U.V Spectroscopy.

Seminar on U,V Spectroscopy

1. Instrumentation and Pharmaceutical Application of U.V Spectroscopy. Shaikh Shakeel Shaikh Quader M.Pharm (Pharmaceutics ) Ali Allana College of Pharmacy Akkalkuwa Dist.Nandurbar ( 2016-17)

2. Content: Introduction Principle Theory Instrumentation Application

3. Introduction Ultraviolet and visible spectrometers have been in general use for the last 35 years and over this period have become the most important analytical instrument in the modern day laboratory. In many applications other techniques could be employed but UV-Visible spectrometry for its simplicity, versatility, speed, accuracy and cost-effectiveness. Absorption spectroscopy in the U.V and Visible region is consider to be one of the oldest physical methods use for the Qualitative, Quantitative analysis and structure elucidation.

4. REGION WAVELENGTH Ultraviolet 200-400 Visible 400-800 The wavelength of U.V radiation starts at the blue end of visible light about 400 And end at 200 A. However, the wavelength of visible radiation of starts at 800 A, and end at 400A.

5. Principle When U.V or visible radiation is passed through a substance under examination, Absorption of energy result in the promotion of electron from the ground electronic state to the exited electronic state. During the process of absorption a large number of photon-molecule collision is possible but only those collision will cause absorption of energy in which the energy of photon matches the energy difference between the ground and exited electronic state of the molecule. In ultra-violet and visible spectroscopy, electronic transition take place.

6. Theory Absorption Laws; Lambert’s law 2) Beer’s Law

7. Lambert’s law Lambert’s law : When a beam of monochromatic radiation is passes through a homogeneous absorbing medium, the rate of decrease of intensity of radiation with thickness of absorbing medium is proportional to the intensity of incident radiation. Mathematically, the law is expressed as: - dl/ dx = kl Where; I= intensity of radiation after passing through a thickness x, of the medium. dl= infinitesimally small decrease in the intensity of radiation on passing through infinitesimally small thickness, dx of the medium. -dl/ dx = rate of decrease of intensity of radiation with thickness of absorbing medium. K= proportionality constant.

8. Beer’s Law Beer’s Law: When a beam of monochromatic radiation is passes through a homogeneous absorbing medium, the rate of decrease of intensity of radiation with thickness of absorbing medium is proportional to the intensity of incident radiation as well as the concentration of the solution. Mathematically, the law is expressed as: - dI / dx = k’ Ic Where; I= intensity of radiation after passing through a thickness x, of the medium. dl= infinitesimally small decrease in the intensity of radiation on passing through infinitesimally small thickness, dx of the medium. -dl/ dx = rate of decrease of intensity of radiation with thickness of absorbing medium. K’= proportionality constant. C= concentration

9. Instrumentation Fig: Diode array U.V Spectrophotometer

10. Fig: Schemic Diagram of and Double beam U.V spectrophotometer.

11. The instrument used in ultraviolet-visible spectroscopy is called a UV/Vis spectrophotometer. It measures the intensity of light passing through a sample and compares it to the intensity of light before it passes through the Reference sample. The ratio is called the transmittance, and is usually expressed as a percentage (%T). The absorbance, , is based on the transmittance. Instruments for measuring the absorption of U.V. or visible radiation are made up of the following components; Radiation Sources (UV and visible) Wavelength selector ( monochromator ) Sample containers Detector Signal processor and readout

12. Radiation sources In U.V spectrometer, the most commonly used radiation source are: Tungstan lamp Hydroger or Deuterium lamp Xenon discharge lamp Mercury arc…

13. Tungstan lamp Tungstan lamp: Functioning same as an electric light bulb. The tungsten filament lamp is commonly employed as a source of visible light. This type of lamp is used in the wavelength range of 350 - 2500 nm. The energy emitted by a tungsten filament lamp is proportional to the fourth power of the operating voltage. This means that for the energy output to be stable, the voltage to the lamp must be very stable indeed. Electronic voltage regulators or constant voltage transformers are used to ensure this stability.

