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A new approach for the isolation of cellulose from aquatic plant tissue and
freshwater sediments for stable isotope analysis
Article in Organic Geochemistry · November 2008
DOI: 10.1016/j.orggeochem.2008.07.014
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Organic Geochemistry 39 (2008) 1545–1561
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Organic Geochemistry
journal homepage: www.elsevier.com/locate/orggeochem
A new approach for the isolation of cellulose from aquatic plant tissue
and freshwater sediments for stable isotope analysis
Holger Wissel, Christoph Mayr 1, Andreas Lücke *
Institute of Chemistry and Dynamics of the Geosphere, Sedimentary Systems (ICG 5), Energy & Environment Research Center Jülich, D-52425 Jülich, Germany
a r t i c l e
i n f o
Article history:
Received 18 February 2008
Received in revised form 24 June 2008
Accepted 25 July 2008
Available online 8 August 2008
a b s t r a c t
Isotope studies of the cellulose of plant tissue and sediments are increasingly important in
ecological and palaeoenvironmental studies. The reliability of analytical results in these
investigations depends on the ability to separate pure cellulose from the organic and inorganic matrix. This study evaluates the performance of a new approach for the isolation of
cellulose based on wet oxidation and cellulose dissolution in cuprammonium solution
(CUAM) with respect to conventional techniques using solely wet oxidation or wet oxidation in combination with density separation. All the methods led to identical changes in
isotopic composition of standard cellulose powder samples, providing evidence that
CUAM-treated cellulose is isotopically indistinguishable from cellulose treated with conventional methods. The performance of CUAM with aquatic plant tissue was at least equal
to those of the other methods used and in numerous cases achieved better results in terms
of cellulose purification and precision of the isotope signal. Compared to conventional
methods, contamination of several samples with small amounts of minerogenic matter
and biogenic opal could be completely removed from the extraction residue with CUAM.
While conventional methods failed to result in isolation of cellulose from two typical
fine-grained lacustrine sediments, extraction residues from CUAM treatments revealed
infrared (IR) spectra resembling those of standard cellulose powder, without evidence of
minerogenic matter, biogenic opal, chitin or refractory organic matter. A series of sedimentary materials ranging from soil, to sediment trap and sediment core material were
extracted with CUAM. IR spectra of all materials tested correspond to cellulose, and carbon
and oxygen isotope compositions of the extracted cellulose fractions agreed with previous
knowledge about the samples. Our results prove that CUAM is a reliable method that yields
clean and pure cellulose for high quality isotope analysis from plant tissue and different
types of sediments alike.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Investigation of the stable isotope composition of light
elements in cellulose (H, C, O) represents an increasingly
important approach for palaeoecological and palaeoclimatological studies. Applications have focussed on the study
* Corresponding author. Tel.: +49 2461 614590; fax: +49 2461 612484.
E-mail address: a.luecke@fz-juelich.de (A. Lücke).
1
Present address: GeoBio-CenterLMU and Department of Earth and
Environmental Sciences, Ludwig-Maximilians-Universität, Richard Wagner-Strasse 10, D-80333 Munich, Germany.
0146-6380/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.orggeochem.2008.07.014
of the C and O isotope composition of cellulose in tree rings
(e.g. Treydte et al., 2006) and, to a lesser extent, in aquatic
plants (e.g. Sternberg et al., 1984) and peat mosses (e.g.
Ménot-Combes et al., 2002). Accordingly, various methods
for the chemical extraction of cellulose from wood have
been developed (Macfarlane et al., 1999; Brendel et al.,
2000; Boettger et al., 2007) that are, in the majority, derivatives of the Jayme–Wise method (Green, 1963).
The use of sedimentary cellulose in palaeohydrology
was first proposed by Edwards and McAndrews (1989).
As a result of technical and methodological advances,
isotope studies of cellulose in organic matter (OM)
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H. Wissel et al. / Organic Geochemistry 39 (2008) 1545–1561
incorporated into lacustrine sediments has attracted
increasing interest during the last decade (Wolfe and Edwards, 1997; Wolfe et al., 2003; Sauer et al., 2001). Compared to wood, the extraction of cellulose from
sediments is, however, exceedingly more laborious and error prone. Freshwater or marine sediments have a complex
composition of abiogenic and biogenic constituents. Sedimentary OM consists of a wide variety of terrestrial and
aquatic plant tissue and organic compounds such as lipids,
pectin, proteins, cellulose and hemicellulose, as well as
refractory OM and humic substances. To our knowledge,
only one method, developed at the University of Waterloo,
was specially designed to solve the challenge posed by the
extraction of sedimentary cellulose (Wolfe et al., 2001,
2007).
Since the reliability of oxygen isotope measurements
depends primarily on the extraction quality, the isolation
of pure cellulose is crucial for sedimentary studies. However, in a number of applications on fine-grained lacustrine
sediments we detected contamination in the cellulose
extraction residues, i.e. minerogenic components, diatom
frustules, chitinous remains or refractory OM with the
Wolfe et al. (2007) method. Similar problems of cellulose
extraction also only partly solved by density separation
were described by Beuning et al. (2002).
In principle, any technique applied to cellulose extraction from sediments should fulfil several fundamental criteria for facilitating optimal accuracy and precision of
subsequent isotope analysis. A physically clean cellulose
preparation devoid of minerogenic residues avoids bias
from matrix–analyte interactions and release of traces of
inorganically bound O during pyrolysis. A chemically pure
cellulose preparation, often conventionally termed a-cellulose, circumvents any bias induced by impurities in the
analyte from other polysaccharides such as, e.g. hemicellulose and chitin. Quantitative and reproducible yields of cellulose evidence the quality of the extraction process and
prevent bias by conceivable isotopic inhomogeneity of
the target cellulose. Furthermore, efficiency in terms of
raw material and sample throughput as well as universality in terms of applicability to OM of diverse origin would
foster the technique.
This study evaluates the performance of a novel approach for the isolation of cellulose from sedimentary matter. The protocol is based on classical approaches of
cellulose extraction with sodium chlorite (NaClO2) and of
cellulose dissolution in cuprammonium solution
([Cu(NH3)4](OH)2; Gupta and Sowden, 1964; Green,
1963; Klemm et al., 1998). Here, we report a number of
experiments with various materials, including cellulose
powders, aquatic plant tissue and fine-grained sediments,
using the developed method (CUAM) in comparison with
the Wolfe et al. (2007) method for sediments (UWEIL, University of Waterloo Environmental Isotope Laboratory) and
a modified Jayme–Wise method used in our laboratory for
woody material (JUEL). Unlike other methods, the new approach achieves a clean and pure fraction of cellulose, not
by removing contaminants, but by separating the target
cellulose from the contaminants by way of cellulose dissolution and subsequent precipitation. The performance of
the different treatments is first compared for standard
cellulose materials, followed by experiments with different
plant tissues and finally by a discussion of results from
fine-grained freshwater sediments.
2. Experimental
2.1. Materials
Commercially available cellulose powders were used as
standards without further treatment. Fresh plant material
from aquatic macrophytes, water mosses and algae was
harvested in the field and brought directly to the laboratory. Further details of the samples are summarized in Table 1. As far as necessary, plant tissue was cleaned
macroscopically through washing in demineralized water,
ultrasonication and hand-picking. Aquatic macrophyte
samples Myriophyllum II and Elodea II were only washed
in demineralized water. The wet material was freeze dried,
homogenized using ultra-centrifugal milling (Retsch,
0.75 mm sieve) at room temperature for 30–60 s and separated into aliquots for replicate chemical extractions. Sedimentary materials were sampled either directly in the
field or, in the case of cores, in the laboratory. Sediments
were treated with aqueous sodium hexametaphosphate
[(NaPO3)6] solution (5%) at room temperature overnight
(16 h) to ensure dispersion. Following dispersion, sediments were sieved at 200 lm to remove coarse organic
debris, washed clean with deionized hot water ( 70 °C)
and freeze dried. The fraction < 200 lm was divided into
aliquots. From each material and each extraction protocol
(UWEIL, JUEL, CUAM) at least three separate extractions
were performed. For UWEIL preparations including the
density separation step, aliquots of HZM surface and AZUL
were additionally sieved at 20 lm to provide a 20–200 lm
fraction.
