Characterization of chitosan.

Influence
of ionic strength and degree of
acetylation on chain expansion
M a r g d t e Rimdo, Miche1 Mnas and Pbam Se Dung
Centre h Rechevches mr le.9 Macrornolhles VtigPtaleJ, CNRS, C L I S U Ch~ I'Uniwrsi~PJ m p h Fourier,
BP 53 X,38041 G r m b b Cedex, F m c e
(Received 13 February 1993; rwkd 20 April 1995)
Thk paper c w w m a new mihad of cbructerirkg chitosm with dferent &gree.t of ucetylatfan (DA = 2,
11.5 md 21%); it mmt~tsOf st& w h c y i ~ nchnwM~ographyusing a rnu/tidetection t O S I m n i al!mhg
&fermimtion of M,[:?;Ia d RG for each point 4 rk chromaragrams.From these rlatu, the Mark-Hawink
p a r m r e r s for intrinsic vtrmf~ymad the expownt whrlyg & a d M me determhd. In the s u m way, !he
role of rhe w h n t iP & W a n d the solwnt AcOH 0.3 M / A ~ 0.2 N M~ lapropwd to r e k tbe premre
ofwgrqutes which perttubs ~raricmoleculur weighr &termination by light scaftertg. The rde of rhe bnic
mncenimrion md of the degree of ~ceiyhrl~ln on the srgness of chitosun i s then disnwed and a theowtical
marmemt 13 proposed aIiowiRg the cbetexrn~wtionof rhe intrinsic persirrenue length (Lp= 50 11) d the
predicliwl of thedimensionsin s n h i h (RGand [tj]). I,t r f d t o be h&pen&ni of DA D1 the r q e iesfed.

IntrorFucdon ex+ as 100 DA) v a n determined br 'H nm.r.,

Chitin md chitman are imprtant polpaccharid~
extracted from different sources (crabs or shrimp shells,
fungi, etc.); these polymers are composed of ffl - 4
D-glueosarnine units with a variable degree of N-
acetylation (DA)'. The distribution of the N-wtyl
considered to be the most sensitive method ;the samplw
were dissolved in D,O after two exchangesin the presence
of CD,-CO,D, The D A was determined from the
integral of the CH, signal at t .97 ppm compared with
the integral of the H-P proton signal considered as an

-
groups along th t polymeric back bone may mtrol the
solubility in a given solvent; chitin (DA I ) is e s p i d l y
soluble in dimethylacetamide-litbium chloride mix-
tures2. When the average DA is lower than approxi-
mately 0.5, the polymers are called chitosans and they
become soluble in aqueous solutions in the presence of
acids such as acetic acid. In fact, chitosan strictly refers
to poly(g1ucosamint) but it is also a family name for
a group of partially deacetyhtd products3.
To characterize the behariour of chitosans in aqueous
solutions and especially their rheology, it is important
to determine their molecular weight and test for the
thmcdynamic properties of the solvent used. Their
behaviour is also controlled by their persistence length
and the electrostatic interactions.
This paper concerns the conditions d solubilization d
chitosans with diflcrmt D A , their molecular weight
distribution and the discussion on their Iccal fiiffness.
The stiffness is characterized by a persistence length,
dewding on tltctrostatic forces, deduced from radrus
of gyration and intrinsic viscosity.
internal standard.
The vjscosi~ymeasurements were prformed with an
Ubbelohde capillary (4 = 0.58 mm) at 15'C f 0.1;two
series of electrolyte solutions were used with different salt
concentrations to screen progressively the electrostatic
interactions. A low s h w viscomcttr (LS30Contraves)
was also u d so test the effect of the shear rate on
viscosity measurements.
The molecular weight distribution and sabsqutnt
characteristics were obtained ftom gel permeation
chromatography (g.p.c. or s.e.c.) on our equipment using
a rnultidesection instrunent (LALLS, refractive index,
viscosity) as descrihd More6, which was also connected
to a muitiangie light scattering detector (MALLS)Dawn
from Wyatt Technology (USA) on line. This equipment
allowed us to determine R, and [qj for each molecular
weight prestnt in the sample.
The dn/dc of chitosan in the solvent usad for g.p.c.
(0.3 M AcOH + 0.2 M AcONa) was found to bt
independent ofDA and equal to 0.163. The columns used
lor chromatagraphy were the Shockx OHpaK B 803 and
805: a11 the solutions were filtered on 0.2 urn Dore
~a&ius membranes before experiments; in sime &es,
0.1 and 0.05 pm membranes were used.

