Ind. Eng. Chem. Res.

1997, 36, 3631-3638 3631

Heterogeneous Cross-Linking of Chitosan Gel Beads: Kinetics,

Modeling, and Influence on Cadmium Ion Adsorption Capacity
Tzu-Yang Hsien and Gregory L. Rorrer*
Department of Chemical Engineering, Oregon State University, Corvallis, Oregon 97331

Chitosan, a linear biopolymer of glucosamine residues, selectively adsorbs transition-metal ions

such as cadmium from dilute solution. In order to process chitosan into a more durable form,
a 5 wt % chitosan solution was cast into spherical gel beads of 3 mm diameter and then reacted
with glutaraldehyde at free amine sites to form imine cross-links between linear chitosan chains.
The rate processes of the heterogeneous cross-linking reaction and the effect of cross-linking on
the cadmium ion adsorption capacity were determined. The cross-linking reaction was complete
within 48 h at 27 °C, and the final extent of cross-linking ranged from 0.07 to 2.40 mol of
glutaraldehyde consumed/mol of amine. Heterogeneous cross-linking was modeled as a shrinking
core process where the molecular diffusion of glutaraldehyde through the cross-linked shell of
the gel bead limited the overall rate of glutaraldehyde consumption. The effective diffusion
coefficient of glutaraldehyde through the cross-linked layer was 4.7 × 10-8 cm2/s. The saturation
adsorption capacity of cadmium ions on the cross-linked gel beads exponentially decreased from
250 to 100 mg of Cd/g of chitosan as the extent of cross-linking increased from 0 to 1.3 mol of
glutaraldehyde consumed/mol of amine. At higher extents of cross-linking, the saturation
adsorption capacity remained at 100 mg of Cd/g of chitosan. Highly porous chitosan beads formed
by freeze-drying of cross-linked gel beads had the same cadmium ion adsorption capacity as the
cross-linked gel beads over the same extents of cross-linking.

