APCI- and APPI-GC/MS-MS for

Characterization of the Macondo Wellhead

Crude Oil and the Oil Spill

Vlad Lobodin

1

National High Magnetic Field Laboratory, Tallahassee, FL

Future Fuels Institute, Florida State University, Tallahassee, FL

Gerald Herbert, AP

April 20, 2010

April 22, 2010

Gerald Herbert, AP

NASA

May 24, 2010

~5 million barrels of crude oil have

leaked from the Macondo well

Michael Spooneybarger, AP

Pensacola Beach, Florida June 23, 2010

the inside of tarballs is saturated

with less weathered petroleum

compounds

Tarballs collected from beach

400 600 800 1000 m/z

Macondo Wellhead Oil

13,700 ± 80 Peaks ≥ 6σ

(+) ESI 9.4 FT-ICR MS

Pensacola Beach

32,232 ± 488 Peaks ≥ 6σ

(+) ESI 9.4 FT-ICR MS

High Resolution FT-ICR

Mass Spectrometry: 20 < C# < 100

Biomarker Region

Biomarker Region

1920

6920

16920

11920

8

6

4

2

0

1920

6920

16920

11920

8

6

4

2

0

1st Dimension

Retention Time

(seconds)

2nd Dimension

Retention Time

(seconds)

Pensacola Beach

Macondo Wellhead Oil

Comprehensive Two-dimensional

Gas Chromatography (GC×GC)

C8-C37, Volatiles

B.M. Ruddy et. al., Energy Fuels, 2014, 28 (6), pp 4043–4050

m/z 500.5 500.4 500.3

N1O1

N1

O1S113C1

N1O1

N1

N1O1 N1S1

A) Macondo Well Oil

10 Peaks across 250 mDa

O113C1 O2

13C1 N1O2

H1C113C1 O1

13C1

O213C1

N1O2

N1O3 O313C1

B) Pensacola Beach

32 Peaks across 250 mDa

(+) ESI 9.4 T FT-ICR MS

C14H30

C16H34

C18H38

C25H52

C30H62

C20H42

50ºC(3 min)- 3ºC/min- 300ºC

GC/MS of “Macondo crude oil” NIST 2779

(Total Ion Chromatogram)

GCxGC/TOF-MS of “Macondo crude oil” NIST 2779

Simulated Distillation for Macondo Well Petroleum (ASTM D-7169)

Temperature (°C)

% R

eco

ve

red

0

10

20

30

40

50

60

70

80

90

100

0 100 200 300 400 500 600 700 800

More than 40 % of the Macondo

petroleum components cannot be

characterized by conventional

GC-based techniques

FT-ICR MS

Reddy, C.M., et al., PNAS, 2011, 1-6

GC Amenable

biomarkers

74% saturated

16% aromatic

10% polar

Macondo well (Deepwater Horizon)

saturated

aromatic

polar (resins / asphaltene)

Reddy et al. (2011) PNAS

“Petroleome”

Elemental composition

Structure

MW distribution

S- 0.3%

V- 52 ppm

Ni- 24 ppm

9.4 Tesla

FT-ICR MS

14.5 Tesla

FT-ICR MS

FT-ICR FACILITIES

N

H N

N

S

C O O H

(+) (-)

(4X difference in pKa)

Analyte Ionization (+) ESI and (-) ESI

500

m/z

105,817 peaks > 6σ

500 < m/z < 2000

750 1000 1250 1500 1750 2000

(+) ESI FT-ICR MS of De-Asphalted Oil”

700.70 700.65 700.60 700.55 700.50 700.45 700.40

93.9 mDa

m/z

1,000 900 800 700 600 500 400 300

(+) ESI FT-ICR MS

Crude Oil

36.4 mDa

8.2 mDa

3.4 mDa

17.1 mDa

C3 / SH4

N / 13CH

C / H12

O / CH4

13C2 / C2H2

N13C / C2H3

8.9 mDa

Broadband Positive ESI FT-ICR Mass Spectrum of Crude Oil

462.1345

m/z 464 463 462

m/z 462.13489

[ C27 H24 N4 58Ni ]+•

(+80ppb)

m/z 462.13432

[ C30 H24 N1 S2 ]+•

(+300ppb)

570 µDa

Theoretical Abundance 2.6%

Experimental Abundance 1.9%

N

N

N

N

O V

DBE = 18

58Ni

Mass e- 548 µDa

Direct Speciation of Metalloporphyrins in Crude Oil

1H = 1.007825

12C= 12.00000

Mass Defect

1H

2H

13C

14N 15

N 12C

16O

19F

17O

18O

31P 32

S 33

S 34S 36

S 35

Cl 37Cl

79Br

81Br

127I

Nuclide

Ma

ss

de

fec

t, m

Da

14N = 14.003074

16O= 15.994915

1. Carbon Number

2. Heteroatom Composition

3. Aromaticity

m/z 704.53510

[C50H72S1]+•

800 700 600 500 400

*

m/z

m/Δm50%

100 - 400 ppb

DBE = C – H

2

N

2 + + 1

McLafferty & Turecek Int. Mass Spectra, 1993

[Z = -2(DBE) + n + 2]

Carbon Number

DB

E

S1 Class

Relative Abundance (% total)

40

30

20

10

0 20 40 60 80

Workflow for High Resolution “Petroleomics”

S CH3

Isomeric structure for S-compounds

S

CH3

C1-dibenzothiophenes (4 isomers)

S

CH3

S

CH3

1-methyl-dibenzothiophene 4-methyl-dibenzothiophene 3-methyl-dibenzothiophene 2-methyl-dibenzothiophene

C1-benzothiophenes (6 isomers)

C2-dibenzothiophenes (26 isomers):

22 dimethyl-dibenzotiophene isomers and 4 ethyl-dibenzotiophene isomers

S

CH3

S

CH3

2-methyl-

benzothiophene

S

CH3

S

CH3

3-methyl-

benzothiophene

4-methyl-

benzothiophene

5-methyl-

benzothiophene

S CH3

6-methyl-

benzothiophene

S

CH3

7-methyl-

benzothiophene

Benzonaphthotiophenes

S

S S Benzo[b]naphtho[1,2-d]thiophene Benzo[b]naphtho[2,3-d]thiophene Benzo[b]naphtho[2,1-d]thiophene

