The thermal evolution of sporopollenin
B.L. Yule, S. Roberts *, J.E.A. Marshall
School of Ocean and Earth Science, Southampton Oceanography Centre, University of Southampton, European Way, Southampton, SO14 3ZH, UK
Received 12 January 1999; accepted 3 May 2000 (returned to author for revision 10 March 1999)
Abstract
Micro Fourier-Transform Infrared (FT±IR) spectroscopy in combination with transmitted and re¯ected light microspectrophotometry relates the chemical and physical properties of sporopollenin during thermal maturation; the physical properties measured being colour, as the chromaticity coordinatesa*,b* andL*, (luminance) and the re¯ec-tance (Rsp) of the sporinite wall layers in the polished section. During maturation, sporopollenin exhibits a wide range of colours before there are any signi®cant changes inRsp. The immature phase is characterised by subtle colour changes through a series of progressively darkening yellows. This coincides with a reduction in the relative proportion of >CO groups and an increase in the relative proportion of aliphatic ±CH2 and ±CH3groups. During the mature phase, functional groups within spores and pollen are thermally cracked to generate hydrocarbons. Their colours change rapidly through orange and brown and the FT±IR data indicate the loss of a considerable portion of the ali-phatic groups and increases in the CC content associated with aromatic rings. Signi®cant structural reorganisation during the spore `oil-window' results in the formation of isolated aromatic rings. A further increase in maturity yields little change in colour but a rapid increase in re¯ectivity. This is caused by the formation of multi-ring aromatic units from isolated aromatic units. The size of these polyaromatic units increases with rank. Investigation of arti®cially matured samples of Lycopodium clavatum spores indicates considerable chemical dierences in >CO, CC and aromatic skeletal structure, in comparison to fossil palynomorphs, although they progress through a similar series of colours. Only the behaviour of the aliphatic CH2, CH3groups, in arti®cially heated samples replicates that seen in samples matured naturally, under geological conditions.#2000 Elsevier Science Ltd. All rights reserved.
Keywords:Sporopollenin; Micro FT±IR; Spores; Pollen
1. Introduction
The outer wall of spores and pollen contains a highly resistant biomacromolecule, sporopollenin, which can survive in geological strata over millions of years with full retention of morphology (Brooks and Shaw, 1978). However, the eects of diagenesis (most importantly temperature and pressure) cause chemical and structural modi®cation. Thermal maturation of the host sediments is re¯ected by the colour change spores and pollen exhibit with increasing depth of burial. Although spore
colour changes are well documented (Staplin, 1969; Fisher et al.,1980; Pearson, 1982; Marshall, 1991; Yule et al., 1998) there is little understanding of how the underlying chemical changes control the physical prop-erties (colour, re¯ectance and ¯uorescence).
Various bulk geochemical analytical techniques have been used to investigate the chemical changes occurring in sporopollenin with progressive rank increase. Infra-red analysis (Brooks, 1971) and 13C nuclear magnetic resonance spectroscopy (Hemsley et al., 1992, 1993, 1995, 1996) showed a loss of aliphatic and oxygen con-taining functional groups as well as increasing aromati-city on increasing thermal maturation. However, these studies used either arti®cially matured samples, heated in conditions quite unlike those occurring in the geological
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* Corresponding author.
environment, or bulk concentrated spore/pollen sam-ples. Concentrating pure spores and pollen from kero-gen samples is not straightforward and often results in a heterogeneous mixture, which includes cutinite, resinite and vitrinite. Furthermore, bulk samples take no account of the chemical dierences between individual spores and pollen. Various pyrolytic and non-selective degradation techniques have also been used to study sporopollenin (Dungworth et al. 1971; Schenck et al. 1981; Davis et al. 1985; van Bergen et al. 1995). The yield of constitutional moieties varies, including n -alkanes,n-alk-1-enes,a,o-alkadienes, alkylphenols and
benzaldehydes, and depends on: (i) the original chemical structure of the sporopollenin; and (ii) the pyrolysis technique applied. Although these products represent, to a certain extent, the initial chemical structure, they reveal no information regarding the connection of the various structural units to each other and may have been formed by secondary reactions.
