- -

GC-MSCommonly done on low resolution (1 dalton) quadrupole instruments in the selected ion ((mass)) mode. e.g. Hewlett-Packard MSD, VG Trio and MD-800 Finnigan INCOS-60 ShimadzuMD 800, Finnigan INCOS 60, Shimadzu QP-500 etc. CCan be done at hiigh resolutiion; accurate mass

What has to be right for successful GC-MS? Everyy sin ggle time yyou tryy!!

•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 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 •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 ….

-

-

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 -System to convert intact molecules to ionsTHE ION SOURCE

•electron impact (EI), chemical ionisation (volatiles) • electrospray or sputtering (non-volatiles eg proteins, DNA)

•Mass Analyser - To filter by mass (and KE) Mass Analyser To filter by mass (and KE) •Ion Detector and ion current amplifier •Dispp ylay mechanisms and Data Acqquisition Syystem

Components of the Mass Spectrometer (2)

•Mass Analyser - To filter by mass (and KE) •si lingle magnett or quaddrupolle -->>

Ion Detector and ion current amplifier•Display mechanisms and Data Acquisition SystemDisplay mechanisms and Data Acquisition System

Formation of the mass sppectrum •Compound ionises in source •Molecular ions fragments as a result of excess energy impparted by the ionisingggy y electrons (standard energy 70eV) ••Ions pushed from source with repellerIons pushed from source with repeller •Accelerated by high voltage •Ions separated by mass analyser •IIons counttedd andd recorddedd

Calibration and leak checkingCalibration and leak checking

P fl k (PFK) i t f•Perfluorokerosene (PFK) - a mixture of fluoroalkanes with ions to > 900 dalton 69, 100, 119, 219, 231 …….. •Hepptacosafluorotributyylamine - a singgle compound MW 614 with 69, 119, 219 ...•Background spectrum should look like air;Background spectrum should look like air;•17,18,28-32 and 40 Da •AArgon usefful to ddetect l t leakksl t t

Characteristic ion series

Sili bl ili t ) 207 281•Silicone bleed ( d (silicon iisotopes) 207, 281, 355 • Alkanes 57, 71, 85, 99, 113 ...••Alkenes 55 69 83 97 111Alkenes 55, 69, 83, 97, 111 …

•Cyclohexanes 83, 97, 111 •Terpanes 123, 149, 191, 205, 217, 231 •Monoaromatics 77 91 105 119 133•Monoaromatics 77, 91, 105, 119, 133 …..

o sat o e e a d st uctu e dete e d ee

Appearance of the Mass Sppectrumpp

•Average spectrum has 250 bits of informationg p •Molecular ion and its isotopic abundances •Ionisation energy and structure determine degreegy eg of fragmentation; 70 eV is standard •Fraggment abundances reflect to relative stabilities of the fragment ions - most abundant are the most stable

Interppretation of the mass sppectrum

•Identify molecular ion •Assess isotope abundances for:

A elements H and F A+1 elements C, N A+2 elements Si S Cl BrA+2 elements Si, S, Cl, Br

•Determine # rings and double bonds Acyclic saturated hydrocarbons CnH2n+2 •Identifyy ‘characteristic’ ions and low mass series

Interppretation of the mass sppectrum

•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

Identify:Identify:

1947 sats Molecular Ion? 100 64

Isotopic features?

Ion series?

256

128128

160

96192192

66

9857 71 850 m/z50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

%

Nuclidic Masses and AbundancesElement Mass Isotope Abundance

carbon 12.00000 13 0034 13.0034

98.9 1 11.1

hydrogen 1.007825 2 0140 2.0140

99.985 0 015 0.015

nitrogen 14.00307 15.000115.0001

99.63 0.370.37

oxygen 15.9949 17.9992

99.8 0.2

fluorine 18.9984 100

lfsulfur 31 9720 31.9720 32.9715 33.9679

94 8 94.8 0.8 4.4

= 412

100 %

80

60

40

20

0

100 %

80

60

40

20

0

A7NO12A#3-1007 Identify 55

Molecular ion69

Odd electron ions; A+1 or A+2?

