Journal of Membrane Science, 16 (1983) 295-308 Elsevier Science Publishers B.V., Amsterdam -Printed in The Netherlands 295 THE CHARACTERIZATION OF N-METHYL. N-ETHYL, N-PROPYL, N-BUTYL AND N-HEXYL CHITOSANS, NOVEL FILM-FORMING POLYMERS* RICCARDO A.A. MUZZARELLI, FABIO TANFANI, MONICA EMANUELLI and SABINA MARIOTTI Instztute of Biochemistry, Faculty of Medicine, University of Ancona, I-60100 Ancona (Italy) (Received June 17, 1982; accepted in revised form September 17,1982) Summary The N-slkyl chitosans in the title were prepared from chitosan (Euphausia superba) by aldimine formation and hydrogenation at room temperature in aqueous media. They are white powders with a certain degree of crystallimty; their substitutron degrees are: acetamido 36%, secondary amine 23-33%, primary amine 31-41%. The chelating ability of N-alkyl chitosans is specific for certain cations such as Cu’*, Hg2+ and Pb2+ for which the capacities are in the range Z-6% by weight, depending on conditrons and choice of N-alkyl chitosan. In the N-alkyl chitosans, the hydrogen bonds are weakened by the presence of substituents and therefore they swell in water. Membranes are easily cast from acetic acid solutions. Circular dichroism and ultraviolet spectrometry measurements indicate that the chelation of metal ions depresses the acetamido group bands and intro- duces new spectral bands due to the chelates. Circular dichroism spectra also reveal the hydration state of the membranes. Applications are foreseen m the pharmaceutical, COS- metic and textile industries. Introduction We have recently reported on the production of N-carboxymethyl chito- sans and N-carboxybenzyl chitosans by aldimine formation and hydrogena- tion [l-3]. Chitosan reacts easily with aldehydes [ 4-71, however aldehyde derivatives of chitosan have been seldom, if at all, reported [8, 91, especially N-n-alkyl chitosans. Hydroxyethyl and ethyl ethers of chitin were described by Danilov and Plisko [lo] ; ethyl chitin was produced by the reaction of ethyl chloride on alkali chltin (see also Ref. [4] p. 72). Ethyl carboxymethyl chitosan (“ethyl carboxymethyl chitin,” according to the original article) was prepared by the American Cyanamid Co. [ 111 in two steps, the first involving ethyl bromide in dimethyl sulphoxide and NaOH to obtain partially ethylated chitosan, and the second, requiring chloroacetic acid and NaOH to further introduce the carboxymethyl substi- tuent. Disadvantages of that procedure were the use of two organic solvents *Paper presented at the Symposium on Membranes and Membrane Processes, May 19-22, 1982, Perugia, Italy. 03767388/83/$03.00 o 1983 Elsevier Science Publishers B.V.
Transcript
Journal of Membrane Science, 16 (1983) 295-308 Elsevier Science Publishers B.V., Amsterdam -Printed in The Netherlands
295
THE CHARACTERIZATION OF N-METHYL. N-ETHYL, N-PROPYL, N-BUTYL AND N-HEXYL CHITOSANS, NOVEL FILM-FORMING POLYMERS*
RICCARDO A.A. MUZZARELLI, FABIO TANFANI, MONICA EMANUELLI and SABINA MARIOTTI
Instztute of Biochemistry, Faculty of Medicine, University of Ancona, I-60100 Ancona
(Italy)
(Received June 17, 1982; accepted in revised form September 17,1982)
Summary
The N-slkyl chitosans in the title were prepared from chitosan (Euphausia superba) by aldimine formation and hydrogenation at room temperature in aqueous media. They are white powders with a certain degree of crystallimty; their substitutron degrees are: acetamido 36%, secondary amine 23-33%, primary amine 31-41%. The chelating ability of N-alkyl chitosans is specific for certain cations such as Cu’*, Hg2+ and Pb2+ for which the capacities are in the range Z-6% by weight, depending on conditrons and choice of N-alkyl chitosan. In the N-alkyl chitosans, the hydrogen bonds are weakened by the presence of substituents and therefore they swell in water. Membranes are easily cast from acetic acid solutions. Circular dichroism and ultraviolet spectrometry measurements indicate that the chelation of metal ions depresses the acetamido group bands and intro- duces new spectral bands due to the chelates. Circular dichroism spectra also reveal the hydration state of the membranes. Applications are foreseen m the pharmaceutical, COS-
metic and textile industries.