More recently, analytical techniques have become available which enable the chemical characterisation of individual spores and pollen. Mastalerz et al. (1993) and Mastalerz and Bustin (1993) used re¯ectance micro-Fourier transform infrared spectroscopy to investigate the evolution of maceral chemistry with rank. Spor-opollenin displayed distinct carboxyl/carbonyl and ali-phatic bands at low rank, which disappeared at higher maturities, alongside dramatic increases in the aromatic content. Cody et al. (1996) used soft X-ray imaging and carbon near-edge-absorption ®ne structure spectroscopy (C-NEXAFS) for the in situ analysis of sporinite in a rank variable suite of organic rich sediments. A sig-ni®cant change in unsaturated carbon environments (presumably aromatic) and losses of aliphatics and hydroxylated aliphatic carbon components was again noted. Additionally, in contrast to previous work, it was shown that carboxyl groups are only present in low and variable concentrations. Cody et al. (1996) suggested that the structural evolution of sporopollenin during diagenesis involved sequential dehydration, Diels±Alder cycle-addition, and dehydrogenation leading to a pro-gressively aromatized bio/geopolymer.
How these chemical changes control sporopollenin colour change is still unknown. Saxby (1982) suggested that yellow to orange spore colours represent the breakdown of carboxyl groups in acids and esters, brown corresponds to oil evolution and black occurs at the point where aliphatic and aromatic carbon bonds break to form methane. However, no chemical analyses were undertaken to verify these hypotheses.
In this paper micro FT±IR spectroscopy, in associa-tion with re¯ected and transmitted light spectrometry, was used in an attempt to link the physical and chemical properties of individual spores. This work is signi®cant for oil generation/thermal maturation studies because there can be considerable variation in the optical
prop-erties of kerogen particles (e.g. vitrinite re¯ectance, spore colour, spore ¯uorescence) within single samples (Rimmer et al., 1989; Marshall, 1991). Hence, an `aver-age' chemical composition from bulk kerogen analysis is neither representative nor comparable to optical mea-surements from single kerogen components. This study links optical thermal maturity measurements during progressive thermal maturation, to the microchemical changes of the individual palynomorphs. This success-fully provides an understanding of the chemical and physical evolution of sporinite at a microscopic level.
2. Equipment and measurement
2.1. Colour measurements
Colour measurements were made using a Zeiss UMSP 50 (universal microspectrophotometer) linked to a com-puter for data acquisition and processing. Illumination was from a 100 W tungsten bulb with a colour tem-perature of 3400 K modi®ed by a conversion ®lter to the 2854 K required for the standard illuminant ``A'' of the Commission Internationale de l'Eclairage (CIE) colour system. The microscope was controlled from the com-puter using a basic programme (`CP', written by J. E. A. Marshall and J. A. Milton). Transmittance measure-ments were taken at 10 nm steps over the range 400±750 nm with a monochrometer slit width of 20 nm and the `CP' programme calculated the 1976 CIE colour co-ordinatesa*,b* andL* from the visible spectrum.
The system is calibrated by taking a measurement through the slide and mounting medium, placed between the light source and the photomultiplier tube. The measurement, therefore, records 100% transmit-tance across the spectrum and de®nes the L* value of the illuminant. Since the measured illumination is monochromatic, the actual power distribution of the halogen bulb is not required. Measurement was taken across an area of the spore using a40 air objective and a 10mm spot diameter. The area chosen (80mm2) was
a homogeneous area of the spore, for example, the sac-cus of a pollen grain or in the inter-radials between the suturae of a simple trilete spore. Measurement was always made on the ubiquitous elements common to most micro¯oras such as bisaccate pollen or simple, single-walled spores or pollen.
2.2. Re¯ectance measurements
was used as for the colour measurements but with the monochrometer ®xed at 546 nm. All measurements were of random re¯ectance, in oil (RI 1.515%) and calibrated with arti®cial YAG (re¯ectance 0.919%) and spinel (re¯ectance 0.413%) standards. A linear calibration is present over the re¯ectance range 0.2±2% with a preci-sion greater than 2%.
2.3. Micro fourier-transform infrared (FT±IR) analysis
Infrared spectra were recorded using a Nicolet FT±IR spectrometer. The spectrometer is coupled to a Nicplan microscope (15 infrared objective) using a ProteÂgeÂTM 460 optical bench and a MCT- A detector which requires cooling with liquid nitrogen toÿ70C or below.
