Note: Descriptions are shown in the official language in which they were submitted.
FL/6-19038/A
N-Substituted chitosan derivatives. pr~cess ~or thelr preparation and the use thereof
The present invention relates to chitosan derivatives the amino groups of which are
substituted by hydroxyacyl that carries a carboxylic acid, phosphonic acid or sul~onic acid
group, or the esters and salts thereof, to a process for their preparation by react~ng a
chitosan with a 4- or ~membe~ed lactone or a 4-membered sultone that carries sulfonic
acid ester, phosphonic acid ester or carboxylic acid ester or CC13 ~OUpS, and subsequently
hydrolysing CCI3 groups, if desired converting ester groups into acid groups and acid
groups thereafter into salts, and tO the use ~hereof for preventing the adherence to and/or
formation of solid coats on inorganic or organic substrates, as well as to the use thereof as
humectants for s~n and mucous membranes.
Chitosan derivatives which ca~y carboxyaLI~yl-substitu~d amino gTOUpS are disclosed in
DE-A-2 222 733. They are prepared by reacting chitosan with a cyclic anhydri~e such as
succinic anhydride. Hydroxyi-substituted anhydrides are not mentioned and even excluded
in the description of ~he synthesis. These chitosan derivatives may be used as chelating
agents, detergents, and ~s additives for cosmetic or phannaceutical compositions.
SulfopIopyl derivatives of chitosan that are obtained by reacting chitosan with
1,3-propanesultone are disclosed in DE-A-3 432 227. These derivatives are used as
additives for cosmetis compositions.
K. Kurita et al., in Polymer Journal 22, No. 5, pages 429434 (199û), descnbe the reaction
of chitosan with ~-butyrolactone t~ prepare ~-hydr~xybutanoyl-sllbstituted chitosan
derivatives. E. Loubaki et al. in Eur. Polym. J, 25, No. 4, pages 379 -84 (1989) describe a
similar modification of chitosan with ~B-propiolactone and ~gluconolactone.
Chitosan derivatives the N-,substituent of which carries hydroxyl groups and acid groups,
and which are thereby capable of salt forma~on, are not yet known in the art. It has now
been ~ound that such chitosan denvatives are obtained in good yield and quality by using
instead of inert lactones those carrying an activadng group in o~-position to the ring
oxygen atom. They are suitable for many u~lities as substitute ~or hyaluronic acid.
- 2 -
In one of its aspects the inveneion relates to oligome.ric and polymeric chitosan derivatives
containing structural units of fonnulae I, II and m ill random dis~ibution
CH20R1 CH20Rl CH20Rl
~O~ H~O~o H~~ro
\~NH/ ~ ~H
Z--R2- X H NHR3 H ~3NH3Ye
I) (m),
wherein the substituents Rl are each independently of one another H or ehe :radical
-ZR2-X, R3 is H or acetyl, Y is the anion O-7.-R2-X, and
(a) Z is -CO- or -SO2-, X is -C02H, -CH2CO2H or -CII2PO(OH)2, and R2 iS
-CHR4CR5(OH~-, R4 is -H, -OH, Cl C4aLkoxy or Cl-C4aLk~YI, and Rs is H or Cl-C4aLlcyl,
or
(b) Z is -CO-, X is -CO2H, and E~2 iS -~6-(~7-CH~O~I)- or
-CHR8-CHRg-CHRlo-CH(OH3-, R6, R7, R8 and Rlo are each independently of one anoeher
-H, -OH, Cl-C4al1yl or Cl-C4alko~sy, and Rg is -H, -OH, Cl-~4aLkyl, Cl-C4aL~coxy or
-CO2H,
and the esters and salts thereof, which chitosan denvatives contain a total of at least
2 stmctural units and, based on 1 mol of the chitosan derivative, 30 to 100 % molar o~
structural units of formula I, 60 to 0 % molar of structural units of formula II, and 30 to
O % molar of struct~al lmits of foImula m, and the sum of the molar percentages is
100%.
In preferred embodiment of the invention, the chitosan derivative contains a total of at
least 4 structural units, oligomers preferal~ly containing 4 to 50, most pre~erably 6 to 30,
s~uctural units. Polymeric chitosan derivatives may typically contain up to 10 000,
preferably up to 8000 and, most preferably, up to 5000, s~ctural units.
In a further prefe~red embodiment of the invention, the chitosan delivative contains 50 to
100 % molar of structural u:nits of formula I, S0 to 0 % molar of structural units of
formula II, and 20 to 0 % molar of structl~l units of folmula m, the sum of the molar
,, .
r~ 3 2 i~
- 3 -
percentages being 100 %. Most preferably, the chitosan deriva~ive contains 60 tolQO % molar of structural units of formula I, 40 to O % molaT of structural units of
forrnula II, and 10 to O % molar of s~lctural units of formula m, the sum of the molar
percentages being 100 %.
Z in definition (a) of the structural units I, II and m is preferably -CO- and X is preferably
-CO2H or -CH2P~(OH)2, or ~ is -SO2- and X is -CO2H.
R4 to Rlo as aLIcyl may be methyl, ethyl, n- or isopropyl or n-, iso- or tert-butyl. Preferably
R4 to Rlo as alkyl are methyl or ethyl.
R4 to Rlo as aLIcoxy may be methoxy, ethoxy, propoxy or butoxy. Preferably R4 to Rlo as
aLkoxy are methoxy.
R4 and R6 to Rlo are preferably H, OH, methyl or methoxy and Rs is H or methyl.
Typical examples of R2 are -C~2CH(OH)-, -CH2C(CH3)(0H)-, -OEI(OH)CEI(r)H)-,
-CH(CH3)CH(OH)-, -CH(OCH3)C~(0~I)-, -CH2CH2CH(C)H)-, -OEI(OH)CH2CH(O~I)-,
-CH(OH)OEI(OH)CH(OH)-, -CH(CH3)~H2CH(OH)-, -CH2C~H(OH)CH(OH)-,
-CH(OCH3)CH(OH)CH(OH)-, -CH2CH(OCH3)CH(OH)-, -CH2CH2CH2CH(OH)-,
-CH2CH2CH(OH)CH(O~l)-,-CH2CH(OH)CH(OH)CH(OH)-,
-CH2CH(COOH)C~I2CH~OH)-, -CH2CH(OH)CH(CH3)CH(OH)- iand
-CH2CH(OH)CH(OC~13)CH(O~I)-. Most preferably R2 iS -CH2CH(OH)- i~md
-CH2C(OEI3)(0H)-.