Introduction
We have recently reported on the production of N-carboxymethyl chito- sans and N-carboxybenzyl chitosans by aldimine formation and hydrogena- tion [l-3]. Chitosan reacts easily with aldehydes [ 4-71, however aldehyde derivatives of chitosan have been seldom, if at all, reported [8, 91, especially N-n-alkyl chitosans. Hydroxyethyl and ethyl ethers of chitin were described by Danilov and Plisko [lo] ; ethyl chitin was produced by the reaction of ethyl chloride on alkali chltin (see also Ref. [4] p. 72).
Ethyl carboxymethyl chitosan (“ethyl carboxymethyl chitin,” according to the original article) was prepared by the American Cyanamid Co. [ 111 in two steps, the first involving ethyl bromide in dimethyl sulphoxide and NaOH to obtain partially ethylated chitosan, and the second, requiring chloroacetic acid and NaOH to further introduce the carboxymethyl substi- tuent. Disadvantages of that procedure were the use of two organic solvents
*Paper presented at the Symposium on Membranes and Membrane Processes, May 19-22, 1982, Perugia, Italy.
03767388/83/$03.00 o 1983 Elsevier Science Publishers B.V.
296
(dimethyl sulfoxide and isopropanol), the waste of large amounts of NaOH, the application of heat (50°C) and the use of dangerous reagents. The pro- duct was presumably a mixture of chitosans modified at the 6-hydroxyl oxygen and at the amine nitrogen.
The present work was therefore undertaken to prepare the lower N-alkyl derivatives of chitosan by reaction of chitosan with n-alkyl aldehydes at room temperature, to obtain the aldimine derivatives which can be easily reduced to secondary amines. N-Methyl chitosan (NMC), N-ethyl chitosan (NEC), N-propyl chitosan (NPC), N-butyl chitosan (NBC) and N-hexyl chito- san (NHC) were then characterized, and their film forming ability was com- pared to that of chitosan. Their interaction with metal ions was studied to reach a deeper understanding of the chelation process leading to selective collection of certain transition metal ions. Possible applications are in the textile, cosmetic and medical fields.
Experimental
Chitosan and reagents Chitosan from antarctic krill (Euphausia superba) was supplied by the
Fisheries Central Board, Szczecin, Poland. Its characteristic properties have already been published [ 121. Chemicals were supplied by Hoechst, Darm- stadt, F.R.G.
Viscome try Measurements were carried out with the Haake Rotovisco RV12 visco-
meter, equipped with a programmer and a recorder; the temperature was controlled with a Haake thermostat-cryostat. The rotor used was model NV. The readings were plots of torque, S, versus test speed, n .The shear rate was calculated as D = M-n (set-I); shear stress as 7 = A- S (Pa) and viscosity as ?? = G/n @Pa-set ). The conversion of viscosity data from SI to CGS units is as follows: viscosity q : Pascal-second (Pa-set); 1 Pa-set = 10 P; shear stress T: Pascal (Pa), 1 Pa = 10 dyn-cm-‘; shear rate D : reciprocal second (l/set). The sample volume was 12 ml. The values were recorded-at.& 10, 30, 40, 50, 60, 70 and 80°C on 1% polymer solutions in 1% acetic acid, 0.1 M in NaCl. Factors (M, A and G) necessary for calculating shear rate, shear stress and viscosity were: A = 1.78 Pa/scale grad; M = 5.41 min/sec, and G = 700 mPa-set/scale grad-min.
Alkalimetry Titrations with 0.1 N NaOH were carried out under nitrogen solution of
polymers (0.5 g) in 0.3 N HCl (20 ml), with the aid of an Amel pH meter and the results were worked out graphically.
Infrared spectrometry Chitosan powders were ground with IR grade KBr in an agate mortar.
297
Spectra were recorded with a Perkin-Elmer IR spectrometer, Model 299-B, on translucent discs obtained by pressing the ground material with the aid of a Perkin-Elmer press.
