Practical applications of solid state 13C NMR to the structural elucidation of sedimentary organic matter

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trends in analytical chemdry,

vol. 9, no. I,1990

Practical applications of solid state 1% NMR to the structural elucidation of sedimentary organic matter Richard L. Patience Sunbury on Thames, U.K and

Michael A. Wilson North Ryde, Australia

The development of solid state methods in 13C NMR has provided the key to the advances made in structure determination of otherwise insoluble and intractable organic matter in sediments. This review covers significant recent developments applied to organic matter in both present day (soils and aquatic sediments) and ancient (in oil shales and coals) sediments.

The development of solid state 1% NMR Considerable technical improvements have been made since the first broadline solid state 13C NMR spectra were obtained around 20 years ago. Increased sensitivity arose with the introduction of cross-polarization (CP) 13C NMR whilst the difficulties in assigning peaks as a result of broad peakwidths were alleviated with the advent of magic angle spinning (MAS). Together, CP-MAS have been combined to produce a technique widely used and reported in the literature. 13C CP-MAS NMR was first applied to coals by Bartuska et al.’ and to kerogens by Resing et al. ‘. These advances led to the development of the concept of ‘degree of aromaticity’ (f,), which is obtained by summation of the signals due to aromatic and aliphatic carbons, as these can be integrated separately. Finally, dipolar dephased 13CCP-MAS NMR was first applied to coal by Murphy et aL3; this led on to the development of more detailed structural work by allowing the signals due to aromatic carbons to be further subdivided into quaternary and tertiary by integration of the dipolar dephased spectrum. Almost parallel studies have occurred in other areas of organic geochemistry such as humic acid, wood, soil and peat chemistry4. For example, the first i3C CP NMR spectrum of a soil was broad and poorly resolved but by late 1981 well-resolved 13C CP-MAS NMR spectra of whole soils had been obtained. Soon after, dipolar dephasing was employed. 01659936/90/$03.00.

Since the early 1980’s there has been a proliferation of papers seeking to apply solid state NMR to environmental problems, coal and shale exploration and processing science, the technique mostly being used for the determination off,, but also to investigate molecular composition in more detail. This paper is a review of the more important applications and developments that have resulted. How 1% NMR works Given the length and scope of this article, we have to assume that readers have a working knowledge of NMR theory - at least for liquids. NMR experiments can be carried out on any state of matter, but ‘high resolution’ spectra are generally only obtained for liquids. In solids, the chemical shift information is complicated by the fact that this property is dependent on molecular orientation (called chemical shift anisotropy). Rapid motion in liquids averages out this effect and an ‘isotropic chemical shift’ is observed, whereas for powdered solids a superimposition of frequencies for all possible orientations is seen, i.e. a very broad line results. To overcome this, MAS can be applied to solids to average the chemical shift anisotropy and leave a spectrum approximately equivalent to that obtained from a liquid, except that the chemical shifts represent the electronic environment in the solid, not the liquid, state. What 13CNMR tells you We shall only discuss here the information to be gained from solid state 13C NMR (13C being the carbon isotope which gives an NMR signal). The results obtained are representative of all carbon isotopes unless 13Chas been added as a label to follow a particular reaction path. The 13C NMR spectrum of a solid can be complex and, if magic angle spinning is employed, the width of lines from individual signals (resonances) may be nearly as narrow as those for solution spectra. However, because of the wide range of structural groups present, this degree of resolution is rarely achieved for organic geochemical samples. Moreover, if high fields (greater than 3 Tesla) are used to increase resolution, spinning side bands appear in spectra (Fig. la). Briefly, the 13C NMR spectrum can be readily @Elsevier

Science Publishers B.V.

trends in analytical chemistry, vol. 9, no. I,1990

a

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15OpP;OO

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PPM

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d

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Fig. I. Typical NMR spectra of a Brown Limestone Formation (Gulf of Suez, Egypt) kerogen. (a) A standard r3C CP-MAS NMR spectrum: I ms CP contact time, 3 s recycle time, 10 Ooo transients recorded. * = spinning sidebands, x = peak due to MgCOs filler. (b) A dipolar &phased spectrum of the same kerogen: conditions as in (a) except for 60 ps dephasing delay time. (c) A spinning sideband suppressed spectrum of the same kerogen: conditions as for (a) except 5500 transients recorded; (i) aliphatic region, (ii) aromatic region, and (iii) carbon-oxygen region. (d) A dipolar dephasedlspinning sideband suppressed spectrum: conditions as for (c) except for 60,u.s dephasing delay time.

