Techniques for Isolation and Characterization of the Geoporphyrins

Chapter 20

Techniques for Isolation and Characterization of the Geoporphyrins and Chlorins J. M. E. Quirke

Downloaded by UNIV OF ARIZONA on January 10, 2013 | http://pubs.acs.org Publication Date: July 6, 1987 | doi: 10.1021/bk-1987-0344.ch020

Department of Chemistry, Florida International University, Tamiami Trail, Miami, FL 33199

The methodologies for the i s o l a t i o n of porphyrins, metalloporphyrins and chlorins from sediments, o i l s , oil shales, bitumens, coals and kerogens are reviewed. The techniques for the analysis and characterization of intact geoporphyrin mixtures are described. The methods for the i s o l a t i o n , and elucidation of the i n d i v i d u a l geoporphyrins are discussed.

The task of reviewing the methods of i s o l a t i o n of the geoporphyrins i s a p a r t i c u l a r l y onerous one, i n view of the many d i f f e r e n t i s o l a t i o n and p u r i f i c a t i o n schemes which have been employed, and the differences i n the aims of the research. Emphasis has been placed on o u t l i n i n g methods which have general application to the analysis of geoporphyrin mixtures. Clearly i t w i l l be essential for the reader to modify a l l the i s o l a t i o n techniques to take into account both the nature of the sample, and the objective of the study i n hand. The significance of the geoporphyrins and their role i n i n d u s t r i a l processses are reviewed elsewhere. 0,2^ The structures of the d i f f e r e n t skeletal types of geoporphyrins, and their abbreviated names are shown i n Figure 1. The strategies used for the i s o l a t i o n of geoporphyrins depend both on the nature of the sample containing the porphyrins, and on the type of studies to be undertaken on the extract. If the study of t o t a l porphyrin mixtures i s the objective, emphasis must be placed on optimising the recovery of the porphyrins. Conversely, i f characterization of individual components i s the goal, the emphasis l i e s i n obtaining samples of the highest possible purity. The i n i t i a l extraction procedures are largely independent of the subsequent studies undertaken. The p u r i f i c a t i o n of t o t a l porphyrin mixtures, and the p u r i f i c a t i o n techniques for the study of i n d i v i d u a l porphyrins are discussed i n turn.

0097-6156/87/0344-0308$07.00/0 © 1987 American Chemical Society

In Metal Complexes in Fossil Fuels; Filby, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


20.

3 5 8 2 4 6 J 1(a) R ,R R ,R = CH ; R ,R ,R ,R

„

=0 C H

π

1

3

Kb)

309

Geoporphyrins and Chlorins

QUIRKE

R ,R

3

3

R ,R

8

4

= CH ; R ,R ,R 2

= CH ; R ,R ,R

2(b) R ,R ,R ,R

= CH ; R ,R ,R

3

5

5

8

5

2

8

2

3

6

7

= C H ; X = OH

4

7

= C H ; X = Η

4

= CH ; R ,R

5

5

4

3

2 ( c ) R ,R ,R ,R 3

2

2

3

8

2

= C H ; R= Η 2

3

2(a) R ,R ,R ,R 3

7

2

5

5

?

= C H ; R = CH CH 2

5

2

Figure 1. Structures of a l k y l geoporphyrin skeletal types and their abbreviated names.


310

METAL COMPLEXES IN FOSSIL FUELS


The Isolation of Nickel and Vanadyl Sediments, Bitumens and Petroleums.

Alkyl

Geoporphyrins

from

The Extraction of Porphyrins from Sediments. The samples must be pulverized for e f f i c i e n t extraction. The porphyrin concentrate i s usually isolated either by soxhlet extraction, or by sonication, or by means of a b a l l m i l l , which has been used extensively by Baker and co-workers. The technique of b a l l m i l l i n g with an appropropriate solvent i s probably the best method as i t avoids the risks of thermal a l t e r a t i o n , and bond cleavage encountered i n soxhlet extraction and sonication respectively. (_5,1_ and references therein) There have been many d i f f e r e n t solvent systems employed for the extraction of geoporphyrins from sedimentary rocks. Toluene-methanol or benzene-methanol (1:1, vol: vol) (3) chloroform (4_), and acetone-methanol (9:1, v o l : v o l ) (_5) are among the most common. Owing to the variety of sediments, i t i s advisable to experiment with solvent systems to optimize recovery. Nevertheless, an i n i t i a l extraction with acetone-methanol followed by extractions either with benzene or with benzene-acetone (1:1, v o l : v o l ) i s generally a very sound choice, provided that the sole objective i s the i s o l a t i o n of the geoporphyrinsother geolipids may be modified by the acetone. (5^) Toluene can be used instead of benzene i n this scheme. Chloroform-pyridine (9:1, v o l : v o l ) i s also a very e f f e c t i v e system provided that a rotary evaporator with a very e f f i c i e n t vacuum system i s available.(6) Extraction of Bitumens and Crude O i l s . There are s i g n i f i c a n t l y d i f f e r e n t problems i n working with bitumens and crude o i l s compared to working with sediments. As these substances are almost exclusively organic, and usually viscous, the extraction techniques described above are generally inappropriate. The porphyrins may be isolated by demetallation, using methanesulfonicacid (MSA). The demetallated geoporphyrins may be rapidly p u r i f i e d as described subsequently. The disadvantages are that the data on the metalloporphyrin d i s t r i b u t i o n s i s l o s t , and that a r t i f a c t s may be generated. Additionally, there may be p r e f e r e n t i a l degradation of some of the porphyrin components under the vigorous demetallation conditions. (7_,&) The porphyrins may be isolated by column chromatography of the entire crude o i l . This method has the advantage that the dangers of generation of a r t i f a c t s i s avoided.(9) There are problems with the aberrant chromatographic behavior of the asphaltenes, which can p r e c i p i t a t e on the column. The n i c k e l porphyrins may be separated from the vanadyl on the alumina (grade I, Brockmann) or the s i l i c a gel (100-200 mesh) column by gradient elution using dichloromethane in hexane. The porphyrins may be isolated by solvent extraction. In t h i s approach, the o i l or bitumen i s dispersed on a s o l i d surface e.g. c e l l u l o s e (_10), alumina (Brockmann Grade I-II) (_7), or even sand (LI), and extracted i n i t i a l l y with either toluene-methanol (1:2, v o l : v o l ) or acetone-methanol (9:1, v o l : v o l ) . A second extraction using benzene-acetone (1:1 v o l : v o l ) i s advisable when working with complex geoporphyrin mixtures.


