COLORIMETRY
Seminar
By
M. Dorathi Priya Santhini
Register No: NSG 18 JUL 02
GUIDE
Dr. Sasi Vaithilingan
Vice Principal and Professor,
Faculty Of Nursing
Vinayaka Mission College Of Nursing,
Puducherry
Vinayaka Mission’s Research Foundation
Sankari Main Road, NH 47, Ariyanoor,
Salem 636 308,
Tamil Nadu, India
PERFORMA FOR SEMINAR TOPIC
1. NAME AND ADDRESS OF Ms. M.DORATHI PRIYA SANTHINI,
THE CANDIDATE Research Scholar
Vinayaka Mission Research Foundation,
Salem.
2. NAME OF THE GUIDE AND DR.SASI VAITHILINGAN
NAME AND ADDRESS OF VP cum Professor,
THE INSTITUTION Faculty Of Nursing,
Vinayaka Mission College Of Nursing,
Puducherry
3. COURSE OF STUDY AND Ph.D (Nursing)
SUBJECT Psychiatric Nursing
4. REGISTRATION NUMBER NSG 18 JUL 02
5. SUBJECT Research Methodology
6. UNIT Unit 3; Microscopy and analytical instrumentation
7. TITLE OF THE SEMINAR Colorimetry
TOPIC
INTRODUCTION:
Colorimetry is a scientific technique that is used to determine the concentration of
colored compounds in solutions by the application of the Beer–Lambert law, which
states that the concentration of a solute is proportional to the absorbance. From: Open-
Source Lab, 2014.
The colorimeter is a device that is mainly used in industries and laboratories for
analyzing the color quality of the products along with color measurement.
The colorimeters are highly sensitive devices that can measure the concentration and
intensity of a particular color that is used in a product.
There are mainly two different types of colorimeters that are used in industries that are
color densitometers and color photometers.
The color densitometers measure the color density of primary colors in a color
combination in a test sample.
The color photometers are used for measuring the reflectance of a color as well as the
transmission.
WORKING PRINCIPLE OF COLORIMETER
The working of colorimeters is mainly based on the Beer-Lambert’s Law.
This law states that the light absorption when passes through a medium are directly
proportional to the concentration of the medium.
When a colorimeter is used, there is a ray of light with a certain wavelength is directed
towards a solution. Before reaching the solution the ray of light passes through a series
of different lenses.
These lenses are used for navigation of the colored light in the colorimeter.
The colorimeter analyzes the reflected light and compares with a predetermined
standard.
Then a microprocessor installed in the device is used for calculation of the absorbance
of the light by the solution.
If the absorption of the solution is higher than there will be more light absorbed by the
solution and if the concentration of the solution is low then more lights will be
transmitted through the solution.
PRINCIPLE OF COLORIMETRY
Colored solutions have the property of absorbing certain wavelength of light
when a monochromatic light is passed through them.
The amount of light absorbed or transmitted by a colored solution is in
accordance with two laws:
Beer’s law
Lambert’s law
BEER’S LAW
When a monochromatic light passes through a colored solution, amount of light
transmitted decreases exponentially with increase in concentration of colored
substance.
i.e. the amount of light absorbed by a colored solution is directly
proportion to the conc. Of substance in the colored solution.
LAMBERT’S LAW
The amount of light transmitted decreases exponentially with increase in path length
(diameter) of the cuvette or thickness of colored solution through which light passes.
i.e. the amount of light absorbed by a colored solution depends on path
length of cuvette or thickness or depth of the colored solution.
The intensity of color is directly proportional to the concentration of the
compound being measured
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.
COLORIMETRY AND WAVELENGTH
Wavelength Color Color
absorbed transmitted
400-450 Violet Green-yellow
450-500 Blue yellow
500-570 Green Red
570-590 yellow Blue
590-620 orange Green-blue
620-760 Red Green
How colours can be identified by a colorimeter?
To analyze the concentration of an unknown sample, there are several specimen
prepared from the test sample and then tested using an efficient colorimeter. After
analysis, the transmittance and concentration of the tested specimen are plotted on a
graph to obtain a graphical representation of the concentration creating a calibration
curve. This curve is then compared with the curve of a known sample, and the
concentration is measured.
Criteria for satisfactory colorimetry estimation
Stability of color
Color may be fade of air oxidation, photochemical decomposition,
temperature.
Intensity of color
The color of the solution should be intense in order to detect small amount
of constituents and for making accurate result in low concentration.
Clarity of the solution
Substance under investigation should be completely soluble in the solvent,
since turbid solution, suspension or colloidal solution absorb as well as
scatter light.
Reproducibility
The intensity of the colored solution must be reproducible. The effect of
order of adding reagent, pH and other variable should be clearly studied
Specificity
Color produced should be specific for the desired constituent. If other
constituents interfere with color reaction they be removed or prevented
from or prevented from functioning through appropriate treatment like use
of other coloring agent, altering the oxidation state.
Validity of Beer’s law
The intensity of color should be proportional to concentration. It can be
easily assessed by plotting absorbance Vs concentration, where a straight
line passing through origin should be obtained.
TYPES OF COLORIMETRY
Visual colorimetry :
Visual colorimetry is one of the oldest form of color measuring technique which
is not used now day, natural or artificial light is used as light is used as light
source and determinations are made with a colorimetry or color comparator
where human eye is used as detector.
Photo-electric colorimetry
Progree in the development of colorimetric method has resulted largely due to
the application of photoelectric cell, which eliminates the difficulties of
complicated visual comparison. In this method human eye is replaced by suitable
photoelectric cell, to afford a direct measure of the light intensity. Instruments
employing photoelectric cell measure the light absorption and not color of
substance
COMPONENTS OF A COLORIMETER
LIGHT SOURCE-TUNGSTEN
TUNGSTEN LAMP:
The light source is usually a tungsten lamp, for wavelength in the visible range
(320 – 700nm) and a deuterium or hydrogen lamps for ultraviolet light (below
350nm).
