Method of making magnetic, crosslinked chitosan support materials and products thereof

Method for magnetizing crosslinked chitosan support material involving the treatment of carboxyl group-containing, crosslinked chitosan gels with solutions of ferrous chloride followed by treatment with dilute aqueous alkali and then oxidation with molecular oxygen gas to produce highly-enriched magnetized chitosan particles, beads, films and/or coatings.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The invention is generally related to a method for forming magnetic, 
crosslinked chitosan support materials, and magnetic, crosslinked chitosan 
products obtained by this method. 
2. Description of the Prior Art 
The application of magnetic separation technologies to biotechnology has 
been proposed and practiced. E.g., see D. Melville et al., "Direct 
magnetic separation of red cells from whole blood", Nature, Vol. 255, Jun. 
26, 1975, p. 706; C. Setchell, "Magnetic Separations in Biotechnology-A 
Review", J. Chem. Tech. Biotechnol., 1985, 35B, 175-182; and undated trade 
brochure by Paesel-Lorei GMBH & Co., "Magnetic Separation with 
Micro-Particles", Flinschstr. 67, W-6000 Frankfurt am Main 60. 
As described in L. Nixon et al., "Preparation and Characterization of Novel 
Magnetite-Coated Ion-Exchange Particles", Chem. Mater., Vol. 4, No. 1, 
1992, 4, 117-121. magnetizable particles for bioseparations have been made 
by incorporating magnetite into ion-exchange gel particles in two 
different modes. In one mode, the magnetite was deposited as a thin 
permeable layer on the surface of the ion-exchange beads, which were 
cross-linked agarose functionalized with carboxymethyl exchange groups or 
sulfopropyl groups (i.e., S-Sepharose), to form a coated type of magnetic 
particle. In a second mode, the magnetite was dispersed into agarose gel 
prior to bead formation. 
R. H. Marchessault et al., Polymer, 33, 4024-4027 (1992), disclose a 
technique for preparing magnetic cellulose fibers and paper obtained by 
synthesizing ferrites in situ. In situ synthesis of iron oxide particles 
was performed by Marchessault et al. via careful oxidation of ferrous 
hydroxide precipitated with caustic from the ferrous ion-exchanged form of 
the matrix. The chemistry yielded magnetic fibers containing small ferrite 
(Fe.sub.3 O.sub.4) particles of about 10 nm in size. Marchessault et al. 
exemplify carboxymethylated cellulose fibers as the material subjected to 
the magnetization scheme disclosed therein, but also suggest that the 
process could be practiced with a wide range of natural biopolymers such 
as polysaccharides and lignocellulosics with amino, carboxyl and sulfonic 
acid groups, such as chitosan, although no reference nor distinction is 
made as between uncrosslinked and crosslinked forms thereof. 
Chitosan is a generally known support material for separation processes. 
Chitosan is the acid-soluble deacetylation product of chitin. For example, 
chitosan is the product of alkaline hydrolysis of abundant chitin produced 
in the crab shelling industry. Chitosan, a biopolymer soluble in dilute 
(0.1 to 10%) solutions of carboxylic acids, such as acetic acid, is 
readily regenerated from solution by neutralization with alkali. In this 
manner, chitosan has been regenerated and reshaped in the form of films, 
fibers, and hydrogel beads. For instance, chitosan beads are prepared in 
one conventional method by precipitating dilute solutions of chitosan in 
acetic acid into alcoholic or aqueous sodium hydroxide followed by solvent 
exchange with water. However, in contrast to beads from cellulose, which 
are insoluble in most organic solvents, acids and bases, chitosan retains 
the solubility in dilute acids of the parent biopolymer. This solubility 
is typically overcome by inducing crosslinking. A conventional chitosan 
crosslinking reaction involves dialdehydes, such as glutaraldehyde, or 
diglycidyl ethers (such as butanediol diglycidyl ether, or epoxides like 
epichlorohydrin). Chitosan beads crosslinked with diglycidyl ethers are 
commercially available under the trade name CHITOPEARL, as manufactured by 
Fuji Spinning, Ltd., Japan. 
Also, various researchers have discussed blending chitosan and cellulose to 
produce biodegradable films (see, for example, U.S. Pat. No. 5,306,550 to 
Nishiyama et al.; Hosokawa et al., Ind. Eng. Chem. Res., 29:800-805 
(1990); Hasegawa et al., J. Appl. Polym. Sci., 45:1873-1879 (1992)). 
Though cellulose contains only trace amounts of carbonyl groups, these 
trace amounts of carbonyl groups are suspected in the art to play an 
important role in crosslinking to chitosan to form a crosslinked polymeric 
network of cellulose and chitosan. 
Also, it has been demonstrated that complexes of chitosan with acetic acid 
(viz., chitosonium acetate) are converted to chitin (i.e., the 
N-acetylamide of chitosan) by a heat-catalyzed amidification or 
dehydration reaction, in U.S. Appln. Ser. No. 08/435,866 to Glasser et 
al., filed May 5, 1995. The amidification reaction described in U.S. 
Appln. Ser. No. 08/435,866 converts acid-soluble chitosan into 
acid-insoluble chitin. 
However, because chitosan is easily solubilized and processed, a great deal 
of research in the hydrogel field has been devoted to experimentation with 
and/or use of chitosan in a wide variety of applications such as 
bioseparations. The art would be highly interested in a facile technique 
to form magnetic-functionalized, crosslinked chitosan support material. 
