GC-MS. Commonly done on low resolution (1 dalton) quadrupole instruments in the selected ion (mass) mode. e.g. Hewlett-Packard MSD, VG Trio and

Transcription

1 GC-MS Commonly done on low resolution (1 dalton) quadrupole instruments in the selected ion (mass) mode. e.g. Hewlett-Packard MSD, VG Trio and MD-8 8, Finnigan INCOS-6 6, Shimadzu QP-5 etc. Can be done at high resolution; accurate mass

2 What has to be right for successful GC-MS? Every sin gle time you try!! GC working with correct column in good condition, right gas flows, autosampler working, correct conditions of injection, temp program, hot interface, no leaks, no bleed, clean injector, clean syringe, clean wash solvents, correct vials and septa... Good vacuum, clean source, right source temp and ionising conditions, appropriate peak resolution & tune, correct slits, correct gain setting, low instrument noise.. DS interface (noise, threshold) set correctly, correct experiment for the job, instrument in calibration, file structure correct, adequate storage space, no bugs before starting sequence Samples correctly ordered in sampler, blanks & external standards included, data archived, QUAN set up correctly. Interpretive skill.

3 Components of the Mass Spectrometer (1) Vacuum System any collisions of an ion with a gas molecules in its path destroys it --> no MS System to convert intact molecules to ions - THE ION SOURCE electron impact (EI), chemical ionisation (volatiles) electrospray or sputtering (non-volatiles eg proteins, DNA) Mass Analyser - To filter by mass (and KE) Ion Detector and ion current amplifier Display mechanisms and Data Acquisition System

4 Components of the Mass Spectrometer (2) Mass Analyser - To filter by mass (and KE) single magnet or quadrupole --> Ion Detector and ion current amplifier Display mechanisms and Data Acquisition System

5 Formation of the mass spectrum Compound ionises in source Molecular ions fragments as a result of excess energy imparted by the ionising electrons (standard energy 7eV) Ions pushed from source with repeller Accelerated by high voltage Ions separated by mass analyser Ions counted and recorded

6 Calibration and leak checking Perfluorokerosene P k (PFK) -a mixture of fluoroalkanes with ions to > 9 dalton 69, 1, 119, 219, Heptacosafluorotributylamine - a single compound MW 614 with 69, 119, Background spectrum should look like air; 17,18,28-32 and 4 Da Argon useful to detect t leaks

7 Characteristic ion series Silicone bleed (silicon isotopes) 27, 281, 355 Alkanes 57, 71, 85, 99, Alkenes , 69, 83, 97, Cyclohexanes 83, 97, 111 Terpanes 123, 149, 191, 25, 217, 231 Monoaromatics 77, 91, 15, 119, 133..

8 Appearance of the Mass Spectrum Average g spectrum has 2 5 bits of information Molecular ion and its isotopic abundances Ionisation o o energy e and structure determine e degree ee of fragmentation; 7 ev is standard Fragment abundances reflect to relative stabilities of the fragment ions - most abundant are the most stable

9 Interpretation of the mass spectrum Identify molecular ion Assess isotope abundances for: A elements H and F A+1 elements C, N A+2 elements Si, S, Cl, Br Determine # rings and double bonds Acyclic saturated hydrocarbons CnH2n+2 Identify characteristic ions and low mass series

10 Interpretation of the mass spectrum Identify molecular ion Molecular Ion corresponds to the loss of one electron from the intact molecule Molecular ion is an odd electron ion Odd electron ions are generally present and have an even mass unless an odd number of nitrogens

11 Identify: 1947 sats 1 64 Molecular Ion? Isotopic features? Ion series? % m/z

12 Nuclidic Masses and Abundances Element Mass Isotope Abundance carbon hydrogen nitrogen oxygen fluorine sulfur

13 1 % A7NO12A# Identify Molecular ion Odd electron ions; A+1 or A+2? low mass series #carbons rings & double bonds 1 % 55 x1. = m/z m/z

14 1947 sats % m/z

15 68 sats % m/z

16 68 sats % m/z

17 1947 sats % m/z

18 1947 sats % m/z

19 1947 sats % m/z

20 Standard Interpretation Procedure Verify masses (calibration); determine elemental compositions & rings+unsaturations ti Mark odd electron ions and verify molecular ion Study appearance of spectrum, molecular stability, labile bonds Identify low mass ion series Identify neutral fragments, logical mass losses Postulate structures of low mass ions Postulate t identity; test t against ref. spectrum or library; rationalize fragmentation pattern McLafferty: Interpretation of Mass Spectra

21 αβ-hopane a fossil hydrocarbon Structure MW = % Mass spectrum

22 Structure MW = 412 C 3 HOPANE % Mass spectrum

23 Triterpanes Da C 3 Ts Tm C 29 C 31 S R Gippsland C 32 C 33 C 29 Ts Dia NW Shelf 3-nor Middle East

24 File A6MA15C:4 SIM-GCMS: Da Scale Factor: % 9 D4 Int. Std. AGSO Standard 221 Da : 55: 1:: 1:5: 1:1: 1:15: TIME (min.)

25 File A6MA15C:4 SIM-GCMS: Da Scale Factor: F t % 9 D3 Rec. Std d. AGSO Standard 234 Da : 55: 1:: 1:5: 1:1: 1:15: TIME (min.)