The carboxylic acid and phosphonic acid group can be estenfied, ~pically with analiphatic, cycloaliphatic, araliphatic or aromatic alcohol which contains 1 to 30, 1 to 20
and, most preferably, 1 to 12, carbon atoms. Representative examples of such alcohols are
alkanols such as me~hanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol,
octanol, nonanol, decanol, undecanol, dodecanol, tetradecanol, octadecanol; po-
lyoxaaLlcanols such as ethylene g,lycol monome~yl ether or monoethyl ether, diethylene
monomethyl ether or monoethyl ether, oligoethylene glycols or oligopropylene glycols or
copolymers thereof containing a total of up to 20, preferably of up to 12, monomer units;
cycloaLlcanols such as cyclopentanol and cyclohexanol; benzyl alcohol and
Cl-cl2alkyl-substiblted benzyl alcohols; phenol and Cl-CI2alkyl-substituted phenols.
- 4 -
The carboxylic acid and phosphonic acid groups may also be in salt ~orm. They may be
metals of the main and subsidiary groups of the Periodic System of the elements, typically
the metals of the third, fourth and fifth main group and the subsidiary groups of the
Periodic System of the chemical elements. Particulalrly suitable metals are Li, Na, K, Mg,
Ca,Sr,Ba,B,Al,Ga,In,Sn,Pb,Sb,Bi,Cu,Ag,Au,Zn,~d,Hg,Sc,Y,La,Ti,~r,Hf,V,
Nb, Ta, Cr, Mo, W, Fe, Co, Ni, Ru, lRh, Pd, Os, k, Pt and ~he lanthanide metals Ce, Pr,
Nd, Pm, Sm, Eu, Gd, Tb, Dy, HO, Er, Tm, Yb and Lu. When using polyvalent metals, the
corresponding cations can act as crosslinkers of the oligomer and polymer chains.
Preferred metals are the alkali metals and aLkaline earth metals. The salts may also be in
the form of amine salts, conveniently of ammonium salts or salts of prim2ry, secondary or
tertiary amines which pre~erably contain 1 to 20 and, most preFerably, 1 to 12, carbon
atoms, or of salts of polyamines containing primary, secondary andlor tertiary amino
groups and, preferably, 2 to 20, most preferably, 2 to 16, carbon atoms, or of salts of a
polymer containing amino groups in structural repeating units.
Typical examples of amines are: methylamine, dimethylamine, trimethylamine,
ethylamine, diethylamine, triethylan~ine, n-propylamine, isopropylamine,
di-n-propylamine, diisopropylamine, tri-n-propylamine, triisopropylamine, n-butylamine,
di-n-butylarnine, tri-n-butylamine, hexylan~ine, dodecylamine, octadecylamin,
icosylamine, morpholinw, N-methylmorpholine, piperidine, N-methylpiperidine, aniline,
N-methylaniline, N-dimethylaniline, pyridin, pyrimidine, ethanolamine, die~hanolamine
and triethanolamine.
Typical examples of polyarnines are: ethylenediamine, N,N'-dimethyle~hylenediarnine,
diethylenetriamine, triethylenetetramine, 1,3-diaminopropane, 1,3-dimethylaminopropane,
1,4-diaminobutane, piperazine, phenylenediamine, naph~hylenediamine,
4,4'-diaminodiphenyl, 4,4'-diaminodiphenyl e~her, 4,4'-diaminodipherlyl thioether and
4,4'-diaminodiphenylmethane.
Typical examples of polymers containing amino groups are poly(aminosaccharides) such
as chitosan itself and polygalactosamine, albumin or polyethylenimine, low molecular
polyamides ca~rying amino end groups and aminoaL~cylated polyacrylamides or polyme-
thacrylamides.
The chitosan derivatives can be prepared in sirnple manner by reacting chitosan with ~-, y-
or ~-lactones or ~B-sultones which contain an esterified carboxyl group or a CCI3 group in
- s -
~-position to the ring oxygen atom, and subsequently hydrolysi:ng the CC13 group and, if
desired, conver~ng the ester groups into acid groups and the acid groups into salts.
In another of its aspects, the invention relates to a process for the preparation ~f the novel
chitosan derivatives containing a total of at least 2 structural units of formulae I, II and III
in random distribution, which process comprises reacting a chitosan containing a total of
at least ? structural Imits of formulae IV and V in random distnbution,
CH20H CH20H
-aV), ~-(V),
H NH2 H NHR3
wherein R3 is acetyl or H, and said chitosan contains 30 to 100 % molar of structural units
of formula IV and 70 to 0 % molar of structural units of forrnula V, based on 1 mol of
chitosan, in the presence of an inert solvent, with at least 30 % molar of a lactone of
formula VI, VIl or YIII, based on said chitosan,
x~ ~R4 R7~_~R6 R~R8
1--11 X/~O~O XlO~o
wherein R4, R5, R6, R7, R8, Rg and Rlo have the mear~ings previously assigned to them, 2;
is =CO or =SO2, Xl is -CC13, -CO2Rll, -cH2cr)2Rll or -c~2Po(oRll)2~ X2 is -ct32Rll,
and Rll is the radical of an alcohol of l to 20 carbon atoms which lacks the hydroxyl
group, and hydrolysing the resultant chitosan derivatives, wherein Xl is -CU3, under
alkaline condidons to form the corresponding carboxylic acid salts, if desired conver~ng
the carboxylic acid salts ancl esters into the colTesponding acids and the acids into salts.
By adjusting the pH of the reaction mixture, the carboxylic acid salts can be conver~ed
into the coIresponding acids,, which are also isolated as such.
Surprisingly, the inventive process makes it possible for the ~lrSt dme to prepare
- 6 -
N-acylated chitosans containing acid and hydrs~xyl groups in the N-acyl group.
Particularly advantageous is the use of the ,B-lactones and ,B-sl1ltones which contain CCl3
groups, as these CCl3 groups can be converted in sirnple manner into the carboxyl group.
The reaction leads under mild conditions to high chemical conve~sions such that even
complete substitutions at the NH2 group of the chitosan can be achieved and side-reactions
substantiaUy avoided.
In yet another of its aspects, the invention relates to ~he intermediate chitosan derivaeives
containing structural units of formulae IX and X in random distribution,
CH20R12 CH20R 12
~0_
Z--R2--X3 H NHR3
(IX) (X)
wheIein the Rl2 substituents are each independently of the other H or ehe radical
-ZR2-X3, 1;~3 is H or acetyl, Z is -CO- or -SO2-, X3 is -CCl3, and R2 is -CHR4CRs(OH)-,
R4 is -H, -OH, Cl-C4aLtcoxy or Cl-C4allyl, and Rs is H or Cl-C4aLlcyl, which chi~osan
derivative contains a total of at least 2 structural units and, based on l mol of s~ud chitosan
derivative, 30 to l00 % molar of structural units of formula IX and 70 to 0 % molar of
structural units of ~ormula X.
