Directory UMM :Data Elmu:jurnal:O:Organic Geochemistry:Vol31.Issue9.2000:
Organic Geochemistry 31 (2000) 859±870
www.elsevier.nl/locate/orggeochem
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 coordinates a*, b* and L*, (luminance) and the re¯ectance (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 in Rsp. 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 ±CH3 groups. 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 aliphatic 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, CH3 groups, 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
* Corresponding author.
E-mail address: [email protected] (S. Roberts).
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 properties (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. Infrared analysis (Brooks, 1971) and 13C nuclear magnetic
resonance spectroscopy (Hemsley et al., 1992, 1993,
1995, 1996) showed a loss of aliphatic and oxygen containing functional groups as well as increasing aromaticity on increasing thermal maturation. However, these
studies used either arti®cially matured samples, heated in
conditions quite unlike those occurring in the geological
0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0146-6380(00)00058-9
860
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
environment, or bulk concentrated spore/pollen samples. Concentrating pure spores and pollen from kerogen 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 nalkanes, 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 microFourier transform infrared spectroscopy to investigate
the evolution of maceral chemistry with rank. Sporopollenin displayed distinct carboxyl/carbonyl and aliphatic 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 signi®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 progressively 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 association 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 `average' chemical composition from bulk kerogen analysis is
neither representative nor comparable to optical measurements from single kerogen components. This study
links optical thermal maturity measurements during
progressive thermal maturation, to the microchemical
changes of the individual palynomorphs. This successfully 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 computer for data acquisition and processing. Illumination
was from a 100 W tungsten bulb with a colour temperature 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 computer using a basic programme (`CP', written by J. E. A.
Marshall and J. A. Milton). Transmittance measurements 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 coordinates a*, b* and L* 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% transmittance 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 a 40 air objective and
a 10 mm spot diameter. The area chosen (80 mm2) was
a homogeneous area of the spore, for example, the saccus 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
Measurements of sporopollenin re¯ectance (Rsp) and
vitrinite re¯ectance (Rv) were made on polished thin
sections, prepared using the method of Hillier and
Marshall (1988). Re¯ectivity measurements were made
using standard methods (e.g. Davis, 1978; Durand et al.,
1986). The same microscope (the UMSP 50) equipment
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
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 precision 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 to ÿ70 C or below.
The system uses an Ever-Glo source and a KBr beamsplitter with a spectral range of 4000±600 cmÿ1. Infrared
spectra were manipulated within OmnicTM software.
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 accumulation 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 analysis 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
cmÿ1 range. The air in the system was not purged during the experiment resulting in CO2 absorbance 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 available in the OMNIC software `matches' the Y-axis
enabling the comparison of the relative intensities of
spectral peaks. The assignments of the main IR characteristic 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
861
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 20 mm. A single short treatment 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 incident light using polished thin sections.
3.1. Arti®cially matured samples
Observations were made on a set of arti®cially
matured Lycopodium clavatum spores. The samples used
were those of Marshall (1991). Arti®cial maturation of
the modern Lycopodium spores 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 25 C intervals
from 50 to 300 C with some additional samples at
intervals of 12 and 6 C 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 at Rv=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 aliphatic ±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 intensity vibrations which dier from spore to spore.
During the `mature' phase (as shown by the microFT±IR spectra from samples with Rv=0.8% and
0.96%) spores and pollen display rapid changes in colour from orange to mid-brown. Signi®cant chemical
changes and structural reorganisation occurs within the
862
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
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-ordinates a*, b* and L* a
Sample No.
Grid reference and location
Formation/age
Rv% a*
b*
L*
Colour
D8657.42
D8653.28
D8659.35
Yaverland 2
Watchet 14
Inn 23
Inn 37
mv 87/66
Papa 18.5
mv 87/64
Inn 8
Inn 9
mv 88/1
mv 87/88
mv 87/91
mv 87/89
mv 85/44
Foula 56
Foula 57
James Ross Island, Antarctic Peninsula
James Ross Island, Antarctic Peninsula
James Ross Island, Antarctic Peninsula
SZ 618 853, Isle of Wight, UK
ST 083 435, Watchet, Somerset, UK
NM 694 454, Loch Aline, Scotland
NM 694 454, Loch Aline, Scotland
NO 545 375, Midland Valley, Scotland
HU 1715 5905, Papa Stour, Shetland
NO 538 394, Midland Valley, Scotland
NM 070 435, Inninmore, Scotland
NM 070 435, Inninmore, Scotland
NO 730 820, Midland Valley, Scotland
NO 446 367, Midland Valley, Scotland
NO 441 370, Midland Valley, Scotland
NO 441 370, Midland Valley, Scotland
NN 656 019, Midland Valley, Scotland
HT 960 415, Foula, Shetland
HT 958 414, Foula, Shetland
Santa Marta Fm, Cretaceous
Santa Marta Fm, Cretaceous
Santa Marta Fm, Cretaceous
Vectis Fm. Cretaceous
Westbury Fm, Late Triassic
Pabba Beds, Jurassic
Pabba Beds, Jurassic
Gedinnian, Early Devonian
Eifelian, Mid Devonian
Gedinnian, Early Devonian
Westphalian B, Carboniferous
Westphalian B, Carboniferous
Gedinnian, Early Devonian
Gedinnian, Early Devonian
Gedinnian, Early Devonian
Gedinnian, Early Devonian
Emsian, Early Devonian
Eifelian, Mid Devonian
Eifelian, Mid Devonian
0.22
0.27
0.37
0.37
0.40
0.56
0.67
0.73
0.75
0.77
0.78
0.80
0.96
1.04
1.04
1.09
1.28
1.50
1.80
19.4
20.5
21.7
27.3
22.8
23.6
23.8
32.2
23.6
28.6
26.5
29.5
26.9
12.6
18.6
13.2
6.3
2.0
6.0
91.78
88.33
88.55
84.76
90.37
87.98
82.98
61.56
58.79
58.15
63.46
71.17
58.8
50.87
48.19
40.67
41.01
37.37
32.78
Pale yellow
Pale yellow
Pale yellow
Pale lemon yellow
Pale yellow
Pale yellow
Lemon yellow
Orange
Orange
Orange
Orange
Yellow orange
Orange
Orange brown
Orange brown
Dark brown
Dark brown
Dark brown
Black
a
4.0
6.3
6.5
10.5
6.4
8.5
9.6
20.6
17.8
20.2
15.5
13.8
19.2
14.8
18.8
17.4
14.9
10.4
11.9
Data sequenced in order of increasing Rv.