2.2. Cellulose extraction protocols
We used three different extraction methods. A protocol
developed at UWEIL especially for lacustrine sediments, a
modified Jayme–Wise protocol used in our laboratory for
woody materials (JUEL) not adapted for sedimentary samples and the new protocol as described here (CUAM). All
protocols consist of several steps.
2.2.1. UWEIL protocol
For practical and safety reasons it was necessary to
slightly modify single steps of the method as described
by Wolfe et al. (2001, 2007). (i) Initially, carbonate was removed by dissolution with HCl (5%) for 2 h at 50 °C in a
water bath. The residue was repeatedly washed with
deionized water. (ii) The removal of lipids, tannins and resins was achieved by solvent extraction in a toluol/ethanol
mixture (2:1) for 24 h at room temperature (3–4 until
completion). Following the last solvent treatment, the mixture was centrifuged and decanted. Acetone was added to
the residue and left for 24 h before the sample was again
decanted and air dried in a fume hood. (iii) Chemical
bleaching of the sample was accomplished by treatment
with sodium chlorite (7%) acidified to pH 4–5 with concen-
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Table 1
Materials used for cellulose extraction experiments and abbreviations used in the text (n.d., not detected)
Abbreviation
Description
Corg (%)
O (%)
Cellulose
Avicel
FlukaCell
IAEA-C3
Cellulose powder, Merck (for column chromatography)
Cellulose powder, Fluka (cellulose from spruce)
Cellulose powder, IAEA Vienna (reference material, milled plates, charge number 337)
43.3
42.2
43.7
47.3
46.8
47.2
Macrophyte
Elodea I
Potamogeton
Callitriche
Myriophyllum I
Myriophyllum II
Elodea II
Whole
Whole
Whole
Whole
Whole
Whole
plant
plant
plant
plant
plant
plant
tissue,
tissue,
tissue,
tissue,
tissue,
tissue,
40.7
37.6
41.3
42.2
35.8
36.4
40.6
36.4
37.6
41.9
39.3
36.4
Aquatic mosses
Fontinalis I
Drepanocladus
‘Aquatic moss’
Fontinalis II
Whole
Whole
Whole
Whole
plant
plant
plant
plant
tissue, stream, Kirnitzschtal, Germany (Fontinalis antipyretica)
tissue, Laguna Potrok Aike, Patagonia, Argentina (Drepanocladus perplicatus)
tissue, Laguna Vizcachas, Patagonia, Argentina, undetermined
tissue (>500 lm), brook, Wennigsen, Germany (Fontinalis antipyretica)
46.6
41.6
41.4
37.3
35.8
39.8
37.6
35.4
Algae
Cladophora
Rhizoclonium
Vaucheria
Whole plant tissue, Lake Holzmaar, Germany (Cladophora glomerata (L.) Kütz.)
Whole plant tissue, Laguna Azul, Patagonia, Argentina (Rhizoclonium spec.)
Whole plant tissue, pond, Wennigsen, Germany (Vaucheria spec.)
39.4
34.4
39.6
37.2
n.d.
n.d.
Sediments
HZM surface
HZM trap
HZM 6500
SAC surface
SAC 1700
POND wen
SOIL for
AZUL
Surface sediment, Lake Holzmaar, Germany, clam-shell bottom sampler
Sediment trap, epilimnion, Lake Holzmaar, Germany
Sediment core, age 6500 cal BP, Lake Holzmaar, Germany, piston core
Lake surface sediment, Sacrower See, Germany, gravity core
Lake sediment core, age AD 1700, Sacrower See, Germany
Surface mud, pond, Wennigsen, Germany
Forest soil, top horizon (A), Wennigsen, Germany
Surface sediment, Laguna Azul, Patagonia, Argentina, gravity core
5.2
7.5
8.3
13.1
7.0
4.2
5.8
12.0
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
pond, Wennigsen, Germany (Elodea canadensis)
stream, Kirnitzschtal, Germany (Potamogeton pectinatus)
stream, Kirnitzschtal, Germany (Callitriche palustris)
Lake Holzmaar, Germany (Myriophyllum spec.)
pond FZ Jülich, Germany (Myriophyllum spec.)
brook, Jülich, Germany (Elodea canadensis)
trated acetic acid (96%) at 60 °C. After 10 h bleaching, the
solution was exchanged and the process continued for another 10 h. Subsequently, the sample was washed (2–3)
with hot deionized water ( 70 °C) to displace the bleach
solution. (iv) Other polysaccharides remaining besides
cellulose were removed by addition of NaOH (17%) and
reaction at room temperature for 45 min. The residue
was washed with cold deionized water and neutralized
with dilute acetic acid. (v) To remove Fe- and Mn-oxyhydrates a leach solution containing 35 g sodium dithionite,
52 g ammonium citrate and 14 g hydroxylamine hydrochloride in 1 l deionized water was used. Leaching for 2 h
at 60 °C was followed by leaching for 24 h at room temperature. Leaching was repeated at room temperature with
exchanged leach solution until the cellulose concentrate
was permanently discoloured. The cellulose residue was
then repeatedly washed with cold deionized water (5–
7) and freeze dried. (vi) Finally, minerogenic components
were removed by heavy-liquid density separation in centrifuge tubes with sodium polytungstate solution adjusted
to a specific gravity of 1.9–2.0. Fe- and Mn-oxyhydrate
leaching and density separation were not used for standard
cellulose powders and plant tissue. For alternative demineralisation, samples were treated with HCl–HF (10%) for
16 h at room temperature.
2.2.2. JUEL protocol
This protocol is a modified version of the Jayme–Wise
protocol after Green (1963), originally designed for wood
powder and complemented with a decarbonization step.
(i) Carbonate was removed by dissolution in HCl (5%) for
2 h at 50 °C in a water bath; the residue was repeatedly
washed with deionized water. (ii) Solvent extraction was
substituted by an initial treatment with NaOH (5%) at
60 °C, repeated twice for 1 h (Rinne et al., 2005). (iii) Chemical bleaching was performed with NaClO2 (7%) acidified to
pH 4–5 with concentrated acetic acid (96%) at 60 °C. After
10 h, the solution was exchanged and the treatment was
repeated for another 10 h. The residue was washed (2–
3) with deionized hot water ( 70 °C) to displace the
bleach solution. (iv) Finally, remaining non-cellulose polysaccharides (hemicellulose) were removed by treatment
with NaOH (17%) for 45 min at room temperature. The
extraction residue was washed with cold deionized water
and neutralized with diluted acetic acid.
2.2.3. CUAM protocol
Cuprammonium solution (cuam, cuoxam) or Schweizer
reagent is an aqueous metal complex solution well known
for its capability for dissolving cellulose in pulp chemistry
(Schweizer, 1857; Saalwächter et al., 2000). Even medium
to highly polymerized cellulose (DPw < 5300) dissolves
quickly and completely in cuprammonium solution at a
copper concentration between 15 and 30 g/l (Klemm
et al., 1998). To verify the copper concentration necessary
for the dissolution of cellulose from natural samples, experiments were carried out with plant tissue and sediments
after conventional sodium chlorite bleaching. The copper
hydroxide concentrations used were 10, 15, 20 and 30 g/l.
