Review

Analyzing cellulose degree

of polymerization and its
relevancy to cellulosic ethanol
Bassem B. Hallac and Arthur J. Ragauskas, Georgia Institute of Technology, Atlanta, Georgia, USA

Received October 2, 2010; revised version received November 8, 2010; accepted November 15, 2010
View online January 28, 2011 at Wiley Online Library (wileyonlinelibrary.com); DOI: 10.1002/bbb.269;
Biofuels, Bioprod. Bioref. 5:215225 (2011)

Abstract: The degree of polymerization (DP) of cellulose in cellulosic biomass and how it changes during enzymatic
and chemical transformations has remained a fundamental property of interest to numerous researchers. Currently,
with increased interest in cellulosic biofuels, more attention is being focused on determining changes in cellulose
DP before and during pre-treatment, as well as the effect of DP on enzymatic deconstruction of cellulose to glucose.
Different sources of celluloses from woody and non-woody biomass have been isolated and the DP has been fre-
quently examined as a key parameter contributing to efficient biomass deconstruction. The isolation and derivatiza-
tion/dissolution of cellulose are crucial steps in determining cellulose DP. This review summarizes approaches to
measuring DP developed over the past six decades and highlights opportunities for further improvements.
2011 Society of Chemical Industry and John Wiley & Sons, Ltd

Keywords: cellulose; degree of polymerization; GPC; biomass; pre-treatment

Introduction cellulosic biomass is converted to ethanol via thermochemi-

cal or biological routes. First-generation biofuels are depend-

T
he necessity to develop alternative transportation
fuels has become a global concern mainly due to three
factors: (i) increase in energy consumption; (ii)finite
petroleum reserves; and (iii) climate change.13 The demand
for energy is expected to grow by more than 50% by 2025,
but the finite amount of petroleum resources available can-
not satisfy this increase.3,4 Biofuels are a sustainable and
renewable source of energy that has become of interest
to many researchers over the past decade.58 The leading
second-generation biofuel is cellulosic bioethanol in which a
ent on the fermentation of starches or sucrose derived from
corn starch and sugarcane, respectively.4,5 However, since
these bioresources have food value and require produc-
tive agricultural lands, research focus has shifted toward
bioethanol produced from lignocellulosic biomass.5 Such
materials do not directly compete with food, have a greater
net energy generation per area of land, and can be grown on
non-agricultural lands in a sustainable manner.9
Lignocellulosic biomass consists primarily of three biopol-
ymers: cellulose, hemicellulose, and lignin, which together

Correspondence to: Arthur J. Ragauskas, Institute of Paper Science and Technology, School of Chemistry and Biochemistry, Georgia Institute of Technology,