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 hydrocarbons 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 `oilwindow' 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 palynomorphs 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 preparation 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 of A and C
factors from the recorded spectrum:
2865 2925cmÿ1
2865 2925 1630cmÿ1
ratio of intensities of aliphatic=aromatic aliphatic bands
A ÿ factor
1705cmÿ1
1705 1630cmÿ1
ratio of intensities of carboxyl=carbonyl aromatic bands
C ÿ factor
Although adsorbance is proportional to the number
of molecules irradiated, deriving quantitative information on the relative proportions of functional groups in
the molecular structure, is complicated by dierent factors. These factors include, transition probability (likelihood 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 the A- 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).
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
863
Fig. 1. Comparison of selected micro- FT±IR spectra of spores from a range of maturities (Rv=0.4±1.8%). During the early stages of
maturation there is a reduction in the relative proportion of >CO bonds and an increase in the relative proportion of CH2/CH3
bonds. The mature phase, where the palynomorphs break down to generate hydrocarbons, is observed in the spectra with Rv=0.80%
and 0.96%. Structural reorganisation results in the loss of aliphatic groups and the development of aromatic structures. Further
increases in maturity result in increasing aromaticity.
Given the limitations described, sample average Aand C-factors have been calculated from approximately
25 spore/pollen individuals per sample and are plotted
against sample average Rv (Fig. 2) to observe the change
in the two parameters with rank. The C-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%),
the C-factor appears to increase slightly with rank.
The A-factor initially increases during the immature
phase (Rv=0.3±0.8%) as the relative proportion of CH2
and CH3 aliphatic groups increases. Subsequent increases
864
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
Table 2
Luminance measurements from a set of commercial (SCI) spore
colour standards showing relationship between luminance (L*)
and colour (Milton 1993)a
Luminance L*
Colour
90
85
80
75
70
60
50
40
35
30
Pale yellow
Pale-lemon yellow
Lemon yellow
Golden yellow
Yellow orange
Orange
Orange brown
Dark brown
Dark brown to black
Black
a
Colour names from Fisher et al., 1980.
Fig. 2. Plot of sample mean A and C-factors versus the vitrinite
re¯ectance of the sample. Both A and C-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.
in maturity (Rv=0.8±1.2%) shows a signi®cant decrease
in A-factor from 0.8 to 0.35 as the palynomorphs thermally crack. In post-mature samples (Rv>1.2%), the Afactor 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 the Afactor 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 CH2 and CH3 peaks relative
to the CC band or low intensity CH2 and CH3 peaks
relative to a very intense CC band. Notably, spores
and pollen rarely have an A-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 formation 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- and C-factor) of individual palynomorphs is shown in Fig. 3.
Fig. 3. Plot of CIELAB luminance (L*) versus A-factor, and
C-factor for samples containing the entire range of colours
displayed by palynomorphs during maturation (lemon yellow
to black). The relationship between L* and A-factor displays:
area 1 Ð an initial cluster of points indicating the immature
stable yellow trend where colour changes are subtle and slow
(L* 90±70). A scatter of data points indicating maturity as the
spore wall is thermally broken down and colour abruptly
changes. Area 2 Ð a ®nal cluster of points resulting from the
post-mature stable brown trend (L* 40±25). The relationship
between C-factor and L* produces signi®cant scattering of
points with a broad trend of decreasing C-factor with decreasing L*.
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,
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
where colour changes are subtle (L* 90±70) to a postmature stable brown trend (L* 40±25). The abrupt colour change between the two trends (L* 70±40), marks
the breakdown of the spore wall to generate hydrocarbons (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 in A-factor [initial
increases in the sample average A-factor with rank (Fig.
2) are not apparent on this graph]. At the onset of spore
thermal cracking (A-factor 0.7) the range of A-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 postmaturity of the palynomorphs and the relationship
between colour and chemistry returns with a narrow
range of A-factor values (0±0.3). Fig. 3 illustrates a clear
interval of chemical instability (A from 0.3 to 0.7) during 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 in A-factor.
No strong relationship exists between L* and C-factor (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 matured Lycopodium
(Fig. 4) display similar bands to those observed in fossil
sporopollenin (CH2, CH3, >CO, CC) but also contain a number of other bands e.g. 1520 cmÿ1. The range
of amide NH+
3 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,
SO4 and PO groups (Banwell, 1983).
Chemical changes are observed in the Lycopodium
spores with increasing thermal maturation. During the
early stages of maturation (25±150 C) there are increases in the relative intensities of CH2, CH3 and >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
865
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 212 C by a
sudden reduction in the aliphatic content coinciding
with an increase in CC and >CO groups. By 250 C
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 samples show some considerable dierences to palynomorphs heated geologically. The Lycopodium spores
retain a high >CO content throughout maturation,
possibly a consequence of sample oxidation during
heating. The Lycopodium spores heated to 250 C are the
same colour (black) as the most mature geological samples (Rv >1.5%) however, the FT±IR spectra show that
there are fundamental dierences between their chemical 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 250 C Lycopodium sample
shows a signi®cant reduction in its aliphatic content.
Additionally, even though the arti®cially matured samples 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 geological 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 implications for the pyrolysis experiments which are used to
derive kinetic reaction constants for thermal maturation
models. For the values of activation energies and frequency factors derived experimentally to have any relevance 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.