SUMMARY OF THE INVENTION 
It is an object of this invention to provide a method for forming magnetic, 
crosslinked chitosan support materials. 
Briefly and in general terms, the invention concerns a method for 
magnetizing crosslinked chitosan support material involving the treatment 
of carboxyl group-containing, crosslinked chitosan gels with solutions of 
ferrous chloride (FeCl.sub.2) followed by treatment with dilute aqueous 
alkali and oxidation with oxygen gas (O.sub.2) to produce highly enriched 
magnetizable chitosan particles, beads, films and/or coatings. 
Before magnetic functionalization is imparted according to the invention, 
the chitosan starting material is crosslinked so that the chitosan can 
better tolerate alkali and acidic conditions encountered during the 
magnetization reaction. The chitosan starting material is crosslinked 
preferably, but not mandatorily, with difunctional, dicarboxylic acids or 
acid anhydrides thereof, which, in addition to their dual carboxylic 
functionality possess functionality capable of independent polymerization 
and thus the formation of an independent polycarboxylic gel. 
A beaded chitosan material crosslinked with a suitable difunctional, 
dicarboxylic acid or acid anhydride thereof, such as itaconic anhydride or 
maleic anhydride or citraconic anhydride, under appropriate heating 
conditions, as described herein, forms a carboxylated, crosslinked 
chitosan gel without disturbing or distorting the preshape of the beads. 
In this invention, these carboxylic acid gel sites are the target of a 
sequence of reaction steps leading to micron-sized magnetic particles via 
a magnetization reaction. The magnetization reaction scheme can be 
repeated one or more times, if desired. 
Bead form magnetic chitosan products of this invention can be oriented, 
made to cluster and aggregate, and collected and harvested using a 
magnetic field produced by an internal or external magnet (permanent or 
electric). The resulting magnetic crosslinked chitosan beads lose their 
magnetism after removal of the magnetic field (superparamagnetism); and 
their magnetic properties do not interfere with other activities, such as 
ion exchange and protein binding. The magnetic crosslinked chitosan beads 
made by this invention can be oriented and collected from complex mixtures 
containing other (nonmagnetic) suspended particles. 
The magnetic chitosan hydrogel material obtained by this invention has 
additional functionality capable of interacting with the products of 
biotechnology and constitute separation materials endowed with great 
versatility. The magnetic crosslinked chitosan beads made pursuant to the 
invention are well-suited for use as softgel beads for bioseparations, 
especially separations of proteins. Also, the magnetic crosslinked 
chitosan beads of this invention can be used for water purification.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION 
The present invention must be practiced with crosslinked chitosan material 
as a starting material to preserve and maintain the preshape of the 
chitosan material during the magnetization reaction scheme. Uncrosslinked 
chitosan, if used, would dissolve when exposed to the dilute solution of 
aqueous alkali employed to cause in situ precipitation of the ferrous 
hydroxide during the second step of the magnetization reaction scheme. 
Therefore, any preshape imparted to the chitosan material as introduced to 
the magnetization reaction scheme would be sacrificed and lost during such 
dissolution. 
Chitosan thus is first crosslinked with acids capable of forming a 
carboxylic acid group containing hydrogel inside a polysaccharide gel by a 
polymerization mechanism which leaves other functionalities largely intact 
(such as by chain growth polymerization). The crosslinked chitosan is then 
magnetized, generally speaking, by a stepwise reaction involving: (a) 
reacting the preshaped gel particles with ferrous chloride, (b) reacting 
the ionic intermediate complex formed by step (a) with dilute aqueous 
sodium hydroxide at room temperature (approx. 20.degree.-28.degree. C.) to 
form (precipitate) ferrous hydroxide (Fe(OH).sub.2) and the corresponding 
sodium salt of the ionic intermediate complex; and (c) oxidizing the 
ferrous hydroxide formed by step (b) in the presence of a stoichiometric 
amount of oxygen gas at 55.degree.-75.degree. C., preferably at 65.degree. 
C., effective to form a fine dispersion of ferric oxide (Fe.sub.3 O.sub.4) 
particles. For purposes of this application, ferric oxide (Fe.sub.3 
O.sub.4) also can be referred to by its trivial names: ferrite and/or 
magnetite. 
It has been discovered by the present investigators that chitosan material 
that is crosslinked in a preliminary operation using dicarboxylic acids or 
acid anhydrides thereof capable of forming an alkali-insoluble (and 
acid-insoluble) polymeric gel containing a sufficient number of carboxylic 
acid groups has the capacity for becoming highly enriched with magnetic 
particles (Fe.sub.3 O.sub.4) by the magnetization reaction and can 
tolerate the acidic and alkaline reagents without sacrificing the preshape 
of the chitosan material. 
More specific details on the methods for crosslinking and magnetizing the 
chitosan are provided below. Methods for crosslinking the chitosan are 
described in U.S. Appln. Ser. No. 08/654,929 (Glasser et al.), filed May 
29, 1996. The crosslinking methods are described herein below. 
Chitosan, as a starting material of the inventive method, is commercially 
available from a wide variety of sources including under the trade name 
VANSON of Redmond, Wash., and PROTAN of Woodinville, Wash. Alternatively, 
the chitosan starting material can be made by any convenient conventional 
method, such as by hydrolyzing chitin in a concentrated solution of sodium 
hydroxide on heating and then recovering chitosan by filtration and water 
washing. The chitosan starting material is soluble in dilute acids but not 
soluble in neutral water or alkali. Chitosan is an amorphous solid which 
is more soluble in water having a pH less than 6, than chitin, but 
chitosan usually requires the use of aqueous organic acids to attain 
solubility. 
