Instrumentation of colorimetry

BYSaravanan.S

M.Pharm (Pharmaceutics) Sri Ramachandra college of pharmacy

What is colorimetry?In analytical chemistry, colorimetry is a technique "used to

determine the concentration of colored compounds in a sample solution” at visible region (400-800 nm)

Principle 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.

Complemetary color Wavelength between 400nm to 760 nm form the visible

spectrum of light light passed through a solution which selectivity absorbs

radiation at fixed wave lengths,then the color of the transmitted light is complementary to that of the absored light.Wavelength Color

absorbedColor

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

V

B G

Y

R O

Criteria for satisfactory colorimetric estimations Stability of color

Color may be fade of air oxidation, photochemical decomposition, temperature.

Intensity of colorThe 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 solutionSubstance under investigation should be completely soluble

in the solvent, since turbid solution, suspention or colloidal solution absorb as well as scatter light.

ReproducibilityThe 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 lawThe 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.

TypesColorimetric analysis is two types

Visual colorimetry Photo-electric 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.

Photoelectric colorimetry

Progree in the development of colorimetric method has resulted largly due to the application of photoeletric cell, which eliminates the difficultes of complicated visual comparition. 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 colorimetry

Light source1. Tungsten lamp filament mode of tungstone sealed in a glass envelope Filed with inert gas. higer temperature shorter the wave length. It operates temperature 2890 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 gaseous tungsten formed by

sublimation.

Carbon arc lampIf sufficient intensity of light is not obtained from tungsten lamp then carbon arc lamp can be use as a source for color measurement.

2. Filtersfilters will absorb light of unwanted wavelength and allow only monochromatic light to pass through

For ex: A green filter absorbs all color, except green light which is allowed to pass through. light transmitted through a green filter has a wavelength from 500-560 nm.

Filter

Absorption filter Interference filter Ex: Glass filter,Gelatin filter

Band pass Cut off filter

Absoption filters1. Absorption filters

These filters are made up of glass, coated with pigments (or) they are made up of dyed gelatin.

Selection:If the colour of the solution is Red, we have to use green filter

and

Prisms 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 prisma

2. Interference filter (or) Fabry-perot filterIt 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 Holder 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

artefacts. Dimension of cuvette is 1cm.

Detectors Detectors are the transducers, which convert light energy to electrical enagery .A detector should be possess follwing characteristics

1.Should be sensitive 2.Should have linear response 3.Its noise level Should be low 4.Should have short response time5.Should stable

Barrier layer cell

Photomultiplyer tube

Barrier Layer cell (BLC) It consists of an iron plate A, upon which a thin layer of

semiconductor selenium is deposited. This in turn covered by a thin film of transparent silver

layer, which is lacquered except for portion of collecting ring for electrical contact.

The iron plate act as anode and silver layer acts as cathode.

Reference Instrumental method of chemical analysis,chatwal.

Instrumental method of analysis, N.Grey, M.Calvin.

Pharmaceutical analysis, Ashutoshkar.

Instrumental method of analysis, B.K.Sharma

Principles and application of ultaviolet and visible spectroscopy, A.Rajasekaran.

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