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  • 1. Instrumentation
  • 2. Chromatography
  • 3. Chromatography • Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures. It involves passing a mixture dissolved in a "mobile phase" through a stationary phase, which separates the analyte to be measured from other molecules in the mixture based on differential partitioning between the mobile and stationary phases.
  • 4. Paper Chromatography Chromatogram of 10 essential oils coloured with vanillin reagent.
  • 5. Development of Chromatogram
  • 6. Paper Chromatography
  • 7. Paper Chromatography Principle Different components of the mixture have different interactions with the mobile phase and stationary phase so the different components of mixture will travel different distances along the paper. This separates the components of the mixture. Processes 1.Add solvent (mobile phase) to chromatography tank 1.Apply spot of mixture to chromatography paper 2.Dry 3.Place in chromatography tank so that spot is just above mobile phase. 4.Components of mixture separate out as the mobile phase moves up through the paper Uses Separates Coloured Substances
  • 8. Thin Layer Chromatography
  • 9. Thin Layer Chromatography
  • 10. Thin Layer Chromatography Principle Different components of the mixture have different interactions in the mobile phase and the stationary phase (a thin layer of silica on the glass plate) so the different components will travel different distances along the silica. This separates the components of the mixture. Processes 1.Add solvent (mobile phase) to chromatography tank 2.Apply spot of mixture to TLC plate 3.Dry 4.Place in chromatography tank so that spot is just above mobile phase. 5.Components of mixture separate out as the mobile phase moves up along the TLC plate Uses Separation of dyes taken from fibres in forensic work
  • 11. Column Chromatography
  • 12. Gas Chromatography
  • 13. Gas Chromatography
  • 14. Gas Chromatography
  • 15. Gas Chromatography Principle Different components of the mixture have different interactions with the stationary phase (liquid supported on a porous bed inside a long coiled column) and mobile phase (inert gas for example nitrogen or argon).The different components will travel at different speeds along the column. This separates the components of the mixture. Processes Injection Uses Transport of the sample along the column Drug tests on athletes Separation in the column Blood alcohol tests Detection
  • 16. High Performance Liquid Chromatography
  • 17. High Performance Liquid Chromatography
  • 18. High Performance Liquid Chromatography (HPLC)
  • 19. High Performance Liquid Chromatography (HPLC) Principle Different components of the mixture have different tendencies to absorb onto very fine particles of a solid in the HPLC column Solvent that is pumped under pressure through column so the different components will travel different speeds along the column. This separates the components of the mixture. Processes 1.Injection Uses 2.Transport of the sample along the column Growth promoters in meat 3.Separation in the column Vitamins in food 4.Detection
  • 20. HPLC of a mixture of compounds
  • 21. All chromatography needs: • support material – stationary phase • solvent (or carrier gas) – mobile phase. Chromatography Type Stationary phase Mobile phase Paper Paper Organic or aqueous solvent. Thin layer Silica gel supported on plastic film Organic or aqueous solvent. G.L.C. High boiling point liquid on inert solid support. Inert gas e.g. nitrogen.
  • 22. Spectroscopy • Spectroscopy is analysis of the interaction between electromagnetic radiation and matter. • Different types of radiation interact in characteristic ways with different samples of matter • The interaction is often unique and serves as a diagnostic "fingerprint" for the presence of a particular material in a sample • Spectroscopy is also a sensitive quantitative technique that can determine trace concentrations of substances.
  • 23. Mass Spectrometry Gas Sample Enters Here Ions accelerate towards charged slit Magnetic Field deflects lightest ions most Filament current Ionises the Gas Ions separated by mass expose film http://antoine.frostburg.edu/chem/senese/101/atoms/faq/how-does-mass-spec-work.shtml
  • 24. Mass Spectrometry
  • 25. Mass Spectrometry Principle Positively charged ions are separated on the basis of their relative masses as they move in a magnetic field Processes Uses Identify compounds e.g. 1.Vaporisation 2.Ionisation 1.in analysis of gases from waste dumps 3.Acceleration 3.in drug testing 4.Separation Measure relative atomic mass 5.Detection Measure relative abundance of isotopes 2.in trace organic pollutants in water
  • 26. Mass Spectrometry
  • 27. Atomic Weights and Mass Spectra
  • 28. GC-MS Chromatography Gas chromatography-mass spectrometry (GC-MS) is a method that combines the features of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample. Applications of GC-MS include 1. drug detection 2. environmental analysis 3. identification of unknown samples.
