Directory UMM :Data Elmu:jurnal:O:Organic Geochemistry:Vol31.Issue12.Dec2000:
Organic Geochemistry 31 (2000) 1755±1763
www.elsevier.nl/locate/orggeochem
Solid-phase ¯uorescence determination of chlorins
in marine sediments
R.F. Chen a,*, Y. Jiang a, M. Zhao b
a
Environmental, Coastal and Ocean Sciences, UMassBoston, 100 Morrissey Boulevard, Boston, MA 02125, USA
b
Department of Earth Science, Dartmouth College, Hanover, NH 03755, USA
Abstract
Chlorophyll degradation products are preserved in marine sediments over timescales of thousands of years. The
production of chlorophyll in the water column is related to biological productivity, so chlorophyll degradation products (chlorins) preserved in marine sediments can be used as indicators of paleoproductivity. A new, rapid, nondestructive method of determining chlorin concentrations in marine sediments is presented. Potential interferences
associated with the solid-phase ¯uorescence (SPF) method are explored using reference materials, yet this method
compares favorably with spectroscopic and high performance liquid chromatographic (HPLC) methods of analysis
using marine sediments from Boston Harbor and the continental shelf o northwest Africa. # 2000 Elsevier Science
Ltd. All rights reserved.
Keywords: Chlorins; Paleoproductivity; Fluorescence; Analysis; Non-destructive
1. Introduction
Biological productivity in the ocean plays an important role in the global cycling of carbon and, therefore,
has a major eect on the global climate (Berger et al.,
1989; Mix, 1989; Falkowski and Wilson, 1992; Falkowski, 1994). Phytoplankton biomass can be estimated
from the concentration of chlorophyll a in seawater, and
attempts have been made to relate chlorophyll concentration determined using satellite data to productivity (Longhurst et al., 1995; Behrenfeld and Falkowski,
1997). Chlorophyll a is transformed rapidly in the water
column and surface marine sediments (e.g. Eckardt et
al., 1992; Harradine et al., 1996), but the macrocyclic
ring, the tetrapyrrole backbone of the chlorophyll a
molecule, is quite stable over long periods of time (e.g.
Treibs, 1936; Eckardt et al., 1991). The green transformation products of chlorophylls, the major pigments
* Corresponding author. Tel.: +1-617-287-7491; fax: +1617-287-7474.
E-mail addresses: bob.chen@ems.umb.edu (R.F. Chen),
meixun. zhao@dartmouth.edu (M. Zhao).
required by all phytoplankton for photosynthesis, have
been termed ``chlorins'' and have been used as an indicator of present day and paleoproductivity (Harris et
al., 1996).
Freshly produced organic matter in the ocean undergoes a number of degradation processes including grazing, microbial degradation, sinking, and sediment
diagenesis before being buried in marine sediments (e.g.
Welschmeyer and Lorenzen, 1985; Hedges and Keil,
1995). Yet, biogenic debris buried in deep-sea sediments
has often been used to explore oceanic productivity in
the past. Several chemical parameters and biomarkers
have been suggested as proxies for paleoproductivity
(e.g. Martinez et al., 1996; Hinrichs et al., 1999), each
with its own strengths and weaknesses. Total organic
carbon in sediments has been used to indicate total
organic carbon rain rate and thus surface production
(Reichart et al., 1997), although inputs from terrestrial
sources might alter this signal (Westerhausen et al.,
1993). Biogenic opal (Stein et al., 1989; Harris et al.,
1996) yields information on the silica-producing phytoplankton community. Barium (McManus et al., 1999),
aluminum and titanium (Dymond et al., 1997), and
0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0146-6380(00)00075-9
1756
231
R.F. Chen et al. / Organic Geochemistry 31 (2000) 1755±1763
Pa/230Th ratios (Kumar et al., 1993; Walter et al.,
1997) have been examined as paleoproductivity indicators. Total organic d13C values (Bouloubassi et al.,
1997) and d13C of individual biomarkers such as C37
alkenones and C22±C33 alkanes have also been studied
(Villaneuva et al., 1997). Common to all these indicators
is the extraction of various components and the
destructive analysis of the sediment sample.
Chlorins have been found to be ubiquitous and
abundant in immature marine and lacustrine sediments
(e.g. Keely and Maxwell, 1991; Prowse and Maxwell,
1991; Keely et al., 1994; Harris et al., 1995; Soma et al.,
1996). They are directly related to photosynthesis and
so if conserved should act as a measure of total primary
productivity (Harris et al., 1996). In order to maximize
our understanding of paleoproductivity, an analysis of
chlorins in marine sediments should have a resolution
at least similar to concurrent measurements of oxygen18 (d18O), total organic carbon (TOC), and biogenic
opal.