The system uses an Ever-Glo source and a KBr beams-plitter with a spectral range of 4000±600 cmÿ1. Infrared spectra were manipulated within OmnicTMsoftware.
The objective magni®cation gives an enlarged image of the sample at the plane of a diaphragm, which restricts the beam as a function of the aperture choice. The upper and lower adjustable apertures (redundant aperturing) allow control of the size and shape of the region to be analysed in the infrared beam. A constant area of 2020 mm was used for analysis. Altering the
area of analysis causes intensity changes in the spectra obtained but the ratio of the peaks relative to each other remains constant. The inferogram is recorded by accu-mulation of 200 scans in 100 s. If necessary, the number of scans can be increased to obtain a better signal to noise ratio. All reported spectra are transmission analy-sis following subtraction of the sample beam from the background (NaCl disc and air).
Spectra are represented in absorbance units as a function of the wavenumber (cmÿ1) in the 4000±600 cmÿ1range. The air in the system was not purged dur-ing the experiment resultdur-ing in CO2absorbance bands at 2400 cmÿ1; this absorbance has been subtracted from the spectrum. The FT±IR spectra are not plotted on a common Y-axis absorbance scale, instead a tool avail-able in the OMNIC software `matches' the Y-axis enabling the comparison of the relative intensities of spectral peaks. The assignments of the main IR char-acteristic group frequencies were primarily determined from the absorption bands of coals and kerogen (Rouxhet et al., 1980; Painter et al.,1982; Rochdi and Landais, 1991).
3. Samples
The fossil spores and pollen were obtained from a variety of samples of Devonian to Cretaceous age (Table 1). These samples were chosen as a subset from a very large collection of demineralised kerogen residues. They are all rich in spores and/or pollen but do not
contain any signi®cant content of AOM (amorphous organic matter) which acts to suppress spore colour. Sampled lithologies are similar, all being dark coloured (grey-green to black) mudrocks. The samples encompass the entire maturity range that can be described by spore colours (i.e. from pale yellow to black).
All the geological samples were initially demineralised in hydrochloric (HCl) and then hydro¯uoric (HF) acids using standard palynological techniques (similar to Phipps and Playford, 1984). The post-HF residues were concentrated by sieving at 20mm. A single short
treat-ment in hot HCl, followed by rapid dilution and sieving was used to remove neoformed ¯uoride contamination. The resulting kerogen isolates were used for FT±IR analysis, colour measurement in transmitted light and sporinite and vitrinite re¯ectance measurement in inci-dent light using polished thin sections.
3.1. Arti®cially matured samples
Observations were made on a set of arti®cially maturedLycopodium clavatumspores. The samples used were those of Marshall (1991). Arti®cial maturation of the modernLycopodiumspores was achieved by heating samples in covered porcelain crucibles, over a range of temperatures, for a constant time period of 60 h. Experimental runs were conducted at 25C intervals
from 50 to 300C with some additional samples at
intervals of 12 and 6C over the greatest range of colour
change.
4. Results
4.1. FT±IR analysis of naturally matured samples
The FT±IR spectra of six representative spores of various rank, whose sample maturity was assessed by vitrinite re¯ectance Rv, exhibit the major chemical changes that occur within palynomorphs during their thermal maturation (Fig. 1). During the early stages of maturity (example spectra atRv=0.4 and 0.67%, Fig. 1) spores and pollen progressively darken through a series of colours ranging from pale yellow to orange. The chemical changes occurring during this immature phase involve a reduction in the relative proportion of >CO groups and an increase in the relative proportion of ali-phatic ±CH2,±CH3 groups and C=C bonds (within or associated with aromatic rings). The skeletal vibration region (1400±700 cmÿ1) exhibits a number of low inten-sity vibrations which dier from spore to spore.
spores and pollen during this phase of maturity. The majority of the ±CH2 and ±CH3 aliphatic groups are lost as the spore wall breaks down to form hydro-carbons i.e. thermal cracking. Structural reorganisation is apparent from the signi®cant increase in the relative intensity of the CC (aromatic) band, the development of weak aromatic vibrations at 3025, 875, 823 and 755 cmÿ1 and the loss of the weak bands in the skeletal vibration region which form a wide peak centred around 1200 cmÿ1. This shows that a fundamental change in the molecular skeletal structure occurs at the spore `oil-window' as C±H and C±C bonds are ruptured and replaced by unsaturated CC bonds and aromatic rings.
