Spectrophotometry by Dr Samson Ojedokun.pptx

Spectrophotometry is a method that hinges on the quantitative analysis of molecules depending on how much light is absorbed by coloured compounds.
Important features of spectrophotometers are spectral bandwidth (the range of colours it can transmit through the test sample), the percentage of sample transmission, the logarithmic range of sample absorption, and sometimes a percentage of reflectance measurement.

1. SPECTROPHOTOMETRY Dr. S.A. Ojedokun Department of Chemical Pathology LTH Ogbomoso 1

2. Outline Introduction Light spectroscopy Principles of light Absorption Principles of Spectrophotometry Classifications/Types Interferences Quality assurance Applications Conclusion References 2

3. Introduction Spectrophotometry is a method that hinges on the quantitative analysis of molecules depending on how much light is absorbed by coloured compounds. Important features of spectrophotometers are spectral bandwidth (the range of colours it can transmit through the test sample), the percentage of sample transmission, the logarithmic range of sample absorption, and sometimes a percentage of reflectance measurement. Photometry is the measurement of the amount of luminous light (luminous intensity) falling on a surface from a source. 3

4. Intro cont’d Spectrophotometry is a scientific analytical technique based on the absorption of light by a solution at a particular wavelength with relevant properties of the solution., e.g., concentration. Spectrophotometry depends on the light-absorbing properties of substances or a derivative of the substance being analyzed. An instrument for measuring the intensity of light in a part of the spectrum, as transmitted or emitted by particular substances is a spectrophotometer 4

5. Light spectroscopy Light is a form of energy propagating into space at a very high speed. As an electromagnetic wave travelling into space – it is radiant energy. The energy of light oscillates periodically between a minimum and a maximum as a function of time – like a wave. The distance between two maxima or two minima, respectively of the electromagnetic wave is defined as the wavelength, given in nanometers (nm). Light behaves like discrete energy packets called photons whose energy is inversely proportional to the wavelength The shorter the wavelength, the higher the energy 5

6. Light spectroscopy cont’d 6

7. Light spectroscopy cont’d Thus, the different components of light are characterised by a specific wavelength. The sum of all components i.e. of all wavelengths, is called a spectrum. More specifically, a spectrum represents a distribution of radiant energy. For instance, the electromagnetic spectrum of visible light ranges from approximately 390nm up to approximately 780 nm with different colours . Each colour has a specific wavelength, e.g. red light has a wavelength of 660 nm, while green light has a wavelength of 520 nm. 7

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9. Light spectroscopy cont’d Optical spectroscopy is based on the interaction of light with matter. The light which is not absorbed by the object is reflected and can be seen by the eye 9

10. Principles of Light Absorption 10 Properties of Light

11. Beer’s Law The absorbance of light is directly proportional to both the concentration of the absorbing medium and the thickness of the medium. In Spectrophotometry the thickness of the medium is called the path length. Beer’s law allows the measurement of samples of differing pathlength and compares the results directly with each other. In basic terms: Absorbance = Concentration × Pathlength 11

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13. Lambert’s Law The proportion of light absorbed by a medium is independent of the intensity of incident light. A sample which absorbs 75% (25% transmittance) of the light will always absorb 75% of the light, no matter the strength of the light source. Lambert’s law is expressed as I/Io=T Where I = Intensity of transmitted light Io = Intensity of the incident light T = Transmittance This allows different spectrophotometers with different light sources to produce comparable absorption readings independent of the power of the light source. 13

14. Beer-Lambert law When light, passes through a transparent cuvette filled with sample solution, the light intensity is attenuated proportionally to the sample concentration. In other words, a highly concentrated sample solution will absorb more light. In addition, the attenuation is also proportional to the length of the cuvette; a longer cuvette will lead to a higher absorption of light. 14

15. Mathematically, 15

16. This relationship is called the Lambert-Beer law where: 1. The sample concentration is c. 2. The path length, d of the cuvette. 3. The extinction coefficient ε (epsilon) is a sample-specific constant describing how much the sample is absorbing at a given wavelength When the path length is 1 cm and the concentration is 1% w/v, the extinction coefficient is called specific absorbance (E ) 16

17. The Lambert-Beer law allows for the determination of the sample concentration from the measured absorbance value. If the extinction coefficient ε and the path length d are known, then concentration c can be calculated from absorbance A as given below: 17

18. Beer’s law holds if the following conditions are met Incident radiation on the substance of interest is monochromatic. The solvent absorption is insignificant compared with the solute absorbance. The solute concentration is within given limits. An optical interference is not present. A chemical reaction does not occur between the molecules of interest and another solute or solvent molecule. The sides of the cell are parallel 18

19. Calibration curve/ standard curve A solution of known concentration is prepared. Serial dilutions up to about 5 solutions of different concentrations. Spectrophotometric absorbance is set at zero using a blank. Measurement of absorbances of the solutions. Plotting of absorbances(y axis) against concentrations (x axis). Determination of the concentration of unknown solution using the graph. 19