The aliphatic content of Lycopodium with maturation
displays the same initial increases in relative intensity
followed by sudden drops during thermal cracking as
the geological samples. As the Lycopodium samples
progress through the same series of colours as naturally
matured samples, the behaviour of the aliphatic groups
may play a role in colour change.
866
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
Fig. 4. Micro- FT±IR spectra of arti®cially heated Lycopodium spores. The observed spectral evolution that occurs with arti®cial heat
treatment of Lycopodium spores is dierent to that occurring naturally. In particular, the arti®cially heated samples do not develop the
aromatic peaks observed in natural samples of the same colour, also skeletal vibrations in the 1400±700cmÿ1 region are dierent. Only
the behaviour of their aliphatic groups mimics that seen in geological samples. For Rsp data see Marshall (1991).
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
progressive change of sporopollenin composition and
chemical structure. Colour and re¯ectance show contrasting behaviour with rank because the major chemical features in¯uencing the physical properties are
dierent. Spore colour appears to be most aected
during the elimination of functional groups and the
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
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 information 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
867
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 narrowly 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
Fig. 5. Plot of the relationship between the re¯ectance and colour of individual spores with increasing maturity. The L* and Rsp
measurement pairs were made on the same single individual spore specimens Two trends are evident from the graph: A large decreases
in L* (yellow series Ð build up of aliphatics; orange colours Ð loss of aliphatics and increase of CC in aromatic rings) coinciding
with a slight increase in re¯ectance. This is followed by a rapid increase in re¯ectance as the aromatic units coalesce to sheets but with
only slight changes in colour (browns). The evolution of spore colour and re¯ectance with maturity is therefore related to a progressive
change of sporopollenin composition and chemical structure.
868
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
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 isolated 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 formation 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; possibly a consequence of within spore heterogeneity at
higher maturity (e.g. at Rsp>0.6%, error in Rsp0.2%).
Considerable spore colour and chemical changes occur
before there are any signi®cant increases in Rsp and 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 sudden increase in Rsp.
5. Discussion
Micro FT±IR spectroscopy in combination with
transmitted and re¯ected light microphotometry relates
the chemical and physical properties (colour and re¯ectance) of sporopollenin during thermal maturation. In
particular, the FT±IR data demonstrate signi®cant chemical 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 functional groups, termed chromophores, which have empty
p* orbitals into which electrons from s or p orbitals can
be excited. Typical chromophores include CO, CC,
NN and NO2 unsaturated groups (Banwell, 1983).
Other substituents which are not themselves chromophores, modify the absorption of molecules containing
chromophores e.g. CH3, Cl, NH2, OH and are termed
auxochromes (Banwell, 1983). Hence, the CH3 aliphatic
content may have an eect on spore colour by modifying the absorption of the CO and CC bonds in
sporopollenin. Alternatively, the yellow to orange spore
colours observed may be related to the strong ¯uorescence 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 yelloworange in colour. At later stages of maturity, non-spectral colours such as brown are associated with absorption 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 modi®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 fundamental 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 treated in the laboratory to chemically represent those subjected 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 palynomorphs provides more de®nitive chemical analysis than
experiments performed on bulk heterogeneous mixtures
the data are still chemically averaged over the entire
individual spore (resolution 40 mm2). Thus the microFT±IR data do not account for possible chemical heterogeneities within the spore itself. Ultrastructural studies 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 displaying layers, lamina or globular units. The possibility of
compositional variations within such units cannot at
present be excluded.
Despite the correlation observed between spore
chemistry and colour, certain other factors may have
aected the observed relationships between the physical
and chemical properties of spores and pollen. For
example, no account of the spore/pollen species was
taken, the selection criteria included only simple, single
walled species. Chemical dierences between species
have been noted, sporopollenin is not a unique substance, each individual species being composed of a different biopolymer (Guilford et al., 1988; Hemsley et al.,
1992). Also those species with thicker exines will appear
darker (lower L*) but there will be no corresponding
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
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 of A-factor in Fig. 3. Additionally, the spores and
pollen may have a dierent precursor chemistry as a
consequence of early diagenetic eects. The physicochemical-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,
CH3 groups.
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 structural reorganisation at the spore `oil-window' has initiated the formation of isolated 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 polyaromatic 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 geologically 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 control their colour.
869
Acknowledgements
BLY was supported by a joint studentship from British Gas Research and Technology Division and the Science Faculty, University of Southampton. The authors
acknowledge the constructive and careful reviews of M.
Mastalerz and J. Potter.
Associate EditorÐM.G. Fowler
References
Banwell, C.N., 1983. Fundamentals of Molecular Spectroscopy, 3rd Edition. McGraw-Hill, London.
Brooks, J. , 1971. Some chemical and geochemical studies on
sporopollenin. In: Brooks, J. et al. (Eds.), Sporopollenin
Academic Press, London, pp. 351±407.
Brooks, J., Shaw, G., 1978. Sporopollenin: a review of its chemistry, palaeochemistry and geochemistry. Grana 17, 91±97.
Carr, A.D., Williamson, J.E., 1989. The relationship between
aromaticity, vitrinite re¯ectance and maceral composition of
coals: Implications for the use of vitrinite re¯ectance as a
maturation parameter. In: Durand, B., Behar, F. (Eds.),
Advances in Organic Geochemistry 1989, Organic Geochemistry. Pergamon, Oxford. vol. 16, pp. 313±323.
Cody, G.D., Botto, R.E., Ade, H., Wirick, S., 1996. The
application of soft X-ray microscopy to the in-situ analysis
of sporinite in coal. International Journal of Coal Geology
32, 69±86.
Davis, A., 1978. The re¯ectance of coal. In: Karr, C. (Ed.),
Analytical Methods for Coal and Coal Products. Academic
Press, London, New York, pp. 27±81.
Davis, M.R., Abbott, J.M., Gaines, A.F., 1985. Chemical
structure of telocollinites and sporinites. Dierentiation
between telocollinites and sporinites by the aromatic structures present in their pyridine extracts. Fuel 64, 1362±1369.
