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Effects of Crystallinity on Dilute Acid Hydrolysis
of Cellulose by Cellulose Ball-Milling Study
Haibo Zhao, Ja Hun Kwak, Yong Wang, James A. Franz, John M. White, and Johnathan E. Holladay
Energy Fuels, 2006, 20 (2), 807-811 • DOI: 10.1021/ef050319a
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 Energy & Fuels 2006, 20, 807-811 807

Effects of Crystallinity on Dilute Acid Hydrolysis of Cellulose by

Cellulose Ball-Milling Study
Haibo Zhao, Ja Hun Kwak, Yong Wang, James A. Franz, John M. White, and
Johnathan E. Holladay*
Pacific Northwest National Laboratory, Institute for Interfacial Catalysis, P.O. Box 999,
Richland, Washington 99352
ReceiVed September 27, 2005. ReVised Manuscript ReceiVed NoVember 23, 2005

The dilute acid (0.05 M H2SO4) hydrolysis at 175 °C of samples comprised of varying fractions of crystalline
(R-form) and amorphous cellulose was studied. The amorphous content, based on XRD and CP/MAS NMR,
and the product (glucose) yield, based on HPLC, increased by as much as a factor of 3 upon ball milling.
These results are interpreted in terms of a model involving mechanical disruption of crystallinity by breaking
hydrogen bonds in R-cellulose, opening up the structure, and making more β-1,4 glycosidic bonds readily
accessible to the dilute acid. However, in parallel with hydrolysis to form liquid-phase products, there are
reactions of amorphous cellulose that form solid degradation products.

