Analytical instruments introduction

spectrometry,laws of photometry,absorption spectroscopy

1. IN 504 Analytical Instruments Reference Text: R S Khandpur “Handbook of Analytical Instrumentation” 1 Presented by; Anju Sunny CUSAT

2. Introduction… What are Analytical Instruments ? Instruments that are used to analyze materials and to establish the composition. Provide, qualitative information quantitative data 2

3. Elements of an Analytical Instrument 3

4. Absorption Spectroscopy Most of the instrumental analysis methods are based on the absorption of electromagnetic radiation in the visible, ultraviolet and infrared ranges. The method based on absorption of radiation of a substance is known as Absorption Spectroscopy or Absorption Spectrophotometer. Advantages: High speed Sensitivity to very small amounts Simple operational method 4

5. Laws of Photometry 1) Lambert’s Law States that each layer of equal thickness of an absorbing medium absorbs an equal fraction of the radiant energy that traverses it. Lambert’s law is expressed as: Transmittance T= I / I Absorbance = Log 10 (1/T) where I  incident radiant energy I  energy which is trans mitted 5

6. Laws of Photometry 2) Beer’s Law States that absorption of light is directly proportional to both concentration of the absorbing medium and the thickness of the medium in the light path. Based on this, for a fixed path length, Absorption spectroscopy can be used to determine the concentration of the absorber in a solution. 6

7. Laws of Photometry 3) Beer - Lambert Law Defines relationship between Absorbance (A) and Transmittance (T). States that the concentration of a substance in solution is directly proportional to the Absorbance, A of the solution. Absorbance, A = ε c b where A   measured absorbance, in Absorbance Units (AU) ε  constant known as the molar absorptivity (function of wavelength) (dm 3 mol -1 cm -1 ) c  concentration of the absorbing species (mol dm -3 ) b  path length through the sample (cm) 7

8. Limitations of Beer - Lambert Law Only applicable to monochromatic radiations . Non-linearity arises at high concentration . Chemical & Instrumental factors which causes non-linearity Deviations in absorpitivity coefficient at high concentration Scattering of light due to particulates in sample Fluorescence or phosphorosence of the sample Changes in the refractive index Shifts in chemical equilibrium Non-monochromatic radiation Stray light 8

9. Types of Absorption Spectrophotometer Commonly used Absorption Spectrophotometer is : UV –Vis - NIR Spectrophotometers ( U ltra V iolet – Vis ible – N ear I nfra R ed Spectrophotometer) This means it uses light in the UV, visible and near-infrared (NIR) ranges. 9

10.  UV - VIS SPECTROPHOTOMETERS   10

11. RADIATION SOURCES  Provide sufficient intensity of light for making a measurement. Blackbody Sources :- A hot material like electrically heated filament which emits a continuous spectrum of light. Discharge lamps :- When electric current pass through a rare gas or metal vapour , the electrons collide with gas atoms, exciting them to higher energy levels and then decay to lower levels by emitting light. Lasers :- Laser beam is highly directional, monochromatic and provide high density energy which can be finely focused. 11

12. Blackbody sources Tungsten filament lamps (350 to 2500 nm)  visible Glowbar lamps (1 to 40 μ m)  infrared Nernst glower lamps (400 nm to 20 μ m)  infrared 12

13. Discharge lamps Hydrogen or deuterium lamps (160 to 380 nm)  ultraviolet Mercury Lamps ( 253.7 nm)  visible and near UV Ne, Ar , Kr, Xe discharge lamps (300 – 13 nm)  near UV to near IR 13

14. Wavelength Selectors or Filtering Arrangement For selection of a narrow band of radiant energy. Requirements of filters:- -- High transmittance at desired wavelength -- Low transmittance at other wavelength It can be; Optical Filters Absorption filter Interference filter Monochromators Prism monochromators Diffraction grating Reflection Gratings 14

15. Absorption Filters Used in the visible range.  Have effective bandwidths from 30 to 250 nm.  Less expensive than interference filters. 15

16. Three types of absorption filters: 1) Coloured glass :- The filters absorb all wavelengths of light except for particular wavelengths which they pass. 2) Dyed gelatin :- do not last long and must be frequently replaced. 3) Sharp cutoff (band pass filter) :- consists of two filters put together. 16

