essential to limit the measuring wavelength to the area of highest absorption.

Figure 22.4 shows that the correct wavelength at which to measure a solution is the one which gives greatest absorption.

The wavelength or colour filter that will produce the maximum absorbance can be selected in two ways:

1. Take readings throughout the spectrum on a typical standard solution of the substance under investigation and establish the peak wavelength.

2. Choose a filter of the complementary colour to the standard solution.

Wavelength Selection:

There are several options open to the manufacturer of a colorimeter when deciding how to select the wavelength, i.e., produce monochromatic radiation (one wavelength band) from polychromatic radiation (white light).

These basic options are:

a. Gelatin filters

b. Interference filters

c. Grating monochromators

d. Prisms

a. Gelatin Filters:

These are low cost selection divides which produce or transmit a wide band of radiation usually a 20 nm. Fortunately most colorimetric analyses have a wide absorption band which allows excellent results to be obtained from a simple colorimeter. The most common type of gelatin filter is constructed by sandwiching a thin layer of dyed gelatin of the desired colour between two thin glass plates.

There are two drawbacks which can be encountered using gelatin filters:

1. They have a wide bandpass (Fig. 22.5), which can lead to non-linearity in standard curves.

2. They absorb approximately 30-40% of all incident radiation thereby reducing energy throughput to the detector.

However, these filters are eminently suitable for most general applications.

(Glass Filters, Coloured glass filters are now more or less historical selection devices in colorimeters and have very wide band passes often up to 150nm. Specific wavelengths can however be achieved by using a combination of glass filters).

To ensure all wavelengths in the visible spectrum are catered for approximately 8 Gelatin filters are required. A typical range of filters will have the following transmission curves.

b. Interference Filters:

These are used to select wavelengths more accurately by providing a narrow bandpass typically of around 10nm. The interference filter also only absorbs approximately 10% of the incident radiation over the whole spectrum thereby allowing light of higher intensity to reach the detector.

The theory of operation of an interference filter is fairly complicated but has been simplified below:

An interference filter comprises of several highly reflecting but partially transmitting films of silver separated by thin layers of transparent dielectric material (often magnesium fluoride (MgF2). This is also referred to as an MD or metallic dielectric filter).

When white (polychromatic) light passes through the dielectric layers multiple reflections appear between the semi-transparent mirrors. However some energy from the light beams passes straight through the filter. It is this wavelength which is desired for analysis. If the dielectric layer thickness is altered slightly the resultant wavelength is changed.