14. Hydroger or Deuterium lamp In this lamps, hydrogen gas is stored under relatively high pressure. When a electric discharge is passed through the lamp, excited hydrogen molecule will be produce which emit U.V radiation. Its Cover the rang 3500-1200A. These are stable, robus and widely use. If deuterium lamp is used instead of hydrogen lamp it increase the emission intensity. Deuterium lamp is more expensive then hydrogen lamp. But are use when higher intensity is required.

15. Xenon discharge lamp In this lamp xenon gas is store under pressure in the rang of 10-30 atmosphere. Xenone lamp posses two tungsten electrodes separated by about 8mm. When an intense arc is form between these two electrodes by applying a low voltage, the U.V light is produced. The intensity of U.V radiation produce by Xenone lamp is mch greater then Hydrogen lamp.

16. Xenon discharge lamp

17. Mercury arc In this, the mercury vapour is under high pressure, and the excitation of mercury atom is done by electric discharge. Not suitable for continuous spectral studies because of the presence of sharp lines and bands. Generally the low pressure mercury arc is very useful for calibration work.

18. Monochromator

19. Most instruments use a monochromator to separate light from the source into discrete wavelength segments Components: – Entrance slit – Collimating/focusing device - mirror or lens, – Dispersing device -filter, grating or prism – Collimating/focusing device - mirror or lens – Exit slit The monochromator are use to dispersing the radiation according to the wavelength. The essential element of the monochromator are an entrance slit, a dispersing element and exit slit. The entrance slit sharply defined the beam of heterochromic radiation.

20. The dispersing element disperses the the heterochromic radiation into its component wavelength whereas exit slit allows the nominal wavelength together with a band of wavelength on either side of it. The position of dispersing element is always adjusted by rotating it to very the nominal wavelength passing through the exit slit. Dispersing element may be prism or grating. Grating is generally made by Glass, Quartz or fused silica. Glass has high resolving power but it is not transparent to radiation having the wavelength between 2000 and 3000 A because glass absorb in this region. Quartz or fused silica are generally used due to their transparency throughout the entire U.V range.

21. b) Prism monochromator

22. Gratings Flat metallic grating Concave grating – more accurate; eliminates mirrors and lenses Holographic – Glass surface with photoresistor is grooved with laser beam; where more accurate distance is achieved; higher density of lines is possible – 6000/mm. This master grating is coated with aluminum to replica of holographic grating.

23. Sample containers (cuvettes)

24. Sample containers The sample call that are to contain samples for analysis should fulfill three main condition: They must uniform in construction. The material of construction should be inert to solvent. They must transmit light of the wavelength used. Most commonly use cell made up of quartz or fused silica. When double-beam instrument are used they needed two cell one as a reference and one for the sample, these two cell must be same in construction to minimize the error called as “Matched cells”.

25. CUVETTES CUVETTES: Virtually all UV spectra can be recorded in the solution phase and the samples are placed in cells or cuvettes. Cells may be made of glass, plastic or quartz. Quartz is transparent in all (200-700nm) ranges and is normally used in UV region. Plastic and glass are only suitable for visible spectra.

26. DETECTORS Signal from detector have to be proportional to the amount of radiation Response of detector have to be linear Detector should have wide linear response range DETECTORS : The four common types of detectors are: 1.Barrier layer cell or photovoltaic cell 2.Phototube or photocell or photo-emissive tubes 3.Photomultiplier tubes(PMT) 4. The linear photodiode array 5. Charge-Coupled Devices (CCDs)

27. Barrier layer cell or photovoltaic cell Also known as photovoltaic. Consist on the semiconductor such as selenium which is deposited on a strong metal base, such as iron. Very thin layer of silver and gold id sputtered over the surface of the semiconductor as the second collector electrode. Radiation falling on the surface produce the electron on the silver interface. Electron are accumulated on silver surface and this accumulation produce the voltage different between silver surface and the bas of cell. Photocurrent is flow which is directly proportional to the intensity of incident radiation beam.

28. Phototubes (PT) Photon Transducers: Covert photon energy to electrical signal (current, voltage, etc.) • Detectors based on photoelectric effect: Phototubes, Photomultiplier tubes • Phototube: – Incident photon causes release of an electron – Photocurrent – Not best for low-light scenarios

29. Phototubes (PT) Its consist on the high sensitive cathode in the form of a half cylinder of metal which is contained in an evacuated tube. The anode is also present in the tube. Inside surface on the photo cell is coated with the high sensitive layer. When the light incident upon the photo cell the surface coating emits the electrons. These are attracted and collected by an anode. The current which is created between the cathode and anode is regarded a measure of radiation falling on the detector.

