MPAT GOKULRAJ UV AND VISIBLE spectroscopy

m.pharm analysis, pharmaceutical analysis, food analysis, cosmetic analysis and evaluation, modern bio analytical techniques, advanced pharmaceutical analysis, phamaceutical validation

1. SUBMITTED TO: MR.T. SREENIVASA RAO ASSISTANT PROFESSOR DEPT. OF PHARMACEUTICAL CHEMISTRY KARNATAKA COLLEGE OF PHARMACY BANGALORE SUBMITTED BY: S.GOKULRAJ M PHARM 1 ST SEMESTER DEPT. OF PHARMACEUTICAL ANALYSIS KARNATAKA COLLEGE OF PHARMACY BANGALORE SUBJECT: MPAT Instrumentation and Application of UV and Visible spectroscopy 1 KARNATAKA COLLEGE OF PHARMACY

2. CONTENT: WHAT IS INSTRUMENT INSTRUMENTATION SOURCE OF LIGHT MONOCHROMATORS SAMPLE CELL DETECTOR APPLICATION 2 KARNATAKA COLLEGE OF PHARMACY

3. What is an instrument: Instrument is a device or tool used for scientific purpose,including the study of both natural phenomena and theoretical research. Instrument is used to measure the absorbance in UV and visible region. Different types of instrument 1. Colorimeter: they measure either absorbance or transmittance or both and have filters for use with different coloured solution. Which are usually inexpensive and Less accurate. The range of wavelength used is usually small.eg 400-800nm 2. spectrophotometer: Which are more expensive than colorimeters. They can be used for wavelength region eg 200-800nm spectrophotometer is an instrument that measure the amount of light absorbed by a sample. The accuracy of the instrument is very high since they employ granting monochromators and photo multiplier tubes. 3 KARNATAKA COLLEGE OF PHARMACY

4. INSTRUMENTATION Different parts are used in the instrumentation process they are, Source of light Monochromators Sample cell Detectors 4 KARNATAKA COLLEGE OF PHARMACY

5. SOURCE OF LIGHT It should be stable It should provide continuous radiation Free from fluctuation. The UV spectrum range from 200nm -400nm. In the visible spectrum range is 400nm -800nm. UV SPECTROSCOPY LIGHT SOURCE: Hydrogen discharge lamp Deuterium lamp Xenon discharge lamp Mercury arc Visible spectroscopy light source: Tungsten lamp Carbon arc lamp. 5 KARNATAKA COLLEGE OF PHARMACY

6. Deuterium lamp Deuterium arc lamps measure in the UV region 200-400nm As Deuterium lamps operate at high temperatures, normal glass housings cannot be used for the casing. Instead, a fused quartz, UV glass, or magnesium fluoride envelope is used. When run continuously typical lamp life for a Deuterium lamp is approximately 1000 hours. Deuterium lamps are always used with a Tungsten halogen lamp to allow measurements to be performed in both the UV and visible regions. It will be more cost. 6 KARNATAKA COLLEGE OF PHARMACY

7. Tungsten lamp Instruments that only use Tungsten halogen lamps as the light source will only measure in the visible region. Most common light source used in spectrophotometer. The lamp consist of a tungsten filament in a vacuum bulb it offers sufficient intensity. It has long life. It will be only used for visible spectroscopy. Eg:400-800nm 7 KARNATAKA COLLEGE OF PHARMACY

8. Monochromator The polychromatic light or hetrochromatic light converted into monochromatic light is called monochromator. Different types of monochromator are used in the instrument they are, Entrance slit ( to get narrow source) Collimator ( to render light parallel) Filters or prism or Grating ( to dispers radiation) Collimator ( to reform the image of entrance slit) Exit slit (to fall On sample cell) 8 KARNATAKA COLLEGE OF PHARMACY

9. Filters Filters are made up of glass coated with pigment or they are made up of dyed Gelatin . They are absorb the unwanted radiation and transmit the wanted radiation. Filters are two types they are, Absorption filter Interference filter MERITS: Simple in construction Cheaper Selection of filter is easy D EMERITS: Less accurate More band pass(30nm) 9 KARNATAKA COLLEGE OF PHARMACY