Chitosan is a glucose amine polymer. The chemical structure of chitosan has 
a repeating structural unit that is represented by Formula (I) as follows: 
##STR1## 
where n is greater than 3. 
The chitosan starting material can theoretically be substituted or 
unsubstituted at the ring hydroxy moiety or the hydroxy methyl moiety. 
These substituents may represent ethers, esters, carbamates, or other 
types. The important feature is that the chitosan has a majority of free, 
primary amine groups along its polymeric backbone to form ionic complexes 
with the dicarboxylic acids or acid anhydrides thereof. Preferably the 
chitosan has a weight-average molecular weight (M.sub.w) ranging from 
about 10.sup.4 to about 10.sup.6 ; although the molecular weight may be 
varied depending on the ultimate use envisaged for the crosslinked 
chitosan product. 
The chitosan starting materials can be porous or nonporous, and have a 
geometrical shape that can be regular or irregular, depending at least in 
part on the ultimate usage of the support material envisaged. 
"For example, the chitosan support material may be in the form of spherical 
beads, films, and fibers. In the case of a porous chitosan starting 
material, the amine groups along its polymeric backbone on internal and 
external surfaces of the starting material should be available for 
reaction to form ionic complexes on the inside and outside of the starting 
material." 
For the sake of convenience and illustration, the description herein will 
occasionally refer to the chitosan support material in the context of 
beads, although it is to be understood that the invention is not 
necessarily limited to that configuration. 
The solid chitosan starting material is dissolved in a weak organic acid, 
such as 1 to 5 wt. % acetic acid, formic acid, and the like, to provide a 
chitosan solution. Bead form chitosan hydrogels are formed from the 
chitosan solution by atomizing the solution of chitosan in aqueous alkali 
(e.g., 0.1 to 2.0N sodium hydroxide solution). The hydrogel beads formed 
are decanted, filtered, washed and stored in distilled water for 
subsequent treatment. The dissolved chitosan starting material, in 
hydrogel form, is solvent-exchanged with a non-aqueous liquid solvent to 
displace water from the chitosan support material (e.g., beads) and create 
anhydrous conditions within the support material (e.g., beads). Suitable 
non-aqueous solvents for this purpose include alcohols (e.g., isopropyl 
alcohol), acetone, dioxane, chloroform, benzene, tetra-hydrofuran, 
toluene, and xylene. This solvent-exchange procedure is performed prior to 
performing any optional functionalization step and the required 
crosslinking step of the invention. The presence of water, especially as a 
medium, is undesired during the amidification reaction as it slows down 
the reaction and lowers the degree of crosslinking attained. Trace amounts 
of water left as residue in the solvent-exchanged beads seldom raise this 
problem. However, if residual traces of water in the solvent-exchanged 
beads interfere with crosslinking (such as indicated by alkali solubility 
in the final product), the traces of water can be more rigorously removed 
and excluded by taking precautions such as employing freshly cut potassium 
or sodium metal as a dessicant in the non-aqueous solvent being used to 
replace the water. Also, moisture indicators, such as benzophenone, 
optionally can be used to assist in determining conditions sufficiently 
anhydrous for reactions to proceed expediently and efficiently. 
Also, the practice of this invention preferably involves usage of 
"never-dried" hydrogel beads from chitosan through the course of the 
inventive method(s). The terminology "never-dried", as used herein, means 
beads which have the bead and pore size of beads which have not been 
subjected to water removal (dehydration) or organic solvent removal to 
leave the beads devoid of liquid solvent, whether aqueous or non-aqueous, 
at any given time before or during processing to permit drying thereof. 
Drying of the beads is undesired as it causes shrinkage and thus size 
reduction in the beads, which effect is not completely reversible upon 
rehydration or upon combination with a polar organic solvent. 
In any event, once solvent-exchanged, the chitosan material is combined 
with a dicarboxylic acid or acid anhydride thereof to form a chitosonium 
ion complex that is water soluble. Although not part of the present 
invention, the chitosonium ion complex, prior to being heated, will behave 
in a manner similar to gum or ionic starch if dissolved in water. 
When the chitosan starting material and the dicarboxylic acid or acid 
anhydride thereof are combined, they interact at the primary amine of the 
chitosan starting material to produce a chitosonium ion complex. For 
example, in the case of maleic anhydride being combined with chitosan, 
chitosonium maleate is formed. The chitosan/dicarboxylic acid complex is 
water-soluble at a pH above 8. 
Once the chitosonium ion complex is formed, heat is applied to induce 
amidification of chitosonium ion complex to form an intermediate product. 
The intermediate product is schematically shown as the product of step 1 
in Reaction Scheme 1 depicted in FIG. 1. Reaction Scheme 1 is a schematic 
representation of the reaction of chitosan with anhydrides. Step 1 is 
fast, and Step 2 requires the presence of polymerizable (i.e., double) 
bonds that react faster than Step 2b. Step 2a and 2b-reactions are 
promoted by the size of the anhydride cycle and by water removal. 
The source of heat can be applied in any convenient manner to the 
chitosonium ion complex. The chitosonium ion complex can be heated in a 
reaction vessel by use of conventional laboratory or industrial heating 
arrangements for liquid reaction baths, as appropriately selected for the 
desired scale of the operation. The heating system can be either an open 
or closed system. 