Petroleum Biomarkers: Hopanes and Steranes

Bacteriohopanetetrol

(hopanoid in prokaryotes) Hopanes

A B

C D

E

1

2

3

4 5

6

7

8

9

10

11

12

13

14 15

16

17

18

19 20

21

22

23 24

25 26

27

28

30

29

31

32

33

34

35

C35H62O4

Cholesterol steroid in eukaryotes

Steranes

A B

C D 1

2

3

4 5

6

7

8

9

10

11

12

13

14 15

16

17

18

19

20 21 22

23

24 25 26

27

28

C27H46O

============================================================ 29

M+•

4% of TIC

C27H46

EI mass spectrum of 17α (H)-22,29,30-tris-norhopane

Petroleum Biomarkers

Steranes Hopanes

Multiple reaction monitoring mode

(MRM)

m/z 191

m/z 217

M+• → m/z 191 M+• → m/z 217

Waters Xevo TQ-S

Ion Source Diagram of APCI-GC/MS-MS

Corona Pin

Capillary

GC Column

Ionization

Chamber

Adapted from Waters Corporation

Ion source

Housing

Mass Spec

Heated Transfer Line

N2+• + M M+• + N2

(Atmospheric

Pressure)

Charge Transfer

Protonation

======================================================

Ionization mechanisms:

Charge Transfer vs. Protonation

By courtesy of Waters Corporation

Phenanthrene 100 pg

100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185

%

0

100

100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185

%

0

100

100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185

%

0

100 179

178

178

179

178

179

m/z

Ionization mechanisms:

Charge Transfer vs. Protonation

“wet” source

“wet” source

“dry” source

M+•

[M+H]+

MW 178

Phenanthrene

M+•

[M+H]+

M+•

APCI-GC/MS-MS of 17α(H)-22,29,30-trisnorhopane

Product (daughter) scan from M+• (m/z 370)

M+•

Collision energy: 15 eV

Collision gas: Ar

C27H46

m/z 50 70 90 110 130 150 170 190 210 230 250 270 290 310 330 350 370

%

0

100 191

95

81 69

149

109

135

121

163

177 355 370

MRM transition: m/z 370 → 191

MS/MS spectrum of 17α(H)-22,29,30-trisnorhopane

MS/MS spectrum

from m/z 370

NIST library EI mass spectrum

17β(H)-22,29,30-trisnorhopane

The first match

Sum of 7 MRM transitions

17α(H)-22,29,30-trisnorhopane C27H46

17α(H),21β(H)-30-norhopane C29H50

17α(H),21β(H)-30-hopane C30H52

ααα 20R-cholestane C27H48

αββ 20R-cholestane C27H48

αββ 20R 24S-methylcholestane C28H50

ααα 20R 24R-ethylsholestane C29H52

17α(H),21β(H)-22R-homohopane C31H54

17α(H),21β(H)-22S-homohopane C31H54

αββ 20R 24R- ethylcholestane C29H52

APCI-GC/MS-MS of NIST2266

(hopanes & steranes standard)

R² = 0.9998

0

500000

1000000

1500000

2000000

0 100 200 300 400 500 600

pg

Calibration curve

17α(H),21β(H)-30-hopane

17α(H)-22,29,30-trisnorhopane

17α(H),21β(H)-hopane

17α(H),21β(H)-22S-homohopane 17α(H),21β(H)-22R-homohopane

17α(H),21β(H)-30-norhopane

APCI-GC/MS-MS of NIST2266. Hopanes.

m/z 370 → 191

m/z 398 → 191

m/z 412 → 191

m/z 426 → 191

αββ 20R-cholestane ααα 20R-cholestane

αββ 20R 24S-methylcholestane

αββ 20R 24R- ethylcholestane

ααα 20R 24R-ethylsholestane

APCI-GC/MS-MS of NIST2266. Steranes.

m/z 372 → 217

m/z 386 → 217

m/z 400 → 217

NIST2779 (Macondo crude oil)

Pricey samples from BP oil spill being sold to scientists

http://www.nola.com/news/gulf-oil-spill/index.ssf/2012/03/federal_government_sells_price.html

By Mark Schleifstein, NOLA.com | The Times-Picayune. March 08, 2012

It's likely to be one of the oddest ironies to emerge from the BP oil spill: the federal

government is selling tiny containers of oil siphoned from the Macondo well at a price

equal to $76.3 million a barrel. By comparison, a barrel of crude oil was selling for

$106 on Wednesday.

Of course, the BP oil is not being sold by the

barrel.

The National Institute of Standards and

Technology, an agency of the U.S. Department of

Commerce, is selling 1.2 milliliter bottles of the oil

to scientists who need it for comparison with

materials collected as part of the federal Natural

Resources Damage Assessment process. The

price: $480 for a set of five.

MS/MS conditions for acquisition of MRM transitions

Compound class MRM transition Dwell time, ms Collision

energy, eV

C27-Hopanes 370.30 > 191.10 50 15

C27-Steranes 372.30 > 217.10 50 20

C27-Steranes 372.30 > 218.10 50 20

C27-Steranes 372.30 > 259.20 50 20

C28-Hopanes 384.30 > 191.10 50 15

C28-Steranes 386.30 > 217.10 50 20

C28-Steranes 386.30 > 218.10 50 20

C28-Steranes 386.30 > 259.20 50 20

C29-Hopanes 398.30 > 191.10 50 15

C29-Steranes 400.30 > 217.10 50 20

C29-Steranes 400.30 > 218.10 50 20

C29-Steranes 400.30 > 259.10 50 20

C30-Hopanes 412.30 > 191.10 50 20

C30-Steranes 414.30 > 217.10 50 20

C31-Hopanes 426.30 > 191.10 50 20

C32-Hopanes 440.40 > 191.10 50 20

C33-Hopanes 454.40 > 191.10 50 20

C34-Hopanes 468.40 > 191.10 50 20

C35-Hopanes 482.40 > 191.10 50 20

APCI/GC-MS/MS of NIST2779 (Macondo crude oil)

