CA1215545A - Method for treating an insoluble composition loaded with th.sup.4.sup. and uo.sub.2.sup.2.sup. to separate th.sup.4.sup. therefrom - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE:

The present invention relates to a method for separating Th4+ from insoluble compositions loaded with Th4+
and UO22+. The insoluble compositions comprise: a suitable insoluble carrier and organic co-ordinating sites covalently fixed to the surface of said carrier, said co-ordinating sites being capable of chelating Th4+ and UO22+.

Description

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The present is a divisional application of Canadian patent application no. 420 567 filed on January 31, 1983.
Canadian patent application no. 420 567 relates to a composition usefu] for the removal of metals, in particular iron, from liquid media. The composition, can for example, be used to lower the iron concentration of a liquid medium to less than 0.1 ~M.
Iron is an essential nutrient for all living things; a large number of cellu:Lar enzymes and other pro-teins require iron in order to function properly. Although iron is amongst the most plentiful of metals, it is diffi-cult for biological systems to acquire; in aerobic environ-ments of substantially netural pH, iron exists as its oxidized Fe3 form which readily hydrates to highly insol-uble Fe(OH)3 polymeric forms. To ensure accessability of iron in their environment, aerobic and facultative micro-organisms synthesize and release into their environment highly selective iron chelating agents called siderophores, the function of which is to provide the microbes with this vital nutrient. The siderophores released by the microbes solubilize iron, putting it into a form readily usable by them. Thus, a free, microbial siderophore is a growth promoting substance for those organisms which can utilise the particular siderophore in question.
As discussed in copending Canadian patent applica-tion no. 409 ~6~ it has been determined that removal of iron (e.g. Fe3 ) from a liquid nutrient medium will substantially restrict the proliferation of microbes provided that the residual iron concentration in the medium is below 0.1 ~M;
and this notwithstanding that the other required nutrients may be present in amounts sufficient for the support of microbial growth.
Thus, it is advantageous to have compositions able ... .
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to remove iron from a liquid, nutrient medium since the absence or limited presence of iron will inhibit microbial growth in such a medium. For èxample, the specific removal of iron from an ophthalmic solution will inhibit microbial growth and spoilage of such a solution. The removal of iron from such a solution would obviate the addition of conven-tional microbial growth inhibitors which can create their own problems such as toxicity, etc.
Removal of iron from liquid media prior to the present invention did present problems (see Neilands, J.
Bacteriology 149 : 880, 1982). Commercially available products (e.g. Chelex 100 sold by Biorad) have a low selectivity for iron. Additionally, other important cations (Mn +, Mg2 ) removed by such commercial products are often desirable components of a liquid medium. Commercial ion exchange products can also liberate sodium or potassium ions into the liquid medium being treated which may not be desirable.
Thus, it would be advantageous to be a~le to remove iron from solution while at the same time avoiding liberating into the solution undesirable ions.
In general, it would be advantageous to be able to remove iron from solution when the presence of iron is undesirable, e.g. when iron is considered a contaminant at concentrations greater thàn 0.1 ~M.
In accordance with copending Canadian patent appli-cation no. 420 567 it has also been determined that insoluble compositions described in copending canadian patent applica-tion no. 409 859 and those that will be discussed below can advantageously be used to remove Th4 and VO2 from solution and, if desired, even to separate Th4 and UO22 .
Copending Canadian patent application no. 420 5~7 in general relates to insoluble compositions, which are capable of removing metal (e.g. selectively) from solution .

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;S~5 (e.g. Fe3 from a liquid nutrient medium so as to lower the Fe content to less than 0.1 ~M); the insoluble composi-tions comprise:
(i) a suitable insoluble carrier and (ii) organic co-ordinating sites covalently fixed to the surface of said carrier, said so-ordinating sites being capable of chelating Fe3+, Th and/or UO2 The necessary co-ordinating sites are provided by covalently fixing an organic chelating compound to a suitable carrier.
Insoluble compositions are disclosed in copending Canadian patent application no. 409 86g which comprise unsubstituted catechol suitably fixed to an insoluble carrier. These compositions can be used to remove Fe from solution. However, if they are stored for a prolonged - period of time loaded with Fe , they undergo a chemical change whereby the compositions gradually loose chelating activity. It is believed that this loss of activity is due to the reciprocal oxidation of the catechol and reduction of Fe (i.e. Fe ~Fe ~. The activity of these composi-tions can be recovered by treatment with a suitable reducing ~, agent such as will be discussed further on.
It has been determined that catechol compounds selected from the group consisting of catechol substituted on the ben~ene ring by one or more electrophilic groups (i.e. the ring is mono or di-substituted) not only have an activity similar to that of unsubstituted catechol but have the additional advantage that they are better able to main-tain their activity notwithstanding prolonged periods of iron loading. Accordingly these compounds can advanta-geously be used in circumstance where it is desired to avoid a reduction treatment.
Thus in accordance with copending Canadian patent ,:

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application no. 420 567 there is provided an insoluble composition comprising (1) one or more catechol compounds covalently fixed to the surface of

(2) a suitable insoluble carrier said catechol compounds being fixed to the surface of said carrier at the benzene ring thereof, said catechol compounds being selected from the group consisting of catechol sub-stituted on the benzene ring by one or two electrophilic substituents.
The above composition can be used to remove Fe Th4+, UO22+ and mixtures thereof from solution.
Copending-Canadian patent application no. 420 567 also provides a method for removing Fe3 , Th4 , UO22+ and mixtures thereof from solution characterized in that the solution is contacted with an insoluble composition as defined above. Thereafter, the composition loaded with metal may be separated from the treated solution, e.g. by filtration. For example, the iron content of a liquid medium may in this way be lowered to less than 0.1 ~M.
In accordance with copending Canadian patent application no. 420 567, there is also provided a method for inhibiting microbial growth in a liquid nutrient medium containing Fe3 b~ lowering the Fe3+ content thereof to less than 0.1 ~M characterized in that said medium is contacted with an insoluble siderophoric composition and thereafter said insoluble siderophoric composition loaded with Fe3 is separated ~rom said medium, said insoluble siderophoric composition comprising:
(1) one or more catechol compounas covalentl~
fixed to the surface of (2) a suitable insoluble carrier said catechol compounds being fixed to the surface of said carrier at the benzene ring thereof, said catechol compounds --`- ~'';

' '~ ' ' ` : ` ' !`: - , ~-3~ r ~ ~S~

being selected from the group consisting of catechol sub-stituted on the benzene ring by one or two electrophilic substituents.
The insoluble siderophoric composition is a composition having a chelating activity with respect to iron, and in particular a selective chelating activity.
The insoluble siderophoric composition can for example be used to specifically remove iron from liquid media such as water, juices, wine, beer, cider, chemical solutions, microbial and tissue culture media, pharmaceuti-cal media, etc.
It has also been determined that microbial siderophores and other organic compounds possessing the same or similar co-ordinating groups or ligands can also, in addition to Fe3 (see copending Canadian patent application no. 4~6 869), remove Th and U02 from solution. If desired, it is also possible to separate Th from U022 if their ions are present in solution in combination.
Thus copending Canadian patent application no.
420 567 in an additional aspect provides a method for removing Th4+, U022~ and mixtures thereof from solution characterized in that the solution is contacted with a composition comprising a member selected from the class 25 consisting of (A) an insoluble composition comprising (1) one or more organic chelating compounds, covalently fixed to the surface of ~2) a suitable insoluble carrier, 30 said organic chelating compounds possessing one or more co-ordinating sites, said organic chelating compounds being selected from the class consisting of microbial siderophores and (B) an insoluble composition comprising : .
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. - , ' -(l) catechol covalently fixed to the surface of (2) a suitable insoluble carrier, said catechol being fixed to the surface of said carrier at the benzene ring thereof.
Compositions as defined above, loaded with ~e Th or UO2 , may possibly be regenerated by chemical means suitable for the removal of the chelated metal; the so regenerated composition can thereafter be recycled for further use.
The insoluble compositions referred to above have a very high affinity for Th and UO2 . They can be used to remove Th and UO2 from solution even if present in trace amounts, e.g. to obtain solutions containing < 0.2 nM
of these ions.
In accordance with the above a mechanism is thus provided for the removal of Fe , Th and UO2 , from liquid media but also fox the preservation of various liquid media through the removal of iron therefrom, i.e. rendering liquid nutrient media highly resistant to microbial growth since any microorganism present cannot proliferate due to the unsufficient amount of iron present.
In accordance with the present invention there is provided a method for treating a composition loaded with Th4 and UO22+ to separate Th4 therefrom, characterized in that said composition is contacted with an aqueous solution containing a suitable Th chelating agent and an organic acid, said organic acid being a carboxylic acid, said solu-tion having a pH greater than 2, said composition comprising a member selected from the class consisting of (A) an insoluble composition comprising (l) one or more organic chelating compounds covalently fixed to the surface of (2) a suitable insoluble carrier, said organic chelating compounds possessing one or more co-. :.
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ordinating sites, said organic chelating compounds being selected from the class consisting of microbial siderophores, and (B) an insoluble composition comprising (l) one or more catechol compounds covalently fixed to the surface of (2) a suitable insoluble carrier, said catechol compounds being fixed to the surface of said carrier at the benzene ring thereof, said catechol compounds being selected from the group consisting of unsubstituted catechol and catechol substituted on the benzene ring by one or two electrophilic substituents.
In accordance with the present invention, it is possible to separate Th and UO2 which are present in a solution. Thus, for example, an aqueous solution containing Th and UO2 and having a pH of about 7 can be contacted with an insoluble composition as defined above, ~preferably a composition incorporating a catechol compound fixed to a silica based carrier), to give rise to a composition loaded with Th and UO2 . This loaded composition can then be recovered and treated as outlined above to separate the Th4 from the composition, to provide a treated composition loaded with UO2 but having a substantially reduced Th4+
content. The treated composition can then be contacted with, for example, an aqueous acidic solution to recover a concen-trated UO2 solution; for example a composition incorporat-ing a catechol compound fixed to a silica based carrier can be treated with an aqueous mineral acid (e.g. HCl) having a pH - 0.8 - 1.5 to recover a concentrated UO2 solution.
The Th4+ and~UO22~ solution can for example, be provided by treating in a known manner, radioactive materials from atomic reactors. For example, Th can be converted to U by treating solid 232Th with neutrons which convert 232Th to 233Th. The 233Th decays to a fissionable type of uranium : .