Atlanta, Georgia 30332, USA. E-mail: arthur.ragauskas@chemistry.gatech.edu

2011 Society of Chemical Industry and John Wiley & Sons, Ltd 215
BB Hallac, AJ Ragauskas
form a complex and rigid structure that is recalcitrant to
biological and chemical degradation.3,10 The biological proc-
ess of converting these materials to bioethanol includes
pre-treatment, enzymatic hydrolysis, and fermentation.
Thermochemical pre-treatment is a favored route for the
conversion process owing to shorter processing times, higher
yields, limited chemical use, and lower energy requirements,
when compared with many biological or mechanical pre-
treatments.11 The purpose of the pre-treatment is to reduce
the recalcitrance of biomass so that cellulose can become
more accessible and reactive to enzymatic hydrolysis, in
which cellulase converts cellulose to fermentable glucose.7,12
Examples of such chemical pre-treatments include dilute
acid, aqueous lime, organosolv, steam explosion, and ammo-
nia fiber explosion (AFEX).7,1316
The efficiency of the enzymatic hydrolysis of cellulose in
biomass is mostly governed by the native biomass structural
features, which can be classified as chemical or physical.17,18
Chemical features include the compositions and structure
of cellulose, hemicellulose, and lignin; while the physical
features consist of accessible biomass surface area, cellu-
lose crystallinity and degree of polymerization (DP), pore
volume, biomass particle size, and physical distribution of
lignin and hemicellulose as well as their association with
cellulose in the biomass matrix.17 Many studies have investi-
gated the relationship between these characteristics and the
enzymatic hydrolysis.11,1730 In pretreated biomass, the pres-
ence of lignin, hemicellulose, and crystalline, high DP cel-
lulose impedes efficient cellulase deconstruction of cellulose.
The reduction in the DP of cellulose significantly improves
its hydrolysis by cellulase because the number of cellulose
chain ends available to the action of exoglucanase in the cel-
lulase complex increases.19,2427,31
The interest in determining the DP of cellulose has
changed over the years. In the early studies (~1950s),
researchers were focused on simply knowing the DP of cel-
lulose isolated from various biomass resources, as a way to
study this biopolymer. Later on (~1970s), with the develop-
ment of the pulp and paper industry, determining the DP
of cellulose became of interest because it was a contributing
factor impacting physical properties including strength of
the final paper products. These studies frequently exam-
ined how the pulping process and bleaching impacted the
216 2011 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. 5:215225 (2011); DOI: 10.1002/bbb
Review: Degree of polymerization of cellulose
structure of cellulose.3236 Nowadays, research focus has cen-
tered on understanding how the effects of the pre-treatment
technologies impact the DP of cellulose and its subsequent
influence on enzymatic hydrolysis of cellulose for the pur-
pose of producing glucose and its fermentation to ethanol.
Over the past decade, a significant volume of research in
evaluating the DP of cellulose from various plant species
with assorted isolation and analysis procedures has been
reported and as interest in cellulosic ethanol grows, this will
only increase. Thus, the objective of this paper is to provide a
summary of the DP of native cellulose from woody and non-
woody biomass, as well as the methods used for isolating cel-
lulose and measuring its DP. Furthermore, the paper reviews
the recent findings on the effect of various pre-treatment
technologies on the DP of cellulose and the techniques to
measure DP.
Cellulose
Cellulose was first discovered by the French scientist
Anselme Payen in 1838, when he noticed a resistant fibrous
solid that remained behind after treating plant tissue with
acids and ammonia. Cellulose is a linear polymer made up
of -D glucopyranose units covalently linked with 14
glycosidic bonds, with the DP varying with the origin and
treatment of the raw material (Fig. 1). 37 The large number
of hydroxyl groups on the cellulose chain forms intra- and
inter-molecular hydrogen bonds, resulting in the crystalline
structure of cellulose.5 Native cellulose in plants is a com-
posite of three crystalline allomorphs: cellulose I, cellulose
I, and para-crystalline cellulose; and two non-crystalline
forms: amorphous cellulose at accessible and inaccessible
fibril surfaces.3842 Cellulose I, a one-chain triclinic unit
cell, is the dominant form in bacterial and algal cellulose,
whereas cellulose I, a monoclinic two-chain unit cell, is
dominant in higher plants, such as wood.41 para-Crystalline
cellulose is the form that is less ordered than cellulose I and
cellulose I but more ordered than amorphous cellulose.41
Accessible fibril surfaces are those in contact with water,
while the inaccessible fibril surfaces are fibril-fibril contact
surfaces and surfaces resulting from distortions in the fibril
interior.43 Cellulose I is meta-stable in nature and can be
converted to the thermodynamically more stable allomorph
(cellulose I) by annealing.41,44 Nishiyama et al. proposed
Review: Degree of polymerization of cellulose
HO
3 OH 1
O HO 2 O
HO 5 2
HO 4 HO
O O
OH 6 OH
OH
Figure 1. Molecular structure of cellulose.
that slippage of the glucan chains is the most likely mecha-
nism for conversion of cellulose I to cellulose I.44 Both
chains in cellulose I and I are organized in sheets packed
in a parallel-up fashion, with the hydroxymethyl groups in
the tg conformation.44
Isolation of cellulose
In order to analyze the DP of cellulose, it must first be puri-
fied and isolated from its native source (i.e. separated from
the extractives, lignin, and hemicelluloses). The common
method for the isolation of cellulose is that described by
Browning, as this method does not alter the chain length of
cellulose significantly.45 First, the air-dry wood meal is pre-
extracted with 95% ethanol in a Soxhlet extractor. Then the
wood is delignified by using glacial acetic acid and sodium
chlorite, which results in the formation of holocellulose
(i.e. a mixture of cellulose and hemicellulose) by a selective
oxidative degradation of lignin. Typically, 4h holocel-
lulose pulping is sufficient, but this involves adding more
CH3COOH and NaClO2 after the first, second, and third
hour. The reaction temperature is usually between 70 and
80C and yields a relatively pure cellulose-hemicellulose
product. Lastly, the cellulose is isolated from holocellu-
lose by extraction with concentrated sodium hydroxide
according to the TAPPI T-203 cm-09 method, in which
holocellulose is treated with 17.5% NaOH by weight at room
temperature for about 1 h. The resulted cellulose is usually
called cellulose. The chemical dosage, temperature, and
period of reaction are factors that affect degradation of cel-
lulose during the isolation process and should be adjusted
depending on the species and their composition.
The acid-chlorite delignification method selectively
removes lignin from biomass with only trace solubiliza-
tion of glucan and hemicelluloses, such as xylan.46 Recently,
there has been a concern about this method that it may
2011 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. 5:215225 (2011); DOI: 10.1002/bbb
BB Hallac, AJ Ragauskas
OH
4 6 OH
5 O HO OH
3 1
O O
n OH
significantly reduce the chain length of cellulose. Kumar
etal. reported a reduction of nearly 75% in the viscosity
average degree of polymerization (DPV) of fi lter paper cel-
lulose after delignification.11 However, it has been noted that
an important consideration for this method is to allow a
few percent of lignin to remain in the preparation, because
complete delignification will result in excessive loss of
polysaccharides.45 Furthermore, Hubbell and Ragauskas
have recently reported that lignin-free fi lter paper samples
showed a DP reduction of nearly 35% after holocellulose del-
ignification, while a reduction of 12% was observed for sam-
ples that contained 30% lignin, and 17% when lignin content
reached 1%.47 Thus, small amount of lignin (~1%) can mini-
mize loss of cellulose DP during holocellulose pulping.
Determining cellulose degree
of polymerization
The acid-chlorite delignification followed by alkali treat-
ment technique for cellulose isolation from lignocellulosic
material has been used by several studies prior to measuring
cellulose DP.11,24,27,4850 Cellulose has to be purified or iso-
lated from its native source in order to adequately determine
its DP.51,52 The presence of hemicelluloses and lignin would
alter the average DP value of cellulose. The two most com-
monly used techniques to measure the DP of cellulose are
the viscometry and the gel-permeation chromatography
(GPC) methods. The determination of the DP of cellulose
begins with its dissolution using a method that does not
significantly affect its original DP. Cellulose is insoluble in
all known common solvents because of its high molecular
weight, intra- and inter-molecular hydrogen bonding, and
the resulting high crystallinity.53 The two most practiced
techniques to dissolve cellulose are: (i) dissolving cellu-
lose in metal complex solutions; or (ii) forming cellulose
derivatives by nitration or tricarbanilation. Metal complex
217
BB Hallac, AJ Ragauskas Review: Degree of polymerization of cellulose