Time, min

50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00

RA

, %

0

100

Time 80.00 82.00 84.00 86.00 88.00 90.00 92.00 94.00

Hopanes: Summed Signals for C27-C35 (M+• → m/z 191)

C35

C34 C33

C32

C31

H30

H29

Ts

Tm

H31S

H31R

H32S

H32R H33S

H33R H34S

H34R H35R

H35S

A B

C D

E

1 2

3 4

5 6

7 8

9

10

11 12

13

14 15

16

17

18

19 20

21

22

23 24

25 26

27

28 29

30

31

32

33

34

35

C29Ts

DH30

M30

m/z 372 → 217

m/z 386 → 217

m/z 372 → 217

βα

βα

αβ αβ

βα

βα

αβ αβ

αααS

βα

βα

αβ αααS

αααR

αβ

Time 50.00 55.00 60.00 65.00 70.00 75.00 80.00

50.00 55.00 60.00 65.00 70.00 75.00 80.00

%

100

50.00 55.00 60.00 65.00 70.00 75.00 80.00

50.00 55.00 60.00 65.00 70.00 75.00 80.00

0

%

100

0

%

100

%

100

0

0

APCI/GC-MS/MS of NIST2779 (Macondo crude oil)

C27 -Diasteranes C27-Steranes

C28 -Diasteranes

C28-Steranes

C29 -Diasteranes C29-Steranes

Sum of C27-C29 Steranes/Diasteranes

A B

C D 1 2

3

4 5

6

7

8

9

10

11 12

13

14 15

16

17

18

19

20 21 22

23

24 25 26

27

28 29

αααS

αββR

αααR αββS

αββR αββS

αααR

αββR αββS

0

0.4

0.8

1.2

1.6

2

βαC27/βαC29

Diasteranes

Ts/Tm

H29/H30

H32S/H32R

H33S/H33R

αββC27/αββC29

Steranes

H30/H31+H32+H33+H34+H35

NIST 2779 (Macondo crude oil)

0

0.4

0.8

1.2

1.6

2

Ts/Tm

H29/H30

H32S/H32R

H33S/H33R H30/(H31+H32+H33+H34

+H35)

αββC27/αββC29

βαC27/βαC29

NIST2779 (Macondo crude oil)

Steranes

Diasteranes

SAM 1-18 collected

1-45 months after

the oil spill

"Megaplume" in the GC600 lease block:

Lat: 27° 22.466' N

Long: 90° 30.689'W

water depth: 1382m

Natural Oil Seeps (GC600, Megaplume)

Natural oils seeps in the Gulf of Mexico - 140,000 tonnes per year (range of

80,000 to 200,000 tonnes).

Natural Oil Seeps. The Gulf of Mexico.

from www.sarsea.org

0

0.4

0.8

1.2

1.6

2

Ts/Tm

H29/H30

H32S/H32R

H33S/H33R H30/(H31+H32+H33+H34+H3

5)

αββC27/αββC29

βαC27/βαC29

Steranes

Diasteranes

Megaplume Oil Seep (GC600)

0

0.4

0.8

1.2

1.6

2

Ts/Tm

H29/H30

H32S/H32R

H33S/H33R H30/(H31+H32+H33+H34+H35)

αββC27/αββC29

βαC27/βαC29

Steranes

Diasteranes

Blue crude (Anadarko Independence Hub)

