MS technique to the structure study of petroporphyrins

Journal of Southeast Asian Earth Sciences, Vol. 5, Nos 14, pp. 11-14, 1991

0743-9547/91 $3.00 + 0.00 Pergamon Press plc

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Application of MS/MS technique to the structure study of petroporphyrins X U LINA a n d WANG PEIRONG Jianghan Petroleum Institute, Hubei, People's Republic of China Ahstract--Petroporphyrins were purified via thin-layer chromatography (TLC) and were de-metallized with p-toluenesulfonic acid after being isolated from crude oil and source rocks of the Liaohe Basin by dry column chromatography. Analysis of de-metallized petroporphyrins by electron ionization mass spectrometry (ELMS) identified the porphyrin classes present and their carbon number ranges, but the spectra were extremely complex. Many fragmental ions were observed for each porphyrin in addition to the molecular ion, and little data on the structure of the individual components were found. To correlate the fragmental ions with their molecular ions and gain more information about the structure and fragmentation of petroporphyrins, MS/MS can be employed. Tandem mass spectrometry (MS/MS) allowed selection of the molecular ions of individual carbon number porphyrins of the DPEP and ETIO types for fragmentation by collision activated dissociation (CAD). Comparison of their daughter spectra with those of porphyrin standards provided the first structural information on individual petroporphyrins. According to the information, the author suggests a method for calculating individual carbon number porphyrin isomers on the basis of quantitative analysis for the nature of petroporphyrins' pyrole-alkyl substituents. This method is useful for the study of the detailed structure of petroporphyrins.

northeastern China at a depth of 1975 m. It is a light green-gray mudstone.

INTRODUCTION PORPHYRINSand their homologs constitute near ubiquitious components of life forms at the cellular level and above. Therefore the study of the structure of porphyrins has important scientific significance for inquiring into the origins of petroleum and life. The search for life outside Earth would also be confirmed via examination of extraterrestrial material for such components. Separation has been necessitated by the fact that the petroporphyrin is a complex mixture composed of several alkyl-porphyrin series with a wide carbon number range. MS/MS is the newly developed mass spectrometric technique of the eighties. It is well suited for analysis of selected compounds in a complex mixture. It has been applied to the analysis of mixtures from a variety of sources, including those from the biological, biomedical, pharmaceutical, industrial, agricultural, environmental, and petrochemical fields (McLafferty 1983). Johnson et al. (1986) reported on the study of geoporphyrins by tandem mass spectrometry. By eliminating the preseparation of high-performance liquid chromatography (HPLC) or gas chromatography (GC), MS/MS simplifies sample preparation and makes more rapid analysis possible. The authors analyse the composition of porphyrins in the source rock from the Du-65 well in the Liaohe Basin in China, for the purpose of clarifying the application of the MS/MS technique in the field of organic geochemistry. Data were obtained on Finnigan MAT triple-stage quadrupole (TSQ-45 model) GC/MS/MS/DS system.

Separation of petroporphyrins The separation scheme of petroporphyrins is shown in Fig. 1. The source rock sample (200 mesh) was extracted in a soxhlet apparatus with benzene and alcohol (9:1 in volume). The extracts were applied to a dry silica gel column (packed with silica G, 80--120mesh, Ocean Chemical Plant of Qingdao) and fractions were obtained by eluting with a mixture of petroleum ether (30-60°C) and toluene (3:7 in volume). The fraction containing metalated porphyrins was further purified with petroleum ether (60--90°C) and chloroform (2: 3 in volume) via thin-layer chromatography (using silica gel GF25a, 10-40 m, Ocean Chemical Plant of Qingdao). A metalporphyrins complex was obtained. A solution of ptoluenesulfonic acid in chloroform was mixed with the metal-porphyrins complex (200-300 mg g-~, MPC) and the mixture then refluxed at 90-95°C for an hour. The de-metallized porphyrin fraction was obtained (Xu Lina 1986).

Mass spectrometry EIMS and MS/MS data were acquired on a triplestage quadrupole mass spectrometer (TSQ-45 with INCOS data system, Finnigan MAT, San Jose, CA) with ion source conditions of 70 eV electron energy, 300 A emission current and 180°C source temperature. The TSQ was primarily operated in the daughter spectrum mode (Fig. 2): a parent ion is mass selected in the first quadrupole (Q1), fragmented by collision activated dissociation (CAD) in the second quadrupole (Q2, rf only), and the resulting daughter ions are mass analysed in the third quadrupole (Q3). Conventional mass spectra were obtained in the Q1 MS mode (Q2 and Q3, no collision

EXPERIMENTAL

Sampling The source rock sample was taken from the Du-65 well in the Liaohe Basin of the Paleogene Formation in ll

12

Xu LINA and WANG PEIRONG SOURCE ROCK

R

R

532

Soxhlet

SOLUBLE EXTRACTS TLC ~

R (DPEP) ...... D P E P - - ETIO

and/orDCC

METALATED PORPHYRINS

518

06/

Demetallation R

DEMETALIZED PORPHYRINS

R

/

Fig. 1. Separation scheme for porphyrins from the source rock.