solutions such as Cuam, Cuen, and Cadoxen have been curve, which is then used to obtain the molecular weight of
used as media to dissolve cellulose. The Cuam solution is a CTC. Despite the differences, researchers have used both
copper complex with ammonia; the Cuen solution is cop- methods to measure the DP of native woody and non-woody
per complex with ethylenediamine that is also known as cellulose.
cupriethylenediamine; and Cadoxen is a cadmium complex Measuring the DP of cellulose viscometrically after nitra-
with ethylenediamine.54 All these solutions have been used tion was a technique developed in the 1940s, in which
for measuring the DP of cellulose either viscosimetrically extractive-free wood meal is treated in a mixture of nitric
or by using GPC;52,54 however, the cupriethylenediamine acid, phosphoric acid, and phosphorous pentoxide in the
solution has been frequently used with the viscosimetric weight ratio of 64:26:10 at 17C for ~40 h, resulting in the
technique.11,24,27,48,50,5557 isolation of cellulose nitrates, which is then solubilized in
DP can be defined in terms of the number average (DPN), either acetone or ethyl acetate.5961 In the 1950s, T. E. Timell
weight average DP (DPW), or viscosity average DP (DPV) extensively investigated this technique and determined the
(Eqns 13).19 DP of several native wood celluloses.6062 A list of the DP
Mn N i Mi of cellulose determined by nitration followed by measure-
DPN = = / MWglu (1)
MWglu Ni ment of viscosity is provided in Table 1.28,5962 An advantage
of this method is that it does not require pre-isolation of
Mw N i Mi2 cellulose through the holocellulose pulping and the base-
DPW = = / MWglu (2)
MWglu Ni catalyzed hydrolysis of the hemicellulose. The DP values
ranged from ~9255500. Table 1 shows that agricultural res-
MV Ni ! idues, such as bagasse and wheatstraw, have lower cellulose
DPV = = / MWglu , where ! = Km Mi"+1 (3)
MWglu Ni DP (~1000) than hardwoods and soft woods, which possess
higher DP cellulose in the range of 40005500.
where Ni is the number of moles of a given fraction i hav-
Today the nitration methodology is rarely used due, in
ing molar mass Mi, MN is the number-average molecular
part, to the uncertainty arising from possible acid hydrolysis
weight, MW is the weight average molecular weight, MV is
of the cellulose chain during derivatization as well as the
the viscosity-average molecular weight, MWglu is the molec-
instability of the derivative.52 The acid hydrolysis of cellulose
ular weight of anhydroglucose (162 g/mol), is viscosity, K m
by nitric and phosphoric acids is more dramatic when deri-
is a constant, and the value of for cellulose and cellulose
vatizing pure cellulose (i.e. without the presence of lignin
derivatives in most cases ranges from 0.751.19 Viscometry
or hemicellulose). For instance, the DP of cotton cellulose
measurements are relatively quick and convenient and are
used to measure the DPV of cellulose.58 However, it has Table 1. DP of native wood and non-woody
three limitations: (i) it provides only M V, which is not an celluloses after nitration using the viscometric
absolute average since it depends of the solvent/temperature method.28,5962
conditions; (ii) it provides no information concerning the Species DP
molar mass distribution (MMD); and (iii) the complex metal Trembling aspen 5000
Beech 4050
solutions used along with the method can degrade cellu- Red maple 4450
lose.58 On the other hand, GPC provides DPn, DPw, all three Eastern white cedar 4250
Eastern hemlock 3900
molecular weights (MN, MW, and MV), as well as the MMD. Jack pine 5000
Similar to viscometry, GPC does not give the absolute molar Tamarack 4350
White spruce 4000
mass because it is calculated based on the molecular weight Balsam fir 4400
of a set of standards which is frequently a set of well-defined White birch 5500
Eucalyptus regnans 1510
polystyrene standards with varying molecular weights.
Pinus radiata 3063
A certain range of standards are used, depending on the Bagasse 925
nature of the analysis, in order to construct a calibration Wheatstraw 1045