0

0.5

1

1.5

2

0

0.5

1

1.5

2

0

0.5

1

1.5

2

0

0.5

1

1.5

2

SAM-1 SAM-2 SAM-3

SAM-4 SAM-6

0

0.5

1

1.5

2

SAM-7

0

0.5

1

1.5

2

SAM-8

0

0.5

1

1.5

2

0

0.5

1

1.5

2

SAM-5

βαC27/βαC29

Diasteranes

Ts/Tm

H29/H30

H32S/H32R

H33S/H33R H30/ΣH31‒H35

C27βα/C29βα

Diasteranes

Ts/Tm

H29/H30

H32S/H32R

H33S/H33R H30/ΣH31‒H35

C27βα/C29βα

Diasteranes

Ts/Tm

H29/H30

H32S/H32R

H33S/H33R H30/ΣH31‒H35

C27βα/C29βα

Diasteranes H29/H30

H32S/H32R

H33S/H33R H30/ΣH31‒H35

Ts/Tm

Ts/Tm

C27βα/C29βα

Diasteranes H29/H30

H32S/H32R

H33S/H33R H30/ΣH31‒H35

C27βα/C29βα

Diasteranes H29/H30

H32S/H32R

H33S/H33R H30/ΣH31‒H35

Ts/Tm

C27βα/C29βα

Diasteranes H29/H30

H32S/H32R

H33S/H33R H30/ΣH31‒H35

Ts/Tm

Ts/Tm

βαC27/βαC29

Diasteranes H29/H30

H32S/H32R

H33S/H33R H30/ΣH31‒H35

C27αββ/C29αββ

Steranes

C27αββ/C29αββ

Steranes C27αββ/C29αββ

Steranes

C27αββ/C29αββ

Steranes

C27αββ/C29αββ

Steranes

C27αββ/C29αββ

Steranes

C27αββ/C29αββ

Steranes C27αββ/C29αββ

Steranes

0

0.5

1

1.5

2

SAM-9

C27βα/C29βα

Diasteranes H29/H30

H32S/H32R

H33S/H33R H30/ΣH31‒H35

Ts/Tm

C27αββ/C29αββ

Steranes

0

0.5

1

1.5

2

SAM-10

0

0.5

1

1.5

2

SAM-11

0

0.5

1

1.5

2

SAM-12

0

0.5

1

1.5

2

SAM-13

0

0.5

1

1.5

2

SAM-14

0

0.5

1

1.5

2

SAM-15

SAM-16

0

0.5

1

1.5

2

0

0.5

1

1.5

2

SAM-17

C27βα/C29βα

Diasteranes H29/H30

H32S/H32R

H33S/H33R H30/ΣH31‒H35

Ts/Tm Ts/Tm

C27βα/C29βα

Diasteranes H29/H30

H32S/H32R

H33S/H33R H30/ΣH31‒H35

C27βα/C29βα

Diasteranes H29/H30

H32S/H32R

H33S/H33R H30/ΣH31‒H35

Ts/Tm

C27βα/C29βα

Diasteranes H29/H30

H32S/H32R

H33S/H33R H30/ΣH31‒H35

Ts/Tm Ts/Tm

C27βα/C29βα

Diasteranes H29/H30

H32S/H32R

H33S/H33R H30/ΣH31‒H35

C27βα/C29βα

Diasteranes H29/H30

H32S/H32R

H33S/H33R H30/ΣH31‒H35

Ts/Tm

C27βα/C29βα

Diasteranes H29/H30

H32S/H32R

H33S/H33R H30/ΣH31‒H35

Ts/Tm Ts/Tm

C27βα/C29βα

Diasteranes H29/H30

H32S/H32R

H33S/H33R H30/ΣH31‒H35

C27αββ/C29αββ

Steranes C27αββ/C29αββ

Steranes C27αββ/C29αββ

Steranes

C27αββ/C29αββ

Steranes C27αββ/C29αββ

Steranes

C27αββ/C29αββ

Steranes

C27αββ/C29αββ

Steranes C27αββ/C29αββ

Steranes

0

0.5

1

1.5

2

SAM-18

C27βα/C29βα

Diasteranes H29/H30

H32S/H32R

H33S/H33R H30/ΣH31‒H35

Ts/Tm

C27αββ/C29αββ

Steranes

0

0.4

0.8

1.2

1.6

2

βαC27/βαC29

Diasteranes

Ts/Tm

H29/H30

H32S/H32R

H33S/H33R

αββC27/αββC29

Steranes

H30/H31+H32+H33+H34+H35

NIST2779 (Macondo crude oil)

Blue crude (Independence Hub)

Megaplume oil seep (GC600)

SAM-10 (Pensacola Beach)

Overlaid spider diagrams

Correlation coefficients

Other case studies: Exxon Valdez oil spill

from www.uaf.edu

Prince William Sound, Alaska

March 24, 1989. 258,000 barrels

25,500 peaks

150 < m/z < 850

850 750 650 550 450 350 250 150

m/z

(+) APPI FT-ICR MS of Macondo crude oil

Relative Abundance (% total) Carbon Number

10

15

5

0

20

DB

E

10 20 30 40

S class (M+•)

50 60

DBE=12

S

DBE=9

R

10 20 30 40 50 60

25

30 HC class (M+•)

DBE=10

S

R

R

(+) APPI FT-ICR MS of Macondo crude oil

Mass Spec

UV-lamp

(Atmospheric

Pressure)

Heated Transfer Line

Capillary

GC Column

Ionization

Chamber

Ion source

Housing

APPI-GC/MS Ion Source Diagram

Kr UV-lamp

Atmospheric Pressure PhotoIonization (APPI)

Spectral distribution of a Krypton lamp

E=10.6 eV, λ= 117 nm

E=10.0 eV, λ= 124 nm

M+• M hν

hν > IE(M)

Ionization energies, IE (eV)

IE(N2) = 15.6 eV

IE(H2O)= 12.6 eV

IE(O2) = 12.1 eV

IE(C6H6) = 9.2 eV

IE(Toluene)= 8.8 eV

IE(Naphthalene) = 8.1 eV

IE (Phenanthrene) = 7.9 eV

IE (Thiophene) = 8.9 eV

IE (DBT) = 8.0 eV

IE(Alkanes) ~ 10 eV

- ē

100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190

%

0

100 178

m/z

APPI-GC/MS. Mass Spectrum of Phenanthrene

M+•

MW 178

IE = 7.9 eV

Phenanthrene (20 pg injected)

M+• M hν

time

5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00

%

0

100 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00

%

0

100

APPI-GC/MS vs APCI-GC/MS of Phenanthrene

APCI-GC/MS

m/z 178 →m/z 152

APPI-GC/MS

m/z 178 → m/z 152

S/N 4160

S/N 32830

APPI-GC/MS of Aromatic compounds

M+•

M+•

M+•

MW 168

IE = 8.1 eV

MW 167

IE = 7.6 eV

MW 184

IE = 7.9 eV

Time 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00

%

0

100

Acenaphthylene

Naphthalene

Acenaphthene

Fluorene Anthracene

Benz[a]anthracene

Fluoranthene

Pyrene

Phenanthrene

Chrysene

Benzo[b]fluoranthene

Benzo[k]fluoranthene

Dibenz[a,h]anthracene

Benzo[g,h,i]perylene

Benzo[a]pyrene

Indeno[1,2,3-cd]pyrene

Boiling T 500 °C

APPI-GC/MS of 610 PAH Calibration Mix A

610 PAH Calibration Std (x 1000 dilution)

Final concentration: 500-1000 ng/mL

APPI/GC-MS/MS of NIST2779 (Macondo crude oil)

S

Me

Me

S

APPI with Argon lamp

Ar UV-lamp

E=11.6 eV, λ= 106.7 nm

E=11.8 eV, λ= 104.8 nm

Spectral distribution

of Ar lamp

0.105 ‒ 9 µm

LiF wavelength transmission range

17α(H),21β(H)-30-hopane

as internal standard Environ. Sci. Technol. 1994, 28, 142-145

APPI(Ar)-GC/MS-MS. PAHs and PASHs ratios.

Depletion of PAHs and PASHs in Environmental

samples from AL-MS shore line.