.

/ R

(a)

R

i1'

(ETIO)

M~2

! 546 3924064.2!).43.4!48. ")11

mixt.

Mol. ion

MS I

CAD

III

IV

MS--I] 100

(b)

1 Patenti o n ~

1I ~

_____~~_

:ill

t5(1

'tN560

500

55o M/Z

Fig. 3. EIMS of the de-metallized porphyrins.

neut.frag. ~ ( + ~ _ __ daughterion

neut.tool. Fig. 2. (a) Principle of analysis for mixture using CAD MS/MS; (b) scheme of collisionally activated dissociation (CAD).

gas, passing all ions, Q1 scanning). MS/MS daughter spectra were obtained at a collision energy of 24.8 eV with argon (1.6 mtorr) as the collision gas. RESULTS AND DISCUSSION Electron ionization mass spectra data of the demetallized porphyrins are shown in Fig. 3 and Table 1. ElMS reveals that the de-metallized porphyrins in source rock from the Du-65 well include DPEP (58.8%) and ETIO (41.2%). The ElMS can only provide the information on porphyrin skeleton types present, and their carbon number ranges, but little data on the structure of the individual components. To correlate the fragment ions with their molecular ions and gain more information about the structure and fragmentation of porphyrin pyrole-alkyl substituents, MS/MS meets the requirement well.

MS~MS of porphyrin standards Four ETIO-porphyrin standards with extended alkyl substituents were subjected to El MS/MS analysis

(Johnson et al. 1986): (a) tetramethyltetra-n-pentylporphyrin (R1, R3, R5, R7 = CH3; R2, R4, R6, R8 = CsHu); (b) the analogous tetra-n-heptylporphyrin; (c) analogous triheptyl-monopentyl-porphyrin; and (d) analogous diheptyldipentylporphyrin (Fig. 4). These daughter spectra of standards indicate: (1) porphyrins with extended alkyl substituents (>C1) will produce MS/MS daughter spectra characterized by the intense daughter ions arising from fl-cleavage of those substituents. Loss of methyl groups in the structure was not observed. Earlier work with porphyrins bearing smaller substituents demonstrated similar fl-cleavage (Sundaraman et al. 1984); (2) alkyl groups with a similar carbon number have approximately equal probability of fl-cleavages. The relative error between the ratio of daughter ion intensity and the ratio of alkyl substituents in a compound is less than 2% [Fig. 4(c) and (d)]. Therefore MS/MS can be used to determine the type and content of n-alkyl substituents of different lengths in a compound.

M S / M S of C34-C36DPEPs in source rock from Du-65 well The C34-C36DPEPs in source rock from the Du-65 well were E1 MS/MS analysis: (a) C34 DPEP (m/z 504); (b) C35 DPEP (m/z 518); (c) C36 DPEP (m/z 532) (Fig. 5). The daughter spectra of the C34-C36DPEPs reveal that the M ÷ ions undergo benzylic cleavage to produce (M--CH3) +, (M--2CH3) + and (M--CH3, --C2H5) ÷ ions. In addition, the C35 and C36 DPEP daughter spectra give (M--3CH3) + and (M--2CH3,

Table 1. Distribution of types for de-metallized porphyrins n*-carbon number of alkyls substituted on porphyrin ring Types

Form. of nucleus

Formula

Dist. of carb. num.

n* range

Max M

Peaks and n*

Relat. abund.

ETIO DPEP

C20N4H14 C22N4H16

310 + 14n 336+ 14n

C29~3s C2:-C39

9-18 3-17

506 532

14 14

41.2 58.8

Application of MS/MS technique

13

M ¸

189 ( --CH, )

646 589

a

- CH~ - C2H~ ( - 2 CH,)

- C, Ho

50:1 ( M' )

m./' z

M' 758

b

-C~, H,

r.

,l 74 ( 3 C H , ) ( - 2CH'

it

,..-.I

(-CH~) 5O3 5 1 8 ( M )

CH~ C~H,

,< Z

M*

73O

7)

C 645 ,]

1

-C,~ H,, ,

673,~

- C , Ho

c CH,

M 702

d6i7

')CH, 173

-C~ Ht~

645

i-

CH,) 532(M-)

I 2CH,) 5102

ITI Z

- C, H9

Fig. 5. E1 MS/MS daughter spectra of the molecular ions, M ÷, of the C34-~C36DPEPs: (a) C34 DPEP (m/z 504); (b) C35 DPEP (m/z 518); (c) C36 DPEP (m/z 532) (argon).

nq,,z

Fig. 4. EI MS/MS daughter spectra of molecular ions, M ÷, of etioporphyrin standards: (a) tetramethyltetrapentylporphyrin; (b) tetramethyltetraheptylporphyrin; (c) analogous triheptylmonopentylporphyrin; (d) analogous diheptyldipentylporphyrin (nitrogen) (2).