Introduction
Biopolymers are a promising new class of low-cost
adsorbents for removal of heavy-metal ions from aque-
ous waste streams (Volesky and Holan, 1996). Of
particular interest is chitosan, a linear polysaccharide
of β-1,4-O-glycosyl-linked glucosamine residues (Figure
1). Chitosan is derived from chitin, a major component
of the shells of crustacean organisms and the second
most abundant biopolymer in nature next to cellulose
(Muzzarelli, 1977). The amine group on each glu-
cosamine unit within the chitosan biopolymer chain
serves as a selective binding site for group III transition-
metal ions. The adsorption of heavy-metal ions on
chitosan, including adsorption isotherms, and the se-
lectivity of group III transition-metal ions over groups
I and II alkali/alkaline earth metal ions are well
documented (Inoue et al., 1988; Jha et al., 1988; McKay
et al., 1989; Udaybhaskar et al., 1990; Kawamura et
al., 1993; Rorrer et al., 1993; Hsien and Rorrer, 1995).
Chemical modifications of chitosan have been reported
to enhance transition-metal ion adsorption capacity
(Tong et al., 1991; Kawamura et al., 1993; Inoue et al.,
1995; Guibal et al., 1995). In the attempt to improve
the material properties of chitosan for engineering and
biotechnological applications, several investigators have
developed porous, chemically cross-linked chitosan beads
for the removal of heavy-metal ions from wastewater
(Kawamura et al., 1993; Rorrer et al., 1993, Hsien and
Figure 1. Acylation and glutaraldehyde cross-linking of amine
Rorrer, 1995), enzyme recovery (Yoshida et al., 1994), groups on chitosan.
and drug delivery (Nishimura et al., 1986; Bodmeier et
al., 1989).
bic substructure to the biopolymer (Hirano et al., 1976,
The amine group on each glucosamine residue within 1977; Fuji et al., 1980). This reduces interchain hydro-
the chitosan chain can serve a reactive site for two gen bonding between chitosan chains and modestly
attractive chemical modifications. First, N-acylation improves the adsorption capacity for heavy metal ions
with nonanyl chloride adds a hydrocarbon side chain at low extents of N-acylation (Kurita et al., 1988; Hsien
to the amine group (Figure 1) and imparts a hydropho- and Rorrer, 1995). Second, reductive animation with
glutaraldehyde forms an imine (-CdN-) cross-link
* Corresponding author. Telephone: 541-737-3370. Fax: between linear chitosan chains (Figure 1). cross-linking
541-737-4600. E-mail: rorrerg@ccmail.orst.edu. reduces the solubility of chitosan in aqueous solvents
S0888-5885(97)00157-7 CCC: $14.00 © 1997 American Chemical Society
3632 Ind. Eng. Chem. Res., Vol. 36, No. 9, 1997
at low pH and can improve resistance to chemical
degradation or long-term biological degradation. The
properties of homogeneously cross-linked chitosan have
been studied (Roberts and Taylor, 1989; Thacharodi and
Rao, 1993). However, no quantitative relationship
between the extent of cross-linking and adsorption
capacity for transition-metal ions has been established.
The limited previous work is incomplete and conflict-
ing: Kurita et al. (1986) and Koyama and Taniguchi
(1986) claimed that homogeneous cross-linking of a
chitosan solution optimized copper ion removal at low
extents of cross-linking, whereas Masri et al. (1978)
claimed that heterogenous cross-linking of chitosan
powder reduced heavy-metal ion adsorption capacity.
Our previous work showed that cross-linking of chi-
tosan gel beads with glutaraldehyde followed by freeze-
drying significantly enhanced final material properties.
For example, cross-linking of 3 mm, 7% N-C9 acylated
chitosan gel beads with 250 mM glutaraldehyde fol-
lowed by freeze-drying resulted in a mesoporous mate-
rial with an internal surface area of 224 m2/g. The
cross-linked, freeze-dried chitosan beads were also
insoluble in low-pH environments (Hsien and Rorrer,
1995). Unlike freeze-drying, the cross-linking process
has many variables associated with it. However, no
studies have considered how cross-linking bath param-
eters affect the heterogeneous cross-linking kinetics and
the final extent of cross-linking. Furthermore, although
cross-linking combined with freeze-drying may produce
highly porous beads, it is not known if the freeze-drying
step improves the adsorption capacity for transition-
metal ions.
To address these research needs, this study has four
objectives. The first objective is to determine the
kinetics of the heterogeneous cross-linking of glutaral-
dehyde with the chitosan gel beads by measuring the
glutaraldehyde consumption as a function of time and
process parameters, most importantly the initial molar
ratio of glutaraldehyde in the cross-linking bath to the
amine groups in the chitosan gel bead. The second