Phen DBT C2-Phen C2-DBT C3-Phen C3-DBT Chrys

≈

100

NIST 2779

(DWH) Jul, 2011 Feb, 2012 Jan, 2014

Depletion is relative to 17α(H),21β(H)-30-hopane (C30-Hopane)

We first utilized AP-GC/MS for a trace analysis of petroleum

biomarkers from the Macondo crude oil and environmental

samples.

We describe an Atmospheric Pressure PhotoIonization

(APPI) source that in combination with GC separation and

MS/MS analysis is an efficient method for characterization of

aromatic compounds in wellhead and spilled oil.

Analysis of petroleum compounds with APGC/MS-MS

provides a sensitive analytical tool for targeted analysis,

source identification of the oil spill, and tracking a fate of oil

spill residues.

SUMMARY

FT-ICR Group, NHMFL

Thank you!

(-) UniSpray TQS mass spectrum of Macondo crude oil

(NIST 2779)

m/z200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900

%

0

100

NEG_UNISPRAY-DWH_1%NH3_B 20 (0.335) Cm (14:143) MS2 ES- 1.28e5421.26

294.15

247.13

490.32566.38

684.51

790.66

858.68

dioctyl sodium sulfosuccinate (DOSS)

[M-H]-

m/z60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440

%

0

100

NEG_UNISPRAY-DWH_MS-MS-421 121 (2.028) Cm (116:128) Daughters of 421ES- 3.73e4421

81

m/z120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

%

0

100

NEG_UNISPRAY-DWH_MS-MS-421 121 (2.028) Cm (116:128) Daughters of 421ES- 904367

227

187

219

291

265

279

338313

298

391

375

404

(-) UniSpray MS/MS spectrum for [M-H]-

ion of dioctyl sodium sulfosuccinate (DOSS)

CID: 20 V; Collision gas: Ar

[M-H]-

[HSO3]-

m/z200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900

%

0

100

POS_UNISPRAY_DWH_1%HCOOH_A 23 (0.386) Cm (23:84) MS2 ES+ 1.82e6360

227

338

305

431

499

478450

567

520

635

586

703

677

839771

(+) UniSpray TQS mass spectrum of Macondo crude oil

(NIST 2779)

1 mg/mL Tol:MeOH (50:50)- 1% HCOOH

APCI-GC/MS conditions

Mass spectrometer – Waters Xevo TQ-S

Tsource 150ºC

Corona 2.5 μA

Cone voltage 30V

Source offset 50V

Auxilliary gas (N2) 200 L/hr

Cone gas (N2) 250 L/hr

Collision gas Ar

Gas chromatograph –7890

Tinj=300ºC

Column: MXT-5 (or MXT-1), 60m × 0.25mm × 0.25 µm

Carrier gas: He

Flow rate: 1.2 mL/min

Split ratio: 1:10

Oven: 50ºC - 20ºC/min -150ºC- 2ºC/min - 350ºC (25 min)

Transfer Line: 380ºC

Injected volume: 1 μL

APPI-GC/MS conditions

Mass spectrometer – Waters Xevo TQ-S

Tsource 150ºC

Kr UV lamp 10 eV (or Ar UV lamp 11.7 eV)

Cone voltage 30V

Source offset 50V

Auxiliary gas (N2) 200 L/hr

Cone gas (N2) 150 L/hr

Collision gas Ar

Gas chromatograph –7890

Tinj=300ºC

Column: MXT-5 (or MXT-1), 60m × 0.25mm × 0.25 µm

Carrier gas: He

Flow rate: 1.2 mL/min

Split ratio: 1:10

Oven: 50ºC - 20ºC/min -150ºC- 2ºC/min - 350ºC (25 min)

Transfer Line: 380ºC

Injected volume: 1 μL

APPI(Ar)-GC/MS-MS. PAHs and PASHs ratios.

NIST2279 (DWH) Jul 17, 2011 Feb 8, 2012 Jan 27, 2014

С2-Phen/C2-DBT 2.9 2.8 2.3 2.8

С3-Phen/C3-DBT 1.9 1.4 1.4 1.5

С2-Phen/C2-DBT and С3-Phen/C3-DBT ratios

SAM-1 (30°17'15.0”N, 87°28'44.6”W))on 04.05.2011,

SAM-2 (30°17'14.6”N, 87°28'50.2”W) on 04.06.2011,

SAM-3 (30°14'25.6”N, 87°44'14.8”W) on 07.17.2011

SAM-4 (29°10'29.3”N, 90°04'33.2”W) on 07.17.2011,

SAM-5 (30°17'18.4”N, 87°28'37.8”W) on 07.19.2011,

SAM-6 (30°14'48.4”N, 87°41'35.2”W) on 11.27.2011

SAM-7 (30°14'25.4”N, 87°44'15.2”W) on 11.28.2011,

SAM-8 (29°56'41.0”N, 88°49'27.0”W) on 11.28.2011,

SAM-9 (29°17'35.5”N, 90°29'17.5”W) on 05.31.2010,

SAM-10 (30°19'32.1”N, 87°10'30.5”W) on 06.23.1010,

SAM-11 (30°14'60.6”N, 88°53'21.1”W) on 02.09.2012,

SAM-12 (30°18'16.4”N, 87°23'20.9”W) on 02.07.2102,

SAM-13 (30°14'34.8”N, 88°42'59.1”W) on 02.08.2012,

SAM-14 (30°14'26.3”N, 87°44'16.9”W) on 08.31.2012,

SAM-15 (30°13'54.4”N, 88°53'47.0”W) on 02.09.21012,

SAM-16 (30°18'30.8”N, 87°22'16.3”W) on 02.07.2012,

SAM-17 (29°10'30.0”N, 90°04'33.6”W) on 07.31.2011,

SAM-18 (30°15'16”N, 88°7'50”W) on 01.27.2014,

Megaplume (27°22'27.96”N, 90°30'41.34”W) - depth – 1200 m, GC600.

Blue crude oil (28°05'89.0”N, 87°59'27.0”W).

Samples’ collection sites and time

Petroleum Biomarkers. Hopanes.