20.

QUIRKE



P u r i f i c a t i o n of n i c k e l and vanadyl a l k y l geoporphyrin

311 mixtures.

P u r i f i c a t i o n of t o t a l porphyrin mixtures. It i s usually essential to carry out extensive chromatography using as wide a range of columns as possible. The chromatographic conditions should be optimized for each sample studied. The solvent conditions described i n the following paragraphs are a good starting point. A l l chromatographic procedures should be carried out on columns which are protected from sunlight to prevent photodecompositiona p a r t i c u l a r l y important precaution when working with porphyrins bearing functional groups. When studying small quantities of porphyrins, i t may be necessary to reduce the number of chromatographic steps to improve sample recovery. Probably the best i n i t i a l p u r i f i c a t i o n step i s gel permeation chromatography (GPC) either using sephadex LH-20 (5,7,12) or Bio beads S-X2 (_13). The chromatograms are developed i n tetrahydrofuran. High molecular weight compounds (ca. >4000 daltons), are removed. Low molecular weight components, and any s a l t s present are retarded. The advantage of GPC as a preliminary step i s that i t reduces aberrant chromatographic behavior i n l a t e r separations owing to the presence of asphaltenes. An alternative approach i s to remove the asphaltenes by p r e c i p i t a t i o n with hexane, and study these substances separately. Column chromatography using either alumina or s i l i c a gel i s by far the most common p u r i f i c a t i o n technique.(7) Generally i t i s advisable to purify on both alumina and s i l i c a columns. It i s not clear whether there i s any advantage to be gained using s i l i c a as the i n i t i a l column followed by alumina or vice versa. A wide range of solvent gradients have been used i n s i l i c a gel chromatography, (e.g.4,5,7,13-17) A gradient elution of a s i l i c a gel column (100-200 mesh, packed i n hexane) from hexane to chloroform i s usually a very e f f e c t i v e system. Most of the n i c k e l porphyrins should elute i n ca. 10-20% CHC1-. The vanadyl porphyrins should elute i n ca. 30-50% CHC1 . There i s s u f f i c i e n t variation between samples, and i n the a c t i v i t y of the s i l i c a gel and humidity that careful monitoring of the eluant by v i s i b l e spectrosopy i s e s s e n t i a l . The vanadyl or n i c k e l geoporphyrin mixtures themselves may be p a r t i a l l y resolved on the s i l i c a gel columns, but the fractions may be recombined i f necessary. It i s important to carry out the separations f a i r l y rapidly as there i s the p o s s i b i l i t y of generating a r t i f a c t s . DPEP porphyrins can undergo hydroxylation at the i s o c y c l i c ring during chromatography on s i l i c a gel.(^8) Usually this i s not a major process, but i t should be considered i f polar geoporphyrins are isolated. Ekstrom and co-workers have p u r i f i e d vanadyl porphyrins using Kieselgel 60 (Merck) eluting with a gradient of chloroform and carbon tetrachloride. (4^) Barwise and co-workers have chromatographed vanadyl porphyrins over "functionalized" s i l i c a . ( 1 9 ) The sulphonic acid groups attached to the s i l i c a help to remove i n t e r f e r i n g nitrogenous bases. Using such columns, i t i s possible to obtain very clean samples; however, both nickel and metal- free porphyrins are lost on the column. In addition, i t i s possible that a r t i f a c t s are generated, therefore residence times on the column should be minimised. There were substantial alterations i n the daughter 3