60 80 100 120 140 160 180 200 220 240 260 280 300 m/z

55 x10.00

60 80 100 120 140 160 180 200 220 240 260 280 300 m/z

83

low mass series97

#carbons#carbons

111

rings & double bonds 125

69

83

97

111 154

168168

266

266

125 182 196 238

1947 sats

57100 57

71

85

55

99

11383127

141 240155 169 18367 197 211

0 m/z50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

%%

68 sats

100 57 71

85

55

113 1276999

9797183

197141

1551696753 81 253 267 282179 211 225

0 m/z5050 60 70 80 9090 100 110 120 130 140 150150 160 170 180 190 190 200 210 220 230 240 250 260 270 280 290 30060 70 80 100 110 120 130 140 160 170 180 200 210 220 230 240 250 260 270 280 290 300

%

68 sats

100 57 71

85

55 113

69 99 183

127

97 141 155

6753 81 169 197 225 239 253 2680 m/z50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

%%

1947 sats

5757100

71

85

55

9969

11383 127

141 254155 169 183 19767 211 2250 m/z50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

%

1947 sats

100 57

71

85

281

69 99

83 113

97 127141 155 169 183 197 211 225 25367

2950 m/z5050 60 70 80 9090 100 110 120 130 140 150 150 160 170 170 180 190 200 210 220 230 240 250 260 270 280 290 30060 70 80 100 110 120 130 140 160 180 190 200 210 220 230 240 250 260 270 280 290 300

%

1947 sats

100 57

55

97

71

69

83

8167 111

99125

53 79 139 153 168 183 207 224 25291 105 2390

65 119 197131m/z

5050 60 70 80 9090 100 110 120 130 140 150 150 160 170 170 180 190 200 210 220 230 240 250 260 270 280 290 30060 70 80 100 110 120 130 140 160 180 190 200 210 220 230 240 250 260 270 280 290 300

%

•

Standard Interpretation ProcedureStandard Interpretation Procedure • Verify masses (calibration); determine elemental

iti & i + t ticompositions & rings+unsaturations • Mark odd electron ions and verify molecular ion • Study appearance of spectrum molecular stability 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 • P t l t id tit t t i t f t libPostulate identity; test against ref. spectrum or library;

rationalize fragmentation pattern

©McLafferty: Interpretation of Mass Spectra

==

αβ-Hopane a fossil hydrocarbon 369

Structure

MW 412 191

369

MW 412

191 %100

Mass spectrum

412 149

123 109

95 81

68

50 100 150 200 250 300 350 400 450 500

412 397

206177

163137 123

369

==

CC HOPANEHOPANECC HOPANEHOPANECC3030 HOPANEHOPANECC3030 HOPANEHOPANE369

Structure

MW 412 191

369

MW 412

191 %100

Mass spectrum

412 149

123 109

95 81

68

50 100 150 200 250 300 350 400 450 500

412 397

206177

163137 123

369

TriterpanesTriterpanes -- 191 Da191 Da C30 ��

GippslandGippslandC29 ��

C31 �� S

Ts Tm S R C32 �� C33 ��

NW ShelfNW ShelfC29Ts

Dia

Middle EastMiddle East30-nor

AGSO Standard 221 Da

File A6MA15C:4 SIM-GCMS: 221.221 Da Scale Factor: 4 9 Scale Factor: 4.9

100

90

80

70

60

50

40

30

20

10

D44

Int.

Std

.

%

50:00 55:00 1:00:00 1:05:00 1:10:00 1:15:00 TIME (min.)

0

d.

AGSO Standard 234 Da

File A6MA15C:4 SIM-GCMS: 234.232 Da S l F t 5 4Scale Factor: 5.4

100 %

90

80

70

60

50

D3

Rec

. Std

30

40

20

10

0

50:00 55:00 1:00:00 1:05:00 1:10:00 1:15:00 TIME (min.)

File A6MA15C:4 SIM-GCMS: 217.196 Da Scale Factor: 4.9

0S 20R

%100

ααα

20S

Bi

cad

TC29

βα

20

+C27

αββ

C29

αββ

20R

29

αββ

20S

0S

β 20

S

R 0R0S α 20

R

C28 +

B

4S +

R

20R

C27

βα

20S

C2

αββ

20R

C28

αββ

20

+ B

icad

T1

C27

αββ

C29

βα

20R

C29

ααα

20S

C29

ααα

20

C27

ααα

2

C27

ααα

0R +

RC

28 β

α 20

S, 2

4

α 20

R, 2

4S +

R

C27

βα

20SBic

ad W

C28

α

C28

ααα

20

Bic

ad R

C

C28

βα

C30

βα

2

αα 2

0RC

30 α

α

AGSO Standard 217 Da

90

80

7070

60

50

40

30

20

10

0 48:00 50:00 52:00 54:00 56:00 58:00 1:00:00

TIME (min.)

