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SPECTROPHOTOMETRY Spectrophotometer is a special form of absorptiometry which mainly deal with the ultra violet (185- 400nm), visible(400- 760nm) and Infra red (0.76- 15µm) region of the electromagnetic spectrum. The U.V- Visible spectroscopy is mainly concerned with - quantitative analysis of compounds - auxiliary tool for structural elucidation - determination of the light absorptive capacity of a chemical system. 3


INSTRUMENTATION COMPONENTS OF SPECTROPHOTOMETER: Radiation source Filters or Monochromators A Pair Of Cuvettes Test Sample Detector Recording System Power Supply 4


RADIATION SOURCE In ultra violet spectrometers the most commonly used radiation sources are - hydrogen discharge lamp - deuterium lamp - xenon discharge lamp - tungsten lamp - mercury lamps When the pressure of the gas is low only line spectra are emitted and if pressure of gas is high , band spectra and continuous spectra will be obtained. 5

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The sources of radiation must meet the following requirements It should be stable. It should provide continuous radiation. It must be of the sufficient intensity for the transmitted energy to be detected at the end of the optical path. 6

Hydrogen Discharge Lamps:

Hydrogen Discharge Lamps In these lamps hydrogen gas is stored under relatively high pressure. The high pressure in the hydrogen lamps causes the hydrogen to emit a continuum rather than a simple hydrogen spectrum. Hydrogen discharge lamp covers a range the range 160-375 nm. These lamps are stable, robust and widely used. 7

Deuterium Lamps:

Deuterium Lamps In case of deuterium lamps the intensity of the radiation emitted is 3 to 5 times the intensity of a hydrogen lamp of comparable design and wattage. It is more expensive than hydrogen lamp. It is used when high intensity is required. It provides a supply of radiation in the wavelength range varying between 320 – 2500nm. Deuterium lamp 8

Tungsten Lamp:

Tungsten Lamp Tungsten lamp is similar in its functioning to an electric light bulb. It provides a supply of radiation in the wavelength range varying between 320- 2500nm. Tungsten lamp 9

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10 It has two short comings -The intensity of radiation at shorter wavelength (<350nm) is small. -Constant intensity should be maintained. Typically the emission intensity varies with wavelength. Xenon Discharge Lamp Xenon gas is stored in lamps at 10 – 30 atm pressure. It contains of two tungsten electrodes that are separated by a distance of about 8mm. It produces greater UV radiation than the hydrogen lamp.

Mercury Arc:

Mercury Arc In this mercury vapor is stored under high pressure and the excitation of mercury atoms is done by electric discharge. The low pressure mercury arc is very useful for calibration. 11


FITERS AND MONOCHROMATORS Filters: Filters isolate a wider band than the monochomators. Filters are of two types - Absorption filters - Interference filters Absorption filters : These filters have a band width that ranges from 30- 250µm. 12 It is frequently necessary to filter (remove) wide bands of radiation from a signal.

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The absorption filters consist of colored glass or a dye suspended in gelatin and sand witched between the two glass plates. The colored glass filter has the advantage of greater thermal stability. Each instrument is provided with a set of 12 filters to cover the range from 390 to 700µm. A narrow spectral band can be obtained by coupling cut off filters with other filters but this combination decreases the intensity of light. 13

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Interference filters: These filters rely on interference phenomenon at desired wavelength thus permitting rejection of unwanted radiation by selective reflection and producing narrow band. Interference filters have a band pass of 100- 150 Å and a peak transmittance of 40- 60%. Schematic cross section of an interference filter 14

Monochromators :

Monochromators Monochromator is used to disperse the heterochromic radiation into its component wavelength and to permit the isolation of desired portion of the spectrum. It consist of an entrance slit, an exit slit and a dispersing device either a prism or a grating Materials of construction should be selected with care to suit the range in which it has to work. For example - quartz for ultraviolet - normal glass for visual range - alkali halides for infra red region 15

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Gratings are cheaper than prism. Refracting Prism : The monochromators based upon the refracting prism need to be encased in specially designed housing within the spectrophotometer. In this type of monochromator the ‘white light’ duly enters the monochromator through an entrance slit before undergoing collimation. 16

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Schematic diagram of prism monochromator The ray of light passes via a refraction prism which eventually disperse the light into its component wavelengths. Finally the ray of light is duly focused by another lens near an exit slit that is located at the focal plane. The refracting prism is carefully rotated by using a stage and a stepper motor to select a desired radiation. 17

Diffraction Grating::

Diffraction Grating: In this instance the white light is made to pass via an entrance slit and is focused subsequently towards a diffraction grating through a concave mirror. Schematic diagram of prism monochromator 18

