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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 phenyl acetate

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

  31. Resolution • R = m/z/Dm/z • Unit resolution for quad and trap • TOF up to 15000 • FT-ICR over 30000 • MALDI, Resolve 13C isotope for a protein that weighs 30000 • Resolve charge states 29 and 30 for a protein that weighs 30000

  32. 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

  33. 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

  34. Quadrupole Mass Ion Filter

  35. Ion Trap

  36. Time of Flight -TOF

  37. 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)

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

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

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

  41. 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?

  42. 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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