Rl2 is preferably H, and X3 is preferably -CCl3. Z is preferably -CO-. R2, R4 and Rs have
the preferred meanings previously assigned to them. Most preferably R2 represents the
radicals -CH2CEI(OH)- and CH2C(C~I33(0H)-. With respect to the number of s~uctural
units and the content of structural units, reference is made to the preferences stated
previously concerning the novel chitosan acids, esters and acid salts.
It has been found useful to activate the commercially available chitosan before the
reaction by dissolving the chitosan in e.g, dilute acetic acid, removing undissolved
constituents by filtration ancl then neutralising with a dilute aqueous base, conveniendy
NaOH, until the pH is c. 8-9, so that the chitosan again precipitates. The salts are removed
7 t'lJ ii ~
by known methods, typically by washing off or dialysis. The product can then be
hyroextracted by repeated centrifugation with a non-solvent, conveniently an ether such as
dioxane, to give products with water contents of c. 3 to 10 % by weight which are highly
swollen and have a high reactivity. Depending on the reaction conditions and on the water
content of the chitosan used, it is possible during the reacdon by hydrolysis to obtain
hydroxycarboxylic acids which lead to chitosan derivatives containing structural units of
formula III by salt for~nation. The reaction can, however, be completely suppressed by
rnild conditions, so that it is also possible to obtain chitosan derivatives which do not
contain structural units of formula m. Under drastic ~action conditions7 and given a high
water and acetyl group content of the chitosan, it is possible to obtain highly swellable
gels as final products which arç readily crosslinked.
In the course of the reaction a limited reaction of the free hydroxyl groups with the
lactones can also take place; but this reaction can be substantially suppressed by ~he
choice of reaction conditions. Chitosan derivatives wherein R1 is hydrogen in the
structural units of formulae I, II and m are preferred.
The amount of structural units of formula II will depend on the one hand on the content of
acetyl groups in the cl~itosan and, on the other, on the reactivity of the lactones used and
on the degree of substitution which is attainable. Commercially available chitosans can
contain up to 70 % mvlar of acetyl group containing s~uctural units of formula V.
Oligomeric chitosans can be typically obtained by hydrolytic degradation of the polymers,
for example with a dllute mineral acid such as hydrochloric acid. The mixtures of
oligomers obtained are then neutralised with e.g. NaOH and freed firom salts and low
molecular constituents by ultra~lltration through a membrane. The resultant mixtures of
oligomers can be used as obtained or fracdonated beforehand in known manner.
Oligomeric chitosans can also be obtained by deacetylation of chitosan oligomers, for
example deacetylated chitobiose or chitohexaose, which are also commercially available.
The lactones suitable for use in the practice of this invention are known, some being
commercially available or obtainable by known methods.
The inventive process may be calTied out by dissolving or suspending the chitosan in a
solvent and then adding the solution or suspension to a solution of the lactone. This step is
preferably carried out at room temperature. Afterwards, the reacdon mixture is s~rred at
f~ U
- 8 -
room or eleYated temperature and the reaction is allowed to go to completion. The reaction
can be calTied out in the temperature range from 0 tO 150C, preferably frorn 10 to 120C
and, most preferably, ~om 20 tO 100C.
The reaction is conveniently carried out excluding moisture, ~or exam~le humidity or
water in solvents. In general, the process is conveniently carried out in a dry inert gas
atmosphcre, typically a rare gas (helium or argon) or nitrogen.
Suitable solvents are typically polar aprotic solvents which can be used singly or as
rnixtures of at least two or more solvents and in which are the chitosan dissolves or swells.
The solvents can also be used as suspension agents. Exemplary of such solvents are ethers
(dibutyl ether, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, ethylene glycol
diethyl ether, diethylene glycol diethyl ether, triethylene glycol dimethyl ether),
halogenated hydrocarbons (methylene chloride, chloroform, 1,2-dichloroethane, 1,1,1-tri
chloroethane, 1,1,2,2-tetrachloroethane), N-aLkylated carboxan~ides and lac~ams
~N,N-dimethylformamide, N,N-diethylformamide, N,N-dimethylacetamide,
tetramethylurea, hexamethylphosphoric triamide, N-methylpyrrolidone,
N-acetylpyrrolidone, N-methylcaprolactam), sulfoxides (dimethyl sulfoxide), sulfones
(dimethyl sulfone, diethyl sulfone, trimethylene sulfone, tetramethylene sulfone),
substituted benzenes (benzonitlile, chlorobenzene, o-dichlorobenzene, 1,2,~tri-
chlorobenzene, nitrobenzene), nitriles (acetonitrile, propionitrile). Also suitable are aroma-
tic-aliphatic ethers such as methyl or ethyl phenyl ether, and ke~ones such as acetone,
methyl ethyl ketone, methyl propyl ketone, dipropyl ketone, dibutyl ketone and methyl
isobutyl ketone.
A group of preferred solvents comprises cyclic ethers, N-aLkylated acid amides and
lactams, sulfoxides and sulfones.
The reaction products can be isolated and purified either by known processes or
subsequently hydrolysed direct andlor converted into salts.
The reaction products contz~ining CCl3 groups are conveniently hydrolysed wi~ aqueous
alkali metal bases, typically KOH or NaOH, and, depending on the adjustment o~ the pH
after the reaction, the acids or their sodium or potassium salts can be isolated in per se
known manner.
L-~ ~ r~
The novel esters can likewise, as described above, be hydrolysed with aqueous alkali
metal bases to the acids or converted into the salts. Novel esters, for example benzyl ester,
can also be converted catalytically with hydrogen in the presence of a noble metal catalyst
into the desired carboxylic acids. It is further possible to convert the ester group into a
readily hydrolysable group, for example with trimethylbromosilane, and then to Temove
this group by hydrolysis to forrn carboxylic acid groups.
The preparatdon of salts from the novel chitosan acids or their aLkali metal salts can be
carried out in per se known manner by reacdon with metal salts of e.g. mineral acids or
carboxylic acids in aqueous solution, in which case - especially when using polyvalent
metal salts - the chitosan salts form insoluble polyelectrolyte gels. Suitable metals are
those previously cited. Suitable metal salts are typically oxides, hydro7~ides, fluorides,
chlorides, bromides, sulfates, nitrites, nitrates, phosphites, phosphates, ~ormates and
acetates.
