High-performance liquid chromatographic and mass spectrometric analyses of porphyrins from deep-sea sediments

Chemical Geology, 35 (1982) 69--85

69

Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

H IG H - P ER F OR MAN C E LIQUID C H R O M A T O G R A P H I C AND MASS S P E C T R OMETR I C ANALYSES OF PO R PH Y RIN S FROM DEEP-SEA SEDIMENTS

J.M.E. Q U I R K E ',2, G. E G L I N T O N '

, S.E. P A L M E R

3,4 and E.W. B A K E R

3

' Organic Geochemistry Unit, University of Bristol, Bristol BS8 1TS (Great Britain) 2Department of Chemistry, University of Durham, Science Laboratories, Durham D H 1 3 L E (Great Britain) 3 College of Science, Florida Atlantic University, Boca Raton, F L 33431 (U.S.A.) 4 Cities Services Company, Energy Resources Group, Exploration and Production Research, Tulsa, O K 74102 (U.S.A.) (Received M a y 12, 1981;revised and accepted October 29, 1981)

ABSTRACT

Quirke, J.M.E., Eglinton, G., Palmer, S.E. and Baker, E.W., 1982. High-performance liquid chromatographic and mass spectrometric analyses of porphyrins from deep-sea sediments. Chem. Geol., 35: 69--85. The nickel, vanadyl and free base porphyrins from sediments of Legs 40 (Angola Basin), 41 (Cape Verde Rise) and 43 (Bermuda Rise) of the Deep Sea Drilling Project were analysed by high-performance liquid chromatography (HPLC). The results compare well with previous mass spectrometric studies. Nickel porphyrin mixtures contain more aetio porphyrins than do the vanadyl mixtures from the same core section. High-carbonnumber porphyrins (> C33) were detected in both nickel and vanadyl porphyrin fractions of a Leg-41 core section (No. 386-63-2) which bad been thermally altered by a volcanic intrusion, but were absent in all the other samples. Preparative HPLC and subsequent mass spectrometric analyses of the isolated fractions reveal a new series of aetio porphyrins which are more polar than any previously detected.

INTRODUCTION

In 1934, p o r p h y r i n s were first isolated f r om oils, shales and bitumens (Treibs, 1934). Subsequently, mass spectrometric (MS) and UV--visible spectrometric studies (e.g., Glebovskala and Vol'kenshtein, 1948; Baker et al., 1967; Baker and Palmer, 1978) showed the porphyri ns occur as two major skeletal classes: d e o x o p h y l l o e r y t h r o e t i o (DPEP) porphyrins [Appendix, (1)] and aetio p o r p h y r i n s [Appendix, ( 2 ) ] . T he porphyri ns are usually present as their nickel or vanadyl complexes; however, c o p p e r porphyri ns (Palmer and Baker, 1978), free base por phyr i ns (e.g., Palmer et al., 1979) and gallium porphyrins ( B o n n e t t and Czechowski, 1980) have also been detected. The advent o f high-performance liquid c h r o m a t o g r a p h y (HPLC) provided

70

a m e t h o d with sufficient resolution to separate mixtures into single-carbonnumber species on a micro scale (HajIbrahim et al., 1978, 1981; Brassell et al., 1980; Mackenzie et al., 1980). This m e t h o d extends MS and UV-~cisible data markedly as it provides information on the number and type of isomers present. Additionally, improved extraction procedures, using thin-layer chromatography, have resulted in the isolation of single-carbon-number porphyrins from the bitumen Gilsonite (Eocene, Uinta Basin, Utah, U.S.A.) in sufficient quantities (> 100 ug) for characterisation by 1H NMR (Quirke et al., 1979, 1980a; Quirke and Maxwell, 1980}. Therefore, it was possible to use the characterised porphyrins from Gilsonite as reference compounds in a program of HPLC coinjection studies of porphyrins from selected geologic sites. Porphyrin mixtures from organic-rich Cretaceous shales of the Angola Basin (Baker et al., 1978), the Cape Verde Rise (Baker et al., 1977) and the Bermuda Rise {Palmer et al., 1979) were chosen for analysis by HPLC (Figs. 1, 2 and 3, respectively). In addition, MS analyses of individual components isolated by preparative HPLC (see Fig. 4) were carried out to extend previous MS and UV--visible data for the total porphyrin mixture (Palmer et al., 1979}. We present the results of the HPLC analyses of the above porphyrin mixtures. We compare the differences in distribution of the nickel and vanadyl porphyrins from the same site as well as the differences in porphyrin distributions from samples with different geological histories from different locations. EXPERIMENTAL