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resolved into three areas, e.g. aliphatic carbon [(i) in Fig. lc]; aromatic carbon [(ii) in Fig. lc]; and carbon attached to oxygen [(iii) in Fig. lc]. This resolution has given rise to the most widely measured parameter from NMR, the degree of aromaticity VI = aromatic C/total C) which is normally measured at low fields. There has been considerable debate about the accuracy of f, values obtained from solid state 13CCP-MAS spectra, but the concensus seems to be that they are a quantitative measurement except in certain circumstances, such as very highly aromatic coals. However, various attempts have been made to subdivide further each of these three components into additional structural features. The most comprehensive approaches seek to resolve aromatic, aliphatic and O-attached carbons into individual functional groups based on the chemical shifts of model compounds5>6. The functional groups which were resolved by the earlier work’ are given in Fig. 2. The main drawbacks are: (i) the difficulty in distinguishing between CH, groups in rings or chains and (ii) the overlap between S-containing functional groups (which are not allowed for) and aromatic or O-containing species. Over the last few years the development of NMR pulse sequences, which involve manipulating the spins of the nuclei so that only certain types of carbons are observed by NMR, has become a challenge for the geospectroscopist. As already noted, the most established of these is the dipolar dephasin technique, but others are now available and in use B. The most promising of these exploits the fact that the lineshape of the spectrum without MAS (chemical shift anisotropy) is also characteristic of the types of structural groups present’ so that a combination of a range of different types of NMR experiments gives detailed information. This, or dipolar dephasing data, or the ‘H spectrum, can be expressed with conventional CP-MAS data to produce two dimensional spectra9 (Fig. 3) in which one axis represents chemical shift information and the other some other parameter so that cross sections across the spectrum give us additional information about the types of carbon present. Organic transformations in sediments The pathways of formation of organic matter deposited as kerogens or coals or as soil organic matter are different in that kerogens and coals are largely deposited in a reducing environment and the latter largely in an oxidising environment. Of course, in reality a continuum exists. The oxidation pathway leads to the formation of carbon dioxide. The reduction pathway leads to methane and elemental carbon (coalification).

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trends in analyticalchemistry, vol. 9, no. I,1990

FUNCTIONALITY

STRUCTURE

CARBONYL

C/H\ C,o=O

CARBOXYL

-

SYMBOL c=o

q ~~RIH

COOR

PHENOL

ArCO

BRANCHED AROMATIC

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ArCC

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CHO

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C

CH

METHINE C METHYLENE (C2)

Cm

METHYLENE

c-c

CH,

ALIPHATIC METHYL

\ ,CHa

CH,al

AROMATIC METHYL

CH,

CH,(C2)

CH,arom

perculate through the profile. Some of these structures are well represented by the Schnitzer model”. Preliminary studies suggest very young soils are highly aromatic. These results also clearly indicate the overriding importance of microbiological activity. In anaerobic environments carbohydrates are degraded, but in aerobic environments there is considerable competition between organisms degrading lignin and carbohydrate. With good drainage there is preferential loss of aromatics in run off, or through oxidation, so that soils tend to be more aliphatic, and consist mainly of polymethylene structures. Size fractionation experiments indicate that this polymethylene accumulates in the smallest size fractions. Moreover, virgin soils tend to accumulate more polymethylene than soils with wheat or permanent pasture, indicating competition of the crop with microorganisms decreases this type of carbon. This suggests the polymethylene is microbially derived, a result which is confirmed by incubating soils with 13C labelled glucose. These experiments show incorporation of label into polymethylene and carboxyl groups but little, if any, into aromatic structures. There is considerable scope for investigating the pathways by which lignin (Fig. 5) is decomposed in soils. Studies with 13C labelled lignin have clearly shown that, during decomposition, demethoxylation occurs but some methyl groups can become attached

Fig. 2. Carbon functionality (and symbols) resolved by solid state 13C NMR.