objective is to develop a physical and mathematical
model for the heterogeneous cross-linking process in
order to predict the extent of cross-linking as a function
of time, bead properties, and cross-linking bath param-
eters. The third objective is to determine how the extent
of cross-linking affects the saturation adsorption capac-
ity of cadmium ions on both chitosan gel beads and
freeze-dried beads. The fourth objective is to determine
if the high internal surface area of the freeze-dried
beads is required for high adsorption capacity of cad-
mium ions. In all studies, both chitosan beads and 7%
N-C9 acylated chitosan beads are compared.
Materials and Methods
Chitosan Gel Bead Synthesis. Chitosan solution
was prepared by dissolving 3.75 g of chitosan powder
(94% deacetylation; Vanson Chemical Co., Redmond,
WA) into 70 mL of a 0.68 M acetic acid solution. The 5
wt % chitosan solution was mixed at 500 rpm with a
4.5 cm marine blade impeller for 30 min and then mixed
on an orbital shaker at 120 rpm and 25 °C for an
additional 72 h to lower the solution viscosity. The
chitosan solution was cast into spherical beads using
the spinnerette system described by Rorrer et al. (1993).
The newly formed beads were dropped into a precipita-
tion bath containing 400 mL of 2 M NaOH, which
neutralized the acetic acid within the chitosan bead and
thereby coagulated the chitosan to a uniform gel. The
gel beads were mixed in the sodium hydroxide solution
for an additional 4 h to ensure complete coagulation.
The residual acetate concentration in the gel bead was
not measured. However, only 70 g of gel beads were
made per 400 mL of a 2 M NaOH solution so that no
more than 6% of the NaOH in the precipitation bath
was neutralized. The total amine and residual aceta-
mide group content was estimated to be 6.13 mmol/g,
assuming that the chitosan initially consisted only of
94% glucosamine residues (mol wt 161.2) and 6%
residual N-acetylglucosamine residues (mol wt 203.2).
For simplicity, both groups are referred to as unreacted
amine groups.
To prepare chitosan with 7% of the amine groups
acylated as N-C9 amides, 70 mL of a 5 wt % chitosan
solution was acylated with 0.28 g of nonanoyl chloride,
CH3(CH2)7COCl (Aldrich Chemical No. 15,683-3, mol wt
176.7), and then recovered in pyridine and purified as
described by Hsien and Rorrer (1995). The same gel
bead casting procedures were followed, using a 1.0 M
aqueous methanolic sodium hydroxide solution (50% v/v)
as the precipitation bath. The free amine and aceta-
mide group content (YB) of the 7% N-C9 acylated
chitosan was taken as 5.77 mmol/g.
Heterogeneous Cross-Linking of Chitosan Gel
Beads. The chitosan gel beads were cross-linked with
aqueous glutaraldehyde (GA), HOC(CH2)3COH (Aldrich
Chemical No. G400-4, 25 wt % in aqueous solution, bp
101 °C). Gel beads (6.40 g) were mixed with an aqueous
glutaraldehyde dialdehyde solution (9.60 g) within a
sealed 125 mL Erlenmeyer flask on an incubated orbital
shaker at 120 rpm and 27 °C. The initial glutaralde-
hyde concentration in the cross-linking bath ranged
from 12.5 to 500 mM. The cross-linking bath was
sampled periodically after 48 h. The pH of the cross-
linking bath was measured periodically with a pH
electrode.
The glutaraldehyde concentration in the cross-linking
bath was determined by a gas chromatograph (GC).
Specifically, a 1.0 µL aliquot of solution sampled from
the cross-linking bath was injected into a Hewlett-
Packard 5890 GC system equipped with a Hewlett-
Packard HP-FFAP column (10 m × 0.53 mm × 1.0 µm)
and FID detector. The analysis conditions were set at
the following: 150 °C injector, 100 °C column, 155 °C
detector, He carrier gas at 10 mL/min. The retention
time of glutaraldehyde was 2.7 min under these analysis
conditions. The concentration of glutaraldehyde was
quantified by the external standard method. The extent
of glutaraldehyde consumption by cross-linking (XT),
defined as the moles of glutaraldehyde consumed per
mole of amine groups initially available in the chitosan
gel bead, was estimated by
XT )
moles of GA monomer consumed by the gel bead
moles of -NH2 initially in the gel bead
)
(CA0 - CA)VC
(1)
mbxBYB
where CA0 is the initial concentration of glutaraldehyde
in the cross-linking bath, CA is the current concentration
of glutaraldehyde in the cross-linking bath, mb is the
mass of beads in the cross-linking bath, VC is volume of
glutaraldehyde solution in the cross-linking bath, xB is
the weight fraction of chitosan in the gel bead, and YB
is free amine group content of the chitosan before cross-
linking. As a control experiment, 9.6 mL of a 250 mM
glutaraldehyde solution containing no gel beads were
mixed on an orbital shaker at 120 rpm and 27 °C for 48