Deep Sea Drilling Project (DSDP) samples The Cretaceous shales were collected from sites 364 (Angola Basin, DSDP Leg 40), 368 {Cape Verde Rise, DSDP Leg 41 ) and 386 (Bermuda Rise, DSDP Leg 43). Leg-40 samples were collected from a present (sub-bottom) depth of 1000--1040 m (Baker et al., 1978); the lithology is carbonate clay. Leg-41 samples were collected from 975 and 979 m, sub-bottom (Baker et al., 1977) The geological history of the area shows igneous activity in the Miocene with a diabase sill at 971 m. The intrusion resulted in a high degree of thermal alteration of sediments in corztact with the sill, and a steep thermal gradient (Baker et al., 1977}. The lithology is calcareous black shale (Baker et al., 1977}. Leg-43 samples were obtained from three organic-rich zones at ~ 740, 914 and 937 m, sub-bottom. The lithologies are blue-green shales at 740 m, calcareous dark-grey shale at 914 m and black shale at 937 m (Palmer et al:, 1979}.

71

Solvents All solvents were distilled before use.

Isolation, demetallation and purification of the porphyrins The isolation and MS analyses of the total metalloporphyrins, and free base porphyrins from DSDP sediments of Legs 40, 41 and 43 have been described previously [Baker et al. (1978), Baker et al. (1977) and Palmer et al. (1979), respectively]. Typically, the isolated metalloporphyrin fraction isolated from ~ 100-g sediment was dissolved in CH2C12 (1 ml), and placed in a reactor vial. The solvent was evaporated under nitrogen, and methane sulphonic acid (0.5 ml) was added to the residue. The capped vial was heated at 100°C, either for 25 min. for nickel complexes, or for 90 min. for vanadyl complexes. The sample was cooled, and extracted with hexane (5 ml) to remove neutral lipids. The acid solution was diluted with CH2C12 (5 ml), and extracted with saturated aqueous sodium acetate (5 ml). The layers were separated, and the aqueous phase washed with CH2C12 (2.5 ml). The combined organic extracts were evaporated, and pyridine (0.5 ml) added to neutralise any residual acetic acid, then the solution was evaporated again. The initial concentration of metalloporphyrins was determined by UV--visible spectrometry, monitoring the bands at 550 nm (e = 34,280) and at 570 nm (e = 26,140) for nickel and vanadyl porphyrins, respectively (Palmer et al., 1979). Yields of demetallated porphyrins were determined by measuring the intensity of the band at 618 nm in the visible spectrum (e = 6,500), and the average yield was 41% for both species. The demetallated porphyrins were purified further by heating under vacuum (~ 10 -6 Tort) at 170°C (30 min.) to remove volatile impurities. HPLC analyses of the porphyrins HPLC analyses were carried o u t on a stainless steel column (25 cm X 4.6mm i.d.) packed with 5-pm Partisil ®. The equipment comprised t w o Waters ® M6000 D pumps, a Waters ® M660 solvent programmer and a LDS ® spectrop h o t o m e t e r II fitted with 8 t~l flow cells and set at 400 nm. The solvents used were hexane--toluene (9 : 1, vol/vol) as solvent A and toluene-~hloroform (1 : 1, vol/vol) as solvent B, using a linear program (10 min.) from 25% B to 75% B with a flow rate o f 1.5 ml min.-' (Quirke et al., 1979). Typically, 2-pl aliquots of sample ( ~ 0 . 2 5 pg/pl) were injected via a 10-t~l syringe. At the end of each day, the column was washed with ethyl acetate to remove polar impurities. This treatment helps to maintain the efficiency of the column.

72

Preparative HPLC of nickel porphyrins from Leg-43 core section 386-60-5 The demetallated nickel porphyrins from core section 43-386-60-5 were separated into 13 fractions by preparative HPLC (see Fig. 4). The mixture (30 × 2/~g) was eluted as described above, and the fractions collected. The solvent was removed and the fractions analysed by MS.

Mass spectrometric analysis Mass spectra were obtained using a Finnegan ® 4000 spectrometer interfaced with a Finnegan ® INCOS 2300 data system. The scan cycle was 2.8 s and the mass ranges m/e 5 0 - 7 5 0 . Operating conditions were: source temperature, 250°C; electron energy, 70 eV; and emission current 400/~A. Samples ( ~ 2 pg) in CH2C12 were placed in a clean glass crucible. The porphyrins vaporised between 175 ° and 280°C. To avoid the effects of differential evaporation, the spectra for each mixture were averaged over the entire volatility range. It was necessary to perform background subtraction (using the scans immediately before and after the appearance of the porphyrins) as considerable amounts of non-porphyrinic material were present. RESULTS AND DISCUSSION

