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GC and GC-MS. Gas Chromatography Function Components Common uses Chromatographic resolution Sensitivity.

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Presentation on theme: "GC and GC-MS. Gas Chromatography Function Components Common uses Chromatographic resolution Sensitivity."— Presentation transcript:

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, N 2 or He, 1-2 mL/min Injector Oven Column Detector

5 Gas tank Oven Column Injector Syringe 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 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  m

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) 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 50  C 220  C 160  C

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 CH 2 + 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 (10 7 ) 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 To mass analyzer filament 70 eV e- anode repeller Acceleration slits GC column EI Source Under high vacuum

26 EI process M + e- M +* f1f1 f2f2 f3f3 f4f4 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 2CH 4 + e-  CH 5 + and C 2 H 5 + CH 5 + + M  MH + + CH 4 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/  m/z Unit resolution for quad and trap TOF up to 15000 FT-ICR over 30000 –MALDI, Resolve 13 C 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

36 MVVTLIHPIAMDDGLR 594.3 594.7 595.0 601.3 595.3 601.0 601.7 602.0 m/z C 78 H 135 N 21 O 22 S 2 +3 Q-TOF 901.4 891.7 902.3 900.6 891.2 892.6 LCQ R = 0.88 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

37 Quadrupole Mass Ion Filter

38 Ion Trap

39 Time of Flight -TOF

40 Where: m i = mass of analyte ion z i = charge on analyte ion E = extraction field t i = time-of-flight of ion l s = length of the source l d = length of the field-free drift region e = electronic charge (1.6022x10-19 C)

41 TOF with reflectron

42 Sector instruments

43 FT-ICRMS 181/MS_FT-ICR_Huffman_Abraham.pdf

44 Mass accuracy Mass Error = (5 ppm)(201.1001)/10 6 =  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) C 9 H 15 NO 4, FM 201.1001 (mono-isotopic) Mass accuracy = {(Mass Error)/FM}*10 6 Mass Error = (5 ppm)(201.1001)/10 6 =  0.0010 amu

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