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GC and GC-MS.

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Presentation on theme: "GC and GC-MS."— 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, N2 or He, 1-2 mL/min Injector Oven Column
Detector

5 Injector Syringe 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 Splitless (100:90) vs. Split (100:1)
Injector Syringe Purge valve open closed 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 s, which means the sample plug is 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 C16:0 C18:0 C18:1 C18:2 C16:1 Polar column RT (min) 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
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) CI (soft 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

28 Ion Chromatogram of Safflower Oil

29

30 CI/ ion-molecule reaction
2CH4 + e-  CH5+ and C2H5+ CH 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

32

33 Mass Analyzers Low resolution High resolution Ultra high 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 Ion trap, Quad
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 Q-TOF m/z LCQ MVVTLIHPIAMDDGLR C78H135N21O22S2+3 R = 0.88 594.3 594.7
595.0 601.3 595.3 601.7 601.0 602.0 m/z 901.4 100 LCQ 891.7 95 90 891.2 85 80 R = 0.88 75 902.3 70 65 892.6 60 55 50 45 900.6 40 35 30 25 20 15 10 5 m/z

37 Quadrupole Mass Ion Filter

38 Ion Trap

39 Time of Flight -TOF

40 zi = charge on analyte ion E = extraction field
                                                       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)

41 TOF with reflectron http://www.rmjordan.com/tt1.html

42 Sector instruments http://www. chem. harvard

43 FT-ICRMS

44 Mass accuracy Mass Error = (5 ppm)(201.1001)/106 =  0.0010 amu
to (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 (mono-isotopic) Mass accuracy = {(Mass Error)/FM}*106 Mass Error = (5 ppm)( )/106 =  amu


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