Porphyrins from DSDP Leg 40 Free base porphyrins from core section 364-41-3. The HPLC trace of the free base porphyrins show mainly DPEP porphyrins with C32 DPEP as the major c o m p o n e n t (Fig. 1; Table I); aetio porphyrins may be present in trace amounts (Table I). The previous MS analysis was substantiated by the HPLC data, although aetio porphyrins were n o t detected (Table II; Baker et al., 1978). Nickelporphyrins from core section 364-41-3. The HPLC trace of the demetallated nickel porphyrins is more complex than that of either the free base or the vanadyl complexes of this core section (Fig. 1, Table I). It contains several poorly-resolved peaks, and two unidentified components (Table I). There is quite good agreement between HPLC and MS data (Tables I and II, respectively). Tentative assignments of the lower-carbon-number aetio porphyrins (~< C28) were made by comparison with porphyrin fractions isolated from Leg 43, core section 386-60-5 (see Table III, see p. 79). It was n o t possible to assign the components precisely because they almost coincide with the higher-carbon-number aetio porphyrins (> C2s, Table I), producing badly-resolved peaks. C32 DPEP was the major component. Vanadyl porphyrins from core section 364-41-3. The HPLC trace and mass spectrum of the demetallated vanadyl porphyrins (Tables I and II, respectively)

73

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75 T A B L E II Literature mass s p e c t r o m e t r i c data for p o r p h y r i n m i x t u r e s f r o m D S D P Legs 40, 41 and 43

DSDP sample Leg

core s e c t i o n

40 40 40 40 41 41 41 43 43 43

364-41-3 364-41-3 364-41-3 364-43-1 368-63-2 368-63-2 368-63-4 386-43-3 386-60-5 386-63-1

Porphyrin species *~

FB Ni VO VO VO Ni Ni VO Ni Ni

C a r b o n No. range .2 aetio

DPEP

-C24--C33 --C~6--C3e C27-C33 C26--C33 -C2s-C33 C:,--C33

C=t--C32 C~e---C33 C~s--C32 C28--C32 C2s--C~ C2s--C34 C2s--C33 C29--C3~ C2s--C33 C2s C33

Major .2 component

R e f e r e n c e .3

C32 C3= C3= C3= C3= C31 C32 C31 C~o C,s

(a) (a) (a) (a) (b) (b) (b) (c) (c) (c)

DPEP DPEP DPEP DPEP DPEP DPEP DPEP DPEP aetio aetio

-- = n o t detected. *~ FB = free base p o r p h y r i n s ; Ni = nickel p o r p h y r i n s ; VO = vanadyl porphyrins. ,2 F r o m low-voltage single-scan mass spectra. ,3 R e f e r e n c e s : a = Baker et al. ( 1 9 7 8 ) ; b = Baker et al. (1977); c = Palmer et al. (1979).

show a less complex porphyrin distribution than do the demetallated nickel porphyrins. The aetio porphyrins are much less abundant, and C32 DPEP (Fig. 1, peak 11) is the major component. An unknown component (Fig. 1, peak 13a) was also abundant.

Vanadyl porphyrins from core section 364-43-1. The sample is slightly deeper than the previous core section (~10 m; Baker et al., 1978), and the HPLC trace of the demetallated porphyrins resembles that of the demetaUated vanadyl porphyrins described above (Fig. 1). C32 DPEP is the major component, and the unknown (Fig. 1, peak 13a) is also abundant (Table I). The HPLC and MS data are in good agreement (Table II). Inter-comparison o f porphyrin samples from Leg 40. In general, the HPLC and MS data for all four porphyrin samples from Leg 40 are in good agreement (Tables I and II, respectively), particularly if the distortions occurring from the use of single-scan mass spectra are taken into account (Eglinton et al., 1979). These distortions can be very marked, e.g. single-scan MS analysis indicated that the porphyrins of La Luna (Mara) shale (Cretaceous, western Venezuela) consist solely of DPEP porphyrins (Didyk et al., 1975) -- however, averaged mass spectra showed the aetio porphyrins to be abundant components (~ 50% of the mixture) (HajIbrahim et al., 1981). Clearly, the nickel porphyrin fraction contains proportionally more aetio porphyrins than do either the free base or the vanadyl fractions from the same core section (see Fig. 5). The two vanadyl porphyrin distributions from Leg 40 are similar, which is not surprising as the two core sections were close together. High-