i.e. 233U. The mixture of these metals can then be solubilized with a suitable agent such as HNO3. The obtained solution, once neutralized to a pH > 4 can then be treated for the separation of the metals as outlined above, i.e. to produce a concentrated solution of the fissionable type of uranium.
Organic chelating compounds, e.g. siderophoric compounds, useful in accordance ~ith the present invention, may also form complexes with certain other transition, rare earth and actinide metals due to the structural (atomic) similarities with iron; however, the complexes are formed at lower affinities than, for example UO2 or iron. Al-though the compositions of the present invention may possibly be used-for the removal of these other metals from li~uid media, the following discussion will be directed to the removal of Fe , Th and UO2 from liquid media.
In accordance with the present invention, a general process for the preparation of an insoluble composi-tion as defined above can be characterized in that organic co-ordinating sites capable of chelating metal are fixed to the surface of a suitable carrier. Any suitable means of fixing organic co-ordinating sites to a carrier can be used provided that the composition obtained has the necessary chelating activity.
If it is desired to produce a siderophoric composi-tion comprising one or more organic siderophoric compounds fixed to a suitable carrier then the process of its prepara-tion may be characterized in that a suitable carrier is reacted with one or more organic siderophoric compounds possessing co-ordinating sites capable of chelating Fe3+ so as to bond said siderophoric compounds to the surface of said carrier, while maintaining the Fe chelating activity of said siderophoric compounds.
The s:iderophoric compounds as indicated above can gl Z~5~5 be microbial siderophores. In this particular case, the process of preparation can be characteri~ed in that a suit-able carrier is reacted with one or more microbial sidero-phores so as to bond said microbial siderophores to the surface of said carrier, said carrier and said microbial siderophores possessing functional groups reactive one with the other so as to bond said microbial siderophores to said carrier while maintaining the Fe3 chelating activity of said microbial siderophores.
Turning now to the chelating compounds, sufficient metal coordination sites to chelate the metal ions (i.e.
Fe +, Th4 and UO22~) may be provided by a single organic chelating (e.g. siderophoric) compound or alternatively by two or more such compounds; the number of compounds partici-pating in the chelation of the metal ions being dependent upon the number of co-ordinating sites which are available from a particular compound fixed to a carrier.
The organic chelating compound used can as indi-cated above be a microbial siderophore. A microbial sidero-phore may have a molecular weight of less than 2500 Daltons, e.g. a molecular weight in the range of 500 to 2500 Daltons.
A microbial siderophores useful in accordance with the pre-sent invention can also possess one or more types of metal co-ordinating sites within its structure. The sites can be provided by groups selected from the class of groups referred to below, e.g. N-substituted hydroxamate groups, catecholate groups etc. Siderophores possessing these groups display high selectivity and very high affinities for Fe3+, Th and UO 2+-The organic co-ordinating sites of suitable organic chelating compounds may, for example, be provided by groups selected from the class consisting of (a) N-substituted hydroxamate groups of formula ' .

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1~5545 ~ ~H
-- -- -- C

(b) phenolate groups of formula ~OH X
~1.

X being an atom of O or N-(c) catecholate groups of formula )m -X' being an atom of O or N- and m being 1, and (d) mixtures or two or more of the above groups.
A representative list of microorganisms and their siderophores is given in following table l:

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i;S45 Table 1 COMMON NAMES OF SIDEROPHORE
ORGANISM NAME OBTAINED THEREFROM
Prokaryotes Enteric species Enterobactin (enterochelin), Aerobactin Agrobacterium tumefaciens Agrobactin Pseudomonas species Pyochelin, Pyoverdine, Pseudobactins, Ferribactin Bacillus megaterium Schizokinen Anab_ena species Schizokinen Arthrobacter species Arthrobactin Azotobacter vinelandii ~ bis-2,3,-dihydroxyben-zoyllysine Actlnomyces species Ferrioxamines Mycobacterium species Mycobactins Eukaryotes (Fungi) Penicillium species, Ferrichromes, Copragen Aspergillus species, Neurospora, Ustillago Rhodotorula species Rhodotorulic acids Ectomycorrhizal species Hydroxamate type The basic chemical structure, trivial names and possible sources o~ some types of microbial siderophores are listed below; the term microbial siderophore of course includes any suitable functional derivatives, analogs or enantioforms of these molecules:
a) Ferrioxamine ~ZlS~4~

H-N ~ f ONH CONH

N - (I N - I - C

~ -~Fé~~
Linear ferrioxamine ~.0 . :, , , :, -. .. .

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H-N~ ~ CONH\ / CONH\ C=O
2 5 ~ C 2)2 (C~ )5 yCH2)2 (C ~ ~¢

I _ I I - ~ N~

C~clic ferrioxamine wherein:
R = H or -COCH3; R' = CH3- or HOOC-(CH2)2-; n = 4 or 5.
For ferrioxamine B, R=H and R' = CH3-. The mesylate salt of deferrioxamine is marketed by Ciba-Geigy as Desferal (U.S. pat. 1964 3,118,823 and 3,153,621; Can. pat. 1962 648981 and 715051).

b) _errichromes H
~C C ~
C ~ / N ~

3 \ ~ H ~ _ ~ 2 3 ~ ~o ~ ~ ~C I _ C 7 ~H

~ C ~ ~ N _ ( H2 ~
H"N\ I /C~

R"' wherein:
R', R" , R"' = H Ferrichrome R = -CH3 (prototype) , .. .

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~Z~5~5 c) Citrate Hydroxamate Der_vati whereln for:
fH3 l H3 R n c=o o--f schizokinen H 2 N-OH HO-N aerobactin COOH 4 (CH2)n (CH2)n arthrobactin H 4 H-l_N_C_CH2_f_CH2_~_N_C_H
R H OH H R
d) Rhodotorulic Acids Ol OH H ~ ~ U-ICl CH3 rhodotorulic acid H3C-C-~ ~ ~ HO H
H H ~__J_-CH2OH
1l OH O ~ ~ N - C CH3 d~ c CH ~ C - N ~ ~ Hl O acid H

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(d) Rhodotorulic Acids (cont. ) fH3 C3~ 33 ll CH
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Fe3+ ) ~ ccpragen / ~ \N

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CH3 /CH2) 2 ~( H2 1,N3 :

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(e) M~obactins NH ~ CO
HO)~\R2 IHR5 ~o ~ CHR4 R ~H C0 R3 f ~NC NH
R ( H )'"`b ~H- CO
wherein:
Rl = methyl, ethyl or alkyl or alkenyl of 11 to 20 carbon atoms R2 = H or methyl R3 = H or methyl R4 = methyl, ethyl, or alkyl of 15 to 18 carbon atoms R5 = H or methyl (f) Fusarinines r ~ lH2 Hl R 1 wherein:
HOt - C - (CH2)3 N C~ ~H2 CH2 O ~ , H / ~ ~

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g ) Enterobact in OH

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C--O
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HO
OH
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(h) Agrobactins ~OH

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N

wherein:
R = H,OH

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( i ) Pseudobactin O O
~ CH2 CH2-lC

H2N N~ ~; L~\~fCH

NH
OH l - \2 \ / ~C/H~H

C~ NH ~ /CH

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A detailed description of the above siderophores is given by Neilands lAnnu. Rev. Biochem. 50: 715-731, 1981), and their coordination chemistry has been ~eviewed by Raymond ~ Carrano (Accounts of chemical Research, vol. 12, No. 5, 1979 at pages 183-190).
Microbial siderophores can be extracted or exam-ple from spent microbial culture media with organic solvents.
Examples of such methods are given in United States Patent Nos. 3,118,823 and 3,153,621 as well as Canadian Patent Nos.
648,981 and 715,o51. For example, siderophores possessing hydroxamate ligands may be obtained in this fashion. Hydro-xamate microbial siderophores are distributed widely through-out the prokaryotic and eukaryotic microbial world, but to date, only bacteria are known which produce typical mono- and dihydroxybenzoic acid-bearing siderophores.
Some microbial siderophores, their analogs and/or their enantioforms have been chemically synthesized in the laboratory:
(i) enterobactin, its enantioform and carboxylic, methyl, and aromatic analogs;
(ii) Nl, N8-bis-2,3-dihydroxybenzoylspermidine;
~iii) ferrichrome and enantio-ferriochrome.
other processes for the preparation of various siderophores are described in Canadian Patent No. 742,670, 746,873, 773,540 and 775,539.
A discussion of the preparation of siderophores by denovo synthesis can be found in Neilands Review 1981 and Neilands et al J. Biol. Chem. 256 ; 3831 - 3238, 1981.
The ferrioxamine B is sold commercially under the designation Desferal which is a trademark of Ciba-Geigy.
The microbial siderophores, ferrioxamine and enterobactin, referred to above are prototypical natural microbial siderophores and each represents the general struc-ture and properties of hydroxamate and catecholate-bearing siderophores respectively. These particular siderophores will be ~,:
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referred to below (e.g. m the examples). For the purposes of this specifica-tion the expression des as it ap~s before ferrio~aune etc ls to be under-stood to refer to ferrioxamune etc wherein co-ordlnatlng sites are unoccupied e.g.
they are not iron loaded.
Turning now to carriers suitable in accordance with the present invention, they must of course be insoluble in the liquid medium of intended use; for example, the carrier c~n be water insoluble. Desirably, the carrier is also inert in the liquid medium of intended use. The carriers can be in particulate or solid form.
The carrier can be an organic or inorganic compound.
For example, the carrier may be a natural or modified natural polymer (e.g. lignin, agar, alignate, glucan, cellulose, dex-tran, cellulose acetate, humic acid, etc.) a synthetic organic polymer te.g. a polyamide, a polyamine, a polyacrylamide, a polyester, a polyurethane, a polyethylene, a polystyrene, a polypropylene, a polycarbonate, a silicone, nylon, latex, a polyfluroolefin, etc.~ or an inorganic material ~a ceramic, a glass, carbon, etc.).
As indicated above the present invention relates to a (siderophoric) composition comprising one or more microbial siderophores which are immobilized or fixed on a suitable insoluble carrier in such a way that the microbial siderophores retain their high chelating affinity for metal ions, i.e.
iron.
A number of known processes are suitable for the binding of microbial siderophores to carriers so as to preserve the iron chelating ~r complexing properties thereof. For example, the commonly used methods for binding enzymes to insoluble carriers can be adapted for the immobilization of microbial siderophores. See, for example aMethods of Enzy-mology~, XXXIV B:30 (Jakoby W.B. Ed.) Academic Press, New York (1974).
Carriers which are suitable for the process of preparing siderophoric compositions using microbial siderophores are those which have active surfaces; the active surfaces have ,. .~ ,; ` ` ' . .~ . :.. . ~ .