The commercial samples of chitosan were from Fiuka Results and diPrmscin
IDA = 1 1 .S%) and Protan (DA = 21%); a nearly fully
deacetrlated chitosan (DA = 2 %) was prepared in our Theorericu' w~~~~~
laboratory4.Their average degree of acetylation (DA% Whm the chitown, isolated in neutral conditions (i.e.

Int. J. Biol. Mamrn01., 1993, VoL IS, October 281

when glucosamine is in the -NH, form) is dissolved equation:
in acidic conditions, the following equilibrium occurs:

The counterion depends on the electrolyte prtsent in the
medium and on the acid used. Nevertheless, dl the
experimental results, when exp& in the -NH, form,
remain independent of the counterion. Therefore,
chitosan in acidic wnditions is a polyelectmlyte whose
charge parameter, 1,is proportional to the linear charge
density which depends on pH and degree of deacetylation';
in this paper, the degree of protonation is considered to be
nearly 1 as the pH is always lower than4.5. lt becomes;
1= 1.38(1 - DA) (1)
AH the properties and espacialIy the dimensions ofthese
charged polysaccharides a n be predicted using the
treatment described previouslys.
Considering the values of R, as a function of the
molecular weight obtained on a g.p.c. chromatogram, it
is possible to extract the persistence length following the
Bwoit-Doty relationg:
where L is the contour kngth of the n r o ~ eLf ; is the
total persistmcc length taking into account the
electrostatic contribution L, (L - L, -1- L,;L, is the
intrinsic p~sistencclength); &;is the clcnmstatis
expansion coefficient assuming that in a moderate
molecular weight range the e l m a s t a t i c contribution
dominates. <,, is calculated at constant d al conoentra-
t
tion For different values of the molecular weight M and of
L,, fo1lowing rhe methods of Yamakawa and Tanakala,
Odijk and Houwaart" and Fixmm and Sk~lnick'~.
Then & is calcu1atad for different L values and the
kt agrecmcnt with the experimental. &a gives L, f o t
the polymer investigated.Therefore, with the L, adopted,
one dedum from the relation (21 that
with a,,the e x p s i o n coefficient ealmlatad from a,,.,. We
can demonstrate that nhe right-hand side of equation (6)
varies as C; lt2.
From the slop of the curves [vb(C,P1ll)OM can a h
deduce the penisknee length L,: different r d u a of L,
are imposed to calculate the ratio [qlr/[~lr and then [qIz
as a function of C;I1'; the k t agreement betweerr
experimental and theomtical values gives L,.
For different valnts oiM, one can deduce theoretically
from equations (4) and (6) the parameters of the
Mark-Houwink equation for a given salt concentration
having fixed the L, value.
The calculated values are plotted in Figures I and 2
For the relations [q] ( M )and &(M);the role of the salt
concentration (equation (6)) is given in F w e 3. T h e
parameters used for the calculation are given in Table 1.
Mokcubr characte~ticsof chirosan
A sc- solvent composed 010.2 M AcONa and 0.3 M
AcOH was found to be suitable to give g m d elution in
B.P.c., avoiding aggregation as mvch as possible, as
shown separately by dynmic light m k n g . This was
tested by the elution of a given wdght of material filteMd
on mcmbrants with difkrent porosities down to 0.05 jm
pore diameters; the refractometric area of the chromato-
graphic signal remains unchanged.
Analysis of g.ps. data allowed us to determine the
molecular weight distribution, the average molecular
weights, Bwand !&, and the average intrinsic viscosity
[q] (Table 2). Analysis of the chromatogams allowed us
to obtain the dependence of l r p ] and RG with the

From the intrinsic v i d t y [q] of -h mph

extrapoIated at infinite d t m-tmtion [q],, the
Yarnabawa-Fujii treatment allows us to also calculate
L,"; assuming that the long range dmostatic
interactions arc screened ( i t * [q], r [q Jabwe use the
relation valid in 8 conditions:

-
where ML is the mass p r unit length, L, L/&,
d, d/2L,, C and d are the contour length and the
-
diameter of the chain and # is a canstant depending on
draining tffact. The best fit between experimental values.
[qImand [qjilecalculatd for each set of M and L, allows
us to choose L,.
The experimental 6epcndence of[q] as a function d
thc ionic strength usually followr the relation:
Id, = C ~ l m+ SC*-"l (5)
Fiprt 1 Dependence of the intrinsic v i d y with Ute
The slope S of the linear depndence between [qj, and molecular weight in log-log plot for ditfermt average dtgrccs
C; (with C, the molar salt concentration) is related of acttylation. Fht stmight linm cornpond to the calculated
to the stiffness of the rnolac~le'~. valucs: (a) 2%; (b) J 1.5 %; (c) 2 1% (equations(4) and (6) wilth
Using Odijk's treatment1s, the intrinsic viscosity For a L, = MA). The experimental v d w arc obtaincd from analysis:
given ionic strength can be related to [q], by the of the g.p.c. chromatograms

#n Int. J. Biol. Macromol, 1993, VoL 15, &tobet

moloeulat weight M ; w t then deduced the Mark-
~ o u w i n kparameters K and a { [ q j = K M a ) and the
~xponentv of the radius of gyration as a function of
molecular weight IR, -- M'). The experimental data are
given in Table 3 and Figures E and 2.
From Figure 1 , it can bt seen that K depends on the
DA in the range tested (2 to 21%) but that a m a i n s
marly constant. The experimental values are compared
with the values calculated as discussed previously using
L, determined from CvJm (Lp= 50 A). The agreement is
F I p 2 Dqmmhcc of the radius of gyration with thc
molecular weight in log-log plot for memt aveFage degrees
of acetylation. The straight lines cormpond to the calculatsd
values: a) 2%; (b) 11.5%; (c) 21% (equation (2) with
L, = 504! ).The expwiratntal valuaare obtained from analysis
of the ~ p - cch-atograms
.
Table 1 Charactahtiesof tkchitwrans u s 4 in thecalmladon
M m,
D A (%) 1 (-NH,form) (-Maform)
11.5 1.22 290000 166
21 1.09 225 000 170
Monomericunit length b
~=161+42DDA.
- 5.15 A; rnass ofthe monomeric unit
Tnbk 2 Characteristi= of the chitosatw obtained hrrm s.e.c.
'Obtained in AcONi/AcOH 0.3 M as solvmt
bObtaincdin NaCI/AcOH 0.1 hl as d v m t
relatively good, at kast in the intermediate mokcular
weight range. Most Importantly, a variation of DA
directly Impases a change in K indeptndmt of other
Factors.
From the Mark-Houwink equation and value&of [q]
obtained in static experiment, it is possible to determine
the vjscometric a m g e molecular weight A&. The vducs
of M, obtained in this solvent are compared in Table 4
with those obtained using the quation adopttd by
Roberts16 in the comsponding solvent NaCl 0 . 2 ~ /
AcOH 0.1 M:
in which the parameters K d a are complettly diffmnt
from that obtained from g.p.c. and arc considered
independent of DA.
It is demonstrated, in particular, that M, values
obtained using the Roberts equation are overestimated;
this may be attributed to the existence of hrge aggregates
due to the thermdynamic quality of this solvent. This
was proved by light scattering and filtration on calibrated
membran~.
The ammat is better whta the equation p r o m
by Lee3 is considered assuming that tbe thermodynamic
properties; of this solvent (0.2M AcOH, 4 M urea, 0.1 M
NaCl) are as good as those adopted in our work.
Nevertheless the K and a parameters were determined
on the basis of M, determined by sedimentation
equilibrium on polydisptrsed samples. It is important to
redetermine [q] in Lee's solvent using monodispersed
samples.
In Figrrre 2, the dependence oT radiw of gymtion of
chitosans is given ss a fundon of the molecular weight
following squations (2) and (3): the calculated valucs for
P
1
rn
-3 Ratio [q]J[q].. rrs a iunction of C;'* i
AcONa(Cs)/AcOH 0.3 M for chitosan DA = 1 15W, Compari-
-
son whh calculated values: (-1
(b)L, 80 A
' (a) L, = $0 A;
and vimmrtry
Fnt. J. Biol. Macromol, 1993, VoL IS, Octobet 283
Chracteri~arionof chitosan: M.R U o et al.
Z, = 50 A agree with the experimental data. l b is shown
that R, depends on W A but the slopes of the curves are
independent of DA. The experimental value (v = 0.54) is
predicted from our m d e l (v = 0.57).