76

carbon-number porphyrins (1> C34) were absent from all the four samples examined, as might be expected from immature samples. Porphyrins from DSDP Leg 41 Nickel porphyrins from core section 368-63-2. The HPLC trace of the demetallated porphyrins was poorly-resolved, and showed a pronounced hump (Fig. 2). Aetio porphyrins were more abundant than DPEP, and the major component was the fully alkylated C29 aetio porphyrin, peak 7 (Fig. 2; Table I). The HPLC data were in reasonable agreement with MS data obtained previously (Baker et al., 1977), which showed high-carbon-number (> C33) DPEP and aetio porphyrins {Table II). MS data (Baker et al., 1977) indicated DPEP n

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Fig. 2. HPLC traces of demetallated nickel (Ni) and vanadyl (VO) porphyrins from core section 368-63-2, and demetallated nickel porphyrins from core section 368-63-4, DSDP Leg 41. Peak numbers refer to Table I. See Section "Experimental" for HPLC conditions.

77 porphyrins were the more abundant species, however UV--visible analyses supported the HPLC. Vanadyl porphyrins from core section 368.63-2. The demetallated vanadyl porphyrin distribution was broadly similar to the nickel porphyrins, being very poorly-resolved (Fig. 2); however, DPEP porphyrins were the dominant species with C31 DPEP the major c o m p o n e n t (Table I). The MS data revealed high-carbon-number components, extending to C37, and C31 DPEP was the major c o m p o n e n t (Table II). Nickel porphyrins from core section 368-63-4. The HPLC trace of the demetallated porphyrins showed a less complex mixture than either of the two previous samples (Fig. 2). The DPEP porphyrins were predominant and C32 DPEP was the major c o m p o n e n t (Table I). The HPLC and MS data were in good agreement. Intercomparison o f porphyrin samples from Leg 41 Although both the nickel and vanadyl porphyrins from core section 368-63-2 were more complex than any of the Leg-40 samples, the trend that the aetio porphyrins occur in greater abundance in nickel samples than vanadyl samples from the same core section was continued (see Fig. 5). The large difference between the nickel porphyrins distributions in core sections 368-63-2 and 368-63-4 is attributed to the effect of the volcanic intrusion (see Fig. 5). The porphyrins from core section 368-63-2 were subjected to more severe thermal conditions, and it is n o t surprising that their HPLC traces closely resemble porphyrin distributions from crude oils (HajIbrahim et al., 1978; Eglinton et al., 1979, 1980). These observations, combined with the MS data for 368-63-2 samples, give weight to the hypothesis that the porphyrins from mature samples are produced, in part, by cracking from kerogen (Mackenzie et al., 1980; Quirke et al., 1980b). The cracking process should produce complex mixtures containing high-carbon-number porphyrins as is observed, and lead to an increase in porphyrin concentration. Thus the higher concentration of nickel (14.2 ppm) and vanadyl porphyrins (28.7 ppm) in core section 368-63-2 relative to the porphyrins of core section 368-63:4 (3.5 ppm., Ni; 0 ppm. VO) is explained (Baker et al., 1977). The broad similarity of the nickel porphyrins from core sections 368-63-4 and 364-41-3 may be attributed to similar maturational histories (Figs. 2 and 1, respectively; see Fig. 5). All three Leg-41 samples show a small C32 DPEP c o m p o n e n t (Fig. 2, peak 12), isomeric with deoxophylloerythroaetio porphyrin [Appendix, (la)], which is absent in Leg-40 samples (Table I). This c o m p o u n d is a major component of Gilsonite porphyrins (Quirke et al., 1979), and contains a six-membered isocyclic ring (J.M.E. Quirke and coworkers, unpublished data, 1982).

78

Porphyrins from DSDP Leg 43 Vanadyl porphyrins from core section 386-43-3. The HPLC data indicate that DPEP porphyrins predominate, with C32 DPEP the major c o m p o n e n t (Fig. 3, peak 11 ; Table I). The aetio porphyrins are minor, poorly-resolved components (Fig. 3) which were not detected by single-scan mass spectrometry (Table II; Palmer et al., 1979). Nickel porphyrins from core section 386-60-5. Averaged MS analyses of both the total nickel and total demetaUated porphyrins indicated that low-carbon-

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Fig. 3. H P L C traces of demetallated nickel (Ni), nickel (Ni) and vanady| (VO) porphyrins from core sections 386-63-1,386-60-5 and 386-43-3, respectively, D S D P Leg 43. Peak numbers refer to Table I. See Section "Experimental" for H P L C conditions.

79 T A B L E III MS analysis o f t h e p o r p h y r i n f r a c t i o n s o f c o r e s e c t i o n 386-60-5 isolated b y p r e p a r a t i v e HPLC F r a c t i o n .1

33 T o t a l Ni T o t a l F B .3 Ai Bi C D E F G Hi J K L Mi

A e t i o c a r b o n No. a b u n d a n c e (%),2

D P E P c a r b o n No. a b u n d a n c e (%)*2

tr. tr.