Tungsten lamp for Visible range
Deuterium/hydrogen
/hydrogen lamp (preferred) for UV Rays.
Black body radiators (Nerst glower) for Infrared radiations
radiations.
Filament mode of tungsten sealed in a glass envelope
Filed with inert gas.
Higher temperature shorter the wave length.
It operates temperature 2890 K
K.
Use full wave length region between 350 nm and 250 nm.
Increasing temperature 6000 K, than the wave length maximum will be shifted to
500 nm.
Expensive, filament operated at high temperature, quartz envelope required.
Life time is limited due to ga
gaseous tungsten formed by sublimation.
CARBON ARC LAMP
If sufficient intensity of light is not obtained from tungsten lamp then carbon arc lamp
can be use as a source for color measurement.
FILTERS
Filters will absorb light of unwanted wavelength and allow only monochromatic light to
pass through
For example a green filter absorbs all color, except green light w
which
hich is allowed to pass
through light transmitted through a green filter has a wavelength from 500
500--560 nm.
Absorption Filters
These filters are made up of glass, coated with pigments (or) they are made up
of dyed gelatin.
MONOCHROMATORS
Early colorimeters used Absorption filters (i.e. glass filter, Gelatin filter) that
transmitted a wide segment of spectrum (50nm or more).
Newer instrument use Interface filters that consist of thin layer of magnesium
fluoride crystals with a semitransparent coating of silver on each side.
Monochromator consists of:
Entrance slit
Absorption/ interface filter and
Prisms or diffraction grating for wavelength selection
Exit slit
PRISM
In a spectrophotometer, light from the source is directed through a convergent lens into
a entrance slit at the focal point of the lens, then through a prism and a second
convergent lens. The dispersion of radiation involves angularly separating the different
frequencies in a wavefront. The mechanism involved is the process of refraction by
prism.
INTERFERENCE FILTER
It has dielectric spacer film made up of CaF2, MgF2 or SiO, between parallel
reflecting silver films.
The thickness of dielectric spacer film can be 1/2 λ. The mechanism is that, the
radiation reflected by the second film and incoming radiation undergoes
constructive interference to give a monochromatic radiation.
SAMPLE HOLDERS
Cuvette are rectangular cell , square cell or circular one
Common one is square, rectangular to avoid refraction artefacts.
Dimension of cuvette is 1cm.
Made up of optical glass for visible wavelength (quartz or fused silica for UV).
Common one is square, rectangular to avoid refraction artifacts.
Optical path (length) of cuvette is always 1cm.
Capacity may be 3ml/2ml/1ml depending upon the thickness of the wall of the
cuvette.
For accurate and precise reading cuvette must be transparent, clean, devoid of
any scratches and there should be no bubble adhering to the inner surface of the
filled cuvette.
DETECTORS
Detectors are the transducers, which convert light energy to electrical energy .
A detector should be possess following characteristics
1.Should be sensitive
2.Should have linear response
3.Its noise level should be low
4.Should have short response time
5.Should stable
TYPES OF PHOTOSENSITIVE DETECTORS:
Barrier layer cells (photocells) – simplest
Photo emmisive cells
Photomultiplier tube (for low intensity lights)
Photoconductive cells (photodiodes) – newest.
READOUT DEVICE
The detector response can be measured by any of the following devices:
Galvanometer
Ammeter
Recorder
Digital readout.
The signal may be transmitted to computer or print out devices.
SOP OF COLORIMETRY
Glass/gel filter is placed in the filter slot.
3/4th of cuvette is filled with distilled water and placed in the cuvette slot
Instrument is switched ‘on’ and allowed to warm-up for 4-5 minutes
Button is adjusted using ‘coarse’ and ‘fine’ knobs to give zero optical activity in
the galvanometer
Blank solution is placed in an identical cuvette and the OD is read (‘B’).
Blank solution is transferred to the original test tube
Test solution is taken in the same cuvette and O.D. is read (‘T’)
Test solution is transferred back to the original test tube
Standard solution is taken in same cuvette and O.D. is read (‘S’)
Standard solution is transferred back to the test tube
Cuvette is washed
STANDARD OR CALIBRATION CURVE
The standard curve is prepared to check whether the method of assaying a
particular substance follows Beer’s Law, i.e. whether the absorbance of the
substance increases in a linear way with its concentration.
The standard curve is constructed by plotting a vertical axis (y – axis, ordinate)
for optical densities (absorbance) and a horizontal axis (x – axis, abscissa) the
concentration of standard solution.
The concentration of the test/unknown can be measured from the graph
(standard curve).
APPLICATION OF COLORIMETRY
Food ingredients,
Building materials,
Textile products,
Beverages,
Chemical solutions and many others.
Estimation of biochemical compounds in blood, plasma, serum, CSF, urine, etc.:
Glucose
Urea
Creatinine
Uric Acid
Bilirubin
Lipids
Total Proteins
Enzymes [e.g. ALT, AST, ALP]
Minerals [Calcium, Phosphorus etc.] etc….
REFERENCES
N.Grey, M.Calvin, Bhatia, (2018).Instrumental method of analysis, CBS Publishers.
K.D.Geetha (2016). Practical Biochemistry
B.K.Sharma (1981) Instrumental method of chemical analysis.Krishna Prakashan
Media, Meerut, UP.
Keith Wilson& John Walker. (2003). Practical Biochemistry Principles &
techniques.5thedition,Cambridge university press.
