publication

1. A Rapid and Simple Method to Isolate Pure
Alpha-Cellulose
O. Brendel,* P. P. M. Iannetta and D. Stewart
Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK
A protocol is described to isolate small quantities of highly purified cellulose for isotopic analysis of 10–
100 mg samples of secondary (Pinus sylvestris L.) and primary (Rubus idaeus L.) plant cell wall material.
Elemental analysis of 350 cellulose samples isolated from pine wood samples estimated the relative carbon
content to be ca. 43.7% Æ 1.2%. This value indicates that the cellulose quality is high and that the protocol is
highly reproducible. High-performance anion exchange chromatography with pulsed amperometric
detection of hydrolysis products quantified the purity of the cellulose as ca. 99% for wood cellulose and
primary cell wall cellulose. DRIFT spectroscopy corroborated this purity and found no evidence of cellulose
degradation. Carbon isotopic composition of the purified cellulose using mass spectrometry was measured
with an accuracy of 0.11% (standard deviation). The method is rapid (56 samples may be routinely
processed within 8 h) and requires only standard laboratory equipment and chemicals. Copyright # 2000
John Wiley & Sons, Ltd.
Keywords: cellulose; delignification; purification; pine wood; raspberry fruit; stable isotopes.
INTRODUCTION
The analysis of stable isotope ratios (d13
C, d2
H, d18
O)
can provide a valuable record of past climatic conditions
(palaeoclimatology; Wilson and Grinsted, 1977; Huntley,
1996) and is used routinely in dendroclimatological
studies of tree-ring indices (Schweingruber, 1988). The
lignin:cellulose ratio of wood (secondary xylem) can
vary considerably across even one ring (early wood-to-
late wood; Wilson and Grinsted, 1977), and for this
reason data is often derived from one wood constituent
such as cellulose (Mazany et al., 1980; Marshall and
Monserud, 1996).
A protocol originally described by Jayme and Wise
(see Green, 1963) using acidified sodium chlorite is
frequently applied to delignify wood as an initial step in
the purification of cellulose for isotopic analysis
(Brenninkmeijer, 1983; Sheu and Chiu, 1995; Loader et
al., 1997). In this paper we describe a modified protocol
for cellulose purification based on the simultaneous
delignification and removal of non-cellulosic polysac-
charides (NCPs) using an acetic acid: nitric acid mixture
(Crampton and Maynard, 1938). This method should
minimize losses due to filtering or change of reaction
vessels. In addition, only standard laboratory equipment
and chemicals are used. The technique has been applied
to secondary and primary cell wall material, and micro-
samples of the extracted cellulose were tested for yield,
purity and reproducibility in carbon isotopic signature.
EXPERIMENTAL
Plant material. Secondary-thickened cell wall material
was obtained from milled radial cores (5 mm; six
consecutive annual rings; n = 350) from the trunk of
mature Scots pine (Pinus sylvestris L.). During the
cellulose extraction of this test material, a pine wood
standard sample was processed for every 27 samples of
test material. Cellulose was also purified from freeze
dried and milled primary cell wall material (Iannetta et
al., 1998) from the fruit of field-grown raspberry (Rubus
idaeus L. cv. Glen Clova) at different stages of ripening
(n = 9; three replicates for the ripening stages, namely,
green, white and red as defined in Iannetta et al., 1999).
Micro-extraction of cellulose. The method detailed in
Fig. 1 follows the chemistry described by Crampton and
Maynard (1938), but has modified processing steps in
order to adjust for small sample sizes and to minimize
losses. All steps of the protocol were performed in a
fume-cupboard. Samples (10–100 mg, routinely 50 mg)
were weighed into 10 mL Pyrex2
tubes and the
combined weight was measured on a balance capable
of recording to 10 mg. Subsequently, 2.0 mL of acetic
acid (80%; v/v) and 0.2 mL of concentrated nitric acid
(69%; v/v) were added. In order to minimize losses, it
was essential that the tube walls remained free of sample
material at all stages of the protocol. Any sample left
adhering to the inner wall of the test tube was washed
downwards to aid complete extraction: this was achieved
by adding liquid extraction reagent in two parts, the first
PHYTOCHEMICAL ANALYSIS
Phytochem. Anal. 11, 7–10 (2000)
CCC 0958–0344/2000/010007–04 $17.50
Copyright # 2000 John Wiley & Sons, Ltd.