From these new conditions adopted to perform g.pc.,
we svccteded in obtaining a good moleculat solution
avoiding aggregation, at least in the intmed iatc range
of molecular weighis; then a m w sct of Mark-Houwink
parameters were p p o s e d in the solvent AcONa
0.2M/AcQR 0.3 M allowing thedetermination d M ,from
intrinsic viscosity measurements.
We have clearly demonstrated that the solvent NaCl
0.2 M/AcOH 0.1 M is not g o d Tor preparing solutions of
partially deacctylated chitin. It must be pointed out that
the values of [ q ] determined by s.c,c. experiments are
not in agreement with static measurements (compare
Tables 2 and 4) although the first solvents were perfectly
filtered through Be membrane and the porous material
in the column; howtver, the polymer concentration was
then corrected to take into account probable low
retention (lower than 10%) which is seen from the
refractomttric signal. T h e m e quality of molecule
dispersion is almost impossible to obtain with static
measurements.
Influence of the p d y m r and d t concentruliorrs on
o~cosiiy
In Ffgwe 4, the variation of the specific viscosity
(1 = ti - qo/'fo with q, the viscosity of the solwnt) with
Jymer cona~ntrationis drawn as a log-log plot for
DA = 2%. The viscosity in the presence of a salt exass
(AcOH 0.3 M / A ~ O N 0.1~M ) varies as follows:
valid in the Newtonian regime. In the dilute regime,
deviation from Newtonian behaviour was never
observed.
The first two tams of equation (8) cornpond to the
dilute regime dwcribod by the Hugsins relation.
Tht Huggjns constant K was determined (k' = 0.30)
and From the departure from Huggins behaviour we
-
deduced the overlap concentration C*; we found
[q] = 1145 ml g-' and C* 1.75 x 10m3g ml-< i.e.
Tnbk 3 Mark-Houwink ~ ~ t rforachitosans
r in AcONtl
0.2 M/ AcCIH 0.3 M
DA (%) K' d Kmk
2 0.082 0.76 0.076
11.5 0.076 0.76 0.MO
21 0.074 0.76 0.068
*Range 10' < M c 6 x 10' (expimental valuw.sctFlgure I )
Tabk 4 Cornparison of the M, v a l w determined in two solvents using K and o given in Table 3 and literature data
D A (%) Cd'(m1g-l) MV
2 750 163000
11.5 980 256 120
21 560 127025
'Solvtnt AcONa 0.2 w/AcOH 0.3 M (this paper)
hSolvent NaClQ.2M I A ~ O H0.1 M (Ref. 16)
'Relation [q] = 0.0893 x .w+"
[Ref. 3)
28d Int. J. Bfol, MacromoL, 1993*Vol. 15,Octobtr
C[qJ= 2. This value d C*[q] is in the same range as
other polysaccharideslT.The value of k' is lower than
the values obtained by Lce (0.54-0.69)3 and indicates a
btter solubility of the chitosan.
The intrinsic viscosity was then determined in solvents
with different ionic coi~centrationsso as to test the role
al the ionic strength on the electrostatic effects due to
the existence of charged -NH; in acid conditions. The
rcsuhs are given in Figure 5 for the different chitosans
in AcONa(CsC,)/AcOH0.3 M. In this figure, the intrinsic
viscosities [qJa are plotted as a function of C; "'and
usually follow equation (5); extrapolation to infinite
salt content gives [qja; the slope k S.
The intrinsic viscosity was also determind in the
solvent AcOH 0.1 ~ ( N a c 1(variable).T h e values d [v],
for the two series of experiments are compared in Table
2. T h e slopes S are plotted as a function of [q]o.,u, the
intrinsic v i w s i t y in 0.1 M salt concentration, following
Smidsrodt4, in Figure 6. The empirical equation, relating
S to the stiffness parameter B, is:
B - -- S
crlJ;.iu
(9)
F i a m 6 shows the experimental data which lie on ithe
-e stmight line. ~or'thetwo soiventsone gets v = 1.3,
which is the usual value, and B is equal to 0.065 f 0.002,
independent of the degree of aoetylation and of the
solvent. In Smidsrod equations (5) and {9), [tlJ is
expressed in dl g- '.
This means that the stiffne58 of chitosan is a
characteristic of the main chain independent of the DA,
in contrast to the recent conclusions of Wang et al.i8-19.
These authors drew conclusions on the role of DA on
chitosan stiffness only from indirect measurements of [q]
innnn
Ouclld
0.77
0.77
0.77 F t 4 Variation of log p, as a function of the omlap
p a r a v e r .log qtf3 ( x ) and log(CCljj + k'IC[vl)') (ab
Dttermlnatron of C*[q]
[db g- '1 MV
-
.UTO
155 1 104930 336 555
1125 3696770 493410
635 917 380 223030