By the post-mature phase (Rv>1.5%) all the spores and pollen within a sample are black. The palyno-morphs still retain some residual ±CH2,±CH3 and >CO groups within their structure. Aromatization and the concomitant growth of polyaromatic units with rank is apparent from the increase in the intensity of the aromatic 3025 cmÿ1 band, and the three peaks in the 800 cmÿ1 region caused by out of plane deformation vibrations of hydrogen atoms attached to aromatic rings (Rouxhet et al. 1980; Painter et al. 1982; Banwell, 1983; Rochdi and Landais 1991).
Increased adsorption within the hydroxyl region (3400 cmÿ1) gives an impression of increasing±OH content with rank. However, water may have become absorbed onto the spore surface during sample pre-paration since the samples are stored in MilliQ water. Before FT±IR analysis the spores are dried on glass
coverslips but water may easily become absorbed onto the particle surface during this process.
A method of representing the maturity of kerogen and bitumen samples from IR spectra was developed by Ganz et al. (1990), through the derivation ofAandC
factors from the recorded spectrum:
Aÿfactor 28652925cm ÿ1
286529251630cmÿ1
ratio of intensities of aliphatic=aromaticaliphatic bands
Cÿfactor 1705cm ÿ1
17051630cmÿ1
ratio of intensities of carboxyl=carbonylaromatic bands
Although adsorbance is proportional to the number of molecules irradiated, deriving quantitative informa-tion on the relative proporinforma-tions of funcinforma-tional groups in the molecular structure, is complicated by dierent fac-tors. These factors include, transition probability (like-lihood of the system changing state from one to another), the concentration or path length (the larger the sample the more energy is absorbed from the IR beam) and the polarizability of the bond (the more polar a bond the more intense will be the spectrum arising from the vibration). For these reasons theA- and
C-factors are therefore not the ratio of the aliphatic/ aromatic and >CO/aromatic contents, but the ratios of the intensities of the FT±IR bands (Ganz et al., 1990). Table 1
Location, stratigraphical age and thermal maturity of outcrop samples. Thermal maturity measured are vitrinite re¯ectance (Rv) and spore colour CIE chromaticity co-ordinatesa*,b* andL*a
Sample No. Grid reference and location Formation/age Rv% a* b* L* Colour
D8657.42 James Ross Island, Antarctic Peninsula Santa Marta Fm, Cretaceous 0.22 4.0 19.4 91.78 Pale yellow D8653.28 James Ross Island, Antarctic Peninsula Santa Marta Fm, Cretaceous 0.27 6.3 20.5 88.33 Pale yellow D8659.35 James Ross Island, Antarctic Peninsula Santa Marta Fm, Cretaceous 0.37 6.5 21.7 88.55 Pale yellow Yaverland 2 SZ 618 853, Isle of Wight, UK Vectis Fm. Cretaceous 0.37 10.5 27.3 84.76 Pale lemon yellow Watchet 14 ST 083 435, Watchet, Somerset, UK Westbury Fm, Late Triassic 0.40 6.4 22.8 90.37 Pale yellow Inn 23 NM 694 454, Loch Aline, Scotland Pabba Beds, Jurassic 0.56 8.5 23.6 87.98 Pale yellow Inn 37 NM 694 454, Loch Aline, Scotland Pabba Beds, Jurassic 0.67 9.6 23.8 82.98 Lemon yellow mv 87/66 NO 545 375, Midland Valley, Scotland Gedinnian, Early Devonian 0.73 20.6 32.2 61.56 Orange Papa 18.5 HU 1715 5905, Papa Stour, Shetland Eifelian, Mid Devonian 0.75 17.8 23.6 58.79 Orange mv 87/64 NO 538 394, Midland Valley, Scotland Gedinnian, Early Devonian 0.77 20.2 28.6 58.15 Orange Inn 8 NM 070 435, Inninmore, Scotland Westphalian B, Carboniferous 0.78 15.5 26.5 63.46 Orange Inn 9 NM 070 435, Inninmore, Scotland Westphalian B, Carboniferous 0.80 13.8 29.5 71.17 Yellow orange mv 88/1 NO 730 820, Midland Valley, Scotland Gedinnian, Early Devonian 0.96 19.2 26.9 58.8 Orange mv 87/88 NO 446 367, Midland Valley, Scotland Gedinnian, Early Devonian 1.04 14.8 12.6 50.87 Orange brown mv 87/91 NO 441 370, Midland Valley, Scotland Gedinnian, Early Devonian 1.04 18.8 18.6 48.19 Orange brown mv 87/89 NO 441 370, Midland Valley, Scotland Gedinnian, Early Devonian 1.09 17.4 13.2 40.67 Dark brown mv 85/44 NN 656 019, Midland Valley, Scotland Emsian, Early Devonian 1.28 14.9 6.3 41.01 Dark brown Foula 56 HT 960 415, Foula, Shetland Eifelian, Mid Devonian 1.50 10.4 2.0 37.37 Dark brown Foula 57 HT 958 414, Foula, Shetland Eifelian, Mid Devonian 1.80 11.9 6.0 32.78 Black