26 File A6MA15C:4 SIM-GCMS: Da Scale Factor: 4.9 C28 ααα 2S C28 αββ 2R + Bicad T C28 αββ 2S + Bicad T1 C28 ααα2r + Bicad R C29 ααα 2S C29 αββ 2R C29 βα 2S +C27 αββ 2R 1 % 9 AGSO Standard 217 Da C3 αα αα 2R C27 βα C28 βα 2S, 24S +R C28 βα 2R, 24S + R C3 βα 2S Bicad W C27 ααα 2 S C27 αβββ 2S C27 αααα 2R C29 βα 2R C29 ααα 2R C27 βα 2S 2R C29 αββ 2S : 5: 52: 54: 56: 58: 1:: TIME (min.)

27 AGSO Standard: steranes SIM vs. MRM SIM-GCMS: 217 Da C28 βα C28 βα C27 C27 C3 βα C27 C28 ααα 2S + Bicad T C28 αββ 2R C28 αββ 2S + Bicad T1 C28 ααα C29 C3 ααα 2R C27 Bicad W C27 C29 βα C29 C29 βα 2S βα 2R 2S, 24S +R 2R, 24S + R ααα 2S C29 βα 2S +C27 αββ 2R αββ 2S ααα 2R 2S 2R 2R + Bicad R ααα 2S C29 αββ 2R αββ 2S ααα 2R MRM-GCMS: 4 -> 217 Da MRM-GCMS: 386 -> 217 Da MRM-GCMS: 372 -> 217 Da

28 File A6MA15C:4 SIM-GCMS: Da Scale Factor: F t % 9 8 C27 αββ 2R C27 αββ 2S S C29 αββ 2R C29 αββ 2S AGSO Standard 218 Da C28 αββ 2R C28 αββ : 5: 52: 54: 56: 58: 1:: TIME (min.)

29 File A6MA15C:4 SIM-GCMS: Da Scale Factor: F t % AGSO Standard 191 Da C 24 Tet C 19 Tri C 23 Tri C 2 Tri C 21 Tri C 22 Tri C 24 Tr i C 25 Tri (R + S) C 26 Tri(R + S) : 28: 3: 32: 34: 36: 38: 4: 42: 44: 46: 48: 5: TIME (min.)

30 File A6MA15C:4 SIM-GCMS: Da Scale Factor: F t % C29H C3H AGSO Standard 191 Da Compounds 1, 2 and 4 are Oleanoid Triterpanes (see Murray et al. (1997), Geochim. Cosmochim. Acta, in press. Tx is Taraxastane 6 5 O Bicad W Ts Bicad T Tm 29,3 C2 28H C29 Dia 1,2 + 28,3 3 C28H 25-nor C29H+4 C3H Dia C29 Ts C29H βα or C3H 3-no C3H βα C31H S C31H R Tx C31H βα C32H S γ C32H R C33H S C33H R C34HS C34HR C35HS C35HR 5: 55: 1:: 1:5: 1:1: 1:15: TIME (min.)

31 File A6MA15C:4 SIM-GCMS: Da Scale Factor: F t % 9 8 Bicad T W = cis-cis-trans Bicadinane T = trans-trans-trans Bicadinane T1,R = isomers of T AGSO Standard 369 Da Bicad W Bicad T1 Bicad R 1 5: 55: 1:: 1:5: 1:1: 1:15: TIME (min.)

32 File A6MA15C:4 SIM-GCMS: Da Scale Factor: F t 1 1% 8 β(h)-homodrimane AGSO Standard 123 Da : 14: 18: 22: 26: 3: 34: 38: TIME (min.)

33 File A6MA15C:4 SIM-GCMS: Da Scale Factor: F t 1.3 AGSO Standard 25 Da 1 % 9 8 C31 2MeH C31 3MeH : 58: 1:: 1:2: 1:4: 1:6: TIME (min.)

34 File A6MA15C:4 SIM-GCMS: Da Scale Factor: F t % Bicad T AGSO Standard 231 Da Bicad W C31 αα αα 4Me2R : 5: 52: 54: 56: 58: 1:: TIME (min.)

35 GC-MS MS-MS True GC-MS-MS requires an instrument with at least two, independently controllable mass selection regions. Various configurations possible, e.g. Triple Sector Quadruple, Hybrid magnet/quadrupole etc. e.g. Autospec-Q, Finnagin MAT 95Q etc. Very high sensitivity and selectivity very powerful for biomarker work.