The oxidation pathway A typical soil spectrum is shown in Fig. 4. Historically, soil organic substances that have been greatly modified by soil processes to form humic substances were believed to have a structure a bit like coal, but in one study Schnitzer and co-workers” found large quantities of oxidised small ring aromatic compounds. The Schnitzer model, which consists almost entirely of aromatic and carboxylic acid structures, has been widely accepted and has been erroneously interpreted as being applicable to all soils. It is now known that organic structure is quite variable, since a wide range of soils of different structure have been examined using NMR techniques. Under conditions of poor humification, e.g. cold climates, carbohydrates are retained4. For example, in some Antarctic soils, 85% of the carbon is carbohydrate. When humification is rapid, e.g. in soils developed in humid areas with good drainage, many soils contain less than 10% aromatic carbon. In general Spodosol B,, horizons (secondary horizons in forest soil containing precipitated organic matter) are very aromatic, because the aromatics selectively precipitate as they

Fig. 3. Two dimensional NMR spectrum showing 13C and ‘H in contour form. The one dimensional spectra are shown along each axis. (From Zilm and Webb’).

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vol. 9, no. I,1990

IL

200

loo 6 (ppm)

0

Fig. 4. Typical “C CP-MAS spectrum of a soil showing resonances from polymethylene (3Oppm), methoxyl(55ppm), carbohydrate (73,103ppm) aromatic (128,150ppm) and carboxyl carbon (172 ppm).

to aromatic rings as methyl substituents. These experiments were performed to model the coalification process but are also applicable to soil processes under anaerobic conditions. They account nicely for the fact that the degree of substitution of aromatic rings in brown coals is much greater than that in woods12. If oxidation also occurs, it is possible to account for many of the structures in Schnitzer’s model. Because of their polarity, these materials will be very mobile and may be lost to the water table, especially in those cases where R’ = H (Fig. 5). One outstanding problem is that recent NMR studies suggest that the phenolic content of humic substances is much lower than that presumed earlier and predicted from Fig. 5; but if some oxidation of diphenolits occurs, cleavage of aromatic rings between quinone functionality yields cis, cis-muconic acid which may subsequently oxidise to carbon dioxide and/or polymerise. Either way, the decrease in phenolic content can be accounted for. Polymuconic acid is synonymous with polymaleic acid-like materials which have been proposed to be present in soils. The reduction pathway and coalification Solid state 13C NMR spectroscopy has been applied most extensively to the study of coal chemistry and much less in petroleum geochemistry. There are three reasons for this: first, because coal is itself an end product which requires processing, whereas the organic matter in shales (kerogen) is merely the precursor of petroleum. Secondly, coal is a generally in-

soluble solid; determination of its structure has prompted the application of sophisticated solid state techniques. In contrast, petroleum is a liquid and therefore more amenable to standard chromatographic and mass spectrometric analysis. Thirdly, coal is dominantly organic and can be analysed directly whereas, in shales, it is often necessary to first concentrate the kerogen. This section covers work on both coals and shales, since coal is merely the end-product of a particular type of depositional setting. Solid state r3C NMR has been used in two ways: (i) simply to provide a measure of the aliphatic/aromatic carbon content (the ‘degree of aromaticity’ parameter); (ii) to obtain more detailed functional group parameters, such as quantifying bridgehead (ring junction) aromatic carbons. This information has been used in a number of different ways, which are described below. The degree of aromaticity (JJ has been determined for a range of coals (type III kerogens), marine shales (type II kerogens) and lacustrine shales (type I kerogens). In general, lacustrine shale kerogens have f, < 20%2p ,13-15.Marine kerogens vary mostly from 20 to 50% in aromatic carbon content13y14but immature marine kerogens tend to have f, values of around 30%. In contrast, coals invariably have f, > 50%4, due to their greater lignin contribution. There is a good correlation between degree of aliphaticity (100-f,) and potential oil yield as measured by the Fischer assay16. In fact, the amount of aliphatic carbon in the TOC is directly proportional to the amount of hydrocarbons that can be generated

When R’=H.organics to water table

are

Lost through

run

off

Fig. 5. Pathways for decomposition of aromatic guaiacyl building units in gymnosperm lignin in soils.