Given the limitations described, sample average A -andC-factors have been calculated from approximately 25 spore/pollen individuals per sample and are plotted against sample averageRv(Fig. 2) to observe the change in the two parameters with rank. TheC-factor displays little change during the early stages of maturity (Rv=0.3± 0.8%) which is possibly a consequence of variable
>CO content (as observed by Cody et al., 1996). The next maturity step (Rv=0.8±1.2%) shows a sudden drop in C-factor from 0.5 to 0.3. At higher rank (Rv>1.5%), theC-factor appears to increase slightly with rank.
in maturity (Rv=0.8±1.2%) shows a signi®cant decrease inA-factor from 0.8 to 0.35 as the palynomorphs ther-mally crack. In post-mature samples (Rv>1.2%), theA -factor continues to decrease but at a diminished rate.
The sudden decrease in A-factor from 0.8 to 0.35 occurs over a narrow maturity range, so spore thermal cracking is clearly a rapid event. The decrease in theA -factor is much larger than that observed in C--factor because signi®cant losses in aliphatic groups coincide with increases in CC content. The A-factor values shown in Fig. 2 are sample averages, but in the mature samples (Rv=0.8±1.2%), the spores and pollen usually have either high intensity CH2and CH3peaks relative to the CC band or low intensity CH2and CH3peaks relative to a very intense CC band. Notably, spores and pollen rarely have anA-factor between 0.3 and 0.4 (9 out of the 200+ spectra processed). This A-factor range may represent an unstable chemical intermediate because the removal of aliphatic groups and the forma-tion of aromatic rings occurs simultaneously.
4.2. Relationship between A and C-factor and colour
Spores change colour with thermal maturation through a series of colours, which can be measured quantitatively, using the 1976 CIE luminance (L*) values (Milton, 1993): see Table 2. The relationship between the colour (L*) and chemistry (A- andC -fac-tor) of individual palynomorphs is shown in Fig. 3.
Measurements were taken from spores and pollen covering the entire maturity range (L*=90±30).
For luminance (L*) versus A-factor, spore colour changes initially through a progressive yellow trend, Fig. 2. Plot of sample meanAandC-factors versus the vitrinite
re¯ectance of the sample. BothAandC-factors drop over the vitrinite re¯ectance range Rv=0.80±1.04% representing the spore `oil-window'. The drop occurs over a narrow maturity range indicating that spore thermal cracking is clearly a rapid event.
Table 2
Luminance measurements from a set of commercial (SCI) spore colour standards showing relationship between luminance (L*) and colour (Milton 1993)a
LuminanceL* Colour
90 Pale yellow
85 Pale-lemon yellow
80 Lemon yellow
75 Golden yellow
70 Yellow orange
60 Orange
50 Orange brown
40 Dark brown
35 Dark brown to black
30 Black
a Colour names from Fisher et al., 1980.
where colour changes are subtle (L* 90±70) to a post-mature stable brown trend (L* 40±25). The abrupt col-our change between the two trends (L* 70±40), marks the breakdown of the spore wall to generate hydro-carbons (Yule et al., 1998). The immature spores plot as an initial cluster of points (Area 1, Fig. 3) where L* drops progressively with little change inA-factor [initial increases in the sample averageA-factor with rank (Fig. 2) are not apparent on this graph]. At the onset of spore thermal cracking (A-factor0.7) the range ofA-factor values decreases signi®cantly within the mature samples where the colour of spore can range from dark yellow to brown.
