Chromatography

Physical method of separation in which the components to be separated are distributed between two phases – stationary ,and mobile.

1. CHROMATOGRAPHY Dr. Smily Pruthi , MD- Biochemistry See video in mobile app – tulip academy of medical sciences

2. Definition Greek chroma "color" and graphein "to write“ Physical method of separation in which the components to be separated are distributed between two phases – stationary ,and mobile Website : www.tamsmed.com

3. History Used and named in the first decade of 20 th century for separation of plant pigment chlorophyll Russian botanist Mikhail Tswett used columns of CaCo3 for separating chlorophyll Website : www.tamsmed.com

4. Contd.. Ion exchange chromatography - Frank Harold Spedding Paper chrom . - Martin and Synge GLC - Martin Website : www.tamsmed.com

5. Mobile phase carries sample through layer/ column containing stationary phase Solutes distribute btwn 2 phases Lower aff . for stationary phase – mobile phase Greater aff – stationary phase Website : www.tamsmed.com

6. Uses 1.Analytical:To examine a mixture & its contents 2.Identification:To determine the identity of components 3.Purification: To separate & isolate the components 4. Quantification: To determine the amount of the components Website : www.tamsmed.com

7. Types Chromatography Planar Column Paper Thin Layer Gas Liquid (TLC) Website : www.tamsmed.com

8. Planar Stat. Phase coated on sheet of paper or bound to glass plate Paper chrom . - Stat. phase is layer of water/ polar solvent coated on paper fibres TLC – Thin layer of particles eg silica gel spread evenly on glass plate/ plastic sheet HPTLC – Thin layer consists of particles with small dia. ( 4.5 μ M) Website : www.tamsmed.com

9. PAPER CHROMATOGRAPHY Website : www.tamsmed.com

10. Column chromatography Support particles on which stat. phase coated/ chemically bonded packed in tube, or stat. phase coated on inner side of tube Mobile phase – liquid/ gas Instrument- gas/ liquid chromatograph Website : www.tamsmed.com

11. HPLC – Stat phase small dia. Particles GC/ MS, LC/ MS – GC/ LC combined with mass spectrometry Website : www.tamsmed.com

12. Chromatogram Graphical presentation of detector response In analytical GC/ LC eluent exits from column & passes through detector & produces series of electric signals plotted as a func of time, dist/ volume Website : www.tamsmed.com

13. X-axis - Retention time - Time taken for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions Y-axis - signal obtained by eg spectrophotometer Optimal system - signal proportional to conc. of analyte Website : www.tamsmed.com

14. Chromatogram With unresolved peaks With resolved peaks Website : www.tamsmed.com

15. Separation Mechanisms Adsorption chromatography Ion exchange Solvent partition Gel filtration Affinity Steric exclusion Website : www.tamsmed.com

16. Ion Exchange Chrom . Based on electrostatic interaction btwn charged biomol & opp charged groups on ion exchange resins Solutes sep. by difference in sign & magnitude of ionic charge Stat. phase – surface of plastic resin/ silica, has func . groups with fixed cation / anionic charge Website : www.tamsmed.com

17. Resins – cation / anion exchanger Cationic exchange – sulphonic / carboxylic groups Anionic ex – Quart Nitrogen containing compounds eg Amberlite Website : www.tamsmed.com

18. Clinical App. Sep of amino acids, proteins, peptides, nucleic acids Separation & removal of inorganic ions from aqueous mixtures. Eg Water purification beds for preparing de ionized water Website : www.tamsmed.com

19. Partition Chromatography Based on diff. distribution of solutes btwn 2 immiscible liquids One liquid serves as stat phase Thin film of liquid adsorbed/ chem. Bonded on surface of support particles Website : www.tamsmed.com

20. Partition Chrom . Gas liquid (GLC) Liquid liquid (LLC) Normal Phase Reversed Phase Polar liquid – stat phase Non polar – stat MC Website : www.tamsmed.com

21. Reversed phase chrom . 1.Ion Suppression Ionic character of weakly acid/ basic analyte suppressed through modificatn of mobile phase pH By neutralizing the ionic group, solute interacts better with non polar stat phase Website : www.tamsmed.com

22. Contd.. 2. Ion – pair chrom . Counter ion added to mobile phase Forms ion pairs with analytes , & neutralizes analyte ions Ion pairs sep. by RPC Used to separate therapeutic drugs & their metabolites Website : www.tamsmed.com

23. Adsorption Chrom . Based on diff in adsorption of solute on solid particle Involves electrostatic, hydrogen bonds & dispersive interactions among the molecule and the adsorbent bed Low reproducibility Website : www.tamsmed.com

24. Affinity Chrom . Based on specific biological interaction btwn analyte & ligand Eg enz – substrate, hormone – receptor, ag – ab etc. Very selective Website : www.tamsmed.com

25. Contd.. Eg Carbo . With lectin columns LDL & VLDL with heparin col. Glycated Hb with phenyl boronate columns Website : www.tamsmed.com