218 2011 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. 5:215225 (2011); DOI: 10.1002/bbb
Review: Degree of polymerization of cellulose BB Hallac, AJ Ragauskas

measured by a viscometer after nitration was reported to TAPPI T 230 om-08 method, 0.2500 g of dry cellulose is
61
be equal to 4700. However, it is well known that the DP added to 25.00 mL of distilled water and 25.00 mL of cupri-
of cotton is ~10,000,5 suggesting that significant hydrolysis ethylenediamine solution, with continuous nitrogen flush-
occurred to the cellulose chains in cotton during derivati- ing. Shaking and heating might be required to solubilize
zation. The viscometric average DP of aspen cellulose was cellulose. The intrinsic viscosity is then measured for the
determined to be 4581 after isolation via sodium chlorite resulting homogeneous solution.
pulping and NaOH hydrolysis.50 This DP is comparable DP values of several native woody and non-woody cel-
with that of aspen cellulose nitrate (DP = 5000); thus, the lulose samples are listed in Table 2. It appears from the data
numbers in Table 1 appear to be reasonable DP values for in Table 2 that viscometry is more popular in determining
native wood celluloses.61 Furthermore, in cotton, a DP of DP of cellulose in lignocellulosic biomass. In fact, Kumar
10000 to 15 000 for secondary wall cellulose and DP of 2500 et al. stated that the viscometry technique is more adequate
to 4000 and 250 to 500 for primary cell wall cellulose were to analyze cellulose DP, given the complexity of lignocel-
reported.63,64 However, primary cell walls contain lower cel- lulosic biomass.11 The cellulose DP range is ~15004500.
lulose (2025%) than secondary cell walls, which contain Hardwoods, such as poplar and aspen, have a cellulose DP
>95% cellulose.65 of 3500 and 4500, respectively. Sweet et al. reported a DP
Cellulose tricarbanilate (CTC) is another cellulose of 1450 for southern pine, which is significantly less than
derivative for DP measurement, which is usually done by the DP values reported for hardwoods. Agricultural resi-
GPC. 52,56,66 CTC is the most utilized derivative for GPC dues varied over a range of 18004000. Cellulose in jute
studies due to following advantages: (i) complete substitu- fiber has a large DP value when compared to corn kernels,
tion; (ii) no depolymerization occurs during derivatization; cotton stalks, wheatstraw, and rice straw. DP of Nalita cel-
(iii) stability of the derivative; (iv) solubility and stability in lulose increased from ~3200 to 3600 as the tree became
THF. 52 Cellulose tricarbanilation is commonly performed older (12 months to 30 months), suggesting that cellulose DP
by reaction of cellulose with phenyl isocyanate in either increases as the tree develops.50
dimethylsulfoxide (DMSO) or pyridine as the solvents.
However, it has been shown that cellulose oxidation and Level-off DP
degradation occur during derivatization of cellulose in the When cellulose is subjected to acid hydrolysis, the DP
67
presence of DMSO but not in the presence of pyridine. decreases rapidly until it reaches the so-called leveling-off
Therefore, pyridine is most commonly used solvent for the
derivatization, since it is important not to detrimentally
affect its chain length during the isolation and prepara- Table 2. DP of native wood and non-woody
celluloses.11,48,49,50,57
tion of samples. Typically, dried cellulose sample (15 mg)
Species Measurement Technique DP
is derivatized by adding anhydrous pyridine (4.00 mL)
Southern Pine CTC and GPC 1450
and phenyl isocyanate (0.50 mL, 4.62 10 3 moles), and DDGS Cuen and Viscometrya 2243
Corn kernels Cuen and Viscometry 1693
kept at 65 C with stirring until the cellulose is completely
Dhaincha Cuen and Viscometry 2520
dissolved. Afterwards, methanol (1.00 mL) is added to Cotton stalks Cuen and Viscometry 1820
the reaction mixture to eliminate the unreacted phenyl Jute fiber Cuen and Viscometry 3875
Wheatstraw Cuen and Viscometry 2660
isocyanate. The mixture is then poured into a 3:7 water- Rice straw Cuen and Viscometry 1820
methanol mixture (100 mL) to precipitate the cellulose Corn stover Cuen and Viscometry 2520
Poplar Cuen and Viscometry 3500
tricarbanilate. The derivatized cellulose is fi nally purified Aspen Cuen and Viscometry 4581
by repeated washing with water-methanol (3 100 mL) Nalita (12 months) Cuen and Viscometry 3181
Nalita (18 months) Cuen and Viscometry 3383
followed by water (2 100 mL).9,56 Nalita (24 months) Cuen and Viscometry 3518
The viscometry technique involves dissolving cellulose Nalita (30 months) Cuen and Viscometry 3611
a
in 0.5 M cupriethylenediamine solution. According to the Viscometry technique gives DPv values according to equation 3.