h. The glutaraldehyde concentration assayed by GC
was constant over the 48 h of incubation time, demon-
strating that the aqueous glutaraldehyde solution was
stable.
The extent of cross-linking was also estimated gravi-
metrically. The glutaraldehyde solution in the cross-
linking bath was filtered from the gel beads. The
filtered gel beads were air-dried at room temperature
for 24 h, weighed, and then oven-dried at 85 °C for 4 h
and weighed again to make sure that the final weight
of the cross-linked product was obtained. The weight
of the un-cross-linked gel beads was also determined
by soaking 6.4 g of gel beads in 9.6 mL of distilled water
for 48 h, followed by filtration and drying as described
above. The retention of cross-linked reagent in the bead
after air drying (XR), expressed as mole of glutaralde-
hyde cross-linked per total mole of amine groups in the
gel bead, was estimated by
wB - wB0
XR ) (2)
mbxBYBMA
where wB0 is the mass of the air-dried gel beads before
cross-linking, wB is the mass of the air-dried cross-linked
beads, and MA is the average molecular weight of a
monomeric unit within the cross-link assembly, which
was assumed equal to 64 g of cross-link/mol of glutaral-
dehyde consumed.
Cadmium Ion Adsorption Capacity. Batch iso-
therm adsorption measurements for cadmium ions on
chitosan beads are described by Hsien and Rorrer
(1995). In the present experiments, 0.79 g of the gel
beads were mixed with 40 mL of a cadmium nitrate (Cd-
(NO3)2) solution within a sealed 125 mL Erlenmeyer
flask at 120 rpm on an orbital shaker at 25 °C for 60 h.
The solution was filtered from the gel beads and
analyzed for cadmium ion concentration by ion chro-
matography (IC) using a Dionex CS-5 column as de-
scribed by Hsien and Rorrer (1995). The final cadmium
loading in the beads was determined by
(C0 - Cf)V
Qf ) (3)
mbxB
where C0 is the initial cadmium ion concentration, Cf
is the final cadmium concentration, Qf is the final
cadmium loading in the beads (mg of Cd+2/g of adsor-
bent), and V is the volume of the cadmium solution. For
all saturation adsorption capacity measurements, C0
was at least 2000 mg of Cd+2/L. Cadmium adsorption
capacity measurements for the cross-linked freeze-dried
chitosan beads (40 mg/40 mL of a cadmium nitrate
solution) were also obtained by the same methods. All
Qf values were corrected to mg of Cd+2/g of chitosan to
facilitate comparison at different extents of cross-
linking.
Other Measurements. The density for the 7%
N-C9 acylated gel beads was 1.06 ( 0.03 (one standard
deviation, 1s) g/cm3 as determined by volumetric dis-
placement. The diameter of the beads was measured
with a digital caliper, and average diameters were 3.3
( 0.2 (1s) and 3.2 ( 0.2 (1s) mm, respectively, for
chitosan beads and 7% N-C9 acylated chitosan beads.
Selected cross-linked chitosan gel bead and 7% N-C9
acylated chitosan gel bead preparations were also
freeze-dried as described by Rorrer et al. (1993). The
diameter of the cross-linked freeze-dried beads was the
same as that of the cross-linked gel beads. The weight
fraction chitosan in the gel beads before cross-linking
Ind. Eng. Chem. Res., Vol. 36, No. 9, 1997 3633
was determined gravimetrically by oven-drying 6.4 g of
gel beads at 85 °C for 24 h. The pH within the chitosan
gel beads before cross-linking was measured by crushing
6.4 g of gel beads down to a pulp with a mortar and
pestle and then immersing a pH electrode directly into
the crushed gel. The pH was typically between 10 and
12. The alkaline gel beads readily dissolved in a 1 M
acetic acid solution at pH 2.4, but cross-linked gel beads
were insoluble in this solution at 27 °C for at least
24 h.
Modeling of Heterogeneous Cross-Linking
Kinetics
The heterogeneous cross-linking of the spherical
chitosan gel beads with glutaraldehyde is carried out
in a well-mixed batch isothermal reactor at constant
volume. The depletion of glutaraldehyde in the cross-
linking bath is balanced by the formation of cross-linked
chitosan as the glutaraldehyde monomer diffuses into
the chitosan gel and reacts between available amine
groups on adjacent chitosan chains to form the cross-
link assembly. Relevant to this process are two sto-
ichiometric parameters, R and β:
initial moles of GA CA0VC
(4)
initial moles of -NH2 mbxBYB
R) )
moles of GA reacted
β) (5)
moles of -NH2 reacted
The fraction of cross-linked amine groups X
h is related
to β and the extent of glutaraldehyde consumption XT
by
h ) XT/β
X (6)
The diffusion-controlled model assumes that (1) the
cross-linked chitosan is found in an outer shell of the
gel bead bounded by r ) R to r ) rC, where r is the radial
position within the bead, R is the outer radius of the
bead, and rC is the moving boundary between the cross-
linked zone and the uncross-linked zone; (2) the thick-
ness of cross-linked layer R - rC increases with time;
(3) the flux of glutaraldehyde into the gel bead is limited
by molecular diffusion of the glutaraldehyde monomer
through the cross-linked shell; (4) the glutaraldehyde
flux approximates the dilute solution diffusion process;
(5) the glutaraldehyde flux at a given value for rC is at
a nominal steady state, even though the glutaraldehyde
concentration in the cross-linking bath decreases with
time; and (6) the glutaraldehyde monomer completely
and rapidly reacts so that its concentration is zero at
rC. Under these six assumptions, the modified shrink-
ing core model (Rao and Gupta, 1982) can be applied to
the heterogeneous cross-linking process.
Major steps in the mathematical development are
overviewed below. According to the shrinking core
model approach, X h given by eq 6 is represented as
h ) 1 - [rC/R]3
X (7)
Rearrangement and differentiation of eqs 1 and 7 with
respect to time yields
dCA h mbxBYBβ
dX
(8)
dt dt VC
)-
dX 3 drC
h
) - 3rC2 (9)
dt R dt
3634 Ind. Eng. Chem. Res., Vol. 36, No. 9, 1997