X-ray diffraction spectrometry Chitosan powders were milled and sieved to pass 200-mesh net and
pressed to become self-sustaining in the frame for exposure to the primary beam of the N&filtered CuK, radiation from a Jeol X-ray diffractometer.
Atomic absorption spectrometry Analyses were carried out with a Perkin-Elmer 305 spectrometer equipped
with flame and hot graphite atomizers, according to standard methods. Metal ion solutions were prepared at 0.25, 0.50, 1.00 and 2.00 mM concentration, they were stirred (50 ml) with the polymer powder (200 mg) on a shaking machine at 80 r.p.m. for 24 hr; readings were taken after 1 and 24 hr.
UV-spec tropho tometry A Perkin-Elmer spectrophotometer, Model 544 was used. Slit 2 nm; ordi-
Circular dichroism spec tropolarime try A Jasco spectropolarimeter, model J 500 A was used, with wavelength
expansion 10 nm/cm, chart speed 1 cm/min, sensitivity 5 ma /cm, time con- stant 4 set, cell pathlength 1 cm. The polymer concentration was 0.05% in acetic acid 0.1%. The metal ion concentrations were 0.2, 0.6, 1.0 and 2.0 m&f, as sulfates, except HgClz and Pb(NOJ),.
Preparation of the N-alkyl chitosans Chitosan powder, 20 g, corresponding to 65 mmol of NH2, was suspended
in water (2 1) and glacial acetic acid (20 ml) was added under stirring. After dissolution, the aqueous solution of the desired aldehyde was added (twice as much as stoichiometric) and 30 min later, enough NaOH to raise the pH value to 4.5. The hydrogenation was performed with NaBH4 (5 g) dissolved in water (50 ml) in small portions for a 1 hr period with continuous stirring. The pH value rose to 5.5 spontaneously within 2 hr, and it was later adjusted to 10, to insolubilize the N-alkyl chitosans. The products were washed with water to neutrality and then extracted with ethanol and diethyl ether in a Soxhlet apparatus to remove excess aldehyde and inorganic products. In the case of hexanal, the dissolution was carried out in a 2:3 water/ethanol mix- ture. The products were lyophilized in an Edwards freeze-dryer at -40°C. The membranes were cast on glass or plastic surfaces of known area from 1% acetic acid solutions containing the desired amount of polymer; their thickness was about 1.5 pm.
Results and discussion
N-Alkyl chitosans are white powders with a certain degree of crystallinity: their X-ray diffraction spectra differ in terms of the 20 values of the first peak as well as their intensities. The’ X-ray diffraction spectra of NMC, NEC and NHC are shown in Fig. 1. The 20 values of the chitosan from which they are obtained are 8”58’ and 19” 58’ [ 121 ; as a consequence of the N- alkylation, the first peak is shifted to progressively lower 28 values as the length of the n-alkyl group increases, while the second peak appears at slight- ly higher 28 values for all samples. The data are: NMC, 8” 36’ and 20” 30’; NEC, 7”40’ and 20”30’; and NHC, $30 and 20”30’.
Fig. 1. X-ray diffraction spectra of NMC, NEC and NHC powders.
The infrared spectra show a progressive increase of the 2830 cm-l band, due to the methylene groups with increasing aliphatic chain length, together with a contribution at 1450 cm-‘. With these exceptions, the spectra of N- alkyl chitosans are coincident with that of chitosan.
The N-alkyl chitosans can be dissolved in dilute (1%) acetic acid solutions; they keep the characteristic filmogenic property of chitosan, ensuring that membranes are easily cast on glass or plastics. Viscometric measurements on the solutions indicate a decrease of viscosity with increasing temperature, with a marked drop at 4O”C, as shown in Fig. 2. NHC shows higher viscosi- ties than its homologues, close to that of chitosan.
The chitosan derivatives are basic substances like chitosan. The alkali- metric curves are similar to that of chitosan (Ref. [4], p. 105) and even though there are alterations due to two different amino groups, it was im- possible to distinguish the two pK values and therefore, in Table 1, one pK value only is reported.