32 52 22

31 61 23

30 68 34

10

29

32

43 23

tr. 11

20

tr. 17

31 72 57

tr. 15

26 11 5

100

100 10

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30 96 95

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100 100

46 53

65

10 100 47 8 10

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100 15 tr.

40 100

30

85

26

25

27 32

5 16

35 15 28

100 38

25

100 40

Ni = nickel p o r p h y r i n s ; F B = d e m e t a l l a t e d p o r p h y r i n s ; tr. ( t r a c e ) = < 5%; s u p e r i o r i ffi ins u f f i c i e n t sample. • i See S e c t i o n " E x p e r i m e n t a l " f o r H P L C a n d MS c o n d i t i o n s , a n d Fig. 4. • 2 A b u n d a n c e s are b a s e d o n t h e m o s t i n t e n s e p o r p h y r i n m o l e c u l a r i o n = 100, a n d are corr e c t e d f o r i s o t o p i c c o n t r i b u t i o n s ( ' ° N i a n d ~3C) as a p p r o p r i a t e . • ~ T h e s p e c t r u m s h o w e d traces o f Cu p o r p h y r i n in similar relative a b u n d a n c e s t o t h e free base m i x t u r e .

number aetio porphyrins were relatively abundant (Table III). The sample was selected for fractionation by preparative HPLC (Fig. 4), and subsequent MS studies to attempt a more precise assignment of the HPLC peaks due to low-carbon-number (< C2s) aetio porphyrin. The studies revealed these species almost coincide with higher-carbon-number aetio porphyrins, yielding unresolved or badly-resolved peaks, e.g. fraction C, (Fig. 4, peak 1 ; Table III) contains b o b a C~s aetio porphyrin (probably with two unsubstituted/3-positions) and a C26 aetio porphyrin. The combined HPLC and MS data (Table III) also give evidence of a new class o f aetio porphyrins more polar than those with fully alkylated or monoor di-unsubstituted/3-positions detected in Gilsonite (Quirke et al., 1979). The mass spectrum of the C31 aetio c o m p o n e n t (peak K) provides little insight into the precise structure. The large ion at m/e 449 (Fig. 6) indicating the presence of at least one ethyl group (Jackson et al., 1965; Quirke et al., 1979). The corresponding C30 aetio porphyrin coelutes with a C32 DPEP porphyrin (Table III, fraction L). Further studies are required to determine the structure of these species. Each of the porphyrins in the isolated fractions

80

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Fig. 4. Preparative HPLC of demetallated nickel (Ni) porphyrins from core section DSDP Leg 43. Peak numbers refer to Table I, peak letters indicate the fractions collected (Table III). See Section "Experimental" for HPLC conditions. 386-60-5,

LEG 40

\

/ 1 FB 100-- ,

1 Ni

1 VO

2 VO

LEG 41

L E G 43 A

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Ni

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Fig. 5. Percentages of aetio (•) and DPEP (=) porphyrins from demetallated nickel (Ni) and vanadyl (VO) complexes, and free base porphyrins (FB) of DSDP core sections: 1 = 364-41-3; 2 = 364-43-1; 3 = 368-63-2; 4 = 368-63-4; 5 = 386-63-1; 6 = 386-60-5; and 7 = 386-43-3.

showed (M--15) ÷ as the major fragment ion, indicating all the low-carbonnumber species (~< C28) contain an ethyl group (Quirke et al., 1979}. f r o m c o r e s e c t i o n 3 8 6 - 6 3 - 1 . The HPLC trace of the demetallated porphyrins resembles t h a t of core section 3 8 6 - 6 0 - 5 (Fig. 3), with the aetio porphyrins predominant (Fig. 5) and C:8 aetio as major c o m p o n e n t (Table I}; however, the isomeric C n DPEP (Fig. 3, peak 1 2 ) was a b u n d a n t (Table I). The MS data support the HPLC data (Table II).

Nickel porphyrins

81 464 lC31 Aetio)

100-

313

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368

/. 49

Conta minet i o n

I

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Fig. 6. Averaged and background-subtracted partial mass spectrum of fraction K from demetallated porphyrins of core section 386-60-5. For HPLC data see Fig. 4. The ions above m/e 500 are artefacts, and observed in all the other preparative HPLC fractions also. See Sec tion "Experimental" for MS conditions.