figure 5 Intrinsic viscosify of chitwm as a furaction of ihe
salt canantration C; in AcOMa(C,)/AcOH 0.3M solvents.
Linear extrapolation to infinite salt content gives [q],
Flgm 6 Repmentation of the dope S (equation (5)) as a
function ofthe intrinvic v i s d t y in 0.1 M salt, m log-log
plot. ( x ) AcOH 0.3 M / A ~ O (C,);
N ~ (A)AcOH 0.1 M/N~CI(C,)
and M on unfractionated samples. Thc value of B
compared with other polymm corresponds to a
persistence length in the range of SO-IOOAI~ in
agreement with out previous conclusion.
Considering the application of Odijk's treatment, the
calculated ratio [qjc/[~Jegives an overestimated value
for L,; we round L, = 80 A (Figure 3). This value is larger
than the L, obtained from [tlJe,[ I J ~ and. ~ Ro.
~ The same
result had been found previously with sodium hyaluro-
nate.B This behaviour seems to indicate that the
calculated expansion c d c i e n t s are overestimated.
Thcse data give a persistence length which is in the
range of that given for cellulose derivativest0 or
polysaccharides, such as ptctins2', a1 or
X
Na-hyaluronate8. The value of L, = 50 found lor
chitosan is in agreement with one of the values (42A)
given ja our previous work, but not with the second one
(300A) also dtduccd in this preliminary work" or the
values found (150400 6 )by Terkojtvich et One
of the reasons for discrepancies may be due to moleedar
weight determination by light scattering on unfraction-
ated samples in aggregating solvents.
Conclusion
This paper gives the first molecular weight diitsibution
of chitosan obtained from compbe analysis using a
multidetection instrument for gel chromatography. From
this analysis, the Mark-Houwink parameters in the 0.3 M
AcOH + 0.2 M AcONa solvent were determined; we also
demonstrated that until now the molecular weights
determined using the experimental K and a parameters
rrom RobertsI6 were largely overestimated.
In addition, the solvent based on 0.1 M AcOR 0.2 M +
NaCl was shown to be a bad solvent, promoting
aggregation which in- the weight average moIecular
weight and alsa, to a lascs extent. the intrinsic viscosity,
when determined in static expcrirnents. This paper
proposes new conditions to characterize chitosan soluble
in an add medium.
From the analysis of the c u m [tf] (ME,RG ( M ) and
[q] (C,), obtained in the absence or aggregates, the
persistenoe length was determined rrnd the intrinsic value
ofthe persistence length L, = 50 A obtained indeptndent
of the degree of acetylation; this value allows coherent
analysis of the di&rent utptrimental results coming from
tight scattering or viscometry. At the same time, this L,
value dlows a good prediction of the values of [ q ] and
R, for different molecular weights or -11 concentrations
and justifies the dependence of the exponents on the
degree of acetylation. This coherence means that the
values of M obtained by g.p.c. are valid.
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