The glutaraldehyde flux into the gel beads is balanced

by the rate of depletion of glutaraldehyde in the cross-
linking bath

dCA dC′A 2 3mb

VC ) -4πNA,RR2n ) DAe r 3 (10)
dt dr RF b

where CA′ is the glutaraldehyde concentration within

the gel bead, DAe is the effective diffusion coefficient of Figure 2. Extent of glutaraldehyde consumption (XT) and pH vs
glutaraldehyde through the cross-linked layer of the gel time at CA0 equal to 75 mM for the heterogeneous cross-linking of
bead, and NA is the molecular diffusion flux of glutaral- the 7% N-C9 acylated chitosan gel beads. The solid line represents
dehyde with continuity condition NA,rR2 ) NA,rr2. The the calculated profile by the diffusion-limited modified shrinking
core model as described in the text.
number of beads in the cross-linking bath (n) is esti-
mated as 3mb/4πR3Fb, where Fb is the density of the
gel bead. If external mass-transfer resistances are
negligible and the rate of cross-linking is limited by the
molecular diffusion of glutaraldehyde through the cross-
linked layer to reaction boundary rC, then it can be
shown that the combination of eqs 8-10 followed by
integration of CA′ over r from r ) R (CA′ ) CA) to r ) rC
(CA′ ) 0) yields

drC DAeCA Figure 3. Extent of glutaraldehyde consumption (XT) and pH vs

rC2
[ ]
(11) time at CA0 equal to 250 mM for the heterogeneous cross-linking
dt 1 1
)-
xBFbYBβ of the 7% N-C9 acylated chitosan gel beads. The solid line
rC R
-
represents the calculated profile by the modified shrinking core
model.
Separation of variables rC and t followed by integration
gives

[] []
rC rC 6DAe
2 3
1-3
R
+2
R
)
xBFbYB βR
∫t
CA dt
2 0
(12)

In terms of X
h , eq 12 becomes

F(X h )2/3 + 2(1 - X

h ) ) 1 - 3(1 - X h) )
Figure 4. Effect of mixing speed on the XT vs time profile at CA0
6DAe
xBFbYBβR2
∫0 CA dt
t
(13)
equal to 250 mM.

48 h were 0.399 ( 0.025 (1s) and 1.26 ( 0.097 (1s) mol

which is a modified shrinking core model for the
heterogeneous cross-linking of the chitosan gel beads
in a well-mixed batch reactor. Alternatively, combina-
tion of eqs 1, 6,7, and 11 yields a differential equation
explicitly in terms of rC
of GA/mol of -NH2 at CA0 equal to 75 and 250 mM,
respectively. Increasing the orbital shaker speed from
120 to 240 rpm had no effect on the cross-linking
kinetics, as shown in Figure 4. Therefore, the hetero-
geneous cross-linking process was not subject to exter-
nal mass-transfer resistances.

[ [ ( ) ]]
βmbxBYB rC 3 The effects of the initial glutaraldehyde concentration
DAe CA0 - 1- on the final extent of glutaraldehyde cross-linking for
drC VC R the chitosan gel beads and 7% N-C9 acylated chitosan