Spectroscopic and chromatographic methods have
both been used to measure chlorin concentrations in
marine sediments (Trees et al., 1985; Meyns et al., 1994;
Harris and Maxwell, 1995). Chromatographic methods
involve the extraction of a sediment sample and quanti®cation and identi®cation of major chlorophyll transformation products by comparing the chromatographic
peaks with those of authentic standards. This procedure
is destructive and requires long analysis times, making
high resolution (n>50) stratigraphic measurements
practically impossible. Spectroscopic methods rely on
extraction and a comparison of the UV±vis absorbance
or ¯uorescence spectrum of the extract with that of
chlorophyll a or authentic chlorin standard. An automated spectroscopic method using on-line extraction
and ¯uorescence determination has allowed high resolution chlorin determinations that may serve as measurements of total primary productivity variations
(Harris and Maxwell, 1995; Harris et al., 1996).
The stable macrocyclic ring structure is aromatic with
highly delocalized electron density, so it can absorb near
the sunlight maximum at sea level, around 420±450 nm.
This unique property has been used to determine chlorophyll concentrations in living plants and as well as
porphyrins in source rocks by measuring their re¯ectance spectra (Holden and Gaey, 1990; Holden et al.,
1991; Yamada and Fujimura, 1991; Gitelson and Merzlak, 1994, 1996). Chlorophylls are also highly ¯uorescent so ¯uorescence spectroscopy of marine sediments
may oer increased sensitivity by reducing the inherent
scattering interference associated with re¯ectance spectra. We present here a new solid-phase ¯uorescence
method for the non-destructive determination of chlorins in marine sediments and compare this new rapid
method with two established methods for chlorin determination.
2. Methods
2.1. Samples
Boston Harbor surface sediments collected by grab
and box core and sediments from the continental shelf
o NW Africa (Ocean Drilling Program Leg 108, Site
658C; Ruddiman et al., 1988) were frozen until analysis.
Samples were analyzed wet or after drying at 60 C for
12 h. Chlorophyll a standards (extracted from Anacystis
nidulans) were obtained from Sigma Chemical and were
free from chlorophyll b interferences. Sediment standards were obtained from Wards Natural Science
Establishment (Rochester, New York, USA). ``Clean''
sediments were prepared by sonicating sediment standards in acetone repetitively until no chlorin was present
in the solvent phase as determined by ¯uorescence with
420 nm excitation.
2.2. Fluorescence spectrophotometry
A Photon Technologies International (PTI) Quantum
Master-1 spectro¯uorometer equipped with a powder
sample holder was used for solid-phase ¯uorescence
analysis. This spectro¯uorometer utilizes a 150 W xenon
lamp excitation source, 2 x 0.25 m excitation monochromators, a single emission monochromator and a
cooled photomultiplier tube detector. A variable angle
solid sample holder allows minimization of scattered
light interference. In addition, a 420 nm narrow band
pass interference ®lter on the excitation side and a 530
nm long wavelength pass ®lter on the emission side were
used to decrease scattering and stray light (see Section 3).
As scattering was dependent on the sample matrix, no
common ``clean'' or ``blank'' sediment could be prepared. Instead, ¯uorescence emission from 570 to 700
nm was smoothed with a second order polynomial ®t,
and the peak above the curve between 620 and 700 nm
was integrated and compared with ``clean'' sediments
spiked with chlorophyll a to provide quanti®cation.
2.3. High performance liquid chromatography
In addition, a high performance liquid chromatography (HPLC) method and a standard spectroscopic
method (Trees et al., 1985) were utilized to provide
comparison with the new method. Sediments were
extracted with acetone by sonication, volume-reduced
by rotoevaporation, and analyzed with a Dynamax
HPLC system equipped with a UV absorbance detector.
An Econosphere C18 5 mm reversed phase column
(2503.6 mm) was used with an isocratic 60:36:4 acetone:methanol:water mobile phase at 1 ml/min for elution; 20 ml injections were used, and the detector was
set at 665 nm to avoid interference from carotenoids
which do not absorb above 600 nm. Assignment of
R.F. Chen et al. / Organic Geochemistry 31 (2000) 1755±1763
components as chlorins was con®rmed by analyzing
collected peaks with an HP 8453 UV±vis spectrophotometer and obtaining the characteristic spectrum
(including absorption at 650 nm) of chlorins rather than
carotenoids. Peaks were quanti®ed with respect to
chlorophyll a standards which provides only a relative
quanti®cation as the ¯uorescence and absorbance characteristics of individual chlorins and chlorophyll a vary
somewhat. While absolute identi®cation of the HPLCproduced chlorin peaks was not provided, UV-vis spectra provide secondary con®rmation of our interpretation.