Dungworth, G., McCormick, A., Powell, T.G., Douglas, A.G.,
1971. Lipid components in fresh and fossil pollen and spores.
In: Brooks, J. et al. (Eds.), Sporopollenin. Academic Press,
New York, pp. 512±544.
Durand, B., Alpern, B., Pittion, J.L., Pradier, B., 1986. Re¯ectance of vitrinite as a control of thermal history of sediments.
In: Burrus, J. (Ed.), Thermal Modelling in Sedimentary
Basins. Editions Technip, Paris, pp. 414±474.
Fisher, M.J., Barnard, P.C., Cooper, B.S., 1980. Organic
maturation and hydrocarbon generation in the Mesozoic
sediments of the Sverdrup Basin, Arctic Canada. Proceedings
IV International Palynological Conference, Lucknow (197677) 2, 581±588.
Ganz, H.H., Kalkreuth, W., Ganz, S.N., OÈner, F., Pearson,
M.J., Wehner, H., 1990. Infrared analysis Ð state of the art.
Berliner Geowissenschaftliche Abhandlungen 120, 1011±
1026.
Guilford, W.J., Schneider, D.M., Labovitz, J., Opella, S.J.,
1988. High-resolution solid 13C NMR spectroscopy of sporopollenins from dierent plant taxa. Plant Physiology 86,
134±136.
Hemsley, A.R., Chaloner, W.G., Groombridge, C.J., Scott,
A.C., 1992. Carbon-13 solid state nuclear magnetic resonance
870
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
of sporopollenins from modern and fossil plants. Annals of
Botany 69, 545±549.
Hemsley, A.R., Barrie, P.J., Chaloner, W.G., Scott, A.C. 1993.
The composition of sporopollenin and its use in living and
fossil plant systematics. Grana. (Supplement 1), 2±11.
Hemsley, A.R., Barrie, P.J., Scott, A.C., 1995. 13C solid-state
NMR spectroscopy of fossil sporopollenins: variations in
composition independent of diagenesis. Fuel 74, 1009±1012.
Hemsley, A.R., Scott, A.C., Barrie, P.J., Chaloner, W.G., 1996.
Studies of fossil and modern spore wall biomacromolecules
using 13C solid state NMR. Annals of Botany 78, 83±94.
Hillier, S.J., Marshall, J.E.A., 1988. A rapid technique to make
polished thin sections of sedimentary organic matter concentrates. Journal of Sedimentary Petrology 58, 754±755.
Kemp, W., 1991. Organic Spectroscopy, 3rd Edition. Basingstoke, Macmillan.
Marshall, J.E.A., 1991. Quantitative spore colour. Journal of
the Geological Society, London 148, 223±233.
Mastalerz, M., Bustin, R.M., 1993. Variation in maceral
chemistry within and between coals of varying rank: an electron microprobe and micro-FTIR investigation. Journal of
Microscopy 171, 153±166.
Mastalerz, M., Wilkes, K.R., Bustin, R.M., 1993. Variation in
vitrinite chemistry as a function of associated liptinite content; a microprobe and FT±IR investigation. Organic Geochemistry 18, 555±562.
Milton, J.A. 1993. The Application of Quantitative Spore Colour Measurement to Thermal Maturity Studies. PhD Thesis,
University of Southampton.
Painter, P.C., Randy, W.S., Starsinic, M., Coleman, M.M.,
Kuehn, D., Davis, A., 1982. Fourier transform IR spectroscopy Ð application to the quantitative determination of
functional groups in coal. American Chemical Society Symposium Series 205, 47±76.
Pearson, D.L., 1982. Approaching a pollen/spore standard.
Palynology 6, 289.
Phipps, D., Playford, G., 1984. Laboratory techniques for
extraction of palynomorphs from sediments. Papers of the
Department of Geology, University of Queensland 11, 1±23.
Rimmer, S.M., Bensley, D.F., Crelling, J.C., 1989. Fluorescence spectral analysis of sporinite in the Lower Kittaning
coal bed. Organic Geochemistry 14, 343±350.
Rochdi, A., Landais, P., 1991. Transmission micro-infrared
spectroscopy Ð An ecient tool for microscale characterization of coal. Fuel 70, 367±371.
Rouxhet, P.G., Robin, P.L., Nicaise, G., 1980. Characterization of kerogens and of their evolution by infrared spectroscopy. In: Durand, B. (Ed.), Kerogen Ð Insoluble Organic
Matter from Sedimentary Rocks. Editions Technip, Paris,
pp. 163±190.
Saxby, J.D., 1982. A reassessment of the range of kerogen
maturities in which hydrocarbons are generated. Journal of
Petroleum Geology 5, 117±128.
Schenck, P.A., De Leeuw, J.W., Van Grass, G., Haverkamp, J.,
Bouman, M., 1981. Analysis of recent spores and pollen and
of thermally altered sporopollenin by ¯ash pyrolysis±mass
spectrometry and ¯ash pyrolysis±gas chromatography±mass
spectrometry. In: Brooks, J.M. (Ed.), Organic Maturation
Studies and Fossil Fuel Exploration. Academic Press, New
York, pp. 225±237.
Scott, A.C., Hemsley, A.R., 1993. Controls upon the ultrastructural preservation of sporinite. Fuel 72, 1145±1149.
Staplin, F.L., 1969. Sedimentary organic matter, organic metamorphism and oil and gas occurrence. Bulletin of Canadian
Petroleum Geology 17, 47±66.
van Bergen, P.F., Collinson, M.E., Briggs, D.E.G., De Leeuw,
J.W., Scott, A.C., Evershed, R.P., Finch, P., 1995. Resistant
biomacromolecules in the fossil record. Acta Botanica Neerlandia 44, 319±342.
Yule, B.L., Roberts, S., Marshall, J.E.A., Milton, J.A., 1998.