An A-factor of 0.3 (Area 2, Fig. 3) marks post-maturity of the palynomorphs and the relationship between colour and chemistry returns with a narrow range ofA-factor values (0±0.3). Fig. 3 illustrates a clear interval of chemical instability (A from 0.3 to 0.7) dur-ing the spore `oil-window' as the palynomorphs undergo a rapid transition from a stable yellow trend to a stable brown trend, with a concomitant variation inA-factor.
No strong relationship exists betweenL* andC -fac-tor (Fig. 3b),C-factor decreases alongside decreases in
L*. The stronger relationship between A-factor and colour than between C-factor and colour suggests that aliphatic groups play a more important role in spore colour change than oxygen containing functional groups.
4.3. FT±IR analysis of arti®cially matured Lycopodium clavatum samples
The FT±IR spectra of arti®cially maturedLycopodium
(Fig. 4) display similar bands to those observed in fossil sporopollenin (CH2, CH3, >CO, CC) but also con-tain a number of other bands e.g. 1520 cmÿ1. The range of amide NH3+ group, (>NH, C±NO2, C±NO) and secondary amides vibrations all overlap 1520 cmÿ1 (Banwell, 1983) so a con®dent assignment of this band cannot be made. Lycopodium spectra also contain a wide range of bands in the region 1400±700 cmÿ1. These may result from vibrations of the whole molecule which are characteristic of the skeletal structure, however, some functional groups can also vibrate in this region e.g.±OH deformations in alcohols and phenols, SO2, SO4and PO groups (Banwell, 1983).
Chemical changes are observed in the Lycopodium
spores with increasing thermal maturation. During the early stages of maturation (25±150C) there are
increa-ses in the relative intensities of CH2, CH3and >CO bands but no change in the bands due to CC bonds. The IR bands that are not present in the fossil samples rapidly diminish and the nitrogen containing functional group, most likely responsible for the peak at 1520 cmÿ1,decreases in intensity. The bands in the skeletal vibration region decrease in intensity until they are no
longer discernible, merging into a broad peak centred around 1100 cmÿ1. These bands are not present in any of the geological samples, so during natural conditions these changes must occur either before burial or very early on in the burial history. The thermal cracking of
Lycopodium is evident between 206 and 212C by a
sudden reduction in the aliphatic content coinciding with an increase in CC and >CO groups. By 250C
the aliphatic bands are dicult to resolve.
Considering that the Lycopodium spores progress through the same series of colours as naturally matured samples (Marshall, 1991; Yule et al., 1998) the change in spectral characteristics of the arti®cially heated sam-ples show some considerable dierences to palyno-morphs heated geologically. The Lycopodium spores retain a high >CO content throughout maturation, possibly a consequence of sample oxidation during heating. TheLycopodiumspores heated to 250C are the
same colour (black) as the most mature geological sam-ples (Rv>1.5%) however, the FT±IR spectra show that there are fundamental dierences between their chemi-cal structures. The post-mature, naturally matured samples retain a signi®cant portion of aliphatic groups, apparent from small peaks at 2925, 2865 and 1445 cmÿ1. Conversely, the 250C Lycopodium sample
shows a signi®cant reduction in its aliphatic content. Additionally, even though the arti®cially matured sam-ples display increases in the CC aromatic band with maturity, it does not reach the relative intensity seen in naturally matured samples. Furthermore, the
Lycopodium samples do not develop the strong bands observed in post-mature geological samples indicative of aromatic structures (3040, 886, 830 and 758 cmÿ1). This suggests that the development of aromatic bonds may require slow structural reorganisation over geolo-gical time, rather than heating over a 60 h period in a laboratory.
