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Colorimetry is the technique frequently used in
biochemical investigations,involves the quantitative
estimation of colors.
Color can be produced by any substance when it
binds with color forming chromogens.
The difference in color intensity results in the
difference in the absorption of light.
The intensity of color is directly proportional to the
concentration of the compound being measured
Wavelength between 400nm to 700nm form the
visible spectrum of light
visible band of light in electromagnetic spectrum
400-420 Visible Violet Green-yellow
420-500 Visible Blue yellow
500-570 Visible Green Red
570-600 Visible yellow Blue
600-630 Visible orange Green-blue
630-700 Visible Red Green
Light falling on a color solution is either absorbed,
reflected or transmitted.
Io=It + Ia
1. The nature of light absorbing substance.
2. Wavelength of light and
3. Amount of light absorbing substance in
the light path, which in turn depends on
the concentration of light absorbing
substance and depth of the solution
through which light passes.
(a) Relation between absorbance & concentration.
(b) Percentage transmission & concentration.
The relationship between concentration of
the compound and color intensity is given
by Beer’s law and Lamberts law
• When monochromatic light passes through a light
absorbing medium, the intensity of the transmitted
light decreases exponentially as the concentration
of the light absorbing material increases.
• Where A is light absorbed and C is concentration
of the solution.
When monochromatic light passes through a
coloured solution, the amount of light absorbed
increases with the increase in thickness of the layer
of the solution through which the light passes.
Where L = length of light path
By combining above equations,we get
A α CL
Where k=constant for coloured solution
For standard solution : As =Ks Cs Ls
For unknown solution : Au =Ku Cu Lu
Au =absorbance of unknown solution
Cu = conc of unknown solution
AS =absorbance of std solution
CS = conc of std solution
But Ks =Ku & Ls =Lu
Au/As = Cu/Cs
Cu= Au/As X Cs
Common source is a tungsten-filament lamp,
higher powered tungsten –halogen (quartz-iodine)
Factor of light source are range, spectral
distribution, stability of radiant energy and
Mono = single. Chromatic- colour
Monochromatic light is the single colour band
Monochromator and filters are used to split
the light from the light source.
Simple filters are either coloured glass or
suitably dyed gelatin sandwiched in a glass.
filters range is 400-680 nm
The selected filters has the color to the complementary to that of the
color of unknown solution
Complementary colors lie across the diameter on the color
wheel and combine to form “white light”, so the color of
a compound seen by the eye is the complement of the
color of light absorbed by a colored compound; thus it
completes the color.
It is maximum absorbance by the solution at
one particular wavelength .
Cuvette are rectangular cell , square cell or
Made up of optical glass for visible
Common one is square,rectangular
to avoid refraction artefacts.
dimension of cuvette is 1cm.
when light falls on these electric elements electric
current is generated which deflects a galvanometer
The meter reading is proportional to the light
intensity ,these photosensitive detectors are also
referred to as photoelectric cells.
One of the common used photo cell is Barrier layer
Current from detector is fed to a sensitive suitable
measuring device, usually galvanometer.
Absorbance scale ranges from 0 to 2 ,while
% transmission scale ranges from 0 to 100.
Zero absorbance = 100% transmission
Infinite absorbance =0 transmission.
Reads ‘%T’ and ‘OD’
Power required 230 V AC ± 10% 50 Hz, 10 VA
Filters Separate glass filters 400, 420,
470, 500, 530, 620, 660 & 700 nm
Readout 2½ digit LED display
Measurement a) Transmittance 'T'-0-100% b)
Absorption Log 'T'-0-2
Light source LED of infinite life
Detector Photo cell
Test tube 12 mm Dia. with 1ml mark and
Warming time 5 minutes
Weight / Body 1 Kg. (approx.) / ABS
Dimension 95 mm (H) x 225 mm (W) x 215
Sample quantity 1ml
Accessories 5 matched test tubes, Dust proof
It is inexpensive .
Very well applicable for quantitative analysis of
Easily carriable and transportable.
Cannot be used for colorless compounds.
It does not work in UV and IR regions.
We cannot set specific wavelength, as we have to
set a range as a parameter.
Similar colors from interfering substances can
produce errors in results .
It is widely used in hospital & laboratory for
estimation of biochemical samples , like plasma,
serum, cerebrospinal fluid ( csf ) , urine.
It is also used to quantitative estimation of serum
components as well as glucose, proteins and other
various biochemical compound.
They are used by the food industry and by
manufacturers of paints and textiles.
They are used to test for water quality, by
screening for chemicals such as chlorine, fluoride,
cyanide, dissolved oxygen, iron, molybdenum, zinc
They are also used to determine the concentrations
of plant nutrients (such as phosphorus, nitrate and
ammonia) in the soil or hemoglobin in the blood
and to identify substandard and counterfeit drugs.
Clinical chemistry –michael l.bishop
A book of medical science- J.ochei
Practical biochemistry- keith wilson & john
Clinical chemistry &molecular diagnostics-