312


spectra of molecular ions of high carbon number (> Coo) vanadyl porphyrins p u r i f i e d on functionalized s i l i c a compared with those of the untreated vanadyl porphyrin f r a c t i o n . In both cases the background on the mass spectra was low.(20) Alumina columns (grade I I , Brockmann) are developed by gradient elution using a variety of solvent mixtures, and are monitored by v i s i b l e spectroscopy.(e.g.5,8,13-15,21,22) A gradient elution from hexane to dichloromethane v i a toluene i s an e f f e c t i v e system. The n i c k e l geoporphyrins are l i k e l y to elute i n 30-50% toluene i n hexane and the vanadyl porphyrins i n 30-60% dichloromethane i n toluene. There i s a possibility of p a r t i a l decomposition of the geoporphyrins, p a r t i c u l a r l y the nickel complexes, on alumina columns thus i t i s advisable to effect the chromatography as rapidly as possible. There have been recent developments i n the role of low and medium pressure l i q u i d chromatography (LPLC and MPLC). Thus Baker and co-workers purify n i c k e l geoporphrin mixtures on alumina columns, eluting with acetone-petroleum ether mixtures. The porphyrins elute i n 3.5-5.0% acetone-petroleum ether.(5) Barwise and Roberts used MPLC on s i l i c a i n their study of the porphyrins of E l Lajun shale; however, the eluting solvents were not divulged.(23) Thin layer chromatography (TLC) i s also very useful i n the f i n a l stages of p u r i f i c a t i o n of t o t a l mixtures. Nickel geoporphyrins have been p u r i f i e d on s i l i c a gel plates either carbon tetrachloride (24) or heptane-dichloromethane (7:3, vol:vol) as eluant. S i m i l a r l y , vanadyl porphyrins may be p u r i f i e d using heptane-tetrahydrofuran (5:1, vol:vol).(25) The separated geoporphyrin mixtures are often demetallated prior to further analysis. (e_.£. 1,1,19,23,26,27) Typically the geoporphyrin mixtures are demetallated by the Erdman procedure using methanesulfonic acid at 100°C (4 hours).(28) The harsh conditions are required to demetallate the vanadyl components, but n i c k e l porphyrins are demetallated i n less than an hour. Yields vary, and there i s the p o s s i b i l i t y of p r e f e r e n t i a l decomposition of geoporphyrin species. Demetallation of n i c k e l porphyrins using either t r i f l u o r o a c e t i c acid (TFA) and 1,2 ethanedithiol (29) or 10% sulphuric acid-TFA at ambient temperature may replace the Erdman demetallation process.(30) Vanadyl porphyrins are demetallated by anhydrous hydrofluoric acid at 0 C.(31) Extensive chromatography of the demetallated porphyrins should be avoided i f the objective of the analysis i s the study of the intact porphyrin mixtures. A good p u r i f i c a t i o n process for i s o l a t i n g the metal-free porphyrins i s described subsequently. A summary of a reasonably e f f i c i e n t p u r i f i c a t i o n procedure for the i s o l a t i o n of intact n i c k e l and/or vanadyl geoporphyrin mixtures i s shown i n figure 2. P u r i f i c a t i o n of Individual Geoporphyrin Components. The initial chromatographic stages of i s o l a t i o n are e s s e n t i a l l y the same as those described previously. The p u r i f i e d metalloporphyrin fractions are e f f i c i e n t l y separated on TLC. The n i c k e l complexes may be separated using hexane-toluene (3:2, v o l : v o l ) , and the vanadyl porphyrins using hexane-tetrahydrofuran (5:1, vol:vol).(25) The resolution may be improved by multiple elutions or by the continuous


20.


QUIRKE

313

Total Porphyrin Extract 1.

Ni

GPC LH-20 Tetrahydrofuran

and VO P o r p h y r i n s A1 0 2

3

(Grade I I ) Column

Hexane/Toluene/CH Cl


2

Ni

Porphyrins

2

*1

Gradient

VO P o r p h y r i n s

Si0 Column

*1

*1

2

Si0

Hexane/CHCl^ G r a d i e n t 2.

TLC

Hexane/CHC1

(Silica)

2.

Heptane/CH C1 2

Column

2

2

3

Gradient

Functionalised

(7:3)

Tol/Tol-EtOAc TLC

Si0

2

Column

Gradient

(Silica)

Heptane-THF (5:1) Pure N i P o r p h y r i n

Mixture

CF C0 H/H S0 3

2

4

(9:1)

or

1.

C F C 0 H / HSCH CH SH

or

1.

CH S0 H,

Demetallated

Toi

2

Pure VO P o r p h y r i n

3

3

Ni

2

3

2

2

or

Mixture

1.

HF 0 C

1.

CH S0 H, 3

3

100°C, 4h

100°C

Porphyrins

Demetallated

VO

Porphyrins

= T o l u e n e ; EtOAc = E t h y l A c e t a t e ; THF = T e t r a h y d r o f u r a n

1. C o n v e n t i o n a l Columns may be r e p l a c e d by MPLC.

2. Chromatograph r a p i d l y

to prevent

unnecessary generation of a r t i f a c t s .

Figure 2. P u r i f i c a t i o n scheme f o r the study of t o t a l n i c k e l and vanadyl a l k y l geoporphyrin mixtures.


*

2



314

elution method. (_14) Nickel geoporphyrins may be partially fractionated on calcium carbonate columns developing the chromatogram i n benzene-petroleum ether (4:1, v o l : v o l ) . (32) Usually i t i s necessary to demetallate the mixtures to effect the i s o l a t i o n of individual porphyrin components. The range of p o l a r i t i e s of the metal-free geoporphyrins i s greater than those of the metalloporphyrins. It i s often possible to i s o l a t e individual metal free porphyrins from metalloporphyrin fractions by preparative TLC on silica gel. (£«Jl/A>!>i^>iZ>H»!^ Toluene-dichloromethane (1:1, vol:vol) i s an e f f e c t i v e solvent system. The mode of separation seems to be as follows :(14,35) 1) Porphyrins bearing i s o c y c l i c rings are more polar than e t i o porphyrins similar carbon number. 2) The p o l a r i t y of each s k e l e t a l class of porphyrin i s inversely proportional to the carbon number. 3) An unsubstituted Β-position causes a reduction i n p o l a r i t y equivalent to an additional 2-3 methylene units. Thus a C^Q e t i o porphyrin with 1 unsubstituted 3-position i s intermediate i n p o l a r i t y between f u l l y β-alkylated C ^ and C ^ etio porphyrins. The separation i s greatly f a c i l i t a t e d by carrying out Friedel-Crafts acetylations on the geoporphyrin mixtures. The reaction s p e c i f i c a l l y converts unsubstituted β-positions on the porphyrin macrocycle into acetyl moieties. The reaction may be carried out on the nickel geoporphyrin mixtures themselves. Vanadyl porphyrin must be demetallated, and converted to the n i c k e l , copper or iron porphyrins before acetylation. The metalloporphyrin i n dichloromethane i s treated at 0°C with anhydrous tin(IV) chloride and acetic anhydride. After standing for a short time, the solution i s neutralized, and the porphyrins are extracted. The acetylated porphyrins are readily separated by TLC from the f u l l y β-alkylated geoporphyrins. The i n d i v i d u a l acetylated porphyrins may then be isolated, and characterized by nuclear magnetic resonance spectroscopy.(36) The method has yet to be employed i n the study of geoporphyrins bearing > 2 unsubstituted β-positions. Isolation of i n d i v i d u a l porphyrins by High Performance Liquid Chromatography (HPLC) - the best f i n a l p u r i f i c a t i o n technique- i s discussed below. A summary of useful i s o l a t i o n procedures i s shown i n Figure 3. The Isolation and Geological Sources.