C28

βα

C28

βα

C28

αββ

C28

+ Bi

cad

C27

C29C27

C28

ααα

C27C

27

C30

βα

ααα

20RC29

C29

βα

C29C27

C C

C30

α

AGSO Standard: steranes SIM vs. MRM SIM-GCMS: 217 Da

MRM-GCMS: 400 -> 217 Da

MRM-GCMS: 386 -> 217 Da

MRM-GCMS: 372 -> 217 Da

βα 2

0S

βα 2

0R

20S

, 24S

++R

20R

, 24S

+ RR

ααα

20S

C29

βαα

20S

+C27

ααββ

20R

αβ

β 20

S

ααα

20R

20R

C28

ααα

20S

+ Bi

cad

T 20

Rαβ

β 20

ST1

20R

+ B

icaad

R

ααα

20S

C29

αββ

200R

αββ

20S

ααα

20R

20S

Bica

d W

File A6MA15C:4 SIM-GCMS: 218.203 Da S l F t 4 1Scale Factor: 4.1

AGSO Standard 218 Da

αββ

20R

S C29

αββ

20R

9 αβ

β 20

S

100 %%

80

90

100

C29

0S

C27

C27

αββ

20S

C

60

70

C28

αββ

2

8 αβ

β 20

R

30

40

50 C28

10

20

0

48:00 50:00 52:00 54:00 56:00 58:00 1:00:00

TIME (min.)

File A6MA15C:4 SIM-GCMS: 191.179 Da S l F t 6 4Scale Factor: 6.4

100 % C24Tet

90

80

70

60

50

40

30

20

10

0

AGSO Standard 191 Da

C23Tri C19Tri

23

C20Tri C21Tri C24Tr

i

+ S

)

S)

C22Tri

C 25

Tri (

R +

C 26

Tri(R

+ S

26:00 28:00 30:00 32:00 34:00 36:00 38:00 40:00 42:00 44:00 46:00 48:00 50:00

TIME (min.)

File A6MA15C:4 SIM-GCMS: 191.179 Da S l F t 25 1Scale Factor: 25.1

100%

90

80

70

60

50

40

30

20

10

0

AGSO Standard 191 Da

C29H

C30H

Compounds 1, 2 and 4 are Oleanoid Triterpanes (see Murray et al. (1997), Geochim. Cosmochim. Acta, in press. Tx is Taraxastane

O or

C30

HC29

Ts

α

Ts Tm

C31H S

C31H R

C32H28H

DiaC29

H+4

d T

γ

30 C

28H

C30

H β

α30

-no

C29

H β

α

C31

H β

α

C32H S

C32H R C33H

S C33H R

C34HSC34HR C35HS C35HR

29,3

0 C

2 C

29 D

ia

C30

H D

25-n

or C

Bic

ad

Bicad W

1,2

+ 28

,3

Tx

50:00 55:00 1:00:00 1:05:00 1:10:00 1:15:00

TIME (min.)

File A6MA15C:4 SIM-GCMS: 369.352 Da S l F t 10 3 Scale Factor: 10.3

Bicad T

AGSO Standard 369 Da

%

90

100

W = cis-cis-trans Bicadinane T = trans-trans-trans Bicadinane

70

80 T1,R = isomers of T

50

60

Bicad W

30

40

Bicad T1

10

20 Bicad T1

Bicad R

0

50:00 55:00 1:00:00 1:05:00 1:10:00 1:15:00

TIME (min.)

File A6MA15C:4 SIM-GCMS: 123.117 Da S l F t 100Scale Factor: 100

100

90

80

70

60

50

40

30

20

10

0

8 β(H) H i8 β(H)-Homoddrimane

AGSO Standard 123 Da

%

10:00 14:00 18:00 22:00 26:00 30:00 34:00 38:00

TIME (min.)

File A6MA15C:4 SIM-GCMS: 205.194 Da S l F t 10 3 Scale Factor: 10.3

AGSO Standard 205 Da

%

90

100

2MeH

70

80 C31

2

50

60

H

30

40

C31

3M

eH

10

20

0

56:00 58:00 1:00:00 1:02:00 1:04:00 1:06:00

TIME (min.)

File A6MA15C:4 SIM-GCMS: 231.212 Da S l F t 2 5

AGSO Standard 231 Da

%

90

100 Scale Factor: 2.5 Bicad T

70

80

Bicad W

50

60

αα 4

Me2

0R

30

40

C31

αα

10

20

0 48:00 50:00 52:00 54:00 56:00 58:00 1:00:00

TIME (min.)