Intercomparison o f porphyrin samples from Leg 43 There was a slight decrease in the ratio of DPEP porphyrins to aetio porphyrins in core section 386-63-1 compared with 386-60-5 (Fig. 5); this is to be expected as 386-63-1 is the deeper of the two core sections (Palmer et al., 1979). In both samples, the aetio porphyrins were relatively more a b u n d a n t than in the vanadyl porphyrins of core section 386-43-3, an observation which provides support for the hypothesis that aetio porphyrins are more prevalent in the nickel than in the vanadyl fractions. High-carbon-number (i> C34) porphyrins were absent in both the nickel fractions, but the high ratio of aetio porphyrins to DPEP porphyrins indicates the samples have been subjected to mild thermal stress. Core section 386-43-3 is an unusual sample because it contains n o t only vanadyl porphyrins, but also nickel and copper porphyrins, which could n o t be separated readily, and chlorins. Vanadyl porphyrins usually occur in more mature sediments than those containing chlorins (Baker and Palmer, 1978). High-carbon-number porphyrins were absent from the vanadyl fraction, and the HPLC trace resembled those of Leg-40 samples (Fig. 1), although the aetio porphyrins were relatively more a b u n d a n t in the Leg-43 sample.

82

Intercomparison o f porphyrin samples from Legs 40, 41 and 43 Although samples were obtained from quite different parts of the Atlantic Ocean, the porphyrin distributions from all three Legs showed similar trends. Nickel porphyrins appear to have a relatively lower DPEP content than do the corresponding vanadyl porphyrins (Fig. 5). The carbon number ranges of nickel porphyrin fractions subjected to similar degrees of thermal stress are in good agreement, e.g., samples 364-41-3 and 368-63-4 (Figs. 1 and 2, respectively; Table II). Vanadyl porphyrin distributions showed analogous trends, although the differences were less pronounced (Table II). These data indicate that the differences in porphyrin distributions of the same species may be attributed largely to variations in the degree of thermal stress, although there are some species [e.g., the C32 DPEP isomer containing a sixmembered isocyclic ring, Appendix, (3)] which may prove to be indicators of input or environment. The decrease in DPEP concentration relative to aetio porphyrins with increasingmaturity (Fig. 5) is in agreement with previous studies (e.g., Baker and Palmer, 1978; Mackenzie et al., 1980). The results confirm the need to separate vanadyl and nickel porphyrins for the inter-comparison of samples. The aetio porphyrins from all three Legs show high abundances of lowcarbon-number (~< C2s) aetio porphyrins, which must contain one or more unsubstituted/3-positions (Baker et al., 1977, 1978; Palmer and Baker, 1978; Palmer et al., 1979). MS analyses of the individual porphyrin fractions from core section 386-60-5 indicated that even the C2s aetio porphyrin(s) contain at least one intact ethyl group, which provides circumstantial evidence for the preservation of the alkyl groups of chlorophyll a. Similar analyses of the porphyrins from Gilsonite had produced analogous results (Quirke et al., 1979). It seems likely that the unsubstituted ~-position(s) are formed via the cleavage of the isocyclic ring, and/or the loss of the vinyl group out of the precursor chlorophylls. Analyses of chlorins in DSDP samples from the Japan trench provide some support for this hypothesis as chlorins in which the isocyclic ring has been cleaved oxidatively have been detected (Baker and Louda, 1980). The porphyrins from the Atlantic sediments show abundant low-carbon-number (~< C2s) aetio porphyrins, however, the fully alkylated aetio porphyrins are not of such significance.In porphyrin mixtures from petroleums and Gilsonite, the distributionsare the reverse of the D S D P samples described above (Quirke et al.,1979; Eglinton et al.,1980; Hajlbrahim et al.,1981). These data are confirmed by GC--MS analyses of the oxidative degradation products of the porphyrins, the maleimides, which show very low abundances of maleimides with unsubstituted ~-positions (e.g.,Hodgson et al.,1972; Didyk et al.,1975; Quirke et al.,1980). It is not clear w h y the distributions of aetio porphyrins should vary so markedly, Perhaps alkylation occurs at the unsubstituted ~-positions in the more mature samples; clearly, this problem is worthy of further investigation.There are insufficientdata on the D P E P porphyrins to draw conclusions as to the precise nature of the individual compounds.

