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.

8. Paper Chromatography
Chromatogram of 10 essential oils coloured with vanillin reagent.

9. Development of Chromatogram

10. Paper Chromatography

11. Paper Chromatography Principle Uses Processes
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
Add solvent (mobile phase) to chromatography tank
Apply spot of mixture to chromatography paper
Dry
Place in chromatography tank so that spot is just above mobile phase.
Components of mixture separate out as the mobile phase moves up through the paper
Uses
Separates Coloured Substances

12. Thin Layer Chromatography

13. Thin Layer Chromatography

14. 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
Add solvent (mobile phase) to chromatography tank
Apply spot of mixture to TLC plate
Dry
Place in chromatography tank so that spot is just above mobile phase.
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

15. Column Chromatography

16. Gas Chromatography

17. Gas Chromatography

18. Gas Chromatography

19. This separates the components of the mixture.
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
Transport of the sample along the column
Separation in the column
Detection
Uses
Drug tests on athletes
Blood alcohol tests

21. High Performance Liquid Chromatography

22. High Performance Liquid Chromatography

23. High Performance Liquid Chromatography (HPLC)

24. 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
Injection
Transport of the sample along the column
Separation in the column
Detection
Uses
Growth promoters in meat
Vitamins in food

25. HPLC of a mixture of compounds

26. All chromatography needs:
support material – stationary phase
solvent (or carrier gas) – mobile phase.

27. 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.

28. Mass Spectrometry
Gas Sample
Enters Here
Filament current Ionises the Gas
Ions accelerate towards charged slit
Magnetic Field deflects lightest ions most
Ions separated by mass expose film

29. Mass Spectrometry

31. Mass Spectrometry Principle Uses Processes
Positively charged ions are separated on the basis of their relative masses as they move in a magnetic field
Uses
Identify compounds e.g.
in analysis of gases from waste dumps
in trace organic pollutants in water
in drug testing
Measure relative atomic mass
Measure relative abundance of isotopes
Processes
Vaporisation
Ionisation
Acceleration
Separation
Detection

32. Mass Spectrometry

33. Atomic Weights and Mass Spectra

34. 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
drug detection
environmental analysis
identification of unknown samples.

36. Infra Red Absorption Spectrometry

37. Infra Red Absorption Spectrometry

38. Infra Red Absorption Spectrometry
IR Source
Sample
Reference
Splitter
Detector
Processor
Printout

39. Absorptions of Bonds in Organic Molecules

40. Infra Red Absorption Spectrometry

41. Infra Red Absorption Spectrometry

42. IR of Methanol

43. Infra Red Absorption Spectrometry

44. 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
Infra red radiation passes through the sample
The sample absorbs infrared radiation at specific wavelengths which are detected
Absorption spectrum is produced
Uses
Identification of compounds e.g. in
Plastics
Drugs

45. Ultraviolet-visible spectroscopy

46. Ultraviolet-visible spectrophotomer

47. Ultraviolet Absorption Spectrometry

48. 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
Uses
Quantitative determination of organic compounds
Drug metabolites
Plant pigments
Processes
Ultraviolet light is passed through the sample and a blank
The sample absorbs ultra violet radiation at specific wavelengths which are detected
Absorption spectrum is produced

49. UV of Benzene

50. Spectra Continuous Spectra Line Spectra Emission Spectra
Absorption Spectra

52. Emission Spectra

54. Atomic Absorption

55. Atomic Absorption Spectra
Hydrogen
Helium
Lithium

56. 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

57. Atomic Absorption Spectrometry

58. Atomic Absorption Spectrometry

59. Atomic Absorption (Magnesium in Water)

60. Atomic Absorption (Lead in Petrol)

61. Atomic Absorption Spectrometry

62. 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
Sample solution is sprayed into the flame, and the sample element is converted into atoms in the element.
Ground state atoms absorb radiation from a source made from the element
Absorption spectrum is produced
Uses
Identification of elements
Concentration of elements
Analysis of heavy metals in water e.g. lead, cadmium

63. Colorimetry
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 the other solutions.
Wavelength selection
Printer button,
Concentration factor adjustment
Lamp
Readout
Sample compartment
Zero control (100% T),
Sensitivity switch.

64. Colorimetry
Filter or Diffraction grating to select appropriate beam of light

65. Colorimetry

66. Colorimetry Principle
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
Light of a particular wavelength is passed through a number of samples of known concentration.
A graph of absorbance against concentration is plotted
The absorbance of the unknown is noted and using the graph the concentration of the unknown can be found
Uses
Analysis
Lead in water
Fertilisers in water
e.g. nitrates and phosphates

68. Spectra Websites