GCGC MSMS MSMSGCGC--MSMS--MSMSTrue GC-MS-MS reqquires an instrument with at least two, independently controllable mass selection reggions.

Various configurations possible, e.g. Triple Sector Quadruple “HybridSector Quadruple,“Hybrid” magnet/quadrupole etc. e.g. Autospec-Q, Finnagin MAT 95Q etFinnagin MAT 95Q etc.

Very high sensitivity and selectivity very powerful for biomarker work.

C28

βα

C28

βα

C28

αββ

C28

+ Bi

cad

C27

C29C27

C28

ααα

C27C

27

C30

βα

ααα

20RC29

C29

βα

C29C27

C C

C30

α

AGSO Standard: steranes SIM vs. MRM SIM-GCMS: 217 Da

MRM-GCMS: 400 -> 217 Da

MRM-GCMS: 386 -> 217 Da

MRM-GCMS: 372 -> 217 Da

βα 2

0S

βα 2

0R

20S

, 24S

++R

20R

, 24S

+ RR

ααα

20S

C29

βαα

20S

+C27

ααββ

20R

αβ

β 20

S

ααα

20R

20R

C28

ααα

20S

+ Bi

cad

T 20

Rαβ

β 20

ST1

20R

+ B

icaad

R

ααα

20S

C29

αββ

200R

αββ

20S

ααα

20R

20S

Bica

d W

eco d o b o a e o ecu a we t

MRMMRM GCGC MSMSMRMMRM--GCGC--MSMSMetastable Reaction Monitoring GC-MSMetastable Reaction Monitoring GC MS Monitoring of ion reactions (e.g. Molecular-Daughter) by selecting metastable ions in thDaughter) by selecting metastable ions in the first field free region.

Not “true” GCMSMS but allows selective recordingg of biomarker molecular weigght groups (e.g. only C30 steranes cf. all steranes at once)).

MRMMRM GCGC MS (2)MS (2)MRMMRM--GCGC--MS (2)MS (2)Metastable Reaction Monitoring GC MSMetastable Reaction Monitoring GC-MS

Higher selectivity and sensitivitHigher selectivity and sensitivity.

Can be done on double focussing highCan be done on double focussing, high resolution instruments such as VG 70E etc but parent ions can only be selected at lobut parent ions can only be selected at low resolution - some interference or “cross talk” between tracebetween traces.

