colorimeter
TRANSCRIPT
COLORIMETER
Dr. Gangadhar ChatterjeeMBBS;MD
Assistant ProfessorRCSM Govt. Medical college, Kolhapur, MH, India
• Colorimeter is instrument which is used in the measurement of the luminious intensity of light.
• Invented by Louis Jules Duboscq in 1870.
INTRODUCTION
• It is the most common analytical technique used in biochemical estimation in clinical laboratory.
• Color can be produced by any substance when it binds with/turns out 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
PRINCIPLE
• Wavelength between 400nm to 700nm form the visible spectrum of light
• visible band of light in electromagnetic spectrum
Relationship between wavelength & colour
Wavelength (nm)
Spectrum region Colour absorbed Colour transmitted
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
Absorption & transmittance of light
Io It
Ia
Transmittance (T)It is ratio of the intensity of the transmitted light over the intensity of the incident light.
Percentage transmission(%T) = It/Ii X100
AbsorbanceAbsorbance is the amount of light absorbed by a sample. It is calculated from T or %T using the following equations.
Relationship between absorbance and transmittance
A = O.D = Log 1/T = Log( 100/ %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.
Transmittance of a solution containing light absorbing substance depends upon
Absorbance
Concentration 00 0
0 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.
A α C• Where A is light absorbed and C is concentration of the
solution.
Basis of colorimetric techniques
• 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.
A α L• Where L = length of light path
Lamberts law
By combining above equations, we get
A α CL
A= KCL
Where k = constant for coloured solution
• For standard solution : As =Ks Cs L s
• 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 & L s =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
Parts of colorimeter
Flow representation of colorimeter
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..
Light source
• 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
Monochromator/wavelength selector
Complementary filters for coloured solutionsThe 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.
λmaxIt 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, rectangular to avoid refraction artifacts.
• dimension of cuvette is 1cm.
Solution holder
cuvettes
Photo sensitive detector
• 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.
Measuring device
• 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.
Advantage It is inexpensive
Very well applicable for quantitative analysis of colored compounds.
Easily cartable and transportable.
COLORIMETER
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 .
COLORIMETER
• 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.
Application
• 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.
Use of test (T), standard (S) and blank (B)In colorimetric estimation , it is necessary to
prepare a blank (B), a standard (S) & test (T).
Test : this solution is prepared by treating a specific volume of specimen (blood,urine, CSF…etc) with reagents.
• Blank : prepared for rule out color produced by reagents alone.
A blank solution or reference solution has everything except the compound to be measured
• Two types of blank :A) Distilled water as blankB) reagent blank (reagent used in the estimation is taken
as blank)
Use of blank
Standard : prepared by treating a solution of the pure substance of known conc. With reagents.
StandardPRIMARY Standard
same substance is used as standard one which is to be
estimated.e.g. pure glucose is taken as
standard in estimation of blood glucose.
SECONDARY Standard
substance taken as standard is different from the
substance to be estimated.substance taken as standard should match the color of
final product.e.g. methyl red is taken as standard in estimation of
serum bilirubin.