functional groups which can bond to a compatible functional group of the chosen siderophoric compound. The functional group can, for example/ be selected from the class consisting of O O
2' (CH2)n-NH~, n being 0, 1, 2 3 etc o - OH
-C-H,-CH~-X, X being a halogen atom, for example, Br, O-~ = N~, -OH , -~-X, X being, as defined above,~C-N3-SO3H,and ~ N2 .
However, any functional group can be used which will react with a functional grGup on the microbial siderophore in ~uestion to bind it to the carrier, the microbial siderophore retaining its iron chelating capacity.
It is possible to put some distance between a microbial siderophore and the surface of the carrier, e.g.
in order to limit the effect on the microbial siderophore of a surface characteristic of the carrier. For example, TEFLON* may be used as a carrier. However, teflon has a highly hydrophobic surface which is non-wetting. There-fore, it is desirable to put some distance between the sur-face of the TEFLON* and the microbial siderophore to allow the siderophore to extend well into an aqueous liquid medium.
A spacer compound may be used to provide a spacer group to space apart a carrier and a siderophore.
A suitable spacer compound is bifunctional; i.e.
it has a functional group which can react with a functional group of the carrier to bind it thereto; and it has also a second functional group which can react with a compatible functional group on the chosen microbial siderophore to bind it thereto: see the above groups. The spacer group may alternatively have a second functional group which while not reactive with a compatible functional group on the siderophore, may be convertible into such a group.
A spacer compound can,for example, in addition to * trademark .
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the above referred to functional groups, include a hydro-carbon chain, the length of which is chosen in accordance with the distance which it is des:ired to place between the carrier and the siderophore. The spacer compound used may be 1-ethyl-3-(3-dimethylaminopropyl~ carbodiimide hydro-chloride salt or glutaraldehyde. However any compound can be used which will space the microbial siderophore from the carrier, the necessary or desired distance provided of course that it is bifunctional. ;
The spacer compound may be bound, to a carrier by making use of conventional reactions involving the formation of ester groups, amide groups, amino groups, diazo groups, ether groups, sulphonamide groups, amidino groups; the reaction may be a carbon-carbon condensation.
Thus a carrier suitable for the process of the present invention may be represented generally by the formula back ~ R - ~cn~ n wherein n is an inte~er, aback is a carrier backbone, R
is a single bond or a suitable spacer group and ~cn is a functional group as defined above. For example ~cn may be a carboxyl group and R may be a group such as O O
(CH2)2 ~ NH (CH2)2 ( 2)2 A useful carrier may need to have its surface treated in order to provide the surface with a suitable functional group which can bond to a microbial siderophore. -Thus silica (e.g. in the form of a silica gel) having surface hydroxyl groups can for example, be pre-treated with a suitable ~-amino(C2 to C10 alkyl) tri (Cl to C5 alkoxy) silane to provide an active surface comprising amino groups. The silane can, for example, be ~-aminopropyl-f''`~ !

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~s~sj triethoxysilane. See, for example, the following patents wherein silica is treated with a silane: Canadian Patent Nos. 1,102,347, 1,103,035 and 1,102,3~6; U.S. Patent Nos. 4,203,952, 3,886,080, 3,904,373, 3,519,538, 3,652,761, 4,230,803 and 4,290,892.
Nylon, for example, is a carrier which requires a pretreatment to provide it with suitable functional groups. Since the nylon contains the amide group, its surface may be sub]ected to partial hydrolysis using known techniques to give free amino and carboxyl functional groups. The aqueous method may proceed as below:

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;5~5 R - C - N-R _ HCl ~ R -I C - OH HN R
Nylon (R being Modified Nylon the rest of the chain) Alternatively, nylon can be reacted in a non-a~ueous medium with, for example, thionylchloride to gi~e rise to the functional group - C = N. If desired/ an ~1 .
lG appropriate spacer group can be rleadily attached through, for example, the use of ethylene Idiamine or another func-tionally equivalent species such as N02 ~ H2 followed by a reduction of -N02 to -NH2 suitable for generation of diazonium salts which are th~n suitable for ooupling to a mucrobial siderophore,e.g. enterobactin. Succinic anhydride can thereafter be used as a further extension of the spacer group; i.e. to form an amide linkage.
Teflon is another useful carrier which must be pretreated in order to provide it with a suitable functional group which can bond to a microbial siderophoreO
Teflon, a tradename for polytetrafluoroethylene from DuPont, is highly inert and is not readily attacked by acids and bases. No easy displacement of the fluorine atomes is known. Fluorine atom can,however,be displaced by ion-radicals such as sodium or potassium napthalene of formula:
NO
H or On reaction between te10n and such sodium or potassium naphthalene, a sodio or potassio species of teflon is formed of formula:
F F
--C -- C ~
Na F

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These organo metallic species of teflon are highly reactive towards many organic functional groups and their general behaviour is similar to the well known Grignard reagents.
Thus, they can be reacted with a dimethyl carbonate to give rise to an alkoxy carbonyl substituted polytetrafluoro-ethylene. This substituted ethylene can subsequently be subjected to hydrolysis to provide a polytetrafluoroethylene with carboxyl substituents. The carboxylated teflon thus generated, can then be used for direct coupling to microbial siderophores (or chelators) such as aDesferal. As indicated above, it may be desired to space the siderophore from the surface of the teflon. If so, ethylenediamine and similar compounds can be readily attached through the carboxyl group by standard procedures. Since the teflon's backbone is very inert to many organic and inorganic reagents, very vigorous reaction conditions can be ~mployed in further derivatization using the carboxylic functional group. See, for example, Methods in ~nzymology Supra.
As indicated above, the microbial siderophore must also possess compatible ~unctional groups which will react with those of the carriers without interfering with the che-lating activity thereof. For example, suitable functional group in the siderophore enterobactin is the 2, 3-dihydroxy benzoic group which is susceptible to diazonium coupling under neutral to almost neutral conditions. The acylamine required in the generation of diazonium salts can be pre-pared from aminopropylsilylated glass.
Examples of suitable functional groups on the microbial siderophores are the amino group, the carboxyl group, the phenolate group and the cathecolate group, etc.
The previous comments relating to carriers, spacer groups etc for microbial siderophores apply to the use of other organic chelating (e.g. siderophoric) compounds, for example a catechol compound. If a catechol compound is , . -. ~ ; ;'' : ' : .

:,- , ' :
~ ; :

~lZ~54~ j used as the chelating compound, it may be bonded to a carrier by diazo coupling or through reaction with a func-tional group on the carrier such as O In these - C - H.
latter cases the functional group on the carrier is directly reactive with the benzene ring of the catechol.
The preparation of a cornposition comprising a substituted catechol suitably fixed to a silica gel carrier can be graphically described as follows:
1 7t silica) - O - Si - OH + NH2 - (CH2) 3 ~ Si - OEt O O
I Et ¦ Et O O
(silica) O - Si - O - Si - (CH2)3 - NH2 I Et Et = ethyl I Et CH2 CH2 fH2 1 1HO CHO~ (silica3-O-Si-O-Si-(CH2)3-N=CH-(CH2) 3-CHO
o Et ~ OH I Et (catechol) OH O O OH
>
(silica)-O-Ii-O-li-(CH2)3-N=CH-(CH2)3-lH
O O R
I Et .. ` ~: :

~9~ 45 where R is ~ or ~ _ I Et NaBH3CN ~silica) -o-si-o-$i- (CH2)3-N-(CH2)4-CH

l Et ~ OH
~ OH
and ¦ It O O H OH
(silica)-O-Si-O-Si-(CH2)3-N-(CH2)4-CH

¦ Et ~ H
OH
The above catechol compositions will hereinafter be referred to as H

Sili- ~ H
and OH
~ OH
Sili- ~

The above obtained catechol compositions, e.g.
OH
Sili ~ OH , can then be treated to introduce onto the benzene ring of the catechol group one or more of the electropholic substituents (i.e. the ring is mono or di-~.

.

~ . .

. .. ,. , :

.

5~

substituted). The substitutents can be chosen for example from the class consisting of Halogen atoms (e.g. Cl and Br), NO, NO2, COOH and O The ring can be mono or di halo - C - H .
substituted or mono substituted with NO or NO2. The sub stituted catechol residues may thus have the following formulae HO OH OH

R
n Rl~
wherein R is a suhstituent as defined above, n may be 1 or 2.
When using a composition as described above, the conditions of use should of course be such as to avoid the break-down or decomposition of the com-position; i.e. conditions such as pH, temperature, pressure, etc.~ should be chosen so as to avoid the break-down of the composition.
As indicated above, an insoluble (siderophoric) composition, in accordance with the present invention, can be used to remove iron from a liquid medium. In use, the (siderophoric) composition is intermixed with a desired liquid medium for a suitable time, which will of course depend upon the amo~mt of (siderophoric) composition used, the initial iron concentration, the desired final iron concentration, etc.
The Fe3 in the medium combines with the (siderophoric~ compo-sition and can thus be physically separated from the medium.
The affinity of siderophoric compounds for iron can be so great that even small amounts of iron can be removed from a liquid medium. The final concentration of iron in a treated nutrient medium can for example be far below that required to support microbial growth.
In the drawings:

,: ' ~' ," :' ;

~2~L~ii545 Figure 1 is a graph illustrating the inhibition of microbial growth due to the removal of iron from a liquid medium, Figure 2 illustrates regeneration of a siderophoric composi-tion by pH manipulation; and Figure 3 illustrates regeneration of a siderophoric composi-tion by a reducing agent.
Figure 1 as indicated above is a graph illustrative of the inhibition of microbial growth in even the most nutri-tional f ,' ' "
, ,- .:. :