Purification

of

Geoporphyrins

from

Other

There have been few studies on the geoporphyrins i n coals and kerogens. The i s o l a t i o n methods employed i n these cases are very different from those described i n the previous sections because of the d i f f e r e n t nature of the samples and the geoporphyrins themselves. The i s o l a t i o n of Porphyrins from Coals. The p r i n c i p a l recent work done i n this area i s that of the Bonnett group, and that of Palmer and co-workers. Neither group use the method of Treibs, who carried out an i n i t i a l extraction of the coal with pyridine.(37,38) The approach of Palmer involved extraction of ground coal p a r t i c l e s (20-mesh) with MSA (100°C, 3 h). The porphyrins were then


20.


QUIRKE

Porphyrin

Extract GPC

Ni

A^O^

Column

Si0

Column

o

Porphyrins 1.

TLC

*1

VO P o r p h y r i n s

(Silica)

1.

Hexane-Tol (3:2)


-Ni

Porphyrin

1.

VO

i : t

Porphyrin

Fractions

(0Ac), 1.

Ac 0/SnCl. 2 4 TLC CH C1

HF 0 C

o

2.

2

*

(Silica)

^Hexane-THF (5:1)

Fractions

Ni

I TLC

or 1.

C H S 0 H , 100°C, 4h 3

3

2

Metal-Free

Porphyrins

2

Alkyl Ni Porphyrins

Ni A c e t y l Porphyrins

TLC Tol/CH Cl 2

TFA/H S0. 2 4

TFA/H S0. 2 4

o

Metal-Free Acetyl Porphyrins 1.

2

(1:1)

HPLC

Metal-Free Alkyl Porphyrins 1.

TLC CHC1

or 1.

or 1

o

TLC

(Silica)

Tol/CH Cl

3

2

or 1.

HPLC

Individual Acetyl Porphyrins

*

2

(1:1)

HPLC

Individual Alkyl Porphyrins -

T o i = Toluene; ΤFA = T r i f l u o r o a c e t i c A c i d ; THF = T e t r a h y d r o f u r a n ; A c 0 = A c e t i c Anhydride. 2

1. See F i g u r e 2.

k 2.

Demetallate

and s e p a r a t e on HPLC

Figure 3. Procedures f o r the i s o l a t i o n of i n d i v i d u a l porphyrins from n i c k e l and vanadyl a l k y l geoporphyrin mixtures.



316


p u r i f i e d using GPC on Sephadex LH-20, before mass spectrometric analyses. (39) The Bonnett approach of an i n i t i a l treatment of the f i n e l y ground coal with 7% H^SO.-CH^OH for 12 hours at room temperature yields the metalloporphyrins intact. The porphyrins were extracted with chloroform, and neutralized. Using these methods, gallium ( I I I ) , iron (III), and manganese (IT) porphyrins were detected i n coals. Gallium porphyrins were isolated and p u r i f i e d by column chromatography on silica gel using gradient elution of benzene-methanol, i n the presence of 0.02-0.03 molar anhydrous ammonia. The porphyrin extracts are further p u r i f i e d on s i l i c a gel TLC p l a t e s , eluting with benzene-methanol (4:1) i n the presence of ca. 0.1 molar anhydrous ammonia and then rechromatographed i n 4.15 molar methanolic ammonia. The gallium porphyrins may be analysed on reverse phase HPLC using a Waters Bondapak column C ^ / P o r a s i l eluting with methanol:water (17:3).(40,41) Gallium porphyrin methyl esters may be analysed on Techsil 5C 18 columns eluting with methanol-water (4:1).(42) Iron porphyrins are isolated somewhat d i f f e r e n t l y . After the i n i t i a l extraction with 7% sulphuric acid-methanol, the porphyrins are chromatographed on s i l i c a gel TLC plates eluting with 15% 1 molar ammoniacal methanol-toluene.(43) Any porphyrin acids should be converted into their methyl esters. The porphyrins may then be demetallated, and analysed by HPLC using Apex ODS columns and eluting with 3% methanol-acetonitrile.(42) The Isolation of Porphyrins from Kerogens. There i s very l i t t l e information on the generation of porphyrins from kerogens even though there have been many hypotheses on the nature of the porphyrins within the kerogen matrix. Van Berkel and F i l b y have made a detailed study on the subject, and their approach to the problem i s summarized here.(44) The bitumen i s removed from the f i n e l y ground shale. The kerogen concentrate i s prepared by a method similar to that of Durand.(45) The concentrate i s sonicated to remove organic solubles associated with the minerals which associate with the kerogen after treatment with hydrofluoric acid. Then the kerogen i s pyrolysed i n toluene at the selected temperature under an atmosphere of nitrogen. The pyrolysed kerogen i s isolated by centrifugation, and then sonicated with toluene or toluene-methanol, and the organic extract i s combined with the f i l t r a t e from the pyrolysis. The porphyrins may be isolated using the methodologies described e a r l i e r . The kerogen may then be pyrolysed again at a higher temperature. Tygically, the pyrolyses temperatures are selected i n the range 100-450 C. Using this method i t i s possible to distinguish the bituminous porphyrins from those porphyrins associated with inorganic mineralsbut not intimately bound into the kerogen matrix- and from the porphyrins associated with the kerogen. It i s also possible to obtain information on the order i n which the geoporphyrins are liberated from the kerogen. The studies indicate that the majority of the porphyrins are liberated at 300°C.


