1. Chromatographic separations Chapter 26 The “stuff” you do before you analyze a “complex” sample

2. It is all about “Reducing Interferences”

3. Chromatography basics Mobile and Stationary phase Mobile and Stationary phase Retention - Migration Retention - Migration Bands or zones Bands or zones Equilibrium! Equilibrium! Column vs. planar Liquid vs. gas vs. SF High vs. low resolution Partition Adsorption Ion exchange Size exclusion

4. ChromatographyChromatography A. Column Chromatograpgy, B. Planar Chromatograpgy

5. Column Chromatography Chromatogram Dilution & Peak broadening!

6. Chromatography: Peak separations

7. Chromatography: Peak Resolution Poor resolution More separation Less band spread

8. Chromatography: Distribution Constant (recommended by IUPAC) (old term: partition coefficient) stationary mobile A mobile ↔ A stationary K ~ constant  linear chromatography >>>K >>> Retention in the stationary phase  Retention times How to manipulate K? C S = n S /V S, C M = n M /V M

9. Chromatography Retention Times t M = retention time of mobile phase (dead time) t R = retention time of analyte (solute) t S = time spent in stationary phase (adjusted retention time) L = length of the column

10. Chromatography: Velocities Linear rate of solute migration! Velocity = distance/time  length of column/ retention times Velocity of solute: Velocity of mobile phase:

11. Chromatography Velocity/Retention time and Kc

12. Chromatography Velocity Relationships

13. Chromatography Retention Factor : are we there yet? Adjusted retention time

14. Relative retention time: RRT = t R /t Rs t Rs = retention time of internal standard

15. Chromatography Selectivity Factor: can you separate from your neighbor B retained more than A   >1 Distribution Constant Retention factor Retention time

16. Chromatography Column Efficiency - Theoretical Plates Plate and Rate Theories   standard deviation  2 /L  variance per unit length. L = length of column packing

17. Chromatography Relation between column distance and retention times

18. ~96%  2  Tangent at Inflection point

19. Chromatography Determining the Number of Theoretical Plates W 1/2

20. Summary of Plate Theory Successfully accounts for the peak shapes and rate of movement Successfully accounts for the peak shapes and rate of movement Does not account for the “mechanism” causing peak broadening Does not account for the “mechanism” causing peak broadening No indication of other parameters’ effects No indication of other parameters’ effects No indication for adjusting experimental parameters No indication for adjusting experimental parameters

21. Rate Theory Zone broadening is related to Mass Transfer processes Zone broadening is related to Mass Transfer processes

22. Column Efficiency Kinetic variables

23. Zone Broadening Flow Rate of Mobile Phase Liquid chromatography Gas chromatography Note the differences in flowrate and plates height scales Why GC normalluy has high H, but also high overall efficiency?

24. Zone Broadening Kinetic Processes Van - Deemter Equation λ and γ are constants that depend on quality of the packing. B is coefficient of longitudinal diffusion. Cs and Cm are coefficients of mass transfer in stationary and mobile phase, respectively.

25. Zone Broadening Kinetic Processes Van - Deemter Equation

26. Zone Broadening Multiple Pathways Eddy Diffusion: band broadening process results from different path lengths passed by solutes. 1.Directly proportional to the diameters of packing 2.Offset by ordinary diffusion 3.Lower mobile-phase velocity, smaller eddy diffusion Stagnant pools of mobile phase retained in stationary phase.

27. Zone Broadening Longitudinal Diffusion The higher the , the smaller the H Much smaller in LC than in GC Column Diffusion

28. Zone Broadening Mass Transfer between Phases Slow equilibrium of solute between mobile and stationary phases Time is required for solute molecules to diffuse from the interior of these phase to there interface where transfer occur

29. Zone Broadening Longitude vs. Mass Transfer Longitude Parallel to the flow Inversely proportional to the flow of the mobile phase Mass Transfer Diffusion tends to be right angles to the flow The faster the mobile phase moves, the larger the band broadening

30. Chromatography Resolution

31. Chromatographic Separations with a twist

32. Chromatographic Definitions

33. Chromatographic Relationships

34. Quantitative Analysis Peak areas Peak areas Peak height Peak height Calibration and standards Calibration and standards Internal Standard method Internal Standard method

35. Summary  Relate to column chromatography  Retention times  Velocities of mobile and component  Height equivalent of theoretical plates  Peak or zone broadening  Resolution