Visible Spectrophotometry and Colorimetry.pptx

Visible Spectrophotometry and Colorimetry.pptx
Color comparison was one of the oldest methods used for quantitative
estimation of elements and substances. The variation of a color of a system with change
in concentration of some components forms the basis of colorimetric analysis. The color
is usually due to the formation of a colored compound by the addition of an appropriate
reagent at specific condition or it may be inherent in the desired constituent itself. The
intensity of the color may then be compared with that obtained by treating a known
amount of the substance in the same manner. Colorimetry is concerned with the
determination of the concentration of a substance by measurement of the relative
absorption of light with respect to a known concentration of a substance. In visual
colorimetry, natural or artificial white light.

1. Visible Spectrophotometry and Colorimetry By Dr. Neetu Soni ,SHUATS, Prayagraj

2. Instrumental visible spectroscopy  is a logical extension of the color tests and visual observations. It is a sensitive, accurate method of measuring the colors or mixtures of colors that our eyes perceive.

3. Spectrophotometry is the quantitative measurement of how much a chemical substance absorbs light by passing a beam of light through the sample using a spectrophotometer. By measuring the intensity of light detected, this method can be used to determine the concentration of solute in the sample.

4. The visible light spectrum is the segment of the electromagnetic spectrum that the human eye can view. More simply, this range of wavelengths is called visible light. Typically, the human eye can detect wavelengths from 380 to 700 nanometers.

5. Spectrophotometer The spectrophotometer refers to an instrument that measures the absorbance of the test sample at a  specific wavelength  by measuring the amount of light transmitted by the sample. This device contains several components like a light source, collimator, monochromator , cuvette , light detector, and digital meter.

6. 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.

7. Theory of spectrophotometry and colorimetry When light (monochromatic or heterogeneous) is incident upon a homogeneous medium, a part of the incident light is reflected, a part is absorbed by the medium, and the remainder is transmitted as such. Color comparison was one of the oldest methods used for quantitative estimation of elements and substances. The variation of a color of a system with change in concentration of some components forms the basis of colorimetric analysis.

8. In visual colorimetry , natural or artificial white light (the whole range of the visible radiation is from 400-760 nm) is generally used as a light source and determinations are usually made with a simple instrument termed as colorimeter or color comparator.

9. Lambert’s and Beer’s law. When light (monochromatic or heterogeneous) is incident upon a homogeneous medium, a part of the incident light is reflected, a part is absorbed by the medium, and the remainder is transmitted as such. If I denotes the intensity of the incident light, It, the transmitted light, I r the reflected light and I a the absorbed light, then it may be written : I = I a + I t + I r I r is very small then I = I a + I t

10. Lambert’s law: When a monochromatic light passes through a transparent medium, the rate of decrease in intensity (I) with thickness (l) of the medium is proportional to the intensity of light. Mathematically, – dI / dl∞I On deduction it stands to I t = I × e – kl (k is a proportionality constant ) On changing the above equation from natural to common logarithm , I t = I × 10 –0.4343kl or I t = I × 10 –Kl (K is called absorption coefficient) The absorption coefficient is generally defined as the reciprocal of the thickness (l cm.) required to reduce 1/10th of its intensity. It/I0 = 0.1 = 10–Kl or K × l = 1 or K = 1/l So unit of K is cm–1

11. The ratio (I t /I ) is termed as Transmittance (T ) The ratio (I /I t ) is termed as Opacity log(I /I t )is termed as the Absorbance (A) of the medium or Optical density (OD).

12. Beer’s law: When a beam of monochromatic light is allowed to pass through a solution, the intensity of the light beam decreases exponentially with the increase in concentration as well as thickness of the solution. The equation becomes: I t = I × 10 –a.c.l (a is called molar absorption coefficient) The value of ‘a’ depends on the unit of concentration. If c is expressed in gm-moles/ litre , l in cm, then a is replaced by ε and is termed as molar extinction coefficient. The equation becomes: I t = I × 10 –ε.c.l A = ε.c.l or , ε = A/ c.l The unit of ε thus becomes litre gm-moles–1cm –1 Absorbance per unit path length (l) per unit concentration (c) is known as specific extinction coefficient

13. Instrumentation All spectrophotomete , Photometers and Colorimeters have the following basic components. Source of continuous radiation over the wavelengths of interest. A Filter or Monochromator for selecting a narrow band of wavelengths from the source spectrum. Sample cell A container for the sample. A Detector for converting radiant energy into electrical energy, and A Read out system device to read out the response of the detector.

14. The radiation source Tungsten filament (300–2500 nm) . Filter   It can transmit or reflect a specified range of wavelength. Filters are classified into three types L ong wave pass filters , S hort wave pass filters     Band pass filters

15. Types of filter from the viewpoint of function ( 1)  Absorption filter   : a filter which absorbs a specific range of wavelengths , for examples,  colored filter glass  and  gelatin filter . ( 2)  Interference filter   : a filter which transmits a specific range of wavelengths by utilizing the interference effect of a thin film .

16. Prisms A prism disperses polychromatic light from the source into its constituent wavelengths by virtue of its ability to reflect different wavelengths to a different extent Two types of Prisms are usually employed in commercial instruments. Namely, 600  cornu quartz prism and 300  Littrow Prism.

17. Grating Gratings are often used in the monochromators of spectrophotometers operating ultraviolet, visible and infrared regions.

18. Monochromator  (1) entrance slit, (2) collimating mirror (to form a parallel beam after the slit), (3) diffraction grating (dispersive element), (4) camera mirror (focuses light from the dispersive element onto the exit slit), and (5) exit slit.

19.   Detectors Detectors are instruments that measure the amount of ultraviolet or visible light absorbed by the sample molecules . In order to detect radiation three types of photosensitive devices are Photovoltaic cell Phototubes Photomultiplier

20. Photovoltaic cell The Photovoltaic cell is the  semiconductor  device that converts the light into electrical energy. The voltage induces by the PV cell depends on the intensity of light incident on it. 

21. Phototubes Phototube is also known as a photoelectric cell. It is filled with gas under low pressure. It contains a light-sensitive cathode and anode inside an evacuated quartz envelope. A photon entering the tube strikes the cathode and leads to the ejection of an electron. Electrons strikes the anode and results in the flow of current. The resulting current is of low intensity and requires amplification. The response in the phototube depends on the wavelength of incident light .

22. Phototubes

23. Photomultiplier tube is used in UV-Vis spectrophotometers . It has an anode, cathode, and many dynodes. Photon when entering the tube, strikes the cathode, resulting in the emission of electrons. The emitted electrons are accelerated towards the first dynode, An electron striking the first dynode will result in the production of several electrons. The process repeats itself continuously from one dynode to another and after passing to 10 dynodes. Photomultiplier Tube

24. A colorimeter is an instrument that compares the amount of light getting through a solution with the amount that can get through a sample of pure solvent.  Visual comparators Multiple standard method Duplication Method Dilution methods Balancing method Colorimeter

25. Dubosq colorimeter

26. Application of colorimetry and spectrophotometry : ( i ) Determination of molar composition of complexes . (ii) Determination of instability constants of complexes . (iii) Determination of pK value of an indicator . (iv) Elucidation of structures of organic as well as inorganic compounds. ( v) Determination of molecular weight of colored substances .