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GC_and_GC_MS.ppt
- 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
- 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. Splitless (100:90) vs. Split (100:1) Injector Syringe Injector Syringe Purge valve open Purge valve closed GC column GC column He He
- 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. 0.32 mm ID Liquid Stationary phase Mobile phase (Helium) flowing at 1 mL/min Open Tubular Capillary Column 15-60 m in length 0.1-5 mm
- 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. C16:0 C18:0 C18:1 C18:2 C16:1 C16:0 C18:0 C18:1 C18:2 C16:1 RT (min) RT (min) Polar column 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 Time (min) 0 60 50C 220C 160C
- 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
- 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. To mass analyzer filament 70 eV e- anode repeller Acceleration slits GC column EI Source Under high vacuum
- 26. EI process • M + e- M+* f1 f2 f3 f4 This is a remarkably reproducible process. M will fragment in the same pattern every time using a 70 eV electron beam
- 28. Ion Chromatogram of Safflower Oil
- 30. 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
- 31. EI spectrum of phenyl acetate
- 33. Mass Analyzers • Low resolution – Quadrupole – Ion trap • High resolution – TOF time of flight – Sector instruments (magnet) • Ultra high resolution – ICR ion cyclotron resonance
- 34. 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
- 35. 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
- 37. Quadrupole Mass Ion Filter
- 38. Ion Trap
- 39. Time of Flight -TOF
- 40. 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)
- 44. 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?
- 45. 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