20. Calibration curve for glucose assay 20

21. Principles of Spectrophotometry The spectrophotometry technique is used to measure light intensity as a function of wavelength using a spectrophotometer. And this is achieved through : Diffraction of the light beam into a spectrum of wavelengths Directing the diffracted light onto an object Reception of the light reflected or returned from the object Detecting the intensities with a charge-coupled device Displaying the results as a graph on the detector and then the display device 21

22. Basic components of spectrophotometer Light source Monochromator Cuvette or sample cell Photo detector Readout device Recorder 22

23. 23 Basic components of a spectrophotometer

24. Lamp source 1. Incandescent lamps UV spectrum Deuterium-discharge lamp Mercury arc lamp Xenon arc lamp Hydrogen lamp Visible and near infrared region Tungsten halogen lamp containing iodine or bromine 2. Laser ( Light amplification by stimulated emission of radiation ) This is a device used in spectrophotometry, which transform light of various frequencies into an extremely intense, focused, and nearly non divergent beam of monochromatic light. 24

25. Important factors for a light source Range Spectral distribution within the range Stability of radiant energy Source of radiant production Temperature 25

26. Monochromator Necessary to isolate a desired wavelength of light and exclude other wavelengths Wavelength isolation is a function of the type of device used and the width of the entrance and exit slits. Devices used to obtain monochromatic light include Filters (colored glass and interference) Prisms Diffraction gratings 26

27. Colored glass filters Least expensive Simple, although not always precise Usually pass a relatively wide band of radiant energy Interference filters Produces a monochromatic light based on the principle of constructive interference of waves Prism A narrow beam of light focused on a prism is refracted as it enters the denser glass. The prism can be rotated, allowing only the desired wavelength to pass through an exit slit. 27

28. Diffraction gratings Most commonly used as monochromators. Diffraction (the separation of light into component wavelengths), is based on the principle that wavelengths bend as they pass a sharp corner. The degree of bending depends on the wavelength Diffraction grating consists of many parallel grooves (15,000-30,000 per inch) etched onto a polished surface. Because the multiple spectra have a tendency to cause stray light problems, accessory filters are used. 28

29. Cuvette/sample cell Are small vessels used to hold liquid samples to be analysed in the light path of the spectrophotometer. Can be round or square Square cuvettes have advantages over round cuvettes in that there is less error from lens effects, orientation and refraction. It can be made of Glass (Visible range), or quartz(UV & Visible range) Light path must be kept constant Cuvettes with scratched optical surfaces should be discarded as they scatter light. 29

30. Photodetector Detects transmitted radiant energy and converts it into an equivalent amount of electricity Types include: Photocell/Barrier layer cell Phototube Photomultiplier tube Photodiode 30

31. Photocells/Barrier layer cell: Least expensive and durable Composed of a film of light sensitive materials: Selenium on a plate of iron covered by a thin transparent layer of silver When exposed to light, electrons in the light sensitive materials are excited and released to flow through highly conductive silver Resistance prevents ions from flowing in the opposite direction towards the iron thus forming a barrier. EMF is generated from the resistance which can be measured. It is temperature sensitive and becomes non linear at very high or very low levels of illumination. Output is not easily amplified so it is used in instruments with illumination levels such that there is no need to amplify the signals. Light-sensitive 31

32. Phototube Similar to photocell but needs an outside voltage to operate it. Contains cathode and anode enclosed in a glass case Cathode: made up of lithium or rubidium that acts as a resistor in the dark but emits electrons when exposed to light. The emitted electrons jump over to the positively charged anode where they are collected and return through an external measurable circuit 32

33. Photomultiplier(pm) tube Detects and amplifies radiant energy, hence more sensitive than the phototube. Incident light strikes the coated cathode emitting electrons The electrons are attracted to a series of anodes known as dynodes each having a successively higher positive voltage These dynodes are of a material that gives off many secondary electrons when hit by a single electron Initial electron emission at the cathode triggers a multiple cascade of electrons within the PM tube The accumulation of light striking the anode produces a current signal measured in amperes They are used in instruments designed to be extremely sensitive to very low light levels and light flashes of very short duration 33

34. Photodiode In a photodiode, absorption of radiant energy by a reverse-biased pn -junction diode ( pn , positive-negative) produces a photocurrent that is proportional to the incident radiant power. Photodiode array (PDA) detectors are available in integrated circuits containing 256 to 2,048 photodiodes in a linear arrangement Each photodiode responds to a specific wavelength, Its excellent linearity (6–7 decades of radiant power), speed, and small size make them useful in applications where light levels are adequate 34

35. Readout devices Analog Uses deflector pin on a meter Zero error is common Parallax error Easily affected by current/light voltage No longer popular Digital Now common with newer spec Limit zero error No parallax error 35