[ ]
(14)
dt 1 1 2
)-
xBFbYBβ - r
rC R C
If DAe and all other constant parameters are known,
then the kinetics of the cross-linking process can be
obtained by numerical integration of eq 14 to obtain rC
vs time. With rC vs time known, predicted values for
X
h , XT, and CA vs time can then be backed out in order
by eqs 7, 6, and 1, respectively.
Results and Discussion
Heterogeneous Cross-Linking of Chitosan Gel
Beads. Representative kinetic data for the heteroge-
neous cross-linking of 3 mm 7% N-C9 acylated chitosan
gel beads at initial concentrations of 75 and 250 mM
glutaraldehyde in the cross-linking bath are shown in
Figures 2 and 3 respectively. In both experiments, the
concentration of glutaraldehyde in the cross-linking
bath went to zero within 48 h, and the pH in the bath
leveled off between 7 and 8. Final values for XT after
gel beads are shown in parts a and b of Figure 5,
respectively. The initial glutaraldehyde concentration
values in terms of R as defined by eq 4 are shown in
Table 1. The extent of cross-linking was estimated as
XT by eq 1 using the measured glutaraldehyde concen-
tration in the cross-linking bath and as XR by eq 2 using
gravimetric data from air-dried gel beads before and
after cross-linking. In all experiments, maximum val-
ues for both XT and XR were obtained within 48 h of
cross-linking. If one glutaraldehyde molecule cross-
links with two amine groups as schematically illustrated
in Figure 6, then an R value equal to 0.5 mol of GA/mol
of -NH2 is required for stoichiometric conversion. At
low extents of cross-linking below this R value, XT and
XR were comparable. However, at higher R values, both
XT and XR continued to increase. Furthermore, XT was
now higher than XR. The reason for this last result is
not clear. However, when the gel beads were dried prior
to measurement of XR, any glutaraldehyde not chemi-
cally bonded to amine groups may have volatilized along
with the water.
 Ind. Eng. Chem. Res., Vol. 36, No. 9, 1997 3635
Homogeneous Cross-Linking of a Chitosan So-
lution. In the homogeneous cross-linking experiment,
6.4 g of a 5.0 wt % chitosan solution was vigorously
stirred into 9.6 g of a 250 mM glutaraldehyde solution
for 10 s, and then mixed on an orbital shaker at 120
rpm and 27 °C. At these conditions, R was equal to 1.2
mol of GA/mol of -NH2. The mixture formed a uni-
formly textured, rigid gel within 1 min. In the homo-
geneous cross-linking reaction, the mixture was acidic
with about pH 2.7, whereas in the heterogeneous cross-
linking reaction, the pH environment was neutral to
basic. Despite the difference in pH environments, this
experiment showed that the homogeneous cross-linking
reaction was very rapid relative to the heterogeneous
reaction and further supported the premise that the
heterogeneous cross-linking process was subject to
mass-transfer resistances associated with the diffusion
of glutaraldehyde through the cross-linked layer of the
Figure 5. Effect of initial glutaraldehyde concentration (CA0) on bead.
the final extent of glutaraldehyde consumption, measured as both Modeling of the Heterogeneous Cross-Linking
XT and XR: (a) 7% N-C9 acylated chitosan gel beads; (b) chitosan Process. Although glutaraldehyde is a common protein
gel beads. Error bars are not shown, as the standard for all points
shown was within 10% of the average value from duplicate cross-linking reagent, the cross-link assembly resulting
samples. from the reaction of aldehyde groups on glutaraldehyde
with the amine groups on the substrate to be cross-
linked is still under debate. Although glutaraldehyde
exists only as a monomer in aqueous solution at acidic
or near neutral pH (Hardy et al., 1969; Kawahara et
al., 1992), glutaraldehyde can polymerize to short-chain
oligomers in alkaline environments. Specifically, sev-
eral investigators (Richards and Knowles, 1968; Peters
and Richards, 1977; Margel and Rembaum, 1980; Gor-
man and Scott, 1980; Kawahara et al., 1992) have
verified that glutaraldehyde polymerizes by aldol con-
densation to form R,β-unsaturated aldehydes. During
cross-linking, an available amine group reacts with the
terminal unsaturated aldehyde moieties on each side
of the glutaraldehyde oligomer to yield a Schiff’s base
with a conjugated double bond (-NdC-Cd), as sche-
matically illustrated in Figure 6 (Peters and Richards,
1977; Gorman and Scott, 1980). Although the size of
Figure 6. Proposed cross-link assemblies for monomeric and the oligomers appears to be dependent on pH, temper-
oligomeric forms of glutaraldehyde, based on the literature cita- ature, and time, two protein cross-linking studies (Korn
tions given in the text. et al., 1972; Monsan et al., 1975) showed that the
dominant stable oligomer consisted of 8 glutaraldehyde
Table 1. Initial pH and GA/-NH2 Ratio in the
Cross-Linking Bath for Data Shown in Figure 5
residues per cross-link assembly or 4 glutaraldehyde
residues per -NH2 group. According to Figure 6, if β
initial GA/-NH2 ratio, R ) CA0VC/mbxBYB is equal to 0.5, then the cross-link assembly consists of
7% N-C9 acylated one glutaraldehyde residue with a molecular weight of
chitosan beads chitosan beads 64; if β is equal to 4, the molecular weight of the cross-
CA0 (mM) initial pH (xB ) 0.068) (xB ) 0.0453) link assembly is 640, and MA is equivalent to 80.
12.5 4.16 0.044 0.072 In this present study, the cross-linking bath is slightly
25.0 4.07 0.088 0.143 acidic to neutral with a final pH between 7 and 8.
75.0 3.68 0.265 0.430 Consequently, glutaraldehyde assumes a monomeric
250.0 3.40 0.885 1.434
500.0 3.25 1.77 2.87 form in the cross-linking bath. However, the chitosan
gel bead is alkaline with an nominal pH of 10-12 before
cross-linking, because a high stoichiometric excess of 2
The above results suggest that not all of the glutaral-
M NaOH is used in the casting bath to precipitate the
dehyde consumed by the gel bead was directly cross-
chitosan during the gel bead casting process. Conse-
linked to -NH2 groups on chitosan. One possibility is quently, the alkaline environment inside the gel bead
that the glutaraldehyde polymerized into short-chain could promote the polymerization of glutaraldehyde to
oligomers before cross-linking. Another possibility is higher oligomers. Previous work strongly suggests that