The heating temperature applied to the reaction bath through all stages of 
the crosslinking reaction (i.e., steps 1 and 2; or steps 1, 2a and 2b) 
preferably is maintained at a constant value. The reaction temperature and 
duration of heating to effect crosslinking of the chitosan according to 
the inventive method will depend in large part upon the non-aqueous 
solvent (and its boiling point) involved and the degree of crosslinking 
desired. The reaction temperature generally will range between 40.degree. 
to 160.degree..degree. C. depending on the particular non-aqueous solvent. 
For example, where isopropyl alcohol is the liquid medium exchanged for 
water in the chitosan hydrogel, the crosslinking reaction temperature 
preferably is approximately 80.degree. C., and the heating time generally 
is about 5 to 50 hours, usually about 6 to 24 hours to effect complete 
crosslinking of chitosan hydrogel beads. 
As to the intermediate product of step 1, the application of heat initially 
converts ammonium organic acid ionic groups of the chitosonium ion complex 
to N-organic acid groups. Thus, a non-crosslinked monocarboxylic acid 
derivative is formed as an intermediate product derived during an 
incipient phase of the heating procedure from the partial amidification of 
chitosonium ion complex. The proportion of the chitosonium ion complex 
functionalized in this way upon completion of the first step (step 1) of 
the heating procedure is variable, although high levels of 
functionalization are typically targeted and achieved in practice. The 
intermediate reaction product of step 1 of the inventive method, i.e., the 
noncrosslinked monocarboxylic derivative, remains insoluble in neutral 
water and becomes insoluble in acids while being soluble in dilute alkali 
as attributable to the carboxyl functionality. 
For example, where maleic anhydride is used, chitosan maleate is formed 
upon completion of step 1 as the initial phase of the heating procedure. 
That is, ammonium maleate groups at primary amines are converted to 
chitosan N-maleayl groups. The resulting intermediate product is alkali 
soluble as attributable to the remaining carboxylic functionality of the 
added maleayl group. 
As seen by reference to Reaction Scheme 1, inter-molecular polymerization 
of the intermediate product of step 1 can proceed along two possible 
pathways depending on whether the dicarboxylic acid reactant or anhydride 
thereof has a polymerizable double bond(s). Advancement by Step 2 requires 
the presence of polymerizable double bonds in the dicarboxylic acid or 
anhydride thereof which will react faster than step 2b. The heating 
condition is maintained such that the polymerizable double bonds react 
with polymerizable double bonds on a neighboring intermediate product. The 
product of step 2 is an inter-molecular network formed from different 
monocarboxylic acid derivatives. 
However, when dicarboxylic acids or anhydrides thereof lacking 
polymerizable double bonds are involved, then the reaction will advance 
through steps 2a and 2b to completion. To effectuate and complete the 
desired crosslinking reaction according to the pathway of steps 2a and 2b, 
the free carboxylic acid group of the non-crosslinked monocarboxylic acid 
derivative, i.e., the intermediate formed at step 1, is reacted with the 
NH.sub.2 group of an adjacent chitosan molecule after first driving off a 
molecule of water. The reactions of Steps 2a and 2b are promoted by the 
size of the anhydride cycle and by the removal of water. 
Therefore, as heating is continued in steps 2a and 2b, crosslinking is 
heat-induced between the monocarboxylic acid derivative intermediate 
product that has completed step 1 with nonfunctionalized chitosonium ion 
complex that is present. As with the intermediate formed by step 1 during 
initial phases of heating, the heating condition is also used to convert 
the anhydride to a monocarboxylic acid by amidification. However, the 
reaction that occurs in this later phase of heating of step 2b converts, 
inter-molecularly, the ammonium groups of a chitosonium ion complex 
(non-functionalized during initial heating) to N-acyl groups by reaction 
with the remaining free carboxyl functionality on a monocarboxylic acid 
derivative intermediate product. The dicarboxylic acid or acid anhydride 
thereof, is thus progressively and ultimately reacted with both carboxylic 
acid functionalities with two equivalents of amines, culminating in 
inter-molecular crosslinking as a second reaction stage reached in a later 
phase of the heating step. In this way, a fully crosslinked, 
non-carboxylic diamide is formed via steps 2a and 2b. 
The resulting amidified, crosslinked chitosan complex of either step 2 or 
step 2b is swellable in aqueous or non-aqueous solvents, and it is 
insoluble in acids, bases or neutral water. Catalysts are not required for 
the first or second stages of the amidification reaction, although it is 
contemplated that certain catalysts could be used to promote either stage 
of this step, such as Lewis acids like Al.sub.2 O.sub.3. 
Since the noncrosslinked intermediate of step 1 is insoluble in dilute acid 
or neutral water but soluble in alkali, while the final product of the 
heating step, i.e., the crosslinked dicarboxylic derivative of step 2 or 
2b, is insoluble in all solvents whether acids, neutral in pH, or bases, 
the progress and extent of the crosslinking (heat-induced amidification) 
reaction of the present invention can be easily monitored and assessed by 
routine solubility tests. 
It is to be borne in mind that the particular dicarboxylic acid anhydride 
depicted in Reaction Scheme 1, is merely representative. The dicarboxylic 
acids and acid anhydrides thereof described herein can be used and will 
inter-act with the chitosan material, and the ionic complex and 
intermediate derivatives thereof, consistent with general scheme shown in 
Reaction Scheme 1. For instance, Reaction Scheme 2 depicted in FIG. 2 
illustrates a carboxylated, crosslinked chitosan gel made using itaconic 
acid, a difunctional dicarboxylic acid, for crosslinking where the 
crosslinked chitosan hydrogel has free carboxylic acid groups. 