2011 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. 5:215225 (2011); DOI: 10.1002/bbb 219
BB Hallac, AJ Ragauskas Review: Degree of polymerization of cellulose

Table 3. Typical level-off DP of some cellulose Table 4. DP of Cellulose after pre-treatment.

materials.69 Biomass Pre-treatment DPa
a
Form of cellulose LODP Corn Stover 11
AFEX 6800
Ramie 350300 ARP 4500
Cotton 250200 Controlled pH 5700
Unbleached sulfite wood pulps 400250 Dilute Acid 2700
Bleached sulfite pulps 280200 Lime 3100
Bleached sulfate wood pulps 190140 SO2 3000
a Poplar11 AFEX 2600
Based on hydrolysis in 2.50 N HCl at 105C for 15 min.
ARP 3100
Controlled pH 1800
Lime 1500
or limiting DP.68 The DP of a cellulose sample is rapidly SO2 500
Bagasse28 O3 800
reduced to a relatively constant value upon being subjected CE 572
to severe hydrolyzing treatments under conditions that do AE 550
Wheatstraw28,71 O3 908
not produce humic substances.69 The resulting cellulose CE 698
fragments will have the maximum length that will allow AE 662
Organosolv (acetic acid) 1594
them to dissolve in the particular hydrolyzing medium
Organosolv (formic and acetic acid) 2182
used.69 The initial rapid DP degradation phase corresponds Organosolv (methanol) 1519
Organosolv (ethanol) 1356
to the hydrolysis of the reactive amorphous region of cel-
Eucalyptus regnans28 O3 1065
lulose; while the slower plateau rate phase corresponds to CE 815
the hydrolysis of the slowly reacting crystalline fraction of Pinus radiata28 O3 2900
Spruce:Pine Dilute Acid 200
cellulose.68 The acid hydrolysis conditions (acid concentra- (50:50)27
tion, temperature, and time) employed are dependent on a
DP was measured using either nitration or Cuen and viscometry
technique. Viscometry was used on corn stover, poplar, and wheat-
the nature of the starting cellulose material.70 Table 3 sum-
straw organosolv studies, while nitration was used on the remaining
marizes typical LODP values associated with the cellulose biomass.
materials as well as the hydrolysis conditions used.69

stover cellulose decreased by ~60% (from ~7200 to ~2800)

