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Mechanical depolymerisation of acidulated cellulose:

Cite this: DOI: 10.1039/c3gc40945g understanding the solubility of high molecular
weight oligomers†
Abhijit Shrotri,a Lynette Kay Lambert,b Akshat Tanksalec and Jorge Beltramini*a

Received 21st May 2013,
Accepted 5th August 2013
DOI: 10.1039/c3gc40945g
www.rsc.org/greenchem
Water soluble oligomers of cellulose were produced by milling acidulated microcrystalline cellulose. Acids
with low pKa were found to be more effective for the treatment. The yield of water soluble fraction was
proportional to the increasing severity of milling or the acid amount. Soluble oligomers were found to
have an average degree of polymerisation of ∼7 monomer units. High resolution NMR spectroscopy was
used to determine the chemical structures of soluble oligomers. It was found that branched oligomers
with α (1 → 6) linkages were formed, which increased their solubility in water and reduced the gene-
ration of monomers and dimers which may degrade during milling or subsequent hydrolysis–hydro-
genation. The soluble oligomers showed excellent reactivity towards hydrolysis–hydrogenation in the
presence of bi-metallic Ni–Pt/alumina catalyst. High yields (∼90%) of sorbitol and mannitol were
obtained with only 1 h of reaction time.

1. Introduction in a slow and sequential cleavage of the cellulose chain. It is

Among all forms of biomass available, lignocellulosic biomass
is considered the most suitable for biofuel and green chemical
production. It is primarily composed of polymers cellulose,
hemicellulose, and lignin.1 Cellulose is one of the most abun-
dant sources of organic carbon. In its pure state it exists as a
large polymer composed of repeating units of anhydro glucose
monomer units linked via β (1 → 4) glycoside bonds. Aqueous
phase hydrolysis of cellulose selectively produces glucose
which can then be reacted to form key platform chemicals.2
Despite worldwide focus, commercial application of this
technology is still not viable. One of the primary roadblocks is
the slow rate of the hydrolysis reaction, which leads to low
yields and long reaction times. Cellulose hydrolysis proceeds
via a heterogeneous mechanism where the reaction rates are
influenced by the physical state of cellulose. Insolubility of cel-
lulose limits the access to β (1 → 4) glycoside bonds, resulting
a
ARC Centre of Excellence for Functional Nanomaterials, University of Queensland,
Brisbane, QLD 4072, Australia. E-mail: j.beltramini@uq.edu.au;
Fax: +61 7 3346 3973; Tel: +61 7 3346 3803
b
Centre for Advanced Imaging, University of Queensland, Brisbane, QLD 4072,
Australia. E-mail: lynette.lambert@cai.uq.edu.au; Fax: +61 7 3365 3833;
Tel: +61 7 3365 9737
c
Department of Chemical Engineering, Monash University, Clayton, VIC 3800,
Australia. E-mail: akshat.tanksale@monash.edu; Fax: +613 9905 5686;
Tel: +61 3 9902 4388
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3gc40945g
also important to note that all research in this field has been
performed exclusively on batch reactor systems.
To facilitate large-scale operation it is imperative to develop
a continuous process for the production of fuels and chemi-
cals from cellulose. A possible solution to overcome these
issues is to solubilise cellulose in water prior to the reaction.
Dissolution of cellulose is known to increase the rate of hydro-
lysis. Handling a solution would be much easier in an indus-
trial scenario and it would also allow the reaction to be
performed in a continuous fixed bed reactor. However, cellu-
lose is known to be insoluble in most conventional solvents.
Cellulose has an ordered crystalline structure, which is
the result of an intermolecular network of hydrogen bonds
between hydroxyl groups of monomer units. This structural
rigidity makes cellulose insoluble in water. Nonetheless, in-
solubility of native cellulose in water is not yet fully under-
stood.3,4 It is commonly accepted that dissolution of cellulose
in aqueous solutions is achieved by disruption of the hydrogen
bond network through decrystallisation and depolymerisation
of cellulose.5 Recently, aqueous solvent systems such as alkali-
urea6 and supercritical water7 were proposed for the dissol-
ution of cellulose. However, a large-scale application of these
processes is questionable due to their high-energy requirement
and the use of large amounts of concentrated alkali solutions.
Modification of cellulose structure via pre-treatment is
widely practised to improve its solubility and reactivity with
heterogeneous catalysis. All pre-treatment processes primarily
aim to decrystallise cellulose to produce amorphous cellulose.