Crosslinked chitosan bead products made as described herein, generally have 
a solids content, by weight, ranging from about 1.0% to about 10.0%, with 
the remainder being constituted by solvent. As saturated with water, it is 
preferred that crosslinked beads with the magnetization functionalization 
described herein, contain 1 to 3 wt. % solids and have an average particle 
diameter size greater than about 0.3 mm, more preferably ranging from 
about 0.5 mm to about 2.0 mm. Also, after crosslinking the chitosan 
material, as described herein, the crosslinked chitosan hydrogel can be 
exchanged back into aqueous medium (i.e., water), and the non-aqueous 
solvent recovered. 
The dicarboxylic acids useful in the invention are organic compounds having 
two carboxyl groups. The carboxyl groups are pendant groups and can 
independently be terminal groups or side groups on the main chain. The 
related acid anhydrides of the dicarboxylic acids that can be used are 
five-or six membered ring structures including a fused ring portion formed 
of the two carboxyl groups. The pair of carboxyl groups in the 
dicarboxylic acids or acid anhydrides thereof can represent the only 
substituents (non-inclusive of hydrogen atoms) attached to the main chain. 
Alternatively, the dicarboxylic acid or acid anhydride thereof can possess 
other substituents in addition to the pair of carboxyl groups as long as 
the additional substituents do not interfere with, or otherwise hinder, 
the amidification reaction. The dicarboxylic acid main chains can be 
saturated or unsaturated at the various carbon-to-carbon bonds forming the 
main chain or backbone. The main chain preferably comprises 1-30 carbon 
atoms. Straight chains are usually preferable over branched chains due to 
steric effects. The carbon-to-carbon bonds forming the ring structure of 
the acid anhydrides can be saturated or unsaturated. 
Preferred dicarboxylic acids and acid anhydrides thereof for use in the 
invention include those possessing polymerizable double bonds, more 
preferably conjugated double bonds. While not desiring to be bound to any 
particular theory at this time, it is postulated that double bonds present 
in the backbone or main chain of the dicarboxylic acid or anhydride 
thereof become polymerization sites when the NH.sub.2 groups are 
exhausted. The polymerization of the double bonds between different 
chitosan molecules forms a network structure which serves to increase 
opportunities for unreacted amines to interact with free carboxyl groups 
in the system, thereby enhancing the advancement of crosslinking. 
Double bonds that alternate with single bonds are said to be conjugated. As 
known, a conjugated system is characterized by a delocalized n-bond spread 
over the bond lengths of several linked carbon atoms due to them all 
contributing p-orbitals to the r-bond system, so that electron-rich atoms 
result at each carbon atom in the conjugated chain as a mesomeric effect. 
Useful conjugated systems include dicarboxylic acids or acid anhydrides 
thereof having isolated double bonds, cumulated double bonds, and 
.alpha.,.beta.-unsaturated carbonyl compounds. 
In an .alpha.,.beta.-unsaturated carbonyl compound, the carbon-carbon 
double bond and the carbon-oxygen double bond are separated by only one 
carbon-carbon single bond. Examples of .alpha.,.beta.-unsaturated carbonyl 
compounds useful in the practice of this invention include, for example, 
maleic acid (cis-HOOCCH.dbd.CHCOOH), fumaric acid 
(trans-HOOCCH.dbd.CHCOOH), itaconic acid ("methylene succinic acid" 
CH.sub.2 .dbd.C(COOH)CH.sub.2 COOH), citraconic acid (CH.sub.3 
C(COOH).dbd.CHCOOH), maleic anhydride, itaconic anhydride, and citraconic 
anhydride. 
The dicarboxylic acid compounds can be made by conventional methods, such 
as by hydrolysis of a dinitrile or a cyanocarboxylic acid, or oxidation of 
dimethylbenzene to yield a phthalic acid. The acid anhydrides can be 
formed by simple heating of the corresponding dicarboxylic acid to produce 
a ring structure, such as in case of maleic anhydride, succinic anhydride, 
and phthalic anhydride. For instance, when maleic acid is heated at 
100.degree. C., or when fumaric acid is heated at 250.degree.-300.degree. 
C., both acids yield the same anhydride, viz., maleic anhydride 
(cis-butenedioic anhydride). 
Dicarboxylic acids and anhydrides thereof lacking carbon-carbon double 
bonds also can be used as they will react as long as COOH and NH.sub.2 
groups are present in equimolar amounts. For instance, other useful 
dicarboxylic acids and anhydrides thereof include oxalic acid 
(HOOC--COOH); malonic acid (HOOCCH.sub.2 COOH), succinic acid 
(HOOC(CH.sub.2).sub.2 COOH), glutaric acid (HOOC(CH.sub.2).sub.3 COOH), 
adipic acid (HOOC(CH.sub.2).sub.4 COOH), phthalic acid (1,2-C.sub.6 
H.sub.4 (COOH).sub.2), isophthalic acid (1,3-C.sub.6 H.sub.4 
(COOH).sub.2), terephthalic acid (1,4-C.sub.6 H.sub.4 (COOH).sub.2), 
succinic anhydride, phthalic anhydride. Other dicarboxylic acids that can 
be used include substituted dicarboxylic acids, such as malic acid 
(HOOC--CH(OH)CH.sub.2 --COOH) or aldaric acids (HOOC--(CHOH).sub.n --COOH 
where n=1 or more) such as tartaric, glucaric, mannaric, xylaric acids, 
and the like; and dimethoxy carboxylates such as dimethoxy succinic acid 
(HOOC--CH(OCH.sub.3)C(OCH.sub.3)--HCH(OCH.sub.3)--COOH) which also will 
undergo removal of methanol during amidification. The dicarboxylic acid 
also can be a polycarboxylic acid such as pectin, xylan, carboxy methyl 
cellulose, and the like. 