2.4. Sediment extract ¯uorescence
Sediment acetone extracts were also analyzed in the
solution phase with the PTI QM-1 spectro¯uorometer
operating in the 90 con®guration. Samples were placed
in a 1 cm2 quartz cell, and the integrated emission from
620 to 700 nm was compared to chlorophyll a standards
1757
for quanti®cation. A 530 nm long wavelength pass ®lter
was used to decrease scattering.
3. Results
Fluorescence excitation scans show a maximum of
410±420 nm for chlorophyll a and chlorins in sediments
(Fig. 1a). Second order scattering below 350 nm was
apparent but does not interfere in normal measurements
at 420 nm excitation where a 420 nm narrow band
interference ®lter was used. Emission scans from 500 to
700 nm were recorded as the chlorophyll/chlorin peak
was broad and shifted slightly (640±670 nm) for dierent samples (Fig. 1b). The apparent peak at 540 nm and
the long wavelength tail extending to 700 nm is a product of light scattering and the long wavelength passes
®lters used (Fig. 1b).
In order to optimize the solid-phase ¯uorescence
method, various experimental parameters were explored.
Fig. 1. (a) Excitation scans of a solvent-extracted sediment, a silt with a low and a high concentration of spiked chlorophyll a, a sand
spiked with chlorophyll a, and a marine sediment from Boston Harbor (Reserve Channel). Emission wavelength is ®xed at 666 nm.
Excitation peaks are at 410±420 nm. (b) Emission scans of marine sediments and a ``clean'' sediment with chlorins extracted. Note the
large scattering tail extending to 700 nm. Excitation wavelength is ®xed at 420 nm. Chlorin peaks are seen between 640 and 670 nm
depending on the composition of the chlorins.
1758
R.F. Chen et al. / Organic Geochemistry 31 (2000) 1755±1763
First, the incident angle of the excitation radiation was
altered aording optimal signal intensity at 25 , similar
to the 22 optimal angle observed in ¯uorescence investigations of fuels in soils (Fig. 2; Apitz et al., 1992a).
This maximum is a result of reducing scattering while
maintaining signi®cant signal and may be somewhat
dependent on the optical con®guration used.
Next, water content also showed an eect on ¯uorescence eciency. Fig. 3 shows dried standard sediments
(sand and kaolinite, not organic-free) spiked with
chlorophyll a (added in acetone and then dried) with
consecutive amounts of deionized water added. Fluorescence response decreases with increasing water content. Possible explanations for this behavior are: (1)
Fig. 2. The eect of incident angle of the excitation beam to the window of the powder sample holder in the PTI QM-1 spectro¯uorometer on the ¯uorescence response of chlorophyll a.
Fig. 3. The eect of water added to dry sediment on the ¯uorescence response of chlorophyll a.
R.F. Chen et al. / Organic Geochemistry 31 (2000) 1755±1763
absorption of light by the organic compounds associated with the standard mineral grains and which dissolve into the water, (2) increased re¯ection of the
excitation light by water, or (3) a hydration of the
pigment on the sediment surface, thus altering its geometry and reducing its quantum yield. Water extractions of the sediments did not show an appreciable
absorption of light at 420 nm or 680 nm compared to
the absorption due to chlorins at relevant concentrations. It is known that the ¯uorescence of chlorophyll
can be either increased or decreased by the association
of the chromophore with various surfaces and by its
interaction with various solvents (Mamleeva et al., 1988;
Komleva et al., 1989). Most probably, water interacts
with the surface of the mineral grain, ``releasing'' the
chlorin from the surface, with a concomitant reduction
in the surface-enhanced ¯uorescence of the chlorin.
Another possibility is that water reduces the re¯ectivity
of the sediment (wet sediments are darker) thus reducing
the excitation and emission light throughput (Lohmannsroben and Schober, 1999).
While chlorophyll a spiked on to all sediment standards yielded good linearity (r2>0.85) over various
concentrations ranges varying from 0±1.5 mg/g for kaolinite to 0±60 mg/g for silt, ¯uorescence intensity actually
decreases with increasing concentration above some
threshold concentration, probably due to chromophore
stacking and quenching. In any case, detection limits
were around 1 mg/g for all standard sediments.
Sediment composition also has a dramatic eect on
¯uorescence response (Apitz et al., 1992b). Response
1759
factors for the various matrices are shown in Fig. 4.
Kaolinite and opal yield the highest sensitivity of detection and bentonite yields the lowest for the standard
minerals measured. This variability in ¯uorescence
response is probably due to complex surface interactions
between the chromophore and the mineral surfaces
(Mamleeva et al., 1988). Formation of complexes would
generally reduce ¯uorescence while certain surfaces
could enhance the pigment ¯uorescence. Thus the solidphase ¯uorescence method may be in¯uenced by variations (especially in the opal and kaolinite fraction) in
sediment composition. A full description of the lithology of the analyzed sediments would aid in the interpretation of the relative abundances of chlorins in those
sediments.