The results showed that cellulose was dissolved at all con-
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centrations tested. Reproducibility of cellulose yield and of
carbon isotope values was best at a copper hydroxide concentration of 15 and 20 g/l. Based on these results, we developed a protocol merging a conventional approach for
bleaching with sodium chlorite with a classical approach
for cellulose dissolution in cuprammonium solution. Initial
bleaching is necessary to make the cellulose in the samples
readily accessible to the metal complex.
In the protocol, (i) chemical bleaching of the sample was
accomplished by treatment with NaClO2 (7%) acidified to
pH 4–5 with concentrated acetic acid (96%) for 10 h at
60 °C. The residue was washed (2–3) in hot deionized
water ( 70 °C) to remove the reactant and freeze dried.
(ii) The cuprammonium solution was prepared in a
1000 ml amber glass bottle; 15 g copper hydroxide
[Cu(OH)2] were dissolved in 900 ml ammonium hydroxide
solution (25%) and 100 ml deionized water. The resulting
blue coloured solution was stirred for 16 h at room temperature and filtered (Klemm et al., 1998, vol. 1, p. 234). The
solution was stored in a cool, dark place (fridge). The dry
sample ( 3 g sediment) was placed in a 50 ml stirrer jar,
ca. 20–30 ml cuprammonium solution were added to each
jar, which was closed with a lid. The jar was placed on a stirrer for 6 h and left for a further 10 h at room temperature to
completely dissolve the cellulose. The supernatant copper
complex cellulose solution was decanted into a centrifuge
tube and centrifuged at 4000 rpm for 25 min. The supernatant was again decanted into another centrifuge tube
loaded with 3 ml H2SO4 (20%) and subsequently filled up
to 50 ml with cold deionized water. The tube was closed
with a lid and the solution carefully shaken before it was left
for 20 min at room temperature. The solution was again
centrifuged at 4000 rpm for 25 min and decanted. Some
drops of deionized water and 3 ml H2SO4 (20%) were added
to the precipitate and the centrifuge pellet (blue) was destroyed. The sample was then allowed to sit (1–5 min) until
the precipitate was completely ‘‘decopperised” (change of
colour from blue to white) and filled up with cold deionized
water. The precipitate was rinsed at least 3 with deionized
water at room temperature and freeze dried.
Treatment with cuprammonium solution might increase the amorphous character of cellulose and therefore
probably water content. To avoid effects on isotope composition induced by additional water adsorbed to cellulose
after CUAM treatment, samples of CUAM cellulose were
stored in a vacuum drier at 40 °C after packing in silver
capsules. Immediately prior to analysis, samples were
placed overnight (16 h) in a vacuum drier at 100 °C.
2.3. IR spectroscopy
IR spectra of standard materials and extracted fractions
were measured using direct transmittance with the KBr
pellet technique. Spectra (4000–200 cm 1, 2.4 cm resolution) were obtained using a Perkin-Elmer PE 783 spectrometer equipped with a coated thermocouple detector.
Transmission spectra were converted to absorbance. The
spectra were normalized relative to the difference in
absorbance at 1560 and 1164 cm 1 to enable comparability of individual spectra. Comparison of band intensities
can only describe qualitative differences. IR spectra are
shifted relative to each other to improve visibility of single
spectra in the graphs, so are given in arbitrary units (A.U.)
on the ordinate. Absorbance values were smoothed by
applying an 11 point moving average filter.
2.4. Mass spectrometry/isotope analysis
Values of d13C were determined using 200–300 lg dry
cellulose weighed into tin foil cups and combusted at
1080 °C using an elemental analyzer (EuroEA, Eurovector)
interfaced on-line to an isotope ratio mass spectrometer
(Isoprime, GV Instruments). Carbon content was determined through peak integration of m/z 44, 45 and 46,
and calibrated against elemental standards. For measurement of d18O values, about 275 lg of cellulose was
weighed in silver capsules, vacuum-dried and pyrolysed
at 1450 °C in a high temperature pyrolysis analyzer (HTO, HEKAtech), and measured on-line with a coupled GV
Instruments Isoprime isotope ratio mass spectrometer.
Oxygen content was determined by peak integration of
m/z 28, 29 and 30, and calibrated against elemental standards. Isotope results are reported as d values in per mil
using conventional notation d = (Rs/Rst 1) 1000 with
Rs and Rst as isotope ratios (13C/12C, 18O/16O) of samples
and international standards (VPDB for carbon, VSMOW
for oxygen), respectively. The overall precision of replicate
analyses is estimated to be better than 0.1‰ for d13C, 0.3‰
for d18O and 5% (rel.) for C and O content.
3. Results and discussion
3.1. Variation in extract yield
The effect of the extraction procedure on the amount of
original sample mass remaining after treatment is shown
in Fig. 1a for different standard cellulose powders. Independent of method, all treatments caused a loss of standard material and quantitative yield (100%) of cellulose
was never accomplished. Highest yields were found for
all materials with CUAM, in which the maximum recovery
reached 92.8% (av. of three replicates). Variations between
replicate treatments were found to be rather high, partly
exceeding differences of 10% in yield, although extractions
were conducted with great care. The range in yield for the
same standard material between the methods could be due
to differences in the chemical treatments and the number
of processing steps involved in the protocols. Moreover,
standard cellulose powders differ with respect to physical
and chemical cleaning steps in their preparation as well
as in degree of polymerization, so can be expected to perform differently with different protocols. Hence, these results should not be overemphasized and may not be
representative for natural samples.
Because of its different functionality, cellulose content
in vascular and non-vascular plants can vary largely from
about 42% to 50% in wood (Sjostrom, 1993) and from 2%
to 39% or even complete absence in green algae (Rho and
Litsky, 1979). The actual cellulose yields found for plant
tissue of aquatic macrophytes, aquatic mosses and green
algae fall in this range, with mean values of replicate treatments from about 7% to 23% (Fig. 1b–d). Highest amounts
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100
80
60
40
20
0
30
25
20
15
10
5
0
30
25
20
15
10
5
0
30
25
20
15
10
5
0
0.20
(a)
Avicel
FlukaCell
IAEA-C3
(b)
Yield (%)
Yield (%)
H. Wissel et al. / Organic Geochemistry 39 (2008) 1545–1561
Elodea I
Potamogeton
Callitriche
Myriophyll. I
Myriophyll. II
Elodea II
Yield (%)
(c)
Fontinalis I
Drepanocladus
Aquatic moss
Rhizoclonium
Vaucheria
Fontinalis II
Yield (%)
Yield (%)
(d)
Cladophora
(e)
0.15
0.10
0.05
0.00
HZM surf
HZM trap
HZM 6500
SAC surf
SAC 1700 POND wen
SOIL for
Materials
Fig. 1. Extraction yield of standard cellulose powders (a), macrophyte
tissue (b), water moss tissue (c), green algae (d) and sediments (e) using
UWEIL (blue bars), JUEL (green bars) and CUAM (brown bars) methods.
Height of bars represents arithmetic mean values of replicate treatments
and thin lines the respective standard deviation (1s).
of cellulose were obtained from aquatic mosses, while
yields for macrophytes were lowest, with cellulose con-
tents of green algae being intermediate. The differences
in yield for the same tissue with the different methods
are not as distinct as those found for the standard cellulose
materials but may reach comparable orders of magnitude
on the basis of absolute cellulose content. None of the
methods stands out from the others in terms of the amount
of cellulose that can be gained from a certain material. The
reproducibility in replicate treatments is considerably improved vs. standard cellulose powders. In the majority of
cases one standard deviation does not greatly exceed a value of 1%. Thus, within the methodological uncertainties,
cellulose yields from plant tissue can comprise a good estimate of relative differences in cellulose content between
plant taxa.