20.

QUIRKE


317

The i s o l a t i o n of Unusual C y c l i c Tetrapyrroles.


With the exception of coals, nickel and vanadyl alkyl porphyrins are the dominant porphyrin species i n sedimentary rocks and petroleums. Nevertheless, other porphyrin species have also been i s o l a t e d . The methodology for the i s o l a t i o n of these components w i l l be discussed. Copper Geoporphyrins. Copper Porphyrins have very similar chromatographic and chemical properties to n i c k e l porphyrins. Thus they are isolated i n the same manner as described previously. They may be demetallated i n the same way as the n i c k e l porphyrins. In a l l i s o l a t i o n procedures, i t i s p a r t i c u l a r l y important to rigorously exclude copper s a l t s , because metal free porphyrins chelate with copper (II) ions very readily.(46) Metal-Free Geoporphyrins. Metal-free porphyrins occur rarely i n geological samples; however, they are perhaps the easiest species to i s o l a t e . The simplest i s o l a t i o n procedure i s to treat the t o t a l organic extract i n ethereal solution with fresh d i l u t e (III>II>l IV>II,I>III IV>II>111>I III>IV>II>I

C

(14) (14) (54) (55)

See Figure I. UV data f o r pure metal free benzo e t i o , and DPEP-7 (5) porphyrins are unavailable. The intensity of band I I I i s diminished i n the 3-unsubstituted e t i o porphyrins compared to the f u l l y substituted components. C

The isomeric DPEP-5 (3) i s similar.



320

Table I I I . Molecular Ions of Geoporphyrins


C No.

20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

Etio Ni VO FB

Porphyrin Molecular ions Isocyclic Benzo Benzo D. Ni VO FB Ni VO FB Ni VO FB

366 380 394 408 422 436 450 464 478 492 506 520 534 548 562 576 590 604 618 632 646

392 406 420 434 448 462 476 490 504 518 532 546 560 574 588 602 616 630 644

375 389 403 417 431 445 459 473 487 501 515 529 543 557 571 585 599 613 627 641 655

310 324 338 352 366 380 394 408 422 436 450 464 478 492 506 520 534 548 562 576 590

401 415 429 443 457 471 485 499 513 527 541 555 569 583 597 611 625 639 653

336 350 364 378 392 406 420 434 448 462 476 490 504 518 532 546 560 574 588

416 430 444 458 472 486 500 514 528 542 556 570 584 598 612 626 640

425 439 453 467 481 495 509 523 537 551 565 579 593 607 621 635 649

360 374 388 402 416 430 444 458 472 486 500 514 528 542 556 570 584

414 428 442 456 470 484 498 512 526 540 554 568 582 596 610 624 638

423 437 451 465 479 493 507 521 535 549 563 577 591 605 619 633 647

358 372 386 400 414 428 442 456 470 484 498 512 526 540 554 568 582

Ni

TetraBD VO FB

-

-

446 460 474 488 502 516 530 544 558 572 586 600 614 628 642

455 469 483 497 511 525 539 553 567 581 595 609 623 637 651

-

a

390 404 418 432 446 460 474 488 502 516 530 544 558 572 586

The molecular ions are based on C, ^N, "^Ni. Higher carbon number porphyrins may be observed at 1 dalton above the calculated value owing to rounding up the p a r t i a l mass to the next integer. I s o c y c l i c = Molecular ions for a l l porphyrins bearing an i s o c y c l i c ring, and tetrahydrobenzo porphyrins (2-6, Figure 1.) Benzo = Benzo porphyrins (8, Figure 1.) Benzo D. = Benzo DPEP porphyrins (9, Figure 1.) TetraBD = Tetrahydrobenzo DPEP porphyrins (7, Figure 1.) FB = Free Base; Ni = Nickel; VO = vanadyl porphyrin.