Finally, the grain size of the same minerals shows an
eect on the ¯uorescence response factors (Fig. 5).
Increasing grain size generally decreases ¯uorescence
response. There are two possible explanations. First, the
more re¯ecting ®ne grained sediments, especially for
opal with respect to quartz, may lead to an increase in
the eective path length of the excitation radiation and
the resulting ¯uorescence emission. The ¯uorescence
response is thus enhanced because light enters and exits
the small grains more eciently. The dramatic decrease
in ¯uorescence response with grain size for opal but not
for sand supports this explanation. It has been shown
that ¯uorescence responses in sediments can be corrected by taking into account sediment re¯ectance
(Lohmannsroben and Schoder, 1999). Second, as grain
size increases, more chlorin may be forced out of the
Fig. 4. The eect of mineral composition on ¯uorescence response of chlorophyll a.
1760
R.F. Chen et al. / Organic Geochemistry 31 (2000) 1755±1763
Fig. 5. The eect of grain size on the ¯uorescence response of chlorophyll a.
sediment grain interstices and would, therefore, be more
prone to stack together, self-quenching the ¯uorescence
emission of the chlorin. This is the opposite eect that
was seen with smaller chromophores, the polynuclear
aromatics, in marine diesel fuel as observed by Apitz
(1992a). In that study, the smaller grain sizes oered
more surface areas for PAH to adsorb and be hidden
from excitation radiation, thus the ¯uorescence response
increased with increasing grain size. The large planar
geometry of the chlorin probably dominates the
adsorption factors in our case. Again, knowledge of
grain size would aid in the calibration of solid-phase
¯uorescence for determining the relative abundances of
chlorins.
Humic substances, major constituents of marine sediments, are known to ¯uoresce (Hayase and Tsubota,
1985; Chen and Bada, 1995), but do not show appreciable interference in the 650±700 nm range. A ¯uorescence emission scan, given the optical con®guration
(including a 530 nm long wavelength pass ®lter), of
humics obtained from Aldrich Chemical yielded an
apparent maximum at 550 nm. The tail of this ¯uorescent peak in the 650±700 nm range was simply background-subtracted along with scattered light and
random noise and did not interfere with quanti®cation
of the chlorins.
To correct for all these possible interferences, a correction factor for the ¯uorescence response of chlorins
could be calculated given information on sediment
water content, mineral composition and grain size.
However, using real marine sediments from a wide
range of environments [Boston Harbor surface sediments and sediments from the continental shelf o NW
Africa (Ocean Drilling Program Leg 108, Site 658C;
Ruddiman et al., 1988)] and wide range of chlorin concentrations (0±50 mg/g), the solid phase ¯uorescence
technique does agree quite well with both the spectroscopic method (Fig. 6a; r2=0.82) and the HPLC
method (Fig. 6b; r2=0.77) for 40 samples. A closer
examination of the HPLC method shows a signi®cant
intercept using this method while the solid phase
method shows no ¯uorescence (Fig. 6b). This may be
due to the HPLC quanti®cation (based on absorbance)
of peaks that are not chlorins or chlorins that are not as
¯uorescent as chlorophyll a. Because the solid and
liquid phase ¯uorescence methods correlate with a
nearly one-to-one relationship (slope=0.84), it would
appear that the HPLC method quanti®es only a fraction
of the ¯uorescent chlorins (slope=0.30), but also
responds to a few peaks that are not ¯uorescent. Initial
investigations into the identity of the HPLC peaks used
for quanti®cation showed that these peaks were not
carotenoids but had absorption spectra similar to
chlorins. However, further investigation of some of
these peaks by comparison with puri®ed standards was
not carried out because detailed characterization of the
individual chlorins in marine sediments is not the focus
of this paper; rather, this new, non-destructive method
for the approximate determination of chlorin concentrations in marine sediments shows promise as a
rapid screening tool for estimating paleoproductivity
changes.
R.F. Chen et al. / Organic Geochemistry 31 (2000) 1755±1763
1761
Fig. 6. (a) Comparison of the concentration of chlorin in marine sediments from Boston Harbor (*) and the continental shelf o
northwest Africa (&) determined by solid phase ¯uorescence and the ¯uorescence of an acetone extract. Chlorins are quanti®ed by
comparison with calibrations using chlorophyll a. (b) Comparison of the concentration of chlorin in the same samples determined by
solid phase ¯uorescence and high performance liquid chromatography.