17. Interference Filters Use optical interference to provide narrow bandwidths of radiation. Consists of a dielectric insulator like MgF 2 or CaF 2 which is sandwiched between two semitransparent metallic films.   These three layers are then sandwiched between two plates of glass or transparent materials. 17

18. Interference Filters - working Thickness of the dielectric layer determines the wavelength of the transmitted radiation.  When the beam of radiation strikes this filter, some of the radiation passes through the first metallic layer while the rest is reflected.  The remaining light then strikes the second metallic layer and some is passed while the rest is reflected. 18

19. Interference Filters - working If the reflected light from the second layer is of the proper wavelength, it is partially reflected from the inside surface of the first layer in phase with incoming radiation of the same wavelength.   The result is that the desired wavelength is reinforced while the others wavelengths, being out of phase, undergo destructive interference. Interference filters are used throughout the ultraviolet and visible regions and about 14 μ m into the IR region. 19

20. Interference Interference  is a phenomenon in which two waves superimpose to form a resultant wave of greater or lower amplitude. Two types ; Constructive interference :- Constructive interference occurs when the phase difference between the waves is a multiple of 2π. Destructive interference :- Destructive interference occurs when the difference is an odd multiple of π. 20

21. 21 Resultant wave Wave 1 Wave 2 Constructive interference Destructive interference

22. Monochromators Principle is based on Refraction. Allows only certain wavelengths to be selected and used. Types; Prism Grating Diffraction Grating Reflection Grating 22

23. Prism Monochromator Basic principle : The isolation of different wavelengths in a prism monochromator is based on refractive index of materials is different for radiation of different wave lengths. Optical elements: Entrance slit Collimating lens Prism or grating Focussing element Exit slit 23

24. Basic Principle 24

25. Basic Setup – Prism Monochromator 25

26. Working - How select one particular wavelength? 26

27. h 1 sin q 1 = h 2 sin q 2 Snell’s Law of Refraction : Also , remember that no refraction occurs if light at normal or θ 1 = 0 So , light must hit prism at an angle. Most common is a 60 o prism of glass or quartz. High resolution prism: mixture of Silicon dioxide, Sodium chloride and Potassium bromide.

28. Grating Monochromator Two types: Diffraction Grating Reflection Grating 29

29. Diffraction /Transmission Grating Basic Principle : Diffraction Diffraction phenomenon is described as the apparent bending of waves around small obstacles and the spreading out of waves past small openings. 30 Interference pattern from two-slit diffraction.

30. Diffraction Grating A diffraction grating consists of a series of parallel grooves or slits on a highly polished reflecting surface. When light is incident on a diffraction grating, diffractive and mutual interference effects occur, and light is reflected or transmitted in discrete directions. The separation of grooves in the direction of radiation is a whole number of wavelength, then the waves would be in-phase and radiation would be reflected undistributed. 31

31. Diffraction Grating 32

32. Relation between λ , d, and θ λ  wavelength of radiation d  distance between grooves θ  angle at which the radiation is reflected m  order of interference N  total no. of grooves 33 m λ = 2 d sin θ Resolving Power= mN

33. Diffraction Grating in Monochromator 34

34. Reflection Grating On contrary to the above, if the plate is mirrored, we get reflection grating which have got vast applications in spectrophotometry . Most commonly used grooved surface with reflective coating (Al, Au, Pt) . 35 n l = d(sin b + sin  )

35. Effect of Slit Width 36

36. Comparison between Grating & Prism 37 Type of Dispersion Size Stray Light l range of use Grating uniform dispersion vs. l Smaller Higher stray light unlimited Prism shorter l better separated larger Less of problem Limited ( l ≤ 350 nm) Increase size of either prism or grating will give better dispersion. Stray light can be removed with filters.