The chitosan gel beads crosslinked with dicarboxylic acids capable of 
simultaneously forming a continuous carboxylic acid gel structure embedded 
inside the continuous chitosan gel are not soluble in any of neutral, 
acidic, or alkaline solutions. These crosslinked chitosan beads contain 
free carboxylic acid, by virtue of partly amidified, noncrosslinked 
intermediate products, as well as unreacted amine functionalities on the 
glucose amine polymer material itself. 
In addition to the techniques for crosslinking chitosan used in this 
invention prior to conducting the magnetization of the beads, the chitosan 
beads optionally can be subjected to activation reactions which result in 
altered functionality and altered solubility in addition to the 
magnetization. For instance, the chitosan beads in solid form, in 
non-aqueous suspension, can be reacted with a quaternizing reagent, such 
as a glycidyl quaternary salt, to form a quaternized functional chitosan 
hydrogel. An example of a suitable quaternizing compound in this regard is 
glycidyl trimethyl ammonium chloride. 
Chitosan beads so derivatized, i.e., quaternized, reveal significant 
solubility in both aqueous dilute acids and neutral water (pH=7). This 
functionalization by quaternization is highly beneficial for the sorption 
properties of chitosan beads and can be used for separations in 
biotechnology (e.g., protein separations). Quaternized chitosan tends to 
be soluble in aqueous environments. However, solubility in water and 
dilute acids is overcome in the present invention when the functionalized 
beads are subsequently subjected to amidification and crosslinking with 
dicarboxylic acids or acid anhydrides thereof according to the present 
invention. Quaternized chitosan beads crosslinked with either itaconic or 
maleic anhydride according to the invention, for instance, have been 
observed to be both 
(a) insoluble in acid, water, or alkali; and 
(b) quaternized so as to possess highly adsorbent functional groups for 
protein adsorption. 
The quaternized chitosan beads in non-aqueous suspension can be further 
functionalized with .alpha.-, .beta.-, or .gamma.-cyclodextrin. The 
cyclodextrin functionalized chitosan beads are highly beneficial for 
chiral separations. They are insoluble in aqueous medium over a wide 
pH-range. The crosslinked-cyclodextrin functionalized chitosan beads 
obtained according to the amidification crosslinking reaction with 
itaconic, citraconic or maleic acid or anhydrides thereof are insoluble in 
acid, water, or alkali, and they possess the functional groups needed for 
chiral separations. 
The quaternary functionalization reaction and the secondary 
cyclodextrin-modification reaction for the chitosan can employ reaction 
conditions (temperature and time) similar to those described above for 
performing the crosslinking reactions for a common type of chitosan salt 
and solvent. Methods for the optional functionalizations of the chitosan 
are described in greater detail in U.S. Appln. Ser. No. 08/654,929 
(Glasser et al.), filed May 29, 1996, which teachings are incorporated 
herein by reference. 
The method of using heat-induced amidification chemistries involving 
dicarboxylic acid or related acid anhydride reagents enables the 
production of crosslinked, highly expanded chitosan beads, optionally 
functionalized in any one of several functionalities in addition to 
magnetization, which is described in greater detail below, in solid form 
without distortion or loss of the original bead shape. 
The crosslinked, optionally functionalized, gel structure is then subjected 
to a magnetization reaction scheme of the present invention involving 
sequential steps using reagents of ferrous chloride, aqueous alkali, and 
oxygen. The magnetization reaction scheme according to this invention is 
illustrated in Reaction Scheme 3 depicted in FIG. 3. 
This method, as illustrated in Reaction Scheme 3, for forming magnetizable, 
crosslinked chitosan support material includes a first step (i.e., Step A) 
of reacting the crosslinked chitosan support material with aqueous ferrous 
chloride at about 20.degree.-28.degree. C., preferably 25.degree. C., for 
about 10 minutes effective to form a Fe (II) chitosonium ion complex. 
Then, the Fe (II) chitosonium ion complex formed by Step A is reacted in a 
second step (i.e., Step B) with dilute aqueous sodium hydroxide at about 
20.degree.-28.degree. C., e.g., 25.degree. C., for about 5 minutes to form 
ferrous hydroxide and a corresponding sodium salt of the chitosonium ion 
complex. In a third step (i.e., Step C), the ferrous hydroxide formed by 
step B is oxidized in the presence of a stoichiometric amount of oxygen 
gas with stirring at about 55.degree.-75.degree. C., preferably 65.degree. 
C., for about two hours effective to form a dispersion of ferric oxide 
particles in the crosslinked chitosan support material. Size is controlled 
by functionality. 
The magnetization reaction scheme can be repeated one or more times, if 
desired, to increase the degree of enrichment with ferric oxide. 
An important outcome of the magnetization reaction involving the 
crosslinked chitosan is that Fe (II)-sites are evenly distributed 
throughout the bead, resulting in an even distribution of Fe.sub.3 O.sub.4 
-particles throughout the bead structure. The treatment with aqueous 
sodium hydroxide at ambient temperatures and with oxygen gas at elevated 
temperatures produces a fine dispersion (inclusion) of Fe.sub.3 O.sub.4 
-particles in a size range of nano- to micrometers (ie, 0.005 to 5 hrs.) 
as has been confirmed by the present investigators by SEM (Scanning 
Electron Microscope) and EDAX Spectral analysis. Micron size iron oxide 
particles produce super-paramagnetism that is required for re-usable 
magnetic gel particles which display magnetic properties only in the 
presence of a magnetic field. This means that they fail to cluster on 
account of their remanent magnetic properties when not exposed to a 
magnetic field. 
