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Chromatographic separations

Chromatographic separations. Chapter 26 The “stuff” you do before you analyze a “complex” sample. It is all about “Reducing Interferences”. Mobile and Stationary phase Retention - Migration Bands or zones Equilibrium!. Column vs. planar Liquid vs. gas vs. SF High vs. low resolution

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Chromatographic separations

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

  2. It is all about “Reducing Interferences”

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

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

  5. Column Chromatography Dilution & Peak broadening! Chromatogram

  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) A mobile ↔ A stationary stationary mobile CS = nS/VS, CM = nM/VM K ~ constant  linear chromatography >>>K >>> Retention in the stationary phase  Retention times How to manipulate K?

  9. Chromatography Retention Times tM = retention time of mobile phase (dead time) tR = retention time of analyte (solute) tS = time spent in stationary phase (adjusted retention time) L = length of the column

  10. Chromatography: VelocitiesLinear 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 = tR/tRs tRs = retention time of internal standard

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

  16. Chromatography Column Efficiency - Theoretical PlatesPlate andRate Theories L = length of column packing s standard deviation s2/L variance per unit length.

  17. Chromatography Relation between column distance and retention times

  18. Chromatography Relation between column distance and retention times ~96%  2 Tangent at Inflection point

  19. Chromatography Determining the Number of Theoretical Plates W1/2

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

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

  22. Column EfficiencyKinetic variables

  23. Zone BroadeningFlow 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 BroadeningKinetic 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 BroadeningKinetic Processes Van - Deemter Equation

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

  27. ChromatographyResolution

  28. ChromatographicSeparations with a twist

  29. Chromatographic Definitions

  30. Chromatographic Relationships

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

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

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