Effect of pre-treatment on DP of cellulose
In the realm of cellulosic ethanol, studies have been reported
to examine the effect of pre-treatment on DP of cellulose,
which in turn affects the efficiency of the downstream enzy-
matic deconstruction step. Table 4 summarizes the DP of
cellulose of various biomass after some pre-treatment proc-
esses, such as AFEX, ammonia recycled percolation (ARP),
controlled pH, dilute sulfuric acid, lime, SO2, ozone (O3),
carbon dioxide explosion (CE), alkaline explosion (AE), and
organosolv.
As to be expected, each pre-treatment has a different effect
on the DP of cellulose and the extent of depolymerization
that occurs. As discussed previously, a benefit of pre-treating
biomass is the reduction of the DP of cellulose without
extensive degradation providing more reducing ends for
the enzymatic hydrolysis.19 Low pH pre-treatments (i.e.
dilute acid and SO2) cause more cellulose depolymerization
than the AFEX and ARP pre-treatments.11 The DP of corn
after dilute acid and SO2 pre-treatments, while alkaline-
based pre-treatments caused ~6% (AFEX) and ~38% (ARP)
reduction in DP.11 A similar effect occurred to poplar cel-
lulose, where the DP of cellulose was reduced by ~86% and
~20% after acidic and basic pre-treatments, respectively.11
For the mixture of spruce and pine (50:50), as the dilute
acid pre-treatment severity increased, the cellulose DP
decreased from 700 until it reached the leveling-off value
of 200.27 Furthermore, for ozone (O3) pre-treated samples,
the decrease in DP was less than that in the carbon diox-
ide explosion (CE) and alkaline explosion (AE) pre-treated
samples. For example, the DP of bagasse cellulose decreased
from 925 to 800, 572, and 550 after O3, CE, and AE pre-
treatments, respectively.28
For organosolv pre-treatment, in which the biomass was
pre-treated in a mixture of organic solvents, water, and
HCl as catalyst, the DP of cellulose varied across the dif-
ferent organic solvents used.71 Ethanol gave the lowest DP;

220 2011 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. 5:215225 (2011); DOI: 10.1002/bbb
Review: Degree of polymerization of cellulose
methanol and acetic acid gave comparable DPs; and formic/
acetic acid system had the least cellulose depolymerization.
The organosolv pre-treatments were carried out under dif-
ferent reaction conditions as shown in Table 5.71 Ethanol
organosolv pre-treatment caused more depolymerization of
cellulose than methanol, formic acid, and acetic acid. The
increase in organic solvent preserved the reduction in DP of
cellulose as shown in the higher DP values in samples 2 and
3 compared to 1, as well as the higher DP of sample 5 com-
pared to sample 4.
The chemical and physical changes that occur to the bio-
mass from pre-treatment are what dictate efficient enzymatic
digestibility. Such changes include decreasing the DP of
cellulose, lignin content, hemicellulose content, lignin-
carbohydrate complexes, and cellulose accessibility.72 It
is difficult to assess the effect of these factors individually
since more than one change usually occurs during pre-
treatment. Therefore, these collective contributions provide
more enzymatically digestible cellulose. Lower DP improves
enzymatic hydrolysis due to two factors: (i) increasing the
number of cellulose chain reducing ends; and (ii) making
cellulose more reactive to the enzymes.42 As the DP of cel-
lulose decreases, the number of reducing ends of cellulose
increases, thus allowing for more exoglucanase effective
activity.24,26 Vljame et al. showed that increasing the
number of endoglucanase-generated chain ends from
0 mol.g1 to 6 mol.g1 in bacterial cellulose, enhanced the
exoglucanase hydrolysis of cellulose, which resulted in an
increase in the amount of solublized total sugars from
0.1 mM to 0.25 mM.73 Furthermore, shorter chains allow
cellulose to be more amenable to enzymatic deconstruction
Table 5. Change in DP of wheatstraw cellulose
after various organosolv pre-treatment.a71
Sample # Solvents used Solvents ratio
(%v/v)
1 Acetic acid-water 65/35
2 Acetic acid-water 80/20
3 Acetic acid-water 90/10
4 Formic acid-acetic 20/60/20
acid-water
5 Formic acid-acetic 30/60/10
acid-water
6 Methanol-water 60/40
7 Ethanol-water 60/40
a
Performed using 0.1% HCl as a catalyst at 85C for 4 h with a
liquor-to-solid ratio of 20:1 (mL/g).
2011 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. 5:215225 (2011); DOI: 10.1002/bbb
BB Hallac, AJ Ragauskas
because they do not form strong hydrogen bonding (i.e.
they form weaker networks permitting greater possibility
for enzyme access.24,26 Depending on the pre-treatment
technology used, the resulting pretreated lignocellulosic sub-
strate will contain different amount of reducing chain ends.
For example, solids resulting from low pH pre-treatments
(dilute acid and SO2,) have higher reducing ends than high
pH pre-treatments (AFEX and lime).11
The study by Puri showed that the enzymatic saccharifica-
tion increased with reduction in cellulose DP.28 For instance,
the extent of enzyme saccharification increased from 28%
for the untreated bagasse to 86%, 78%, and 85% after O3, CE,
and AE pre-treatment, respectively. The mixture of spruce
and pine samples also showed higher enzymatic saccha-
rification as the cellulose DP decreased.27 At DP 200, 40%
saccharification was achieved as compared to 5% saccharifi-
cation at DP 400.27 Although changes in the structure of cel-
lulose undoubtedly contributed to the reduced recalcitrance
observed for these pre-treated biomass samples, additional
changes in lignin and hemicellulose also contributed as well;
and thus future research is needed to determine their rela-
tive importance.
Light scattering measurements
of cellulose DP
In order to obtain absolute molecular weight of cellulose
and thus DP, light scattering techniques are usually applied.
Size exclusion chromatography (SEC) coupled with a multi-
angle laser light scattering (MALLS) is the system typically
used for the light scattering measurement. The mobile phase
and the solvent used to dissolve cellulose is either lithium
chloride/N,N-dimethylacetamide (LiCl/DMAc) or lithium
chloride/1,3-dimethyl-2-imidazolidinone (LiCl/DMI). In
DP such system, no cellulose derivatization is required. The
former solvent system has few disadvantages that led to
1594 the development of the latter solvent system.74 LiCl/DMAc
1763
1952 causes (i) aggregation of cellulose in the solution, which is
2182 dependent on the concentration of cellulose or LiCl;75,76 (ii)
2289 incomplete dissolution of certain cellulose samples, such as
tunicate cellulose77 and soft wood bleached kraft pulps;78 and
1519
1356 (iii) detrimental degradation of cellulose upon heating dur-
ing dissolution in LiCl/DMAc.79 On the other hand, LiCl/
DMI has shown superior advantages over LiCl/DMAc, such
221
BB Hallac, AJ Ragauskas Review: Degree of polymerization of cellulose