The resulting superparamagnetic chitosan hydrogel beads are insoluble in 
acid, alkali, and neutral solutions; and they remain virtually unaltered 
by the magnetization reaction other than in color. The magnetized, 
crosslinked beaded chitosan product of the invention assume a light brown 
textured hue. The amine functionalities of the chitosan backbone 
substantially remain intact. 
The magnetic beads respond to the application of a magnetic field by being 
attracted; by becoming oriented in relation to the magnetic field; by 
migrating in the direction of the magnetic pole; and by becoming 
collectable and harvestable in complex solutions and suspensions of 
nonmagnetic particles. 
The invention thus provides a technique for magnetizing chitosan hydrogel 
beads that have been previously crosslinked with difunctional, 
dicarboxylic acids or acid anhydrides thereof, those acids and acid 
anhydrides thereof being capable of supporting an independent 
polymerization mechanism and producing a highly carboxylated hydrogel 
inside, and attached to, the chitosan hydrogel, by a stepwise and 
sequential treatment with ferrous chloride, alkali, and oxygen. The 
resulting magnetic beads have particulate ferrite (Fe.sub.3 O.sub.4) 
inclusions in a size producing highly desirable magnetic properties 
without remanence. 
The magnetic, crosslinked hydrogel beads made by this invention possess 
additional functionality capable of interacting with the products of 
biotechnology as separation materials with great versatility. For example, 
the beads can be used in separations of proteins. The beads also can be 
used in waste water purification systems as a filter material. The 
magnetized, crosslinked beaded chitosan product of the invention can be 
oriented and collected from complex mixtures containing other 
(nonmagnetic) suspended particles. 
The magnetized, crosslinked chitosan materials formed by the inventive 
methods can be effectively used as chromatographic supports such as in 
biotechnology applications, as well as in waste water treatment. 
This invention will now be understood more readily with reference to the 
following examples. However, these examples are intended merely to 
illustrate the invention and are not to be construed as limiting the scope 
of the invention. In the examples, all weights, percentages, and amounts 
therein are by weight unless indicated otherwise. 
EXAMPLES 
Preparation of Chitosan Starting Material 
For purposes of the following examples, chitosan starting material was 
prepared as follows. Chitosan flakes were dissolved in 1 to 5 wt. % acetic 
acid to provide a chitosan concentration between 0.5 to 3 wt. % by 
stirring at room temperature. The dissolved chitosan was centrifuged to 
remove extraneous matter. The solution of chitosan was atomized into a 0.1 
to 2N aqueous sodium hydroxide solution to form chitosan hydrogels in bead 
form. The chitosan beads were removed from the sodium hydroxide solution 
by decantation and filtration. The beads were washed with distilled water 
in a glass column by standing in water for 30 minutes and then replacing 
with fresh distilled water several times to obtain chitosan beads at a 
neutral pH. The beads were kept in water and stored in a closed container. 
The solids contents of the beads were between 1 and 5 wt. % as determined 
by lyophilization and thermogravimetric analysis (TGA). 
Example 1 
An aqueous suspension of chitosan beads prepared from the Preparation 
Procedure for Chitosan Starting Material was transferred to a sintered 
glass Buchner funnel and excess water was removed by suction. 100 g of the 
moist chitosan beads (solids content 2.0 wt. %) were immersed in 200 mL 
isopropyl alcohol and allowed to exchange with water inside the beads by 
stirring for 30 minutes. The isopropyl alcohol was filtered away and then 
replaced with a fresh batch of 200 mL of isopropyl alcohol. The mixture 
was stirred for 30 minutes. The solvent exchange process was repeated 3 
times to exchange almost all water from the beads to isopropyl alcohol. 
The isopropyl alcohol-exchanged chitosan beads (never-dried) were suspended 
in 200 mL isopropyl alcohol and 4.0 g itaconic anhydride was added to the 
bead suspension. The reaction mixture was refluxed for 16 hours at a 
temperature of 82.degree. C., and then cooled to room temperature. The 
crosslinked chitosan beads obtained were washed twice with 200 mL portions 
of isopropyl alcohol and then three times with 250 mL portions of 
distilled water by stirring for 30 minutes each. The crosslinked chitosan 
beads were stored in water. The solids content of the chitosan itaconate 
beads was 5.9 wt. % as determined by TGA. The crosslinked itaconate beads 
produced were insoluble in dilute acetic acid, neutral pH water, and 
dilute sodium hydroxide solution. 
The crosslinked chitosan was then magnetized according to the following 
procedure. 
12.0 g of the moist crosslinked beads were reacted with 20 mL aqueous 
ferrous chloride (0.028 g/mL) at about 25.degree. C. for 10 minutes. The 
Fe (II) chitosonium ion complex product was reacted with 40 mL 0.112N 
aqueous sodium hydroxide at 25.degree. C. for five minutes to form ferrous 
hydroxide precipitate and a corresponding sodium salt of the chitosonium 
ion complex. The temperature of the reaction mixture was raised to 
65.degree. C. under a nitrogen atmosphere and stirred for 30 minutes. In a 
final step, the ferrous hydroxide formed by the previous step was oxidized 
in the presence of a stoichiometric amount of oxygen gas under a nitrogen 
atmosphere with stirring at 65.degree. C. for two hours effective to form 
a uniform distribution of ferric oxide particles in the matrix of the 
crosslinked chitosan support material. 