Dierent chemical reactions occur under oxidative experimental conditions compared to those occurring under natural geological conditions. This has implica-tions for the pyrolysis experiments which are used to derive kinetic reaction constants for thermal maturation models. For the values of activation energies and fre-quency factors derived experimentally to have any rele-vance geologically, the same reaction must be occurring in both environments and by the same mechanism. However, at `fast' laboratory heating rates dierent sporopollenin maturation reactions are clearly taking place.
4.4. Spore colour and re¯ectance
The re¯ectance and colour of individual spores and pollen were measured to investigate the relationship between these two physical properties. The evolution of spore colour and re¯ectance with maturity is related to a
conversion of the structure to saturated rings after the breaking of C±C and C±H bonds. Conversely, the re¯ectance of a maceral depends upon its refractive and absorptive indices. Refractive index is a function of atomic density and increases with aromatization. Absorption, however, is dependent on the size of the aromatic units and increases with condensation (Carr and Williamson, 1989). Colour and re¯ectance values, therefore, provide dierent but complementary infor-mation on the structure of the sporopollenin.
The re¯ectance of a palynomorph was measured using the microspectrophotometer which was then recon®gured for transmitted light to measure the colour of the same individual spore. A strong relationship is
observed between L* and Rsp (Fig. 5) and two trends are apparent on the graph. At lower maturity there is a large decrease in L* (95±70) coinciding with a slight increase in Rsp (0.05±0.15), with the data points nar-rowly constrained. During this immature phase, the FT±IR data show that there is gradual loss of >CO groups and initial increase in the relative proportion of aliphatic groups causing a slow and subtle change in colour. Further maturation (L* 70±50), involves the loss of a signi®cant proportion of aliphatic groups with the colour changing rapidly through oranges and browns. There are only slight increases in re¯ectance over this region but the FT±IR data show a signi®cant increase in the proportions of CC bonds associated with aromatic
rings. As re¯ectance is dependent on the number of aromatic rings within a structural unit, the rise in CC aromatic bonds may be caused by the formation of iso-lated aromatic rings (initiated by the rupture of C±C, C± H bonds), as re¯ectance is unlikely to be aected by the formation of single rings. Hence the slight increase in
Rsp from 0.05 to 0.2 may be the result of limited for-mation of multi-ring aromatic units.
At higher maturity there is a change in gradient on the graph corresponding to a rapid increases in Rsp (0.15±1.6) occurring alongside slight decreases in L* (50±30), and an increased variance of data points; pos-sibly a consequence of within spore heterogeneity at higher maturity (e.g. atRsp>0.6%, error in Rsp0.2%). Considerable spore colour and chemical changes occur before there are any signi®cant increases inRspand the optimum oil-generation of sporopollenin is apparently reached before the rapid increase of Rsp. The sudden increase in Rsp occurs towards the end of the mature phase, after the CC bonds have been ruptured to form hydrocarbons and the resulting structure contains unsaturated bonds in aliphatic chains between isolated aromatic rings. Multi-ring structural units could be formed from isolated aromatic rings if the remaining bonds between the rings are converted into aromatic units. A molecular reorganisation of this kind would produce molecules with polycyclic aromatic units, which have a high re¯ectance and could account for the sud-den increase inRsp.
5. Discussion
Micro FT±IR spectroscopy in combination with transmitted and re¯ected light microphotometry relates the chemical and physical properties (colour and re¯ec-tance) of sporopollenin during thermal maturation. In particular, the FT±IR data demonstrate signi®cant che-mical alterations in the palynomorphs, especially during their `thermal cracking'. These progressive chemical changes in sporopollenin composition and chemical structure control the evolution of spore colour and re¯ectance.
The results suggest that aliphatic groups play an important role in colour change. However, aliphatic groups do not absorb in the visible region (Kemp, 1991). Absorption in the visible region occurs through the presence of molecules which contain various func-tional groups, termed chromophores, which have empty
p* orbitals into which electrons fromsorporbitals can be excited. Typical chromophores include CO, CC, NN and NO2 unsaturated groups (Banwell, 1983). Other substituents which are not themselves chromo-phores, modify the absorption of molecules containing chromophores e.g. CH3, Cl, NH2, OH and are termed auxochromes (Banwell, 1983). Hence, the CH3aliphatic
content may have an eect on spore colour by modify-ing the absorption of the CO and CC bonds in sporopollenin. Alternatively, the yellow to orange spore colours observed may be related to the strong ¯uores-cence of sporopollenin. Kemp (1991) noted that many organic molecules ¯uoresce and if the ¯uorescence `tails' into the visible spectrum it can absorb the violet end of the white light making the molecules appear yellow-orange in colour. At later stages of maturity, non-spec-tral colours such as brown are associated with absorp-tion distributed over a wide wavelength range, and black is the result of absorption throughout the visible spectrum. This is likely to be a consequence of CC bonds distributed, in a number of environments, throughout the sporopollenin structure. Further work is needed to con®rm if either ¯uorescence or CH3 mod-i®cation of CO and CC absorption is the principal control of spore colour change.