A characteristic property of chitosan, the specificity of which is enhanced in the N-alkyl chitosans, is the chelating ability. Figure 3 shows some results in terms of capacity of NHC as a function of the metal ion concentration. The capacity values are particularly high in the cases of Cu2+, Hg2’ and Pb”,
299
r vIscosIIY /
0 10 20 30 40 M CELSIUS DEGREES **
Fig. 2. Viscosity data of ehitosan, NHC, NMC, NBC, NEC and NPC (sequence at 25°C) recorded at 512 r.p.m. on 1% solutions in 1% acetic acid with 0.1 M NaCl.
TABLE 1
Substitution degrees and pK values for N-alkyl chitosans (Acetamido % obtained from IR data and alkalimetry; secondary amine %, obtained from CD data and alkalimetry; primary amine %, obtained by difference; pK computed graphically from titration curves)
*It was experimentally impossible to distinguish the two pK values.
whilst for other cations they are much lower than for chitosan; the collection
process appears also to be slower with these derivatives, because the values for Cr3+, Ni2+ and Co2+ obtained after 1 hr contact are lower than those after 24 hr. The data for the other N-alkyl chitosans are similar to those of NHC in Fig. 3: for example, the capacities for Pb2+ are 36,41,11,16 and 58 mg Pb/g polymer at a concentration of 1.5 mA2, in going from NMC to NHC, respec- tively. The collection percentages from 0.25 m.A4 metal ion solutions are reported in Table 2.
Circular dichroism spectra were recorded on all N-alkyl chitosans; it was observed that the band height is proportional to the polymer concentration. Table 3 reports the spectropolarimetric data on chitosan and N-alkyl chito-
0 3.5 I .1 1.5 M
4:
K5iz 80
Hg Pb
30 5s
20 4”
10 t- -I 20
0 L-IL. 0 0 0.5 I .D I .5 0 0.6 i .” 1.5 Iy
Fig. 3. Capacities of NHC for metal ions versus metal ion concentration at 25” C after 24 hr (lower curve for 1 hr, otherwise coincident). Final pH values: Cr, 6.6-6.3; CO, 7.0;
sans alone and in the presence of CrK(S04h, HgCl,, Pb(N03)3, CoSO, and CuSO,, each salt being at concentrations of 0.2, 0.6, 1.0 and 2.0 mM. All solutions were clear. A general trend was the decrease of the 210 nm band with increasing cation concentration; its position was also shifted to higher values, up to 220 nm.
In the presence of Pb2+, no positive band appears and even low concentra- tions of Pb (0.2 r&f) shift the negative band from 210 nm to higher values; this band is absent with higher Pb*’ concentrations. In the presence of Co 2+, the spectra are not significantly altered, in agreement with the low capacities and collection percentages for this cation. Conversely, Cr3+ introduces signi- ficant alterations.
TA
BL
E
2
Col
lect
ion
perc
enta
ges
of
met
al
ions
(a
ll su
lfat
es,
exce
pt
HgC
l, an
d Pb
( N
O,)
,)
on N
-alk
yl
chito
sans
(2
00
mg
in
50 m
l 0.
5 ~.
LW
at
20”
C)
unde
r m
echa
nica
l st
irri
ng,
afte
r 1
and
24 h
r ._
~
.-
.---
_.
Col
lect
ion
perc
enta
ge
._.
Cr
‘+
Mn”
+
co2+
N
i2+
C
Xla
+
Znz
+ C
d2+
H
g2+
Pb
”’
1 24
1
24
1 24
1
24
1 24
1
24
1 24
1
24
1 24
_
N-M
ethy
l ch
itosa
n 88
89
24
13
7
13
47
50
100
100
30
24
15
24
97
92
54
50
N-E
thyl
ch
itosa
n 94
96
20
16
6
9 23
28
97
10
0 32
27
14
10
97
92
51
51
N-P
ropy
l ch
itosa
n 96
10
0 31
13
0
0 16
16
10
0 10
0 10
6
33
16
92
88
74
57
Ar-
But
yl
chito
san
95
100
31
13
0 0
22
20
100
100
34
27
34
16
93
87
44
45
N-H
exyl
ch
itosa
n 60
92
16
27
10
12
23
47
66
98
41
15
15
20
85
81
39
35
-_
_
__.
TABLE3
g
Cbw.&rdichroism
da
ta o
n chitosan and on N-alkylchitosanssolutions (O.&j%in 0.1% acetic aeid)in the presence of metal ions et VatiOuS concentrations, N
In Table 3, the data on C!u’+ refer to two pH values; 4.5 and 6.0. At the latter, the solution is not quite clear and two new bands characterize the spectrum: a negative one at 2’75-290 nm and a positive one at 240.-245 nm, both with intensities and position values increasing with Cu2+ concentration. The pH value affects the cation-polymer interaction and the CD spectra are consequently altered.