Spore colour scale using colour image analysis. Organic
Geochemistry 28, 139±149.
www.elsevier.nl/locate/orggeochem
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 coordinates a*, b* and L*, (luminance) and the re¯ectance (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 in Rsp. 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 ±CH3 groups. 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 aliphatic 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, CH3 groups, 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
* Corresponding author.
E-mail address: [email protected] (S. Roberts).
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 properties (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. Infrared analysis (Brooks, 1971) and 13C nuclear magnetic
resonance spectroscopy (Hemsley et al., 1992, 1993,
1995, 1996) showed a loss of aliphatic and oxygen containing functional groups as well as increasing aromaticity on increasing thermal maturation. However, these
studies used either arti®cially matured samples, heated in
conditions quite unlike those occurring in the geological
0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0146-6380(00)00058-9
860
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
environment, or bulk concentrated spore/pollen samples. Concentrating pure spores and pollen from kerogen 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 nalkanes, 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 microFourier transform infrared spectroscopy to investigate
the evolution of maceral chemistry with rank. Sporopollenin displayed distinct carboxyl/carbonyl and aliphatic 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 signi®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 progressively 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 association 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 `average' chemical composition from bulk kerogen analysis is
neither representative nor comparable to optical measurements from single kerogen components. This study
links optical thermal maturity measurements during
progressive thermal maturation, to the microchemical
changes of the individual palynomorphs. This successfully 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 computer for data acquisition and processing. Illumination
was from a 100 W tungsten bulb with a colour temperature 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 computer using a basic programme (`CP', written by J. E. A.
Marshall and J. A. Milton). Transmittance measurements 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 coordinates a*, b* and L* 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% transmittance 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 a 40 air objective and
a 10 mm spot diameter. The area chosen (80 mm2) was
a homogeneous area of the spore, for example, the saccus 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
Measurements of sporopollenin re¯ectance (Rsp) and
vitrinite re¯ectance (Rv) were made on polished thin
sections, prepared using the method of Hillier and
Marshall (1988). Re¯ectivity measurements were made
using standard methods (e.g. Davis, 1978; Durand et al.,
1986). The same microscope (the UMSP 50) equipment
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
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 precision 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 to ÿ70 C or below.
The system uses an Ever-Glo source and a KBr beamsplitter with a spectral range of 4000±600 cmÿ1. Infrared
spectra were manipulated within OmnicTM software.
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 accumulation 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 analysis 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
cmÿ1 range. The air in the system was not purged during the experiment resulting in CO2 absorbance 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 available in the OMNIC software `matches' the Y-axis
enabling the comparison of the relative intensities of
spectral peaks. The assignments of the main IR characteristic 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
861
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 20 mm. A single short treatment 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 incident light using polished thin sections.
3.1. Arti®cially matured samples
Observations were made on a set of arti®cially
matured Lycopodium clavatum spores. The samples used
were those of Marshall (1991). Arti®cial maturation of
the modern Lycopodium spores 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 25 C intervals
from 50 to 300 C with some additional samples at
intervals of 12 and 6 C 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 at Rv=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 aliphatic ±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 intensity vibrations which dier from spore to spore.
During the `mature' phase (as shown by the microFT±IR spectra from samples with Rv=0.8% and
0.96%) spores and pollen display rapid changes in colour from orange to mid-brown. Signi®cant chemical
changes and structural reorganisation occurs within the
862
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
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-ordinates a*, b* and L* a
Sample No.
Grid reference and location
Formation/age
Rv% a*
b*
L*
Colour
D8657.42
D8653.28
D8659.35
Yaverland 2
Watchet 14
Inn 23
Inn 37
mv 87/66
Papa 18.5
mv 87/64
Inn 8
Inn 9
mv 88/1
mv 87/88
mv 87/91
mv 87/89
mv 85/44
Foula 56
Foula 57
James Ross Island, Antarctic Peninsula
James Ross Island, Antarctic Peninsula
James Ross Island, Antarctic Peninsula
SZ 618 853, Isle of Wight, UK
ST 083 435, Watchet, Somerset, UK
NM 694 454, Loch Aline, Scotland
NM 694 454, Loch Aline, Scotland
NO 545 375, Midland Valley, Scotland
HU 1715 5905, Papa Stour, Shetland
NO 538 394, Midland Valley, Scotland
NM 070 435, Inninmore, Scotland
NM 070 435, Inninmore, Scotland
NO 730 820, Midland Valley, Scotland
NO 446 367, Midland Valley, Scotland
NO 441 370, Midland Valley, Scotland
NO 441 370, Midland Valley, Scotland
NN 656 019, Midland Valley, Scotland
HT 960 415, Foula, Shetland
HT 958 414, Foula, Shetland
Santa Marta Fm, Cretaceous
Santa Marta Fm, Cretaceous
Santa Marta Fm, Cretaceous
Vectis Fm. Cretaceous
Westbury Fm, Late Triassic
Pabba Beds, Jurassic
Pabba Beds, Jurassic
Gedinnian, Early Devonian
Eifelian, Mid Devonian
Gedinnian, Early Devonian
Westphalian B, Carboniferous
Westphalian B, Carboniferous
Gedinnian, Early Devonian
Gedinnian, Early Devonian
Gedinnian, Early Devonian
Gedinnian, Early Devonian
Emsian, Early Devonian
Eifelian, Mid Devonian
Eifelian, Mid Devonian
0.22
0.27
0.37
0.37
0.40
0.56
0.67
0.73
0.75
0.77
0.78
0.80
0.96
1.04
1.04
1.09
1.28
1.50
1.80
19.4
20.5
21.7
27.3
22.8
23.6
23.8
32.2
23.6
28.6
26.5
29.5
26.9
12.6
18.6
13.2
6.3
2.0
6.0
91.78
88.33
88.55
84.76
90.37
87.98
82.98
61.56
58.79
58.15
63.46
71.17
58.8
50.87
48.19
40.67
41.01
37.37
32.78
Pale yellow
Pale yellow
Pale yellow
Pale lemon yellow
Pale yellow
Pale yellow
Lemon yellow
Orange
Orange
Orange
Orange
Yellow orange
Orange
Orange brown
Orange brown
Dark brown
Dark brown
Dark brown
Black
a
4.0
6.3
6.5
10.5
6.4
8.5
9.6
20.6
17.8
20.2
15.5
13.8
19.2
14.8
18.8
17.4
14.9
10.4
11.9
Data sequenced in order of increasing Rv.
