1. Separations - s ee text for chapters on each topic 1. Solvent Extraction 2. What is Chromatography 3. Efficiency of Separation 4. Why Bands Spread 5. Electrophoretic Separations (gel and capillary) 6. Electrochromatography

2. Separations – t wo-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): e.g. Consider K p =2 and single vs 5 extractions (V 1 = 100 mL; V 2 (total) = 1 L) Single: q = 100/[100+(K*1,000) = 0.048 (4.8% unextracted or 95.2% extracted) Multiple: q=[100/(100+(K*200)] 5 = 0.00032 ( 0.032% unextr. or 99.998% ext.)

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

6. Extraction of metal chelates

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

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

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) Developing chamber Solvent front

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

18. Visualizing Spots  May be visible  If not, try UV light I 2 Mark spots with pencil when visualized Lichen extract

19. Retardation Factor, R f

20.  R f 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 LSC liquid solid ? ? ?

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 (SiO 2 ) Alumina (Al 2 O 3 ) 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  R f 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) Even at enormous pressure the flow rate is 0.1-10 mL/min Detector is double-beam UV-Vis spectrometer, constantly scanning a selected range of ’s Can also hook-up the column directly to a MS to give LC-MS

29. HPLC Data

30. HPLC – use of solvent gradients

31. 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, C 30 H 62  Dimethyl silicone oil, (CH 3 ) 3 -Si-O-[Si(CH 3 ) 2 -O] n -Si(CH 3 ) 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’= (t r - t m ) / t m t solute is in stationary phase = ---------------------------------------- t solute is in mobile phase

39. Qualitative Analysis  Retention times, t R Difficult to develop data base because of the variety of factors which alter t R  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. Band broadening and resolution van Deempter equation 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. Pressure reducers Flow rate valve Mobile phase is called “carrier gas” (He, Ar, N 2 )

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 H 2 + O 2 → 2 H 2 O Proceeds without ionic intermediates Cathode - Amp

48. Flame Ionization Detector (FID) CH 4 + 2 O 2 → CO 2 + 2 H 2 O Proceeds by forming a variety of ionic intermediates Cathode - Amp

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 t R 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.

51. Quantitative Analysis  The area under each peak is proportional to the percent (by mass) of the analyte in the original sample A 1 : A 2 : A 3 : … = %1 : %2 : %3 : … Get areas by  Numerical or electronic integrator  Does not account for different detector response to different molecules  Alternatively, prepare a calibration curve for each analyte Area versus concentration

52. GC-MS  GC is excellent for separating mixtures  MS is excellent for qualitative analysis  Make the MS the detector for the GC Always use a capillary column No “regular” detector at the end The column is plumbed directly into the MS ion source subsystem

53. Experiment starts when sample is injected into GC At that instant, start the MS recording spectra sequentially

54. Experiment starts when sample is injected into GC It takes about 0.6 s to record a MS. Thus, about 100 spectra are recorded (and stored) every minute It may take 15-45 min for all the components to elute from the GC column Thus the MS may record and store 1500 – 4500 spectra during the lifetime of the experiment

55. Most of these are blanks (only carrier gas coming through) It may take 15-45 min for all the components to elute from the GC column When an analyte component appears, its band may take 10 s to completely elute During that time, perhaps 17 or so spectra are recorded. The peaks will get more and more intense, and then will wane

56. Quantitative Analysis  For each chromatographic peak Calculate the Total Ion Count (TIC) Plot TIC versus time  Has the appearance of a regular GC  Perform the same sorts of quantitative analysis as you would do on any other chromatogram

57. Qualitative Analysis w/ MS  Sort through the 1500 – 4500 spectra and pick out the ones that aren’t blank  Of the sets of non-blanks, pick out the most intense from each set  Identify the component based on the MS fragmentation patterns (MS is covered in a separate section of the course)

58. Problem Samples  Substances with enormous molecular masses (e.g.: polymers) Not volatile Not soluble  Much forensic evidence falls in this category Paint Grease Tars Fibers Plastics

59. Pyrolysis GC (PGC)  The analyte is pyrolyzed (heated to 500 – 1,000 o C in the absence of oxygen) Giant molecules fragment (often into radicals)  Take GC of the fragments (pyrogram)  Will not identify the analyte, but can be used to individualize it Compare complex pyrograms of known and questioned samples

60. Pyrolysis GC Data (with FIDetector)