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(3) Jj: i s esggntial to correct for isotopic contributions from C, Ν and Ni (where appropriate) i n quantitation to d i s t i n g u i s h between molecular ions and coincident isotope peaks from other components. Such correction procedures have been described. (_7) Chemical Ionisation Mass Spectrometry (CIMS) may provide a better solution to the quantitation problem once the problem of r e p r o d u c i b i l i t y i s overcome. In CIMS using hydrogen or methane as reagent gases, porphyrins may either fragment to form their individual pyrrole rings or y i e l d the desired molecular ion without any major fragments.(57,58) The mode of fragmentation appears to be dependent on the temperature of the source. Analysis of geoporphyrins mixtures using Fast Atom Bombardment (FAB) methods may prove valuable, although the technique may not be s u f f i c i e n t l y sensitive for quantitative work. The techniques of GC-MS and HPLC-MS are both methods of great potential. The developments i n GLC using s i l y l a t e d s i l i c o n porphyrin derivatives by Eglinton and co-workers are p a r t i c u l a r l y exciting. McFadden et a l . , have carried out HPLC-MS analysis on geoporphyrin mixtures. The data confirm the potential of the method. The only impediment l i e s i n the e f f i c i e n t interfacing of the HPLC and the MS.(59) Mass spectrometric analyses of t o t a l mixtures gives only limited structural data. Analyses of metastable ions gives information on the nature of the substituents on the porphyrin macrocycle. In this way, Titov and co-workers were able to confirm the presence of geoporphyrins with extended a l k y l substituents «C ).(60) Tandem mass spectrometric (MS/MS) i s another valuable a n a l y t i c a l t o o l . (6J[,62) Using this technique i n the EI mode i t i s possible to obtain daughter ions and neutral losses from the molecular ions of the porphyrin components without prior separation of the mixture. These data provide information on the type of the substituents on a l l the porphyrins of single carbon number. The predominant mode of cleavage of the molecular ions i s 3-cleavage of the substituents. The data indicate that high carbon number O C 3 3 ) geoporphyrins have more than one s i t e of extended alkylation, and there may be several isomeric porphyrinic species present. (63-65) The daughter spectra of the molecular ions may prove to be valuable "fingerprints" i n correlation studies. The technique of CIMS/MS has great p o t e n t i a l for the analysis of geoporphyrin mixtures also, but there are problems of reproducibility at present.(20) 12

Chromatographic Methods. HPLC i s the most valuable a n a l y t i c a l technique. The porphyrins eluting from the various columns are monitored by v i s i b l e spectrophotometry. The choice of detecting wavelength i s dependent on the nature of porphyrin species being studied. (Tables 1,11). Demetallated porphyrin mixtures are usually analysed using normal phase s i l i c a columns. Maxwell, Barwise and co-workers have achieved the best separations of such mixtures to date. (e_._g 23). F i n a l l y , after over four years since the o r i g i n a l publications, the chromatographic conditions are to be published i n "Journal of Chromatography". Sundaraman and co-workers have succeeded i n the e f f i c i e n t separation of vanadyl porphyrin mixtures; unfortunately the precise



322


conditions employed have not been divulged.(66) Nickel porphyrins are not usually analysed i n the metallated state; however, Fookes reported the separation of the n i c k e l porphyrins of J u l i a Creek o i l shale into 30 fractions on a C-18 column eluting with methanol.(24) Eglinton and co-workers have made notable advances i n the GLC and GC-MS analyses of porphyrins.(e.g. 67-70) Although i t i s possible to chromatograph several metalloporphyrin species inluding gallium, aluminium and rhodium (III) complexes, analysis of the s i l i c o n ( I V ) derivatives i s the preferred method.(68) The procedure i s summarized below. The demetallated porphyrins i n dry toluene are converted into their s i l i c o n derivatives by treatment with hexachlorodisilane, for 2 hours at ambient temperature. The porphyrins are p u r i f i e d on alumina TLC, eluting with dichloromethane, and the hydroxy ligands are s i l y l a t e d using BSTFA-pyridine. The chromatography i s carried out on either OV-1 or CPSil 5 c a p i l l a r y columns, using helium as the c a r r i e r gas (50-120 cm sec flow rate). The i n i t i a l oven temperature i s 60 C, and the temperature i s increased l i n e a r l y to ca. 300°C. The advantages of GC-MS analysis of geoporphyrins f o r fingerprinting of crude o i l s and correlation studies are obvious. Clearly, GC-MS-MS would be a yet more powerful a n a l y t i c a l t o o l . In addition to i t s role as a p u r i f i c a t i o n technique, GPC has been used i n the study of the molecular weight d i s t r i b u t i o n s of geoporphyrin mixtures. Blumer and co-workers studied the porphyrins of the Serpiano shale, and obtained evidence for very high molecular weight porphyrin species, and even dimeric porphyrins.(71,72) More recently, Fish and co-workers have used size exclusion HPLC to investigate the nature of the nickel and vanadium compounds i n petroleums. The eluates from the 50-100 u Spherogel column were analysed by graphite furnace atomic absorbtion. Using this technique i t i s possible to deduce the molecular weight range of such species.(73) Degradative Methods. The degradation of porphyrin mixtures to maleimides (2,5-pyrrolediones) using chromic acid gives valuable information on the substituent patterns of the geoporphyrins. (9^,74,75) Each p y r r o l i c subunit i s converted to a maleimide bearing the o r i g i n a l pair of substituents. The i s o c y c l i c ring-bearing pyrroles are converted to maleimide carboxylic acids.(76) The method compliments chromatographic and mass spectrometric analyses of such geoporphyrin mixtures. An e f f i c i e n t procedure i s outlined below. (9^) Treatment of the demetallated porphyrin mixture i n t r i f l u o r o a c e t i c acid with chromium trioxide i n sulphuric acid at 0 C for 2 hours yields the maleimides. These compounds may be analysed by GLC or GC-MS using OV-1 or carbowax c a p i l l a r y columns programmed from 60°C to 260°C at 4°C miη . The maleimides usually show molecular ions, and a c h a r a c t e r i s t i c fragment ion, m/z 125. To obtain better peak shapes, the maleimides may be s i l y l a t e d p r i o r to analysis. There are three other degradative techniques, which may be used for the analysis of geoporphyrin mixtures. Oxidation of porphyrins using lead (IV) dioxide (4h, ambient temperature) to the corresponding 5,10,15,20-tetraoxoporphyrinogens i s a potentially