Yields for sediments could not be compared between
the different methods because of methodological problems
(i.e. impurities) with UWEIL and JUEL that are discussed
below. The yields with CUAM for diverse sedimentary
materials from seston to soil (Fig. 1e) are about two orders
of magnitude lower than those found for plant tissue.
These rather low cellulose contents agree well with results
from studies on natural sediments and soil with other
methods of cellulose quantification (Uzaki and Ishiwatari,
1983; Ogier et al., 2001; Martens and Loeffelmann, 2002).
The yields were low, independent of the carbon content
of the sample. Despite rather low cellulose content in the
samples, the reproducibility within the replicates is quite
satisfactory (1s 6 0.01%). The small amounts of cellulose
in these materials illustrate the demanding methodological requirements that have to be met for the reproducible
and quantitatively comparable extraction of cellulose from
sediments of various kinds.
3.2. Chemical and isotopic characterisation of extraction
residues from standard cellulose powders
Fig. 2 shows the IR spectra of the untreated and CUAMtreated commercial cellulose powders. They exhibit sev-
1.8
1.6
Absorbance (A.U.)
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
-0.4
4000 3750 3500 3250 3000 2750
1800
1600
1400
1200
1000
800
600
400
-1
Wavenumber (cm )
Fig. 2. IR absorbance spectra of unprocessed and CUAM-processed standard cellulose powders: Avicel (untreated: light blue, treated: dark blue), IAEA-C3
(untreated: orange, treated: brown) and FlukaCell (untreated: light green, treated: dark green).
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eral absorption bands and peaks characteristic of cellulose
(Silverstein and Webster, 1998; Pandey, 1999; Pandey and
Pitman, 2003; Liu et al., 2004). At high frequencies a strong
O–H stretching (H-bonded) absorption is seen around
4000–3000 cm 1, and a prominent C–H stretching absorption at 2900 cm 1. The peaks in the fingerprint region between 1800 and 800 cm 1 have been assigned to
absorbed O–H and conjugated C–O at 1640 cm 1, C–H
deformation in carbohydrates at 1426 cm 1, C–H deformation in cellulose at 1373 cm 1, CH2 wagging at 1317 cm 1,
O–H deformation at 1200 cm 1, C–O–C asymmetric vibration at 1162 cm 1, glucose ring stretch at 1107 cm 1, C–O
stretch at 1058 cm 1, C–O stretch at 1019 cm 1 and C–H
deformation in cellulose at 894 cm 1.
Compared with the controls, several of these characteristic bands changed in absorbance or wavenumber in the
CUAM-treated spectra. Common to all standard celluloses
the peaks at 1110 and 1429 cm 1 are concealed, whereas
absorbance is increased at 894 cm 1 and in the band from
350 to 550 cm 1 (Fig. 2). For Avicel and IAEA-C3 maximum
absorbance of H-bonded O–H stretching is shifted to higher wavenumber (from 3365 to 3444 cm 1) and the C–H
stretch is slightly shifted to lower wavenumber (from
2900 to 2895 cm 1). Similar changes in absorbance characteristics of standard celluloses are also effected by UWEIL
and JUEL (not shown). The peak shifts observed are attributed to the alkaline treatment, either by NaOH or NH3, that
is part of all the extraction protocols (Oh et al., 2005a,b).
Alteration in absorbance at 1650 and 3364/3444 cm 1, increases for Avicel and IAEA-C3 and decreases for FlukaCell,
indicate changes in crystallinity and amount of adsorbed
water as induced by chemical treatment (Hurtubise and
C/O
1.1
Krässig, 1960; Liu et al., 2004; Oh et al. 2005a). Similarly,
coeval changes in absorbance in the bands at 1426 and
894 cm 1 are referred to as index for changes in cellulose
crystallinity (A1426/A894) by Oh et al. (2005a,b). In summary, the chemical treatments caused alteration of the
spectra of standard celluloses in relation to the amount
of adsorbed water and crystallinity. These changes were,
however, not specific but common to all three methods.
Further drying at higher temperature (> 100 °C) would
not necessarily lead to the removal of a larger amount of
adsorbed water below the thermal decomposition threshold (Carrillo et al., 2004).
According to the stoichiometric C and O content of cellulose the theoretical C/O ratio is 0.90. Among the untreated standard cellulose powders only FlukaCell has
exactly this theoretical value, while values for Avicel and
IAEA-C3 are slightly higher (Fig. 3a). Alteration of the ratio
by the treatments is generally small to moderate, with the
exception of IAEA-C3, where all methods, and especially
the JUEL protocol, led to lower values. This could reflect
distinctive characteristics for this material, lower degree
of polymerization, probably in conjunction with an increased amount of absorbed water caused by the chemical
treatment.
The d13C and d18O values of the treated standard cellulose powders are shown in Fig. 3b and c. All three methods
cause insignificant changes in the carbon isotope compositions although a slight unidirectional move to depleted
values is visible vs. untreated cellulose. A similar but stronger shift is evident for the oxygen isotopes, with offsets
reaching values between 0.5‰ and 1.0‰ depending on
material and method. The depleted values are possibly
1.1
(a)
1.0
1.0
0.9
0.9
0.8
0.8
-23
-24
-24
-25
-25
18
δ O (‰)
13
δ C (‰)
(b)
-23
33
32
31
30
29
28
27
(c)
Avicel
FlukaCell
IAEA-C3
33
32
31
30
29
28
27
Materials
Fig. 3. C/O ratio (a), carbon isotope composition (b) and oxygen isotope composition (c) of standard cellulose powders. Comparison between untreated
cellulose (black) and cellulose with three different extraction methods, UWEIL (blue), JUEL (green) and CUAM (brown). Shown are mean values (closed
circles) of replicate treatments of the same material together with the overall mean (open squares) and standard deviation (bars) for all measurements of
the respective material.
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associated with changes in water content evident from IR
spectroscopy and, however less likely, exchange between
cellulose water and demineralised water. Specific isotope
labelling experiments would be needed to verify this
hypothesis. Nevertheless, all the methods cause carbon
and oxygen isotope changes indistinguishable from each
other. The results prove that the use of cuprammonium
solution (alkali dissolution/acid precipitation) does not result in exceptional changes in the isotopic composition of
cellulose extracted with the CUAM method compared to
conventional methods.
3.3. Chemical and isotopic characterisation of extraction
residues from aquatic plant tissue
3.3.1. Aquatic macrophytes
Fig. 4 shows an example of IR spectra for an aquatic
macrophyte of the genus Myriophyllum (Myriophyllum
II), comparing the plant tissue with extraction residues
from the different treatments. The spectrum of the untreated tissue reveals several major bands relating,
according to Kačuráková et al. (2000), Giordano et al.
(2001) and Gorgulu et al. (2007) to a range of molecular
components. The strong absorption in the region 3700–
3000 cm 1 arises mainly from N–H and O–H stretching
modes of proteins and polysaccharides, intermolecular
H bonding and adsorbed water. The peak around
2917 cm 1 is caused by CH2 and CH3 symmetric and
asymmetric stretching in lipids and proteins. In the fingerprint region, the Amide I band at ca. 1650 cm 1 is assigned to C@O stretching in proteins and pectin. The
strong absorbance of the Amide I band partly disguises
the C@O stretching from phospholipids and hemicellulose
at 1722 cm 1. Various conspicuous bands in the region
1200–950 cm 1 are assigned to C–O stretching, OH
stretching and P@O stretching, indicating a combination
of different cell wall polysaccharides.