4. Discussion
Solid-phase ¯uorescence determination of chlorins in
marine sediments appears to be a rapid, non-destructive
method of estimating paleoproductivity changes. The
method has been shown to give similar results to
extraction methods using ¯uorescence spectroscopy
(Harris and Maxwell, 1995; Fig. 6a) and HPLC (Trees
et al., 1985; Fig. 6b). In the concentration range of 1±50
mg/g chlorin, the SPF method shows some (
www.elsevier.nl/locate/orggeochem
Solid-phase ¯uorescence determination of chlorins
in marine sediments
R.F. Chen a,*, Y. Jiang a, M. Zhao b
a
Environmental, Coastal and Ocean Sciences, UMassBoston, 100 Morrissey Boulevard, Boston, MA 02125, USA
b
Department of Earth Science, Dartmouth College, Hanover, NH 03755, USA
Abstract
Chlorophyll degradation products are preserved in marine sediments over timescales of thousands of years. The
production of chlorophyll in the water column is related to biological productivity, so chlorophyll degradation products (chlorins) preserved in marine sediments can be used as indicators of paleoproductivity. A new, rapid, nondestructive method of determining chlorin concentrations in marine sediments is presented. Potential interferences
associated with the solid-phase ¯uorescence (SPF) method are explored using reference materials, yet this method
compares favorably with spectroscopic and high performance liquid chromatographic (HPLC) methods of analysis
using marine sediments from Boston Harbor and the continental shelf o northwest Africa. # 2000 Elsevier Science
Ltd. All rights reserved.
Keywords: Chlorins; Paleoproductivity; Fluorescence; Analysis; Non-destructive
1. Introduction
Biological productivity in the ocean plays an important role in the global cycling of carbon and, therefore,
has a major eect on the global climate (Berger et al.,
1989; Mix, 1989; Falkowski and Wilson, 1992; Falkowski, 1994). Phytoplankton biomass can be estimated
from the concentration of chlorophyll a in seawater, and
attempts have been made to relate chlorophyll concentration determined using satellite data to productivity (Longhurst et al., 1995; Behrenfeld and Falkowski,
1997). Chlorophyll a is transformed rapidly in the water
column and surface marine sediments (e.g. Eckardt et
al., 1992; Harradine et al., 1996), but the macrocyclic
ring, the tetrapyrrole backbone of the chlorophyll a
molecule, is quite stable over long periods of time (e.g.
Treibs, 1936; Eckardt et al., 1991). The green transformation products of chlorophylls, the major pigments
* Corresponding author. Tel.: +1-617-287-7491; fax: +1617-287-7474.
E-mail addresses: bob.chen@ems.umb.edu (R.F. Chen),
meixun. zhao@dartmouth.edu (M. Zhao).
required by all phytoplankton for photosynthesis, have
been termed ``chlorins'' and have been used as an indicator of present day and paleoproductivity (Harris et
al., 1996).
Freshly produced organic matter in the ocean undergoes a number of degradation processes including grazing, microbial degradation, sinking, and sediment
diagenesis before being buried in marine sediments (e.g.
Welschmeyer and Lorenzen, 1985; Hedges and Keil,
1995). Yet, biogenic debris buried in deep-sea sediments
has often been used to explore oceanic productivity in
the past. Several chemical parameters and biomarkers
have been suggested as proxies for paleoproductivity
(e.g. Martinez et al., 1996; Hinrichs et al., 1999), each
with its own strengths and weaknesses. Total organic
carbon in sediments has been used to indicate total
organic carbon rain rate and thus surface production
(Reichart et al., 1997), although inputs from terrestrial
sources might alter this signal (Westerhausen et al.,
1993). Biogenic opal (Stein et al., 1989; Harris et al.,
1996) yields information on the silica-producing phytoplankton community. Barium (McManus et al., 1999),
aluminum and titanium (Dymond et al., 1997), and
0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0146-6380(00)00075-9
1756
231
R.F. Chen et al. / Organic Geochemistry 31 (2000) 1755±1763
Pa/230Th ratios (Kumar et al., 1993; Walter et al.,
1997) have been examined as paleoproductivity indicators. Total organic d13C values (Bouloubassi et al.,
1997) and d13C of individual biomarkers such as C37
alkenones and C22±C33 alkanes have also been studied
(Villaneuva et al., 1997). Common to all these indicators
is the extraction of various components and the
destructive analysis of the sediment sample.
Chlorins have been found to be ubiquitous and
abundant in immature marine and lacustrine sediments
(e.g. Keely and Maxwell, 1991; Prowse and Maxwell,
1991; Keely et al., 1994; Harris et al., 1995; Soma et al.,
1996). They are directly related to photosynthesis and
so if conserved should act as a measure of total primary
productivity (Harris et al., 1996). In order to maximize
our understanding of paleoproductivity, an analysis of
chlorins in marine sediments should have a resolution
at least similar to concurrent measurements of oxygen18 (d18O), total organic carbon (TOC), and biogenic
opal.
