Apurva colorimeter
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Transcript of Apurva colorimeter
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