The spectra of the extraction residues show notably different absorbance patterns, indicating the effectiveness of
the respective chemical treatments. The excellent match
between Myriophyllum II treated via JUEL and CUAM and
the target cellulose spectrum (cf. Fig. 2) testifies to the
complete removal of contaminants and the yield of pure
cellulose with these protocols. In contrast, the UWEIL-treated spectrum of Myriophyllum II shows atypical strong
bands at 1093 and 465 cm 1 and reduced absorbance at
3440 and 2900 cm 1. The distinctive peak at 465 cm 1
and the higher wave number of the band at 1093 cm 1
vs. cellulose (1063 cm 1) can best be explained by biogenic
opal impurity in the extraction residue. Biogenic opal,
characterised by strong absorbance at 1091 and
464 cm 1 (cf. Fig. 10b), disguises the bands typical for cellulose and dominates the spectrum of the Myriophyllum II
extract for UWEIL. The opal contamination is most likely
caused by frustules of epiphytic diatoms that were not removed by mechanical cleaning and alkaline treatment.
Thus, Myriophyllum II is an example where the UWEIL
method is insufficient for complete removal of biogenic silica. Because of the opaline dominance in the spectrum we
can not further evaluate whether organic components also
contaminate the cellulose in such cases. The challenge of
opal contamination of aquatic macrophyte tissue can not
solely be solved by mechanical pre-cleaning since the IR
spectrum of the thoroughly cleaned Myriophyllum I (not
shown) is, similar to Myriophyllum II, also strongly influenced by opal bands.
The C/O values of macrophyte tissue from different genera decrease to varying degrees with all the treatments but
emerge overall at 0.85–0.95 (Fig. 5a). The difference between the untreated and the treated tissue varies from
genus to genus, most likely due to variable contents of
hydrocarbons and oxygen-bearing contaminants. The
UWEIL method gave unexpectedly low values (av. 0.78)
for Myriophyllum I for unknown reasons. The carbon iso-
2.0
1.8
Absorbance (A.U.)
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
4000 3750 3500 3250 3000 2750
1800
1600
1400
1200
1000
800
600
400
-1
Wavenumber (cm )
Fig. 4. IR absorbance spectra of whole tissue and extraction residue of an aquatic macrophyte, genus Myriophyllum retrieved from Jülich pond
(Myriophyllum II). Untreated tissue (black) in relation to tissue treated using UWEIL (blue), JUEL (green) and CUAM (brown) methods.
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C/O
1.1
1.0
0.9
0.9
0.8
0.8
0.7
0.7
13
δ C (‰)
(b)
-16
-20
-20
-24
-24
-28
-28
-32
-32
26
18
1.1
1.0
-16
δ O (‰)
(a)
(c)
26
23
22
21
20
19
18
23
22
21
20
19
18
Elodea I
Potamogeton
Callitriche
Myriophyllum I Myriophyllum II
Elodea II
Materials
Fig. 5. C/O ratio (a), carbon isotope composition (b) and oxygen isotope composition (c) of macrophyte cellulose. Comparison between the untreated bulk
material (black) and cellulose for different extraction methods, UWEIL (blue), JUEL (green) and CUAM (brown). Shown are mean values (closed circles) of
replicate treatments of the same material together with the overall mean (open squares) and standard deviation (bars) for all measurements of the
respective material.
tope composition of macrophyte tissue showed large variations between genera (Fig. 5b). Independently, the different treatments caused only small deviations from the
untreated control, with negligible differences between
the single variants. The largest deviation is for Potamogeton, with a 13C enrichment of 1.0‰ after all treatments.
Removal of non-cellulose compounds from plant tissue
should, in general, increase the d18O value of the residue
because, e.g. lipids are isotopically lighter than cellulose
(Boutton, 1996; van Dongen et al., 2002). As expected, ‘‘cellulose” fractions from the extraction treatment consistently show an 18O enrichment of 3–5‰ vs. bulk tissue
(Fig. 5c). Enrichment seems to be independent of the bulk
value and is greatest for Elodea I. In general, observed
enrichments are similar to those reported for wood cellulose (Barbour et al., 2001). The variations between different
treatments are small and generally of the order of 0.5‰.
The exception, the UWEIL-treated Myriophyllum II sample,
shows 1‰ less enrichment than the other variants. As indicated from IR spectroscopy, the most likely reason is contamination of the cellulose by biogenic opal. At
temperatures of 1450 °C, the typical temperature for organic matter pyrolysis, biogenic opal is dehydrated and
partly decomposed, thereby contributing opal-bound oxygen to the oxygen bound in cellulose (Lücke et al., 2005).
3.3.2. Aquatic mosses
IR spectra of extraction residues from the different
treatments of Fontinalis II are shown as examples for aquatic mosses in Fig. 6. The spectra of residues generally
resemble that of standard Fluka cellulose at high frequencies. Below 1200 cm 1 and especially in the band from 750
to 350 cm 1, discrepancies arise between the spectra of
UWEIL and JUEL on one hand and CUAM on the other. In
this region a peak at 471 cm 1, accompanied by a smaller
one around 519 cm 1, indicates minerogenic impurities
in both the UWEIL and JUEL fractions (cf. Fig. 10a). Moreover, absorbance at 1360 cm 1 is reduced vs. 1063 cm 1
for both the UWEIL and JUEL fractions. The argument for
minerogenic contamination is also supported by a shift in
maximum absorbance in the 1060 cm 1 region from
1063 cm 1 in Fluka cellulose to 1022 cm 1 in the UWEIL
extract. Such contamination is absent from the CUAMtreated extraction residue of Fontinalis II.
Extracts from aquatic mosses have C/O values around
0.9 (Fig. 7). Only for Fontinalis II treated with UWEIL and
JUEL do C/O values decline towards 0.8. Fontinalis I is characterised by a rather high initial ratio of almost 1.3. This
might be the cause of the rather high offset of 2.5‰ between the carbon isotope composition of bulk and cellulose fraction of Fontinalis I. Extracted cellulose from every
other moss tissue is enriched by 0.8–1.3‰. Differences in
the carbon isotope ratios of cellulose extracted with the
different methods are small and are within the analytical
uncertainty. Like macrophyte tissue, the cellulose fractions
of aquatic mosses are enriched by 1.3–4.8‰ in oxygen isotope composition compared to bulk tissue. Although oxygen isotope differences between the treatments are
generally small and are well within the analytical uncertainty, the Fontinalis II residue from the CUAM method
shows a significant enrichment vs. both the other variants.
As the oxygen isotopic composition of minerogenic matter
is depleted relative to organic matter this has to be interpreted as inorganic contamination of the UWEIL and JUEL
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1.4
1.2
Absorbance (A.U.)
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
4000 3750 3500 3250 3000 2750
1800
1600
1400
1200
1000
800
600
400
-1
Wavenumber (cm )
Fig. 6. IR absorbance spectra of extraction residues of an aquatic moss, genus Fontinalis (Fontinalis II). Shown are spectra for tissue treated using UWEIL
(blue), JUEL (green) and CUAM (brown) methods in relation to standard cellulose FlukaCell (grey).
1.3
1.3
(a)
1.2
1.1
1.1
1.0
1.0
0.9
0.9
0.8
0.8
-28
-30
-28
-30
(b)
-36
-36
-38
-38
-40
-40
18
δ O (‰)
13
δ C (‰)
C/O
1.2
-42
26
24
22
20
18
16
14
-42
26
24
22
20
18
16
14
(c)
Fontinalis antipyretica I
Drepanocladus
Aquatic moss
Fontinalis antipyretica II
Materials
Fig. 7. C/O ratio (a), carbon isotope composition (b) and oxygen isotope composition (c) of cellulose from aquatic mosses. Comparison between the
untreated bulk material (black) and cellulose from different extraction methods, UWEIL (blue), JUEL (green) and CUAM (brown). Shown are mean values
(closed circles) of replicate treatments of the same material together with the overall mean (open squares) and standard deviation (bars) for all
measurements of the respective material.
treated tissue, as also indicated by IR spectroscopy (Fig. 6).