that unreacted glutaraldehyde was trapped within the the oligomer has a putative chain length of 8 glutaral-
gel bead. To determine if any unreacted glutaraldehyde dehyde residues, or a β value of 4. Therefore, the
was freely absorbed within the gel, cross-linked gel proposed physical model for the heterogeneous cross-
beads were mixed with distilled water at 27 °C and 120 linking process of glutaraldehyde with the chitosan gel
rpm within an orbital shaker. After 48 h, less than 0.1% bead consists of three steps: (1) molecular diffusion of
of the glutaraldehyde originally taken up by the gel bead the glutaraldehyde monomer through the cross-linked
was found in the distilled water, suggesting that the shell of the gel bead to the moving reaction boundary
glutaraldehyde was irreversibly retained within the rC; (2) rapid polymerization of the glutaraldehyde
cross-linked bead. monomer to glutaraldehyde oligomers; (3) rapid reaction
3636 Ind. Eng. Chem. Res., Vol. 36, No. 9, 1997
Figure 7. F(X h ) vs ∫CA dt plot for the estimation of the effective
diffusion coefficient DAe for glutaraldehyde in the 7% N-C9
acylated chitosan gel beads. The solid line represents the least-
squares linear fit to eq 13. Input parameters: CA0 ) 250 mM, R
) 0.16 cm, xB ) 0.055, YB ) 5.77 mmol of -NH2/g of chitosan, β
) 4 mol of GA reacted/mol of -NH2 reacted, Fb ) 1.06 g/cm3. From
the least-squares slope, DAe ) 4.7 × 10-8 ( 9.3 × 10-10 cm2/s (r2
) 0.990).
Table 2. Effect of β on DAe for the Data Shown in
Figure 7
effective diffusion coefficient
β DAe ( 1s (r2), cm2/s DAe/DA°
0.5 6.3 × 10-7( 5.5 × 10-8(0.918) 0.070
1.0 3.4 × 10-7 ( 1.4 × 10-8 (0.950) 0.038
2.0 9.9 × 10-8 ( 1.6 × 10-9 (0.993) 0.011
4.0 4.7 × 10-8 ( 9.3 × 10-10 (0.990) 0.0052
of the terminal, conjugated -CHO groups on glutaral-
dehyde oligomer with available -NH2 sites to form the
imine (-NdC-) cross-link. The process is mathemati-
cally modeled according to the modified shrinking core
model described earlier.
Using the modified shrinking core model in the form
of eq 13, the effective diffusion coefficient of monomeric
glutaraldehyde through the cross-linked shell of the 3
mm 7% N-C9 acylated gel bead was estimated. A plot
of F(Xh ) vs ∫CA dt at β equal to 4 and CA0 equal to 250
mM yielded a straight line with a regression coefficient
of 0.990, demonstrating that the modified shrinking core
model was appropriate for data analysis (Figure 7).
From the least-squares slope, DAe was estimated to be
4.7 × 10-8 ( 9.3 × 10-10 (1s) cm2/s. Since the value of
β could not be determined absolutely, DAe was reesti-
mated for different values of β (Table 2). As β increased,
DAe decreased. The regression coefficient for the data
fit was optimal at β equal to 2 and 4. The molecular
diffusion coefficient of monomeric glutaraldehyde in
water at infinite dilution (DA°) is 9.0 × 10-6 cm2/s at 27
°C by the Wilke-Chang correlation. All DAe values
were 1-2 orders of magnitude lower than the molecular
diffusion coefficient, showing that the cross-linked layer
provides a significant barrier to diffusion. If the as-
sumption that glutaraldehyde polymerization only oc-
curs at the reaction boundary is not valid, then DAe
could represent a composite diffusivity of glutaraldehyde
monomers, dimers, trimers, and higher oligomers in the
gel bead during the cross-linking process.
In comparing Figures 2 and 3, the time required for
cross-linking to achieve completion cross-linking de-
creased as CA0 decreased. This observation was also
predicted by the diffusion-limited modified shrinking
core model. The calculated XT vs time curves using DAe
equal to 4.7 × 10-8 cm2/s at β equal to 4 were consistent
with the measured curves, except at very short cross-
linking times at the lower initial glutaraldehyde con-
centration of 75 mM. Although the heterogeneous cross-
linking reaction was modeled as diffusion controlled, at
very short times or low values of R the process could be
reaction rate controlled, leading to cross-linking rates
Figure 8. Comparison of representative cadmium ion (Cd2+)
adsorption isotherms for both gel and freeze-dried 7% N-C9
acylated chitosan beads prepared at an extent of cross-linking
equivalent to 0.4 mol of GA/mol of -NH2.
Figure 9. Effect of cross-linking on the saturation cadmium ion
(Cd2+) adsorption capacity for chitosan beads: (a) 7% N-C9
acylated gel and freeze-dried chitosan beads; (b) chitosan gel beads.
The solid line is the nonlinear least-squares fit of the gel bead
data to eq 16.
that could be higher than those proposed by a diffusion-
limited model.
Effect of Cross-Linking on Cadmium Ion Ad-
sorption Capacity. Representative cadmium ion ad-
sorption isotherms at pH 6.5 for cross-linked gel chito-
san beads and freeze-dried, cross-linked chitosan beads
are provided in Figure 8. Both gel beads and freeze-
dried beads possess a stepped isotherm. The physical
processes underlying the shape of this two-step isotherm
are described by Rorrer et al. (1993) and Hsien and
Rorrer (1995). Figure 8 shows that both gel and freeze-
dried beads have comparable maximum cadmium ion
adsorption capacities. However, for the gel beads, the
location of the second step on the isotherm occurs at a
significantly lower cadmium ion concentration than that
for the freeze-dried beads, suggesting that the cadmium
ions penetrate more readily into the gel bead. Further-
more, although the freeze-dried, cross-linked chitosan
beads have high internal surface areas exceeding 150
m2/g (Hsien and Rorrer, 1995), the highly-porous struc-
ture of the freeze-dried beads did not improve the
saturation cadmium ion adsorption capacity relative to
the cross-linked chitosan gel beads.
The effect of cross-linking on the saturation cadmium
ion adsorption capacity at pH 6.5 for both chitosan beads
and 7% N-C9 acylated gel beads is shown in Figure 9.
The saturation cadmium ion adsorption capacities were
obtained from the last plateau of the two-step isotherm.
The cadmium ion adsorption capacities of the gel beads
decreased exponentially as the extent of cross-linking
increased, leveling off at XT values greater than 1.0 mol
of GA/mol of -NH2. The freeze-dried 7% N-C9 acylated
chitosan beads showed a similar trend.