1. Introduction
Cellulose is the most abundant renewable carbon source
available and can potentially meet our future energy needs if
cellulose can be efficiently converted to monomeric sugars. A
single cellulose molecule is an unbranched chain of glucose
units linked head to tail. Compared to starch, the hydrolysis of
native cellulose is much more difficult. Cellulose forms two
crystalline forms, IR and Iβ, in statistically variable proportions.1
In crystalline cellulose, each cellulose chain approximates to a
flat ribbon, with alternate glucose units facing in opposite
directions.2 All the cellulose chains lie parallel, hydrogen-bonded
edge to edge. The sheets of chains so formed are stacked on
top of one another with a stagger, along the microfibril, which
differs between the IR and Iβ forms. These structures are well
characterized by synchrotron X-ray and neutron fiber diffrac-
tion.3,4 The weak C-H‚‚‚O hydrogen bonding is thought to be
the force that holds sheets of chains together in stacks.2,3
R-Cellulose was used in our study because it is more abundant
in nature than β-cellulose. The crystal structure and hydrogen
bonding in cellulose greatly limit the access to β-1,4-glycosidic
bonds by reactants and catalysts. Water is excluded almost
completely from the crystalline regions in cellulose.5-7 This
limitation makes cellulose hydrolysis much slower than that of
starch.
The amorphous part of cellulose is more readily accessible
by water and other reactants.8 Molecular dynamics simulations
* Corresponding author. Tel: 509-375-2025. Fax: 509-372-4732. E-
mail: john.holladay@pnl.gov.
(1) Atalla, R. H.; Vanderhart, D. L. Science 1984, 223, 283.
(2) Jarvis, M. Nature 2003, 426, 611.
(3) Nishiyama, Y.; Sugiyama, J.; Chanzy, H.; Langan, P. J. Am. Chem.
Soc. 2003, 125, 14300.
(4) Nishiyama, Y.; Langan, P.; Chanzy, H. J. Am. Chem. Soc. 2002,
124, 9074.
(5) Child, T. F. Polymer 1972, 13, 259.
(6) Hatakeyama, T.; Ikeda, Y.; Hatakeyama, H. Makromol. Chem. 1987,
188, 1875.
(7) Nakamura, K.; Hatakeyama, T.; Hatakeyama, H. Text. Res. J. 1983,
11, 682.
(8) Vittadini, E.; Dickinson, L. C.; Chinachoti, P. C. Carbohydr. Polym.
2001, 46, 49.
by Mazeau and Heux show that the total number of hydrogen
bonds per repeat units is eight in the IR crystalline form and is
only 5.3 in the amorphous form.9 They also show that the
crystalline form of cellulose has much larger cohesive energy
density than noncrystalline forms.9 This suggests that noncrys-
talline forms of cellulose will be more reactive.
The acid-catalyzed hydrolysis of crystalline cellulose is a
heterogeneous reaction, and models are well described in the
literature.10-13 Even with continued research and development,
breakthroughs are needed to raise the glucose yields above 65%.
Two factors limiting glucose yield are glucose degradation14
and cellulose modification15 under various reaction conditions
since temperatures higher than 200 °C are often used in tradi-
tional dilute acid cellulose hydrolysis processes. To reduce
glucose degradation and cellulose modification, less harsh
reaction conditions should be used in cellulose hydrolysis, but
this will greatly decrease hydrolysis rate. Pretreating cellulose
to increase the noncrystalline fraction will increase the number
of accessible β-1,4-glycosidic bonds and likely reduce reaction
temperature and time. Mild reaction conditions could greatly
reduce glucose and cellulose degradation during reactions. The
reprecipitated cellulose formed from that dissolved in 65%
concentrated sulfuric acid solution can be hydrolyzed much
faster than its original form because it is less crystalline.16
Increasing the noncrystalline cellulose fraction may also ac-
celerate cellulose enzyme hydrolysis processes due to an
increase of accessible β-1,4-glycosidic bonds.
In this paper, we physically decreased cellulose crystallinity
by the ball milling of R-cellulose. Although this pretreatment
(9) Mazeau, K.; Heux, L. J. Phys. Chem. B 2003, 107, 2394.
(10) Mok, W. S.; Antal, M. J. Ind. Eng. Chem. Res. 1992, 31, 94.
(11) Torget, R. W.; Kim, J. S.; Lee, Y. Y. Ind. Eng. Chem. Res. 2000,
39, 2817.
(12) Xiang, Q.; Kim, J. S.; Lee, Y. Y. Appl. Biochem. Biotechnol. 2003,
105, 337.
(13) Saeman, J. F. Ind. Eng. Chem. 1945, 37, 43.
(14) Corner, A. H.; Wood, B. F.; Hill, C. G.; Harris, J. F. J. Wood Chem.
Technol. 1985, 5, 461.
(15) Bouchard, J.; Abatzoglou, N.; Chornet, E.; Overend, R. P. Wood
Sci. Technol. 1989, 23, 343.
(16) Xiang, Q.; Lee, Y. Y.; Pettersson, P. O.; Torget, R. W. Appl.
Biochem. Biotechnol. 2003, 105-108, 505.

