By

Apurva Jha

Colorimeter is instrument which is used in the measurement of the luminious intensity of light.

Invented by Louis Jules Duboscq in 1870.

colorimeter

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

Wavelength (nm)

Spectrum region

Colourabsorbed

Colourtransmitted

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

Io It

Ia

A= O.D = Log 1/T

= Log( I00/ %T)

= Log100-Log%T

i.e. O.D. = 2-Log (%T)

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) .

Absorbance

Concentration 0

0 00 Concentration

% transmission

(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

Beer’s 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.

• AαC• 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.

AαL Where L = length of light path

By combining above equations,we get

A α CL

A= KCL

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 solutionAS =absorbance of std solutionCS = conc of std solution

But Ks =Ku & Ls =Lu

Au/As = Cu/Cs

Cu= Au/As X Cs

Light source

Monochromator/ wavelength selector

• Filter

Solution/sample holder

• Cuvette

Photosensitive detector system

Measuring device

Colorimeter

(1) Wavelength selection, (2) Printer button(3) Concentration factor adjustment, (4) UV mode selector (Deuterium

lamp)(5) Readout(6) Sample compartment(7) Zero control (100% T), (8) Sensitivity switch

Common source is a tungsten-filament lamp, higher powered tungsten –halogen (quartz-iodine) lamp.

Factor of light source are range, spectral distribution, stability of radiant energy and temperature..

Mono = single. Chromatic- colour

Monochromatic light is the single colour band of light.

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

Color Wheel(ROYGBIV)

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 circular one

Made up of optical glass for visible wavelength.

Common one is square,rectangularto avoid refraction artefacts.

dimension of cuvette is 1cm.

cuvettes

when light falls on these electric elements electric current is generated which deflects a galvanometer needle.

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 cell.

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.

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 position mark

Warming time 5 minutes

Weight / Body 1 Kg. (approx.) / ABS

Dimension 95 mm (H) x 225 mm (W) x 215 mm (L)

Sample quantity 1ml

Accessories 5 matched test tubes, Dust proof

Reads ‘%T’ and ‘OD’

Advantage It is inexpensive .

Very well applicable for quantitative analysis of colored compounds.

Easily carriable and transportable.

Disadvantage

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 and hydrazine.

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 walker

Clinical chemistry &molecular diagnostics-Tietz

Water blank

Reagent blank

Standard solution