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 hydrocarbons 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 `oilwindow' 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 palynomorphs 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 preparation 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 of A and C
factors from the recorded spectrum:
2865 2925cmÿ1
2865 2925 1630cmÿ1
ratio of intensities of aliphatic=aromatic aliphatic bands
A ÿ factor
1705cmÿ1
1705 1630cmÿ1
ratio of intensities of carboxyl=carbonyl aromatic bands
C ÿ factor
Although adsorbance is proportional to the number
of molecules irradiated, deriving quantitative information on the relative proportions of functional groups in
the molecular structure, is complicated by dierent factors. These factors include, transition probability (likelihood 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 the A- 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).
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
863
Fig. 1. Comparison of selected micro- FT±IR spectra of spores from a range of maturities (Rv=0.4±1.8%). During the early stages of
maturation there is a reduction in the relative proportion of >CO bonds and an increase in the relative proportion of CH2/CH3
bonds. The mature phase, where the palynomorphs break down to generate hydrocarbons, is observed in the spectra with Rv=0.80%
and 0.96%. Structural reorganisation results in the loss of aliphatic groups and the development of aromatic structures. Further
increases in maturity result in increasing aromaticity.
Given the limitations described, sample average Aand C-factors have been calculated from approximately
25 spore/pollen individuals per sample and are plotted
against sample average Rv (Fig. 2) to observe the change
in the two parameters with rank. The C-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%),
the C-factor appears to increase slightly with rank.
The A-factor initially increases during the immature
phase (Rv=0.3±0.8%) as the relative proportion of CH2
and CH3 aliphatic groups increases. Subsequent increases
864
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
Table 2
Luminance measurements from a set of commercial (SCI) spore
colour standards showing relationship between luminance (L*)
and colour (Milton 1993)a
Luminance L*
Colour
90
85
80
75
70
60
50
40
35
30
Pale yellow
Pale-lemon yellow
Lemon yellow
Golden yellow
Yellow orange
Orange
Orange brown
Dark brown
Dark brown to black
Black
a
Colour names from Fisher et al., 1980.
Fig. 2. Plot of sample mean A and C-factors versus the vitrinite
re¯ectance of the sample. Both A and C-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.
in maturity (Rv=0.8±1.2%) shows a signi®cant decrease
in A-factor from 0.8 to 0.35 as the palynomorphs thermally crack. In post-mature samples (Rv>1.2%), the Afactor 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 the Afactor 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 CH2 and CH3 peaks relative
to the CC band or low intensity CH2 and CH3 peaks
relative to a very intense CC band. Notably, spores
and pollen rarely have an A-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 formation 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- and C-factor) of individual palynomorphs is shown in Fig. 3.
Fig. 3. Plot of CIELAB luminance (L*) versus A-factor, and
C-factor for samples containing the entire range of colours
displayed by palynomorphs during maturation (lemon yellow
to black). The relationship between L* and A-factor displays:
area 1 Ð an initial cluster of points indicating the immature
stable yellow trend where colour changes are subtle and slow
(L* 90±70). A scatter of data points indicating maturity as the
spore wall is thermally broken down and colour abruptly
changes. Area 2 Ð a ®nal cluster of points resulting from the
post-mature stable brown trend (L* 40±25). The relationship
between C-factor and L* produces signi®cant scattering of
points with a broad trend of decreasing C-factor with decreasing L*.
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,
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
where colour changes are subtle (L* 90±70) to a postmature stable brown trend (L* 40±25). The abrupt colour change between the two trends (L* 70±40), marks
the breakdown of the spore wall to generate hydrocarbons (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 in A-factor [initial
increases in the sample average A-factor with rank (Fig.
2) are not apparent on this graph]. At the onset of spore
thermal cracking (A-factor 0.7) the range of A-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 postmaturity of the palynomorphs and the relationship
between colour and chemistry returns with a narrow
range of A-factor values (0±0.3). Fig. 3 illustrates a clear
interval of chemical instability (A from 0.3 to 0.7) during 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 in A-factor.
No strong relationship exists between L* and C-factor (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 matured Lycopodium
(Fig. 4) display similar bands to those observed in fossil
sporopollenin (CH2, CH3, >CO, CC) but also contain a number of other bands e.g. 1520 cmÿ1. The range
of amide NH+
3 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,
SO4 and PO groups (Banwell, 1983).
Chemical changes are observed in the Lycopodium
spores with increasing thermal maturation. During the
early stages of maturation (25±150 C) there are increases in the relative intensities of CH2, CH3 and >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
865
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 212 C by a
sudden reduction in the aliphatic content coinciding
with an increase in CC and >CO groups. By 250 C
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 samples show some considerable dierences to palynomorphs heated geologically. The Lycopodium spores
retain a high >CO content throughout maturation,
possibly a consequence of sample oxidation during
heating. The Lycopodium spores heated to 250 C are the
same colour (black) as the most mature geological samples (Rv >1.5%) however, the FT±IR spectra show that
there are fundamental dierences between their chemical 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 250 C Lycopodium sample
shows a signi®cant reduction in its aliphatic content.
Additionally, even though the arti®cially matured samples 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 geological 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 implications for the pyrolysis experiments which are used to
derive kinetic reaction constants for thermal maturation
models. For the values of activation energies and frequency factors derived experimentally to have any relevance 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.
The aliphatic content of Lycopodium with maturation
displays the same initial increases in relative intensity
followed by sudden drops during thermal cracking as
the geological samples. As the Lycopodium samples
progress through the same series of colours as naturally
matured samples, the behaviour of the aliphatic groups
may play a role in colour change.
