Colorimetry

colorimetry

1. Instrumentation of colorimetry BY Saravanan.S M.Pharm (Pharmaceutics) Sri Ramachandra college of pharmacy

2. 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)

3. 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.

4. 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. Wave length Color absorbed Color 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

5. Criteria for satisfactory colorimetric estimations 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, suspention or colloidal solution absorb as well as scatter light.

6. 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.

7. Types Colorimetric 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 .

8. 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

9. Components of colorimetry

10. Light source 1. Tungsten lamp filament mode of tungstone sealed in a glass envelope F iled with inert gas. higer temperature shorter the wave length. I t operates temperature 2890 K. U se full wave length region between 350 nm and 250 nm. I ncreasing temperature 6000 K, than the wave length maximum will be shifted to 500 nm. E xpensive, filament operated at high temperature, quartz envelope required. L ife time is limited due to gaseous tungsten formed by sublimation.

11. 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.

12. 2. Filters filters 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 . F ilter Absorption filter Interference filter Ex: Glass filter,Gelatin filter Band pass Cut off filter

13. Absoption filters 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

14. 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. T he dispersion of radiation involves angularly separating the different frequencies in a wavefront. T he mechanism involved is the process of refraction by prisma

15. 2. Interference filter (or) Fabry-perot filter I t has dielectric spacer film made up of CaF 2 , MgF 2 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 .

16. 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.

17. Detectors Detectors are the transducers, which convert light energy to electrical enagery .A detector should be possess follwing characteristics S hould be sensitive S hould have linear response I ts noise level S hould be low S hould have short response time S hould stable Barrier layer cell Photomultiplyer tube

18. 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.

19. 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. WWW.Pharmatwiter.com WWW.Pharmainfo.in

20. Thank you