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This can be achieved easily by milling crystalline cellulose in a
ball mill.8 Along with reducing the crystalline structure,
milling also reduces particle size and increases the effective
surface area. Milling efficiency is a function of various para-
meters including milling time, ball size and the sample to ball
ratio. Irrespective of the conditions used, amorphous cellulose
thus produced exhibits similar properties. Amorphous cellu-
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lose has fewer hydrogen bonds per repeating monomer unit
than crystalline cellulose.9 β (1 → 4) Glycoside bonds in amor-
phous cellulose are more easily accessed by water and other
reactants for hydrolysis.10–12 Amorphous cellulose is also
known to undergo hydrolysis at a lower temperature.13 Despite
various advantages amorphous cellulose is insoluble in water
and reaction rates are governed by heterogeneous pathways.
Very recently catalytic depolymerisation of cellulose to oligo-
saccharides by milling of acid treated cellulose was proposed
by Meine et al.14 However, there is a lack of understanding of
the mechanism of depolymerisation during the treatment.
Moreover, the significance of the organic solvent used during
their acid impregnation is unclear.
The focus of this research is on accomplishing complete
solubility of cellulose in water without the use of organic sol-
vents. We also aim to determine the factors influencing the
cellulose treatment and propose a mechanism of oligomer for-
mation. Here we present a pre-treatment method involving a
combination of acid impregnation in aqueous media and ball
milling to depolymerise cellulose into water soluble oligomers
at room temperature. Parameters affecting the solubility of cel-
lulose were analysed to optimise the process with an aim to
achieve 100% solubility, while maintaining a high degree of
polymerisation to prevent degradation of monomers. Struc-
tural changes in cellulose were investigated by X-ray diffraction
and Scanning Electron Microscopy (SEM). Nuclear Magnetic
Resonance (NMR) spectroscopy was used to resolve repeat unit
structures of soluble fractions of cellulose. A combination of
1D and 2D NMR techniques, including 1H, 13C NMR, gradient
selected correlation spectroscopy (gCOSY),15 one-dimensional
total correlation spectroscopy (1D-TOCSY),16 multiplicity-
edited heteronuclear single quantum coherence (HSQC-edit),17
heteronuclear 2 bond correlation (H2BC),18 heteronuclear
single quantum coherence total correlation spectroscopy
(HSQC-TOCSY),17 and heteronuclear multiple bond correlation
(HMBC),19 was used to elucidate the molecular bonding struc-
ture of the soluble oligomers. To the best of our knowledge, a
detailed NMR study of cellulose oligomers produced via ball
milling has not been reported up to now.
To validate the positive effects of soluble oligomers when
compared to insoluble cellulose, we tested the catalytic conver-
sion of cellulose and soluble oligomers. The cellulose sub-
strates were reacted over a supported metal catalyst to
selectively produce sorbitol and mannitol (hexitols). Sorbitol
has been identified as a key platform chemical produced from
cellulose,20 which can be utilised to produce hydrogen and
liquid hydrocarbon fuels.21,22 After Fukuoka et al.23 first
reported that sorbitol can be produced from cellulose using
Ru/C catalysts, we recently showed that bi-metallic Ni–Pt
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catalyst supported on alumina and beta-zeolite can also be
used for this reaction.24 The catalyst showed a comparable
yield and selectivity towards hexitols during hydrolysis of
microcrystalline cellulose. However, depolymerisation of cellu-
lose into glucose monomer units is the rate limited step due to
low solid–solid interaction.23 Therefore, it is expected that
using soluble oligomers will enhance the reaction rate, result-
ing in a high yield of hexitols.
2. Experimental
2.1 Cellulose treatment
The required amount of acid was diluted in water to a total
volume of 25 ml. 10 g of Sigmacell 20 μm micro-crystalline cel-
lulose (MCC) was added to this solution and stirred for a few
minutes. The resulting slurry was air dried at 50 °C for
48 hours to remove excess water to produce acidulated MCC. It
was then milled in a planetary ball mill with cellulose to a ball
weight ratio of 1 : 10. The mill was operated at 300 rpm, with a
20 minute pause after 15 minutes of continuous milling. The
pause allowed dissipation of the heat produced during
milling, maintaining the milling temperature below 40 °C
throughout the treatment. The milling time reported here
refers only to the active milling time.
Solubility was determined by dissolving 0.5 g of the treated
cellulose in 15 ml of water under ambient conditions. Undis-
solved solids were separated via centrifugation and weighed
after drying. Solubility was calculated by subtracting the
weight of undissolved solids from the weight of total cellulose
added and reported in weight percentage.
2.2 Characterisation and analysis
The average degree of polymerisation (DP) of cellulose was
determined by methods described elsewhere.25 Typically, the
number of reducing groups in a sample was determined by
assaying the sample using 2,2′-bicinchoninate method (BCA).
A BCA solution was prepared fresh by mixing equal volumes of
solutions A and B. Solution A was prepared by dissolving
0.097 g of disodium 2,2′-bicinchoninate, 2.71 g of Na2CO3, and
