This document discusses colorimetry and its application in trace mineral analysis. Colorimetry is a common analytical technique used in biochemical estimation that involves the quantitative estimation of color. The principle involves how colored substances absorb light in relation to their concentration. Colorimeters measure this using principles like Beer's law and Lambert's law. The document outlines the basic structure of a colorimeter, including its light source, filters, sample cuvette, and detector. It also discusses other analytical techniques like spectrophotometry, atomic absorption spectrophotometry, and flame emission photometry that are used in trace mineral analysis.

1. AAPPPPLLIICCAATTIIOONN OOFF
CCOOLLOORRIIMMEETTRRYY IINN TTRRAACCEE
MMIINNEERRAALL AANNAALLYYSSIISS
CCoouurrssee :: VVBBCC 881122
AAddvvaanncceess iinn TTeecchhnniiqquueess iinn BBiioocchheemmiissttrryy
Prepared By:
Irshad A.
PhD Scholar
Department of MSC and Tech
VCRI-Namakkal

2. WWHHAATT IISS CCOOLLOOUURR??????
• Colour is one aspect of appearance
• Electromagnetic radiation between 380-780 nm
• Colour = light source + object properties + eye + brain

4. OOBBJJEECCTTSS……....WWhhyy iitt llooookk lliikkee??
Objects are characterized by the amount
Incident
of light they emit and reflect or transmit
Light
at each wavelength of interest
When light is incident on an object a part
of it is absorbed, a part is reflected and a
part may be transmitted
The object may also emit light
All these characteristics contribute to the
observed colour/appearance Reflected
Light
Absorbed
Light
Transmitted
Light

5. A compound will absorb light when the radiation posesses the
energy needed to move an electron from its lowest energy
(ground) state to some excited state. The particular energies of
radiation that a substance absorbs dictate the colours that it
exhibits.
Conversely the colour of a compound can help us to determine
its electronic configuration.
White light contains all wavelengths in this visible region.
When a transparent sample (like most aqueous solutions)
absorbs visible light, the color we perceive is the sum of the
remaining colours that are transmitted by the object and strike
our eyes.

7. CCOOLLOORRIIMMEETTRRYY
It is the most common analytical technique used in biochemical
estimation in clinical laboratory.
It involves the quantitative estimation of colour.
A substance to be estimated colorimetrically, must be coloured
or it should be capable of forming chromogens (coloured
complexes) through the addition of reagents.
Coloured substance absorb light in relation to their colour
intensity.
The colour intensity will be proportional to the conc. of coloured
substance.

8. CCOOLLOORRIIMMEETTEERR
The instruments used
colorimeters
or
Photometers
or
Absorptiometers

9. PPrriinncciippllee ooff CCoolloorriimmeettrryy
Colored solutions have the property of absorbing certain
wavelength of light when a monochromatic light is passed through
them.
Light Intensity Change : By Absorbance or Transmittance
Quantity : Using Absorbance

10. PPrriinncciippllee ooff CCoolloorriimmeettrryy
The amount of light absorbed or transmitted by a colored
solution is in accordance with two laws:
BBeeeerr’’ss llaaww
LLaammbbeerrtt’’ss llaaww
“Lambert-Beer’s Law”

11. BBeeeerr’’ss llaaww
 When a monochromatic light passes through a coloured solution,
amount of light transmitted decreases exponentially with
increase in concentration of coloured substance.
 i.e. the amount of light absorbed by a coloured solution is
directly proportion to the conc. of substance in the coloured
solution.
A α c

12. BBeeeerr’’ss llaaww
The amount of radiation absorbed may be measured in a number of
ways:
1. Transmittance, T = P / P0
2. Absorbance, A = log10 P0 / P
So, if all the light passes through a solution without any absorption,
then absorbance is zero, and percent transmittance is 100%.
If all the light is absorbed, then percent transmittance is zero, and
absorption is infinite.

13. LLaammbbeerrtt’’ss llaaww
The amount of light transmitted decreases exponentially with
increase in pathlength (diameter) of the cuvette or thickness of
coloured solution through which light passes.
i.e. the amount of light absorbed by a coloured solution depends
on pathlength of cuvette or thickness or dept of the colored
solution.
Independent of the wavelength of incident light

14. LLaammbbeerrtt--BBeeeerr’’ss LLaaww
• Amount of light transmitted through a coloured solution
decreases exponentially with increases in conc. of coloured
solution & increase in the pathlength of cuvette or thickness of
the colored solution.