as stability of cellulose and solubility of tunicate cellulose
and kraft pulps.74,80
SEC-MALLS analysis has been mostly performed on
cellulose-rich samples, because dissolving native wood is
still difficult to achieve.80 Table 6 summarizes some of the
samples analyzed by SEC-MALLS with their respective DP
values.8082 The samples were dissolved in 8% LiCl/DMI. As
shown in Table 6, the samples include native cellulose, such
as cotton lint cellulose, algal cellulose, and bacterial cellu-
lose, and a variety of bleached and unbleached pulps (kraft
or sulfite).
Tencel is regenerated cellulose fiber prepared from dis-
solving wood pulp using the N-methylmorpholine-N-oxide
system, while Bemliese is regenerated cellulose fiber pre-
pared from cotton linters using the cuprammonium solvent
system.82 Tencel and Bemliese were mercerized (M) (alkali-
treated) with 20% NaOH, and acid hydrolyzed in 1 M HCl at
105C for 3 h (i.e. H-M-Tencel and H-M-Bemliese).82
Future development
One way the field of cellulose DP analysis can develop is
through providing a process to readily fractionate biomass
so that cellulose can be extracted, dissolved, and analyzed
for DP values. Ionic liquids (ILs) have shown potential for
such application. Many studies have demonstrated the abil-
ity of ILs to dissolve cellulose without significantly altering
Table 6. DP of several cellulose-rich samples
after dissolution in LiCl/DMI using the SEC-
MALLS method.8082
Species
Bleached SW kraft pulp
Bleached HW kraft pulp
Bleached spruce sulfite pulp
Cotton lint cellulose
Algal cellulose
Bacterial cellulose
Unbleached SW kraft pulp
Oxygen-bleached SW kraft pulp
Chlorine-bleached SW kraft pulp
Alkali-extracted SW kraft pulp
ClO2-bleached SW kraft pulp
Unbleached SW sulfite pulp
Unbleached HW kraft pulp
H-M cotton linter
H-Tencel
H-Bemliese
H-M-Tencel
H-M-Bemliese
its chain lengths (DP). ILs, such as 1-butyl-3-methylimida-
zolium chloride ([bmim] Cl), 1-allyl-3-methylimidazolium
chloride ([amim] Cl), and 1-ethyl-3-methylimidazolium
acetate ([emim] Ac), have been used to dissolve pulps (DP =
650 and 1000), and cotton linters (DP = 1600).8386 Moreover,
few studies have reported the solubility of several lignocel-
lulosic biomass in ILs, including spruce, silver fir, beech,
chestnut, pine, poplar, eucalyptus, and oak.8688 Typically,
wood sawdust/powders/shavings (0.15 mm) are suspended
in IL, heated at about 80120C for ~624 h under constant
stirring.8688 The dissolved cellulose is then regenerated by
precipitating it in water. The resulting cellulose is virtually
free of lignin and hemicelluloses. In such methodology,
there will be no need for delignification and derivatization of
cellulose and its application to molecular weight determina-
tion of cellulose is being rapidly pursued by several groups.
This advantage can allow for rapid analysis of cellulosic
biomass and further understanding of the plant cell wall
polymers. As mentioned earlier, concerns have been raised
about the acid-chlorite delignification step as cellulose might
be degrading during this process. Eliminating sample prepa-
ration steps is usually favorable; thus using ILs requires no
delignification or base-catalyzed hydrolysis of hemicelluloses.
Furthermore, the ability of ILs to fractionate biomass to its
major three biopolymers (lignin, cellulose, and hemicel-
luloses) permits more analyses to be carried out in the same
sample preparation procedure. Besides determining DP of
cellulose after dissolution in ILs, a recent study has shown
that lignin content in hardwood and softwood can also be
measured via UV-Vis spectroscopy (at 400 nm) after dissolu-
DP tion of biomass in [bmim] Cl IL.89 In addition, another study
4500
reported the ability for direct dissolution and NMR analysis of
3000
3000 plant cell walls polymers using perdeuterated pyridinium IL
2600
and DMSO-d6.90 Such ILs include: 1-allylpyridinium chloride
4300
7300 ([Apyr] Cl), cyanomethylpyridinium chloride ([Cmpyr] Cl),
3700 and pyridinium chloride ([Hpyr] Cl).90 In all likelihood, as
3140
2510 our knowledge of ionic liquids grows, especially in regards to
3170 the solubilization of biomass, these new solvents will assume
2300
1640 greater importance for DP analysis of cellulosic biomass.
1590
64
44 Conclusions
35
46 Determining the DP of cellulose has been the focus of
41 many researchers over the years. The current interest in

