Instrumentation of uv visible spectrophotometer

A spectrophotometer is an apparatus for measuring the intensity of light in a part of the spectrum, especially as transmitted or emitted by particular substances. The instrumentation of the Spectrophotometer is described in this presentation.

1.   بِسْمِ اللهِ الرَّحْمٰنِ الرَّحِيْمِ Instrumentation of UV Visible Spectrophotometer Prepared By: Talha Liaqat Department Of Chemistry KFUEIT, RYK

2. What is an instrument ? I nstrumentation Instrumentation is a collective term  for measuring instruments that are used for indicating, measuring and recording physical quantities. A scientific instrument is a device or tool used for scientific purposes, including the study of both natural phenomena and theoretical research.

3. Components: Instrumentation of UV-Visible Spectrophotometer The modern UV/Visible Spectrophotometer consists of: Light source or radiation source Monochromator Sample Holders/Cuvettes Optical chopper Detector Amplifier Recording devices

4. Spectrophotometer

5. 1) Light S ource O r R adiation S ource Light source is the most important component in UV-Visible spectrophotometer . The requirements for a spectrophotometer light source include : It must be of sufficient intensity for the transmitted energy t Should be stable It should provide continuous radiations To be detected at the end of optical path Low cost

6. Tungsten Lamp Similar in functioning to an electric bulb It provides a supply of radiations in the wavelength range of 320-2500nm Continuous source of light When tungsten filament is heated to incandescence by an electric current , light is produced . The glass bulb enclosing the filament contains a low pressure of inert gas, usually argon Small amounts of halogen like iodine is added to improve the intensity (Tungsten-Iodine Lamp)

7. Tungsten Lamp

8. H ydrogen D ischarge L amp In these lamps, a pair of electrodes is enclosed in a glass tube filled with hydrogen gas under relatively high pressure . it is a continuous source . Covers a range 160-375nm . Stable and wisely used.

9. Deuterium Discharge Lamp Similar to hydrogen discharge lamp Deuterium is filled in place of hydrogen The intensity of radiation emitted is 3-5 times the intensity of a hydrogen lamp More expensive then hydrogen lamp Used when high intensity is required The deuterium lamp has a short emission wavelength of 400 nm, or less

10. Deuterium Discharge Lamp

11. 2 ) Monochromator: A monochromator is an optical device that separates polychromatic light (such as sunlight or light coming from a lamp) into a range of individual wavelengths (monochromatic light ). It allows a narrow band of these individual wavelengths to be selected . The light of the desired wavelength band is then directed onto a sample, a detector or other components of the optical system.

12. Working of Monochromator The separation of light into its individual wavelength components is called dispersion . An element with this property is called a dispersive element.  In order to select a narrow band of these wavelengths , a slit can be used to block the unwanted wavelengths. The narrower the slit, the narrower the wavelength band.  In general, a monochromator consists of a dispersive element, an entrance slit, mirrors to produce a parallel beam similar to sunlight, an exit slit, and mirrors to extract the monochromatic light.

13. Types Of Monochromators: Prism Monochromators: The dispersive element in prism Monochromators is a prism. Prisms have a high light utilization efficiency and do not produce higher order light and very little stray light However , dispersion is dependent on wavelength ( high for UV, low for IR) and temperature . Grating Monochromators: The dispersive element in grating Monochromators is a reflecting diffraction grating. It provides a constant dispersion for all wavelengths and a low dependence on temperature. However, they produce relatively large amounts of scattered light and require the use of filters to block higher order light.  Due to their superior dispersion properties, diffraction gratings are often used in modern instruments.

14. Difference between Prism & Grating M onochromators

15. 3) Sample Holders / cuvettes The cells or cuvettes are used for handling liquid samples. The cells generally used are made of quartz. Quartz is transparent in both UV and visible region. Samples are placed in cells for measurements. Generally, the square cell with an optical path length of 10 mm is used. However, in cases where absorption is low, a cell with a longer optical path length is used, whereas when absorption is high, a cell with a shorter optical path length is used.

16. Optical Chopper: A chopper directs the source’s radiation, using a transparent window to pass radiation to the sample and a mirror to reflect radiation to the blank. The chopper’s opaque surface serves as a shutter, which allows for a constant adjustment of the spectrophotometer. The photographic insert shows a typical instrument. The unit in the middle of the photo is a temperature control unit that allows the sample to be heated or cooled.