The preparation of salts from the novel chitosan acids and ammonia, mono- or polyamines
or polymeric polyamines can be calTied out in ~he same manner. Water-insoluble
polyelectrolyte gels are also obtained when using polyamines or polymeric polyamines.
The novel chitosan derivatives can be isolated by known methods, typically filtration,
whereas soluble products can be precipitated beforehand by addition of non-solvents or by
adjusting the pH. The products can be isolated by dialysis or by passage over ion exchange
resins. For purification, the products can be washed and then dned, but without a complete
drying being necessary, and the purified products may have water contents of up to
c. 40 % by weight and more. The products can also be milled to powders. Lyophilisadon
is particularly useful, as buLlcy and cottonwool-like products are obtained which are
especially readily soluble and very reac~ve.
The novel chitosan deriva~ves are solid products which are readily soluble or highly
swellable in aqueous or aqueous-aLkaline media or in polar organic solvents. The free
acids or their salts with non-toxic cations are physiologically acceptable and
biodegradable. The products are suitable for a wide range of utili~ies.
The novel chitosan derivatives, especially the salts and acids, are polyampholytes with
film-forming and chelating properties also in the presence of alkaline ear~h metal ions.
Owing to their pronounced chelating action for heavy metal ions even in low
2 ~ 3
- 10-
concentrations, the products can be used for removing such cations from contallunat~d
water, for example for removing iron or copper ions from mains water. In addition, they
can be used as chelating agents in the food industry, the pharmaceutical industry and the
textile industry, as well as ,detergents singly or in conjunction with cationic, anionic or
neutral detergents.
The novel chitosan derivatives have a sorprisingly good complexing ac~on for metal ions,
whereby the precipitation of polymeric metal salts, especially in the case of polyvalent
cations, can be favourably influenced or prevented. Furthermore, they have a modulating
action in crystallisation processes, especially on the formation of seed crystals, their
growth and the morphology of the resultant crystals and their size distribotions, as well as
on the aggregation and adhesion properties. They are therefore suitable for water treatment
to prevent the formation of deposits in water-conducting systems (water treatment plants),
for exarnple on the walls of containers, membranes or conduits. They can also be used for
the pretreatment of textiles, conveniently cotton. The novel chitosan derivatives also
prevent the formation of deposits of inorganic and/or organic components. They are
therefore also suitable for use as additives for dental care products for the prevention of
dental plaque, as well as additives for detergent formulations.
The invention fi~her relates to a process for the prevention of the adherence to andfor
formation of solid deposits on inorganic or organic substrates, which comprises add;ng to
a fluid or a composition that is in contact with an inorganic or organic substrate at least
one of the novel chitosans, but preferably 0.01 to 20 % by weight, most preferably 0.1 to
10 % by weight.
The novel chitosan derivatives have film-forming properties. The evaporation of aqueous
solutions leads to the folmation of transparent, solid and water-containing films which are
permeable to air and moisture. By vir~ue of this property and their hydropectic action, they
are also suitable for use as humectants for the skin or mucous membranes in cosmetic and
pharmaceutical compositions, as agents for maintaining articular mobility (lubricant action
simlar to that of hyaluronic acid), and for surgical dressings. Typical cosmeticformulations are skin and hair care products and deodorants. The novel chitosan
derivatives, especially gels made therefrom and also the esters, are fur~er suitable ~or the
preparation of compositions with controlled release of the chemical agent over aprolonged period.
h ~ 5 ~ 3
- 11 -
The novel chitosan derivatives also have a viscosity increasing and dispersing action in
aqueous solutions. They are thus suitable for use as additiv~s in suspensi~ns, emulsions
and aqueous solutions, for example in the manufacLure of foodstu~fs or active sobstance
concentrations as well as in dye and pigment forrouLltions.
I he novel chitosan derivatives can also have biocidal activity, typically bacteriostatic,
fungistatic or algicidic activity.
Especially preferred, and a further object of the invention, is the use of the novel chitosan
derivatives, preferably the acids or alkali metal salts, for preventing the adhesion to and/or
formation of solid deposits on inorganic or organic substrates. The deposits, which often
have a crusty consistency, may be composed of inorganic and/or organic components,
typically salts and polymers, also of biological origin. The substrates may be inorganic
andlor organic materials or biologlcal materials, for example glass, ceramics, metals and
alloys, natural or synthetic plastic materials, paper, textiles, leather or vegetable or animal
organs or tissues. Yet a further object of the invention is the use of the novel chitosan
derivatives as humectants ~or the skin or mucous membranes.
It has also surprisingly been ~ound that the novel chitosan derivatives can be crosslinked
with polyepoxides to give products which are swellable, but insoluble~ in water with good
mechanical properties.
The invention filrther relates to crosslinlced chitosan derivatives obtainable by reaction of
novel chitosan derivatives with at least one polyepoxide that con~ains on ave~age at least
two epoxy groups in the molecule.
Suitable polyepoxides are typically glycidyl compounds containing on average two epoxy
groups in the molecule. Particularly suitable glycidyl compounds a~e those having two
glycidyl groups bonded to a hetero atom ~e.g. sulfur, preferably oxygen or nitrogen),
J~-methylglycidyl groups or 2,3-epoxycyclopentyl groups. Typical examples are preferably
bis(2,3-epoxycyclopentyl) ether; diglycidyl ethers of polyhydric aliphadc alcohols,
typically 1,4-butanediol, or polyaLkylene glycols such as polypropylene glycols; diglycidyl
ethers of cycloaliphadc pol~yols such as 2,2-bis(4-hydroxycyclohexyl)p~opane; diglycidyl
ethers of polyhydric phenols such as resorcinol, bis(p-hydro~yphenyl)methane,
2,2-bis(p-hydroxyphenyl)propane (=diomethane), 2,2-bis(4'-hydroxy-3',5'-dibromophen-
yl)propane, 1,3-bis(p-hydroxyphenyl)ethane; bis(B-methylglycidyl) ethers of the above
- 12 - ~ 3 l ~3
dihydric alcohols or dihydric phenols; diglycidyl esters of dicarboxylic acids such as
phtha]ic acid, terephthalic acid, ~4-tetrahydrophthallc acid and hexahydrophthalic acid,
N,N-diglycidyl derivatives of primary amines and amides and heterocyclic nitrogen bases
that carry hVO N-atoms, and N,N'-digiycidyl derivatives of disecondary diamides and
diamines, including N,N-diglycidylaniline, N,N-diglycidyltoluitline,
N,N-diglycidyl-p-aminophenyl methyl ether, N,N'-dime-
thyl-N,N'-diglycidylbis(p-aminophenyl)methane; N',N"-diglycidyl-N-phenylisocyanurate;
N,N'-diglycidylethylene urea; N,N'-diglycidyl-5,5-climethylhydantoin, N,N'-di-
glycidyl-5-isopIopylhydantoin, N,N-methylenebis(~;~',N'-diglycidyl-5,5-dimethylhytlan-
toin), 1,3-bis(N-glycidyl-5,5-dimethylhydantoin)-2-hydroxypropane; N,N'-diglycidyl-
5,S-dimethyl-6-isopropyl-S,~dihydrouracil, ~iglycidylisocyanurate.