1.21 g of NaHCO3 in 50 ml of water. Solution B was prepared
by dissolving 0.062 g of CuSO4·5H2O and 0.063 g of L-serine in
50 ml of water. 2 ml of a BCA solution was mixed with less
than 1 mg of cellulose dispersed in 2 ml of water. The result-
ing mixture was incubated at 75 °C for 30 min with constant
stirring using a magnetic stirrer. After cooling to room temp-
erature the sample was centrifuged to remove cellulose, the
supernatant was then analysed for absorbance at 560 nm wave-
length using a Shimadzu UV-Vis spectrophotometer (UV-2450).
A calibration curve was prepared using glucose solutions with
concentrations of 0–150 μM. Total glucosyl monomer units in
the sample were determined by dissolving cellulose in 72%
sulphuric acid and diluting to obtain 0.2 g L−1 cellulose con-
centration. 0.10 ml of a 40 wt% phenol solution was then
added to 2 ml of a cellulose solution. Then 5 ml of 95% sul-
phuric acid was added rapidly and the mixture was allowed to
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stand for 10 min prior to shaking for 20 min at 25 °C. The
resulting mixture was transferred to a cuvette and tested for
absorbance at 460 nm. The average DP was calculated by divid-
ing the number of glycosyl monomer units by the number of
reducing ends for each sample.
Powder X-ray diffraction (XRD) of cellulose was carried out
using a Rigaku Miniflex X-ray diffractometer with Fe-filtered
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Co radiation. Measurements were made from 10 to 50 degree
2θ with a step size of 0.02 degree 2θ. Samples for the Scanning
Electron Micrograph (SEM) were prepared by loading them
onto a carbon stub and coated with a platinum or carbon
Sputter Coater. Images were taken using a JEOL 6300 SEM
operating at 5–10 kV.
High resolution NMR spectra of completely soluble
samples were obtained using a Bruker Avance III 900 spectro-
meter operating at 900.13 MHz and 226.34 MHz for 1H and
13
C, respectively, using a 5 mm triple-resonance inverse (TCI)
cryoprobe with the sample temperature of 25 °C. 15 or 30 mg
of the treated cellulose sample was dissolved in 650 μL of
deuterium oxide; the spectra were referenced internally to DSS
(sodium 2,2-dimethyl-2-silapentane-5-sulfonate) at 0 ppm for
both 1H and 13C. One-dimensional TOCSY experiments using
gradients and a selective refocusing pulse were performed
using mixing times of 30–80 ms. Two-dimensional experi-
ments performed included gradient-selected COSY, multi-
plicity-edited HSQC, HSQC-TOCSY and H2BC. The HMBC
spectrum, phase-sensitive in the indirect dimension, was
acquired with the long-range coupling delay set for 4 Hz.
2.3 Catalytic test
Catalytic conversion of cellulose and soluble oligomers was
performed in a Parr high pressure batch reactor at 50 bar H2
pressure (at room temperature) and 200 °C for 1 h. 20 g L−1 of
a cellulose solution was prepared in 100 ml of deionised water,
and 0.5 g of reduced Ni–Pt/alumina catalyst (see ESI†) was
added to the reactor before applying pressure and heat. After
the reaction was complete, the resulting solution was filtered
using a 0.45 mm filter to recover the catalyst and unconverted
cellulose. The product solution was analysed using a Shi-
madzu Prominence HPLC system equipped with a Rezex RCM
Monosaccharide column (7.8 × 300 mm) with 8% Ca cross
linking with an oven temperature of 65 °C and a water (mobile
phase) flow rate of 0.6 ml min−1. The products from the reac-
tion were detected using a Shimadzu low temperature ELSDII
detector at 30 °C and 370 kPa N2 pressure.
3. Results and discussion
3.1 Effect of acid strength and milling time on the solubility
and degree of depolymerisation
MCC was acidulated with various acids and milled to study the
effect of acid strength on solubility (Table 1). In the absence of
acid, milled MCC was sparingly soluble in water (entry 1). Solu-
bility was influenced by the strength of acid present during
milling (entries 2–7). Low strength ( pKa > 0) acetic acid, oxalic
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Table 1 Effect of various acids on cellulose solubility after milling of acidulated
cellulose
Concentrationa Milling Solubility
Entry Acid pKa (mmol g−1) time (h) (%)
1 None — — 5 1.5
2 C2H4O2 4.76 0.16 5 1.6
3 H2C2O4 1.27 0.16 5 3.6
4 H3PO4 2.12 0.16 5 2.5
5 HCl −8.0 0.16 5 3.4
6 HNO3 −1.3 0.16 5 12.3
7 H2SO4 −3.0 0.16 5 34.6
a
Millimoles of acid per gram of dry cellulose.
acid and phosphoric acid had little effect on solubility,
whereas high strength ( pKa < 0) acids improved the solubility
significantly. H2SO4 was the most active catalyst resulting in
34.5% solubility, followed by HNO3, which resulted in 12.3%
solubility. HCl was the exception with low pKa and low solubi-
lity. This could be due to loss of HCl in vapour form during
the drying step. The amount of acid on MCC before milling
was measured by observing the change in pH when 1 g of
acidulated MCC (0.25 mmol of acid) was dispersed in 20 ml
of water. The observed pH value of 2.95 corresponds to
0.0224 mmol of HCl which suggests that only 10% HCl was
left on the surface after drying. In contrast, dispersion of
H2SO4 impregnated cellulose resulted in pH of 1.83 which is
close to the expected value of 1.77 for 0.25 mmol of acid dis-
persed in 20 ml of water. This supports our observation of
higher solubility when H2SO4 was used even though its pKa is
higher than that of HCl. We also observed that when sulphuric
acid was used, solubility was proportional to an increase in
