1. 1 Principles of Chromatography Chap. 20

2. 2 Analytical Separations and Chemical Problem Solving If you, a researcher of a food company are asked to find the source of an uncertain musty odor being released from a plastic food package, what should you do?

3. 3 Chromatography The most powerful tool in analytical chemistry Coupled with mass spectrometry Chomatography is a physical method of separating solute mixtures in which the components (solutes) to be separated are distributed between the two phases, one of which is stationary while the other, the mobile phase. Chromatography operates on the same principle as extraction, but one phase is held in place while the other moves past it.

4. 4 In 1903 M. Tswett first applied adsorption chromatography to the separation of plant pigments

5. 5 The mobile phase in chromatography is either a liquid (LC) or a gas (GC). The stationary phase is most commonly a viscous liquid coated on the inside of a capillary tube or on the surface of solid particles packed into the column In any case, the partitioning of solutes between the mobile and stationary phases gives rise to separation.

6. 6 Chromatography The process of passing liquid or gas through a chromatography column is called elution

7. 7 Columns are either packed or open tubular. A packed column is filled with particle containing stationary phase. An open tubular one is a narrow hollow capillary with stationary phase coated on the inside walls.

8. 8 Types of Chromatography

9. 9 Use of a solid stationary phase and a liquid or gaseous mobile phase. Equilibration between the stationary and the mobile phase accounts for separation of different solutes. Adsorption chromatography

10. 10 A liquid stationary phase forms a thin film on the surface of a solid support. Solute equilibrates between the stationary liquid and the mobile phase. Partition chromatography

11. 11 Anions and cations are covalently attached to the stationary solid phase, usually a resin. Solute ions of the opposite charge are attached to the stationary phase by electrostatic force. Ion-exchange chromatography

12. 12 Separates molecules by size. No attractive interaction between two phases Molecular exclusion chromatography

13. 13 The most selective kind of chromatography. The immobolized molecule might be an antibody to a particular protein. Affinity chromatography

14. 14 The Chromatogram A chromatogram is a graph showing the detector response as a function of elution time. The retention time, t r, for each component is the time needed after injection of the mixture onto the column until that component reaches the detector.

15. 15

16. 16 A Measure of Column Efficiency Number of theoretical plates (N) ( 이론단수 ) The name comes from the theory of distillation in which separation can be performed in discrete stages called plates. Large N ==> narrow peak ==> better separation N is dimensionless. W 1/2 :The width of half-height N = 5.55 tr2tr2 W 2 1/2

17. 17 Height Equivalent to Theoretical Plates (HETP) HETP (H) = Length of column N Unlike N, HETP has a dimension. It is expressed in length. Smaller HETP is desirable which means the narrower bandwidth. Small H ==> narrow peak ==> better separation

18. 18 Efficiency of Separation Resolution ( 분리도 ) =  tr tr W av The average width of the two peaks Separation between peaks (in units of time or volume)

19. 19 Phenomenon of Band Broadening Intra column band broadening –Longitudinal Diffusion –Finite Equilibration Time Between Phases –Eddy Diffusion

20. 20 Longitudinal Diffusion ( 세로확산 ) Diffuses away from the concentrated center of its zone. Longitudinal diffusion: diffusion takes place along the axis of the column The greater flow rate, the less time is spent on the column and the less longitudinal diffusion occurs.

21. 21 Finite Equilibration Time Between Phases Finite time required for solute to equilibrate between the mobile and stationary phases. Although some solute is stuck in the stationary phase, the remainder in the mobile phase moves forward, thereby resulting in spreading of the overall zone of solute.

22. 22 The slower the linear flow, the more complete equilibration is and the less zone broadening occurs. Decreasing the thickness of the stationary phase reduces HETP because solute can diffuse faster from the stationary phase to the mobile phase. HETP is decreased by increasing temperature, which increases the diffusion coefficient of solute in the stationary phase. Finite Equilibration Time Between Phases

23. 23 Van Deemter Equation It tells us how the column and flow rate affect the plate height. A,B, and C are constant for a given column and stationary phase. The u x is the linear flow rate. Longitudinal Diffusion Eddy DiffusionEquilibration time H = A + B/u x + Cu x

24. 24

25. 25 Eddy Diffusion Different sample molecules take different flow paths. The smaller the stationary phase particles, the less serious this problems is. This process is absent in an open tubular column.

26. 26

27. 27

28. 28

29. 29 Information of Molecular ion: unbroken to its fragment ions Sensitive qualitative and quantitative Fragmented cations from gaseous organic molecule by ionization are separated according to their mass in electric or magnetic field Mass Spectrometry

30. 30

31. 31 Electric ionization (Electron Impact Ionization : EI) Ionization

32. 32 Ion-molecule reaction(s) between a reagent gas and the sample at a relatively high pressure Most common reagent gases – methane, isobutane, ammonia Chemical ionization ; CI Mechanism CH 4 + e- CH 4 + + 2e- CH 4 + + CH 4 CH 5 + + CH 3 CH 5 + + M CH 4 + MH + M(C 2 H 5 ) +

33. 33 Total ion and Selected Ion Chromatograms Total ion Chromatograms Selected Ion Chromatograms Fig. 21-15 Liquid Chromatography of opium alkaloids found in street heroin

34. 34 Information in a Mass Spectrum Norminal Mass Fragmentation Patterns

35. 35 Isotope Patterns and the Nitrogen Rule

36. 36 Volatile Flavor Components of Candy Sample preparation Analysis of GC/MS