The FT±IR data show that arti®cially matured spores are chemically distinct from palynomorphs subjected to geological conditions. At high maturities there are fun-damental dierences in the molecular skeleton and the arti®cially matured samples do not develop the bonds indicative of an aromatic structure. These dierences highlight the problems of using samples thermally trea-ted in the laboratory to chemically represent those sub-jected to natural burial conditions. This is also true of models which use rapid pyrolysis at high temperatures to represent the reactions occurring in the geological environment.
Although micro FT±IR analysis from single palyno-morphs provides more de®nitive chemical analysis than experiments performed on bulk heterogeneous mixtures the data are still chemically averaged over the entire individual spore (resolution40mm2). Thus the
micro-FT±IR data do not account for possible chemical het-erogeneities within the spore itself. Ultrastructural stu-dies using scanning electron microscopy and transmission electron microscopy (Scott and Hemsley, 1993) showed that, in cross-section, `fossilised' spore walls are not always homogeneous, sometimes display-ing layers, lamina or globular units. The possibility of compositional variations within such units cannot at present be excluded.
variation in re¯ectance or chemistry, thus aecting the relationship between L*, Rsp and chemistry. This may explain the range of colour (L*) observed for a single value ofA-factor in Fig. 3. Additionally, the spores and pollen may have a dierent precursor chemistry as a consequence of early diagenetic eects. The physico-chemical-microbiological factors operating in the ®rst metre of burial are important in remoulding the spore pollen chemical structure. Such dierent conditions of oxidation, acidity, microbiological attack will cause chemical dierences between species depending on their susceptibility.
Sporopollenin maturation can be used as a proxy for oil-generation because the chemical compositional changes are similar to those occurring during the maturation of amorphous organic matter (the dominant component of oil-prone kerogen). Sporopollenin composition and optical properties show a step function at the oil-window and, unlike vitrinite, allow direct observation of the generation of liquid hydrocarbons.
6. Conclusions
Spores and pollen change colour from pale yellow to mid browns before there are any signi®cant changes in re¯ectance,Rsp. In the immature phase, colour changes are subtle and slow through a series of progressively darkening yellows. This coincides with a reduction in the relative proportion of >CO groups and an increase in the relative proportion of aliphatic CH2, CH3groups.
During the mature phase the chemical constituents of spores and pollen are thermally cracked to generate hydrocarbons. Their colour changes rapidly through a series of orange and brown colours and the FT±IR data show the loss of a considerable portion of the aliphatic groups and an increase in the CC content associated with the formation of aromatic rings. Signi®cant struc-tural reorganisation at the spore `oil-window' has initi-ated the formation of isoliniti-ated aromatic rings.
Further increases in maturity are re¯ected in a rapid increase in re¯ectivity (but little colour change). This is caused by the formation of multi-ring aromatic units from isolated aromatic units. The size of these polyaro-matic units increases with rank.
A set of arti®cially matured Lycopodium clavatum
spores show considerable chemical dierences (in >CO, CC and aromatic skeletal structure) to geo-logically matured samples even though they progress through the same series of colours. Only the behaviour of the aliphatic CH2, CH3 groups replicates that seen naturally. It is suggested that the aliphatic and CC (aromatic) content of spores and pollen appear to con-trol their colour.
Acknowledgements
BLY was supported by a joint studentship from Brit-ish Gas Research and Technology Division and the Sci-ence Faculty, University of Southampton. The authors acknowledge the constructive and careful reviews of M. Mastalerz and J. Potter.
Associate EditorÐM.G. Fowler
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