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Thus, the diffraction grating disperses the light into its component wavelengths, and thereby results into the reflection of the light upon a second concave mirror. The emerged light may now be reflected duly and focused critically by the concave mirror. The grating is strategically mounted on a stage that may rotated carefully through a stepper motor. With the careful rotation of the diffraction grating various selected wavelength may be obtained. 19


CUVETTES Cuvettes are generally made of - quartz if work has to be done in ultra violet region - fused glass in case of violet region Glass absorption cell and silica cells are suitable for absorbance measurement in the visible region. Cuvettes may be rectangular or cylindrical in shape . Cuvette 20


TEST SAMPLE The sample to be analyzed is placed between the source and the monochromator or between the detector and monochromator. The walls of the cell in which the test sample is placed should be transparent to the radiation used. Cortex glass and quartz are transparent for the wavelength range between 210 – 300 nm and are most commonly used. The solvents for the test solution must be one that does not absorb the radiant energy within the region of electromagnetic spectrum being studied. 21


DECTECTORS Detectors are used to measure accurately the absorption of an analyte via the intensity of the transmitted light. In order to detect radiations three types of photosensitive devices are mainly used - Photovoltaic cell or Barrier layer cell - Phototubes - photomultiplier cells 22

Photovoltaic cell :

Photovoltaic cell It is also called as barrier layer cell. It consists of a iron electrode upon which a layer of semi conducting material like selenium is deposited. 23 Schematic diagram of photovoltaic cell

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The radiations falling on the surface produces electrons at the selenium silver interface. The electrons are accumulated on the silver surface which produces an electrical voltage difference between the silver surface and the base of the cell. If the external circuit has a low resistance a photo current will flow which is directly proportional to the intensity of incident radiation. The current output of the cell depends upon the wavelength of the incident light 24 The base plate of iron acts as one electrode and surface of selenium covered by gold or silver acts as second collector electrode.


Phototube Phototubes consists of a semi – cylindrical cathode and a wire anode sealed inside a evacuated transparent envelope. The concave surface of the cathode emits electrons when irradiated with light thus when a potential is applied across the electrode, electrons are emitted. Schematic diagram of Phototube 25

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These electrons flow to the anode and a photo - current results. The number of electrons ejected from the photo - emissive surface is directly proportional to the radiant power of the beam striking the surface. Photo tubes are generally operated at a potential of about 90 V. Phototubes are more sensitive than photovoltaic cells. They are employed for measuring low intensities of illumination . 26

Photomultiplier Tubes:

Photomultiplier Tubes A photomultiplier tube consists of a electrode covered with a photo emissive material. Schematic diagram of photomultiplier tubes 27

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By a photomultiplier tube, the overall amplification factor of about 10⁶ can be achieved. Most photomultiplier tubes have about 10 dynodes. Each dynode is maintained at 75 to 100 V more positive than the preceding dynode. Upon striking the dynode each photoelectrons causes the ejection of several additional electrons which in turn are accelerated towards the next dynode. The foresaid phenomenon continues until the entire cascade of electrons is collected ultimately at the so called collecting electrodes. 28


RECORDING DEVICE The signal from the detector is finally received by the recording device. The recording is done by a recording device. This type of arrangement is only done in recording UV spectrophotometer. 29


POWER SUPPLY The power supply serves as a triple function It decreases the line voltage to the instrument operating level with a transformer. It converts A.C to D.C with a rectifier if D.C is required by the instrument. It delivers a constant voltage to the source lamp and instrument. 30


SINGLE BEAM SPECTROPHOTOMETER In this system UV radiation is given off by the source. Convex lens : Gathers the beam of radiation and focuses it on the inlet slit. Inlet slit : Permits light from the source. Monochromator : Splits the light according to wavelength. Exit slit : Allows light of required wavelength to pass through. 31

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Different detectors respond differently at different wavelengths. For eg the IP28 is not useful at 800 nm but the R136 and gallium arsenide detectors respond in this range. The detectors selected must operate over the desired range of the experiment. The problems in the instrument variation can be largely overcome by using the double beam system. 32 Schematic diagram of single beam spectrophotometer


DOUBLE BEAM SPECTROPHOTOMETER 33 Schematic diagram of double beam spectrophotometer

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Advantages : It not necessary to continually replace the blank with the sample or adjust to zero at each wavelength as in single beam unit. Any errors due to variation in the intensity of the source and fluctuation in the detector is minimized. 34


REFERENCE 35 Pharmaceutical analysis volume II by Ashutosh kar , pg 423- 438 Instrumental method of chemical analysis by H. G Kaur, pg 153- 156 Instrumental method of chemical analysis by Gurudeep and Sham .K. Anand, pg 2.167 - 2.172 Practical pharmaceutical chemistry, fourth edition – part two by A. H. Beckett and J. B. Stenlake, pg 264 - 274



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