Bacteriohopanetetrol

(hopanoid in prokaryotes) Hopanes

A B

C D

E

1

2

3

4 5

6

7

8

9

10

11

12

13

14 15

16

17

18

19 20

21

22

23 24

25 26

27

28

29

30

31

32

33

34

35

Over 150 distinct, naturally-occurring hopanoids have been identified in soils, sediments, and other

organic matter. Hopanoids have a fixed stereochemistry and differ in the orientation about Carbon-

17 and Carbon-21 (α or β) and Carbon-22 (R or S).

17β, 21β(H) is biological configuration

The order of thermodynamic stability of the 17-21 hopane isomers is

17α(H),21β(H) > 17β(H),21α(H) > 17α(H),21α(H) > 17β(H),21β(H)

22R is biological configuration.

17α(H),21β(H) –hopanes are the most stable. 17β(H),21α(H) are called moretanes.

22S/(22S+22R) ~ 0.58-0.62 for C31-hopane

C35H62O4

Cholesterol

Petroleum Biomarkers. Steranes.

steroid in eukaryotes Steranes

A B

C D 1

2

3

4 5

6

7

8

9

10

11

12

13

14 15

16

17

18

19

20 21 22

23

24 25 26

27

28 29

C27H46O

5α,14α,17α(H)–cholestane-20R

biological configuration

5α,14β,17β(H)

stable configuration

14β,17β(H)/[14α,17α(H) + 14β,17β(H)] ~ 0.7 – Endpoint configuration

Diasteranes (rearranged steranes) - rearrangement product from sterol precursors through

diasterenes. The rearrangement involves migration of C-10 and C-13 methyl groups to C-5 and

C-14 and is favored by acidic conditions, clay catalysis, and/or high temperatures. Diasteranes

increase relative to steranes with thermal maturation and they are low in clay-poor carbonate

source rocks and related oils. 13β(H),17α – diasteranes 20S or 20R

Assigned Hopanes

18α(H)-22,29,30-trisnorhopane (Ts),

17α(H)-22,29,30-trisnorhopane (Tm),

17α(H),21β(H)-30-norhopane (H29),

18α(H),21β(H)-30-norneohopane (C29Ts),

17α(H)-diahopane (DH30),

17β(H),21α(H)-hopane (moretane, M30),

17α(H),21β(H)-hopane (H30),

17α(H),21β(H)-22S-30-homohopane (H31S),

17α(H),21β(H)-22R-30-homohopane (H31R),

17α(H),21β(H)-22S-30,31-bishomohopane (H32S),

17α(H),21β(H)-22R-30,31-bishomohopane (H32R),

17α(H),21β(H)-22S-30,31,32-trishomohopane (H33S),

17α(H),21β(H)-22R-30,31,32-trishomohopane (H33R),

17α(H),21β(H)-22S-30,31,32,33-tetrakishomohopane (H34S),

17α(H),21β(H)-22R-30,31,32,33-tetrakishomohopane (H34R),

17α(H),21β(H)-22S-30,31,32,33,34-pentakishomohopane (H35S),

17α(H),21β(H)-22R-30,31,32,33,34-pentakishomohopane (H35R).

Assigned Steranes and Diasteranes

13β(H),17α(H)-20S-diacholestane (C27βαS),

13β(H),17α(H)-20R-diacholestane (C27βαR),

13α(H),17β(H)-20S-diacholestane (C27αβS),

13α(H),17β(H)-20R-diacholestane (C27αβR),

5α(H),14α(H),17α(H)-20S-cholestane (C27αααS),

5α(H),14β(H),17β(H)-20R-cholestane (C27αββR),

5α(H),14β(H),17β(H)-20S-cholestane (C27αββS),

5α(H),14α(H),17α(H)-20R-cholestane (C27αααR),

13β(H),17α(H)-20S-24-methyldiacholestane (C28βαS),

13β(H),17α(H)-20R-24-methyldiacholestane (C28βαR),

13α(H),17β(H)-20S-24-methyldiacholestane (C28αβS),

13α(H),17β(H)-20R-24-methyldiacholestane (C28αβR),

5α(H),14α(H),17α(H)-20S-24-methylcholestane (C28αααS),

5α(H),14β(H),17β(H)-20R-24-methylcholestane (C28αββR),

5α(H),14β(H),17β(H)-20S-24-methylcholestane (C28αββS),

5α(H),14α(H),17α(H)-20R-24-methylcholestane (C28αααR),

13β(H),17α(H)-20S-24-ethyldiacholestane (C29βαS),

13β(H),17α(H)-20R-24-ethyldiacholestane (C29βαR),

13α(H),17β(H)-20S-24-ethyldiacholestane (C29αβS),

13α(H),17β(H)-20R-24-ethyldiacholestane (C29αβR),

5α(H),14α(H),17α(H)-20S-24-ethylcholestane (C29αααS),

5α(H),14β(H),17β(H)-20R-24-ethylcholestane (C29αββR),

5α(H),14β(H),17β(H)-20S-24-ethylcholestane (C29αββS),

5α(H),14α(H),17α(H)-20R-24-ethylholestane (C29αααR).

Biomarker ratios (Ts/Tm, H29/H30, H32S/H32R, H30/Σ(H31-H35), H33S/H33R,

C27αββ/C29αββ steranes, and C27βα/C29βα diasteranes)