30

by distillation (Fischer assay) or pyrolysis. The precise numerical relationship between aliphatic carbon content and carbon generated as oil has been explored13. In general, for every aliphatic carbon atom, slightly less than one carbon atom is converted into oil. The degree of aliphaticity is thus a good measure of source rock petroleum potential. The structure of the oil, however, is very dependent on the rate of heating, residence time, the type of kerogen and coal and the effects of mineral matter since these factors can affect the degree of aromatisation4’17. The degree of aromaticity in coals or kerogens increases as a result of increasing thermal stress, whether from natural or laboratory heating (see e.g. ref. 14). The rate of increase is highest during petroleum generation, as aliphatic hydrocarbons are cleaved off. A number of attempts have been made to subdivide further the aromatic and aliphatic portions of an NMR spectrum into individual functional groups (e.g. CH, groups or aromatic C-H). Most of the applications have been on coals394Y18719, but there has been some work on shales596. In many cases, the results are very difficult to compare, because the extent to which the structural analysis has been pushed is variable and the structural parameters calculated have been equally disparate. However, the most comprehensive attempts at structure elucidation have been those of Trewhella et al.’ and, more qualitatively, Witte et aL6. Recently, dipolar dephasing has been developed to detect very mobile petroleum-like entities trapped within an or anic matrix by cross-linking or other mechanismsz~2’. Similar sorts of information on mobility can be obtained using other NMR experiments’, and there seems to be considerable scope for studying petroleum reservoirs in rocks by NMR techniques. Conclusions Two sets of experiments now face the organic geospectroscopist. First, the development of easily interpretable structural information that tells us more than just simple functional and structural groups such as aromaticity, number of methyl or carboxy1 groups, etc. Progress has been made in measuring the degree of substitution of aromatic rings, and their cluster size. NMR intrinsically gives information on molecular motion as well as structural parameters and so one development might be enough to determine the range of molecular mobilities of different structural groups, such as mobile and rigid polymethylene. Molecular weight data and information on inhomogeneity, through estimating areas in

trendsin analyticalchemistry,vol. 9, no. I,1990

which spin diffusion is operative, may tell us about various phases in geochemical samples. Success has been obtained in this area from the study of polymers. Second, there is scope for understanding the infinite variety of natural processes occurring in nature in which organic matter is transformed to coals, kerogen and soils. Even using current technology, few practical problems have been tackled, although vast amounts of money have been spent using other techniques to determine how petroleum is formed from kerogen or algal coals and why soil organic matter plays such an important role in changing both the physical and chemical properties of soils. The challenge is there for the geospectroscopist employing NMR and the results should be of great practical importance. Acknowledgements We would like to thank Iain Poplett (BP Research) for acquiring the NMR data shown in Fig. 1, Iain Poplett and Mike Taylor (BP Research) for helpful comments and BP Research for permission to publish this review. References 1 V. J. Bartuska, G. E. Fuel, 56 (1977) 354. 2 H. A. Resing, A. N. (1978) 450. 3 P. D. Murphy, T. J. (1982) 1233, 4 M. A. Wilson, NMR

Maciel, J. Schaefer and E. 0. Stejskal, Garroway and R. N. Hazlett, Fuel, 57 Casady and B. C. Gerstein,

Fuel, 61

Techniques and Applications in Geochemistry and Soil Chemistry, Pergamon, Oxford, 1987. 5 M. J. Trewhella, I. J. F. Poplett and A. Grint, Fuel, 65 (1986) 541. 6 E. G. Witte, H. J. Schenk, P. J. Mueller and K. Schwochau,

Org. Geochem., 13 (1988) 1039. 7 P. Tekely, D. Nicole, J. Brondeau and J.J. Delpuech, J. Phys. Chem., 90 (1986) 5608. 8 N. K. Sethi, R. J. Pugmire, J. C. Facelli and D. M. Grant, Anal. Chem., 60 (1988) 1574. 9 K. W. Zilm and G. G. Webb, Fuel, 65 (1986) 721. 10 M. Schnitzer and S. U. Khan, Humic Substances in the Environment, Marcel Dekker, New York, 1972. 11 P. G. Hatcher, M. Schnitzer, A.M. Vassallo and M. A. Wilson, Geochim. Cosmochim. Acta, 53 (1989) 125. 12 P. G. Hatcher, Energy Fuels, 2 (1988) 48. 13 F. P. Miknis, D. A. Netzel, J. W. Smith, M. A. Mast and G. E. Maciel, Geochim. Cosmochim. Acta, 46 (1982) 977. 14 A. J. G. Barwise, A. L. Mann, G. Eglinton, A. P. Gowar, A. M. K. Wardroper and C. S. Gutteridge, Org. Geochem., 6 (1984) 343. 15 S. Derenne, C. Largeau, E. Casadevall and F. Laupetre,

