Instrumentation

Chromatography

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.

Paper Chromatography

Chromatogram of 10 essential oils coloured with vanillin reagent.

Development of Chromatogram

Paper Chromatography

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

Thin Layer Chromatography

Thin Layer Chromatography

Thin Layer Chromatography

PrincipleDifferent 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.

Processes1.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

UsesSeparation of dyes taken

from fibres in forensic work

Column Chromatography

Gas Chromatography

Gas Chromatography

Gas Chromatography

PrincipleDifferent 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.

Gas Chromatography

Processes

Injection

Transport of the sample along the column

Separation in the column

Detection

UsesDrug tests on athletes

Blood alcohol tests

High Performance Liquid Chromatography

High Performance Liquid Chromatography

High Performance Liquid Chromatography (HPLC)

PrincipleDifferent 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.

High Performance Liquid Chromatography (HPLC)

Processes

1.Injection

2.Transport of the sample along the column

3.Separation in the column

4.Detection

UsesGrowth promoters in meat

Vitamins in food

HPLC of a mixture of compounds

All chromatography needs:

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.

• support material – stationary phase• solvent (or carrier gas) – mobile phase.

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.

Mass Spectrometry

Gas SampleEnters Here

Filament current Ionises the Gas

Ions accelerate towards charged

slit

Magnetic Field deflects lightest ions most

Ions separated by mass expose film

http://antoine.frostburg.edu/chem/senese/101/atoms/faq/how-does-mass-spec-work.shtml

Mass Spectrometry

PrinciplePositively charged ions are separated on the basis of their relative masses as they move in a magnetic

field

Mass Spectrometry

Processes1.Vaporisation2.Ionisation

3.Acceleration

4.Separation

5.Detection

UsesIdentify compounds e.g.

1.in analysis of gases from waste dumps

2.in trace organic pollutants in water

3.in drug testing

Measure relative atomic mass

Measure relative abundance of isotopes

Mass Spectrometry

Atomic Weights and Mass Spectra

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.

Infra Red Absorption Spectrometry

Infra Red Absorption Spectrometry

Infra Red Absorption Spectrometry

IR Source

Sample

Reference

Splitter Detector Processor Printout

Absorptions of Bonds in Organic Molecules

http://en.wikipedia.org/wiki/Infrared_spectroscopyhttp://www2.ess.ucla.edu/~schauble/MoleculeHTML/CO2_html/CO2_page.htmlhttp://www2.ess.ucla.edu/~schauble/MoleculeHTML/CH4_html/CH4_page.html

Infra Red Absorption Spectrometry

Infra Red Absorption Spectrometry

IR of Methanol

Infra Red Absorption Spectrometry

Infra Red Absorption Spectrometry

PrincipleMolecules 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 sample2.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

Ultraviolet-visible spectroscopy

Ultraviolet-visible spectrophotomer

Ultraviolet Absorption Spectrometry

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

Processes1.Ultraviolet light is passed through the sample and a blank

2.The sample absorbs ultra violet radiation at specific wavelengths which are detected

3.Absorption spectrum is produced

UsesQuantitative determination of organic compounds

1.Drug metabolites

2.Plant pigments

UV of Benzene

Spectra

Continuous Spectra

Line Spectra

Emission Spectra

Absorption Spectra

Emission Spectra

Atomic Absorption

Atomic Absorption Spectra

Hydrogen

Helium

Lithium

• bottom step is called the ground state • higher steps are called excited states

Summary: • line spectra arise from transitions between

discrete (quantized) energy states

Energy Staircase Diagram for Atomic Hydrogen

Atomic Absorption Spectrometry

Atomic Absorption Spectrometry

Atomic Absorption(Magnesium in Water)

Atomic Absorption(Lead in Petrol)

Atomic Absorption Spectrometry

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 element3.Absorption spectrum is produced

Uses1.Identification of elements

2.Concentration of elements

3.Analysis of heavy metals in water e.g. lead, cadmium

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.

1. Wavelength selection2. Printer button, 3. Concentration factor adjustment4. Lamp5. Readout6. Sample compartment7. Zero control (100% T), 8. Sensitivity switch.

Colorimetry

Filter or Diffraction grating to select appropriate beam of light

Colorimetry

Processes1.Light of a particular wavelength is passed through a number of samples of known concentration.2.A graph of absorbance against concentration is plotted3.The absorbance of the unknown is noted and using the graph the concentration of the unknown can be found

UsesUsesAnalysis 1.Lead in water2.Fertilisers in water

e.g. nitrates and phosphates

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.

Colorimetry

• http://www.le.ac.uk/spectraschool/

Spectra Websites