* Correspondence to: O. Brendel, INRA Centre de Nancy, 54280
Champenoux, France.
E-mail: brendel@nancy.inra.fr
Contract/grant sponsor: Scottish Office Agriculture, Environment and
Fisheries Department; Contract/grant number: FF461/FF505.
Contract/grant sponsor: Ministry of Agriculture, Fisheries and Food; Contract/
grant number: 2112 TSF.
Received 7 October 1998
Revised 25 May 1999
Accepted 25 May 1999

2. to suspend the sample and the second to rinse the tube
wall. Samples were then suspended by careful vortexing.
The tubes were sealed using screw-caps fitted with Teflon
liners and placed into a heating block pre-heated to
120°C for 20 min (extraction). Once cooled, 2.5 mL of
ethanol (99%; v/v; AnalaR1
quality) was added and the
samples were centrifuged (5 min at 2000 rpm). The
supernatant was then carefully decanted and the pellets
were washed sequentially as follows: (1) with 2Â 2.5 mL
ethanol, to remove extraction breakdown products; (2)
with 2 Â 2.5 mL deionized water, to remove traces of
nitric acid (omission of this water wash resulted in
samples with increased nitrogen content); (3) with 2 Â
2.5 mL ethanol; and (4) with 2 Â 2.5 mL acetone (general
purpose grade). Steps (3) and (4) allowed more thorough
washing and sample dehydration. Between each wash,
samples were pelleted by centrifugation and the super-
natant discarded.
In order to free the sample tubes for further extractions,
it was necessary to transfer the purified cellulose into
1.5 mL microfuge tubes using 0.4 mL of acetone.
Subsequently the inner walls of the sample tubes were
rinsed with a further 0.6 mL of acetone and the washings
transferred to the microfuge tubes. The 1 mL sample was
then centrifuged for 10 min in a vacuum-evaporator and
the remaining acetone decanted. Samples were re-
centrifuged in the vacuum-evaporator until no further
weight loss could be recorded. Samples were kept in
sealed bags containing anhydrous silica gel. For quanti-
tative analysis the samples can be dried to constant
weight in the Pyrex test tubes.
Mass spectrometry. For the %C, %N and d13
C analysis
of each sample, 1 mg of purified cellulose was weighed
into a tin cup and processed using a Europa Scientific
(PDZ Europa Ltd., Crewe, Cheshire, UK) Tracermass
CF-IRMS (continuous flow-isotope ratio mass spectro-
meter; Handley et al., 1993). Two routine standards of
1 mg wheat-flour were analysed at the end of every
sample batch processed in the MS.
Acid hydrolysis of cellulose and estimation of cellulose
purity by high performance anion exchange chroma-
tography (HPAEC). Three random samples from each
plant source were used to estimate the purity of the
isolated cellulose. The cellulose was totally hydrolysed
using an adaptation of the method of Fry (1988).
Sulphuric acid (11 M) was added to the isolated cellulose
(50 mL per mg of pellet) contained in an Oakridge (BDH,
Poole, Dorset, UK) tube, which was then sealed and
shaken at 225 rpm for 1 h at 25°C. A volume (11Â) of
sterile distilled water was then added and the mixture
hydrolysed by heating to 120°C for 1 h. After cooling,
25 mL of 1% (w/v) bromophenol blue was added and the
solution partially neutralized by stirring rapidly and
adding a volume (2Â) of barium hydroxide (0.18 M).
