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Chapter 21

Chapter 21. Principles of Chromatography. Chromatography is the most powerful tool for separating & measuring the components of a complex mixture. Quantitative & qualitative analysis. 21.1 What is Chromatography?-1. Solvent Extraction : transfer of a solute from phase 1  phase 2

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Chapter 21

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  1. Chapter 21 Principles of Chromatography

  2. Chromatography is the most powerful tool for separating & measuring the components of a complex mixture. Quantitative & qualitative analysis

  3. 21.1 What is Chromatography?-1 • Solvent Extraction : transfer of a solute from phase 1 phase 2 S (in phase1)  S (in phase 2) Partition coefficient

  4. 21.1 What is Chromatography?-2 • Chromatography : same as extraction • One phase: held in place stationary phase. solid material (packing material) Another phase : fluid phase mobile phase. sample: gas (GC) liquid (LC)

  5. 21.1 What is Chromatography?-3 • A solute equilibrates between a mobile and a stationary phase. • The more it interacts with the stationary phase, the slower it is moved along a column. Xm  Xs Ks = • Solutes with a large Ks value will be retained more strongly by the stationary phase.

  6. 21.1 What is Chromatography?-4

  7. 21.1 What is Chromatography?-5 • The science & art of separation • Originator : adsorption chromatography by M.Tswett in 1903 • Eluent, eluate, elution.

  8. 21.1 What is Chromatography?-6 Elution : always (100%) dilution

  9. 21.1 What is Chromatography?-7 • Types of Chromatography Is divided into categories on the basis of the mechanism of interaction of the solutev.s.the stationary phase.

  10. 21.1 What is Chromatography?-7 polar s.p. for GC & LC for GC

  11. 21.1 What is Chromatography?-8 resin-SO3- gel filtration resin-N(CH3)3+ by size

  12. 21.1 What is Chromatography?-9 Most selective one pH, and ionic strength Ask Yourself 20-A p.432

  13. 21.2 How do we describe a chromatogram -1 • Chromatogram : A graph showing the detectors response as a function of elution time : band’s shapes, position, resolution.

  14. 21.2 How do we describe a chromatogram -2 • For individual band : • Retention time (tr) :The time needed after injection for an individual solute to reach detector. • An ideal chromatographic peak Gaussian shape.w½ = 2.35σ, w = 4σ

  15. 21.2 How do we describe a chromatogram -3

  16. 21.2 How do we describe a chromatogram -3

  17. 21.2 How do we describe a chromatogram -4 • For pairs of bands • Efficiency : two factors contribute to how well components are separated : the widths of the peaks : the wider the peak, the poorer separation. the spacing in time : the further apart, the better separation.

  18. 21.2 How do we describe a chromatogram -5 • Theoretical plates (N): (from distillation) the more plates on a column, the more equilibration steps, and the better the separation. Number of plates on column : N = 5.55(tr/w½)2 Plate height : H = L/N The smaller plate height  narrower peaks  better separation

  19. Resolution (Rs) 21.2 How do we describe a chromatogram -6

  20. 21.2 How do we describe a chromatogram -7 Qualitative: • Co-chromatography • Mass spectrometer • IR spectrophotometer Quantitative: • The area of peak Internal standard d) Qualitative & Quantitative analysis

  21. 21.2 How do we describe a chromatogram -8 e) Scaling up (rule at p.452) • Analytical chromatography: long & thin column. For a small scale: separate, identify, or measure. • Preparative chromatography: short, fat column. For large scale : purify

  22. 21.3 Why do bands spread ? -1 • Why broadening? • diffusion • slow equilibration of solute between the m.p and s.p. • irregular flow paths.

  23. 21.3 Why do bands spread ? -2 • Longitudinal diffusion : the faster the flow  the less a band spends in column. the less time for diffusion.  broadening

  24. m.p. s.p. 21.3 Why do bands spread ? -3 • solute requires time to equilibrate between phases. (s.p.m.p.) with temp. broadening  u Can’t equilibrate rapidly enough.

  25. 21.3 Why do bands spread ? -4

  26. 21.3 Why do bands spread ? -5 • An optimum rate :flow rate for the best separation.

  27. 21.3 Why do bands spread ? -6 • Multiple paths

  28. 21.3 Why do bands spread ? -6 • Plate height equation

  29. 21.3 Why do bands spread ? -7 Plate height equation

  30. 21.3 Why do bands spread ? -8 • open tubular columns Packed column (A, B, C  0 in van Deemter’s eqn.) Open tubular column (A = 0 in van Deemter’s eqn.)  resolution (∵ H & column length)  sample capacity (∵ less s.p.)

  31. 21.3 Why do bands spread ? -9 • Funny shapes

  32. 20.4 Chemical Analysis by Chromatography -2

  33. 21.4 Mass Spectrometry Transmission Quadrupole Mass Spectrometer

  34. 21.4 Mass Spectrometry • Ionization: 1) Electron ionization 2) Chemical ionization

  35. 1)Electron ionization M + e- M+ + e- + e- 70 eV -55 eV 0.1eV Molecular ion break into fragments. Base peak: most intense peak.

  36. 2) Chemical ionization CH4 + e- CH4+ + 2e- CH4+ + CH4CH5++ CH3 CH5++ M  CH4 +MH+ CH4+ CH3+ + H CH3+ + CH4C2H5++ H2

  37. Total ion Chromatograms • Selected ion Chromatograms: • Simplify analysis • improve S/N

  38. 21.5 Information in a mass spectrum Rxn : CH3(CH2)2CH2–OH + Br- CH3(CH2)2CH2–Br 1–Butanol 1–Bromobutane

  39. 21.5 Information in a mass spectrum CH3 15 CH2 14 Br 79 Fragmentation Patterns C4H979Br+50.0% C4H981Br+

  40. 21.5 Information in a mass spectrum

  41. 21.5 Information in a mass spectrum Isotope Patterns CnHxOyNz 12C/13C Intensity =n x 1.1% Ex: C6H6 (M+1)/M+ = 6 x 1.1 % Nitrogen Rule: A compound: odd nominal mass / odd number of N atoms; even nominal mass/ even number of N atoms

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