10.1021/ef050319a CCC: $33.50 © 2006 American Chemical Society

Published on Web 12/23/2005
808 Energy & Fuels, Vol. 20, No. 2, 2006
is not viable in its practical nature because of the high energy
input, the purpose of our work was to correlate hydrolysis
reactivity with decreasing cellulose crystallinity. Cellulose
structures after ball milling were characterized by X-ray
diffraction (XRD) and cross polarization/magic-angle spinning
13C solid-state nuclear magnetic resonance (CP/MAS NMR).
We examined the influence of crystallinity on hydrolysis in
dilute sulfuric acid at a relatively low reaction temperature.
2. Materials and Methods
2.1. R-Cellulose. R-Cellulose was purchased directly from
Sigma-Aldrich (product no. C6663). Ball-milling experiments were
performed on an US Stoneware ball-mill machine. A ZrO2 ball
(mass of 1 kg and diameter of 1 cm) and 30 g of R-cellulose were
loaded into a polypropylene bottle (500 mL). Spinning speed was
set at 60 rpm. Five grams of cellulose samples were taken out at a
desired time (1, 2, and 6 days) and used in our experiments.
2.2. X-ray Diffraction Method (XRD). XRD measurements
were performed on a Philips PW3040/00 X′Pert MPD system. The
diffracted intensity of Cu KR radiation (wavelength of 0.1542 nm,
under conditions of 50 kV and 40 mA) was measured in a 2θ range
between 10° and 50°.
2.3. CP/MAS 13C Solid-State NMR. Cross polarization/magic-
angle spinning (CP/MAS) 13C solid-state NMR experiments were
performed on a Chemagnetics CMX100 spectrometer operating
under a static field strength of 2.3 T (100 MHZ 1H) at 25 °C. The
contact time for CP was 1 ms with a proton pulse of 5.5 µs and
decoupling power of 45 kHz. The MAS speed was 3 kHz. The
delay time after the acquisition of the FID signal was 2 s. The
chemical shifts were calibrated by using the hexamethylbenzene
methyl resonance at 17.3 ppm. The cellulose crystallinity index
(ICr), was determined from the peak areas assigned to C4 crystalline
(86-92 ppm) and C4 noncrystalline (79-86 ppm) material. The
cellulose crystallinity index is defined as ICr ) A86-92 ppm/(A79-86
ppm + A86-92 ppm) × 100% by deconvolution using a Lorenzian line
shape, as discussed by Liitiä et al.17
2.4. Reaction Test. Reaction tests were performed using sealed
tubular reactors. The reactors (∼15 cm3 internal volume) were
constructed of Hastelloy C-276 tubing (0.5-in. OD × 6-in. length)
capped with Swagelok end fittings. In our experiments, 0.8 g of
cellulose and 8.0 g of 0.05 M sulfuric acid solution were loaded
into a reactor. Reactors were heated to 175 °C in a fluidized sand
bath, held for a selected reaction time, and taken out and quickly
quenched in cold water bath. The liquids and solid residues were
separated by filtration. HPLC was used to separate and quantify
liquid products. Solid residues were washed with deionized water,
dried at 105 °C overnight in a vacuum drier, and analyzed by CP/
MAS 13C solid-state NMR. We defined the yield so that for a 100%
glucose yield all the initial cellulose is converted to glucose.
2.5. HPLC Analysis of Liquid Products. Liquid products were
analyzed by HPLC using a Bio-Rad Aminex HPX-97H column. A
refractive index detector was used in our analysis.
3. Results and Discussions
3.1. Ball-Milling Effects on Cellulose Crystallinity. Figure
1 shows cellulose XRD patterns for samples unmilled and ball-
milled for 1, 2, or 6 days. The strongest peak, at 2θ ) 22.6°,
originates from the cellulose crystalline plane 002.18,19 The
intensities of peaks from other crystal planes also decrease as
ball-milling time increases (Figure 1). Clearly, mechanical ball
milling reduces the long-range order. In addition, we estimated
(17) Liitiä, T.; Maunu, S. L.; Hortling, B.; Tamminen, T.; Pekkala, O.;
Varhimo, A. Cellulose 2003, 10, 307.
(18) Park, C. H.; Kang, Y. K.; Im, S. S. J. Appl. Polym. Sci. 2004, 94,
248.
(19) Newman, R. H. Solid State Nucl. Magn. Reson. 1999, 15, 21.
Zhao et al.
Figure 1. XRD patterns of as-received R-cellulose and ball milling
for 2, 4, and 6 days as indicated.
the lateral dimensions of our cellulose samples to be 7.3 nm
using the Scherrer equation:20
L ) Kλ/(β cos θ)
where the X-ray wavelength is λ, the angle between incident
and diffracted rays is 2θ, and K is a constant with a value of
0.94.
Figure 2 shows the cellulose CP/MAS NMR spectra with
and without ball milling. The milling procedure is expected to
affect the crystallinity of the cellulose, which is confirmed by
our XRD data. Consistent with this expectation, the peak ratios
of C4(86-92 ppm)/C4(79-86 ppm) and C6(63-67 ppm)/C6(56-63 ppm) also
decrease with increased ball-milling time, which clearly suggests
that more disordered cellulose is produced in the ball-milling
process.
Even though CP/MAS NMR has been widely used to study
cellulose structures, the underlying reasons for the shape of the
C4 resonance (79-92 ppm) remain controversial. Two peaks
are detected in most cellulose samples. The assignment of the
peak between 86 and 92 ppm to the crystalline region is well
accepted. The peak between 79 and 86 ppm is usually assigned
either to the crystal surface or the disordered component in
cellulose;21 however, it may also comprise a mixture of the two,
since native cellulose always includes some noncrystalline
regions.1,22 Recently, Kono et al. prepared IR- and Iβ-rich
cellulose samples with no noncrystalline region.23 Their CP/
MAS NMR spectra show almost no signal between 79 and 86
ppm, which strongly suggests that this peak must originate from
noncrystalline regions. The crystallinity index I which is the
ratio between the C4 area of crystalline cellulose and the total
C4 peak areas, can be used to define the crystallinity of
cellulose.17,24 The cellulose crystallinity decreased from 0.77
for the as-received material to 0.52 after ball milling for 6 days.
(20) Murdock, C. C. Phys. ReV. 1930, 35, 8.
(21) Atalla, R. H.; Vanderhart, D. L. Solid State Nucl. Magn. Reson.
1999, 15, 1.
(22) Vanderhart, D. L.; Atalla, R. H. Macromolecules 1984, 17, 1465.
(23) Kono, H.; Yunoki, S.; Shikana, T.; Fujiwara, M.; Erata, T.; Takai,
M. J. Am. Chem. Soc. 2002, 124, 7506.
(24) Lenholm, H.; Larsson, T.; Iversen, T. Carbohydr. Res. 1994, 261,
119.
Crystallinity and Dilute Acid Hydrolysis of Cellulose Energy & Fuels, Vol. 20, No. 2, 2006 809