37. Stray light Stray light is light of wavelengths different from those wanted. Sources of stray light: incompletely removed higher order wavelengths in grating instrument scattering of light by dust reflection of light by lens and grating mountings Prevention of stray light: use a double monochromator first monochromator selects wavelengths and passes them to a second which refines the wavelengths paint all components except for reflecting surfaces which are desired black enclose system to keep out dust 38

38. Bandwidth is defined as the frequency band around the carrier frequency containing 99 percent of the signal power. The bandwidth for an individual AM station is about 10,000 Hz. The bandwidth for an individual FM station is about 200,000 Hz . Signal bandwidth frequency bandwidth fc =carrier frequency fc+fm=upper sideband fc+fm=lower sideband

39. Resolution It is the smallest amount of input signal change that the instrument can detect reliably. 40

40. Photosensitive Detectors Purpose is quantitative measure of radiation intensities. In photosensitive detector, the light energy is converted into electrical energy . Electric current produced by this can be measured with a sensitive galvanometer. Requirements of a good detector High sensitivity Linear response over the wavelength range of interest Fast response Little or no signal in absence of light (dark current) 41

41. Photosensitive Detectors Different types: Photovoltaic Cell Photo-emissive Cell Silicon Diode Detectors Photo Diode Array (PDA) 42

42. Photovoltaic or Barrier Layer Cell 43

43. Photovoltaic Cell Advantages : Robust in construction Need no external power source Good for portable instrument Sensitive to almost the range of wavelength of the spectrum. Disadvantage : Shows fatigue (decrease in response with continued illumination), Difficult to amplify signal, since small internal resistance of selenium (Ohm’s law: I=(V/R)). 44

44. Photo-emissive Cell Requires an external power supply to facilitate flow of electrons. Amplifier circuits are employed for the amplification of the current. Three types; High vacuum Photo-emissive cell Gas-filled Photo cell Photomultiplier Tube (PMT) 45

45. High vacuum Photo-emissive cell 46

46. High vacuum Photo-emissive cell The spectral response depends upon the nature of the substance coating at the cathode. Cesium-Silver oxide cell  sensitive to Near InfraRed wavelength . Potassium-Silver oxide/ Cesium-Antimony  sensitive to visible & UV wavelength. Current  number of photons. Smaller current than photovoltaic cell, but can be amplified. 47

47. Gas-filled Photo cell Sometimes inert gas like Ar , is present in the tube. As e - collide with gas, more e - and ions produce results in an increase in current. Presence of the small quantities of the gas prevent the phenomenon of saturation current, when higher potential difference are applied between the cathode & anode. 48

48. Photomultiplier Tube (PMT) 49 photochathode anode high voltage voltage divider network dynodes light electrons e -

49. Photomultiplier Tube - Working It is a very sensitive device in which electrons emitted from the photosensitive cathode strike a second surface called dynode which is positive with respect to the original cathode. Additional electrons are generated at each dynode. If the above process is repeated several times, so more than 10 5 to 10 7 electrons are finally collected for each photon striking the first cathode. The amplification depends upon the number of dynodes and accelerating voltage . 50

50. Photomultiplier Tube Advantages : very sensitive to low intensity. very fast response. Disadvantages : need a stabilized high voltage power supply. intense light causes damages. Large & expensive 51

51. Silicon Diode Detectors 52 Silicon diode/Photo diode can be powered from a low voltage source. And signal can be amplified by a low noise op-amp. This type is not as sensitive as PMTs, but are small and robust. Commonly used semiconducting materials; Si Ge InAs – Indium Arsenide InSb – Indium Antimonide

52. Silicon Diode Detectors 53 Diagram (Refer Note)

53. Photo Diode Array (PDA) 54 Diagram (Refer Note)

54. 55 Scanning Double Beam Spectrophotometer

55. 56 Scanning Double Beam Spectrophotometer In double beam arrangement, the light alternately passes through the sample and reference (blank), directed by rotating half-sector mirror (chopper) into and out of the light path. When light passes through the sample, the detector measures the P . When the chopper diverts the beam through the blank solution, the detector measures P . The electronic circuit at the detector automatically compares P and P to calculate absorbance

56. 57 Advantages of double beam instruments Automatic correction for changes of the source intensity and changes in the detector response with time or wavelength because the two beams are compared and measured at the same time. Automatic scanning and continuous recording of spectrum (absorbance versus wavelength).

57. 58 Applications of Ultraviolet/Visible Spectrophotometry Molecular spectroscopy based upon UV-Vis radiation is used for identification and estimation of inorganic, organic and biomedical species. Molecular UV-Vis absorption spectrophotometry is employed primarily for quantitative analysis. UV/Vis spectrophotometry is probably more widely used in chemical and clinical laboratories throughout the world than any other single method.