866
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
Fig. 4. Micro- FT±IR spectra of arti®cially heated Lycopodium spores. The observed spectral evolution that occurs with arti®cial heat
treatment of Lycopodium spores is dierent to that occurring naturally. In particular, the arti®cially heated samples do not develop the
aromatic peaks observed in natural samples of the same colour, also skeletal vibrations in the 1400±700cmÿ1 region are dierent. Only
the behaviour of their aliphatic groups mimics that seen in geological samples. For Rsp data see Marshall (1991).
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
progressive change of sporopollenin composition and
chemical structure. Colour and re¯ectance show contrasting behaviour with rank because the major chemical features in¯uencing the physical properties are
dierent. Spore colour appears to be most aected
during the elimination of functional groups and the
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
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 information 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
867
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 narrowly 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
Fig. 5. Plot of the relationship between the re¯ectance and colour of individual spores with increasing maturity. The L* and Rsp
measurement pairs were made on the same single individual spore specimens Two trends are evident from the graph: A large decreases
in L* (yellow series Ð build up of aliphatics; orange colours Ð loss of aliphatics and increase of CC in aromatic rings) coinciding
with a slight increase in re¯ectance. This is followed by a rapid increase in re¯ectance as the aromatic units coalesce to sheets but with
only slight changes in colour (browns). The evolution of spore colour and re¯ectance with maturity is therefore related to a progressive
change of sporopollenin composition and chemical structure.
868
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
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 isolated 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 formation 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; possibly a consequence of within spore heterogeneity at
higher maturity (e.g. at Rsp>0.6%, error in Rsp0.2%).
Considerable spore colour and chemical changes occur
before there are any signi®cant increases in Rsp and 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 sudden increase in Rsp.
5. Discussion
Micro FT±IR spectroscopy in combination with
transmitted and re¯ected light microphotometry relates
the chemical and physical properties (colour and re¯ectance) of sporopollenin during thermal maturation. In
particular, the FT±IR data demonstrate signi®cant chemical 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 functional groups, termed chromophores, which have empty
p* orbitals into which electrons from s or p orbitals can
be excited. Typical chromophores include CO, CC,
NN and NO2 unsaturated groups (Banwell, 1983).
Other substituents which are not themselves chromophores, modify the absorption of molecules containing
chromophores e.g. CH3, Cl, NH2, OH and are termed
auxochromes (Banwell, 1983). Hence, the CH3 aliphatic
content may have an eect on spore colour by modifying the absorption of the CO and CC bonds in
sporopollenin. Alternatively, the yellow to orange spore
colours observed may be related to the strong ¯uorescence 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 yelloworange in colour. At later stages of maturity, non-spectral colours such as brown are associated with absorption 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 modi®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 fundamental 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 treated in the laboratory to chemically represent those subjected 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 palynomorphs provides more de®nitive chemical analysis than
experiments performed on bulk heterogeneous mixtures
the data are still chemically averaged over the entire
individual spore (resolution 40 mm2). Thus the microFT±IR data do not account for possible chemical heterogeneities within the spore itself. Ultrastructural studies 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 displaying layers, lamina or globular units. The possibility of
compositional variations within such units cannot at
present be excluded.
Despite the correlation observed between spore
chemistry and colour, certain other factors may have
aected the observed relationships between the physical
and chemical properties of spores and pollen. For
example, no account of the spore/pollen species was
taken, the selection criteria included only simple, single
walled species. Chemical dierences between species
have been noted, sporopollenin is not a unique substance, each individual species being composed of a different biopolymer (Guilford et al., 1988; Hemsley et al.,
1992). Also those species with thicker exines will appear
darker (lower L*) but there will be no corresponding
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
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 of A-factor in Fig. 3. Additionally, the spores and
pollen may have a dierent precursor chemistry as a
consequence of early diagenetic eects. The physicochemical-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,
CH3 groups.
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 structural reorganisation at the spore `oil-window' has initiated the formation of isolated 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 polyaromatic 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 geologically 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 control their colour.
869
Acknowledgements
BLY was supported by a joint studentship from British Gas Research and Technology Division and the Science Faculty, University of Southampton. The authors
acknowledge the constructive and careful reviews of M.
Mastalerz and J. Potter.
Associate EditorÐM.G. Fowler
References
Banwell, C.N., 1983. Fundamentals of Molecular Spectroscopy, 3rd Edition. McGraw-Hill, London.
Brooks, J. , 1971. Some chemical and geochemical studies on
sporopollenin. In: Brooks, J. et al. (Eds.), Sporopollenin
Academic Press, London, pp. 351±407.
Brooks, J., Shaw, G., 1978. Sporopollenin: a review of its chemistry, palaeochemistry and geochemistry. Grana 17, 91±97.
Carr, A.D., Williamson, J.E., 1989. The relationship between
aromaticity, vitrinite re¯ectance and maceral composition of
coals: Implications for the use of vitrinite re¯ectance as a
maturation parameter. In: Durand, B., Behar, F. (Eds.),
Advances in Organic Geochemistry 1989, Organic Geochemistry. Pergamon, Oxford. vol. 16, pp. 313±323.
Cody, G.D., Botto, R.E., Ade, H., Wirick, S., 1996. The
application of soft X-ray microscopy to the in-situ analysis
of sporinite in coal. International Journal of Coal Geology
32, 69±86.
Davis, A., 1978. The re¯ectance of coal. In: Karr, C. (Ed.),
Analytical Methods for Coal and Coal Products. Academic
Press, London, New York, pp. 27±81.
Davis, M.R., Abbott, J.M., Gaines, A.F., 1985. Chemical
structure of telocollinites and sporinites. Dierentiation
between telocollinites and sporinites by the aromatic structures present in their pyridine extracts. Fuel 64, 1362±1369.
