Chromatography is a technique used to separate mixtures based on how their components interact with stationary and mobile phases. There are several types of chromatography including paper chromatography, thin layer chromatography, gas chromatography, high performance liquid chromatography, and column chromatography. Chromatography uses a mobile phase to transport the analyte through a stationary phase, separating the mixture's components as they travel different distances. Spectroscopy analyzes interactions between electromagnetic radiation and matter. Different types of spectroscopy including mass spectrometry, infrared spectroscopy, ultraviolet-visible spectroscopy, atomic absorption spectroscopy, and colorimetry are used to identify substances based on their unique spectra.

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
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

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
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

15. Column Chromatography

16. Gas Chromatography

17. Gas Chromatography

18. Gas Chromatography

19. 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

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
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

25. HPLC of a mixture of compounds

26. 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.

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
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

29. Mass Spectrometry

31. 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

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
1.
drug detection
2.
environmental analysis
3.
identification of unknown samples.

36. Infra Red Absorption Spectrometry

37. Infra Red Absorption Spectrometry

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

39. 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

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
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

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
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

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
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

63. 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

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

65. Colorimetry

66. 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

68. Spectra Websites
• http://www.le.ac.uk/spectraschool/