Spectroscopic and chromatographic methods have
both been used to measure chlorin concentrations in
marine sediments (Trees et al., 1985; Meyns et al., 1994;
Harris and Maxwell, 1995). Chromatographic methods
involve the extraction of a sediment sample and quanti®cation and identi®cation of major chlorophyll transformation products by comparing the chromatographic
peaks with those of authentic standards. This procedure
is destructive and requires long analysis times, making
high resolution (n>50) stratigraphic measurements
practically impossible. Spectroscopic methods rely on
extraction and a comparison of the UV±vis absorbance
or ¯uorescence spectrum of the extract with that of
chlorophyll a or authentic chlorin standard. An automated spectroscopic method using on-line extraction
and ¯uorescence determination has allowed high resolution chlorin determinations that may serve as measurements of total primary productivity variations
(Harris and Maxwell, 1995; Harris et al., 1996).
The stable macrocyclic ring structure is aromatic with
highly delocalized electron density, so it can absorb near
the sunlight maximum at sea level, around 420±450 nm.
This unique property has been used to determine chlorophyll concentrations in living plants and as well as
porphyrins in source rocks by measuring their re¯ectance spectra (Holden and Gaey, 1990; Holden et al.,
1991; Yamada and Fujimura, 1991; Gitelson and Merzlak, 1994, 1996). Chlorophylls are also highly ¯uorescent so ¯uorescence spectroscopy of marine sediments
may oer increased sensitivity by reducing the inherent
scattering interference associated with re¯ectance spectra. We present here a new solid-phase ¯uorescence
method for the non-destructive determination of chlorins in marine sediments and compare this new rapid
method with two established methods for chlorin determination.
2. Methods
2.1. Samples
Boston Harbor surface sediments collected by grab
and box core and sediments from the continental shelf
o NW Africa (Ocean Drilling Program Leg 108, Site
658C; Ruddiman et al., 1988) were frozen until analysis.
Samples were analyzed wet or after drying at 60 C for
12 h. Chlorophyll a standards (extracted from Anacystis
nidulans) were obtained from Sigma Chemical and were
free from chlorophyll b interferences. Sediment standards were obtained from Wards Natural Science
Establishment (Rochester, New York, USA). ``Clean''
sediments were prepared by sonicating sediment standards in acetone repetitively until no chlorin was present
in the solvent phase as determined by ¯uorescence with
420 nm excitation.
2.2. Fluorescence spectrophotometry
A Photon Technologies International (PTI) Quantum
Master-1 spectro¯uorometer equipped with a powder
sample holder was used for solid-phase ¯uorescence
analysis. This spectro¯uorometer utilizes a 150 W xenon
lamp excitation source, 2 x 0.25 m excitation monochromators, a single emission monochromator and a
cooled photomultiplier tube detector. A variable angle
solid sample holder allows minimization of scattered
light interference. In addition, a 420 nm narrow band
pass interference ®lter on the excitation side and a 530
nm long wavelength pass ®lter on the emission side were
used to decrease scattering and stray light (see Section 3).
As scattering was dependent on the sample matrix, no
common ``clean'' or ``blank'' sediment could be prepared. Instead, ¯uorescence emission from 570 to 700
nm was smoothed with a second order polynomial ®t,
and the peak above the curve between 620 and 700 nm
was integrated and compared with ``clean'' sediments
spiked with chlorophyll a to provide quanti®cation.
2.3. High performance liquid chromatography
In addition, a high performance liquid chromatography (HPLC) method and a standard spectroscopic
method (Trees et al., 1985) were utilized to provide
comparison with the new method. Sediments were
extracted with acetone by sonication, volume-reduced
by rotoevaporation, and analyzed with a Dynamax
HPLC system equipped with a UV absorbance detector.
An Econosphere C18 5 mm reversed phase column
(2503.6 mm) was used with an isocratic 60:36:4 acetone:methanol:water mobile phase at 1 ml/min for elution; 20 ml injections were used, and the detector was
set at 665 nm to avoid interference from carotenoids
which do not absorb above 600 nm. Assignment of
R.F. Chen et al. / Organic Geochemistry 31 (2000) 1755±1763
components as chlorins was con®rmed by analyzing
collected peaks with an HP 8453 UV±vis spectrophotometer and obtaining the characteristic spectrum
(including absorption at 650 nm) of chlorins rather than
carotenoids. Peaks were quanti®ed with respect to
chlorophyll a standards which provides only a relative
quanti®cation as the ¯uorescence and absorbance characteristics of individual chlorins and chlorophyll a vary
somewhat. While absolute identi®cation of the HPLCproduced chlorin peaks was not provided, UV-vis spectra provide secondary con®rmation of our interpretation.
2.4. Sediment extract ¯uorescence
Sediment acetone extracts were also analyzed in the
solution phase with the PTI QM-1 spectro¯uorometer
operating in the 90 con®guration. Samples were placed
in a 1 cm2 quartz cell, and the integrated emission from
620 to 700 nm was compared to chlorophyll a standards
1757
for quanti®cation. A 530 nm long wavelength pass ®lter
was used to decrease scattering.