acid concentration (Fig. 1A). Similarly milling for a longer dur-
ation also increased the solubility of MCC. It is important to
note that neither acid impregnation nor milling was effective
on its own because we did not observe any change in solubility
under such conditions. We also found that complete solubility
can be achieved under different conditions by varying the
severity of the acid amount and milling time.
MCC used in these experiments has a DP of ∼200 monomer
units.25 Dissolution of cellulose in water can only take place
via depolymerisation into smaller oligomer fractions. DP was
analysed at each stage to analyse its effect on overall solubility
(Fig. 1B). Acidulation of MCC reduced its degree of polymeri-
sation without milling, suggesting some hydrolysis of cellulose
before the milling. Milling without acid impregnation also
resulted in depolymerisation of cellulose. However, rapid
depolymerisation of the cellulose chain into soluble oligomers
was observed only when milling was performed on acidulated
MCC. The resulting oligomers had a DP of less than 15. We
found that higher milling time and acid loading improved
solubility without significantly changing the DP. Samples with
100% solubility exhibited an average DP of 6–9 monomer units
which did not decrease further by increasing the severity of
the treatment. HPLC analysis of the completely soluble oligo-
mer solutions showed only 1.0 wt% glucose concentration.
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Fig. 1 (A) Solubility and (B) degree of polymerisation of cellulose after milling in the presence of increasing acid concentration. Milling time: (●) 0 h, (○) 2.5 h,
(▼) 5 h, (△) 7.5 h, (■) 10 h.
This confirmed that within the limits of our experiments,
depolymerisation of cellulose did not lead to the formation of
large amounts of glucose. This is advantageous since glucose
formation may lead to formation of glucose degradation
products.
3.2 Structural and chemical changes due to acid catalysed
milling
We observed that for cellulose samples with DP < 10, an
increase in solubility was unrelated to the change in DP,
suggesting other contributing factors. All further characteris-
ations to determine these factors were performed on the as-
received Sigmacell cellulose (C-0/0), cellulose milled for 10 h
without acid impregnation (C-0/10) and cellulose milled for
10 h with 0.25 mmol g−1 H2SO4 (C-0.25/10). The last sample
was the only one of the three to be completely soluble in water
under ambient conditions.
Milling MCC in a ball mill resulted in the formation of
amorphous cellulose. XRD peaks characteristic of microcrystal-
line cellulose (C-0/0) centred at 26.2° and 18.1° 2θ were
replaced by a broader peak with a maximum at 22.0° 2θ for
C-0.25/10 (see ESI Fig. S1†), suggesting complete conversion of
cellulose into amorphous form. Although the degree of crystal-
lisation in C-0/10 was also very low, the sample was sparsely
soluble in water. Therefore, we conclude that cellulose crystal-
linity is not a determining factor in solubility.
To test whether the impregnated H2SO4 was chemisorbed
on the cellulose surface or physisorbed in the pores, we dis-
persed never-milled acidulated cellulose powder into water.
We observed that the change in pH of water was instantaneous
due to the acid leaching into water. The lower pH was main-
tained even after filtering out the cellulose powder. SEM EDS
of acidulated MCC was performed to study surface distribution
of acid. Observation of the relative intensity of sulphur on the
cellulose surface suggested a uniform distribution of sulphuric
acid on the smooth surface and pits of cellulose particles (see
ESI Fig. S2†). These findings suggest that acid is loosely
bonded to cellulose and is evenly distributed on the surface.
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Fig. 2 SEM micrograph of cellulose: (a) C-0/0, (b) C-0/10 and (c) C-0.25/10.
The absence of acid in C-0/10 caused agglomeration of milled
cellulose flakes to form spherical particles of 10 μm size after
milling (Fig. 2b). Despite being disordered, amorphous cellu-
lose exhibits hydrogen bonding between molecules which pro-
motes agglomeration of particles.9 It is evident from the SEM
image of C-0.25/10 (Fig. 2c) that the acid present on the cellu-
lose surface interacts with the oligomers, inhibiting the
formation of hydrogen bonds, and reduces the tendency of
agglomeration. Rapid dispersion of smaller particles and
reduced intermolecular bonding are likely to promote the solu-
bility of oligomers formed during acid catalysed milling.
Authors believe that changes in the physical structure alone
may not result in 100% solubility of cellulose because we know
that oligomers with more than six glucopyranose monomer
units (GMUs) are insoluble in water under ambient con-
ditions.26,27 Using a solution state high resolution NMR study
of the dissolved C-0.25/10 sample, it was possible to determine
the configuration of oligomers present. The 1H NMR spectrum
of C-0.25/10 dissolved in D2O is presented in Fig. 3. Peaks
between δH 4.4 and 5.6 (δH is 1H chemical shift in ppm) are
indicative of anomeric protons. Apart from the water peak at
δH 4.78, several peaks for anomeric protons were detected in
this region pertaining to GMUs with β (1 → 4) internal linkages
(i), β (1 → 4) linked sugars with non-reducing ends (nr) and
reducing ends in β and α configurations.28 The dominant
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linked monomers with a non-reducing end (Table 2; entries