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valuable method. These compounds fragment i n EIMS to give the individual p y r r o l i c species, together with d i - and t r i - p y r r o l i c fragments from the t e t r a p y r r o l i c macrocycle.(77) Treatment of the porphyrins with hydriodic acid gives the i n d i v i d u a l pyrroles, which may then be analysed by GC-MS. The method has been superceded by the developments i n CIMS.(78) The oxidative degradation of porphyrins by potassium permanganate to form 2,5-pyrrole dicarboxylic acids could be used for geoporphyrin analyses, but i t too has been supercedeed by CIMS.(79) Characterisation of Individual Porphyrin Components. This i s the area of geoporphyrin chemistry i n which the greatest advances have been made. The developments i n NMR i n p a r t i c u l a r have resulted i n a c l a r i f i c a t i o n of the T r e i b s hypothesis on the o r i g i n of the geoporphyrins, and confirmed that these compounds are derived from naturally-occurring chlorophylls. In addition, i t i s possible to determine i n some cases the precise precursor of s p e c i f i c geoporphyrins.(51,80)


1

Spectrophotometric Methods. UV/Visible spectrophotometry provide information on the s k e l e t a l type of porphyrin present. Otherwise i t is of l i t t l e value i n s t r u c t u r a l elucidation. The most important role f o r this technique i s i n the characterisation of the geochlorins.(12) Mass Spectrometric Methods. High resolution EIMS yields the molecular formula of a porphyrin. The primary mode of fragmentation of the porphyrins i n the EIMS i s cleavage 3 to the porphyrin ring. Thus determination of the daughter ions of the molecular ion provides limited s t r u c t u r a l information.(56,63,64) The CIMS analysis of porphyrins using hydrogen as reagent gas and a source temperature of 200°C reveals pyrrole, dipyrrole and t r i p y r r o l e fragments, which can be used to sequence the pyrrole units within the porphyrin macrocycle, and requires very little sample (ca. 1 yg). (58,80 More reproducible results are obtained by using ammonia as reagent gas.(82) This i s usually the best a n a l y t i c a l technique when working such small quantities of porphyrin that NMR i s i n f e a s i b l e . The method w i l l probably be further improved by using CIMS-MS with ammonia as the reagent gas. Nuclear Magnetic Resonance Methods. The use of NMR has revolutionized the study of the geoporphyrins. In fact i t i s by far the most important method currently employed for the analysis of individual geoporphyrins. The technique i s i d e a l for such studies as i t can be effected on f a i r l y small samples (C«) substituents. The chemical s h i f t of the water peak may be varied by a l t e r i n g the pyridine concentration. Porphyrins bearing i s o c y c l i c rings are i d e a l l y suited for nOe analysis. Geochlorins w i l l also be readily analysed by the nOe technique because these compounds bear a reduced p y r r o l i c ring which ensures that the meso protons are well resolved. In addition the substituents on the reduced p y r r o l i c ring are very readily assigned which w i l l greatly f a c i l i t a t e such studies. Porphyrins bearing an unsubstituted 3-position may be analysed d i r e c t l y by the nOe method, but better results are obtained i f the porphyrins are initially acetylated by the Friedel-Crafts acylation reaction.(86,87) Usually the technique i s of l i t t l e value for the characterisation of f u l l y B-alkylated etio porphyrins as the meso protons coincide. There have been two other approaches to the analysis of porphyrins by NMR. A etio porphyrin i n Gilsonite was i d e n t i f i e d as etioporphyrin-III \Ia) by converting the porphyrin into i t s "mercury sandwich" complex i n which two porphyrin macrocycles are interspersed between three mercury ions. The mercury sandwich complex of each of the four porphyrin type isomers has a different H NMR spectrum. These metal-free type isomers have v i r t u a l l y i d e n t i c a l NMR spectra.(L7) The method i s limited because i t i s essential to obtain spectra of a l l the possible isomers for comparison. Also the complex readily decomposes to the 1:1 complex; hence, the CDCl^ solvent should be passed through alumina to remove traces of acid. Krane and co-workers used aggregation studies to assign the structure of the C~ etioporphyrin i n Marl slate as etioporphyrin-III.(91) This approach i s l i k e l y to be the method of choice for the analysis of porphyrins which are not amenable to nOe analysis. The chemical s h i f t s of some important geoporphyrinic substituents are shown i n F i g . 4. Degradative Techniques. The maleimide degradation may be used to confirm the NMR data. ( 14,1_7,82) Otherwise, the method i s of l i t t l e value for precise structure determination.



range

3

CH CH

2

The

3

1

o f v a l u e s i s a approximate.

3.1-3.5 M e t a l and m e t a l

f r e e s p e c i e s have s i m i l a r

δ-value.

Figure 4. H NMR chemical shift data for common geoporphyrin substituents and exocyclic rings.