Fuel, 66 (1987) 1084. 16 G. E. Maciel, V. J. Bartuska and F. P. Miknis, Fuel, 57 (1978) 505. 17 F. P. Miknis, T. F. Turner, L. W. Ennen and D. A. Netzel, Fuel, 67 (1988) 1568. 18 K. D. Schmitt and E. W. Sheppard, Fuel, 63 (1984) 1241.

trendsi; analyticalchemistry,vol. 9, no. I,1990

19 D. E. Lambert and M. A. Wilson, ACS Div. Fuel Chem., 30 (1985) 256. 20 A. Soderquist,

D. J. Burton, R. J. Pugmire, A. J. Beeler, D. M. Grant, B. Durand and A. Y. Hut, Energy Fuels, l(l988) 50. 21 M. A. Wilson, R. J. Pugmire, K. W. Zilm, K. M. Goh, S. Heng and D. M. Grant, Nature, 294 (1981) 648. Dr. Richard L. Patience is a Senior Geochemist with BP. After completing a PhD in organic geochemistry at the University of Bristol in 1979, he left the subject for many years and only re-

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turned to the geochemical fold in 1985 on joining BP. He is currently a member of the editorial boards of the Journal of Chromatography and the Journal of Geochemical Exploration, and a contributing editor of TrAC. Dr. Michael Wilson currently holds a joint position as Senior Principal Research Scientist at CSIRO Division of Coal Technology and Senior Lecturer at the University of New South Wales, Sydney, Australia. Dr. Wilson is a member of the editorial board of the journal Organic Geochemistry and author of over 130 papers including the monograph ‘NMR Techniques and Applications in Geochemistry and Soil Chemistry’.

HPLC Analysis of Basic Drugs Series. IV

Use of silica with reversed-phase type eluents for the analysis of basic drugs and metabolites Brian Law Macclesfield, U.K. The use of silica with simple methanol-buffer eluents offers a powerful and versatile approach to the analysis of a wide range of basic drugs and their metabolites. Chromatographic per$ormance is good and the retention characteristics, particularly of drug metabolites, can be easily predicted. The system is particularly suited to bioanalysis, offering good selectivity with respect to endogenous compounds.

Introduction In a number of areas of drug analysis, e.g. emergency drug screening and forensic toxicology, there is a requirement for versatile chromatographic methods which can be rapidly applied to bioanalytical problems. To fulfill this need a wide range of chromatographic methods [thin-layer chromatography, gas chromatography and high-performance liquid chromatography (HPLC)] have been developed for the analysis of the various therapeutic drug classes’. A similar problem exists in the area of pharmaceuticals discovery and development. The problem here is further compounded by the novelty of the drug materials studied. In the early stages of pharmaceuticals discovery, it is often necessary to provide absorption, metabolic and pharmacokinetic data on a range of potential drug candidates. To

have maximum impact on the drug discovery programme, this information needs to be fed back to the medicinal chemist as rapidly as possible. A lengthy method development process is obviously out of the question, and this is occasionally made less desirable by the need to assay only a small number of samples. What is required therefore are ‘off-the-shelf’ HPLC methods, applicable to a wide range of drug types and metabolites, that can be rapidly and effectively applied with the minimum development . For the analysis of basic compounds (pK, I 8) the use of silica with reversed-phase type eluents would appear to be the method of choice. In a number of respects this approach offers several advantages over the use of reversed-phase (RP) HPLC which frequently requires complex strategies, e.g. ion-pair chromatography, or the use of silanol masking agents, to ensure good chromatographic performance . Compared with these reversed-phase based systems for example, the eluents used with silica are relatively simple, usually consisting of an organic solvent, e.g. methanol and an aqueous buffer. Although a number of variants have been used (e.g. refs. 2-7), the preferred eluent in this laboratory is methanol-ammonium acetate buffer (9:1), pH 9.1, the buffer being prepared from ammonia (65 ml, 25%) acetic acid (11 ml) and water (924 ml). De-