1 and 2).29 A cluster of doublets was observed between δH 4.6
and 4.7, all of which were identified as GMUs with β reducing
ends. The most abundant of these species were GMUs with β
reducing ends linked at C4 (Table 2; entry 3), followed by
monomeric β-glucose. Similarly, overlapping species of GMUs
with α reducing ends (J ∼ 3.4 Hz) were identified between δH
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5.18 and 5.25. GMUs with an α reducing end in the oligomer

chain comprised the dominant species (Table 2; entry 4), fol-
lowed by monomeric α-glucose.30 These findings suggest that
monomers are primarily linked with β (1 → 4) bonds as
1
Fig. 3 The anomeric region of the H NMR spectrum of C-0.25/10 expected from cellulose based oligomers.
(30 mg ml−1) at 298 K. Peaks between δH 4.93 and 5.02 are not characteristic of
anomeric protons of any GMU present in cellulose. It was
doublet (J = 8 Hz) at δH 4.52 in our experiment was character- difficult to determine the coupling constant for these
istic of internal β (1 → 4) linked glucose monomers. Likewise, H1 multiplets in the one-dimensional proton spectrum because
the doublet (J = 8 Hz) at δH 4.50 was assigned to the β (1 → 4) of the overlap of different oligomer peaks. By examining the