The theoretical maximum loading of cadmium ions
in the un-cross-linked chitosan gel bead is estimated by
Qe ) YBMCd/ν (15)
where MCd is the molecular weight of cadmium (112 mg
of Cd/mmol of Cd), and ν is the chelation coordination
number for cadmium (2 mol of -NH2/mol of Cd2+).
From eq 15, the value of Qe is 347 mg of Cd/g of chitosan.
The cadmium ion chelation site for the cross-linked
outer shell is an imine group (-NdC-), whereas the
chelation site for the un-cross-linked core is an amine
group (-NH2). Both N-containing moieties are strong
transition-metal ion chelators (Kantipuly et al., 1990).
However, even the un-cross-linked gel bead had a
saturation adsorption capacity of only 257 mg of Cd/g
of chitosan, suggesting that the metal was not uniformly
loaded in the bead, a phenomena described by Nestle
and Kimmich (1996) for transition-metal ion adsorption
into biopolymer gel beads. At higher extents of cross-
linking where the cadmium adsorption capacity levels
off, the adsorbed cadmium may be localized in the cross-
linked layer of the bead. This behavior further suggests
the empirical correlation
Qf ) Qf,c + (Qf,o - Qf,c)e-XT/KX (16)
where Qf,o is the cadmium loading for the un-cross-
linked chitosan gel bead, Qf,c is the cadmium loading
within the cross-linked zone of the gel bead, and KX is
a proportionality constant. The adsorption data for the
7% N-C9 acylated chitosan gel beads given in Figure
9a were fitted to eq 16 with Qf,o equal to 257 mg of Cd/g
of chitosan. Best-fit values of Qf,c equal to 100 ( 13.5
(1s) mg of Cd/g of chitosan and KX equal to 0.325 ( 0.094
(1s) mol of GA/mol of -NH2 were obtained by nonlinear
regression with a regression coefficient of 0.94.
When the homogeneously cross-linked chitosan de-
scribed earlier was used for cadmium ion adsorption
measurements, the saturation cadmium ion adsorption
capacity was 75 mg of Cd/g of powdered air-dried
material, or 50 mg of Cd/g of chitosan, assuming MA
equal to 64 g/mol of GA reacted. This value was
comparable to the heterogeneously cross-linked chitosan
gel beads.
Conclusions
Chitosan, a linear biopolymer of glucosamine resi-
dues, is a highly selective biosorbent material for heavy-
metal ions such as cadmium. Chemical cross-linking
of amine (-NH2) groups on chitosan gel beads with
glutaraldehyde (GA) renders the bead insoluble in acid
solution and imparts other desirable material proper-
ties, including high internal surface areas upon freeze-
drying. The overall goals of this study were to experi-
mentally and mathematically describe the kinetics of
heterogeneous cross-linking of chitosan gel beads with
glutaraldehyde and to determine how the extent of
cross-linking affects the saturation cadmium ion ad-
sorption capacity.
The kinetics of the heterogeneous cross-linking pro-
cess for 3 mm chitosan gel beads were measured at
initial glutaraldehyde concentrations ranging from 12.5
to 500 mM at 27 °C and pH 7-8. Final extents of cross-
linking ranged from 0.07 to 2.40 mol of GA consumed/
mol of amine (-NH2). Glutaraldehyde consumption was
considerably higher than the theoretical value of 0.5 mol
of GA/mol of -NH2. This information, combined with
considerable literature precedence for glutaraldehyde
Ind. Eng. Chem. Res., Vol. 36, No. 9, 1997 3637
polymerization at high pH, suggested that glutaralde-
hyde polymerized to an oligomeric cross-link assembly
within the alkaline gel bead. Furthermore, the hetero-
geneous cross-linking of chitosan gel beads was modeled
as a “shrinking core process” where the molecular
diffusion of glutaraldehyde through the cross-linked
layer of the gel bead limits the overall rate of cross-
linking. The diffusion-limited modified shrinking core
model adequately described the kinetics of the hetero-
geneous cross-linking process as a function of gel bead
properties, cross-linking reagent properties, and cross-
linking bath reaction parameters. The effective diffu-
sion coefficient of glutaraldehyde cross-linking reagent
through the cross-linked layer of the gel bead was 4.7
× 10-8 cm2/s, 2 orders of magnitude lower than the
molecular diffusion coefficient for glutaraldehyde in
water.
The saturation adsorption capacity of cadmium ions
onto cross-linked chitosan gel beads exponentially de-
creased from 250 to 100 mg of Cd/g of chitosan as the
extent of cross-linking increased from 0 to 1.3 mol of
GA/mol of -NH2. At higher extents of cross-linking, the
saturation adsorption capacity remained at 100 mg of
Cd/g of chitosan. Highly porous 7% N-C9 acylated
chitosan beads formed by freeze-drying of cross-linked
gel beads had the same saturation cadmium ion adsorp-
tion capacity as the cross-linked gel beads over the same
extents of cross-linking. Consequently, it the cross-
linking process which determines both the material
properties and the maximum cadmium ion adsorption
capacity of the chitosan beads. However, the rates of
cadmium ion uptake may still be different for each bead
preparation. This question will be addressed in future
work.
Notation
CA ) glutaraldehyde concentration (GA) in the cross-
linking bath, mmol/L (mM)
CA0 ) initial glutaraldehyde concentration in the cross-
linking bath, mmol/L (mM)
CA′ ) unreacted glutaraldehyde concentration within the
gel bead, mmol/L (mM)
Co ) initial concentration of cadmium ions in the batch
adsorption experiment, mg of Cd2+/L
Cf ) final concentration of cadmium ions in batch adsorp-
tion experiment, mg Cd+2/L
DAe ) effective diffusion coefficient of glutaraldehyde
through the cross-linked layer of the gel bead, cm2/s
DA° ) molecular diffusion coefficient of glutaraldehyde in
water at infinite dilution, cm2/s
KX ) proportionality constant in eq 16, mol of GA/mol of
-NH2
MA ) average molecular weight of a monomeric glutaral-
dehyde residue within the cross-link assembly, g of cross-
link/mol of glutaraldehyde reacted
mb ) total mass of the gel beads within the cross-linking
bath, g
MCd ) molecular weight of cadmium, 112 mg/mmol
NA ) flux of the glutaraldehyde monomer through the gel
bead, mol/cm2 s
Qe ) theoretical maximum loading of adsorbed cadmium
on uncross-linked chitosan, mg of Cd2+/g of chitosan
Qf ) loading of adsorbed cadmium on the chitosan bead,
mg of Cd2+/g of chitosan
Qf,o ) loading of adsorbed cadmium on the un-cross-linked
chitosan gel bead, mg of Cd2+/g of chitosan
Qf,c ) loading of adsorbed cadmium on the cross-linked
layer of the chitosan gel bead, mg of Cd2+/g of chitosan
r ) radial position within the gel bead, cm
R ) outer radius of the gel bead, cm
3638 Ind. Eng. Chem. Res., Vol. 36, No. 9, 1997
rC ) radial position within the gel bead defining the
boundary between the outer cross-linked layer and the
inner un-cross-linked core, cm
t ) cross-linking time, h
V ) volume of cadmium ion solution in the batch adsorp-
tion experiment, cm3
VC ) volume of glutaraldehyde solution in the cross-linking
bath, cm3
wB ) mass of dried cross-linked beads after cross-linking,
g crosslinking of chitosan for enhanced cupric ion adsorption. J.
wBO ) mass of chitosan in gel beads before cross-linking, g
xB ) weight fraction of chitosan in the gel bead, g of
chitosan/g of gel
X
h ) extent of cross-linking defined by eq 6, mol of -NH2
reacted/mole of -NH2 before reaction
XR ) extent of cross-linking defined by eq 4
XT ) extent of cross-linking defined by eq 1, mol of
glutaraldehyde consumed by the gel bead/total mol of
-NH2 groups within the gel bead
YB ) amine group content of chitosan, mol of -NH2/g of
chitosan before cross-linking
R ) initial molar ratio of glutaraldehyde to amine groups
in the cross-linking bath, mol of GA/mol of -NH2
β ) mol of GA consumed to form a cross-link assembly/
mol of -NH2 cross-linked
Fb ) density of the gel bead, g/cm3
ν ) chelation coordination number for cadmium, 2 mol of
-NH2/mol of Cd2+
Acknowledgment
The authors gratefully acknowledge support of this
research by the U.S. Environmental Protection Agency
through the Exploratory Research Grants Program
(Grant No. 818626-01-0). The authors also thank Van-
son Chemical Co. (Redmond, WA) for the donation of
flaked chitosan.
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Received for review February 17, 1997
Revised manuscript received June 9, 1997
Accepted June 11, 1997X
IE9701579
X Abstract published in Advance ACS Abstracts, August 1,
1997.