3. Results
Fluorescence excitation scans show a maximum of
410±420 nm for chlorophyll a and chlorins in sediments
(Fig. 1a). Second order scattering below 350 nm was
apparent but does not interfere in normal measurements
at 420 nm excitation where a 420 nm narrow band
interference ®lter was used. Emission scans from 500 to
700 nm were recorded as the chlorophyll/chlorin peak
was broad and shifted slightly (640±670 nm) for dierent samples (Fig. 1b). The apparent peak at 540 nm and
the long wavelength tail extending to 700 nm is a product of light scattering and the long wavelength passes
®lters used (Fig. 1b).
In order to optimize the solid-phase ¯uorescence
method, various experimental parameters were explored.
Fig. 1. (a) Excitation scans of a solvent-extracted sediment, a silt with a low and a high concentration of spiked chlorophyll a, a sand
spiked with chlorophyll a, and a marine sediment from Boston Harbor (Reserve Channel). Emission wavelength is ®xed at 666 nm.
Excitation peaks are at 410±420 nm. (b) Emission scans of marine sediments and a ``clean'' sediment with chlorins extracted. Note the
large scattering tail extending to 700 nm. Excitation wavelength is ®xed at 420 nm. Chlorin peaks are seen between 640 and 670 nm
depending on the composition of the chlorins.
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R.F. Chen et al. / Organic Geochemistry 31 (2000) 1755±1763
First, the incident angle of the excitation radiation was
altered aording optimal signal intensity at 25 , similar
to the 22 optimal angle observed in ¯uorescence investigations of fuels in soils (Fig. 2; Apitz et al., 1992a).
This maximum is a result of reducing scattering while
maintaining signi®cant signal and may be somewhat
dependent on the optical con®guration used.
Next, water content also showed an eect on ¯uorescence eciency. Fig. 3 shows dried standard sediments
(sand and kaolinite, not organic-free) spiked with
chlorophyll a (added in acetone and then dried) with
consecutive amounts of deionized water added. Fluorescence response decreases with increasing water content. Possible explanations for this behavior are: (1)
Fig. 2. The eect of incident angle of the excitation beam to the window of the powder sample holder in the PTI QM-1 spectro¯uorometer on the ¯uorescence response of chlorophyll a.
Fig. 3. The eect of water added to dry sediment on the ¯uorescence response of chlorophyll a.
R.F. Chen et al. / Organic Geochemistry 31 (2000) 1755±1763
absorption of light by the organic compounds associated with the standard mineral grains and which dissolve into the water, (2) increased re¯ection of the
excitation light by water, or (3) a hydration of the
pigment on the sediment surface, thus altering its geometry and reducing its quantum yield. Water extractions of the sediments did not show an appreciable
absorption of light at 420 nm or 680 nm compared to
the absorption due to chlorins at relevant concentrations. It is known that the ¯uorescence of chlorophyll
can be either increased or decreased by the association
of the chromophore with various surfaces and by its
interaction with various solvents (Mamleeva et al., 1988;
Komleva et al., 1989). Most probably, water interacts
with the surface of the mineral grain, ``releasing'' the
chlorin from the surface, with a concomitant reduction
in the surface-enhanced ¯uorescence of the chlorin.
Another possibility is that water reduces the re¯ectivity
of the sediment (wet sediments are darker) thus reducing
the excitation and emission light throughput (Lohmannsroben and Schober, 1999).
While chlorophyll a spiked on to all sediment standards yielded good linearity (r2>0.85) over various
concentrations ranges varying from 0±1.5 mg/g for kaolinite to 0±60 mg/g for silt, ¯uorescence intensity actually
decreases with increasing concentration above some
threshold concentration, probably due to chromophore
stacking and quenching. In any case, detection limits
were around 1 mg/g for all standard sediments.
Sediment composition also has a dramatic eect on
¯uorescence response (Apitz et al., 1992b). Response
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factors for the various matrices are shown in Fig. 4.
Kaolinite and opal yield the highest sensitivity of detection and bentonite yields the lowest for the standard
minerals measured. This variability in ¯uorescence
response is probably due to complex surface interactions
between the chromophore and the mineral surfaces
(Mamleeva et al., 1988). Formation of complexes would
generally reduce ¯uorescence while certain surfaces
could enhance the pigment ¯uorescence. Thus the solidphase ¯uorescence method may be in¯uenced by variations (especially in the opal and kaolinite fraction) in
sediment composition. A full description of the lithology of the analyzed sediments would aid in the interpretation of the relative abundances of chlorins in those
sediments.
Finally, the grain size of the same minerals shows an
eect on the ¯uorescence response factors (Fig. 5).