The magnetized crosslinked chitosan itaconate beads were washed several 
times with distilled water by stirring for 30 minutes each. The yield of 
moist magnetized chitosan beads was 13.6 g. The ferric oxide content of 
the magnetized chitosan itaconate beads was 11.3 wt. % of dried beads as 
determined by TGA. The whole procedure of the magnetization of crosslinked 
chitosan beads was considered one cycle. 
2.0 g of magnetized crosslinked chitosan beads (cycle 1) was stored in 
water and the remaining of the magnetized beads from cycle 1 (11.6 g) was 
further magnetized with the same procedure for cycle 1 magnetization to 
increase the degree of enrichment. The yield of cycle 2 magnetized 
chitosan itaconate beads was 10.2 g. The ferric oxide content of the 
magnetized chitosan itaconate beads (cycle 2) was 12.5 wt. % of dried 
beads as determined by TGA. 
2.0 g magnetized crosslinked chitosan beads (cycle 2) was stored in water 
and the remaining of the magnetized beads from cycle 2 (8.2 g) was further 
magnetized with the same procedure for cycle 1 magnetization to further 
increase the degree of enrichment. The yield of cycle 3 magnetized 
chitosan itaconate beads was 8.0 g. The ferric oxide content of the 
magnetized beads (cycle 3) was 17.5 wt. % of dried beads as determined by 
TGA. 
2.0 g magnetized crosslinked chitosan beads (cycle 3) was stored in water 
and the remainder of the magnetized beads from cycle 3 (6.0 g) was further 
magnetized with the same procedure for cycle 1 magnetization to further 
increase the degree of enrichment. The yield of this cycle 4 magnetized 
chitosan itaconate beads was 5.6 g. The ferric oxide content of the 
magnetized beads (cycle 4) was 24.3 wt. % of dried beads as determined by 
TGA. 
2.0 g magnetized crosslinked chitosan beads (cycle 4) was stored in water 
and the rest of the magnetized beads from cycle 4 (3.6 g) was further 
magnetized with the same procedure for cycle 1 magnetization to further 
increase the degree of enrichment. The yield of cycle 5 magnetized 
chitosan itaconate beads was 3.3 g. The ferric oxide content of the 
magnetized beads (cycle 5) was 25.9 wt. % of dried beads as determined by 
TGA. 
The magnetized, crosslinked chitosan beads formed were then examined by SEM 
and EDAX (Energy dispersive X-ray) Spectral analysis which verified the 
inclusion of ferric oxide in the micron size range on the surfaces and 
within the crosslinked chitosan beads. The spike observed in the data 
taken by the EDAX Spectral analysis corresponded identically to the 
location of the signal taken for an iron control, which confirmed the 
magnetized state of the beads. 
Example 2 
The same procedure for crosslinking of chitosan beads as in example 1 was 
used except that maleic anhydride was used instead of itaconic anhydride. 
The crosslinking reaction time was again 16 hours. The crosslinked 
chitosan maleate beads had a solids content of 4.7 wt. % according to TGA. 
The crosslinked chitosan maleate beads produced were insoluble in dilute 
acetic acid, neutral pH water, and dilute sodium hydroxide solution. 
The crosslinked chitosan maleate beads were magnetized for five cycles in 
the same manner as Example 1. The cycle 1, 2, 3, 4 and 5 magnetized 
crosslinked chitosan maleate beads had a ferric oxide content 2.2, 2.8, 
4.9, 6.5, and 8.0 wt. % of the dried beads, respectively. 
Example 3 
The same procedure for crosslinking of chitosan beads as in example 1 was 
used except that citraconic anhydride was used instead of itaconic 
anhydride. The reaction time was again 16 hours. The crosslinked chitosan 
citraconate beads had a solids content of 3.8 wt. % according to TGA. The 
crosslinked chitosan citraconate beads produced were insoluble in dilute 
acetic acid, neutral pH water, and dilute sodium hydroxide solution. 
The crosslinked chitosan citraconate beads were magnetized for five cycles 
in the same manner as Example 1. The cycle 1, 2, 3, 4 and 5 magnetized 
crosslinked chitosan citraconate beads had a ferric oxide content 2.2, 
2.8, 4.9, 6.5 and 8.0 wt. % of the dried beads, respectively. 
Example 4 
The same procedure for crosslinking of chitosan beads as in Example 1 was 
used except that citraconic acid was used instead of itaconic anhydride. 
The crosslinking reaction time was again 16 hours. The crosslinked 
chitosan citraconate beads had a solids content of 2.4 wt. % according to 
TGA. The crosslinked chitosan citraconate beads produced were insoluble in 
dilute acetic acid, neutral pH water, and dilute sodium hydroxide 
solution. 
The crosslinked chitosan citraconate beads were magnetized for five cycles 
in the same manner as Example 1. The cycle 1, 2, 3, 4 and 5 magnetized 
crosslinked chitosan citraconate beads had a solids content of 3.4, 6.7, 
6.7, 9.6 and 10.1 wt. % of the dried beads, respectively. 
While the invention has been described in terms of its preferred 
embodiments, those skilled in the art will recognize that the invention 
can be practiced with modification within the spirit and scope of the 
appended claims.