CHROMATOGRAPHY

1. CHROMATOGRAPHY

2. CHROMATOGRAPHY • This method is used for separation of mixtures of compounds between two phases: • Stationary ( solid or liquid located on neutral medium) • Mobile( liquid or gas) • This method is used for separation of mixture of compounds with different speed of migration a given compounds in porous medium with different ability of adsorption or ion exchange or different solubility in solvents.

3. Metods of chromatography • GC, Gas chromatography) • LC, Liquid chromatography) • HPLC, High Pressure liquid chromatography • TLC, Thin layer chromatography)

4. Classification of chromtographic methods • 1. Because of physico – chemical forces: • Adsorption chromatography • Ion exchange chromatography • Partition chromatography • Gel (size exclusion) chromatography • Affinity chromatography • Capillary chromatography • 2. Because of techniques of preparation: • Column chromatography • Planar chromatography ( thin layer, paper, gel) • 3. Because of density of mobile phase: • GC, Gas Chromatography) • LC, Liquid Chromatography) • 4. Because of density of mobile and stationary phases: • Gas – liquid chromatography ( GLC) • Liquid – liquid chromatography ( LLC) • Gas – solid chromatography (GSC) • Liquid - solid chromatography (LSC)

5. Investigated compound is located in so called mobile phase and migrate with it across stationary phase, it means porous medium wich is adsorbent, ion exchanger or molecular sieve.

6. Chromatography is used for: • Separation of compounds from mixtures • Purification • Identification • Quantitative and qualitative analysis • Choice of the method depends on: • Amount of analysed mixtures • Kind of compounds • Complexity of separation

7. Explanation: • Compound is placed on stationary phase. • Mobile phase passes through the stationary phase. • Mobile phase solubilizes the components. • Mobile phase carries the individual components a certain distance • through the stationary phase, depending on their attraction to both of the phases.

8. Illustration of Chromatography Stationary Phase Separation Mobile Phase Mixture Components

10. Liquid Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase) Gas Chromatography – separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase) Paper Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase) Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase)

11. Adsorption Chromatography • Adsorption of molecules which are in contact with solid adsorbent can be caused by • Physical forces ( physical adsorption) • Chemical interaction (chemisorption). • Classification of adsorbents: • Because of adsorption activity: • Weak ( starch, saccharose) • Middle ( calcium carbonate, sodium carbonate) • Strong ( aluminium oxide, activated silica acid) • 2. Because of chemical properties: • acidic ( SiO2) • basic ( CaO) • neutral ( charcoal) • Amphoteric ( Al2O3)

12. 3. Because of chemical nature: • Organic • Inorganic • Mixed • Specyfic • 4. Because of polarity : • Highly polar ( aluminium oxide, silica oxide) • Weak polar (calcium carbonate) • Non polar ( Charcoal) • Adsorption ability depends on used solvent, adsorbent preparation and its activity • Component of the mixture is adsorbed stronger if its polarity is less different than • polarity of adsorber. • Loading on the column with polar adsorber more polar solvent than components • of separated mixture cause elution of these components from the column

13. Solvents used in chromatography are ordered with increasing • ability of elution adsorbed polar compounds: • Heksane ( the least polar) • Carbon tetrachloride (CCl4) • Benzene • Diethyl ether • Acetone • Chloroform • Ethyl acetate • Ethanol • Methanol • Water

14. Thin Layer Chromatography – TLC (partition) Separation of the mixture depends on the difference on partition coefficient of the mixture of components between two non miscible phases, from which one is a liquid coated on a medium (stationary phase), and a second one – mobile phase (liquid,gas). Nernst law of partition: K = Cs/ Cc K – Coefficient at equilibrium state, depends only on temperature and properties of solutes in solutions, and does not depend on amount of dissolved compound. Cs – concentration of compound dissolved in stationary phase Cc - concentration of compound dissolved in mobile phase

15. Thin Layer Chromatography – TLC (partition) • Simple an d fast method. • Processes responsible for separation: • adsorption • partition • ion exchange • - Combination of three above. • Advantageous of TLC: • short time of separation • simplicity • cheap