When mercury is present, chitosan behaves differently from N-alkyl chito- sans; as shown in Fig. 4, the chelation of Hg2+ originates a positive band at 235 nm which is absent in the N-alkyl chitosans. The UV spectra of the solu- tions of chitosan with mercuric ions show an absorption band at 235 nm, corresponding to the band appearing in the CD spectrum in Fig. 4. The 196 nm band in the UV spectrum is still present when the Hg’+ concentra- tion is 1.0 mM, but is absent when the Hg2+ concentration is 2.0 mM. This fact is in agreement with the persistence of the 218 nm band in the CD spec- trum when Hg2+ . is at 1.0 mM concentration and its absence when Hg” is at 2.0 mM concentration (Fig. 5). The situation is summarized as follows:
lfh lo-4 I I I I I I i I
0.8
0.6
0.4
Fig. 4. Circular dichroism spectra of chitosan solution in 0.1% acetic acid in the presence of HgCl, at various millimolarities, pH 5.5 t 0.1.
Fig. 5. UV spectra of a 0.05% chitosan solution in 0.1% acetic acid in the presence of 1.0 and 2.0 mM HgCl, against the same HgCl, solution. Dilution for measurement: 1, fivefold; 2, twofold. pH 5.5 f 0.1.
304
CD uv
1.0 m&l Hg” (-) 218 and (+) 235 nm interval 196 to 240 nm 2.0 mM Hg2+ (+) 125 nm interval 208 to 260 nm
The interaction between metal ions and chitosan or N-alkyl chitosans can be followed by using the polymers in form of membranes instead of solutions (the N-alkyl chitosan membranes swell to a large extent upon immersion in water). The CD spectra, recorded on membranes suitably held in the cell filled with water or solution, strictly correspond to those recorded on N- alkyl chitosan solutions (Table 3).
Although the chitosan membranes do not undergo swelling to the same extent as the N-alkyl chitosan membranes, remarkable optical alterations occur when they dry and vice versa. For instance, Fig. 6 shows the alteration of the CD spectra in relation to the hydration state of the chitosan-Cu mem- brane during the drying process which takes 40 min at room temperature.
+ IlIllIII~ I III i 1 ~ II’1
-
Fig. 6. Circular dichroism spectra of a chitosan-Cu membrane: 1, in water; 2, wet; 3, moistened; 4, dry. Instrument sensitivity: 10, 5, 5, 5, respectively.
The chitosanp0.r membrane in water shows two negative bands, at 215 and 280 nm, the intensities of which are about twice those evidenced during drying. The “dry” membrane no longer possesses the 215 nm band but a novel band at 310 nm; this phenomenon is reversible.
The N-alkyl chitosan membranes in water show a negative band at 210- 215 nm in the CD spectra and intense absorption at 196 nm in the UV spec- tra. On the other hand, the CD spectra of the N-alkyl chitosans in Cu form possess negative bands around 210 and 280 nm; correspondingly, the UV spectra show strong absorption at 196 nm with a shoulder at 280 nm. These
spectra indicate that the polymers are not fully engaged in complex forma- tion, under the conditions adopted.
On the other hand, the CD spectra taken on N-alkyl chitosan-Cu mem- branes in a 1000 p.p.m. C&O4 solution, (shown in Fig. 7) indicate that the polymers are completely engaged in the chelation of Cu2+. In these spectra, in fact, there is no band in the region 180-240 nm, while there are new bands at 305 nm for chitosan and NMC, and at 270, 265, 270 and 273 nm for NEC, NBC, NPC and NHC, respectively. In agreement with these findings, the UV spectra (in Fig. 8) exhibit strong bands centered at 275 nm on all N-alkyl chitosans (280 nm for NMC), due to copper chelates.