Complete neutralization was achieved after the addition
of 2 g of barium carbonate and shaking at 225 rpm for 1 h
at 25°C. Insoluble barium sulphate and barium carbonate
were removed by centrifugation for 15 min at 3000 g and
the supernatant retained. Traces of residual barium salt
were removed by freezing and then thawing the extract
followed by re-centrifugation. Finally, the supernatant
was shaken at 25°C with 5 g of ion-exchange resin
[Dowex 50W-8X(H); 16–40 mesh] to remove any soluble
barium ions. The supernatant (25 mL) was diluted to
prepare a 500 mL aliquot which was used in HPAEC
analysis.
HPAEC (Dionex, Camberley, Surrey, UK) was
performed using a GD40 gradient pump and eluant-
organizer, both of which were pressurized with helium,
and an ED40 pulsed amperimetric detector (PAD-II). An
aliquot (25 mL) from a 500 mL sample was removed by an
auto-sampler (AS 3500) and bound onto a CarboPac PA1
column with an inline filter (5 mm:35 mm). The bound
sample was eluted with 200 mM sodium hydroxide at a
flow rate of 1 mL/min over a 20 min period. Neutral sugar
(glucose) standards were also applied (0–2 mg) at the
beginning, middle and end of each test.
Diffuse reflectance Fourier-transform infrared
(DRIFT) spectroscopy. DRIFT spectra were acquired
Figure 1. The protocol for puri®cation of cellulose from micro-
samples of plant cell wall material using acetic acid:nitric acid
for simultaneous deligni®cation and removal of non-cellu-
lose polysaccharides.
8 O. BRENDEL ET AL.
# 2000 John Wiley & Sons, Ltd. Phytochem. Anal. 11: 7–10 (2000)

3. exactly as described by Stewart et al. (1995). For
comparative purposes a commercial cellulose was
obtained from Sigma (Poole, UK).
RESULTS AND DISCUSSION
A concern in using this technique was the possibility that
traces of nitric acid could contaminate the purified
cellulose, causing elevated and variable nitrogen con-
tents. However, the average nitrogen content of the pine
wood cellulose was close to the minimum sensitivity of
the elemental analyser (ca. 0.15%), indicating that the
washing process was effective in removing virtually all
traces of nitric acid from the isolated cellulose.
Pure cellulose has a theoretical relative carbon content
(RCC) of 44.45% and any reduction in chain length
would lower the RCC. Consequently, the anticipated
range of RCCÁfor cellulose would be ca. 41–45%. The
theoretical RCC of lignin from a conifer is lower (ca.
37%) than that of cellulose and thus its presence as a
contaminant in the purified cellulose would lower the
cellulose RCC. Monoterpenes have a carbon content of
about 88% and thus the presence of wood-resin residues
would increase the RCC. MS analysis of standard pine
wood samples showed a RCC of 42.6 Æ 1.9% (Table 1;
n = 14) while Scots pine samples yielded an RCC within
the expected range at 43.7 Æ 1.2% (n = 350). The carbon
isotopic composition (d13
C) of standard pine wood
cellulose showed a standard deviation (SD) of Æ 0.11%
(Table 1; n = 14). This low SD indicates the high
reproducibility of the described technique and the value
compares favourably with estimates in the published
literature of Æ0.10% and Æ0.11% (Loader et al., 1997;
n = 12) derived using a two-step protocol to extract
holocellulose, which involves a sodium chlorite treat-
ment as the first stage followed by treatment with sodium
hydroxide to remove NCPs and isolate a-cellulose.
The cellulose yield from hardwood using the sodium
chlorite:sodium hydroxide method was 30 Æ 5% and
29 Æ 4% (Loader et al., 1997). These yields are
significantly below the published values (40–50%) for
cellulose content of hardwoods (Robson and Hague,
1993). Using the acetic acid:nitric acid method, we
estimate an a-cellulose yield of 41 Æ 3%, a value which
is within the range of 40–45% published for softwoods
(Robson and Hague, 1993). Compared to the results of
Loader et al. (1997), the improved yield reported here
may be a consequence of using a one-step protocol to
minimize cellulose loss. In addition, statistical analysis of
cellulose yields revealed that it is acceptable to use a
range of starting material (10–100 mg) for cellulose
isolation since there was no significant correlation
between yield and starting sample weight (r2
= 0.004;
pslope = 0.78).
