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Principle : 

2 Principle The principle involved in working of UV spectrophotometers is Beer-Lambert’s law. Mathematical equation for Beer-Lambert’s law is: A=εct Where A=Absorbance or optical density or extinction coefficient ε=Molecular extinction coefficient c=Concentration of drug (m mol/lit) t=Path length (normally 10mm or 1cm)

Instruments used: : 

3 Instruments used: Colorimeters Spectrometers Spectrophotometers


4 PARTS OF UV INSTRUMENTATION Radiation source Wavelength selector Sample cell Detector Readout device

Block diagram of model spectrophotometer : 

5 Block diagram of model spectrophotometer

Radiation sources: : 

6 Radiation sources: Hydrogen (or) Deuterium discharge lamps Incandescent filament lamps Basic requirements: They must provide energy over the wavelength region. They should maintain a constant intensity over the time interval during which the measurements are made.

Wave length selectors: : 

7 Wave length selectors: Filters Absorption filters Interference filters Monochromators Prisms (Refractive ,Reflective) Gratings (Diffraction ,Transmission)

Interference filter (Fabry-Perot) : 

8 Interference filter (Fabry-Perot) Merits: Inexpensive. More accurate than absorption filters. Demerits: Peak transmission is low. Higher resolutions cannot be obtained.

Prism monochromator : 

9 Prism monochromator

Diffraction grating : 

10 Diffraction grating Mechanism :The rays which are incident upon the gratings gets reinforced with the reflected rays and hence the resulting radiation has wavelength which is governed by the equation: mλ = b (sin i ± sin r) Where λ = wavelength of light produced b = grating spacing i = angle of incidence r = angle of reflection m = order (0,1,2,3 etc)

Sample cells : 

11 Sample cells The sample holders in UV instruments should be made of quartz as it does not absorb UV radiation. Sample volume: Small volume cells(0.5 ml or less) Large volume cells(5-10ml) Shape of cell: Mostly rectangular. Path length (internal distance): Normally 1 cm. Material: Quartz

Radiation detectors and indicators : 

12 Radiation detectors and indicators Photovoltaic cells or Barrier layer cells Photo emissive (vacuum) Tubes Photomultiplier tubes

Photovoltaic or Barrier layer cells : 

13 Photovoltaic or Barrier layer cells MECHANISM: Electrons are transferred at the interface from the semiconductor to metal when the semiconductor is irradiated. The electric current produced is proportional to the radiance power of the incident beam and to the area of photosensitive being irradiated.

14 Merits: At low level of illumination it produces photo current proportional to the radiant power received on it. It is relatively cheap and widely used in filter photometers and cheap spectrophotometers. Demerits: Slow response. Fatigue effect. While using it there should be no fluctuations in power and low external electric resistance.

Photo tubes or Photo emissive cells : 

15 Photo tubes or Photo emissive cells It consists of a photosensitive cathode containing an alkali metal oxide, such as cesium oxide and an anode mounted in an evacuated glass tube. The spectral response of phototube depends upon the nature of material coated on photocathode. Ex; Coatings of Cs-Sb (200-650nm) Cso2-Ag(250-1200nm)

Slide 16: 

16 Mechanism: Radiant energy striking the photocathode causes photoelectrons to be emitted and the number of electrons is proportional to the radiant power of the incident beam .The velocity of electrons is directly proportional to frequency of incident radiation energy .These electrons are collected by the anode and a photocurrent is generated. Merits : More sensitive when compared to photovoltaic cell. Demerits: High voltage(90-100v) is required. Current produced is small and hence amplification is required.

Photomultiplier tubes : 

17 Photomultiplier tubes .Mechanism: It has a number of photo emissive electrodes each charged at a successively higher potential and so arranged that the electrons ejected from the photocathode travel successively from one electrode to the next. In each step the photocurrent is increased by the secondary emission of electrons.

Slide 18: 

18 Merits: It is highly sensitive to light. Best suited where weaker or low radiation is received.


19 UV INSTRUMENTS-TYPES Colorimeters Spectrophotometers

Double beam colorimeter : 

20 Double beam colorimeter

Slide 21: 

21 SPECTROPHOTOMETERS TYPES: Single beam spectrophotometers Double beam spectrophotometers

Single beam spectrophotometer : 

22 Single beam spectrophotometer

Double beam spectrophotometer : 

23 Double beam spectrophotometer


24 APPLICATIONS Qualitative analysis : Detection of impurities. Structure elucidation of organic compounds. Prediction of functional groups of organic compounds.

Slide 25: 

25 Quantitative analysis: Determination of molecular weight. Quantitative analysis of pharmaceutical substances. Assay of medicinal substances.

References : 

26 References Instrumental methods of analysis Willard , Merritt 2. Textbook of pharmaceutical analysis Ravishankar Pharmaceutical analysis-Industrial methods Nirali Prakashan Spectroscopy B.K.Sharma 5. Industrial methods of instrumental analysis Chatwal

Slide 27: 

27 Thank you

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