Preferred epoxides are those that are soluble in strongly polar solvents and, more
particularly, those that are soluble in wa~er.
A preferred group of polyepoxides comprises glycidylated aliphatic diols and poly-
oxaalkyIene diols, novolaks, hydantoins, aminophenols, bisphenols and aromatic diarnines
or cycloaliphatic epoxy compounds. Particularly preferred polyepoxides are glycidylated
aliphatic diols and polyoxaaLkylene diols, cresol novolaks, diglycidyl ethers of bisphe-
nol A and bisphenol F, or rnixtures thereof.
Particularly prefeIred a~ water-soluble glyridyl aliphatic diols and polyoxaalkylenediols,
as the rnixing with the novel chitosan derivatives can be calTied out in simple manner in
aqueous systems.
To prepare novel crosslinked products it is also possible to use in addition cu~ing
accelcrators. Typical examples of curing accelerators are 3-ethyl-4-methylimidazole,
triamylammonium phenolate); mono- or polyphenols (phenol, diomethane, salicylic acid);
and phosphoric acid. Curing accelerators and catalysts are normally used in an amount of
0.1 to 10 % by weight, based on the polyepoxide.
The amount of polyepoxide will depend mainly on the desired degree of crosslinking and
the properties associated therewith. Advantageously up to SO %, preferably up to 35 %
and, most preferably, up to 25 %, of the stluctural units of the novel chitosans are
crosslinked.
The preparation of the crosslinked derivatives can be carried out in a manner known per
se~ conveniently by rnixing the components together with an optional solvent or
suspension agent which is then removed by heating. The mixture can be thermally
crosslinked, typically by heating to 50-200C.
The crosslinked chitosan derivatives are particularly suitable for the preparation of water
swellable and mechanical stable moulded articles, such that shaping can be combined with
the preparation. It is thus possible to prepare f;lms and foils which can be used as
membranes or surgical dressings, or to make capsules or encapsulations for chcrnical
agents the release of which to the environrnent is delayed and continuous.
The following Examples illustrate the invention in more detail.
A) Preparation of inte~mediates
Example A1. Reacdon of a chitosan with R(-)-4-trichloromethyl-2-oxetanone.
a) Activation of the chitosan
To activate the chitosan, a commercial product (~luka: average molecular weight
M ~ 75 000, acetyl group CQntent 4.5 %) is dissolved in 5 % acetic acid and undissolved
constituents are removed by filtration. The product is precipi~ated again by addition of
2 N sodium hydroxide solution to the fil~ate until the pH is 8-9. The swollen white
product is freed from salts by repeated centrifugation, decantation and resuspension in
distilled water or by dialysis of the aqueous suspension. Afterwards the product is dried by
repeated centrifugation with dioxane up to a water content of 9.8 %. Titration of a
lyophilised sample of this material with 0.1 N HC1, taking into account the water content
of 9.8 %, gives a base content of 5.11 mes~/g. The dioxane-containing activated polymer
gel is used for most of the subsequent reactions.
b) Reaction with R~2~-trichloromethy1-2-oxetanone
546.2 g of the chitosan gel (25 g of chitosan = 0.115 mol) are suspended in 500 rnl of dry
N-methylpyrrolidone (NMP) with the addition of 25 g of LiCI and the suspension is added
in increments at room temperature to a solution of 58.9 g of R(-)-4-(trichloromethyl)-2-
oxetanone (0.310 mol) in 1 1 of NMP. The resultant suspension is stirred for 1 hour under
nitrogen and excluding moisture in a sulfonation flask fitted with reflux condenser, blade
7 ~
- 14 -
stirrer and internal thermometer, then heated to 55C and kept at this temperature ~or
24 hours. After cooling, the clear gel obtained is precipitated, with efficient s~Ting, with
S litres of acetone. T} e ~me suspension is ~lltered over a sintered suction filter and washed
free of chloride with distilled wa~er. The product is suspended once more in methanol and
again collected by ~lltra~don. A small sample is cautiously dried under vacuum and the
ClJN ratio of elemental analysis is 3.06.
Examples A2-A10: The products listed in Table 1 are prepared in accordance with
Example Al. In Examples A2 and A5 R-t~ichloromethyl-2-oxetanone is used. In
Example A3 S- trichloromethyl-2-oxetanonine and in Example A4 R,S-4-trichloro-
methyl-2-oxetanone is used; and in Examples A9 and A10 S-4-trichlorome-
thyl-~methyl-2-oxetanone is used. The solvent is dirnethyl sul~oxid ~MSO)
(Examples A2 to A4 100 ml, Examples A5 to A7 800 rnl, Example A8 400 rnl,
Examples A9 and Alû 200 ml). The amount OI LiCl is ~ g (Examples A2 to A4), 40 g(Exarnples A~ to A7), 20 g (Example A8~, 10 g (Examples A9 and A10). Further
particulars will be found in Table 1. In Example A9 the reaction time is 32 hours a~ 80C
and in Example A10 24 hours at 80C and 24 hours at 1û0C. In the last column ~he
content of Cl and N is given in m eq/CCI3/g m eq N/g and in brackets the CVN rado.