Cellulose extracted from pine wood and raspberry fruit
that had been hydrolysed using sulphuric acid and
analysed using (Dionex) HPAEC, revealed only one
major peak (b99% total peak area) at the retention time
of glucose. Thus, plant cell wall material processed in the
manner described (Fig. 1) can provide cellulose
approaching 100% purity.
The efficacy of the extraction method and the absence
of deleterious cellulose degradation were reflected in the
DRIFT spectra of a wood sample before and after
extraction (Fig. 2). The extraction procedure removed all
of the lignin-associated absorbances (1595, 1510 and, to a
lesser extent, 1440 cmÀ1
) and almost completely elimi-
nated the NCP-associated absorbances (1210–
1040 cmÀ1
; Stewart, 1997). In fact, the general resolution
of the absorbances over this area improved significantly
following extraction, producing a spectrum with a line
shape very similar to isolated cellulose (Michell, 1990)
and approaching that of a commercial crystalline
cellulose (Fig. 2). It should be noted that traces of
residual NCP were still evident and reflected in the
presence of an acetyl ester carbonyl (C==O) absorbance at
1740 cmÀ1
and, to a lesser extent, the corresponding
carbonyl-derived C—O stretch at 1250 cmÀ1
. These
acetyl esters would be derived from the glucomannans
and/or arabino-4-O-methylglucoronoxylans reported to
be predominant in Pinus sylvestris (Timell, 1965).
It is significant that there are no peaks unique to the
spectrum of the extracted sample. This indicates that
little or no cellulose degradation occurred. In particular,
there is no evidence of increased acid or aldehyde
Table 1. Values obtained following %N, %C and d13
C
analysis of cellulose isolated from separate extrac-
tions (n = 14) of a standard pine wood sample
Standard %N %C d13
C(% %)
1 0.149 43.23 À25.63
2 0.133 44.03 À25.37
3 0.132 42.17 À25.44
4 0.098 44.62 À25.25
5 0.113 41.24 À25.35
6 0.090 43.69 À25.39
7 0.075 44.86 À25.37
8 0.101 40.60 À25.56
9 0.198 45.15 À25.57
10 0.175 42.54 À25.47
11 0.130 41.50 À25.41
12 0.125 41.03 À25.40
13 0.109 38.31 À25.41
14 0.072 42.85 À25.30
Mean 0.121 42.56 À25.42
Standard
deviation 0.036 1.91 0.11
Figure 2. The DRIFT spectra of untreated and extracted wood
(Pinus sylvestris L) and commercial cellulose (for method of
sample preparation and spectroscopic protocol see the
Experimental section).
CELLULOSE PURIFICATION 9
# 2000 John Wiley & Sons, Ltd. Phytochem. Anal. 11: 7–10 (2000)

4. absorbances (1660–1600 cmÀ1
), the presence of which
would indicate oxidative degradation — a possible
consequence of exposure to aqueous nitrogenous oxi-
dants (Singh, 1990).
In summary, the technique described here may be
considered an improvement upon other published
methods. The protocol is simple and may be performed
rapidly, requiring only standard laboratory materials and
apparatus. A single sample can be processed in about 1 h
(excluding the vacuum-evaporation step). With a batch
size of 28 samples (a restriction occasioned by limitations
in centrifuge availability in our laboratory), experience
has shown that two batches can be comfortably processed
in 8 h. Furthermore, while negating the risk of toxic
chlorine dioxide gas inherent in the use of sodium
chlorite for delignification, the quantity of nitric acid
used is minimal (11.2 mL to process 56 samples) and the
waste composition (approximately 50% ethanol, 20%
water, 20%, acetone, 10% acetic acid and 1% nitric acid)
should reduce disposal problems.
Acknowledgements
The authors wish to thank the Scottish Office Agriculture Environment
and Fisheries Department (grant FF461/FF505), the Ministry of
Agriculture, Fisheries and Food (grant 2112TSF, P.P.M.I.), and Drs
I. Morrison and L. Shepherd (SCRI, Invergowrie, Dundee, Scotland).
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