:, lZ~ S
solutions (e.g. bacteriological broth medla) on removal of iron therefrom.
In particular, fiqure 1 illustrates the lnability of the bacterium Neisser_a meningitidis to ~row in a complex highly nutritional medium (neisseria defined medium - NDM) from which only iron has been extxacted with ferrioxamine immobilized on agarose, the agarose having previously been activated by cyanogen bromide fo;r couplin~ to ferrioxamine.
Thus, one gram of the above siderophoriC composition was contacted with 20~ ml NDM at 22C for a time period of 20 min. before recovering the siderophoric composition.
Prior to treatment, the NDM contained about 3.6 ~M of iron;
after treatment, it contained less than 0.1 ~M iron. The treated medium was then divided into two portions and FeC13 was added to one of them. A control consisting of untreated NDM and the two portions were then inoculated with microbes and maintained at a pH o 7.4 and a temperature of 37C.
As can be seen ln figure 1, unhibited growth occurs in the control (0). However, in the treated medium, (~), cells are unable to undergo anymore than one or two divisions due to the absence of the vi~al nutrient iron. On the other hand if exog~nous iron is added back to the treated medium full growth is again realized ~
Liquid media to be treated to remove Fe3 can have, for example, a pH in the range of 4.5 to 9. During the contact with the siderophoric composition, the temperature of the mixture can for example range from 1C to 50C and the contact can occur under atmospheric pressure~ Examples of different media which can be treated with the composition axe listed in table 2 which follows:

::

~' :: :

5X4~

Table 2 Classes of liquid media Spe ific example thereof Liquid foods - fruit and vegetable juices, clear meat broth (e.g. consommé), culture media for microbial, plant and animal cells Breverages - wine, beer, natural and synthetic juices, cider, drinking water 10 Pharmaceutical - Buffer solutions for lavage (e.g.
ophthalmic solution, peritoneal lavage), water used in the manu-facture of various solutions and preparations, antibiotic solutions, 15 Cosmetics (liquid) - those susceptible to microbial degradation, contamination, or spoilage Industrial water and - cooling tower, process and waste waste water water 20 Natural water - removal of actinides (e.g. Th4 , UO22 ) and chromium A siderophoric composition can, as indicated above, for example~ be used to remove iron from microbial fermenta-tion cultures to stop further growth of microbes in the fermenter. Thus, a siderophoric composition in accordance with the present invention may be used to treat wine in order to inhibit microbial growth therein.

The compositions described above may also be used to remove iron from cosmetic solutions to prevent contamina-tion by the growth of microbes. Components for cosmetic .
~ ' . ~.... . . .
, .. : : .:

,. - .. : :
~: , "
~ -. . .

~z~

solutions are often obtained from natural sources and are sus-ceptible to microbial degradation.

A siderophoric composition may also be used fo5 the removal of iron from drinking water, pharmaceutical io ' o y ic ~ ~o I ~ i JA S, // ~

- 32a -. . : .. . .
. .. .
: . . ..
...

,.
.:

~2~15~i4~

Although the microbial siderophores are selective for iron, they can also bind metals that are classified as actinides e.g. uranium. Thus, the present invention addi-tionally provides means for removing such hazardous metals as plutonium from contaminated water; and a rapid means to collect (concentrate) the radioactive heavy metals (e.g.
plutonium) to determine the concentration thereof in standard water volumes. Such metals are selectively removed from water due to their structural similarity (i.e. atomic) to iron.
As indicated previously, compositions as described above, may possibly be regenera-ted for further use by the removal of the metal therefrom by suitable chemical means. In this way, the composition can be economically used since it can be recycled for repeated use.
The regeneration,for example, of a~siderophoric) composition loaded with iron, may be carried out either through the manipulation of the pH of a medium surrounding the iron-loaded(siderophoric)composition and/or by treating the iron-loaded composition with ~ suitable reducing agent. In either case, appropriate conditions should be chosen which will not decompose the composition or destroy the iron binding capacity thereof.
If regeneration is affected by manipulation of the pH, the pH must be brought to or beyond a point at which the iron is released.
In general, when making use of a microbial sidero-phore, a pH of 1 or lower should be avoided; the use of mineral acids should also be avoided. The pH can be manipu-lated through the use of organic acids (for example, acetic acid, succinic acid, citric acid, isocitric acid,ketomalonic acid, malic acid, oxalic acid or pyruvic acid).
If a catechol compound is used a mineral acid may be used to manipulate the pH.

: - 33 -- '' ~' .
':

';

s~

Figure 2 illustrates the xegeneration of a (sidero-phoric)composition by the manipulation of pH. The designa-tion (~) repr~sents enterobactin immobilized on a polyacryl-amide carrier whereas the designation ~O~ represents des-ferrioxamine immobilized to the same type of carrier. The pH was lowered in the presence of lS mM citrate and 0.05M

/

. ~ .

: .. . ~ .
.- ' ::, , : - :. .
,; . ' -''`': '~
.~

~z~ s tris-sodium acetate. The lowering of the pH was accom-plished by an addition of appropriate amounts of acetic acid.
Alternatively, an iron loaded compositian may be treated with a suitable reducing agent to release the iron.
It is possible to use suitable dithionites or ascorbates as the reducing agents, e.g. sodium or potassium dithionite and sodium or potassium ascorbate. The dithionites can be used for the reduction of (siderophoric) compositions which include hydroxamate ligands whereas the ascorbates can be used for the reduction of siderophoric compositions containing phenolate/catecholate group ligands. Other useful reducing agents include hydroxylamine and hydroquinone.
The compositions wherein the organic chelating compound includes catecholate ligands (e.g. siderophoric compositions consisting of enterobactin fixed to glass) and especially the compositions containing diazo linkages must be subjected to mild reduction conditions such as provided using ascorbic acid. The compositions which include hydroxamate groups (Desferal), can be reduced with 1.0 molar sodium dithionite.
Other reducing agents may possibly be used to regenerate a composition; however, the reducing agent used must be chosen on the basis that it will not destroy the integrity or metal-binding (e.g. iron-binding) capacity of the composition. Sodium dithionite (Na2S2O4), hydroxylamine (including its acid addition salts) and hydroquinone are as indicated above examples of useful reducing agents. In particular the reducing agent can be hydroxyl amine chloride.
The regeneration of a (siderophoric) composition may take place in the presence of a suitable organic acid that will complex with the iron that is released. Suitable acids are di or tricarboxylic organic acids that will chelate the liberated iron ions.

, .
.: ., , - : -~: L5S4~ ~

Figure 3 illustrates the repeated regeneration of tsiderophoric) compositions consisting of enterobactin ( ~ ) desferrioxamine ( ~ ) bound to polyacrylamide carriers, the reducing agent consisting of sodium ascorbate. The regenera-tion solution had a pH of 7 in the presence of 15 mM sodium citrate and 0.05 M sodium acetate.
Compositions as definecl above which are loaded with Th and/or UO2 may possibly be regenerated in the same manner as for iron loaded composi-tions. When regenerating a composition loaded with UO22 by manipulation of pH a relatively low pH (e.g. pH - 0.8) may be needed to remove this ion; accordingly it may be necessary to resort to the use of a mineral acid to recover the ion e.g. HCl. Less severe condition may be used to recover the Th e.g. by using a wash solution having a higher pH than that necessary for the removal of UO22 .
Since the Th may be recovered at a higher pH
than the UO22 it is possible to manipulate the pH of the solution to be treated such that a composition in accordance with the present invention will preferentially take up the U2 thereby separating UO22 from Th4 .
Alternatively as indicated above, an insoluble composition as defined above may be used to take up both UO2 and Th . The so obtained composi-tion may then be treated with an aqueous solution containing a suitable Th4 chelating agent and an organic acid, the pH
of the solution being greater than 2.
Preferably the pH of the aqueous so~ution should be about 4 to about 6.
Some of the above referred to microbial siderophores in free form (i.e. not fixed to a carrier) may possibly be used to chelate Th for the above method. Alternatively the ion chelating agents disclosed in Archibald, F.A. and I.W.
DeVoe 1980, Iron acquisition by Neisseria meningitidies in vitro Infect. Xmmun 27:322-334, are suitable for chelating ,.'~' :", , ' ' ., .
.. .:
: . , , , :

, ~554~
Th4+.
The chelating agent and the organic acid may both be a tricarboxylic acid: they may be the same. It is possible to provide the necessary tricarboxylic acid by adding a mineral acid (e.g. HCl) to an aqueous solution containing an alkali metal salt of a suitable tricarboxylic acid e.g.
Na3 citrate.
Suitable Th chelating agents may be selected from among organic acids having the following general struc-10 tureS

R R
R - C - COOH R - C - ~X~ O - C - COOH
(CH2 ) p (Cl H2 ) p 1CH2 ) p 15C - CCOH R'--C - ox~ R - C--COOH
R - C - COOH , (IH2)k and (~H2)k R - C - COOH R - C - COOH
R R

~herein R' and each R are independently selected from the group consisting of hydrogen and suitable organic residues;
R' may also alternatively be OH or alkoxy (Cl to C10); p and k are the same or different and are 0 or an integer;
R', R, p and k are selected such that the chelating acti-vity of the three COOH groups is not interfered with. Rcan for example be selected from alkyl (e.g. Cl to C10);
Halogen (e.g. Cl, Br); p and k may possibly be 1 to 10.

Preferably each R is H and p and k are 0.
Suitable chelating agents are citric acid, iso_ citric acid, cis-aconitic acid and oxalosuccinic acid. The alkali metal salts o~ these acids could of course be used ln conjunctlon with a sultable source of protons li.e. H ) e.g. a mineral acid such as HCl.

.

5~S

The organic acids may be selected from the same group of acids as outlined above for the Th4+ chelating agents. However, other organic acids may be used such as the mono and di carboxylic acids (e.g. acetic acid, mzlonic acid etc.).
A pH for the aqueous solution of greater than 2 is necessary in order to inhibit the simultaneous removal of UO22+ from the composition i.e. at a pH lower than 2 UO22+
car, ~e recovered from the composition. Thus once Th is separated from the composition it is possible to recover a concentrated solution containing UO2 by subsequently treating the composition with an aqueous solution having a low pH. As indicated above since it may be necessary to use a mineral acid in order to recover UO2 the composition in such case can be based on a catechol compound; the catechol compound is preferably fixed to a silica gel if the UO22 is to be recovered and the composition reused.
The insoluble compositions as described above thus provides for the advantageous removal of metals (e.g. iron) from liquid media. Such media remain essentially unchanged except for the absence of metal.
The liquid media referred to herein may be aqueous, organic or mixtures thereof.
Reference will now be made to a number of examples which deal with embodiments of the present invention.
Example 1: kctivation of silica gel (glass) The activation methods were analogous to those as described by H. Weetal ~ A.M. Filbert, Methods of Enzymology XXXIV B:59-72 1974.