2 ~ ~ 2 .~ 1 3
- 15 -
Table 1
~x. Chitosan Amount of Degree of Cl/N content
No. oxetanone subst. (%)
A2 S.Og=0.031mol 11.7g=0.062mol 92 8.43/2,71
sigma; 6.3% acetyl ~3.11)
A3 5.0g=0.031 mol 11.7 g=0.062mol lOS) 9.02/2.57
Fluka;M 2x106; (3.5
4,5% Ace~l
A4 S.0 g=0.031 mol 11.7 g=0.062 mol 1~ 9.22/2.49
Fluka; M 2x106; (3.7)
45% acetyl
A5 20 g--0.124 mol 47.10 g=0.248 mol 9S 8.09/2.48
Fluka; M 2x106; (3.26)
4.5% acetyl
A6 20 ~,~.124 mol 47.10 g~.248 mol 94 8.47/~.50
Fluka; M 7.5x105; (3.38)
4 % acetyl
A7 20 g=0.124 mol 47.10 g~.248 mol 97 8.26/2.49
Fluka;M 7x104; (3.31)
4.3 % ace~l
A8 10 g=0.062 mol 23,6 g=0,124 mol 100 7~95/2,57
chitosan of Ex. A6, (3.01
activated
A9 5.0 g=0.031 mol 12.6 g=0.062 mol 55 6.06/3.24
Fluka;M 2x106; (1.84)
4.5 % acetyl
A10 S.0 g=0.031 mol 12.6 g=0.062 mol 61 4.94/2.99
of activated chitosan (1.65)
acc. Ex. Al, but from
dioxane lyophilised
pC
- 16-
Example A 11:
a) 20 g of chitosan ~luka, M 7.5x105) is partially de,graded with 1~0 ml of HCl (37%) at
75C over 75 rninutes to chitosan oligomers [A. Domard et. al., Int. J. Biol. Macromol. 11,
297 (1989~]. The mixture of oligomers obtained is neutralised to pH 8.5 with 2N NaOH
and freed from salts and very low molecular constituents by ultrafiltration through a
membrane with a cut-off level of 1000. After Iyophilisation, a rnixture of oligomers
having an average degree of polymerisadon of c. 6-20 is obtained.
b) 0.7 g (4.34x10-3 mol) of the dried mixture of oligomers is suspended in 15 ml of dry
DMSO together with 750 mg of LiCl and the suspension is then added, with stirring, to a
solution of 1.65 g (8.64x10-3 mol) of R,S-4-(tTichloromethyl)-2-oxetanone in 15 ml s)f
DMSO containing 750 mg of LiCI. The mixture is stirred in an atmosphere of dry nitrogen
for 3 hours at room temperature and then -for 10 hours at 50C. The oligomeric reaction
product is precipita~ed with the lû-fold arnount by volume of dichloromethan. Piltration
over a glass suction filter and washing off with CH2Cl2 and methanol until the filtrate is
free from chloride, followed by vacuum drying, gives 950 mg ~f a brownish powdered
product which has a Cl/N ratio of 1.52.
Exarnple A12: In accordance with the general procedure described in Exarnple Al 1,
150 mg (7.59x104 mol) of chitohexaose, prepared ~om cornrnercially available
chitohexaose hexahydrochloride (lkara Chem. Ind. Tokyo) by neutralisation with tri-
ethylarnine, are reacted in 10 ml of dry N-methylpyrrolidone with 2&8 mg
(1.52x10-3 mol) of R(-)-4-trichloromethyl-2-oxetanone. Yield: 246 mg (theory 26S mg) of
a beige product which is saponified direct (cf. Exarnples B3 and B4).
Exarnple A13: In accordance with the general procedure described in Example Al, 2.0 g
(1.24x10-2 mol) of a chitosan product which contains 50 % of amino groups and 50 % of
ace~lamino groups (chitin 50TM, Protan A/S, Norway)are reacted in N-methylpyrrolidone
(80 ml) with the addition of 4 g of LiCl with 2.36 g (1.24x10-2 mol) OI
R(-)-4-(trichloromethyl)-2-l~xetanone. To bring the reaction to completion, the reaction
mixture is sti~ed for 18 hours at 80C and for a further 8 hours at 105C, giving 1.88 g
(59 % of theory) of a brownish beige powdered product which has a CVN ratio of 0.55,
corresponding to a degree of substitution of 36.6 %.
Example A14: In acordance with the procedure described in Example Al, S g of chitosan
-17- ~2~ S~;3
are activated and the dioxane-containing gel (69.1 g) is suspended in 100 ml of DMSO in
which 5 g of LiCl has been dissolved. The suspension is added under N~ to a solution of
14 g (6.2x10-~ mol) of R,S-4-trichloromethyl-,B-sultone ~D. Borrrnann et. al., Chem. Ber.
99, 1245 (1966)] in 100 mL of dry DMSO and 5 g of LiCl and the mixture is stirred at
room temperature. After 15 minutes the clear yellowish gel obtained is s~rred for 12 hours
at room temperature. Afterwards the reaction mixtun~ is precipitated in 2 litres of acetone
with vigorous stirring. For purification, the yellowish product is stirred twice in fresh ace-
tone and then swollen in 200 mL of methanol and in ]L litre of water to ~emove LiCI. The
product is filtered on a glass suction filter, washed free of chloride with water, IyophiLised,
and the lyophilisate is dried under 104 mbar for 24 hours. Yield: 6.9 g (58 % of theory) of
a pale yeLLow polymer powder with a ClIN ratio of 3.09 m and a C~IS ratio of 3.31 as
found by elementaL analysis.
Example A15: In acordance with ~he general procedure described in ~xample A14, 2 g
(1.24x10-2 mol) of chitin 50 in 80 ml of dry DMSO containing 4 g of LiCl are reacted with
2.79 g (1.24x10-2 moi) of trichloromethyl-,B-sultone. The reaction mix~lre is reacted for
24 hours at 50C and ~hen for 5 hours at 80C. Yield: 2.44 g of a white polymer which has
a Cl~N ratio of 1.1.
B) Preparation of the chitosan derivatives
Example B 1 The product of Example A1 is subjected direct and moist with methanol to
s2ponification. This is done by suspending the product in 750 ml of water and adding a
solution of 34.1 g of NaOH in '~54 rnl of water over 45 minutes while cooling with ice.
The viscous suspension so obtained is sdrred for a further 14 hours at 0-5C, then ~or
another 8 hours at room temperature. Titration of the reaction mixture with lN HC1 shows
that 3.2 equivalents of the base have reac~ed, i.e. the reaction is complete. The product
solution is adjusted to pH 8.0 with 2N HCI and filtered on- a glass suction filter To remove
inorganic salts, the solution is subjected to ultra~lltration and the chloride-free soludon is
then lyophilised. The white, water-soluble product of stryopor-like consistency is dried
under a high vacuum. Yielcl: 29.9 g (69.5 % of theory); water content 19.11 % by weight;
carboxyl content 3.57 m eq,/g (theory 3.61).
Example B2: To prepare the sodium salt, the calculated amount of 2N sodiurn hydroxide
solution is added to a 3 % aqueous solution of the product obtained in E3xample B1. The
solution is dialysed and then lyophilised, giving a quantitative yield of a white highly
2 ~ G~
- 18 -
porous product that is readily soluble in water.
