Chromatography and Instrumentation

1. Chromatography and Instrumentation

2. Chromatography Components Mixture • Analyze • Identify • Purify • Quantify Separate Chromatography is used by scientists to: • Analyze– examine a mixture and its components • Identify– determine the identity of a mixture or components based on known components example – to identify the mixture found in a fibre at a crime scene. • Purify– separate components in order to isolate one of interest for further study • Quantify – determine the amount of the a mixture and/or the components present in the sample. Example – drugs in urine or blood samples

3. Explanation of Chromatography Explanation: • Mixture is placed on stationary phase • Mobile phase passes through the stationary phase • Mobile phase dissolves the parts of the mixture • Mobile phase carries the individual parts a certain distance through the stationary phase, depending on their solubility in both of the phases

4. Definition of Chromatography Definition: Chromatography is a way to separate parts of a mixture by using the different solubilites of the parts for a mobile phase compared to the stationary phase Terminology: • Mobile Phase – gas or liquid that carries the parts • Stationary Phase – the part of the apparatus that does not move with the sample

5. Types of Chromatography Paper Chromatography Thin Layer Chromatography Column Chromatography Gas Chromatography High Performance Liquid Chromatpgraphy

6. Paper Chromatography Paper = Stationary phase Liquid in jar= Mobile phase

7. Thin Layer Chromatography Glass plate with solid absorbant layer like alumina/ silica on it = Stationary phase Solvent Liquid in jar = Mobile phase Used as way to identify fibres in forensic work

8. Column Chromatography Solid filling a glass column= Stationary phase Liquid flowing downwards under gravity = Mobile phase Used as a purification technique

9. Definition of Chromatography Chromatography is a way to _______parts of a ________by using the different ___________of the parts for a mobile phase compared to the stationary phase Terminology: • Mobile Phase – • Stationary Phase –

10. Paper Chromatography Stationary phase = ? Mobile phase = ?

11. Thin Layer Chromatography Stationary phase= Mobile phase = Common application?

12. Column Chromatography Stationary phase = Mobile phase = Application?

13. HPLC – High Performance Liquid Chromatography • A highly improved form of column chromatography. Mobile phase = • Solvent of high purity • Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures - much faster.

14. HPLC – High Performance Liquid Chromatography Stationary phase • Solid particles - butsmaller particle size which gives a much greater surface area for interactions between the stationary phase and the molecules flowing past it. This allows a much better separation of the components of the mixture – even for parts that are similar to each other.

15. HPLC – High Performance Liquid Chromatography • Better detection methods can be used - highly automated and extremely sensitive. Used for measuring levels of growth promoters and vitamins in meat

16. Gas Chromatography • Mobile phase is a gas • Stationary phase is a high boiling point liquid absorbed onto a solid. • How fast a particular compound travels through the machine will depend on how much of its time is spent moving with the gas as opposed to being attached to the liquid in some way.

17. Gas Chromatogrpahy • The injector for the sample is contained in an oven • It is hot enough so that all the sample boils and is carried into the column as a gas by the carrier gas

18. Gas Chromatography • The coiled column is packed with finely ground SOLID • This is coated with a high boiling LIQUID • Column is cooler than the injector oven

19. How separation works on the column What happens to the parts of the mixture? • May remain in the gas phase. • May condense on the stationary phase. • May dissolve in the liquid on the surface of the stationary phase. Hence different components will have different retention times and separation occurs

20. Gas chromatography • The time taken for a particular compound to travel through the column to the detector is known as its retention time

21. HPLC – High Performance Liquid Chromatography • A highly improved form of column chromatography. • How is the mobile phase different than in Column chromatography?

22. HPLC – High Performance Liquid Chromatography Stationary phase • What is different about it compared to normal column Chromatography?

23. HPLC – High Performance Liquid Chromatography • What else is different about this method? Used for?

24. Gas Chromatography • Mobile phase? • Stationary phase?. • How fast a particular compound travels through the machine will depend on…

25. Gas chromatography • Retention time means… • An application is…

28. Illustration of Paper Chromatography Stationary Phase Separation Mobile Phase Mixture Components

29. Parts of mixtures identified using Rf values: Rf = Distance moved by spot (x) Distance moved by solvent (y) • A characteristic of a particular solute in a particular solvent.

30. Seperating inks in a pen using Paper chromatography Saturate tank with vapour before the experiment Draw a line with pencil on the bottom of chromatography paper Solvent should be below line level Add mixture to the line using a capillary tube, and dry before use. Calculate Rf value of any components present

31. Thinking about this experiment.. • Why is a paper chromatography tank not used for a considerable time after the chromatography solvent has been added? • Why are two lines usually drawn on a paper chromatogram? • What type of mixture is suitable for seperation using paper chromatography? • When two substances are found to have different Rf values, what does this mean? • When two substances are found to have different Rf values, what does this mean?

32. Instrumentation Part 2: Spectroscopy

33. Spectroscopy • Uses a range of techniques to examine the composition, structure and bonding of elements and compounds. • Mass Spectrometer • Infra red Spectroscopy • Ultraviolet absorption Spectroscopy

34. Mass spectrometerin determining relative atomic masses The mass spectrometer can be used to measure relative atomic masses. It is also commonly used to determine concentrations of drugs in urine samples

35. Higher Level Fundamental processes that occur in the mass spectrometer • Vaporisation • Production of positive ions • Acceleration • Separation • Detection

36. Higher Level Stage 1 - Vaporisation The liquid sample is injected into the instrument. There is a vacuum inside the chamber so the liquid turns into a gas – becomes vaporised.

37. Higher Level Stage 2 – Ionisation The vaporised sample passes into the ionisation chamber. The particles in the sample are bombarded with a stream of electrons. The collisions will knock one or more electrons out of the sample particles to make positive ions.

38. 3 - Acceleration Higher Level Negatively charged plates in the accelerator attract the positively charged ions out of the ionisation chamber and into the accelerator As the ions pass through the plates they accelerate and an ion beam passes into the separator. Negatively charged plates

39. Higher Level 5. Detection By changing the strength of the magnetic field ions of different masses are focused on the detector The signal is amplified and can be viewed on a computer screen The signal is sent to a recorder which traces out a mass spectrum. The mass spectrum is interpreted by the scientist.

40. Higher Level A mass spectrum

41. 2. Infra-Red Spectroscopy • Works by molecules absorbing Infrared energy waves which cause particular bonds to vibrate and stretch if present • Allows the identification of functional groups in a molecule

42. 3. Ultraviolet Spectroscopy • Works on the principle that molecules absorb ultraviolet energy waves, and the amount of energy absorbed will depend on the amount of substance present – even for colourless molecules Use – analysing the amount of drugs in a medicine