A smaller, but still apparent, depleted oxygen isotope value
appears for CUAM-treated ‘aquatic moss’ vs. UWEIL and
JUEL (0.5–0.7‰). The spectrum of CUAM ‘aquatic moss’ reveals a comparatively strong absorption at 1635 cm 1 and
an absorbance ratio between 3348 and 1060 cm 1 (A3348/
A1060) that is atypically high for cellulose (spectrum not
shown). Increased absorbance at these values can be
caused by adsorbed water, but could also indicate trace
amounts of proteins and polysaccharides (3348,
1635 cm 1) in the cellulose fraction. As this effect is only
observed in one case and not evident for tissue of other
moss genera, we presume contamination by undefined organic matter containing proteins and polysaccharides that
were probably removed in the NaOH step of UWEIL and
JUEL.
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3.3.3. Freshwater algae
Typical IR spectra for whole tissue and extraction residues are exemplified by the freshwater chrysophyte Vaucheria (Fig. 8). The whole plant tissue is characterised by
several bands similar to macrophyte tissue. Strong absorbance in the region 3700–3000 cm 1 is due to O–H stretching and N–H stretching modes of water, carbohydrates and
proteins, CH2 and CH3 stretching bands with strong absorbance at 2959, 2924 and 2856 cm 1 indicating lipids;
strong C@O and C@N stretching (Amide I and Amide II)
at 1645 and 1537cm 1 are attributed to proteins; the peak
at 1247 cm 1 is assigned to P@O stretching in nucleic acid
and peaks in the region 1200–900 cm 1 are attributed to
C–O–C stretching of cell wall polysaccharides (Giordano
et al., 2001; Sigee et al., 2002; Dean et al., 2007; Gorgulu
et al., 2007).
The IR spectra of the treated material reveal clear differences between the methods in as much as the spectra of
the UWEIL and JUEL extracts indicate organic impurities,
while only the CUAM extract reveals a pure cellulose spectrum for Vaucheria (Fig. 8). The pattern of multiple absorbance peaks around 2900 cm 1 for the UWEIL and JUEL
material is indicative of traces of lipids in the cellulose
fraction. The spectrum of the JUEL material additionally
shows maximum absorbance in the region 3700–
3000 cm 1 at 3342 cm 1. Together with comparably reduced absorbance around 3250 cm 1, including a secondary maximum at 3270 cm 1, this could indicate unknown
impurities in the JUEL residue (Bahmed et al., 2003; Ramírez-Coutiño et al., 2006). In the fingerprint region peaks at
1426, 1370 and 1336 cm 1, in combination with a relative
reduction in absorbance at 1650 cm 1 for the UWEIL and
JUEL extract, might be explained by hemicellulose impurities (Proniewicz et al., 2001; Pandey and Pitman, 2003;
Olsson and Salmén, 2004). However, this can also be
caused by differences in the crystallinity of cellulose extracted from Vaucheria. This is also indicated by the in-
creased peak ratio of 1425 cm 1 over 896 cm 1 (A1425/
A896) pointing towards a higher crystallinity for UWEIL
and JUEL extracted cellulose vs. that from CUAM (Wada
and Okano, 2001; Oh et al., 2005a,b).
C/O values of cellulose extracted from green algae tissue
vary between 0.85% and 0.96% (Fig. 9). Reference values for
untreated Rhizoclonium and Vaucheria are missing due to
lack of material after a failure of the analytical system in
the analysis of oxygen isotope ratios. Results of earlier
low temperature pyrolysis (1080 °C) measurements are given instead as an indicator of the general tendency between raw material and cellulose. The carbon isotope
composition of extraction residues of the different treatments are indistinguishable for Rhizoclonium and Vaucheria, while CUAM cellulose from Cladophora is
considerably less enriched in 13C (0.9‰). A similar picture
emerges for the oxygen isotope compositions. CUAM cellulose from Cladophora is 2‰ more enriched than UWEIL
and JUEL cellulose. The impurities in the cellulose fraction
of Vaucheria visible in the IR spectra (Fig. 9) for UWEIL and
JUEL treatments did not apparently impact on the isotopic
compositions. Either they did not differ in isotopic signature from the cellulose or their contributions to the total
extract were tiny. IR spectra of Cladophora reveal evidence
of either impurities in the fingerprint region or increased
crystallinity (A1425/A896) for UWEIL and JUEL and an additional opal component in the UWEIL extract (spectra not
shown). The latter is clearly not visible in the JUEL extract
spectrum despite the comparably low C/O values for both
UWEIL and JUEL extracts indicating excess oxygen, potentially from biogenic opal. Overall, reasons for the isotopic
similarity between UWEIL and JUEL and the dissimilarity
to CUAM, respectively, observed for Cladophora and Vaucheria remain ambiguous.
In summary, it appears that the performance of the
CUAM method with aquatic plant tissue is in most cases
at least equal to conventional cellulose extraction meth-
2.0
1.8
1.6
Absorbance (A.U.)
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
4000 3750 3500 3250 3000 2750
1800
1600
1400
1200
1000
800
600
400
-1
Wavenumber (cm )
Fig. 8. IR absorbance spectra of whole tissue and extraction residue of a yellow-green alga, genus Vaucheria. The spectrum of untreated tissue (black) is
shown in relation to tissue treated obtained using UWEIL (blue), JUEL (green) and CUAM (brown).
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1.10
C/O
1.05
1.10
(a)
1.05
1.00
1.00
0.95
0.95
0.90
0.90
0.85
0.85
0.80
13
δ C (‰)
-16
0.80
(b)
-16
-18
-18
-20
-20
-22
-22
-24
18
δ O (‰)
28
-24
(c)
28
26
26
*
24
24
*
22
22
20
18
20
Cladophora
Rhizoclonium
Vaucheria
18
Materials
Fig. 9. C/O ratio (a), carbon isotope composition (b) and oxygen isotope composition (c) of cellulose from freshwater algae. Comparison between the
untreated bulk material (black) and cellulose obtained using three different extraction methods UWEIL (blue), JUEL (green) and CUAM (brown). Shown are
mean values (closed circles) of replicate treatments of the same material together with the overall means (open squares) and standard deviations (bars) for
all measurements of the respective material; * indicates that untreated oxygen isotope values for Rhizoclonium and Vaucheria were generated by pyrolysis at
1080 °C and illustrates the tendency but not the absolute value of change between raw material and cellulose.
ods. Thus, no artificial fractionation effect for carbon or
oxygen is introduced by the CUAM treatment. Moreover,
the CUAM method provided reliable cellulose d18O values
even for samples for which the UWEIL and the JUEL methods could not completely remove impurities from the extracted cellulose.