Sample Ts/Tm H29/H30 H32S/H32R H33S/H33R H30/Σ(H31-H35) C27αββ/C29αββ

steranes

C27βα/C29βα

diasteranes

NIST2779 1.42±0.05 0.52±0.04 1.48±0.03 1.46±0.06 0.65±0.03 0.68±0.06 0.94±0.02

SAM-1 1.49±0.02 0.56±0.03 1.36±0.01 1.42±0.07 0.65±0.06 0.68±0.02 0.95±0.03

SAM-2 1.43±0.05 0.58±0.02 1.41±0.13 1.35±0.05 0.62±0.05 0.66±0.04 0.89±0.04

SAM-3 1.49±0.10 0.55±0.01 1.36±0.03 1.34±0.10 0.66±0.02 0.72±0.03 0.91±0.04

SAM-4 1.47±0.03 0.59±0.02 1.39±0.03 1.40±0.12 0.61±0.01 0.73±0.03 0.93±0.02

SAM-5 1.49±0.08 0.57±0.01 1.31±0.07 1.37±0.05 0.62±0.03 0.69±0.02 0.90±0.02

SAM-6 1.56±0.03 0.50±0.05 1.32±0.03 1.33±0.03 0.68±0.01 0.71±0.01 0.97±0.02

SAM-7 1.53±0.09 0.53±0.06 1.35±0.09 1.30±0.01 0.60±0.01 0.67±0.05 0.94±0.05

SAM-8 1.30±0.09 0.62±0.06 1.40±0.09 1.32±0.10 0.46±0.06 0.42±0.02 0.56±0.03

SAM-9 1.43±0.11 0.49±0.03 1.39±0.11 1.49±0.11 0.67±0.05 0.74±0.06 0.96±0.02

SAM-10 1.46±0.07 0.53±0.05 1.42±0.07 1.46±0.08 0.66±0.06 0.64±0.07 0.90±0.08

SAM-11 1.42±0.10 0.52±0.05 1.41±0.08 1.32±0.12 0.60±0.02 0.66±0.07 0.91±0.09

SAM-12 1.48±0.08 0.56±0.06 1.50±0.12 1.39±0.11 0.54±0.05 0.58±0.06 0.89±0.08

SAM-13 1.50±0.07 0.51±0.05 1.46±0.08 1.36±0.09 0.60±0.07 0.62±0.06 0.96±0.06

SAM-14 1.49±0.09 0.49±0.05 1.45±0.13 1.52±0.14 0.63±0.06 0.64±0.05 1.00±0.07

SAM-15 1.33±0.05 0.59±0.05 1.57±0.15 1.52±0.15 0.55±0.05 0.58±0.06 0.92±0.09

SAM-16 1.49±0.10 0.54±0.06 1.46±0.14 1.40±0.12 0.60±0.06 0.54±0.05 0.92±0.07

SAM-17 1.37±0.05 0.54±0.03 1.41±0.09 1.49±0.07 0.46±0.04 0.40±0.03 0.60±0.04

SAM-18 1.47±0.12 0.59±0.06 1.52±0.09 1.45±0.14 0.55±0.05 0.59±0.06 0.81±0.80

Megaplume 1.00±0.05 1.01±0.05 1.48±0.05 1.44±0.08 0.52±0.04 0.84±0.07 0.82±0.08

Blue crude 0.15±0.04 0.74±0.05 0.54±0.05 0.94±0.05 2.00±0.10 0.11±0.03 0.84±0.06

M+•

4% of TIC

C27H46

EI mass spectrum of 17α (H)-22,29,30-tris-norhopane

M+•

7% of TIC

C27H48

EI mass spectrum of ααα 20R-cholestane

GC/MS of NIST 2779 (Macondo crude oil)

(Ion Chromatogram at m/z 191 and 217)

Ion Chromatogram at m/z 191

Ion Chromatogram at m/z 217

Hopanes

C30

C31

C32

C33

C29

C34 C35

Steranes

C28

C29

C29

C27

C29

C27

C29

C30

C27 C28 C29

C29

m/z 217

m/z 191 Ts

Tm

Constant neutral loss scan

Multiple reaction monitoring mode

Precursor (parent) ion scan

Product (daughter) ion scan

MS/MS Modes

C H 3

C H 3

C H 3

C H 3 C H 3

R M+•

+

•

•

+

+

R= H m/z 217

R= CH3 m/z 231

R= C2H5 m/z 245

R= C3H7 m/z 259

C H 3

C H 3

R

C H 3

C H 3

C H 3

C H 3

C H 3

R

C H 3

C H 3

C H 3

C H 3

C H 3

R

H

Fragmentation scheme for steranes

APCI-GC/MS-MS of 17α(H)-22,29,30-trisnorhopane

Product (daughter) scan from M+• (m/z 370)

M+•

Collision energy: 20 eV

Collision gas: Ar

C27H46

m/z 50 70 90 110 130 150 170 190 210 230 250 270 290 310 330 350 370

%

0

100 191

95

81 69

149

109

135

121

163

177 355 370

50 70 90 110 130 150 170 190 210 230 250 270 290 310 330 350 370

%

0

100 217

121

95

81

73

107 135

149

175 161

189 203

357

262 372

APCI-GC/MS-MS of ααα 20R-cholestane

Product (daughter) scan from M+• (m/z 372)

M+•

Collision energy: 20 eV

Collision gas: Ar

C27H48

m/z

MS/MS spectrum of ααα 20R-cholestane

MS/MS spectrum

from m/z 372

NIST library EI mass spectrum

of Cholestane

The first match

C35H72

C14H30

C16H34

C18H38

C25H52

C30H62

C20H42

Pr

Ph

Isoprenoid indices

Pr/Ph=1.33

n-C17/Pr=1.58

n-C18/Ph=1.67

Reported Pr/Ph ratio for MC252 is 0.9

Environ. Res. Lett. 2012, 7, 035302

GC/MS of “Macondo crude oil” NIST 2779

(Total Ion Chromatogram)

+

+ •

R= H m/z 217

R= CH3 m/z 231

R= C2H5 m/z 245

R= C3H7 m/z 259

R= H m/z 218

R= CH3 m/z 232

R= C2H5 m/z 246

R= C3H7 m/z 260

M+•

C H 3

C H 3

R

C H 3

C H 3

R

C H 3

C H 3

C H 3

C H 3 C H 3

R

Characteristic ions for steranes

(+) APPI FT-ICR MS of Macondo crude oil

25,500 peaks

150 < m/z < 850

850 750 650 550 450 350 250 150

m/z

Relative Abundance (% total)