Figure 2. CP/MAS 13C solid-state NMR spectra of as-received

R-cellulose and ball milling for 1, 2, and 6 days as indicated. The Figure 3. Glucose yields at different reaction times from cellulose
crystallinity index, Icr, is indicated on each curve and is calculated as ball-milled for different numbers of days.
ICr ) A86-92 ppm/(A79-86 ppm + A86-92 ppm) × 100%.

3.2. Reactivity of Ball-Milled Cellulose. To evaluate the

reactivity of ball-milled cellulose, cellulose hydrolysis was
carried out at low temperature (175 °C) with a dilute sulfuric
acid (0.05 M) catalyst. Other experiments (not shown) suggest
that when the as-received R-cellulose is used in the reaction,
crystallinity and structure are not altered under these reaction
conditions. Hydrolysis occurs to a small extent and produces
hydroxymethylfurfuraldehyde (HMF) and levulinic acid, pre-
sumably via glucose formation followed by glucose degradation.
Figure 3 shows the glucose yield at various reaction times
from the hydrolysis of as-received and ball-milled cellulose
samples. The glucose yield increases with ball-milling time and
reaction time. The latter observation implies that the rate of
glucose formation exceeds the rate of glucose degradation.
Under various reaction conditions glucose (C6 molecule) can
react to form HMF (C6 molecule), which can further react to
form levulinic acid (C5 molecule). To better understand the
cellulose hydrolysis process, we plotted total C5 and C6 product
yields with reaction time in Figure 4. This combined number
represents real hydrolysis yields by including glucose degrada-
tion products. Increasing cellulose ball-milling time from 0 to
6 days more than doubles the total C5 and C6 produced. The
yield also increases with increasing reaction time, but the rate
drops, particularly for reaction times exceeding 20 min. The
decreasing rate may be due, in part, to changes in the cellulose Figure 4. Total C5 + C6 products yields at different reaction times
since it darkened with increasing reaction time. Perhaps a small from cellulose ball-milled for different numbers of days.
amount of cellulose degrades or glucose degradation products
adsorb on the cellulose surface. Amounts of degradation It has been reported that cellulose acid hydrolysis rates are
products adsorbed on exposed surfaces could be sufficient to limited in two general wayssintrinsic cleavage of glycosidic
inhibit the hydrolysis rate without being detectable in CP/MAS linkages and physical access to these linkages. When the
NMR. The comparison of Figures 3 and 4 suggests that cellulose that dissolved in 65% H2SO4 solution was precipitated,
increasing ball-milling time increased the total conversion of the so-called reprecipitated cellulose (amorphous) was hydro-
cellulose, but it also increased the yield of glucose degradation lyzed 100-fold faster than crystalline R-cellulose.16 The repre-
products because glucose degradation will increase with increas- cipitated cellulose is claimed to be hydrolyzed at the same rate
ing glucose concentration in the reactants. as corn starch under identical reaction conditions.16 Consistent
810 Energy & Fuels, Vol. 20, No. 2, 2006 Zhao et al.