Table 2 13
C and 1H chemical shifts and coupling constants for sugar residues present in C-0.25/10 dissolved in D2Oa

Proton shift δH Carbon shift δC Coupling constant

Anomeric configuration Illustration (ppm) (ppm) (Hz)

β (1 → 4) Linked (i) H1 4.52 C1 105.0 JH1,H2 8.0

H2 3.35 C2 75.6
H3 3.65 C3 76.7
H4 3.67 C4 80.9
H5 3.62 C5 77.5
H6 3.82 C6 62.5
H6′ 3.97

β (1 → 4) Linked (nr) H1 4.50 C1 105.2 JH1,H2 8.0

H2 3.31 C2 75.8
H3 3.50 C3 78.1
H4 3.41 C4 72.1
H5 3.49 C5 78.6
H6 3.73 C6 63.2
H6′ 3.91

β Reducing end H1 4.65 C1 98.4 JH1,H2 8.0

H2 3.27 C2 76.6
H3 x C3 76.9
H4 x C4 81.2
H5 x C5 77.4
H6 x C6 62.7
H6′ x

α Reducing end H1 5.21 C1 94.5 JH1,H2 3.4

H2 3.56 C2 73.9
H3 3.70 C3 x
H4 3.94 C4 81.4
H5 3.86 C5 72.8
H6 3.81 C6 62.6
H6′ x

α (1 → 6) Linked H1 4.98 C1 101.3 JH1,H2 4.0

H2 3.55 C2 74.1
H3 3.79 C3 75.6
H4 3.41 C4 72.3
H5 x C5 x
H6 x C6 63.2
H6′ x
a
δH and δC are 1H and 13C chemical shifts in ppm. (i) Internal sugar residue in an oligomer chain linked at both the C1 and C4 positions.
(nr) Sugar residue at the non-reducing end of an oligomer chain linked only at C1 position. (x) Assignment not possible due to overlap of
multiple peaks.