Example B3: The saponification of the product of Example A12 with 5 equivalents of
NaOH in aqueous suspension according to Example B 1 gives the sodium salt of
c hitohexaose-hexamalamide in a yield of 135 mg (73 % of theory) after the product has
been freed from salts in a dialysis tube with a cut-off level of M = 1~00 (Spec~apor
No. 6).
Example B4: The corresponding hexacarboxylic acid is prepared from the salt of
Example B3 by dissolving the sodium salt in 5 ml of water. The solution is acidi~led with
0. lN HCl to pH 3.0, followed by fresh dialysis as described in Example B3. Yield: 93 mg
(77.5 % of theory) of a slighdy yellowish powder which has a carboxyl group content of
3.~2 m eq/g.
Example B5: In acordance with the general plocedure described in Example A14, 10.0 g
(0.062 mol) of activated chitosan (138.2 g of dioxane-containing gel) are reacted in 400 ml
of dry DMSO and 16 g of LiCI with 13.7 g (0.062 mol) of 4-(diethylphosphonomethyl)-2-
oxetanone (J.G. Dingwall et. al., J. Chem. Soc., Perkin Trans I 1986, p. 20813. The
reaction rnixture is stirred for 24 hours at 60C and then worked up, giving 7.5 g of a
white polymer powder which has a P/N ratio of 0.12.
Example B6: In a 100 ml sulfonation flask equipped with stirrer, reflux condenser and
thermometer, 3 g (0.0145 mol) of ben~yl 2-oxetanone-4-carbw~ylate are dissolved in
25 ml of dry dioxane. To this solution are added 2.31 g (0.0132 mol) of activated chitosan
(according to Example A1; 33.1 g of dioxane-containing chitosan gel) at room
temperature ~nd the reac~iion mix~ure is stirred for 18 hours. The reaction mixture is then
heated for 8 hours to 60C and for a further 48 hours to 100C The consumption of
,B-lactone in the reaC1iOn mixture as an indication of the progress of the raection can be
monitored by thin-layer chromatography on silica gel with toluene/ethyl acetate 1:1 as
eluant. The reaction product is subsequently precipitated in ~00 ml of diethyl ether,
collected by filtration and washed with 3x100 ml of diethyl ether. The product is
suspended in 50 ml of dioxane and then lyophilised, and the lyophilisate is dried under
10-2 mbar for 24 hours over phosphorus pentoxide. Yield: 2.85 g (~8.8 % of theory) of a
white polymer powder whic:h according to elemental analysis is substituted to a degree of
66.2 %.
d ~
- 19-
Example B7: To remove the benzyl protective g;oup from the product of Example B6, 2 g
of the product are dissolved in 100 ml of dry tet;ahydrofuran and, after addition of 40 mg
of palladium acetate, hydrogenated at 50C and 50 bar hydrogen pressure for 21 hours~
~he catalyst is afterwards removed by filtration on a glass suction filter. The polymeric
acid is extracted from the filter residue with 0.1N NaOH and combined with the filtrate,
which is also adjusted to pH 8. After dialysis through a membrane with a cut-off level of
1000 and subsequent lyopnilisation there are obtaine~d 1.48 g (83 % of th~ory) of a white
powder which is the sodium salt of the polymeric acid. No more signals of the benzyl
group can be detected in tne lH-NMR spectrum of the polymer in DMSO-d6.
Example B8: In acordance with the general procedure described in Example B 1, the
product of Example A14 is saponified with sodium hydroxide solution. This is done by
adding a soluition of 2.85 g (r/'1.12x10-3 mol) of NaO~l in 23 ml of water to a suspension
of S g (12.93x10-3 mol) of the product in 5 ml of water and stirling the rnixture for
2 hours, whereupon the suspension becomes a clear solution which is s~rred for a further
12 hours at 0-5C. Titration of the reaction mixture with 0. lN HCl snows that
3.o4 equivalents of sodium hydroxide have been consumed. For working up, the reaction
mixture is freed from minor amolmts of gel by filtration on a glass suction filter, adjusted
to pH 3.0 with 2N HCI solution while cooling with ice, and subseq~uen~ly dialysed against
dis~led water through a membrane with an exclusion limit of 1000 Dal~on. The dialysate
is lyophilised and the product is dried under 10-2 mbar for 24 hours. Titration of the
yellowish polymer obtailled in 65.3 % yield (2.61 g) with 0.1 N NaOH shows a carboxyl
group content of 1.67 m eq/g.
Examples B9-B17. In acordance with the general procedure described in Example Bl, the
products listed in Table 2 are saponified with NaOH and the acids isolated.
Table 2
Exarnple Educt of Carboxyl group content
No. Example No. ~m eq/g)
B9 A2 2.8
B 10 A4 3.05
Bll A3 3.1
- 20 -
B12 A5 2.~
B13 A6 3.58
B 14 A7 3.36
B15 A8 3.65 (100 % of theory)
B16 A9 1.73 (50 % of theory)
B17 A13 0.98 (50 % of theory)
Exa~8: Crosslinking of chitosan-mala~Dic ac;d with metal ions or polyamines to
form hydrogels.
1 ml of a 3 % aqueous solution of the following reagents is added at room temperature to a
0.5 % aqueous solution (1 ml) of the sodium salt obtalned in ~xample B~. The
crosslinking is indicated by the ~ormation of an insoluble precipitate in the form of a
hydrogel:
Salt Consistency
aluminium acetate white hydrogel
- iron(III) nitrate yellow hydrogel
polyethyleneimine white hydrogel
chitosan C~13COOH white hydrogel
albumin (from beef
blood) white hydrogel
Fxample B 19: TIiethanolamine salt of chitosan malamic acid.
400 mg (0.00144 mol) of the product of Example Bl a~e dissolved at room temperature in
100 ml of dis~lled water and undissolved constituents a~e removed by ISltration on a glass
suction filter. Then 215 mg (û.00144 mol) of triethanol~mine are added dropwise to the
cle~r solution. The viscous solution can be applied in a thin layer (1000 ~m) with a doctor
knife to glass plates or polyester sheets and air d~ied. The thin, glass-clear films obtained
bond well to glass, but can be easily removed from a polyester substrate.
Lyophilisation of the solution gives the triethanolamine salt of chitosan malamic acid in
the form of a white, buL~cy powder which is readily soluble in water
Example B20: Crosslinking of chitosan malamic acid with diepoxides to form hydrogel
films.
