Separations - s ee text for chapters on each topic

1. Separations- see text for chapters on each topic • Solvent Extraction • What is Chromatography • Efficiency of Separation • Why Bands Spread • Electrophoretic Separations (gel and capillary) • Electrochromatography

2. Separations – two-phase separation: partitioning Single phase separation: electrophoresis; ultracentrifugation ; diffusion; mass spectrometry; excited state reactions

3. Solvent extraction Partition coefficients and undissociated species Partition coefficient:

4. Multiple extractions For multiple extraction with each extraction using same volume of V, : the fraction unextracted after n extractions For “infinite number of extractions (i.e., the limit):

5. B +H+ BH+ B Extraction of acid/base species

6. Extraction of metal chelates

7. 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Counter current extraction (equilibrium-based partitioning)

8. For large values of n: 1 2 3 Calculations after many extractions fraction present in the r th tube after n extractions r: tube #

9. Counter current vs. “chromatography”

10. Chromatography Separating Molecules

11. Science of Chromatography • Separation (relative speeds of molecules) depends on • Polarity of solvent • Polarity of substrate • Other molecular properties (b.p., chirality, etc.) • Like moves fastest with like • Polar molecules move fastest with polar solvents

12. Chromatography • Two phases • Stationary phase • Solid or liquid • Spatially fixed throughout experiment • Mobile phase • Liquid or gas • In motion relative to the stationary phase • Chromatography is classified as to type of mobile phase and stationary phase • LC (liquid mobile) . . . . liquid or solid stationary • GC (gas mobile) . . . . usually liquid stationary

13. Types of chromatography • adsorption • partition • ion-exchange • molecular exclusion (size exclusion) • affinity • chiral separations

14. Subtypes of Chromatography • Paper chromatography • Thin layer chromatography (TLC) • Gas chromatography (GC) • High-performance liquid chromatography (HPLC) • And several others

15. Liquid Chromatography

16. Paper Chromatography Spotting sample (pencil mark near spot) Solvent front Developing chamber

17. Final position of solvent front (pencil line) Original spot (pencil line)

18. Visualizing Spots • May be visible • If not, try • UV light • I2 • Mark spots with pencil when visualized Lichen extract

19. Retardation Factor, Rf

20. Rf is relatively imprecise • Cut out spots, extract solute and run further tests to complete identification • In paper chromatography, • The mobile phase is a • The stationary phase is a • The chromatographic classification is liquid ? solid ? LSC ?

21. Cellulose Absorbed water in the polar regions is actually the stationary phase Thus, paper chromatography’s actual classification is LLC

22. Thin Layer Chromatography (TLC) • Stationary layer is a thin layer of a solid bonded to an inert plastic or glass binder • Silica (SiO2) • Alumina (Al2O3) • Binder is often plaster of Paris

23. Silica Surface O’s converted into O-H’s Small particles mean very enormous surface area Alumina works similarly

24. TLC is an example of LSC • TLC is exactly like paper chromatography in terms of the actual procedure • Rf values are more stable than on paper • In forensics, TLC is routinely used for identifying and/or individualizing • Inks • Dyes • Drugs

25. Thin Layer Chromatography • What components are in the unknown from the case?

26. Thin Layer Chromatography • Identifying gasoline by separation of dye additives in the fuel

27. High Performance Liquid Chromatography (HPLC) • Directly analogous to GC • Column is relatively short (10-30 cm), with inside diameter of 4-10 mm • Column is very tightly packed • LSC • Packed with microparticles (3-10 m) of silica or alumina • LLC • Packed with microparticles coated with a liquid

28. Liquid solvents (up to four) replace carrier gas in GC experiment Column is at room temperature Liquid pressure is enormous (in excess of 6000 psi = 45 bar) Can also hook-up the column directly to a MS to give LC-MS Detector is double-beam UV-Vis spectrometer, constantly scanning a selected range of l’s Even at enormous pressure the flow rate is 0.1-10 mL/min

29. HPLC Data

30. HPLC – use of solvent gradients

31. Ion Chromatography

32. Ion Chromatography

33. Size Exclusion Chromatography

34. Gas Chromatography

35. Gas Chromatography (GC) • Column • Contains stationary phase • Long coil (2-3 m) of tubing • Relatively narrow internal diameter (2-4 mm) • Glass or metal • Stationary phase • May be solid (GSC) • Silica, alumina • May be liquid at operating temperatures (GLC) • Squalane, C30H62 • Dimethyl silicone oil, (CH3)3-Si-O-[Si(CH3)2-O]n-Si(CH3)3 • Coated onto inert beads or granules

36. Equilibration between Phases • Molecules divide up between those in mobile phase and those in the stationary phase • Rapid equilibrium established • Avoid column overloading • The ratio in each phase depends on • Temperature • Polarities of column material, mobile phase, and molecules • “other” factors

37. Gas Chromatograms • Complex mixtures can have many peaks • Broad peaks (later) • High backgrounds can come from molecules that decompose as they move through the column or from column degradation etc. (column bleed)

38. Identification of species: retention times Capacity factor:k’= (tr - tm) / tm t solute is in stationary phase = ---------------------------------------- t solute is in mobile phase

39. Qualitative Analysis • Retention times, tR • Difficult to develop data base because of the variety of factors which alter tR • Nature of stationary and mobile phases • Length and diameter of column • Flow rate of carrier gas • Temperature, etc. • Compare original chromatogram to a known chromatogram • Use GC to separate components, then identify them in some other fashion (IR, MS, etc.) • Detector must be nondestructive

40. van Deempter equation Band broadening and resolution • plates ( ‘theoretical plates’) • plate height, H • Height equivalent of a theoretical plate, HETP • number of plates • resolution Improved resolution with larger N, longer retention on column (i.e., larger k’)

41. Band Broadening Description –assumes Gaussian peaks

42. Mobile phase is called “carrier gas” (He, Ar, N2) Flow rate valve Pressure reducers

44. Column housed in oven User selects temperature Must be sufficient to volatilize all components Lower temperatures lead to better resolution but longer retention times

45. GC Improvements • Capillary Columns • Fused silica • 10-60 m long • 0.1-0.3 mm internal diameter • Coated with a liquid (GLC) • Temperature programming

46. Types of columns • Packed • high capacity • common in many types of chromatography adsorption, LC (e.g., HPLC), affinity, frontal • Open tubular • less capacity • better resolution (less band broadening) • preferred approach for most GC applications

47. Flame Ionization Detector (FID) 2 H2 + O2→ 2 H2O Proceeds without ionic intermediates Amp - Cathode

48. Flame Ionization Detector (FID) CH4 + 2 O2→ CO2 + 2 H2O Proceeds by forming a variety of ionic intermediates Amp - Cathode

49. Gas Chromatograms • Complex mixtures can have many peaks • Broad peaks (later) • High backgrounds can come from molecules that decompose as they move through the column or from column degradation etc. (column bleed)

50. Temperature • Isothermal runs can suffer from: • Very broad peaks after long tR at low temps. • Poorly Resolved peaks at short retention times at high temps. • Use programmed temperature ramp • Start at low temp • Allows resolution of early eluting cmpds. • Ramp to high temp • Prevents band broadening of late eluting cmpds.