Instrumentation

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.laboratory techniquesseparation of mixturesanalyte

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

Development of Chromatogram

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

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

Column Chromatography

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. Gas Chromatography Processes Injection Transport of the sample along the column Separation in the column Detection Uses Drug tests on athletes Blood alcohol tests

High Performance Liquid Chromatography

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. High Performance Liquid Chromatography (HPLC) Processes 1.Injection 2.Transport of the sample along the column 3.Separation in the column 4.Detection Uses Growth promoters in meat Vitamins in food

HPLC of a mixture of compounds

All chromatography needs: 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 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

Mass Spectrometry

Principle Positively charged ions are separated on the basis of their relative masses as they move in a magnetic field Mass Spectrometry Processes 1.Vaporisation 2.Ionisation 3.Acceleration 4.Separation 5.Detection Uses Identify 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.gas-liquid chromatographymass spectrometry Applications of GC-MS include 1. drug detection 2. environmental analysis 3. identification of unknown samples.

Infra Red Absorption Spectrometry

IR Source Sample Reference Splitter Detector ProcessorPrintout

Infra Spectrophotometer

Absorptions of Bonds in Organic Molecules

Infra Red Absorption Spectrometry

IR of Methanol

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

Ultraviolet-visible spectroscopy

Ultraviolet-visible spectrophotomer

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 2.The sample absorbs ultra violet radiation at specific wavelengths which are detected 3.Absorption spectrum is produced Uses Quantitative 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 (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 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

Colorimetry A colorimeter is a device used to test the concentration of a solution by measuring its absorbance of a specific wavelength of light.colorimeter 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.solutionsdistilled watercuvette 1.Wavelength selection 2.Printer button, 3.Concentration factor adjustment 4.Lamp 5.Readout 6.Sample compartment 7.Zero control (100% T), 8.Sensitivity switch.

Colorimetry Filter or Diffraction grating to select appropriate beam of light

Colorimetry

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 Analysis 1.Lead in water 2.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

Spectra Websites

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