Carbon Number

10

15

5

0

20

DB

E

10 20 30 40 50 60

25

30 HC class (M+•)

DBE=10

(+) APPI FT-ICR MS of Macondo crude oil

C1-phenathrene has 5 isomers

R

CH3 CH3

1-methylphenanthrene 2-methylphenanthrene

CH3

3-methylphenanthrene

CH3

4-methylphenanthrene

CH3

9-methylphenanthrene

C2-phenanthrene has 30 isomers:

25 isomers for dimethyl-

phenanthrenes

5 isomers for ethyl-phenanthrenes

C#= 25-30

DBE

HC class (M+•)

DBE Distribution

Relative Abundance (% total)

Carbon Number

10

15

5

0

20

DB

E

10 20 30 40

S class (M+•)

50 60

C#=19

DBE=12

DBE=6 S

R

25

30

S

C3

S R

(+) APPI FT-ICR MS of Macondo crude oil

DBE=12

DBE=9

S

R

C2-dibenzothiophenes (26 isomers): 22 dimethyl-dibenzotiophene isomers and

4 ethyl-dibenzotiophene isomers

S CH3

Isomeric structure for S-compounds

S

CH3

C1-dibenzothiophenes (4 isomers)

S

CH3

S

CH3

1-methyl-dibenzothiophene 4-methyl-dibenzothiophene 3-methyl-dibenzothiophene 2-methyl-dibenzothiophene

C1-benzothiophenes (6 isomers)

C2-dibenzothiophenes (26 isomers):

22 dimethyl-dibenzotiophene isomers and 4 ethyl-dibenzotiophene isomers

S

CH3

S

CH3

2-methyl-

benzothiophene

S

CH3

S

CH3

3-methyl-

benzothiophene

4-methyl-

benzothiophene

5-methyl-

benzothiophene

S CH3

6-methyl-

benzothiophene

S

CH3

7-methyl-

benzothiophene

Benzonaphthotiophenes

S

S S Benzo[b]naphtho[1,2-d]thiophene Benzo[b]naphtho[2,3-d]thiophene Benzo[b]naphtho[2,1-d]thiophene

DBE

S class (M+•)

DBE Distribution

S

R

DBE=12

S

R

DBE=9

DBE=6 S

R

DBE=3 S

R

Steranes Hopanes

Cholesterol

Petroleum Biomarkers

Eukaryotes Prokaryotes

APCI. Mechanism of Ionization (I)

Charge Transfer

• The nitrogen in the source is ionized by corona discharge by the

following series of reactions:

N2 + e ¯ → N2+• + 2e ¯

N2+• + 2N2 → N4

+• + N2

• If the nitrogen is dry the N2+• and N4

+• act as reagent ions with charge

transfer being the most likely pathway for ionization.

N2+•/ N4

+• + A → A+• + xN2 charge transfer

Where A represents an analyte molecule

• Charge transfer results in the formation of radical cations and is

particularly useful for the ionization of non-polars.

APCI. Mechanism of Ionization (II)

Proton Transfer

• In the presence of water vapour the following reactions then occur to

generate ionized water clusters:

N4+• + H2O → H2O

+• + 2 N2

H2O+• + H2O → H3O

+ + OH•

H3O+ + H2O + N2 → H+(H2O)2 + N2

H2O + H+(H2O)2 → H+(H2O)3 + N2

• The last reaction can then proceed further with successive additions

of water

• Ionization of the analyte then occurs by proton transfer

H3O+ + A → AH+ + H2O proton transfer

• Proton transfer is the main ionization pathway associated with APCI

in LC/MS.

30 40 50 60 70 80 90 100 110 120 130 140 150 160 170

%

0

100 152

128

178

APPI-GC/MS-MS of Phenanthrene (m/z 178)

m/z

M+•

[M-C2H2]+•

[C10H8]+•

C14H10 -C2H2

Collision energy (Ekin) = 27 eV

Collision gas - Ar

Eint = Ekin Mgas

Mion + Mgas

Product (daughter) scan from M+• (m/z 178)

MS/MS spectrum of Phenanthrene (m/z 178)

MS/MS spectrum

from m/z 178

NIST library EI mass spectrum

of Phenanthrene

The first match

Wavelength Transmission/Absorption Range

0.11 ‒ 7.5 µm

N2 < 0.1 O3 0.17-0.35 0.45-0.75

O2 < 0.245 H2O < 0.21 0.6-0.72

Gas Absorption wavelengths (μm)

MgF2 (window) wavelength transmission range

Sum of 7 MRM transitions

APPI(Ar)-GC/MS-MS of NIST2266

(hopanes & steranes standard)

20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00

%

0

100

time

IE(Alkanes) ~ 10 eV

αββ 20R-cholestane

ααα 20R-cholestane

17α (H)-22,29,30-

trisnorhopane

αββ 20R 24S-

methylcholestane

αββ 20R 24R-

ethylcholestane

17α(H),21β(H)-30-

hopane 17α(H),21β(H)-30-

norhopane

ααα 20R 24R-

ethylsholestane

17α(H),21β(H)-

22S-homophane

17α(H),21β(H)-22R-

homopane

20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00

%

0

100

time

Ar-lamp

hν =11.7 eV

S

17α(H),21β(H)-hopane

50 ng/mL

Peak area - 218

50 ng/mL

Peak area - 218 m/z 412 → m/z 191

dwell time – 250 ms

m/z 178 → m/z 152

dwell time – 250 ms

m/z 184 → m/z 152

dwell time – 250 ms

585 ng/mL

Peak area - 4900

APPI(Ar)-GC/MS-MS of Phenanthrene,

Dibenzothiophene, and 17α(H),21β(H)hopane

DB

E

Carbon number

0 70 80 90 60 50 40 30 20 10 100 110 120

80

60

40

20

100

120

0

C# = 112

DBE = 100

C# = 54

DBE = 46 C# = 24

DBE = 19

C20

DBE = C – H

2

N

2 + + 1

Double Bond Equivalent (DBE)

n-hexane, C6H14, DBE=0 Cyclohexane, C6H12, DBE=1 Benzene, C6H6, DBE=4

V.V. Lobodin, et al. Anal. Chem. 2012, 84, 3410-3416