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Fig. 4 (A) HSQC-TOCSY and (B) HMBC spectra of C-0.25/10 (30 mg ml−1) in D2O at 298 K (13C shifts – vertical axis; 1H shifts – horizontal axis).
cross-peak to H2 in a gradient-COSY experiment it was noted
that the coupling to H2 was approximately 4 Hz, which is diag-
nostic of H1 being in the α configuration. In a HSQC-TOCSY
experiment, a proton signal in a glucose ring shows correlations
to all carbons in a sugar residue due to the magnetisation trans-
fer between the coupled protons in the ring. In this spectrum
(Fig. 4A) the signal at δH 4.98 shows correlations to a number of
carbon signals. In particular, the cross-peak at δH 4.98, δC 63.2
was assigned as the C6 methylene. In an HMBC experiment, a
proton signal will show cross-peaks to long-range coupled
carbon atoms (2-bond and 3-bond). Therefore, an anomeric
proton will potentially exhibit correlations to H2, H3 and H5 in
the sugar ring (intra-residue couplings). It can also display a
three-bond correlation across the anomeric bond to the linked
sugar residue (inter-residue coupling). In the expansion of the
HMBC experiment (Fig. 4B), the proton at δH 4.98 exhibits a pro-
minent correlation at δC 68.6 which must be an inter-residue
cross-peak as it does not appear in the HSQC-TOCSY spectrum.
In the multiplicity-edited HSQC spectrum (Fig. S3†), the cross-
peak at δC 68.6 is clearly due to a CH2 group because it is the
opposite phase to the CH sugar cross-peaks. Hence it was deter-
mined that these GMUs are α (1 → 6) linked sugars. Chemical
shifts were assigned for the major component present in this
cluster. The chemical shifts were found to be in agreement with
those of the α (1 → 6) linked glucose unit of the disaccharide,
isomaltose31 (see ESI†). The C4 of this major component had a
chemical shift of 72.3 ppm, which is indicative of a non-redu-
cing end. HSQC-TOCSY showed a low intensity component,
which had a carbon signal at 81.3 ppm, indicative of a C-4
linkage. This suggests that the α (1 → 6) branching consisted of
one or more GMUs. One monomer unit branch was the most
abundant species.
The approximate composition of sugar residues based on
the anomeric proton shifts was calculated by integrating all
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peaks between δH 4.4 and 5.5 ppm in the 1H spectrum. We
found that 68% of GMUs were linked via β (1 → 4) bonds,
while 12% and 7% of GMUs had β and α reducing ends,
respectively. This ratio is in accordance with the known equili-
brium ratio of 64 : 36 for β- and α-glucose in aqueous solu-
tion.32 Approximately 9% of GMUs were linked via α (1 → 6)
bonds and approximately 4% of sugar residues corresponded
to small and broad peaks which could not be identified.
Concluding from the above results we propose that depoly-
merisation of cellulose is catalysed by the presence of acids
impregnated on glycosidic bonds and the required energy for
bond cleavage is supplied by the impact of balls on the cellu-
lose powder during milling. Furthermore, the appearance of a
high percentage of sugar units with the α (1 → 6) linkage is
interesting as these linkages are not a part of a cellulosic struc-
ture and are not formed during depolymerisation of cellulose
using conventional techniques. We believe that this was
caused due to repolymerisation of monomers and low mole-
cular weight oligomers during the milling process which is cat-
alysed by sulphuric acid. These new linkages create branching,
which promotes the solubility of high molecular weight oligo-
mers which are not completely soluble under ambient con-
ditions. The repolymerisation phenomenon also prevents
formation of glucose, which would otherwise undergo degra-
dation to form undesirable compounds.
3.3 Comparison of catalytic conversion of dissolved
oligosaccharides versus solid cellulose into hexitols
Bi-metallic Ni–Pt catalyst supported on γ-alumina was used for
catalytic conversion of dissolved oligosaccharides and solid
MCC in water suspension. The BET surface area of alumina
was 220 m2 g−1 with an average pore size of 6 nm. Ni and Pt
were co-impregnated on the support with 10 wt% Ni and
1 wt% Pt, which gave an atom ratio of 22 : 1. The average metal
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Fig. 5 Yield of sugar alcohol (bars) and conversion (▲) after catalytic hydrolysis
and hydrogenation of cellulose.
surface area of the catalyst as measured by CO-TPD was
4.2 m2 g−1. Although the hydrogenation activity of Ni catalyst
is lower than that of Ru and Pt, it is significantly economical.
Here the hydrogenation activity of Ni is promoted by addition
of a small amount of Pt. The relative yield of hexitol from cellu-
lose is similar to noble catalyst with 3–5% metal loading.24,33
Using soluble oligosaccharides allows access to all glycoside
bonds present, increasing the rate of hydrolysis, and therefore
higher polyol yield is achieved at lower reaction times. Partial
hydrolysis due to depolymerisation also reduces the reaction
time during the catalytic test. ∼80% yield of hexitols was
obtained with the dissolved C-0.25/10 sample after only 1 h of
reaction (Fig. 5, C-0.25/10(A)).
Glucose produced as a result of hydrolysis undergoes rapid
hydrogenation to form sorbitol. Sulphuric acid present on the
surface of the oligosaccharides also catalyses the hydrolysis
reaction, thereby increasing the rate of conversion substan-
tially. However, sulphuric acid also catalysed dehydration of
sorbitol to form 1–4 anhydro sorbitol. To avoid the
dehydration reaction, the C-0.25/10 sample was neutralised
with Ba(OH)2 and the resulting BaSO4 precipitate was separ-
ated by centrifugation. Yields of sorbitol and mannitol
increased to ∼90% (Fig. 5, C-0.25/10 (B)) after the removal of
sulphuric acid, whereas 1–4 anhydro sorbitol yields decreased
from 10.8% to 1.4%. The results prove that 1 h reaction time
was sufficient for complete conversion of oligosaccharides in
the absence of sulphuric acid, while increasing the hexitol
yield by avoiding the dehydration reaction. In comparison, the
yield of polyols from untreated cellulose (C-0/0) and milled
cellulose (C-0/10) was substantially lower.
4. Conclusions
Water soluble oligomers were produced by milling acidulated
cellulose. Sulphuric acid was found to be the most active
towards producing the water soluble fraction in our
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experiment. Rapid depolymerisation of cellulose was observed
during treatment which was proportional to acid concentration
and milling time. Soluble fractions were found to have a DP of
less than 10 monomer units. These units were primarily linked
via β (1 → 4) glycosidic bonds along with α (1 → 6) linkages
which were formed during the treatment. Complete decrystalli-
sation was also observed along with the formation of highly
dispersed particles of size less than 1 μm. Formation of
α (1 → 6) linkage was found to be responsible for enhancing
the solubility of larger oligosaccharides and reducing the
degradation of glucose. Soluble oligomers were highly reactive
towards the hydrolysis and hydrogenation reactions. High
yields of hexitols were obtained using Ni–Pt bimetallic catalyst
supported on alumina. Authors believe that this process is a
possible solution for increasing cellulose reactivity without the
use of hazardous conditions. It has been argued that the
energy required for this process is comparable to other
methods on an industrial scale14,34 and understanding the
mechanism of depolymerisation will result in improving the
efficiency, making it feasible for commercial application.
Acknowledgements
This work was supported by the National and International
Research Alliances Program (NIRAP) funded by Queensland
State Government, Australia. The authors would like to
acknowledge the work of Ms Jun Jhang from the ARC Centre of
Excellence for Functional Nanomaterials at the University of
Queensland, who helped with SEM-EDS experiments.
References
1 A. Wiselogel, S. Tyson and D. Johnson, in Handbook on
Bioethanol: Production and Utilization, ed. C. E. Wyman,
Taylor & Francis, 1996, pp. 105–118.
2 G. W. Huber, S. Iborra and A. Corma, Chem. Rev., 2006,
106, 4044–4098.
3 B. Medronho, A. Romano, M. Miguel, L. Stigsson and
B. Lindman, Cellulose, 2012, 19, 581–587.
4 W. Glasser, R. Atalla, J. Blackwell, R. Malcolm Brown Jr.,
W. Burchard, et al., Cellulose, 2012, 19, 599–599.
5 A. Pinkert, K. N. Marsh and S. Pang, Ind. Eng. Chem. Res.,
2010, 49, 11121–11130.
6 J. Cai and L. Zhang, Macromol. Biosci., 2005, 5, 539–548.
7 M. Sasaki, Z. Fang, Y. Fukushima, T. Adschiri and K. Arai,
Ind. Eng. Chem. Res., 2000, 39, 2883–2890.
8 T. Paakkari, R. Serimaa and H. P. Fink, Acta Polym., 1989,
40, 731–734.
9 K. Mazeau and L. Heux, J. Phys. Chem. B, 2003, 107, 2394–
2403.
10 M. S. Bertran and B. E. Dale, J. Appl. Polym. Sci., 1986, 32,
4241–4253.
11 T. Jeoh, C. I. Ishizawa, M. F. Davis, M. E. Himmel,
W. S. Adney, et al., Biotechnol. Bioeng., 2007, 98, 112–122.
Green Chem.