  • 29. Infra Red Absorption Spectrometry
  • 30. Infra Red Absorption Spectrometry
  • 31. Infra Red Absorption Spectrometry Sample IR Source Splitter Reference Detector Processor Printout
  • 32. Absorptions of Bonds in Organic Molecules http://en.wikipedia.org/wiki/Infrared_spectroscopy http://www2.ess.ucla.edu/~schauble/MoleculeHTML/CO2_html/CO2_page.html http://www2.ess.ucla.edu/~schauble/MoleculeHTML/CH4_html/CH4_page.html
  • 33. Infra Red Absorption Spectrometry
  • 34. Infra Red Absorption Spectrometry
  • 35. IR of Methanol
  • 36. Infra Red Absorption Spectrometry
  • 37. Infra Red Absorption Spectrometry Principle Molecules of a substance absorb infra red light of different frequencies. The infra red radiation is absorbed by vibrations of the bonds in the molecules. The combination of frequencies absorbed is peculiar to the molecules of that substance (fingerprinting technique). Processes 1.Infra red radiation passes through the sample 2.The sample absorbs infrared radiation at specific wavelengths which are detected 3.Absorption spectrum is produced Uses Identification of compounds e.g. in Plastics Drugs
  • 38. Ultraviolet-visible spectroscopy
  • 39. Ultraviolet-visible spectrophotomer
  • 40. Ultraviolet Absorption Spectrometry
  • 41. Ultraviolet Absorption Spectrometry Principle •Absorption of ultraviolet radiation by molecules results in the promotion of electrons from their ground state energy levels to higher energy levels •Absorbance is directly proportional to concentration Processes 1.Ultraviolet light is passed through the sample and a blank Uses Quantitative determination of organic compounds 2.The sample absorbs ultra violet radiation at specific wavelengths which are detected 1.Drug metabolites 3.Absorption spectrum is produced 2.Plant pigments
  • 42. UV of Benzene
  • 43. Spectra Continuous Spectra Line Spectra Emission Spectra Absorption Spectra
  • 44. Emission Spectra
  • 45. Atomic Absorption
  • 46. Atomic Absorption Spectra Hydrogen Helium Lithium
  • 47. Energy Staircase Diagram for Atomic Hydrogen • bottom step is called the ground state • higher steps are called excited states Summary: • line spectra arise from transitions between discrete (quantized) energy states
  • 48. Atomic Absorption Spectrometry
  • 49. Atomic Absorption Spectrometry
  • 50. Atomic Absorption (Magnesium in Water)
  • 51. Atomic Absorption (Lead in Petrol)
  • 52. Atomic Absorption Spectrometry
  • 53. Atomic Absorption Spectrophotometer Principle Atoms in the ground state absorb light of a particular radiation characteristic of an element. Absorbance is directly proportional to concentration Processes 1.Sample solution is sprayed into the flame, and the sample element is converted into atoms in the element. 2.Ground state atoms absorb radiation from a source made from the element 3.Absorption spectrum is produced Uses 1.Identification of elements 2.Concentration of elements 3.Analysis of heavy metals in water e.g. lead, cadmium
  • 54. Colorimetry 1. 2. 3. 4. 5. 6. 7. 8. Wavelength selection Printer button, Concentration factor adjustment Lamp Readout Sample compartment Zero control (100% T), Sensitivity switch. A colorimeter is a device used to test the concentration of a solution by measuring its absorbance of a specific wavelength of light. To use this device, different solutions must be made, and a control (usually a mixture of distilled water and another solution) is first filled into a cuvette and placed inside a colorimeter to calibrate the machine. Only after the device has been calibrated can you use it to find the densities and/or concentrations of
  • 55. Colorimetry Filter or Diffraction grating to select appropriate beam of light
  • 56. Colorimetry
  • 57. Colorimetry Principle 1.If a solution is coloured then the intensity of the colour is proportional to the concentration. 2. The percentage of light absorbed by the coloured solution in the colorimeter is proportional to the concentration. Processes 1.Light of a particular wavelength is passed through a number of samples of known concentration. 2.A graph of absorbance against concentration is plotted 3.The absorbance of the unknown is noted and using the graph the concentration of the unknown can be found Uses Uses Analysis 1.Lead in water 2.Fertilisers in water e.g. nitrates and phosphates
  • 58. Spectra Websites • http://www.le.ac.uk/spectraschool/