-. ~
- ~ - . :.. . . .: .. :, ~ ~ - .. .
: ..
,.

i54S

(a) Pretreatment 100 grams of silica gel designated Sigma S-4133 (sold by Sigma Chemical Company) of 100 to 200 mesh (70 to 140 microns) chromatographic grade and of pore diameter of about 25 angstrom was suspended in a * trademark 3 :

.

~L2~ ;S4~5 mixture of 50 ml of 70% HNO3 and 300 ml of distilled water. The suspension was refluxed with mixing for about 1 hour.
The gel was allowed to settle and the liquid layer drawn off. The gel was then washed repeatedly with distilled water until the wash water was about neutral pH.

(b) Amine activated silica gel:

The above pretreated silica gel (wet) was used for the following amination without drying. A 10%
solution of ~-aminopropyltriethoxysilane in distilled water 1500 ml~ was added to the above obtained silica gel. The pH was then adjusted to 3.45 with 6N hydrochloric acid. The suspension was then maintained under stirring at a temperature of 75C for about 3 hours. The gel was then filtered and washed with 500 ml of distilled water and dried in an oven at 100 to 110~C.
- The above amination can be described graphically as follows:
Et f OH O
30glass ~ Si-OH ~ Et-O-Si-CH -CH -C~ -NH P >
I I 12 2 2 2 aqueous _~ O O75C.
Et , ~ ~
.
,. . -~: ,, . . . . .. ......

5~
Il Et I ~ , if desired, glass ~ Si-O-Si-CH2-CH2-CH2-NH2 ~ >
~ I o treated to add group H Et -Si-OH
-Si- OH
H O to block reactive group ~ I of Et.
glss J Si_O_si_c~2_cH2_cH2_NH2 H
--si--I Et = ethyl The above obtained amineactivated silica gel will hereinafter be reerred to as glass JNH2 .

(c) Aminoiarylcarbonyl activated silica gel: -50 grams o glass ~NH2 was suspended in 300 ml of ethanol free chloroform. 2.5 qrams p-nitrobenzoyl chloride ~Eastman KodakJ was subsequently admixed therewith.

:

. - , , . , :

:: : ``- ~ : :
: :
:, a~ss~

Thereafter, 30 ml of dry triethylamine was added and the resultant mixture was refluxed for 20 hours. The beads were allowed to settle and the liquid phase drawn off. The beads were then wash repeatedly with chloroform then repeatedly with ethanol and finally with distilled water with ethanol.
The wet water-washed gel was subjected to a trea~ment for the reduction of the nitroaryl group to an aminoaryl group by adding the gel to a solution of 50 gm of sodium dithionite in 250 ml. of distilled water. The whole suspension there-after being refluxed with mixing for 45 min. The suspensionwas then filtered while still hot and washed repeatedly with dilute hydrochloric acid and then washed with distilled waterO
After thorough washing with distilled water, the gel was dried in an oven at 70 to 80C; this type of activated~carrier can be used for subsequent diazotization and coupling.
The above reaction can be represented graphically as follows: NO2 ~ - Chloroform H O
glass ~ NH2 ~ ~ ¦ CHCl~ -~ glass f N-e ~ NO
J ~ Triethylamine 2 C=0 Et3N
Cl a Reduction H IOI r_~
Na2S24 in~ glass ~ N-C ~ NH2 ~

: . ' :' ' ' :

' ' :

~lSS~

(d) Carboxyl ~ctivated silica gel:

50 gm of glass ~ H2 (see above~, were suspend~d in 250 ml of water cold in an ice bath. 50 ml of 1 N sodium hydroxide was added followed by lS gm of solid succinic anhydride. After mixing or 2 hours, the pH was adjusted with the addition of 1 N sodium hydroxide to a - pH of 5-6. This adjustment of pH with sodium hydroxide was repeated hourly three additional times and the mixture was left overnight. The suspension was thereafter filtered and washed thoroughly with distilled water and dried at 100 to 120C.
. The above reaction can be repr~sented graphically as follows:

H - H
glass ~ N-H ~ CH -CH NaOH~ glass f N-C-CH2 ~ . 1 2 1 2 aqueous. I

HO--C=O

. . .~, . . .

., , . ; - .. ,. ;: ,, - ~,. ;
:.

: . . .

. :.
- ~ : .
.; .. : :

S

(el Aldehyde activated silica gel:

20 gm of glass f NH2 ( see above) were suspended in 50 ml of 0.1 M sodium phosphate at a pH of 7 followed by the addition of 10 ml purified 8% glutaraldehyde.
The mixture was subjected to vacuum and mixed occasionally.
The reaction was allowed to proceed for 3 hours and the mixture was thereafter filtered, washed thoroughly with distilled water and dried under vacuum.
The reaction outlined above can be represented gra~hically as follows:

H H
glass ~ N-H + O=l-CH2-CH2-CH2-C=O

glass ~ ~=c-cN2-cN2-CN2-C=O

.~ :

.

'. ' - . .

,. :

s~ss Example 2: Activation of polyacr.ylamide (azide coupling) 60 ml of ethylene diamine activated polyacrylamide gel (per Inman, Methods in Enzymology XXXIV
B:35 1974) was diluted with water to make up to 100 ml volume. About 1.~ g of p-nitrobenzoyl azide was dissolved in 100 ml of tetrahydrofuran and the obtained solution was subjected to filtration.
The obtained filtrate was added immediately to the aqueous gel suspension referred to above.
1.5 ml of triethylamine was then added to the suspension. The gel mixture was then stirred gently for 30 min. An additional portion of 1.2 grams of p-nitrobenzoyl azide in 50 ml of tetrahydrofuran was added followed by another 1 ml of triethylamine. Gentle stirring was continued for an additional hour. The obtained gel was filtered and washed tho~ughly with 1:1 tetrahydrofuran: O.2 M sodium chloride and then resuspended in 0.2 M sodium chloride. 3 ml of acetic anhydride was added to the suspension which was then mixed for 1 hour, the gel being thereafter washed withO.l M NaCI.
The obtained gel can be used directly for diazo coupling.
The reaction can be represented graphically as follows-:

. .

: . ~.. :

~z~s~s l3 Ç=~
N-CH2-CH2 NH2 ~ ~ B f N-CH2-CH2~ ~ NO
~ triethylamune 2 ~ l~ H O
,,~ B~ CH2- CH2-N-C~}NH2 wherein:B = polyacryla~ide backbone ~ie carrier) Example 3: Activation of agarose with cyanogen bromide.

Agarose swollen in water was mixed with an equal volume of water. Finally divided CNBR
~50-300 mg per ml of agarose) was added atonce to the stirred suspension. The pH of the suspension was immediately adjusted and maintained a~ pH 11 with sodium hydroxide. The temperature of the suspension was maintained at 20C ~nd the reaction allowed to proceed for about 30 min. Thereafter, the gel was then washed rapidly with a large amount of ice cold water followed~by washing with an appropriate huffer. The~obtained activated gel should be used as soon as possibIe.
The chemical reactlons involved in the agarose activation can be indicated graphically as ~ollows: ;

::

.

: . ::

: , ' ': ~
,: -. ..

s~

agarose agarose _ ~ OH e ~
+ CN Br OH ~ C=N-H ~ H20 _ - OH t Cyanogen bromide, by analogous procedures, can be used to activate other polysaccharides;
for example, alginate, glucans, cellulose, agax, dextrans, etc.

Example 4: Immobilization of ferrioxamine or enterobactin 1 mM HCI-washed, water swollen CNBR activated carrier, obtained in accordance with Example 3,was mixed with ferrioxamine or enterobactin 120 mg/ml)in an NaHCO3 buffer (O.l M, pH 8.3~ containing 0.5 M NaCl.
The mixture was agitated overnight at 4C. The gel was then washed with 0.1 M acetate buffer pH

4.0 containing 0.5 ~ NaCl to remove excess uncoupled ferrioxamine or anterobactin.

Example 5: Immobilization of Desferal to polyacrylamide gel -la) Biogel*P-150polyacrylamide from BioRad was linked through ethylenediamine and then succinic anhydride following published procedures as described by ~- * trademark -. :i .
.: ; : ,. `' -': :
" '; ~', ~ ~ ' ~21~;~i4~

Inman (John R. Inman, Covalent Linkage of Functional Groups, Ligands, and Proteins to polyacrylamide beads in Methods of Enzymology , XXXIV B, 30 ~Jakoby, W.B. ed. ? Academic Press, New York (1974) and Biochemistry 8:4074 (1969)~ (see examples 16 and 17) infra. Unreacted free amino groups in ethylene-diamine were blocked by reaction of acetic anhydride at the end of the reaction period.
Test for presence of any free amino group was through the TNBS (trinitrobenzene sulfonic acid) test. The addition of aceti~ anhydride was repeated twice until TNBS test were negative,.

This activated, extended polyacrylamide was immediately used for coupling.