Dungworth, G., McCormick, A., Powell, T.G., Douglas, A.G.,
1971. Lipid components in fresh and fossil pollen and spores.
In: Brooks, J. et al. (Eds.), Sporopollenin. Academic Press,
New York, pp. 512±544.
Durand, B., Alpern, B., Pittion, J.L., Pradier, B., 1986. Re¯ectance of vitrinite as a control of thermal history of sediments.
In: Burrus, J. (Ed.), Thermal Modelling in Sedimentary
Basins. Editions Technip, Paris, pp. 414±474.
Fisher, M.J., Barnard, P.C., Cooper, B.S., 1980. Organic
maturation and hydrocarbon generation in the Mesozoic
sediments of the Sverdrup Basin, Arctic Canada. Proceedings
IV International Palynological Conference, Lucknow (197677) 2, 581±588.
Ganz, H.H., Kalkreuth, W., Ganz, S.N., OÈner, F., Pearson,
M.J., Wehner, H., 1990. Infrared analysis Ð state of the art.
Berliner Geowissenschaftliche Abhandlungen 120, 1011±
1026.
Guilford, W.J., Schneider, D.M., Labovitz, J., Opella, S.J.,
1988. High-resolution solid 13C NMR spectroscopy of sporopollenins from dierent plant taxa. Plant Physiology 86,
134±136.
Hemsley, A.R., Chaloner, W.G., Groombridge, C.J., Scott,
A.C., 1992. Carbon-13 solid state nuclear magnetic resonance
870
B.L. Yule et al. / Organic Geochemistry 31 (2000) 859±870
of sporopollenins from modern and fossil plants. Annals of
Botany 69, 545±549.
Hemsley, A.R., Barrie, P.J., Chaloner, W.G., Scott, A.C. 1993.
The composition of sporopollenin and its use in living and
fossil plant systematics. Grana. (Supplement 1), 2±11.
Hemsley, A.R., Barrie, P.J., Scott, A.C., 1995. 13C solid-state
NMR spectroscopy of fossil sporopollenins: variations in
composition independent of diagenesis. Fuel 74, 1009±1012.
Hemsley, A.R., Scott, A.C., Barrie, P.J., Chaloner, W.G., 1996.
Studies of fossil and modern spore wall biomacromolecules
using 13C solid state NMR. Annals of Botany 78, 83±94.
Hillier, S.J., Marshall, J.E.A., 1988. A rapid technique to make
polished thin sections of sedimentary organic matter concentrates. Journal of Sedimentary Petrology 58, 754±755.
Kemp, W., 1991. Organic Spectroscopy, 3rd Edition. Basingstoke, Macmillan.
Marshall, J.E.A., 1991. Quantitative spore colour. Journal of
the Geological Society, London 148, 223±233.
Mastalerz, M., Bustin, R.M., 1993. Variation in maceral
chemistry within and between coals of varying rank: an electron microprobe and micro-FTIR investigation. Journal of
Microscopy 171, 153±166.
Mastalerz, M., Wilkes, K.R., Bustin, R.M., 1993. Variation in
vitrinite chemistry as a function of associated liptinite content; a microprobe and FT±IR investigation. Organic Geochemistry 18, 555±562.
Milton, J.A. 1993. The Application of Quantitative Spore Colour Measurement to Thermal Maturity Studies. PhD Thesis,
University of Southampton.
Painter, P.C., Randy, W.S., Starsinic, M., Coleman, M.M.,
Kuehn, D., Davis, A., 1982. Fourier transform IR spectroscopy Ð application to the quantitative determination of
functional groups in coal. American Chemical Society Symposium Series 205, 47±76.
Pearson, D.L., 1982. Approaching a pollen/spore standard.
Palynology 6, 289.
Phipps, D., Playford, G., 1984. Laboratory techniques for
extraction of palynomorphs from sediments. Papers of the
Department of Geology, University of Queensland 11, 1±23.
Rimmer, S.M., Bensley, D.F., Crelling, J.C., 1989. Fluorescence spectral analysis of sporinite in the Lower Kittaning
coal bed. Organic Geochemistry 14, 343±350.
Rochdi, A., Landais, P., 1991. Transmission micro-infrared
spectroscopy Ð An ecient tool for microscale characterization of coal. Fuel 70, 367±371.
Rouxhet, P.G., Robin, P.L., Nicaise, G., 1980. Characterization of kerogens and of their evolution by infrared spectroscopy. In: Durand, B. (Ed.), Kerogen Ð Insoluble Organic
Matter from Sedimentary Rocks. Editions Technip, Paris,
pp. 163±190.
Saxby, J.D., 1982. A reassessment of the range of kerogen
maturities in which hydrocarbons are generated. Journal of
Petroleum Geology 5, 117±128.
Schenck, P.A., De Leeuw, J.W., Van Grass, G., Haverkamp, J.,
Bouman, M., 1981. Analysis of recent spores and pollen and
of thermally altered sporopollenin by ¯ash pyrolysis±mass
spectrometry and ¯ash pyrolysis±gas chromatography±mass
spectrometry. In: Brooks, J.M. (Ed.), Organic Maturation
Studies and Fossil Fuel Exploration. Academic Press, New
York, pp. 225±237.
Scott, A.C., Hemsley, A.R., 1993. Controls upon the ultrastructural preservation of sporinite. Fuel 72, 1145±1149.
Staplin, F.L., 1969. Sedimentary organic matter, organic metamorphism and oil and gas occurrence. Bulletin of Canadian
Petroleum Geology 17, 47±66.
van Bergen, P.F., Collinson, M.E., Briggs, D.E.G., De Leeuw,
J.W., Scott, A.C., Evershed, R.P., Finch, P., 1995. Resistant
biomacromolecules in the fossil record. Acta Botanica Neerlandia 44, 319±342.
Yule, B.L., Roberts, S., Marshall, J.E.A., Milton, J.A., 1998.
Spore colour scale using colour image analysis. Organic
Geochemistry 28, 139±149.