200 250 300 nm 350
Fig. 7. Circular dichroism spectra of chitosan-Cu and N-alkyl chitosan-Cu membranes in a 1000 p_p.m. CuSO, solution at pH 5.1 f 0.1. Instrument sensitivity: 1 for NPC, NBC and NHC; 2 for chitosan and 5 for NMC and NEC.
These results are in agreement with those previously reported on chitosan- Cu membranes [ 13 ] and throw new light on the chelation process. Recent findings concerning C$’ and Pb2’ [ 141 indicate that discrete aggregates of oxides and hydroxides are formed on the chitosan surface. The growth of oxide aggregates, presumably promoted by the initially formed chitosan- Pb chelates, within thin membranes, would explain the observed decay of the mechanical properties.
When the N-alkyl chitosan-Pb membranes are used under the experimen- tal conditions adopted for the chitosan-Cu membranes [ 131, opacity and rupture occur immediately, presumably due to the excessive growth of lead compounds within the membrane. In the past we have reported on the collec- tion of metavanadate, molybdate, tungstate and phosphate on chitosan,
306
J am 33 n3 Im
Fig. 8. UV spectra of chitosan-Cu and N-alkyl chitosan--Cu membranes in a 1000 p.p.m CuSO, solution against the same solution.
leading to the production of chitosans, the inorganic portion of which ex- ceeded the organic in weight [15] (see also Ref. 141, p. 197). TheN-alkyl chitosan chelates of a few metal ions presumably initiate and promote the deposition of inorganic compounds.
Conclusions
N-Alkyl chitosans can be easily prepared under mild conditions. The alkyla- tion procedure that we have developed is the same that we used to produce N-carboxymethyl chitosan and N-carboxybenzylchitosan, and therefore it lends itself to the preparation of chitosan derivatives carrying both alkyl and carboxyalkyl groups.
The aqueous solutions of N-alkyl chitosans are slightly less viscous than the corresponding chitosan solutions: membranes are simply obtained by evaporation and neutralization.
N-Alkyl chitosans are chelating polymers, especially suitable for the col- lection of Cr3+, Cu2+, Hg2+ and Pb2+ ions for which they exhibit selectivity,
Since the negative band in the CD spectra at 210 nm is reportedly due to the acetamido group 1151, the alteration of the spectra following the interaction with the metal ions can be explained by the involvement of the acetamido group in the chelate.
307
The primary and secondary amino groups are principally responsible for the metal ion chelation, to which water molecules and hydroxyl ions also contribute; however the acetamido groups presumably play an important role in chelation whenever the water molecules present in the complex are removed (by drying, for example) and the secondary amine carries large hydrophobic groups.
The presence of alkyl groups on the amine function of chitosan seems to weaken the hydrogen bonds of chitosan with consequences on the crystal structure and on the physico-chemical properties. The engagement of the acetamido group in the complex further affects the hydrogen bonds; as a consequence the N-alkyl chitosan-lead membranes swell to such an extent as to rupture easily under osmotic pressure.
The selectivity shown by N-alkyl chitosan membranes toward certain metal ions is due to the preferential complexation of those metal ions able to react with the acetamido groups. N-Alkyl chitosans possess a reduced proportion of free amino groups in addition to relatively inaccessible secondary amino groups; therefore collection of metal ions which preferentially react with the primary amino groups is depressed in terms of extent and velocity.
The present study presents, for the first time, extended information on a series of N-alkyl chitosans. The data relevant to the metal ion chelation con- stitute a substantial contribution to the comprehension of the chelation process. In view of their behavior with water, the N-alkyl chitosans are pro- posed as novel semi-synthetic biopolymers useful in the production of mem- branes of medical interest, defoggers, deodorants, supports for delayed re- lease of fragrances and drugs. Their physical properties make them suitable for application in membrane and textile technology, as plasticizers of chito- san and for the production of N-alkyl chitin fibers, [ 11,17, 181.
Acknowledgements
The present work was carried out with the financial contribution of the Consiglio Nazionale delle Ricerche, Progetto Finalizzato Chimica Fine e Secondaria, Rome (Contract No. 82-00656.95). The skilful technical assistance of Dr. M.G. Ponzi Bossi, Dr. G. Laterza and Mrs. M.G. Muzzarelli is acknowl- edged.
References
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