15. Standard CCuurrvvee,, CCaalliibbrraattiioonn ccuurrvvee

16. Limitation ooff LLaammbbeerrtt--BBeeeerr’’ss LLaaww

17. AApppplliiccaattiioonnss ooff CCoolloorriimmeettrryy

18. SSttrruuccttuurree ooff CCoolloorriimmeetteerr

19. Structure ooff CCoolloorriimmeetteerr--LLiigghhtt SSoouurrccee
1. Tungsten filament lamp - Wavelength Range : Part of the UV and
the whole of the Visible range (350 ~ 2,500nm)
2. Deuterium Arc Lamp - UV Region, Wavelength Range :190~420nm
3. Xenon Lamp - Wavelength Range : 190~800nm

20. SSttrruuccttuurree ooff CCoolloorriimmeetteerr
Slit : it is adjustable which allows only a beam of light
to pass through. it prevents unwanted or stray light
Condensing lenses: light after passing through slit falls
on condenser lens which gives a parallel beam of
light.

21. Structure ooff CCoolloorriimmeetteerr-- FFiilltteerr
• made of coloured glass. Filters are used for selecting light of
narrow wavelength.
• filters will absorb light of unwanted wavelength and allow only
monochromatic light to pass through.
For ex: a green filter absorbs all colour, except green light which is
allowed to pass through.
 Light transmitted through a green filter has a wavelength from
500-560 nm.
• Filter used is always complimentary in colour to the colour of
solution.

23. MMoonnoocchhrroommaattoorr

24. Structure of Colorimeter- CCuuvveettttee((ssaammppllee hhoollddeerr))
the monochromatic light from the filter passes through the
coloured solution placed in a cuvette.
it is made up of special glass/plastic/quartz/ fused silica material.
• it may be square/rectangular/round shape with fixed diameter
(usually 1 cm) & having uniform surface.

25. SSttrruuccttuurree ooff CCoolloorriimmeetteerr-- DDeetteeccttoorr
Detector
• Detector are photosensitive elements which converts
light energy into electrical energy.
• The electrical signal generated is directly proportional to
intensity of light falling on the detector.
Output : the electrical signal generated in photocell is
measured by galvanometer, which displays percent
transmission & optical density.

26. SSttrruuccttuurree ooff CCoolloorriimmeetteerr-- DDeetteeccttoorr

27. HHooww ttoo eessttiimmaattee aa ssaammppllee....??
Use of test (T), standard (S) and blank (B)
Test (T): this solution is prepared by treating a specific
volume of specimen (blood,urine, CSF…etc) with
reagents.
Standard (S): prepared by treating a solution of the pure
substance of known conc. with reagents.
For ex : pure glucose is taken as standard in estimation of
blood glucose.

28. HHooww ttoo eessttiimmaattee aa ssaammppllee....??
Blank : prepared for rule out colour produced by reagents
alone.
Two types of blank :
A) Distilled water as blank
B) reagent blank (reagent used in the estimation is taken as
blank)
Calculation :
• conc. Of substance in mg /100mg or gm/100ml of sample.
OD of test- OD of blank × conc. of standard × 100
OD of standard – OD of blank vol. of test sample

29. Application ooff ccoolloorriimmeettrriicc aassssaayy
Used in determination of amount of many substances in blood,
urine, saliva, CSF & other specimens.
Ex for common colorimetric assay are :
Determination of blood glucose,
Blood urea,
Serum creatinine,
Serum proteins,
Serum cholesterol,
Serum inorganic phosphate,
Urine creatinine
Glucose in CSF

30. OOtthheerr MMeetthhooddss
a. Spectrophotometry
b. Molecular fluorescence technique
c. Atomic spectroscopy
I. Flame emission photometry- most
common in trace mineral analysis
II. Atomic fluorescence
III. Plasma emission spectroscopy
**Entirely restricted to visible region of the of the spectrum
d. Atomic absorption spectrophotometry
e. Magnetic resonamce spectroscopy- Study of molecular
stucture and biochemical reactions

31. Flame emission photometry

32. FFllaammee eemmiissssiioonn pphhoottoommeettrryy

33. FFllaammee eemmiissssiioonn pphhoottoommeettrryy

34. AAttoommiicc AAbbssoorrppttiioonn SSppeeccttrroopphhootteemmeettrryy

35. RReeffeerreennccee
David Holme and Hazel Peck (1998) Analytical Biochemistry, 3rd edition, Prentice
Hall, p-504.
Ayoola P.B. and Olayiwola A. O. (2014) Trace elements and major minerals
evaluation of earthworm casts from a selected site in southwestern Nigeria.
ARPN Journal of Agricultural and Biological Science 9(6)216-218
Lab. Manual 9, Manual of methods of analysis of foods food safety and standards
authority of India, Ministry of health and family welfare government of India
new delhi, 2012.
Andzouana Marcel and Mombouli Jean Bievenu, (2012). Proximate, Mineral and
Phytochemical Analysis of the Leaves of H. myriantha and Urera trinervis.
Pakistan Journal of Biological Sciences, 15: 536-541.

36. DDiissccuussssiioonn