3.4. Chemical and isotopic characterisation of extraction
residues from sediments
Unlike plant tissue, sediments and soil are composed of
a mixture of inorganic and organic matter of different origins. Moreover, the organic fraction is itself highly diverse
since it consists of the remains of vascular and non-vascular plants, refractory organic matter and biominerals. The
spectroscopic fingerprint of such common inorganic and
organic constituents in sediments and soil is illustrated
in Fig. 10. The IR spectrum of illite represents an example
of the large group of clay minerals. Characteristic absorbance peaks appear at 3694 and 3617 cm 1 and in the fingerprint region at 1107, 1034, 1008, 912 cm 1 and at 538,
471 and 430 cm 1 (Fig. 10a). The location and intensity of
these peaks may vary for other inorganic components (e.g.
muscovite, montmorillonite, smectite), nevertheless, the
overall pattern of spectral intensity is similar (Kovac
et al., 2004). The spectrum of biogenic opal derived from
diatom frustules (Fig. 10b) typically shows two prominent
peaks at 1091 and 464 cm 1, distinguishing opal from clay
minerals and quartz (Kovac et al., 2004; Moschen et al.,
2006). In contrast to opal, the spectrum of chitin is com-
plex and has typical bands allowing differentiation between chitin and cellulose (Fig. 10c and d). Characteristic
for chitin are peaks in the high frequency region at
3440 cm 1 (O–H stretch, N–H stretch), 3272 cm 1 (N–H
stretch) and 3108 cm 1 (Amide II band overtone) together
with a broad multi-peak band around 2923 cm 1. In the
fingerprint region chitin is readily distinguished from cellulose by the Amide bands I and II at 1627–1658 and
1554 cm 1, respectively, and the characteristic bands at
1381 and 1314 cm 1 (Shigemasa et al., 1996; Bahmed
et al., 2003; Ramírez-Coutiño et al., 2006).
We evaluated the performance of the UWEIL method
including the density separation step and the CUAM method on sedimentary materials using a fine-grained surface
sediment from the mesotrophic/eutrophic Lake Holzmaar.
Prior to density separation the IR spectrum reveals definite
evidence of impurities in the UWEIL extraction residue
(Fig. 11, light blue line). Minerogenic particles are indicated by bands at 1025, 528 and 470 cm 1, while peaks
at 3272 and 3108 cm 1, as well as at 1627/1658 and
1554 cm 1, represent chitinous remains. The spectrum of
the sample after density separation shows, however, almost complete accordance with the former spectrum,
without significant improvement (Fig. 11, dark blue line).
Clearly, cleaning by density separation was not capable
of further purifying the cellulose by removing embodied
organic and inorganic impurities. For comparison, we
tested the effect of demineralization using HF (10%), but
with even less success with respect to the removal of minerogenic matter (Fig. 11, grey line). The extraction quality
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H. Wissel et al. / Organic Geochemistry 39 (2008) 1545–1561
6
clay mineral (illite)
4
2
0
Absorbance (A.U.)
2
biogenic opal (Fragilaria)
1
0
0.4
chitin
0.2
0.0
0.4
cellulose
0.2
0.0
4000
3500
3000
1800 1600 1400 1200 1000
800
600
400
-1
Wavenumber (cm )
Fig. 10. IR absorbance spectra of common constituents of lake sediments representing potential contaminants in the fraction of extracted cellulose. Shown
are spectra of untreated illite (brown) as representative for fine clastic contaminants (clay minerals), of biogenic opal from a freshwater diatom (yellow) and
of standard chitin as representative for zooplankton chitin (green) in comparison to CUAM-treated FlukaCell (blue). Mark the axis break on the abscissa.
1.0
0.8
Absorbance (A.U.)
0.6
0.4
0.2
0.0
-0.2
-0.4
-0.6
-0.8
4000 3750 3500 3250 3000 2750
1800
1600
1400
1200
1000
800
600
400
-1
Wavenumber (cm )
Fig. 11. IR absorbance spectra of the extraction residue from the Holzmaar surface sediment gained by different treatments. Shown are results for UWEIL
treatment before (light blue) and after density separation (dark blue), demineralisation with HF with subsequent UWEIL treatment without density
separation (grey) and CUAM treatment (brown).
differs remarkably for the CUAM-treated residue from the
same raw material. Here, the IR spectrum reveals the
absorbance pattern of pure cellulose without any detectable impurities (Fig. 11, brown line). This is fundamental
for d18O analysis, since the presence of minerogenic matter
in the cellulose extract risks biasing the oxygen isotope
signature of the cellulose. Recently, Gong et al. (2007)
demonstrated that even preheated mineral samples can release several percent water at temperatures used for online pyrolysis (1450 °C). Moreover, in the treatment of sed-
iments the skill for complete removal of chitin from cellulose is critical since zooplankton chitin (e.g. Cladocera and
Chironomidae), comparable in thermal stability to cellulose, has to be expected in almost any sedimentary
material.
An example of a lacustrine sediment poor in minerogenic matter but rich in biogenic opal and OM is represented by the surface sediment from Laguna Azul
(Fig. 12). The IR spectrum of the UWEIL extraction residue
is characterised by a comparably broad and unstructured
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1.6
1.4
1.2
Absorbance (A.U.)
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
-0.4
-0.6
4000 3750 3500 3250 3000 2750
1800
1600
1400
1200
1000
800
600
400
-1
Wavenumber (cm )
Fig. 12. IR absorbance spectra of the extraction residue from the Laguna Azul surface sediment gained by different treatments. Shown are results for UWEIL
treatment before (light blue) and after density separation (dark blue), demineralisation with HF with subsequent UWEIL treatment without density
separation (grey) and CUAM treatment (brown).
band around 1084 cm 1 and peaks at 954, 790 and
465 cm 1 typical for biogenic opal. In this case, density
separation removes opaline impurities almost completely,
presumably because the majority of biogenic opal is derived from massive sponge spicules (Fig. 12, dark blue
line). Only the still visible peak at 465 cm 1 might indicate
remaining traces of biogenic opal or minerogenic matter.
However, the spectrum of the UWEIL residue including
density separation is again dominated by peaks derived
from chitin and not from cellulose. Similarly, the HF treatment could remove minerogenic and opaline particles but
the spectrum is still dominated by chitin (Fig. 12, grey
line). Unfortunately, further evaluation of the composition
of the extraction residue using IR spectroscopy with respect to other organic contaminants like hemicellulose or
refractory OM is therefore impossible. In comparison to
UWEIL, the IR spectrum of the CUAM aliquot shows absorbance peaks indicative neither of opal and chitin nor of
minerogenic matter. Again, the CUAM treatment was capable of successfully removing contamination to give a pure
cellulose extract from the Laguna Azul sediment (Fig. 12,
brown line). The discrepancy in cellulose purification between UWEIL and CUAM as expressed qualitatively by
the IR spectra is also apparent in the carbon isotope composition of the extracts. The offset between the Laguna
Azul surface sediment extraction residue of UWEIL with
density separation ( 19.0‰) and CUAM ( 17.3‰)
amounts to 1.7‰ (bulk 24.4‰).
We used a variety of additional sedimentary materials
to further verify the performance of the CUAM extraction.
This included sediment trap material, lacustrine surface
sediments, a core sediment of Holocene age (6500 cal
BP), a pond surface sample and a forest soil. The IR spectra
provide evidence that the extraction in all cases was
successful and that the CUAM treatment always isolated
a pure and clean cellulose fraction (Fig. 13). The C/O ratio
of the CUAM extracts from the sediments shows some
variability in replicate analyses (Fig. 14). This is especially
the case for samples HZM trap, SAC surface and POND wen,
for no discernible reason. Nevertheless, the respective
overall mean C/O ratio is close to the theoretical value of
0.90 for cellulose. The increased variability in the elemental composition is not reflected in the carbon and oxygen
isotope compositions of the extraction residues, for which
reproducibility is rather good. Carbon isotope enrichment
in cellulose relative to bulk OM from terrestrial sediments
is in the range reported for woody plants, whereas enrichment reaches 6‰ for cellulose from freshwater sediments.
This difference is much larger than that observed for macrophytes and algae and reflects the diverse composition of
bulk sedimentary OM. Nevertheless, absolute carbon isotope signatures of cellulose from aquatic macrophytes or
algae and of cellulose from aquatic sediments are comparable and tend to be enriched relative to terrestrial OM.