10. Prisms The polychromatic light convert into monochromatic light to use the prism. Prism is made up of glass, quartz, fused silica. Quartz Or fused silica is used for uv spectrum method. Glass prism is used for visible spectrum because it will be produce the rainbow. The material of prism is normally glass. These are found in inexpensive instruments. They are two types Refractive type Reflective type 10 KARNATAKA COLLEGE OF PHARMACY

11. Grating Grating are the most efficient ones in converting a polychromatic light to monochromatic light in the real sence . Grating are commenly used in spectrophotometers. Gratings are rulings made on glass, Quartz or alkyl halides depending upon the instrument. Grating are mostly used in expensive instrument because it will be very accurately separate the light source. The band pass is 1nm. They are two types of grating Diffraction grating Transmission grating 11 KARNATAKA COLLEGE OF PHARMACY

12. 1. Diffraction Grating More refined dispersion of light is obtained by means of diffraction gratings. These consist of large number of parallel lines (grooves). They are made up of master grating with epoxy resin. To make the surface reflective, a deposit of aluminium is made on the surface. 2. Transmission grating It is similar to diffraction grating but refraction takes place instead of reflection. Refraction produces reinforcement. this occurs when radiation transmitted through grating reinforces with the partially refracted radiation 12 KARNATAKA COLLEGE OF PHARMACY

13. Sample cell Sample cells are also called as a cuvettes. Sample cells or cuvettes are used to hold a sample solution. the sample cell may either be rectangular or cylindrical in nature. The cuvette are made up of glass,plastic,quartz . In uv region the cells are prepared from quartz or fused silica. In visible region the cells are prepared from glass. The surface of the absorbption cells must be kept clean.no fingerprints or blotches should be present on the cells. Cleaning carried out washing with distilled water or with dilute alcohol,acetone . 13 KARNATAKA COLLEGE OF PHARMACY

14. Detectors Detectors are used to detect the sample. Detector used in uv visible spectrophotometers can be called as photometric detectors. Device which converts light energy into electrical signal, that are displayed on readout devices. The transmitted radiation falls on the detector which determines the intensity of radiation absorbed by sample. It should give quantitative response. It should have high sensitive and low noise level. It should have short response time. The most commonly used detectors are, Barrier layer cell or photovoltaic cell. Photo tubes or photo emissive cell Photo multiplier tube 14 KARNATAKA COLLEGE OF PHARMACY

15. 1. Barrier layer cell/Photovoltaic cell These cells are cheapest. The detector has a thin film metallic layer coated with silver or gold and acts as an electrode. It also has a metal base plate which acts as another electrode. These two layers are separated by a semiconductor layer of selenium. When light radiation falls on selenium layer, electrons become mobile and are taken up by transparent metal layer. This creates a potential difference between two electrodes & causes the flow of current. When it is connected to galvanometer, a flow of current observed which is proportional to the intensity and wavelength of light falling on it. 15 KARNATAKA COLLEGE OF PHARMACY

16. 2.Photo Tubes/Photo emissive Tubes Consists of a evacuated glass tube with a photocathode and a collector anode. The surface of photocathode is coated with a layer of elements like cesium , silver oxide or mixture of them. When radiant energy falls on photosensitive cathode, electrons are emitted which are attracted to anode causing current to flow. More sensitive compared to barrier layer cell and therefore widely used. 16 KARNATAKA COLLEGE OF PHARMACY

17. 3. Photo Multiplier Tubes The principle employed in this detector is that, multiplication of photoelectrons by secondary emission of electrons. In a vacuum tube, a primary photo-cathode is fixed which receives radiation from the sample. Some eight to ten dynodes are fixed each with increasing potential of 75-100V higher than preceding one. Near the last dynode is fixed an anode or electron collector electrode. Photo-multiplier is extremely sensitive to light and is best suited where weaker or low radiation is received 17 KARNATAKA COLLEGE OF PHARMACY

18. APPLICATION Qualitative analysis Quantitative analysis Detection of impurities Identification of functional group Determination of structure Molecular weight estimation Used in pharmaceutical industries in drug testing Clinical labs Food and beverage companies. 18 KARNATAKA COLLEGE OF PHARMACY

19. Reference Text book of PHARMACEUTICAL ANALYSIS 4th edition Dr. Ravi Sankar. Instrumental methods of Chemical Analysis by Gurudeep R Chatwal & Sham K Anand 19 KARNATAKA COLLEGE OF PHARMACY

20. THANK YOU 20 KARNATAKA COLLEGE OF PHARMACY