36. Classifications Electromagnetic forms UV, ViS , & IR Geometry designs Scanning & Array Optical pathways Single & double beam 36

37. A. Based on electromagnetic form 1. UV spectrophotometry Uses light over the UV range (180-400nm) A prism of suitable material and geometry will provide a continuous spectrum in which the component wavelengths are separated in space In addition to prisms, diffraction gratings are also employed for producing monochromatic light Quartz cuvettes used to hold samples 37

38. 2. ViS Spectrophotometry Uses the visible range (~400-700nm) of the electromagnetic radiation spectrum Plastic or glass cuvettes can be used for visible spectrophotometry 3. Infrared spectroscopy ( IR) Infrared spectrum refers to a spectrum greater than 760nm, which is the most commonly used spectral region of organic compounds, and can analyze a variety of conditions (gas, liquid, solid) of the sample. 38

39. 39 B. Based on Geometry designs 1. Scanning spectrophotometer The working principle of a conventional scanning spectrophotometer is based on the measurement of the transmittance value at each single wavelength. The transmittance at this specific wavelength is recorded. The whole spectrum is obtained by continuously changing the wavelength of the light (i.e. scanning) incoming onto the sample solution by rotating the grating

40. Scanning spectrophotometer 40

41. 41 2. Array spectrophotometer In this configuration, the sample in the cuvette simultaneously absorbs different wavelengths of light. The transmitted light is then diffracted by a reflection grating located after the cuvette Subsequently, the diffracted light of various wavelengths is directed onto the detector. The detector, with its long array of photosensitive, semiconductor material, allows for simultaneous measurement of all wavelengths of the transmitted light beam. This design is also known as "reverse optics"

42. Array spectrophotometer 42

43. 43 C. Based on optical paths 1. Single beam configuration The light beam is directly guided through the sample onto the detector. A cuvette containing only solvent has to be measured first to determine the blank value. After measuring the blank value, the solvent cuvette is replaced by a cuvette containing the sample to measure the absorption spectrum of the sample.

44. 2. Double-beam configuration In a double-beam configuration, the light beam is split into a reference and a sample beam. Simultaneous in time: The light beam of the lamp is split into two beams of equal intensities. Each beam passes through a different cuvette; one is the reference cuvette, whereas the second cuvette contains the sample solution. The intensities of both beams are measured simultaneously by two detectors. 44

45. Simultaneous-in-time 45

46. 46 b) Alternating in time: This configuration is achieved by directing the light path with an optical chopper (OC), which is a rotating sectional mirror. The light is directed alternately through a sample and a reference cell. A unique detector measures both light beams one after the other.

47. Interference Interference is phenomenon that leads to changes in intensity of the signal in spectrophotometry. Types of interference Optical interference is a phenomenon in which two wavelengths superimpose to form a greater or lower wavelengths. Chemical interference arises out of the reaction between different interferents and the analyte. Physical interference are due to physical properties of the sample e.g. impurities in the solution 47

48. Quality assurance Wavelength accuracy Checked using standard absorbing solutions or filters with maximal absorbance of known wavelength. Stray light Refers to any wavelength outside the band transmitted by the monochromator. Most common causes are light reflection from scratches on optical surfaces or dust particles in the light path. Stray light is detected and eliminated by using cutoff filters. Linearity Refers to the difference between the actually measured value and the value derived from the equation. It is checked using coloured solutions of different concentrations labelled with expected absorbances. 48

49. Applications of Spectrophotometry Chemical reactions End point reaction Kinetics reaction Fixed time Two-point absorbance Fixed wavelength 49

50. Bio applications Use in clinical laboratory analysis Dissolution/ in vitro releases assay of drugs Quantification of DNA, RNA and proteins Dye, ink and paint industries Heavy metal and organic matter from environmental/agricultural samples 50

51. Other applications Quantifying concentrations of compounds. Determining the structure of a compound. Finding functional groups in chemicals. Determining the molecular weight of compounds. Determining the composition of materials 51

52. Conclusion The use of spectrophotometers spans various scientific fields, such as  physics,   materials science,   chemistry,   biochemistry,   chemical engineering, and  molecular biology semiconductors, laser and optical manufacturing, printing and forensic examination, as well as in laboratories for the study of chemical substances.  Spectrophotometry continues to enjoy wide popularity due to the common availability of its instrumentation and simplicity of procedures, as well as speed, precision and accuracy of its technique. 52

53. References Tietz fundamentals of clinical chemistry, 2008 fifth edition chapter 4 pg 63-80. Cosimo A. De Caro, Haller Claudia. UV/VIS Spectrophotometry - Fundamentals and Applications. 2015 https://www.researchgate.net/publication/321017142 The principles of use of a spectrophotometer and its application in the measurement of dental shades Chapter 11 Spectrophotometer Mass spectrometry https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/massspec/masspec1.htm 53

54. 54 Thank you