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GC and GC-MS

GC and GC-MS. Gas Chromatography. Function Components Common uses Chromatographic resolution Sensitivity. Function. Separation of volatile organic compounds Volatile – when heated, VOCs undergo a phase transition into intact gas-phase species

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GC and GC-MS

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  1. GC and GC-MS

  2. Gas Chromatography • Function • Components • Common uses • Chromatographic resolution • Sensitivity

  3. Function • Separation of volatile organic compounds • Volatile – when heated, VOCs undergo a phase transition into intact gas-phase species • Separation occurs as a result of unique equilibria established between the solutes and the stationary phase (the GC column) • An inert carrier gas carries the solutes through the column

  4. Components • Carrier Gas, N2 or He, 1-2 mL/min • Injector • Oven • Column • Detector

  5. Syringe Injector Detector Gas tank Column Oven

  6. Injector • A GC syringe penetrates a septum to inject sample into the vaporization camber • Instant vaporization of the sample, 280 C • Carrier gas transports the sample into the head of the column • Purge valve controls the fraction of sample that enters the column

  7. Syringe Syringe Injector Injector Purge valve closed Purge valve open Splitless (100:90) vs. Split (100:1) He He GC column GC column

  8. Split or splitless • Usually operated in split mode unless sample limited • Chromatographic resolution depends upon the width of the sample plug • In splitless mode the purge valve is close for 30-60 s, which means the sample plug is 30-60 seconds • As we will see, refocusing to a more narrow sample plug is possible with temperature programming

  9. Open Tubular Capillary Column 0.32 mm ID Mobile phase (Helium) flowing at 1 mL/min Liquid Stationary phase 0.1-5 mm 15-60 m in length

  10. FSOT columns • Coated with polymer, crosslinked • Polydimethyl soloxane (non-polar) • Poly(phenylmethyldimethyl) siloxane (10% phenyl) • Poly(phenylmethyl) siloxane (50% phenyl) • Polyethylene glycol (polar) • Poly(dicyanoallyldimethyl) siloxane • Ploy(trifluoropropyldimethyl) siloxane

  11. Polar vs. nonpolar • Separation is based on the vapor pressure and polarity of the components. • Within a homologous series (alkanes, alcohol, olefins, fatty acids) retention time increases with chain length (or molecular weight) • Polar columns retain polar compounds to a greater extent than non-polar • C18 saturated vs. C18 saturated methyl ester

  12. C18:2 C16:0 C18:1 C18:0 C16:1 RT (min) Polar column C18:2 C18:1 C16:0 C18:0 C16:1 RT (min) Non-polar column

  13. Oven • Programmable • Isothermal- run at one constant temperature • Temperature programming - Start at low temperature and gradually ramp to higher temperature • More constant peak width • Better sensitivity for components that are retained longer • Much better chromatographic resolution • Peak refocusing at head of column

  14. Typical Temperature Program 220C 160C 50C 0 60 Time (min)

  15. Detectors • Flame Ionization Detectors (FID) • Electron Capture Detectors (ECD) • Electron impact/chemical ionization (EI/CI) Mass spectrometry

  16. FIDs • Effluent exits column and enters an air/hydrogen flame • The gas-phase solute is pyrolized to form electrons and ions • All carbon species are reduced to CH2+ ions • These ions collected at an electrode held above the flame • The current reaching the electrode is amplified to give the signal

  17. FID • A general detector for organic compounds • Very sensitive (10-13 g/s) • Linear response (107) • Rugged • Disadvantage: specificity

  18. ECD • Ultra-sensitive detection of halogen-containing species • Pesticide analysis • Other detectors besides MS • IR • AE

  19. Mass Spectrometry

  20. What kind of info can mass spec give you? • Molecular weight • Elemental composition (low MW with high resolution instrument) • Structural info (hard ionization or CID)