83 A n assessment o f HPLC and MS

The combined use of HPLC "fingerprinting" and MS determination is more powerful than application of either technique alone, leading to a better appreciation of the complexity of porphyrin distributions, including the presence of structural isomers. Clearly, for correlation purposes, it is essential to examine the nickel and vanadyl porphyrin mixtures separately as the data show that the compositions of the two metal complexes isolated from the same sediment are quite different. The HPLC requires further development, in particular, an improvement in the resolution of the aetio porphyrins is required. This would provide better quantitative data on the low-carbonnumber (~< C28) aetio compounds which are prevalent in many immature sediments (Baker and Palmer, 1978). The HPLC--MS analysis of core section 386-60-5 is an initial step, in particular, it confirms the broad similarity between the Gilsonite porphyrins and the DSDP porphyrins, and it indicates that the peak assignments are valid. The use of non-aqueous reverse-phase (NARP) HPLC columns may prove a valuable modification as preliminary studies have shown enhanced resolution of petroporphyrin mixtures (HajIbrahim et al., 1981). CONCLUSIONS

These HPLC analyses of DSDP porphyrin samples have complemented previous MS studies by showing the presence of porphyrin isomers, including a new series of relatively polar aetio porphyrins (core section 386-60-5). In addition, these studies clearly demonstrated the value of averaging mass spectra of porphyrin mixtures to prevent distortions. HPLC proved particularly valuable for the comparison of nickel and vanadyl porphyrin distributions from the same core section, and illustrated the greater abundance of aetio porphyrins in the nickel complexes. The differences in distribution for the porphyrins of the three DSDP legs may largely be attributed to differences in maturation. Loss of functional groups from chlorophyll to produce porphyrins with unsubstituted ~-positions is a major degradative pathway in sediments. Acknowledgements

The authors thank the Natural Environment Research Council for providing mass spectrometric facilities (GR3/2951) and HPLC facilities (GR3/ 2420), and the Nuffield Foundation for a grant in support of data processing facilities. We (G.E. and J.M.E.Q.) wish to thank the National Aeronautics and Space Administration (sub-contract from NGL 05-003-003 University of California at Berkeley) for financial support. One of us (S.E.P.) is grateful to the Natural Environment Research Council for a Senior Visiting Fellowship. We are grateful to Morris Ashby, Ltd. and the American Gilsonite Co. for

84 the gifts o f Gilsonite, We are m o s t grateful t o Mrs. A.P. G o w a r B.Sc. and Mr. G.J. S h a w B.Sc. f o r assistance in the d e t e r m i n a t i o n o f mass spectra.

APPENDIX Structural formulae of alkylporphyrins, types 1--3 .2 R2

R3

R2

R3

R3 ~NH

R

T

~

R

s

Rs~,

~

R5

7 R

6 R (2}

Rv

R

T

~

R

5

R6 (3)

[1} Types 1,2 and 3 --R I , R 2 ,... , R s -- H or alkyl for formulae (I), (2) and (3). T y p e l a - - R ' , R 3,R s , R 7 = C H ~ ; R 2,R 4 , R ~ --C2H s. REFERENCES Baker, E.W. and Louda, J.W., 1980. Products of chlorophyll diagenesis in Japan trench sediments, Deep Sea Drilling Project sites 438, 439 and 440. In: E. Honza et al. (Editors), InitialReports of the Deep Sea Drilling Project, LV1, LV11. U.S. Government Printing Office, Washington, D.C., pp. 1397--1408. Baker, E.W. and Palmer, S.E., 1978. Geochemistry of porphyrins. In: D. Dolphin (Editor), The Porphyrins, Vol. 1. Academic Press, N e w York, N.Y., pp. 485--551. Baker, E.W., Yen, T.F., Dickie, J.P., Rhodes, R.E. and Clark, L.F., 1967. Mass spectrometry of porphyrins, II. Characterization of petroporphyrins. J. Am. Chem. Soc., 89: 3631-3639. Baker, E.W., Palmer, S.E. and Huang, W.Y., 1977. Intermediate and late diagenetic tetrapyrrole pigments, Leg 41 : Cape Verde Rise and Basin. In: Y. Lancelot, E. Seibold et al. (Editors), Initial Reports of the Deep Sea Drilling Project, XLI.. U.S. Government Printing Office, Washington, D.C., pp. 825--837. Baker, E.W., Palmer, S.E. and Huang, W.Y., 1978. Chlorin and porphyrin geochemistry of DSDP Leg 4 0 sediments. In: H.M. Bolli, W.B.F. Ryan, et al. (Editors), Initial Reports of the Deep Sea Drilling Project, XL. U.S. Government Printing Office, Washington, D.C., pp. 639--647. Bonnett, R. and Czeehowski, F., 1980. Gallium porphyrins in bituminous coal. Nature (London), 283: 465--467. Brassell, S.C., Comet, P.A., Eglinton, G., McEvoy, J., Maxwell, J.R., Quirke, J.M.E. and Volkman, J.K., 1980. Preliminary lipid analyses of cores 14, 18 and 28 from Hole 4 1 6 A . In: Y. Lancelot and E.L. Winterer (Editors), Initial Reports of the Deep Sea Drilling Project, L. U.S. Government Printing Office, Washington, D.C., pp. 647--664. Didyk, B.M., Alturki, Y.I.A., Pillinger, C.T. and Eglinton, G., 1975. Petroporphyrins as indicators of geothermal maturation. Nature (London), 256: 563--565. Eglinton, G., HajIbrahim, S.K., Maxwell, J,R., Quirke, J.M.E., Shaw, G.J., Volkman, J.K. and Wardroper, A.M.K., 1979. Lipids of aquatic sediments. Recent and ancient. Philos. Trans. R. Soc. London, Set. A, 2933: 69--91.