%100

e

C28 389 → 234 Da M th d t d d

AGSO Standard

80

90

H)-c

hole

stan

389 → 234 Da 8.6

Method recovery standard

60

70

3-C

D3 -

5α-(

H

30

40

50

10

20

30

0

10

48:00 50:00 52:00 54:00 56:00 58:00 1:00:00

C27372 → 217 Da372 → 217 Da18.3

%

90

100

27 β

α 20

S AGSO Standard

70

80

90

C2

0R

50

60

C27

βα 2

ααα

20R

β 20

Sαβ

β 20

R

20S

30

40 C27

α

C27

αββ

C27

C

27 α

αα 2

10

20

48:00 50:00 52:00 54:00 56:00 58:00 1:00:00 0

C28386 → 217 Da5.2

AGSO St d d %

90

100

βα 2

0S

28 α

ββ 2

0S

0R

{

AGSO Standard

70

80

90 C

28 β

0R

C2

C28

αββ

20

C28

βα

20R

50

60

70

C28

ααα

2

α 20

S

C

{

30

40

50

C28

ααα

10

20

30

48:00 50:00 52:00 54:00 56:00 58:00 1:00:00 0

AGSO %

90

100

0R C29

404 → 221 Da GCMS Internal Standard

AGSO Standard

70

80

90

gmas

tane

20404 → 221 Da

13.4

50

60

70

D 4 -

ααα-

sti

30

40

50

10

20

0 48:00 50:00 52:00 54:00 56:00 58:00 1:00:00

AGSO Standard %

90

100

βα 2

0SC29 400 → 217 Da

70

80

90

C29

β

βα 2

0R

C29

αββ

20S

ββ

20R

S

400 → 217 Da 13.4

50

60

70

C29

β

C29

ααα

20R

C

C29

α

C29

ααα

20S

30

40

CC

10

20

48:00 50:00 52:00 54:00 56:00 58:00 1:00:00 0

R+S

)C

30αβ

β20

(RC

AGSO Standard

%

90

100

20S

C30

βα

20S

C30

βα

20R

C30 414 → 217 Da 0 9

AGSO Standard

70

80

90

30 α

αα 2

0R

C30

ααα

C C0.9

50

60

70 C

30

40

50

10

20

30

48:00 50:00 52:00 54:00 56:00 58:00 1:00:00 0

R S+ SAGSO Standard

%

90

100

30 2

α M

e 20

R

4α M

e ββ

20S

β M

e ββ

20R

+

C30 414 → 231 Da 2 2

70

80

90

+ D

ino

C3 +

20S

Me

20R

e 20

S C

30 3

β2.2

50

60

70

4α M

e 20

R +

C30

4α

Me

2 C

30 4

α ββ

20S C

30 3

βMe

30

40 C30

4

3β M

e 20

R

C30

2αM

e 2

10

20 C30

48:00 50:00 52:00 54:00 56:00 58:00 1:00:00 0

%

90

100 C31 426 → 205 Da

AGSO Standard

70

80

90

2α M

e

426 → 205 Da 4.7

50

60

70

C31

2

1 3β

Me

30

40

50

C31

10

20

56:00 58:00 1:00:00 1:02:00 1:04:00 1:06:00 0

File:A6MA16 #1-986 Acq:17-MAR-1996 00:09:04 GC EI+ MRM Autospec-UltimaQ Sample Text:AGSOSTD 1/2A File Text:Ultra-1, 50m, H2@20psi

E MRM OZOILS STD S:6 F:2

Exp:MRM_OZOILS_STD

Bicad T

48:00 50:00 52:00 54:00 56:00 58:00 1:00:00

%

90

100 C30 412 → 369 Da AGSO Standard

70

80

90 100

50

60

70

30

40

50

RT1

10

20

30

Bic

ad R

Bicad W B

icad

0

AGSO Standard Ts

100 %

90 C27 370 191 D Tm + Bic T1 370 → 191 Da 23.2

ic T

)

Bi

C27

17β

(H)

90

80

7070

60

5050

40

3030

20

1010

0

48:00 50:00 52:00 54:00 56:00 58:00 1:00:00

AGSO Standard

%

90

100 C28 384 → 191 Da

70

80

90 384 → 191 Da 7.6

50

60

70 29,30 C28H

30

40

50

C28

H

10

20

30

28,3

0 C

48:00 50:00 52:00 54:00 56:00 58:00 1:00:00 0

AGSO Standard %

90

100 C29 H

C29 398 → 191 Da

AGSO Standard

70

80

H398 → 191 Da 40.2

50

60

30

40

C29Ts

10

20

29

C29

Dia

C29H βα

29 D

iane

o

48:00 50:00 52:00 54:00 56:00 58:00 1:00:00 0

C2

C30H 100 %

90

80

70

60

50

40

30

20

10

0 56:00 58:00 1:00:00 1:02:00 1:04:00 1:06:00

C30 412 → 191 Da

AGSO Standard

412 → 191 Da 35.4

Compounds 1 - 6 are Oleanoid Triterpanes (see Murray et al. (1997), Geochim. Cosmochim. Acta, in press. Tx is Taraxastane

ia

30 C30H

Oleanoid triterpanes

C30

H D

i

C30H βα

O 30-nor C30H

γ1 2 3 2

4 6

Tx 3 Tx

426 → 191 Da

80

70

60

50

40

30

20

10

0 56:00 58:00 1:00:00 1:02:00 1:04:00 1:06:00

%

90

100 C31HS

C31 426 → 191 Da

AGSO Standard

C31HR15.9

C31

Dia

SC

31 D

ia R

Note: This trace is from run A6FE24:5using MRM-OZOILS 50

90

80

7070

60

5050

40

30

20

10

0 45:00 50:00 55:00 1:00:00 1:05:00

AGSO Standard %

90

100 C34HS

C34 468 → 191 Da

using MRM OZOILS_50 AGSO Standard

468 → 191 Da

C34HR

-Note: This trace is from run A6FE24:5using MRM-OZOILS 50 AGSO St d d %

90

100 C35 482 → 191 Da

C35HS using MRM OZOILS_50 AGSO Standard

70

80

482 → 191 Da

50

60 C35HR

30

40

10

20

0 45:00 50:00 55:00 1:00:00 1:05:00

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12.458 Molecular Biogeochemistry Fall 2009

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