Paper
12 H. Zhao, J. H. Kwak, Y. Wang, J. A. Franz, J. M. White,
et al., Energy Fuels, 2005, 20, 807–811.
13 Y. Yu and H. Wu, Ind. Eng. Chem. Res., 2010, 49, 3902–
3909.
14 N. Meine, R. Rinaldi and F. Schüth, ChemSusChem, 2012, 5,
1449–1454.
15 S. Braun, H.-O. Kalinowski and S. Berger, 150 and more
Published on 06 August 2013. Downloaded by University of Missouri at Columbia on 22/08/2013 17:39:04.
basic NMR experiments: a practical course, Wiley-VCH, Wein-
heim, New York, 1998.
16 G. Xu and J. S. Evans, J. Magn. Reson., Ser. B, 1996, 111,
183–185.
17 T. D. W. Claridge, High-resolution NMR techniques in organic
chemistry, Pergamon, Amsterdam, New York, 1999.
18 N. T. Nyberg, J. Ø. Duus and O. W. Sørensen, J. Am. Chem.
Soc., 2005, 127, 6154–6155.
19 D. O. Cicero, G. Barbato and R. Bazzo, J. Magn. Reson.,
2001, 148, 209–213.
20 T. Werpy and G. Petersen, Top Value Added Chemicals from
Biomass Volume I-Results of Screening for Potential Candi-
dates from Sugars and Synthesis Gas, National Renewable
Energy Laboratory (NREL), 2004.
21 D. M. Alonso, J. Q. Bond and J. A. Dumesic, Green Chem.,
2010, 12, 1493–1513.
Green Chem.
View Article Online
Green Chemistry
22 G. W. Huber, R. D. Cortright and J. A. Dumesic, Angew.
Chem., Int. Ed., 2004, 116, 1575–1577.
23 A. Fukuoka and P. L. Dhepe, Angew. Chem., Int. Ed., 2006,
45, 5161–5163.
24 A. Shrotri, A. Tanksale, J. N. Beltramini, H. Gurav and
S. V. Chilukuri, Catal. Sci. Technol., 2012, 2, 1852–1858.
25 Y. H. P. Zhang and L. R. Lynd, Biomacromolecules, 2005, 6,
1510–1515.
26 J. B. Taylor, Trans. Faraday Soc., 1957, 53, 1198–1203.
27 G. L. Miller, Methods Carbohydr. Chem., 1963, 3, 134–139.
28 H. Sugiyama, K. Hisamichi, T. Usui, K. Sakai and
J. i. Ishiyama, J. Mol. Struct., 2000, 556, 173–177.
29 L. A. Flugge, J. T. Blank and P. A. Petillo, J. Am. Chem. Soc.,
1999, 121, 7228–7238.
30 A. Teleman, K. Kruus, E. Ämmälahti, J. Buchert and
K. Nurmi, Carbohydr. Res., 1999, 315, 286–292.
31 D. S. Wishart, D. Tzur, C. Knox, R. Eisner, A. C. Guo, et al.,
Nucleic Acids Res., 2007, 35, D521–D526.
32 S. J. Angyal, Angew. Chem., Int. Ed. Engl., 1969, 8, 157–166.
33 P. L. Dhepe and A. Fukuoka, Catal. Surv. Asia, 2007, 11,
186–191.
34 S. M. Hick, C. Griebel, D. T. Restrepo, J. H. Truitt,
E. J. Buker, et al., Green Chem., 2010, 12, 468–474.
This journal is © The Royal Society of Chemistry 2013