  21. How does it work? • Gas-phase ions are separated according to mass/charge ratio and sequentially detected

  22. Parts of a Mass Spec • Sample introduction • Source (ion formation) • Mass analyzer (ion sep.) - high vac • Detector (electron multiplier tube)

  23. Sample Introduction/Sources Volatiles • Probe/electron impact (EI),Chemical ionization (CI) • GC/EI,CI Involatiles • Direct infusion/electrospray (ESI) • HPLC/ESI • Matrix Assisted Laser Adsorption (MALDI) Elemental mass spec • Inductively coupled plasma (ICP) • Secondary Ion Mass Spectrometry (SIMS) • surfaces

  24. EI, CI • EI (hard ionization) • Gas-phase molecules enter source through heated probe or GC column • 70 eV electrons bombard molecules forming M+* ions that fragment in unique reproducible way to form a collection of fragment ions • EI spectra can be matched to library stds • CI (soft ionization) • Higher pressure of methane leaked into the source (mtorr) • Reagent ions transfer proton to analyte

  25. EI Source Under high vacuum filament 70 eV e- To mass analyzer GC column anode Acceleration slits repeller

  26. EI process M+* • M + e- f1 f2 f4 f3 This is a remarkably reproducible process. M will fragment in the same pattern every time using a 70 eV electron beam

  27. Ion Chromatogram of Safflower Oil

  28. CI/ ion-molecule reaction • 2CH4 + e-  CH5+ and C2H5+ • CH5+ + M  MH+ + CH4 • The excess energy in MH+ is the difference in proton affinities between methane and M, usually not enough to give extensive fragmentation

  29. EI spectrum of phenylethyl acetate

  30. Ammonia CI Spectrum of Phenylethyl acetate

  31. Mass Analyzers • Low resolution • Quadrupole • Ion trap • High resolution • TOF time of flight • Sector instruments (magnet) • Ultra high resolution • ICR ion cyclotron resonance

  32. Resolution • R = m/z/Dm/z • Unit resolution for quad and trap • TOF up to 15000 • FT-ICR over 30000 • MALDI, Resolves 13C isotope for a protein that weighs 30000

  33. High vs low Res ESI • Q-TOF, ICR • complete separation of the isotope peaks of a +3 charge state peptide • Ion abundances are predictable • Interferences can be recognized and sometimes eliminated • Ion trap, Quad • Unit resolution

  34. MVVTLIHPIAMDDGLR 594.3 594.7 C78H135N21O22S2+3 Q-TOF 595.0 601.3 595.3 601.7 601.0 602.0 m/z 901.4 LCQ 100 891.7 95 90 891.2 85 80 R = 0.88 902.3 75 70 892.6 65 60 55 50 900.6 45 40 35 30 25 20 15 10 5 0 m/z

  35. Quadrupole Mass Ion Filter

  36. Ion Trap

  37. Time of Flight -TOF

  38. Where: • mi = mass of analyte ion • zi = charge on analyte ion • E = extraction field • ti = time-of-flight of ion • ls = length of the source • ld = length of the field-free drift region • e = electronic charge (1.6022x10-19 C)

  39. TOF with reflectronhttp://www.rmjordan.com/tt1.html

  40. Sector instrumentshttp://www.chem.harvard.edu/mass/tutorials/magnetmovie.html

  41. FT-ICRMS • http://www.colorado.edu/chemistry/chem5181/MS_FT-ICR_Huffman_Abraham.pdf

  42. Mass accuracy • Mass Error = (5 ppm)(201.1001)/106 =  0.0010 amu • 201.0991 to 201.1011 (only 1 possibility) • Sector instruments, TOF mass analyzers • How many possibilities with MA = 50 ppm? with 100 ppm?

  43. Exact Mass Determination • Need Mass Spectrometer with a high mass accuracy – 5 ppm (sector or TOF) • C9H15NO4, FM 201.1001 (mono-isotopic) • Mass accuracy = {(Mass Error)/FM}*106 • Mass Error = (5 ppm)(201.1001)/106 =  0.0010 amu

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