~b) Coupling of Desferal to activated polyacrylamide gel .
700 mg Desferal was dissolved in 5 ml of deionized water and followed by addition of163 mg ferric chloride. A deep red solution was formed. 20 ml activated gel, obtained above, was washed twice with ethanol by centrifigation and decantation.
The total solid shrank to a very small volume a~ter the second volume of ethanol (20 ml~ was added.
The 5 ml of ferric complexed Desferal was added~
at 20C to this shrunken gel and the mixture was mixed by swirling. The gel gradually swelled to yive a solid mass. 5 ml of H20 was added to help disperse the gel and the pH of the suspension ;~

. . . - , -:~ - . ~
" ' ' .' ~','',1''~' ~z~ss~

was adjusted to 4.2 with NaOH ~lN). 200 mg EDC
(l-ethyl-3-(3-dimethylamino propyl) carbodiimide), 200 mg was added in one portion and the pH of the suspension monitored and kept at 4.3 to 4.6 by addition of HCl ~1 N), :Eor 3 hrs. A further portion of 200 mg EDC was added and the mixture, allowed to stand overnight at room tempexature. The gel was filtered on a sinte:red flass funnel and washed with 0.2 M NaCl.
The reaction can be graphically represented as follows:

PA
CH2 O H o O
H-CI - C-~-CH2-CH2-N-C-CH2-CH2-C-OH + H2N-CH2-Def + C2H5N=C=N

Cla PA C H -N-C=O

~_c_c_N-cH2-cH2-N-c-cH2-cH2-c-~-cH2-De f ~ C\3 NH

Cle wherein: PA = polyacrylamide backbone or carrier and Def = desferal~

. , i , ': ~ ' Example 6: Immobilization of ferrioxamine or enterobactin to polyacrylamide carrier -A swollen polyacrylamide gel acyl azide derivative freshly prepared as in accordance with example 2 was suspended in a solution containing the following: 0.1 M CaC12, 0.001 N HCl acid, ferrioxamine or enterobactin at 0.3 mg/ml (pH 4.0).
The pH was immediately adjusted to 9.O and the mixture was stirred for 60 min. at 0C. In the case of enterobactin, the solutions are 50% ethanol.
The coupled gel waswashed with large volumes of Q.05 M Tris-acetate-0.15 M citrate, pH 7Ø

Example 7: Immobilization of Desferal to silica gel (glass beads) -About 700 mg ~or approximately one mMole) of Desferal was dissolved in 25 ml of water followed~
by the addition of 170 mg of ferric chloride.
The pH of the solution was then adjusted to 4.3 with one normal hydrochloric acid and to ~his was added 25 gm of succinylated silica gel characterized by the formula:

glass ~ N - C - C~2 - CH2 ~ C-N~2

- 5~ -,, , i54S

200 mg of l;DC: HCl was added to thls mixture which was thereafter agitated for 3 hours at room temperature. The pH of the solution was then adjusted to 4.3 and the mixture allowed to stand S overnight. After filtration, the obtained compo~lte was washed thoroughly with distilled water until the washing water was colorless. Thereafter, the obtained composite was dried under vacuum.

Example 8: Immobilization of enterobactin to aminoarylcarbonyl activated silica gel-5 gm of aminoarylcarbonyl activated silica gel obtained as in` step (c) of example 1, was mixed with 10 ml of 1 M sodium nitrite and cooled in an ice bath. 1 ml of concentrated hydrochloric acid was added dropwise to the cooled solution. The mixture was maintained in the ice bath for an - - additional 45 min. to allow ~or complete diazoti zation. The mixture was then filtered in the cold, washed with cold distilled water and, while still cold, the gel was added to a solution of about 30 mg of enterobactin in 10 ml of ethanol including about 2 ml of saturated borax solution. The reaction was allowed to proceed for 1 hr in an ice bath. The composite was then recovered by filtration and washed with an ethanol water mixture containing 0.1 normal hydrochloric acid and thereafter with ethanol until the filtrate obtained .
, .
:

:~
:

~12~5'~5 was colorless. The obtained composite was then dried under vacuum.

The coupling reaction can be represented graphically as below:

7 I NaNo2 I R
glass f N-~ ~ H2 ~ glass f N-C ~ N2 H O
+ EnterobaCtin~ glass ~ ~-C- ~ N=7 C=O HO

H~ ~
OH ~C=o Enterobactin xample 9: Immobilization of cathechol to polyacrylamide gel:
diazo coupling -20 ml of the gel obtained in accurdance withExample 2, was suspended in 20 ml of 0.1 normal HCl and cooled in an ice bath. While the suspension was maintained in contact with the ice bath, 2 ml of 1 M sodium nitrite was added dropwise with agitation u~ the suspension. The resultant .. , .. ~... , ~ , ............ ..

.,. , ~ ~ , s~s mixture was kzpt ln the ice bath fsr an additional 30 min. and centrifuged to remove ~he li~uid phase.
The gel was then washed twice with ice-cold distilled water and then placed in contact with 20 ml of an ice-cold solution of 10% catechol in saturated borax.
The reaction was allowed to proceed overnight at 4C The composite was recovered by filtration and it was washed repeatedly with water containing 0.1 normal HCl. The reaction can be indicated schematically as follows:
H H
f N-(CH2)2-N-C- ~ NH2 + NaNO2 ~ HCl H H O
15 PA ~ N-(CH2)2 ~ N ~ + ~ Borax.
OH
OH

PA f N-(CH2)2-N-C ~ N=N ~

OH OH

wherein PA is polyacrylamide carrier or backbone.

.
.
0 Example 10:Immobili~ation of catechol to aminoaryl carbonyl activated silica gel-20 gm of aminoarylcarbonyl activated silica gel , ' .. :

~L~lS~4~

obtained in accordance with example 1 (c) was suspended in 20 ml of ice-cold water. 10 ml of 1 M sodium nitrite was added to the suspension while maintaining the suspension in an ice bath.
10 ml of 2 normal hydrochloric acid (ice-cold) was slowly added dropwise to the suspension.
The reaction was allowed to proceed at between 0 and 4C for about 1 hour and it was then washed with ice-cold distilled watex. The solid was then added to 10 ml o~ 10~ cathecol in saturated borax solut:ion lice-cold) with mixing.
20 ml of water was added to facilitate mixing and the reaction was allowed to proceed in an ice bath for an additional one hour. The - composition was then allowe~ to settle and the liquid siphoned off. The composition was washed with distilled water containing 0.1 normal hydrochloric acid and ethanol. The composition was repeatedly washed until the washing water was colorless. The composltion was then recovered and dried under a vacuum.
The reactions involved with the catechol can be represented generally as below:

glass ~ N-C ~ 2 ~ HC1 ~ ~

::

glass f N-C- ~ N=N- ~

OH OH
.

~L2~SS45 5 Example 11: Immobilization of catechol to aldehyde activated silica gel -5 gm of aldehyde activated silica gel (activated as in example 1 (e) above) was suspended in lO ml of 10~ cathecol in saturated borax solution for 1 hour. The mixture was then subjected to vacuum evaporation and then heated while still under vacuum at 70C for one hour. The mixture was then cooled to room temperature and water was added thereto, the mixture then being heated at 70C for an additional hour. The mixture was then cooled to room temperature and 500 mg of sodium borohydride was added and the mixture was maintained at 70C for a further hour.
The resultant reaction mixture was then cooled in an ice bath and 5 ml of glacial acetic acid was added dropwise with mixing. The reaction - was allowed to proceed for an additional 30 min.
~5 and the mixture was then fiItered. The recovered composition was then washed repeatedly with distilled~water and ethanol alternately and then - dried under vacuum.
The general chemical reactions ~re believed to be as follows:

,, .~

- : ~: . ..... ... .. . .. .

.

L 5 ~ 4 5 ~ I ~ Borax glass ~ N = CH-CH -CH -CH -C=O + O
J 2 2 2 ~ ~ solution I OH
OH

glass f N-CH-CH2-CH2-CH2-C- ~ Na H4 OH OH Sodium Borohydride glass7LN-cH2-cH2- C~I2 C1~2 X~;~
H
OH OH

Example 12: Removal of iron from wine -: 20 The inability of wine to spoil after extraction - of iron using the composition of the present invention is illustrated in table 3 below. This protection from spoilage is retained even when the wine is vigourously aerated (agitated continu-ally in open flasks at 25C~. The treatment of - the commercial wine samples consisted of vigou-rously aerating the sample after inoculating with ac.etobacter xylinum, aeration continuing for a period of about 30 days at 25C.

,: -: .~
:~. ,' : :-` : ' 31~lS5~
Table3 TREATMF.NT OBSERVATIONS
None By three week~, wine was foul smelling (including acetic acid smell); turbid from bacterial growth Filter sterilize~no bacteriaNo spoilage (0.45 ~m pore size) added) Iron extraction with No spoilage siderophoric composition of the present invention Addition of iron ions By three weeks, wine was foul to wine subjected to smel]ing (including acetic acid iron removal by treat- smell); turbid from bacterial ment with the sidero- growth phoric composition of the present invention me following examples (i.e.-13&14) illustrate the procedure which may be used to recycle siderophoric compositions in accordance with the present invention i.e. recycling after - removal of bound iron. The reuse of the siderophoric composition of the present invention makes it economically attractive.

Example 13: Re~eneration of an active iron-free siderophoric composition comprising enterobactin fixed to silica gel -The siderophoric composition subjected to regeneration can in general be represented by the following graphic formula:
H O
glass f N - C ~ N=N-enterobactin The above siderophoric composition loaded with iron, was subjected to treatment with an equal volume of 0.1 M sodium citrate and 0.1 M ascorbic .

-. . .

acid, the treatment lasting for a period of about 12 hours. The treatment was repeated twice for a recovery of loaded iron in the range of 95~.

Repeated loading and unloading of the siderophoric composition with Fe3 results in retention of up to 95~ of the original iron-binding capacity.

On average, each gram of iron-loaded siderophoric composition included about 212 to 232 micrograms of iron per gram of composition. In the above procedure, about 80-100 mg~ of siderophoric composition were subjected to regeneration.

Example 14: Regeneration of a siderophoric composition comprising a catechol fixed to a silica gel -. O
(a) The siderophoric composition can be represented generally by the following graphic formula:

glass f N - C ~ N=N ~
OH H

The siderophoric composition was subjected to the samP treatment as in Example 13. The iron-loaded siderophoric composition contained from~
110 micrograms to 163 micrograms of Fe per gram of the composition. Retention of iron-binding capacity after regeneration was up to 95%.

~.
~- - 58 - ~ ~
.

...... :

:
~ - . . ... : .. . ~ : , : :, . . ~ , :

, ; , . - , : ~ ,, ~ : -., ~
,. .
:--. ~

54~ .

(b) The siderophoric composition having the following general structure was subjected to a reductive regeneration:

glass ~ N-CH2-CH2-CH2-1H ~

OH OH

The iron loaded siderophoric composition contained from 43 micrograms to 56 micrograms or iron per gram of siderophoric composition. The sidero-phoric composition was regenerated using 5 ml of 0.1 M sodium dithionite. Treatment in this way resulted in the recovery of 85 to 93~ of the iron-binding capacity ofthe siderophoric composition.