16. Preparation of chromatographic plates • Glass plates (1-2 mm thick) • For microanalytical purpose - 0.2 mm thick • Loading investigated solution on a plate: • - Using micropipette – load one small drop of a solution about 1 cm above the edge of the plate such way to reach the smallest dot. • Chromatography chamber must be : • - Leak proof • - Saturated by solwent

17. Developing of chromatogram • Developing occurs during migration of solvent • from start to the end on about 10 cm.distance • Solvent migrates because of: • capillary forces ( up technique) • gravity forces ( down technique) • Development of chromatogram • After withdrawal from the chamber and drying – act on the plate by special reagent (detector) which can react with components of the mixture and give coloured products (iodine, concentrated sulphuric acid, detector which can give fluorescence inUV).

18. Since the sample is separated in the column, different peaks on the chromatogram correspond to different components in the sample mixture. The chromatograms above show the results of separations of protein mixtures by ion exchange chromatography. The lettered peaks correspond to different proteins (A = ovalbumin, B = conalbumin, C = cytochrome c, D = lysozyme). The separation corresponding to the chromatogram on the left was performed at pH 5.85, while the one on the right was performed at pH 6.5. It is evident that operation conditions such as pH and temperature have a significant effect on the output.

19. Chromatography application for quantitative analysis – Rfcoefficient. Substance location on chromatogram is characterized by Rfvalues. (ratio of fronts) = retention factor Distance from START line to the middle of substance spot - A Rf = Distance from START line to the End line of solvent - B END B A START

20. Rf - is characteristic value for each substance. This values are provided in text books and are for use in substance characterization.

21. GAS CHROMATOGRAPHY • Fast and effective method of separation for volatile compounds mixtures. • Separated compound is carry out by gas (mobile phase) through the column filled with stationary phase. • Depends on stationary phase chromatography can be divided as : • Partition gas chromatography ( stationary phase – liquid on stationary carrier) • Adsorption gas chromatography ( stationary phase – adsorbent) • Capillary gas chromatography ( stationary phase – liquid are directly on the column walls.)

22. Schematic of gas chromatograph Sample’s injection Chromatogram =result Column Oven Carrier gas Column –stationary phase on stationary carrier. Capillary column with about 0.25 mm diameter does not need carrier material.

23. GAS CHROMATOGRAPHY cont. Carrier gas ( hydrogen, helium, nitrogen, argon, carbon dioxide) is introduced to the column. Investigated sample or mixture is injected to the column. Stream of gas is carrying samples to be separated. Compounds are divided according to their retention factor between gas and liquid phase. Separated substances are measured and registered at the outlet of the column using detector system. Operating temperature is 0oC – 400oC. Stationary phase – organic liquids with small vapor pressure. Carrier – materials with large specific area and small adsorption properties ( diatomaceous earth, clays).

24. Detectors in gas chromatography • Quantitative measurement of mixture components can be done by • using different chemical and physical properties of the compounds. • Therefore there are the following type of detectors: • Thermal conductivity detector – katarometr. • Flame-ionization detector - (FID) • β- ionization detector –radioactive . Its energy induces elements of carrier gas. • Recombination detector – is radioactive. • Detector signals are registered by sensitive writing system. • As a result we are obtaining CHROMATOGRAM.

25. Chromatogram – it is graph showing relationship detector signal vs time or gas volume. Absorbancja Czas Peak area is proportional to the amount of single analyzed sample.

26. Gas chromatograph

27. Chromatography column in gas chromatograph

28. GCMS

29. Retention parameters • Retention time tR – time from the moment of peak appearance to maximum • of substance amount • 2. Retention volume vR – volume of carrier gas necessary to elute • compound in specific retention time. • Relative retention–retention obtained by standard added to • investigated substance. • Distribution ratio K: • cL nL nG • K= = : • cG vL vG • cL cG – concentration of substances in liquid and gas phase • nL, nG – amount of moles • vL, vG – volume of liquid and gas phase

30. Distribution ability of column is increasing with length of the column, type and amount stationary phase, column temperature, speed and pressure of carrier gas. Gas chromatography is appled in qualitative and quantitative chemistry.

31. Column chromatography – another type of chromatography Chromatographic columns