CH C=0

1.6-1.9

9.0-10.5

3.7-4.3

3

CH CH

2

Meso-H


to LA

326



Synthetic and Chromatographic Methods. The method of structure elucidation by comparison of the isolated compound with a l l the possible s t r u c t u r a l isomers i s impractical except for research groups s p e c i a l i s i n g i n porphyrin synthesis. (91-96) A t y p i c a l t o t a l synthesis w i l l take over twenty steps from readily available starting materials, and involves a moderate y i e l d cyclisation step.(91-96) The method was successfully used to determine the structure of the desethyl C~Q etio porphyrin (1 b).(97) Four isomers of the compounds were synthesized, and separated by HPLC on 10 y ODS-HC-Sil-XI eluting with a c e t o n i t r i l e , and the structure of the geoporphyrin was assigned by co-injection. The only occcassion when i t i s essential to employ this strategy i s i f the structure determination of porphyrins cannot be readily determined by other techniques. Crystallographic Methods. The major d i f f i c u l t y i n using the technique i s that i t i s very d i f f i c u l t to grow crystals of s u f f i c i e n t quality, when working with small amounts (< 1 mg) of sample. Nevertheless the nickel complexes of deoxophylloerythroetioporphyrin and deoxophylloerythrin (2a, 2b) have been characterized by X-ray crystallography.(4 98^ χ

Characterisation of Bound Porphyrins. There have been very few studies on the nature of the porphyrins within the organic substances or inorganic mineral matrix. The techniques available for such studies have been mainly applied to model compounds. Bergaya and van Damme have studied the s t a b i l i t y of metalloporphyrins adsorbed onto a clay surface.(99) The porphyrin species were investigated by using UV-visible diffuse reflectance and diffuse transmission spectra. The diffuse transmittance spectra were i d e n t i c a l with conventional transmission spectra. Provided that the sample i s a highly dispersed suspension, i t i s also possible to obtain quantitative data. The diffuse reflectance spectra are not as e a s i l y compared; however a direct comparison gives enough information for distinguishing the nature of the porphyrin species present. For rigorous work the two parameters are correlated through the Kubelka-Munk function: (l-R ) /2R = k/s Z

O0

0o

Roo = the reflectance of a layer s u f f i c i e n t l y thick that further concentration does not change the reflectance, k = absorption c o e f f i c i e n t (molar extinction times concentration) s = scattering c o e f f i c i e n t . Cady and Pinnavaia used a similar approach; however the v i s i b l e spectroscopic data were obtained by dispersing the m i c a - s i l i c a t e bearing the porphyrin i n a nujol mull. Infra red studies were effected by running the spectra of the s o l i d material as thin films supported between KBr plates.(100) Electron spin resonance spectroscopy (ESR) i s a powerful technique for the study of bound vanadyl porphyrins. T y p i c a l l y , the spectra show eight weak p a r a l l e l lines (three may be hidden) and


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eight perpendicular l i n e s , which are readily recognized. The basic methodology, and interpretation of the data has been reviewed recently by Lin.(101) Using the method, evidence has been obtained for the presence of non-porphyrinic vanadyl compounds i n petroleums and tar sands.(102,103) Goulon e_t a l . have reported x-ray absorption spectroscopic analysis on asphaltenes which indicated that the vanadium was associated with porphyrins, and that the porphyrin content may be greater than indicated by v i s i b l e spectroscopy.(104,105)


Summary There i s s t i l l no one scheme which i s i d e a l for the i s o l a t i o n of porphyrins from a l l types of geological samples. This i s not surprising because of the substantial variations i n the sedimentary environment. The development of HPLC, and the advances i n mass spectrometry have been major assets i n the characterisation of t o t a l geoporphyrin mixtures. In the future, GC, ÇC-MS, HPLC-MS w i l l y i e l d even more information. The advent of H NMR has provided the geochemist with by f a r the most important tool f o r ^ t h e s t r u c t u r a l elucidation of i n d i v i d u a l geoporphyrins. No doubt, C NMR studies w i l l become feasible as the s e n s i t i v i t y of the instruments i s improved. MS-MS w i l l also be a valuable technique for the study of i n d i v i d u a l components within intact geoporphyrin mixtures. The major area of the f i e l d which i s yet to be thoroughly investigated i s the study of the bound geoporphyrins. It i s e s s e n t i a l that there are more studies i n t h i s area i f the chemistry of the geoporphyrins i s to be f u l l y understood. Acknowledgments I am g r a t e f u l to Dr. J.F. Branthaver (Western Research I n s t i t u t e , Laramie), Dr. R.H. F i l b y and Mr. G.J. van Berkel (Washington State University, Pullman), Dr. K.M. Smith and his students (University of C a l i f o r n i a , Davis) for useful discussions i n the preparation of t h i s manuscript. References 1. F i l b y , R.H.; Van Berkel, G.J., t h i s volume. 2. Branthaver, J.F., this volume. 3. Thomas, D.W.; Blumer, Μ., Geochim. Cosmochim. Acta 1964, 28, 1147-1154. 4. Ekstrom, Α.; Fookes, C.J.R.; Hambley, T.; Loeh, H.J.; Miller, S.A.; Taylor, J.C. Nature 1983, 306, 173-174. 5. Louda, J.W.; Baker, E.W. In " I n i t i a l Reports of the Deep Sea D r i l l i n g Project "; Yeats, R.S.; Haq, B.U. et al., Eds.; U.S. Government Printing Office: Washington, 1981; Vol.LXIII, pp.785-818. 6. Quirke, J.M.E.; Dale, T.; Britton, E.D.; Yost, R.A.; Trichet, J.; Belayoumi, H. Org. Geochem. submitted. 7. Baker, E.W.; Palmer, S.E. In "The Porphyrins"; Dolphin, D., Ed.; Academic: New York, 1978; Vol.I, pp. 485-622. 8. Baker, E.W. J . Am. Chem. Soc. 1966, 88, 2311-2315.



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