--C2 Hs) ÷ ions respectively. Loss of over three C groups was not observed. These daughter spectra reveal: (1) the C34--C36DPEPs contain ethyl and propyl, besides methyl (the substitute compound has one more carbon atom than the lost fragments because of fl-cleavage; (2) the ratio of ethyl/propyl can be estimated by the intensities of ions in the daughter spectra (see Table 2 and Fig. 5). The result indicates that the ratio of ethyl/propyl is 9.23 for C36DPEP, 8.11 for C35 DPEP and 9.05 for C34 DPEP. The author suggests a method for calculating individual carbon number porphyrin isomers on the basis of quantitative analysis for the nature of petroporphyrin pyrole-alkyl substituents. A method for petroporphyrin

517t

calculating

isomer

composition

of

Since the MS/MS analysis of the standards demonstrated an exclusive cleavage, it can be assumed that the losing frequency of a given alkyl group may be from various isomers of porphyrin, and actually concords

with the relative content of the group in those porphyrins. Therefore we can calculate the composition of petroporphyrin structure isomers. Calculation includes two steps. Firstly, according to the carbon number of alkyl substituents, the number of substituted places and alkyl types of all possible isomers can be listed. Secondly, the reasonable combination of isomers can be defined by all kinds of possible alkyl arrangement. For example, out of the C36 DPEP nucleus, there are fourteen carbon atoms (n = 36-22 = 14), seven substitution positions (cf. Fig. 3 and Table 1), and three kinds of alkyl groups (isomers A, B, C, D; see Table 3). When A: B: C: D = 6:0:1 : 1 (molecular rate), the ratio of ethyl/propyl in isomers is equal to 9.20. The relative error between this ratio (9.20) and the estimated ratio (9.23, cf. Table 2) is 0.3%. The result indicates that C36 DPEP contains heptaethylporphyrins (75%), triethyldimethyldipropylporphyrins (12.5%) and monoethyltrimethyltripropylporphyrins (12.5%). By analogy, C35 DPEP contains hexethylmonomethylporphyrins (63.6%), tetraethyldimethylmonopropylporphyrins (18.2%), diethyltrimethyldipropylporphyrins (13.6%) and tetramethyltripropylporphyrins (4.6%); the C34 DPEP contains pentethyldimethylporphyrins (62.5%) triethyltrimethylmonopropylporphyrins (25%) and monoethyltetra-

Table 2. Estimate of the ratios of ethyl/propyl in the CM-C36 DPEPs Relat. abundance %

Mol. ions

m/z

532

--CH3

--2CH 3 --3CH 3

100

6

0

95

27

13

100

45

0

C36

rn/z 518 C35

m/z

504 C34

---'CH~

--2CH 3

---C2 H 5

----C2H 5

5~---CH 3

Z - C:H5

24 (12+12) 38 (19 + 19) 36 (18 + 18)

3 (2+1) 0

120 (I00 + 6 + 12 + 2) 154 ( 9 5 + 2 7 + 13+ 19) 163 ( 1 0 0 + 4 5 + 18)

13 (12 + 1) 19

0

18

~~:-~C 1

C2

E--C2

C3

120/13 9.23 154/19 8.11 163/18 9.05

Xu LINA and WANG l~mONG

14

Table 3. Calculation of isomer composition of the C34~236 DPEPs (substituted places: 7) Isomers Mol. ions

m/z 532 C36 n 14

m/z 518 C35 n 13

m/z 504 C34 n 12

Project

(C2H5)

(C3H7)

(CH3)

Mol. rate

Ethyl

Propyl

C2/C3

Mol. %

A B C D A B C D A B C

7 5 3 1 6 4 2 0 5 3 1

0 1 2 3 0 l 2 3 0 1 2

0 1 2 3 1 2 3 4 2 3 4

6 0 I 1 14 4 3 1 5 2 1

42 0 3 1 84 16 6 0 25 6 1

0 0 2 3 0 4 6 3 0 2 2

46/5 = 9.20 estimated (9.23) error 0.3% 106/13 = 8.15 estimated (8.11) error 0.5% 32/4 = 9.00 (9.05) error 0.5%

75 0 12.5 12.5 63.6 18.2 13.6 4.6 62.5 25 12.5

methyldipropylporphyrins (12.5%). Distribution of all alkyl groups must obey the rule of minimum space effects. CONCLUSION

According to the structural information of porphyrin pyrole-alkyl substituents provided by EI MS/MS daughter spectra, the composition of porphyrin isomers of a definite carbon number can be calculated.

REFERENCES Johnson, J. V., Britton, E. D., Yost, R. A., Qurike, J. M. E. and Cuesta, L. L. 1986. Tandem mass characterization for geoporphyrins of higher carbon number. Anal. Chem. $8, 1325 1329. McLafferty, F. W. (Ed.) 1983. Tandem Mass Spectrometry. Wiley, New York. Sundaraman, P., Gallegos, E. J., Baker, E. W., Stayback J. R. B. and Johnston, M. R. 1984. Hydrogen chemical ionization tandem mass spectrometry of metalloporphyrins, Anal. Chem. 56, 2552-2556. Xu Lina. 1986. The study of separation method for petroporphyrins. J. Jianghan Petrol. Inst. 8, 117--126.