85 Eglinton, G., HajIbrahim, S.K., Maxwell, J.R. and Quirke, J.M.E., 1980. Petroporphyrins: Structural elucidation and the application of HPLC fingerprints to geochemical problems. In: A.G. Douglas and J.R. Maxwell (Editors), Advances in Organic Geochemistry 1979. Pergamon, Oxford, pp. 193--203. Glebovskaia, E.A. and Vol'kenshtein, M.V., 1948. Spectra of porphyrins in petroleums and bitumens. Zh. Obshch. Khim., 18: 1440--1452. HajIbrahim, S.K., Tibbetts, P.J.C., Watts, C.D., Maxwell, J.R., Eglinton, G., Colin, H. and Guiochon, G., 1978. Analysis of carotenoid and porphyrin pigments of geochemical interest by high-performance liquid chromatography. Anal. Chem., 50: 549--553. HajIbrahim, S.K., Quirke, J.M.E. and Eglinton, G. Petroporphyrins, V. Structurally-related porphyrin series in bitumens, shales and petroleums. Evidence from HPLC and mass spectrometry. Chem. Geol., 32: 173--188. Hodgson, G.W., Strosher, M. and Casagrande, D.T., 1972. Geochemistry of porphyrins -Analytical oxidation to maleimides. In: H.R. yon Gaertner (Editor), Advances in Organic Geochemistry 1971. Pergamon, Oxford, pp. 151--161. Jackson, A.H., Kenner, G.W., Smith, K.M. Aplin, R.T., Budzikiewicz, H. and Djerassi, C., 1965. Pyrroles and related compounds, VIII. Mass spectrometry in structural and stereochemical problems; LXXVI. The mass spectra of porphyrins. Tetrahedron, 21: 2913--2924. Mackenzie, A.S., Quirke, J.M.E. and Maxwell, J.R., 1980. Molecular parameters of maturation in the Toarcian shales, Paris Basin, France, II. Evolution of metalloporphyrins. In: A.G. Douglas and J.R. Maxwell (Editors), Advances in Organic Geochemistry 1979. Pergamon, Oxford, pp. 239--248. Palmer, S.E. and Baker, E.W., 1978. Copper porphyrins in deep-sea sediments: a possible indicator of oxidised terrestrial organic matter. Science, 201: 49--51. Palmer, S.E., Huang, W.Y. and Baker, E.W., 1979. Tetrapyrrole pigments from Bermuda Rise: DSDP Leg 43. In: B.E. Tucholke and P.R. Vogt, et al. (Editors), Initial Reports of the Deep Sea Drilling Project, XLIII. U.S. Government Printing Office, Washington, D.C., pp. 657--661. Quirke, J.M.E. and Maxwell, J.R., 1980. Petroporphyrins, III. Characterisation of a C n aetio porphyrin from Gilsonite as the his [porphyrinato-mercury(II) acetato] mercury(II) complex Origin and significance. Tetrahedron, 36: 3453--3456. Quirke, J.M.E., Eglinton, G. and Maxwell, J.R., 1979. Petroporphyrins, I. Preliminary characterisation of the porphyrins of Gilsonite. J.Am. Chem. Soc., 101: 7693--7697. Quirke, J.M.E., Eglinton, G., Maxwell, J.R. and Sanders, J.K.M., 1980a. Petroporphyrins, IV. Nuclear Overhauser enhancement 'H NMR studies of deoxophylloerythroetio porphyrins from Gilsonite. Tetrahedron Lett., 21: 2987--2990. Quirke, J.M.E. Shaw, G.J., Soper, P.D. and Maxwell, J.R., 1980b. Petroporphyrins, II. The presence of porphyrins with extended alkyl side chains. Tetrahedron, 36: 3261-3267. Treibs, A., 1934. Chlorophyll und H~rninderivative in bituminSsen Gesteinen, ErdSlen, Erdwachsen und Asphalten. Ann. Chem., 510: 42--62. -

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