Example 15: A siderophoric composition comprising ferrioxa-- mine fixed to silica gel -The siderophoric composition was obtained in accordance with the procedure outlined in Example 7. The siderophoric composition had an iron binding capacity of about 740. micro-grams of iron per gram of siderophoric compo-sition. 12 gm of the above composition when exposed to 5 ml of a 500 ~M Fe3 solution was : able to remove or recover 79.1% of the iron;
on being exposed to about 5 ml o~ a 5 ~M
Fe3 solution about 98.8% of this iron was removed or recovered from the solution by the:
siderophoric composition.

., .
_ 59 _ - , ''' "'' '' ': ' ,:' Example 16: Activation of Biogel P 150 with Ethylenediamine:

PA
~H2 H-C-C-NH2 + H2N-CH2-CH2 NH2 -~ CH2 ~A
PA = polyacrylamide back bone:

All operations were carried out in a well ventilated hood. 200 ml. anhydrous ethylene diamine in a 500 ml. 3 necked round bottom flask was heated with a heating mantle and the final temperature was reached and adjusted and maintained at 90C + 2C. The glass was also equipped with a condenser with outlet protected by a drying tube, a mechanical stirrer and the third neck was used for addition of materials and temperature monitoring~
10 gm of Biogel - P 150 was added through the thermometer neck in one portion and the mixture - was stirred and heated at 90C ~ 2C for a : period of 3 to 4.hours. The solid gel swelled : to a`great volume and the evolution of ammonia can be ascer.tained by wette.d pH paper at the drying-tube outlet. At the end of the reaction, the mixture was poured with mechanical stirring on to a mixture of 400 ~1l ice and water (1:13.
Any gel adhering to the flask can be washed down `: i .~

.-, ~ , . ,:

. ,, ,; :. ~

by jets of water. The gel was filtered while the mixture was still cold. The gel was promptly washed repeatedly with 0.2 M NaCl and 0.00 1 N HCl until the filtrate gave a negative TNBS test ~Trinitrobenzenesulfonic Acidl. The total gel volume was about 170 ml. i.e. wet gel.

Example 17: Succinylation: Carboxylic arm extension:

~A PA
CH2 ~ H CH2-CH ~H2 R 7 H-C - C-N-CH~-CH2-NH2 ~ O=C\ f =O ~ H-f C-N-cH2-cH2-N-H
PA C~2 ~OOE~

PA = polyacrylamide back-bone.

50 ml. wet gel (Biogel P-150-Ethylenediamine activated) is suspended in 50 ml. 0;1 N NaOH
in a 250 ml. beaker. External cooling in an ice bath and gentle mechanical stirring is also provided. 1.0 gm. succinic anhydride (10 mmole) was added in one portion and the mixture stirred in the cold for 2 hrs. A
further 1 gm. portion of succinic anhydride was added with further cooling and stirring for an additional hr. During the addition of the second portion of succinic anhydride, the mixture pH is monitored intermittently . . I
~" .,1 . . . .
,............. .

5~

with a pH meter and additional amounts of 1 N NaOH were added to ma:intain a pH of 3.5 to 4Ø . A third portion of 1 g. succinic anhydride was added and the monitoring pro-cedure was same as the previous addition. The TNBS Test showed that there were still free amino group on the gel and these were blocked by addition 10 ml. acetic anhydride and stirred for 30 min. The mixture was eventually washed thoroughly with 0.1 M NaCl. TNBS Test was negative for the gel.

For the following examples 18 to 21, the formula for the starting catechol composition is indicated as a matter of convenience as comprising a single isomer; the composi-tion used was however a mixture of OH OH ~ OH

Sili ~ OH and Sili ~

Example 18: Preparation of a chloro substituted catechol composition ~ ÇH OH Cl ff Sili - ~ OH ~ Sili- ~ OH ~ S1li- ~ ~

(silicat) ~ HCl (Mono chloro) (dichloro) To one batch (prepared from 500 gm silica) of silicat was added 100 ml Commercial sodium hypochlorite (4 - 6~ solution) and the mixture was stirred and cooled in an ice bath. 50 ml of glacial acetic acid was added and the mixture - ,- . . ,,. ~ :

.

- ~
' '''`: i~ ':- ':

was stirred and evacuated simultaneously. Coo-ling in the ice bath continued for another 30 minutes. Then 50 ml concentrated hydrochloric acid was added and the mixture allowed to come to room temperature gradually. Stirring and evacuation continued for a further two hours and the mixture was then diluted with a large volume of water and filtered on a Buchner funnel. The solid was washed thoroughly with water first. Then it was soaked in 5%
aqueous ammonia and washed with water again thoroughly. Then it was soaked in 5~ hydro-chloric acid and washed with water again. The solid is stored as a suspension in the acidified form for subsequent utilization~

Example 19: Preparation of a bromine substituted catechol `-composition Sili - ~ N ~ Sili _ ~ CH + Sili -(Silicat) (Mono bromo~ (dibrom3) To one batch (prepared from 500 ~m. silica) of silicat was~added lO gm. sodium ~acetate and the mixture was cooled in an ice bath. This was fol~owed bv the addition of 5 ml. of liquid bromine and~the mixture was stirred, evacuated~
and cooled in an ice bath simultaneously. The reaction was allowed to proceed in this manner for tw~ hours and the obtained mixture was then washed thoroughly with water to eliminate all unreacted bromine.
The mixture was then soake in 5 %-ammonium .

: ~ :
~ - 63 -:
~:

. ,: .. . : :

hydroxide, washed with water and then soaked in5% hydrochloric acid. It was finally washed thoroughly with water and stored in water in an acidified aqueous environment.

Example 20: Preparation of NO substituted catechol compo-sition (Sillcat~ - Sili - ~ ~ & sili -One batch (prepared from 500 gm. silica) of silicat was cooled sufficiently in an ice bath and followed by the addition of 7.0 gm.
sodium nitrite (NaNO2). The suspension was stirred, evacuated and cooled in an ice bath simultaneously. The reaction was allowed to proceed for 30 minutes. This was followed by the addition of lD ml. concentrated hydro-chloric acid and a perceptible evolution of nitrogen oxide was observed. The reaction was processed as before for another two hours.
The mixture was then washed thoroughly with water, soaked in 5% ammonium hydroxide which brings about a deepening of the color of the solid to a redder shadeO The ~iltrate and aqueous washings were reddish orange and washing with water continued until the washings were colorless. The solid was then soaked in 5~
hydrochloric acid which changes the col~r f~om reddish to a more orange shade and the suspension was washed thoroughly with water and stored in this acidified form.

35 Example 21: Preparation of NO~ substituted catechol composition :
.

. . : . - .

- ~

5~

Sili- ~ OH - 3 Sili ~ OH ~ Sili-(Silicat) 2 One batch (prepared from 500 gm. silica) of silicat was cooled in an ice bath thoroughly.
To this cooled suspension was added continuously 250 ml. commercial concentrated nitric acid and the mixture was stirred, evacuated and cooled in ice bath for 30 min. Then 250 ml.
concentrated sulfuric acid was added very cautiously with careful stirring. The mixture was warmed up considerably and it was cooled - in the ice bath. The reaction was allowed to proceed for another two hours. The mixture was then diluted with a large amount of water and then filtered, washed with water, followed by soaking with 5% ammonium hydroxide, giving a nuxture reddening in shade and orange filtrates and washings . Thorough washing with water was c:ontinued until the washing was colorless. It was then soaked in 5% hydrochloric acid and which brought about a lightening of color from reddish to orange and washed with water again thoroughly. It was stored in this aci-difisd form for subsequent utilization.

Example 22: Removal of U02 from solution using a compo sition consisting of desferrioxamine fixed to a silica gel.

A bed consisting of 4 gms of a composition consisting of desferrioxamine coupled to a silica gel with glutaraldehyde 3see example n 1 (e) was placed into a column. The compo-- :

:` ` : : ,`
:' ~ ` .
.,, ~ ' ~

:: :

5~

sition was then treated with 100 ml of an aqueous 0.5 M sodium dithionite solution to remove any bound metal e.g. iron. The com-position was then washed with 100 ml of an aqueous 0.01 M sodium acetate solution of pH 5.9 at 20~C. The composition was then con-tacted at 20C with a total of 80 ml of an aqueous solution ( pH 6.9 ) containing Uranyl acetate at a concentration of 600~M
the solution beincJ brought into contact with the composition at the rate of 2.3 ml/min.
No uranyl ion emerged from the column until a total of 62 ml of the solution had been applied. The composition took up a total ~f 34.6 ~ mole of UO2 2 or 8.64 ~ mole of UO22~(2-0 mg f U2 2 )per gram of composition. The loaded composi-tion was thereafter treated with an aqueous 6mM Fe solution (`pH 6.9 ). The composition failed to develop a red colour indicative of the binding of Fe3 thereto; i.e.~omposition has higher affinity for UO2 2 than for Fe3+.

Example 23: Removal f UO2 from a citrate buffered solution using a composition consisting of catechol fixed to a silica gel.

110 ml. of a buffered uranyl acetate solution ~Uranyl acetate 600 ~M ; sodium acetate 60 mM~
of pH 6.9 was contacted with 4 gms (dry wgt) o a catechol-glutaraldehyde-silica composition (see example n 11). The composition was > 99.9~ efficient in removing Uranyl ion from solution for the first 50 ml of solution contacted with the composition; i.e. the com-position took up 7.3 ~ mol UO2 2+/gm of compo-' ' ~:
::
:

~2~SS~i sition. The efficiency of the composition thereafter progressively decreased. The overall binding capacity of the composition was found to be about 14 ~M (3.3 mg) UO~ /gm of compo-sition. Overall the concentration of the Uranyl ion in the solution was lowered to less than 6 ~M from 600 ~M.

Example 24: Removal of UO2 in trace amounts from dis-tilled water using a composition consisting of catechol fixed to a silica gel.

42 1. of water containing 285 nM UO2 acetate (~ pH 6.9) was contacted with 30 gm (dry weight) of a catechol-glutaraldehyde-silica composi-tion ~see ex. no. 11) placed in a column,at a rate of 4.5 ml/min; the composition was pre-condi~ibned by contact~ith 100 ml ~f aqueous 0.1 M sodium - acetate solution pH 6.9. The loadea ccm~osition was then washed with 50 ml aqueous 2 N HCl to wash out the UO2 . It was determined that the amount of UO2 removed from the water was such that the 285 nM solution was lowered to < 0.2 nN; i.e. an efficlency of > 99.9%, Example 25: Removal of UO2 from artlficial sea water using a composition consisting of catechol fi~ed to a silica gel.