4~
- 21 -
A solu~ion of 0.01 ml of the diglycidyl ether of 1,4-butane diol in 1 ml of water is added at
room temperatu}e to 22 ml of a 0.3 % aqueous solution of the product of Example B2. The
resultant clear solu~on is cast to a ~llm on a polyester substrate as described in
Example Bl9. The film is dried to give a clear film which is crosslinked by heating for
3 hours to 100C and which can be hardened to a coating which is swellable, but no longer
soluble, in water.
Example B21: Preparation of a polyelectrogel.
Equimolar amounts of a 0.024 molar aqueous soludon of a product of Fxample B2 and a
0.062 molar aqueous solution of the chitosanlacetic acid salt are mixed, with vigorous
stirnng. The resultant precipitate is collected by filtration, washed with water until neutral
and lyophilised. The bully polyelectrolyte powder is dried under 10-2 mbar for 24 hours. It
contains solely chitosan and malic acid as components and, despite the sharp drying, has a
residual water content of 14.8 % by weight. The product swells in water to a glass-clear
and very bulky gel which is able to bind large amounts of water.
Example B22. The solution used in Example B21 is cast to a film and placed in a solution
of chitosan acetate to give a water-insoluble polyelectrolyte ~llm. Tlle ~llm is glass-clear,
strongly refractive and contains ~he 50- to 100-fold amount by volume of water. The ~llm
has a hybrid structure in which solely the polyelectrolyte gel with chitosan as polymeric
counterion is formed on the surface of the primary film of chitosan-malamic acid.
Laminate filrns of reverse structure with sirnilar properlies, namely with chitosan as
primary film and chitosan-malamic acid as polymeric counterion, are prepared by the
same procedure.
C) Use Examples
Exarnple Cl: Crystallisation of sodium chloride under the influence of acid
polysaccharides
A solution of 204 g of sodium chloride in 600 ml of distilled water is filtered through a
membrane ISlter and put into each of 6 glass beakers covered with a filter paper and
provided with a blade stirrer. The stirring rate is adjusted to 30 rpm. Then to each of the
6 solutions is added 1 ml of a solution that contains 1 mg of one of the polysaccharides
listed below. The concentration of polysaccharide in the crystallisation solution is thus
10 ppm. Then the solutions are stirred for 4 days at room temperature and afterwards the
2 ~
- 22 -
crystals obtained are isolated by ~lltration. Forrn, size, size distIibution, agglomeration and
adhesion of the crystals are assessed by microscopy and scanning electron
photomicrographs. The results are reported in Table 3.
Table 3
Polysaccharide Gystalformand '3ize distribution Adhesion
size
. _
hyaluronic acid cubes of broad range growth on glass
different size, S ~ to 2 mm wall and
mrn agglomerates stirrer
irregular growth
i-carrageenan as above, broad range growth on glass
agglomerates S ~1 to 2 mm wall and
and cubes of mm size stirrer
x-carrageenan as above, broad range growth on glass
agglomerates S ,u to 2 rr~n wall and
and cubes of rnm size stirrer
chondroitin- round less growth on glass
4-sulfate agglomerates heterogeneous walland
of many single 30-300 llm s~rrer
crystals,
cube structure
chitosan-mal- regular, well uniform no growth on
amic acid formed ~win 50-100 ~,lm glass par~s
acc. E~. B 1 to quadruplet
aggregates of
octahedron structure
blanlc test cube broad range growth on
agglomerates S ~L to 2 mm glass wall and
of different size stirrer
.~ , .
- 23 -
Example C2: Crystallisa~on of CalCiUIII carbonate under the influence of polysaccharides
A saturated solution of calcium hydrogencarbonate is prepared by suspending 700 mg of
CaC03 in 7QO ml of distilled water, introducing C02 until solution is almost complete, and
filtration. The undissolved constituents are removed by filtration and the solution is put
into each of 6 glass beakers as described in Example Cl and to each of which are added
10 ppm of the polysaccharides listed below. The solutions are heated for 1 hour to
8~85C and the CaC03 precipitates are evaluated as described in Example Cl.
Polysaccharide Cnstal form and Size distribution adhesion
sl~e
blank test a,o,gregates of 30-300 ~m hard crusts
needles on some glass
parts
hyaluronic ac aggregates of 30-3001,lm hard crusts
needles on some glass
parts
i-carrageenan aggregates of 30-300~,1m hard crusts
needles on some glass
parts
x-carrageenan aggregates of 30-3QO~Lm hard CIUStS
needles on some glass
parts
chondroitin- grou th of 30-300~,1m hard crusts
4-sulfate needles aggregates on some glass
parts
chitosan-mal- amorphous 30-100 ~lm no crusts,
amic acid plates 5-10 llm all in suspen-
acc. ~x. Cl thic'k sion
- 24 -
Example C3:
a) In accordance with the general procedure describe d in Example Cl, solutions having
the following ion concentrations are prepared:
375 ppm Ca 220 ]ppm HCO3e
183 ppm Mg 85 ppm Co32~
After addition of 2 and 8 ppm respec~ively of a polysaccharide, the solutions are heated for
30 minutes to 70C and filtered after cooling. The uncrystallised calcium concentration in
the filtrates is determined by atomic absorption analysis (or titration wi~h a 0.01 M
solution of ethylenediaminetetraacetic acid~ and expressed in percentage inhibition.
b~ In similar manner, the inhibi~ion of ~e barium sulfate concentration with different
polysaccharides is determined by the standardised Downell test.
The test is callied out by starting from a solution which contains 8.9 ppm of Ba2~~,
20 000 ppm of Na(: 1 and 8 ppm of a polysaccharide. The solution is adjusted with acetate
buffer to pH 5.5 and Icept a~ 25C. Then 270.5 ppm of so42- are added, precipitated BaSO4
is removed by filtration after 4 hours and, as described in a), ~he residual Ba2~ con-
centration in the filtrates is determined.
The results of the inhibition of crystallisation are reported in Table 4.
- 25 -
Table 4
Polysaccharide Concentration in ppm Inhibition of crystallisa~ion (%)
Ca/MgCO3 BaSO4
_ _ _ _ . _
i-c~rageenan 8 10 0
sodium 8 0 0
chondroitin-
4-sulfate
sodium 8 0 0
chondroitin-
~sulfate
x-carrageenan 8 8.6 0
xanthane 8 10.6 0
polygalactu- 8 20 0
ronic acid
alginic acid 8 : 27.6 0
chitosan-mal- 2 14.6 24.8
amic acid
acc.Ex.B2
chitosan-mal- 8 43.6 67.2
amic acid
acc. Ex. B2
.
- : - .~ .
