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 1)Solvent Extraction : transfer of a solute from phase 1  phase 2 S (in phase1)  S (in phase 2) Partition coefficient

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

5. 21.1 What is Chromatography?-3 b) 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. c)The science & art of separation d)Originator : adsorption chromatography by M.Tswett in 1903 e)Eluent, eluate, elution. 21.1 What is Chromatography?-5

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

9. 3)Types of Chromatography Is divided into categories on the basis of the mechanism of interaction of the solute v.s. the stationary phase. 21.1 What is Chromatography?-7

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

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

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

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

14. 2) For individual band : a)Retention time (t r ) : The time needed after injection for an individual solute to reach detector. b)An ideal chromatographic peak  Gaussian shape. w ½ = 2.35σ, w = 4σ 21.2 How do we describe a chromatogram -2

15. 21.2 How do we describe a chromatogram -3

17. 3)For pairs of bands a)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. 21.2 How do we describe a chromatogram -4

18. b)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(t r /w ½ ) 2 Plate height : H = L/N The smaller plate height  narrower peaks  better separation 21.2 How do we describe a chromatogram -5

19. c)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. e) Scaling up (rule at p.452) 1.Analytical chromatography: long & thin column. For a small scale: separate, identify, or measure. 2.Preparative chromatography: short, fat column. For large scale : purify 21.2 How do we describe a chromatogram -8

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

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

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

25. 21.3 Why do bands spread ? -4

26. 21.3 Why do bands spread ? -5 4)An optimum rate : flow rate for the best separation.

27. 21.3 Why do bands spread ? -6 5)Multiple paths

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

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

30. 21.3 Why do bands spread ? -8 7)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 8)Funny shapes

32. 20.4 Chemical Analysis by Chromatography -2

33. 21.4 Mass Spectrometry Transmission Quadrupole Mass Spectrometer

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

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 CH 4 + e -  CH 4 + + 2e - CH 4 + + CH 4  CH 5 + + CH 3 CH 5 + + M  CH 4 + MH + CH 4 +  CH 3 + + H CH 3 + + CH 4  C 2 H 5 + + H 2

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

38. 21.5 Information in a mass spectrum Rxn : CH 3 (CH 2 ) 2 CH 2 –OH + Br -  CH 3 (CH 2 ) 2 CH 2 –Br 1–Butanol 1–Bromobutane

39. CH 3 15 CH 2 14 Br 79 C 4 H 9 79 Br + 50.0% C 4 H 9 81 Br + 21.5 Information in a mass spectrum Fragmentation Patterns

40. 21.5 Information in a mass spectrum

41. Isotope Patterns C n H x O y N z 12 C/ 13 C Intensity = n x 1.1% Ex: C 6 H 6 (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