17. 4 ) Detectors The purpose of detector is to convert the transmitted radient energy into equivalent amount of electrical energy. The detector converts the incoming light into an electrical current that can be quantitated. The higher the current, the greater the intensity of the light. For each wavelength of light passing through the spectrometer, the intensity of the light passing through the sample cell is measured. Modern-day Spectroscopic Detectors Can Be Classified Into 3 Basic Categories : Phototube P hotomultiplier tube Diode array detector

18. A phototube comprises of a light-sensitive cathode and an anode inside an evacuated quartz envelope. A potential difference of approximately 100 V is applied between the two electrodes . A photon entering the tube strikes the cathode and results in ejection of an electron which strikes the anode and results in flow of current. The current is generally of low intensity and needs to be amplified. The response of the phototube is dependent on wavelength of incident light. Phototube

19. Photomultiplier Tube The photomultiplier tube is inherently more sensitive than the photo tube. It comprises of a photosensitive cathode, anode and several dynodes . Photons entering the tube strike the cathode resulting in emission of electrons. The electrons are accelerated towards the first dynode which is 90 V more positive than the cathode . The electrons striking the first dynode resulted in several electrons for each incident electron. The process repeats itself from one dynode to next and after about 10 dynodes each photon results in production of 106 to 107 electrons . Photomultipliers have high sensitivity for UV and visible radiation and have fast response times.

20. Photomultiplier tube

21. The diode array detector is a multichannel detector capable of simultaneous measurement of all wavelengths of dispersed radiation. It comprises of an array of silicon photodiodes on a single silicon chip (generally 1024). The individual diodes are subsequently scanned for response . The diode array detector is less sensitive than the photomultiplier tube but offers the advantage of simultaneous measurement of different wavelengths. A diode array detector is more rugged than the photomultiplier tube as alignment problems are non-existent. There are no optical performance variations with wavelength change as in case of scanning monochromator instruments. Diode Array Detector

22. Diode A rray Detector

23. 5 ) Amplifier: The alternating current generated in the photocells is transferred to the amplifier . The amplifier is coupled to a small servometer . Generally current generated in the photocells is of very low intensity . T he main purpose of amplifier is to amplify the signals many times so we can get clear and recordable signals. 6)Recording devices: Most of the time amplifier is coupled to a pen recorder which is connected to the computer . Computer stores all the data generated and produces the spectrum of the desired compound.

24. Instrument Design: Single beam spectrophotometer is an analytical instrument in which all the light waves coming from the light source passes through the sample. Therefore , the measurements are taken as the intensity of light before and after the light pass through the sample. They are more compact and optically simpler than double beam spectrophotometers. A single beam spectrophotometer measures the concentration of an analyte in a sample by measuring the amount of light absorbed by that analyte . The Beer Lambert Law comes into operation. This law states that The concentration of an analyte is directly proportional to the absorbance . Single Beam Spectrophotometer:

25. Single Beam Spectrophotometer

26. Double Beam spectrophotometer: The double-beam spectrophotometer is a device that uses two rather than one beam of light to measure how light is absorbed during spectrophotometry. Unlike single beam units, the device allows for simultaneous measurement of a sample beam and a reference beam, t he light beam coming from the light source splits into two fractions. One fraction acts as the reference (the reference beam) while the other fraction passes through the sample (sample beam). The sample beam can measure the absorbance of the sample . The reference beam can measure the absorption (the sample beam can be compared with the reference beam).

27. Double Beam Spectrophotometer

28. Single beam spectrophotometer is an analytical instrument in which all the light waves coming from the light source passes through the sample. Double beam spectrophotometer is an analytical instrument in which the light beam coming from the light source splits into two fractions.   Light Beam Single beam spectrophotometer uses a non-split light beam. Double beam spectrophotometer uses a light beam that is split into two fractions before passing through the sample. Measurement The measurements taken from single beam spectrophotometers are less reproducible because a single light beam is used. The measurements taken from double beam spectrophotometers are highly reproducible because electronic and mechanical effects on both sample and reference beams are equal. Single Beam vs Double Beam Spectrophotometer Single Beam Spectrophotometer Double Beam Spectrophotometer

29. Thank you !