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Filters are made of glass or dyed gelatine between glass

plates and have a limited transmission band at which they

transmit maximally. To understand the use of light filters

consider a bluish-green solution which absorbs light in the

red part of the spectrum. Such a solution when illuminated

by white light absorbs red color wavelengths and emits

bluish-green light together with a small amount of red.

The greater the concentration of the solution the smaller

the amount of red light transmitted. The most sensitive

readings of the galvanometer will therefore be obtained

by allowing only the transmitted red light to activate the

photoelectric cell. The red filter achieves this by stopping

the transmission of bluish-green light and allowing only

the red light to pass through the solution.

Clinical Chemistry 463

In the more expensive type of equipment, a diffraction

grating or a prism is used to obtain the required

wavelength.

In diffraction grating, the white light is dispersed

into a continuous spectrum. By turning a wavelength

adjustment, the grating is rotated and different parts of the

spectrum are allowed to fall onto the photocell.

In glass prism spectrophotometers, light is focused

onto the prism. Light passes through and forms an

extended spectrum. On adjusting the exit slit (wavelength

adjustment) light can pass through the cuvette, and

illuminate the photocell.

Cuvettes and Flow-through Cells

These are used to hold colored solutions and must be

scrupulously clean, with no dirty finger marks or spillage

of fluid on the outside of the optical side. Spillage of fluid

or dirty finger marks will absorb light and interfere in the

measurement of the color. Scratches on the glass must be

avoided and if badly scratched it must be discarded.

In order to speed up laboratory work, a more recent

development in colorimetry is the introduction of followthrough cells. These cells enable colorimetric readings to

be speeded up considerably, since the cells or cuvettes can

be drained without being removed from the colorimeters.

Photoelectric Cell

A photoelectric cell consists of photoelectric elements;

light falling on these elements generates an electric current

which deflects a galvanometer needle, the deflection being

proportional to the light intensity.

Galvanometer

The galvanometer measures the output of the

photosensitive element, and in good instruments a very

sensitive instrument is used.

Requirements of Colorimetric Analysis

When colorimetric determinations are made, it is essential

to ensure that the color being measured is only due to the

substance under investigation and is not due to any of

the reagents used. It is, therefore, essential to include the

following solutions.

1. Test solution

This contains the unknown concentration of the substance

together with the reagents used in the test.

2. Standard solution

This is usually identical to the test solution, except that

it contains a known amount of the substance being

determined and is approximately equal in concentration

to that expected in the test.

3. Blank solution

This solution is identical to both the test and standard

solution and it is carried through the complete test

procedure and contains all the reagents used, but

without any test or standard substance. Any color given

by the reagents used in the analysis can be detected and

eliminated.

In order to be sure that the absorbance is due solely to

the substance under test, the reading given by the ‘blank’

solution must be considered with the reading obtained

from the ‘test’ and ‘standard’ solutions. The photoelectric

absorptiometer is set to read zero absorbance with distilled

water. The blank, test and standard absorbance readings

are recorded, rechecking the zero absorbance between

each reading. The blank reading is then subtracted from

the test and standard reading as follows:

 Test – Blank _________________ × concentration of standard

Standard – Blank

This procedure will usually ensure that only the substance under investigation is being measured. Satisfactory

results are only obtained with absorbance ranging from

0.2 to 0.8, so that if possible the determination should

be modified in order that the lower and upper limits of

deflection fall within this range.

Sources of Error in Photometry

Errors in photometry can be attributed to three sources.

1. Inherent properties of the solution being measured

2. Instrument

3. Operator.

Inherent Properties of the Solution

The factors, which may be included in this group, influence

the absorption of light by the solution and can be the cause

of deviations from Beer’s Law.

a. Chemical Nature of the Solvent and Solution

Deviations from Beer’s law may occur either as a result

of a shift in the shape of a given portion of the absorption

curve as the concentration changes or because of the

absence of a linear relationship between optical density

and concentration. A shift in the shape of a portion of the

absorption curve can indicate a chemical transformation

of a portion of the colored component being analyzed into

a second component of a different color. The production

of a second colored component may also occur due to

an impurity in the solvent in which the original colored

464 Concise Book of Medical Laboratory Technology: Methods and Interpretations component is dissolved. For example, iodine dissolved in

carbon tetrachloride is deep purple but dissolved in alcohol

is brown. The presence of only 1% alcohol as an impurity

in carbon tetrachloride is sufficient to change the color and

hence, the shape of the absorption curve of iodine in carbon

tetrachloride. Thus, the absolute purity of the solvent is very

important in spectrophotometric work. This is particularly

true for analysis carried out in the ultraviolet region. The

breakdown of a linear relationship between optical density

and concentration can be due to the dissociation of a

colorless substance to give colored ions, or vice versa.

b. Exposure to Light

Certain compounds tend to bleach or discolor or get colored

when exposed to light. Such photochemical reactions are

likely to occur when the test sample is stored in a warm,

brightly-lighted room. This may occur while the sample

is in the photometer, if the intensity of illumination is too

high,