30. Phototubes(PT)

31. Photomultiplier tubes(PMT) The photomultiplier tube is a commonly used detector in UV-Vis spectroscopy. It consists of a photo-emissive cathode (a cathode which emits electrons when struck by photons of radiation), several dynodes (which emit several electrons for each electron striking them) and an anode. A photon of radiation entering the tube strikes the cathode, causing the emission of several electrons. These electrons are accelerated towards the first dynode (which is 90V more positive than the cathode).

32. Photomultiplier tubes(PMT) The electrons strike the first dynode, causing the emission of several electrons for each incident electron. These electrons are then accelerated towards the second dynode, to produce more electrons which are accelerated towards dynode three and so on. Eventually, the electrons are collected at the anode. By this time, each original photon has produced 106 - 107 electrons. The resulting current is amplified and measured. Photomultipliers are very sensitive to UV and visible radiation. They have fast response times. Intense light damages photomultipliers; they are limited to measuring low power radiation.

33. The linear photodiode array The linear photodiode array is an example of a multichannel photon detector. These detectors are capable of measuring all elements of a beam of dispersed radiation simultaneously. A linear photodiode array comprises many small silicon photodiodes formed on a single silicon chip. For each diode, there is also a storage capacitor and a switch. The individual diode capacitor circuits can be sequentially scanned. They are useful for recording UV absorption spectra of samples that are rapidly passing through a sample flow cell, such as in an HPLC detector.

34. The linear photodiode array

35. Charge-Coupled Devices (CCDs) Charge-Coupled Devices (CCDs) are similar to diode array detectors, but instead of diodes, they consist of an array of photo-capacitors.

36. Recording system The signals from the photomultiplier tube is finally received by the recording system. The recording is done by the recorder pen.

37. Application The Ultra-violet spectroscopy has been mainly applied for the detection of functional group, the extent of conjugation, detection of polynuclear compound by comparison etc. The wavelengths of absorption peaks can be correlated with the types of bonds in a given molecule and are valuable in determining the functional groups within a molecule.

38. Some important application is as follow: Extent of conjugation: Conjugation may be between two or more C=C or triple bond, between C-C or C-O bond, between double bond in aromatic rings. 2. Identification of unknown compound: Unknown compound can be identified by their characteristic absorption peaks. 3. Detection of functional group. 4. Distinction of conjugation and non conjugation compound. 5. Examination of poly nuclear carbon atom. 6. Elucidation of the structure of Vit A and Vit K. 7. Preference over two Tautomeric form. 8. Detection of impurity.

39. 9. Identification of compound in different solvent. eg : Chloral hydrate shows absorption maximum at 290 mu in Hexane while the absorption is disappear in aqueous solution. 10. Determination of configuration of Geometrical isomers: Cis compound is absorb at different wavelength as compare to trans compound. eg : Cis - stilben and Trans- stilben 11. Determination of strength of hydrogen bonding.

40. 12. Chemical Reaction A common method of analysis is to change the required component by adding a chemical reagent which reacts with it specifically to form a highly absorbing compound. An example of this is shown in Figure 16. A quantity of the reagent added to the mixture reacts only with one component and both increases its absorption and changes the wavelength of the absorption maximum so that there is no longer interference between the components. The analysis is then reduced to a simple case and its sensitivity is improved. Many hundreds of such specific reagents are now available for all sorts of analyses and sample matrices and are thoroughly detailed in the literature.

41. Improving sensitivity by adding a chemical reagent

42. REFERENCES Chatwal Gurdeep R & Anand Sham K “ Instrumenral Method of Chemical Analysis” Fifth edition, 2007, Published by Himalaya Publication House, page No: 2.107-2.148 Sharma Y.R & Chand.K “Elementary Organic Chemistry Organic Spectroscopy, Principle and Chemical Applications”, Forth Edition 2009, Published by S.Chand & Company Ltd. Page No: 09-64 http://learnspectroscopy.blogspot.com wikipedia.org www.molecularinfo.com www. shu.ac.uk.com