Increasing grain size generally decreases ¯uorescence
response. There are two possible explanations. First, the
more re¯ecting ®ne grained sediments, especially for
opal with respect to quartz, may lead to an increase in
the eective path length of the excitation radiation and
the resulting ¯uorescence emission. The ¯uorescence
response is thus enhanced because light enters and exits
the small grains more eciently. The dramatic decrease
in ¯uorescence response with grain size for opal but not
for sand supports this explanation. It has been shown
that ¯uorescence responses in sediments can be corrected by taking into account sediment re¯ectance
(Lohmannsroben and Schoder, 1999). Second, as grain
size increases, more chlorin may be forced out of the
Fig. 4. The eect of mineral composition on ¯uorescence response of chlorophyll a.
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R.F. Chen et al. / Organic Geochemistry 31 (2000) 1755±1763
Fig. 5. The eect of grain size on the ¯uorescence response of chlorophyll a.
sediment grain interstices and would, therefore, be more
prone to stack together, self-quenching the ¯uorescence
emission of the chlorin. This is the opposite eect that
was seen with smaller chromophores, the polynuclear
aromatics, in marine diesel fuel as observed by Apitz
(1992a). In that study, the smaller grain sizes oered
more surface areas for PAH to adsorb and be hidden
from excitation radiation, thus the ¯uorescence response
increased with increasing grain size. The large planar
geometry of the chlorin probably dominates the
adsorption factors in our case. Again, knowledge of
grain size would aid in the calibration of solid-phase
¯uorescence for determining the relative abundances of
chlorins.
Humic substances, major constituents of marine sediments, are known to ¯uoresce (Hayase and Tsubota,
1985; Chen and Bada, 1995), but do not show appreciable interference in the 650±700 nm range. A ¯uorescence emission scan, given the optical con®guration
(including a 530 nm long wavelength pass ®lter), of
humics obtained from Aldrich Chemical yielded an
apparent maximum at 550 nm. The tail of this ¯uorescent peak in the 650±700 nm range was simply background-subtracted along with scattered light and
random noise and did not interfere with quanti®cation
of the chlorins.
To correct for all these possible interferences, a correction factor for the ¯uorescence response of chlorins
could be calculated given information on sediment
water content, mineral composition and grain size.
However, using real marine sediments from a wide
range of environments [Boston Harbor surface sediments and sediments from the continental shelf o NW
Africa (Ocean Drilling Program Leg 108, Site 658C;
Ruddiman et al., 1988)] and wide range of chlorin concentrations (0±50 mg/g), the solid phase ¯uorescence
technique does agree quite well with both the spectroscopic method (Fig. 6a; r2=0.82) and the HPLC
method (Fig. 6b; r2=0.77) for 40 samples. A closer
examination of the HPLC method shows a signi®cant
intercept using this method while the solid phase
method shows no ¯uorescence (Fig. 6b). This may be
due to the HPLC quanti®cation (based on absorbance)
of peaks that are not chlorins or chlorins that are not as
¯uorescent as chlorophyll a. Because the solid and
liquid phase ¯uorescence methods correlate with a
nearly one-to-one relationship (slope=0.84), it would
appear that the HPLC method quanti®es only a fraction
of the ¯uorescent chlorins (slope=0.30), but also
responds to a few peaks that are not ¯uorescent. Initial
investigations into the identity of the HPLC peaks used
for quanti®cation showed that these peaks were not
carotenoids but had absorption spectra similar to
chlorins. However, further investigation of some of
these peaks by comparison with puri®ed standards was
not carried out because detailed characterization of the
individual chlorins in marine sediments is not the focus
of this paper; rather, this new, non-destructive method
for the approximate determination of chlorin concentrations in marine sediments shows promise as a
rapid screening tool for estimating paleoproductivity
changes.
R.F. Chen et al. / Organic Geochemistry 31 (2000) 1755±1763
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Fig. 6. (a) Comparison of the concentration of chlorin in marine sediments from Boston Harbor (*) and the continental shelf o
northwest Africa (&) determined by solid phase ¯uorescence and the ¯uorescence of an acetone extract. Chlorins are quanti®ed by
comparison with calibrations using chlorophyll a. (b) Comparison of the concentration of chlorin in the same samples determined by
solid phase ¯uorescence and high performance liquid chromatography.
4. Discussion
Solid-phase ¯uorescence determination of chlorins in
marine sediments appears to be a rapid, non-destructive
method of estimating paleoproductivity changes. The
method has been shown to give similar results to
extraction methods using ¯uorescence spectroscopy
(Harris and Maxwell, 1995; Fig. 6a) and HPLC (Trees
et al., 1985; Fig. 6b). In the concentration range of 1±50
mg/g chlorin, the SPF method shows some (