with the ball-milling results, this is attributed to minimal

intermolecular hydrogen bonding in the reprecipitated cellulose.
Schwanninger and co-workers25 suggested that changes in
the FTIR spectrum of cellulose after ball milling for 1 min were
caused by the disruption of hydrogen bonds. However, our XRD
and CP/MAS NMR experiments (not shown) exhibit no
evidence for alteration of the cellulose structure after 1 h of
ball milling. The FTIR spectra may be more sensitive than XRD
and CP/MAS NMR to disruption of the weak C-H‚‚‚O
hydrogen bonding that holds sheets of chains together in a stack.
Ball milling for longer times delivers mechanical stress sufficient
to be detected by XRD and CP/MAS NMR.
Efficiently disrupting hydrogen bonding, making many more
β-1,4-glycosidic bonds accessible to reactants and catalysts, is
critical to increasing the cellulose hydrolysis rate. Sasaki and
co-workers26 found that cellulose is rapidly dissolved and
depolymerized in supercritical water at 300-320 °C with no
acid. They suggested that supercritical water completely disrupts
the intramolecular and intermolecular hydrogen-bond linkages.
Dissolving the cellulose eliminates one of the restrictions of
the cellulose physical state on the hydrolysis reaction, and the
reaction changes from heterogeneous to homogeneous. Con-
centrated acids can also dissolve cellulose and eliminate
physical-state effect on the hydrolysis reaction, but cost and
environmental concerns make these processes unattractive or Figure 5. CP/MAS 13C solid-state NMR spectra before and after
impractical. Safe, effective solvents or other methods for reaction (5 min, 10 min) for a ball-milled (6 days) sample.
breaking hydrogen bonds in cellulose are needed to make
cellulose hydrolysis more competitive.
3.3. Cellulose Crystallinity Change in the Hydrolysis.
Cellulose cystallinity and structure were not altered under our
reaction conditions when R-cellulose was used in the reactions
although noncrystalline regions exist in R-cellulose. The possible
reason is that these noncrystalline regions are surrounded by
crystalline regions. Figure 5 shows cellulose CP/MAS NMR
spectra both before and after reaction for a ball-milled (6 days)
sample. The cellulose crystallinity increases with increasing
reaction times. This is expected because the amorphous fraction
will be hydrolyzed selectively leaving crystalline cellulose intact.
These results are consistent with the idea that the noncrystalline
material formed by ball milling is located preferentially at the
surface of crystalline material where it can be accessed easily
by acid and water.
3.4. Stability of Ball-Milled Cellulose under Hydrolysis
Conditions. Figure 6 shows cellulose CP/MAS NMR spectra
before and after hydrolysis. The curves are for R-cellulose, and
the samples are coded as follows: A: as-received; B: as-
received and hydrolyzed 40 min; C: ball-milled 2 days and 40
min reaction; D: ball-milled 6 days and 40 min reaction. The
CP/MAS NMR spectra of A and B are indistinguishable even
though, visually, the color changed from white to yellow during
hydrolysis. Evidently, the extent of hydrolysis was not detectable
by CP/MAS NMR. Sample C (ball-milled 2 days) gave
Figure 6. Cellulose CP/MAS NMR spectra before and after hydrolysis.
distinguishing features in the CP/MAS NMR spectra of the The curves are for R-cellulose, and the samples are coded as follows:
remaining solid. Small peaks at 20 and 25 ppm are characteristic A: as-received; B: as-received and hydrolyzed 40 min; C: ball-milled
of methyl groups, and the broad features near 150 ppm are 2 days and 40 min reaction; D: ball-milled 6 days and 40 min reaction.
attributable to CdC double bonds. The peak at 43 ppm remains CdO groups. Consistent with these findings, the color of
