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Clinical Chemistry

C H A P T E R

COLORIMETRY

Colorimetry is the science that deals with the

measurement of the capacity of a chemical, colored

system to absorb light. Since, it makes specific quantitative

measurements, it is very useful and widely used in

laboratories in the form of colorimeter or spectrophotometer. To understand colorimetry, it is essential to have

some knowledge and understanding of what is meant by

Light, Color and Beer’s Law.

¾ Light is a form of energy (radiant energy)

¾ It moves in space in the form of waves like the

electromagnetic waves.

¾ The peak of the wave is called the CREST.

¾ The lowest point of the wave is called the Trough.

¾ The distance between two identical points on a wave

cycle is called the wavelength.

¾ The unit of measure for wavelength is nanometer

(nm).

¾ Wavelengths are also expressed as lambda (λ).

The colors are the wavelength what we see. It is the

wavelength that determines the color of the light. The

human eye can only see the wavelengths of energy between

about 400 and 750 nm. This is called the visible spectrum.

The total light spectrum can be divided into 3 distinct

regions—the ultraviolet region, the visible region and the

infrared region. The wavelengths of the various regions

and colors are shown below.

Light whose wavelength is 400 nm is violet. Light with

wavelength less than 400 nm is not visible to the human eye

and is known as ultraviolet. Light with wavelengths of more

than 700 is not visible and is known as infrared light. The

visible spectrum occurs between the wavelengths of 400

and 700 nm. Here we have the colors of violet, blue, green,

yellow, orange and red or the “rainbow”. Thus, white light

is seen colorless, it is composed of all colors of the visible

spectrum.

The color of a substance will depend on the wavelength

absorbed by the substance and which are transmitted to

the observer’s eye.

Beer’s Law

When a colored solution is illuminated with monochromatic light, its absorbance is directly proportional to

the concentration of the colored solution when the light

path is constant.

Absorbance α Concentration

 (when light path length is constant)

Lambert’s Law

When a colored solution is illuminated with light, its

absorbance is directly proportional to the light path when

concentration of the solution is constant.

Absorbance α Length,

 (when concentration is constant).

If we combine both we get the Beer Lambert’s Law;

“When a colored solution illuminated with monochromatic light, it’s absorbance is proportional to the

concentration of the colored solution and the length of the

light path.”

Absorbance α Concentration X Length

 (A) (C) (L)

 A = ∈ CL

Where ∈ is the molar absorption coefficient.

In all the colorimetric determinations, a reference

standard of known concentration is used and its color

intensity is compared with color intensity of the test sample,

462 Concise Book of Medical Laboratory Technology: Methods and Interpretations

At= ∈CtL

As = ∈ Cs L (t = test, s = standard)

Since, the same cuvette is used for the test and standard,

L is constant.

At = Ct

As = Cs

If concentration of the standard, i.e. Cs is known

then Ct = At × Cs ______

 As

Calculation of Absorbance (A)

Usually colorimeters measure transmittance rather than

absorbance. Transmittance and Absorbance has an inverse

relationship.

T is the ratio of intensity of emergent light (le) to the

intensity of incident light (lo)

T = Ie / Io

A = –log T%

A = 1 / Log T% or A = – log T%

A = – log T%

 = log 1/T%

 = log 100–log T = 2.0–logT

In clinical chemistry, there are two ways of expressing

the amount of light absorbed by a solution. These are:

Amount Transmission (%T)

Percent transmission is the amount of light, which passes

through a colored solution compared to the amount of

light, which passes through a blank or colorless solution.

As the concentration of the colored solution increases

the amount of light absorbed increases while the %T

decreases.

Optical Density (OD)

The OD may be calculated from the %T and is the units

preferred in clinical chemistries, the reason OD is usually

preferred is that there is a direct relationship between

the concentration of a solution and the OD, i.e. as the

concentration of a solution increases, the absorbance or

OD also increases.

By taking the unknown, we can use the derived formula

from Beer’s law to find the concentration of the unknown,

which is:

Concentration unknown OD unknown _______________________ = ______________

Concentration standard OD standard

 OD unknown i.e. Concentration = ______________________ × concentration

 OD standard of standard of unknown

PHOTOMETER

The basic components of a photometer are as shown

above. They are:

¾ Light source

¾ Filter or monochromator

¾ Cuvette

¾ Photodiode cell (receiving light signals)

¾ Galvanometer.

Light Source

A tungsten filament lamp is usually used as source

for light (radiant energy). Its intensity varies upon the

type of photometers. A uniform voltage supply is very

important for a stable source of light. Ageing of the lamp

or accumulation if dirt can result in range in absorbance

reading.

Wavelength Selectors

In most instruments filters are used for this purpose. The

filter chosen is usually complementary to the color of the

solution to be measured (see table below).

Color of solution Usual filter

Blue Yellow

Bluish-green Red

Purple Green

Red Bluish-green

Yellow Blue

Yellowish-green Violet