26. Steric Exclusion Chrom . Gel filtration / molecular sieve /size exclusion/ molecular exclusion chromatography Molecules separated on basis of size Materials used for stat phase - cross linked dextran , poly- acrylamide , agarose . Website : www.tamsmed.com

27. Beads of these materials are porous with pore sizes that allow small molecules to be trapped Larger molecules remain in the mobile phase & get eluted from the column Website : www.tamsmed.com

28. Resolution Measure of successful chromatographic separation Rs < 0.8 ⇒ Incomplete separation Rs > 1.25 ⇒Acceptable Website : www.tamsmed.com

29. Improved by change in Column retention factor ( distribution of solute btwn stat & mobile phase) Column efficiency ( Ease of physical interaction btwn solute & column packing material) Column selectivity ( Chem. Interaction btwn solutes & column packing) Website : www.tamsmed.com

30. TLC Thin layer of sorbent, eg silica gel spread uniformly over glass plate/ plastic sheet Sample added as spot at edge Plate placed in solvent tank with lower edge in and sample band just over mobile phase After mobile phase travels certain dist, plate removed & dried Website : www.tamsmed.com

31. Separation maybe achieved in descending/ radial mode Separated components identified by diff procedures eg UV illumination, spraying with colour generating reagents HPTLC – small dia stat phase particles used → ↑ eff & reproducibility Website : www.tamsmed.com

32. Retention Factor Describes relative migration of a compound Rf =Dist. From application point to solute center / Dist. from application point to solvent front Website : www.tamsmed.com

33. Website : www.tamsmed.com

34. Advantages of TLC Technically easy Relative low cost Capacity to analyze multiple samples in single run Website : www.tamsmed.com

35. Gas Chrom . Used for separating and analyzing compounds that can be vaporized without decomposition Mobile phase is a inert gas like argon, hydrogen, helium, nitrogen, called carrier gas Stat phase is microscopic layer of liquid on inert solid support inside a glass or metal column, eg methyl silicone polymers Website : www.tamsmed.com

36. More volatile solute elutes earlier than less volatile one Effluent from column carries separated sample constituents to detector, which produces a signal displayed as a series of peaks Volume and time at which unknown substance gets eluted used to identify the substance by comparing with ref. material values. Website : www.tamsmed.com

37. Gas Chrom . Parts Website : www.tamsmed.com

38. Chromatographic column Packed capillary Filled with support particles Int dia 1 – 4 mm Length – 1 m or more Glass / stainless steel Carrier gas - Nitrogen Inner wall of silica tube coated with thin film of liquid phase ID 0.1 – 0.5 mm Length 10 – 150 m Hydrogen / Helium Website : www.tamsmed.com

39. Injector Packed Col Glass microsyringe injects 1 – 10 μ L sample through septum Septum – interface btwn injector & chrom . sys Analyte swept into col. By carrier gas Problems Septum leaks Heat → septum decomp products in column→ghost peaks in chromatogram Website : www.tamsmed.com

40. Capillary Col Low sample capacity Split mode – small portion of sample enters column Used when sample contains high conc. of solute Splitless mode – Most of sample enters col Used when sample contains low sol. conc. Temp control – Col. placed in oven Website : www.tamsmed.com

41. Detectors Flame ionization detector ( FID ) MC used Col. Effluent mixed with H2 & air, & burned in flame Electron released detected by electrode Current generated used to identify & quantify solute Photo ionization detector ( PID )- UV light instead Others – Thermal conductivity detector, mass spectrometer Website : www.tamsmed.com

42. Practical Considerations in GC Sample Extraction Eg To extract barbiturates from serum, serum is acidified to convert barb. Into an organic solvent Sample Derivatization To convert non volatile compounds into volatile forms via chem. Modification eg esterification , oximation , acylation Also used to ↑ sens / spec of some reacns Website : www.tamsmed.com

43. Analyte identification Retention time at which unknown solute elutes compared with that of ref compound Appearance of rep peak at same time ⇒ similar constituents Quantification Electric signals from detector used to produce quan . information Website : www.tamsmed.com

44. Liquid Chromatography Parts Chrom . Column Solvent Reservoir Pump Injector Detector ( spectrophotometer / fluorometer / electrochem . Detector) Computer Website : www.tamsmed.com

45. Diag. Representation Website : www.tamsmed.com

46. Columns Stainless steel ID 0.3 -0.5 mm Length 50 – 250 mm Column Packings 1. Bonded phase – Stat phase bonded chemically to silica particles by silica ester/ silicone polymeric linkage Website : www.tamsmed.com

47. Mech / chem stable Avail for ion exchange chromatography Eg octa decylsilane bonded to silica 2. Polymeric packing eg polystyrene divinyl benzene 3. Chiral packings – to separate enantiomers 4. Restricted access packing – outer layer of particles coated with hydrophilic network, pores coated with hydrophobic network Website : www.tamsmed.com

48. Practical Considerations Sample concentration / purification – solid / liquid phase extraction Sample derivatization – eg labelling a. acids with flourescent tags before / after chrom . Solvent degassing Dissolved gas bubbles generated while solvents pass from reservoir into column Create unstable electric signals Removed by vaccuum degassing / Helium purging Website : www.tamsmed.com