unassigned. The CP/MAS NMR spectra indicate the cellulose cellulose after hydrolysis becomes much darker with increasing
degradation during hydrolysis increase as with ball-milling time. ball-milling time.
The intensities of peaks due to cellulose degradation increase, Mazeau and Heux used molecular dynamics simulation to
and new small peaks near 200 ppm indicate the formation of calculate the cohesive energy density of cellulose.9 The cohesive
energy density of IR cellulose is 46 kcal/mol larger than that of
(25) Schwanninger, M.; Rodrigues, J. C.; Pereira, H.; Hinterstoisser, B. amorphous cellulose, which suggests that crystalline cellulose
Vib. Spectrosc. 2004, 36, 23.
(26) Sasaki, M.; Fang, Z.; Fukushima, Y.; Adschiri, T.; Arai, K. Ind. is much more stable than amorphous cellulose. Confirming that
Eng. Chem. Res. 2000, 39, 2883. amorphous cellulose is not thermodynamically stable, early DSC
Crystallinity and Dilute Acid Hydrolysis of Cellulose
studies27 also show that the transition from amorphous to
crystalline structure is exothermic.
Water evolution from dehydration is usually the first step
for cellulose degradation,28 and this process can be catalyzed
by acid. Dehydration involves OH groups, and free OH groups
may react with lower activation energy than those that are
hydrogen-bonded. Since amorphous cellulose has more free OH
groups, this form will be more vulnerable to undesirable
degradation. These findings suggest that less harsh conditions
or shorter reaction times may have to be used to reduce
degradation of amorphous cellulose during acid hydrolysis.
4. Conclusions
Mechanical ball milling significantly increases the noncrystal-
line fraction in crystalline R-cellulose. The decreased crystal-
linity is evidenced by decreased crystalline peak intensity in
XRD and a crystallinity index decrease in CP/MAS NMR. The
crystallinity index of cellulose decreased from 0.773 to 0.523,
and the cellulose hydrolysis rate increase more than doubled
after R-cellulose was ball-milled for 6 days. The increased
hydrolysis rate was attributed to disruption of hydrogen bonding
and a concomitant increase in the number of β-1,4-glycosidic
(27) Kimura, M.; Hatakeyama, T.; Nakano, J. J. Appl. Polym. Soc. 1974,
18, 3069.
(28) Scheirs, J.; Camino, G.; Tumiatti, W. Eur. Polym. J. 2001, 37, 933.
Energy & Fuels, Vol. 20, No. 2, 2006 811
bonds accessible to the acid involved in hydrolysis. During
hydrolysis, the crystallinity increases since the noncrystalline
cellulose was preferentially hydrolyzed.
As-received R-cellulose is very stable under our hydrolysis
reaction conditions; after hydrolysis, CP/MAS NMR of the
remaining solid exhibited nothing other than cellulose. The
results differ for ball-milled samples; after hydrolysis for 40
min, CP/MAS NMR spectra of the remaining solid contained
new features attributable to CH3, CdO and CdC groups. This
could be due to increased numbers of free OH groups resulting
from ball milling. The increased number of free OH groups
increases the number of dehydration sites in cellulose, allowing
for an increase of the average cellulose degradation rate.
Acknowledgment. The authors thank Dr. James F. White, Dr.
Charles H. F. Peden, and Dr. Thomas H. Peterson for helpful and
informative discussions and thank Alan R. Cooper and Danielle S.
Muzatko for initial HPLC analysis. This work was supported by
the Laboratory Directed Research and Development Program at
the Pacific Northwest National Laboratory (PNNL), a multiprogram
national laboratory operated by Battelle for the U.S. Department
of Energy under Contract DE-AC06-76RL01830. Part of the
research described in this paper was performed at the Environmental
Molecular Science Laboratory, a national scientific user facility
located at PNNL.
EF050319A