222 2011 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. 5:215225 (2011); DOI: 10.1002/bbb
Review: Degree of polymerization of cellulose
determining cellulose DP is to correlate the relationship
between changes in DP during pre-treatment technologies
to the efficiency of the enzymatic hydrolysis in the process of
converting cellulosic biomass to biofuels. The general con-
sensus is that shorter cellulose chains (low DP) can be more
readily hydrolyzed enzymatically. The key parameter in the
methods to determine cellulose DP is that the isolation and
derivatization/dissolution techniques must not significantly
alter the native DP of cellulose. Viscometry and GPC are
the two commonly used techniques to measure DP of cel-
lulose. Viscometry provides the viscosity average DP (DPV),
while GPC gives both the number average (DPN) and weight
average DP (DPW). For detailed analysis on the molecular
weight/DP of cellulose, GPC is the technique to use because
it allows analyzing the distribution of the molecular weights
and acquiring more insights on the nature/length of the
cellulose chains. However, given the complex structure of
lignocellulosic materials, it has been recommended to use
viscometry. In conclusion, the need to find a DP measure-
ment technique that can minimize alteration of the native
structure of cellulose is a major future need in the field of
cellulosic ethanol. The technique should be able to fraction-
ate biomass so that cellulose can be extracted, dissolved, and
analyzed for DP values without the need to derivatize it.
Acknowledgements
The authors would like to acknowledge the fi nancial support
from the PSE Fellowship program at IPST@GT. Th is work is
part of the first authors requirements for the degree of PhD
at Georgia Institute of Technology.
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Bassem Hallac
Bassem Hallac is a PhD candidate in the
School of Chemistry and Biochemistry at
Georgia Institute of Technology. His research
is focused on fundamentally understanding
the biochemical conversion of lignocellulosic
biomass to sugars, specifically the structural
transformations that occur to the plant cell
wall biopolymers during pre-treatment.
Art Ragauskas
Art Ragauskas held the first Fulbright Chair
in Alternative Energy and is a Fellow of the
International Academy of Wood Science and
TAPPI. His research program at GA Tech is
seeking to understand and exploit innovative
applications for natures premiere renew-
able biopolymers for biomaterials, biofuels,
biopower, and biobased chemicals.
225