36 AGSO Standard: steranes SIM vs. MRM SIM-GCMS: 217 Da C28 βα C28 βα C27 C27 C3 βα C27 C28 ααα 2S + Bicad T C28 αββ 2R C28 αββ 2S + Bicad T1 C28 ααα C29 C3 ααα 2R C27 Bicad W C27 C29 βα C29 C29 βα 2S βα 2R 2S, 24S +R 2R, 24S + R ααα 2S C29 βα 2S +C27 αββ 2R αββ 2S ααα 2R 2S 2R 2R + Bicad R ααα 2S C29 αββ 2R αββ 2S ααα 2R MRM-GCMS: 4 -> 217 Da MRM-GCMS: 386 -> 217 Da MRM-GCMS: 372 -> 217 Da

37 MRM-GC GC-MS Metastable Reaction Monitoring GC-MS Monitoring of ion reactions (e.g. Molecular- Daughter) by selecting metastable ions in the first field free region. Not true GCMSMS but allows selective recording of biomarker molecular weight groups (e.g. only C 3 steranes cf. all steranes at once).

38 MRM-GC GC-MS (2) Metastable Reaction Monitoring GC-MS Higher selectivity and sensitivity. Can be done on double focussing, high resolution instruments such as VG 7E etc but parent ions can only be selected at low resolution - some interference or cross talk between traces.

39 1 % C Da Method recovery standard d 8.6 e 3-CD 3-5α-(H H)-cholestan AGSO Standard : 5: 52: 54: 56: 58: 1::

40 1 % 9 8 AGSO Standard C Da 18.3 C27βα 2R C27 ααα 2S C27 αββ 2R C27 αβββ 2S C27 ααα 2R C27 βα 2S : 5: 52: 54: 56: 58: 1::

41 AGSO Standard d % C Da 5.2 C28 αββ 2S { C28 αββ 2R C28 βα 2S R C28 βα 2R C28 ααα 2 { C28 αααα 2S : 5: 52: 54: 56: 58: 1::

42 1 % C Da 13.4 GCMS Internal Standard gmastane 2 R D 4 -ααα-sti AGSO Standard : 5: 52: 54: 56: 58: 1::

43 AGSO Standard 1 % C29 βα 2S C29 βα 2R C29 ααα 2S C29 α ββ 2R C29 αββ 2S C29 ααα 2R C Da : 5: 52: 54: 56: 58: 1::

44 AGSO Standard 1 % C Da.9 C3 βα 2S C3 βα 2R 2S C3 ααα C3 αββ 2(R R+S) 3 ααα 2R C : 5: 52: 54: 56: 58: 1::

45 C3 2αMe 2S C3 3βMe 2S C3 3β β Me ββ 2R + S AGSO Standard C3 2α Me 2R + 4α Me ββ2ss 1 % C Da C3 C3 4α Me 2S C3 4α ββ Me 2R 3β Me 2R C3 4α Me 2R + Dino : 5: 52: 54: 56: 58: 1::

46 1 % C Da 4.7 C31 2α Me C31 3β Me AGSO Standard : 58: 1:: 1:2: 1:4: 1:6:

47 File:A6MA16 #1-986 Acq:17-MAR-1996 :9:4 GC EI+ MRM Autospec-UltimaQ Sample Text:AGSOSTD 1/2A File Text:Ultra-1, 5m, S:6 F:2 Exp:MRM_OZOILS_STD STD 1 % C Da 1 Bicad T AGSO Standard Bicad W T1 Bicad Bicad R 1 48: 5: 52: 54: 56: 58: 1::

48 1 % 9 8 C Da 23.2 Ts Tm + Bic T1 AGSO Standard ic T Bi C27 17β(H) 48: 5: 52: 54: 56: 58: 1::

49 AGSO Standard 1 % C Da ,3 C28H ,3 C28H 1 48: 5: 52: 54: 56: 58: 1::

50 1 9 8 % C Da 4.2 C29 H AGSO Standard C 29 Ts C29 Dia C2 29 Dianeo C29H βα 48: 5: 52: 54: 56: 58: 1::

51 1 % C3H AGSO Standard C Da 35.4 Compounds 1-6 are Oleanoid Triterpanes (see Murray et al. (1997), Geochim. Cosmochim. Acta, in press. Tx is Taraxastane Oleanoid triterpanes C3H Di ia 3-nor C3H O C3H βα γ Tx 56: 58: 1:: 1:2: 1:4: 1:6:

52 1 % 9 8 C Da 15.9 C31HS C31HR AGSO Standard C31Dia S C31 Dia R 56: 58: 1:: 1:2: 1:4: 1:6:

53 Note: This trace is from run A6FE24:5 using MRM-OZOILS OZOILS_5 1 % 9 8 C Da C34HS AGSO Standard 7 6 C34HR : 5: 55: 1:: 1:5:

54 Note: This trace is from run A6FE24:5 using MRM-OZOILS OZOILS_5 1 % C35HS AGSO Standard d 9 C Da C35HR : 5: 55: 1:: 1:5:

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