Since an evaluation of oxygen isotope enrichment compared to bulk OM could not be reasonably accomplished,
only the oxygen isotope values of sedimentary cellulose
are presented in Fig. 14. For all extracted sedimentary samples, reproducibility of oxygen isotope values within replicates was excellent (Boettger et al., 2007). Highest values
are observed for cellulose from terrestrial sediments,
expressing strong evaporative enrichment of 18O in these
environments. In comparison, d18O values for cellulose
from freshwater sediments are up to 5‰ lower. Moreover,
cellulose in surface sediment of Sacrower See is enriched in
18
O vs. Lake Holzmaar surface sediment. Actual values of
the oxygen isotope composition of lake water reveal that
the water body of Sacrower See is indeed enriched compared to that of Lake Holzmaar by ca. 5‰ (Lücke et al.,
1998; Bluszcz et al., unpublished results). About half of this
isotopic difference in lake water is compensated in the cellulose, probably by admixture of enriched OM from immmersed macrophytes/shore vegetation to the surface
sediments of Lake Holzmaar.
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H. Wissel et al. / Organic Geochemistry 39 (2008) 1545–1561
2.4
2.2
2.0
Absorbance (A.U.)
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
4000 3750 3500 3250 3000 2750
1800
1600
1400
1200
1000
800
600
400
-1
Wavenumber (cm )
Fig. 13. IR absorbance spectra of the extraction residue of CUAM-treated sediments. Spectra are arranged from top to bottom for Holzmaar surface
sediment (HZM surface), Holzmaar sediment trap (HZM trap), Holzmaar core sediment (HZM 6500), Sacrow surface sediment (SAC surface), Sacrow core
sediment (SAC 1700), pond sediment (POND wenig) and forest soil (SOIL for). The spectra of CUAM-treated FlukaCell (grey) is included for reasons of
comparison.
1.05
C/O
1.00
1.05
(a)
1.00
0.95
0.95
0.90
0.90
0.85
0.85
0.80
13
δ C (‰)
-22
18
-22
-24
-24
-26
-26
-28
-28
-30
-30
-32
31
-32
31
30
δ O (‰)
0.80
(b)
(c)
30
29
29
28
28
27
27
26
26
25
25
HZM surface
HZM trap
HZM 6500
SAC surface
SAC 1700
POND wen
SOIL for
Materials
Fig. 14. Carbon to oxygen ratio (a), carbon isotope composition (b) and oxygen isotope composition (c) of cellulose from sedimentary materials gained by
CUAM treatment (brown). Shown are mean values (closed circles) of replicate treatments of the same material together with the overall means (open
squares) and standard deviations (bars) for all measurements of the respective material.
4. Conclusions
A combination of classical approaches for bleaching
with sodium chlorite (NaClO2) and cellulose dissolution
in cuprammonium solution ([Cu(NH3)4](OH)2) resulted in
a novel approach for the quantitative extraction of recent,
sub-fossil and fossil cellulose from organic matter, especially freshwater sediments. We have evaluated the perfor-
mance of this novel approach (CUAM) against conventional
methods for cellulose extraction (UWEIL, JUEL) used in palaeoenvironmental applications.
Standard cellulose powders were used to exclude preparative artefacts with respect to isotope composition,
potentially introduced by dissolution and precipitation of
cellulose in cuprammonium solution employed in the
CUAM protocol. Wet chemical treatment led to a reduction
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H. Wissel et al. / Organic Geochemistry 39 (2008) 1545–1561
in C/O values for Avicel and IAEA-C3 independently of the
extraction method used. This might indicate an increase in
the amount of water absorbed to the cellulose by the treatments. The effects on the isotopic composition of standard
celluloses are, however, negligible to small for all the
methods used. Carbon isotope values are slightly reduced
compared to the control, while oxygen isotope compositions are systematically depleted relative to the respective
control. Within the analytical uncertainties, all three methods led to identical changes in isotopic composition of
standard cellulose powders, so CUAM is indistinguishable
from conventional methods with respect to any operational artifacts. In accord with the isotopic results, IR investigations indicate a certain change in crystallinity and
water content of standard cellulose treated using CUAM.
However, these changes did again not exceed those introduced with the UWEIL and JUEL methods. To further verify
possible systematic deviations in isotopic composition of
cellulose due to any laboratory treatment, an in depth
study would be desirable to evaluate the effects on the
oxygen isotope composition of cellulose extracted from
natural samples.
Extractions with aquatic plant material of diverse genera from different families revealed the capacity of the
CUAM method to isolate cellulose from whole plant tissue.
IR spectroscopy as well as isotope composition demonstrate that the CUAM method afforded clean and pure cellulose from any sample with one exception (‘aquatic
moss’). In all other cases, the CUAM performance was at
least equal to those of the other methods and in numerous
cases achieved better results in terms of cellulose purification. Contamination of several tissue samples with small
amounts of minerogenic matter and biogenic opal could
not be removed from the extraction residue with the
UWEIL and JUEL methods and consequently led to partly
erroneous isotope results. Underestimation in oxygen isotope composition caused by these impurities was as great
as 2‰ compared to CUAM cellulose. Like results reported
by Rinne et al. (2005) for woody samples, our experiments
show that solvent extraction of resins and tannins is dispensible in cellulose extraction from fresh plant material
and organic sediments.
The main aim of the study was an evaluation of the
capability of the CUAM protocol to give clean and pure cellulose from sedimentary materials for isotopic studies.
First, two typical fine-grained lacustrine sediments were
used in an intercomparison exercise based mainly on IR
spectroscopy of the resulting extraction residues. Neither
the JUEL method (as a priori expected) nor the UWEIL
method, including density separation or HF treatment,
was successful in isolating a pure cellulose fraction from
the bulk sediments. The spectra revealed contamination
of the extraction residues by minerogenic matter and biogenic opal as well as chitin. Bands characteristic of chitin
dominated the spectra, especially in the fingerprint region
and mainly disguised those of cellulose. Accordingly, beside other impurities, chitinous remains of zooplankton
were still present in the extraction residues from JUEL
and UWEIL in quantities large enough to bias the determination of the isotopic composition of the cellulose. Only
extraction residues of the CUAM-treated samples revealed
1559
IR spectra resembling those of standard cellulose powders
without evidence for contamination. Second, the CUAM
extraction was verified with a series of sedimentary materials such as soil, sediment trap and sediment core material. The IR spectra of the extracts demonstrated that the
extraction was successful in providing clean and pure cellulose from these sediments. C and O isotope compositions
of the extracted celluloses are in accord with previous
knowledge about the samples and substantiate the capability of the CUAM method.
We have shown that CUAM is a reliable cellulose
extraction method that yields clean and pure cellulose,
without inorganic and organic contamination, for isotope
analysis. Plant tissue and mixed sediments can be treated alike, granting full comparability of isotopic results
from cellulose between different types of material. The
CUAM method is potentially superior to other methods
for the treatment of sedimentary material and can most
likely be used not only for soils and freshwater sediments but also for marine sediments and organic rich
deposits like peat and lignite. The advantage rests in
the complete removal of contamination from the target
through isolation of cellulose by controlled dissolution
and precipitation. Nevertheless, rigorous quality controls
for the extraction process is recommended since the
composition of organic matter in sediments can be
highly variable and age and burial depth may affect
the feasibility of the method.
Acknowledgements
We are indebted to Mrs. A. Richter for IR spectroscopic
analysis. D. Enters provided sediments from the Sacrower
See. Determination of aquatic plants by B. Messyasz is
gratefully acknowledged. We thank T.W.D. Edwards and
P. Finch for valuable comments. Financial support was provided by the German Federal Ministry of Education and Research in the framework of the German Climate Research
Programme DEKLIM (Grant 01 LD 0001).
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the on-line version, at doi:10.1016/
j.orggeochem.2008.07.014.
Associate Editor—G. D. Abbott
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