COLORIMETER
Colorimetry is a common technique used for the
etc. in biological samples after their modification estimation of substances like glucose, urea, creatinine
into a colored
compound.
Light
It is a form of energy. Some of the
properties of light can be explained by considering it as a
particles, whereassome other properties can be stream of
explained by considering it as a wave.
Wavelength of light
When light is considered as wave the distance
between two
Wavelength of that particular type of light. It is designated by successive crests or troughs is called the
(nanometers) or A° (Angstroms). (lambda) and expressed in terms of nm
When ordinary light (white light) is passed
through a prism, it is split into seven different
(colors). This property is called Dispersion. components
The components from bottom to top will have the
folowing order- Violet (V), Indigo (), Blue (B), Green
(G), Yellow(Y), Orange (0), and Red(R). This
vişible light will cover a wàve-length range from
to700nm. 400nm
Electromagnetic Spectrum
When all the types of radiations are arranged in the
increasing order of their wavelengths, it constitutes
the electromagneticspectrum.
Transmittance (T)
Assume that light, whose intensity is lo is passed
through a solution. Let the intensity of the emergent
light be le. Then transmittance (T) can be defined
as the ratio of intensity of the emergent light
of the incident original light, i.e. T= le/lo. to that
Absorbance (A)
It is the negative logarithm of transmittance to the base
ten.
A=-log1oT
Optical Density (OD)
It is the negative logarithm of percentage
transmittance to the base ten.
OD=-log10T%
Department of Biochemistry, Anna Medical College.
Principles oF Colorimetry
Spectrophotometry and colorimetry techniques are based on the estimation of light absorbing nature of
the substances in solution. In colorimetry, only the colored compounds or the compounds capable
of
forming color complexes by reacting with reagents can be analyzed. These techniques are based on two
laws:
Beer's law
Absorbance of the solution is directly proportional to the concentration of the solution (i.e. A & C) or
transmittance of a solution decreases exponentially with the increase in the concentration of the
solution (i.e. T=e-kc)
Lambert's law
Absorbance of a solution is directed proportional to the thickness of the optical path (i.e. A & t)
Components of colorimeter
Polychromatic Light Monochromatic light
Light Slit Lens Filter Cuvette Photo cell Output
Source
Source of light: The tungsten lamp is the source of visible light in colorimeters. Spectrophotometers
will have two sources of light-a tungsten lamp, which emits visible light (Wavelength ranges from
400nm to 700nm) and a Deuterium lamp which emits ultraviolet (UV) radiations (wavelength range
200 to 400 nm).
Monochromator: In colorimeters replaceable colored glass flters are used to get the monochromatic
light. The multiwavelength radiation from the source passes through the filter and radiation of a narrow
band width comes out.
Slit: This is to allow a narrow beam of selected monochromatic light to pass through the sample
solution.
Cuvette: The container to keep the test solution which has to be filled 3/4" of its height. It may be
made of glass or quartz.
Department of Biochemistry, Anna Medical College.
Photocells: The photocells convert quanta of radiation to electrical energy
detected and recorded.
which may be amplified,
Detector: Usually photomultiplier tubes (PMT) which are based on photo
ionic effect are used as
detectors. (Photo ionic effect: Light photons impinging on a metal surface ín
vacuum cause emission of
electrons in proportion to the intensity of the radiation). The light that
comes out of the cuvette falls on
this and getsconverted into an electrical signal.
Recorder: The electrical signal from PMT is amplified and then recorded
by the galvanometer. Usually,
the recorders are calibrated in such a way that they
directly give the absorbance or transmittance
values.
Applications: These techniques are commonly employed in diagnostic and research
the quantitative estimation of different compounds in laboratories for
biological fluids. Examples: Blood glucose, urea,
cholesterol, creatinine, bilirubin, CSF protein etc.
Spectrophotometer:
Aspectrophotometer is a more sophisticated version of
colorimeter. It differs from colorimeter
in the following ways:
a. It can measure the concentration of a colored or
colorless solution
b. Measurement can be made both in UV as
well as visible range.
C. Monochromator used is prism or grating and not
manually changed filters.
Other advanced variants of Spectrophotometry include:
Absorption spectroscopy, Flame photometry, Nephelometry and Turbidimetry.
Department of Biochemistry, Anna Medical College.