30 gm (dry weight) of a catechol-glutaraldehyde- `
silica composition (see ex. N 11) was placed in a column and contacted with 100 ml of an aqueous 0.1 M sodium acetate solution at pH 6.9.
900 ml of 13 ~M UO2 acetate in artificial sea water (forty fathoms) adjusted to pH 6.6 was thereafter contacted ~ ,. :
- 67 - ~

~L2~S;S~

with the composition. The composition was then washed with 200 ml of distilled water. The washed com~osition was then contacted with 100 ml aqueous 2 N HCl to wash out the UO22 . Of the 12 ~ Mole of UO2 in the 900 ml of sea water 11.52 were recovered from the composition that could be eluted with the 2N HCl; (i.e.
recovery efficiency of ~96%).

Example 26: Elution of UO2 from a composition consistiny of catechol fixed to a silica gel to obtain the UO22 in a concentrated solution.

4 gm (dry wgt) of a catechol-glutaraldehyde-silica composition (see ex n 11) was loaded with ~022 by contact with 50 ml of an aqueous solution (600 ~M UO2 acetate in 0.1 M Na acetate~ at pH 6.9. The loaded composition was washed with 100 ml of distilled water and then washed with 50 ml aqueous 2M HCl to wash out the UO2 . 99.94% of the ~022 bound to the composition was removed.

Example 27: Removal of Th from a buffered solution using a composition consisting of catechol fixed to a silica gel.

4 gm (dry weight) of a catechol-glutaraldehyde-silica composition (see ex. n 11) was pIaced in a column and treated with 100 ml of an aqueous 0.1 M
Naacet.ate solution pH 6.9.~The composition was then contacted with 100 ml of an aqueous solution t600 ~M Th tNO3)4 in 6 mM Na citrate) pH 6.9 at a rate of 3 mljmin. The results were nearly identical to those in ex. 22.

:. . .. . .

,. . . . ,,, ~ . .- :. j.

: : ' . .. `: : :

~2~5'~
Example 28: Separation of UO2 and Th bound to a composi-tion consistlng of catechol fixed to a silica gel.

30 gms (dry wt~ of a catechol-glutaraldehyde-silica composition ~see example n 11) was loaded with UO22 iand Th4 by contacting it with 100 ml of ,an aqueous solution containing Th(NO3)4 3mM
UO2(acetate)2 3mM
Na3(Citrate~ 60 mM
pH 6.9 The loaded composition was washed with distilled water and thereafter placed in a column. The composition was then washed with 200 ml of aqueous 60mM Na Citrate pH 6.9 . The compo-sition was then eluted with a continuous pH
gradient starting from pH 6.9 and finishing at pH 0~8. The gradient was prepared by starting with an aqueous O.lM sodium citrate solution of pH 6.9, thereafter mixing said citrate solution with an aqueous 0.3N HCl (the citratP in lowex and lower amounts the HCl in higher and higher amounts) until finally finishing with said aqueous 0O3N
HCl solution pH 0.8. Over all a tot~l of 150 ml of each solution was used. The Th was selec-tively eluted at a pH of-about 4.45. The uo~2 was eluted at the lower end of the pH gradient.

', " ' ' `' '' '.` .,, " '

Claims (37)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for treating an insoluble composi-tion loaded with Th4+ and UO22+ to separate Th4+ therefrom characterized in that said composition is contacted with an aqueous solution containing a suitable Th4+ chelating agent and an organic acid, said organic acid being a carboxylic acid, said solution having a pH greater than 2, said composition comprising a member selected from the class consisting of (A) an insoluble composition comprising (1) one or more organic chelating compounds, covalently fixed to the surface of (2) a suitable insoluble carrier, said organic chelating compounds possessing one or more co-ordinating sites, said organic chelating compounds being selected from the class consisting of microbial siderophores and (B) an insoluble composition comprising (1) one or more catechol compounds covalently fixed to the surface of (2) a suitable insoluble carrier, said catechol compounds being fixed to the surface of said carrier at the benzene ring thereof, said catechol compounds being selected from the group consisting of unsubstituted catechol and catechol substituted on the benzene ring by one or two electrophilic substituents.

2. A method for treating an insoluble composition loaded with Th4+ and UO22+ to separate Th4+ therefrom charac-terized in that said composition is contacted with an aqueous solution containing a suitable Th4+ chelating agent and an organic acid, said organic acid being a carboxylic acid, said solution having a pH greater than 2, said insoluble composition comprising (1) one or more organic chelating compounds, covalently fixed to the surface of (2) a suitable insoluble carrier, said organic chelating compounds possessing one or more co-ordinating sites, said organic chelating compounds being selected from the class consisting of microbial siderophores.

3. A method as defined in claim 2, wherein said solution has a pH of about 4 to about 6.

4. A method as defined in claim 2, wherein said chelating agent is a tricarboxylic acid and said organic acid is a tricarboxylic acid.

5. A method as defined in claim 3, wherein said chelating agent is a tricarboxylic acid and said organic acid is a tricarboxylic acid.

6. A method as defined in claim 3, wherein said chelating agent and said organic acid are selected from the group consisting of citric acid, iso-citric acid, cis-aconitic acid and oxalosuccinic acid.

7. A method as defined in any one of claims 4, 5 and 6, wherein said microbial siderophores have a molecular weight of less than 2500 Daltons.

8. A method as defined in any one of claims 4, 5 and 6, wherein said microbial siderophores have a molecular weight in the range of 500 to 2500 Daltons.

9. A method as defined in any one of claims 4, 5 and 6, wherein said microbial siderophores have a molecular weight in the range of 500 to 2500 Daltons and wherein said co-ordinating sites are provided by one or more members of the class consisting of (a) a N-substituted hydroxamate of formula (b) a phenolate group of formula X being an atom of O or N-, and (c) a catecholate group of formula X' being an atom of O or N- and m being 1.

10. A method as defined in any one of claims 4, 5 and 6, wherein said microbial siderophores have a molecular weight in the range of 500 to 2500 Daltons and wherein each of said co-ordinating sites is provided by a N-substituted hydroxamate groups of formula

11. A method as defined in any one of claims 4, 5 and 6, wherein said microbial siderophores have a molecular weight in the range of 500 to 2500 Daltons and wherein each of said co-ordinating sites is provided by a catecholate group of formula

12. A method as defined in any one of claims 4, 5 and 6, wherein said organic chelating compounds are selected from the class consisting of desferrioxamines.

13. A method for treating an insoluble composi-tion loaded with Th4+ and UO22+ to separate Th4+ therefrom characterized in that said composition is contacted with an aqueous solution containing a suitable Th4+ chelating agent and an organic acid, said organic acid being a carboxylic acid, said solution having a pH greater than 2, said insoluble composition comprising (1) one or more catechol compounds covalently fixed to the surface of (2) a suitable insoluble carrier, said catechol compounds being fixed to the surface of said carrier at the benzene ring thereof, said catechol compounds being selected from the group consisting of unsubstituted catechol and catechol substituted on the benzene ring by one or two electrophilic substituents.

14. A method as defined in claim 13, wherein said solution has a pH of about 4 to about 6.

15. A method as defined in claim 13, wherein said chelating agent is a tricarboxylic acid and said organic acid is a tricarboxylic acid.

16. A method as defined in claim 14, wherein said chelating agent is a tricarboxylic acid and said organic acid is a tricarboxylic acid.

17. A method as defined in claim 14, wherein said chelating agent and said organic acid are selected from the group consisting of citric acid, iso-citric acid, cis-aconitic acid and oxalosuccinic acid.

18. A method as defined in any one of claims 15, 16 and 17, wherein said catechol compounds are selected from the group consisting of catechol substituted on the benzene ring by one or two substituents selected from the class consisting of halogen atoms, NO and NO2.

19. A method as defined in any one of claims 15, 16 and 17, wherein said catechol compounds are selected from the group consisting of catechol substituted on the benzene ring by one or two halogen atoms.

20. A method as defined in any one of claims 15, 16 and 17, wherein said catechol compounds are selected from the group consisting of catechol substituted on the benzene ring by one or two halogen atoms selected from chlorine and bromine.

21. A method as defined in any one of claims 15, 16 and 17, wherein said catechol compounds are selected from the group consisting of catechol monosubstituted by a substituent selected from NO and NO2.

22. A method as defined in any one of claims 15, 16 and 17, wherein said catechol compound used is unsub-stituted catechol.

23. A method as defined in claim 3, wherein said microbial siderophores have a molecular weight of less than 2500 daltons.

24. A method as defined in claim 3, wherein said microbial siderophores have a molecular weight in the range of 500 to 2500 daltons.

25. A method as defined in claim 3, wherein said microbial siderophores have a molecular weight in the range of 500 to 2500 daltons and wherein said co-ordinating sites are provided by one or more members of the class consisting of (a) a N-substituted hydroxamate group of formula (b) a phenolate group of formula X being an atom of O or N-, and (c) a catecholate group of formula X' being an atom of O or N- and m being 1.

26. A method as defined in claim 3, wherein said microbial siderophores have a molecular weight in the range of 500 to 2500 daltons and wherein said coordinating sites are provided by one or more N-substituted hydroxamate groups of formula

27. A method as defined in claim 3, wherein said microbial siderophores have a molecular weight in the range of 500 to 2500 daltons and wherein said coordinating sites are provided by one or more catecholate groups of formula

28. A method as defined in claim 3, wherein said organic chelating compounds are selected from the class consisting of desferrioxamines.

29. A method as defined in claim 14, wherein the catechol compound used is unsubstituted catechol.

30. A method as defined in claim 14, wherein said catechol compounds are selected from the group consisting of catechol substituted on the benzene ring by one or two substituents selected from the class consisting of halogen atoms, NO and NO2.

31. A method as defined in claim 14, wherein said catechol compounds are selected from the group consisting of catechol substituted on the benzene ring by one or two halogen atoms.

32. A method as defined in claim 14, wherein said catechol compounds are selected from the group consisting of catechol substituted on the benzene ring by one or two halogen atoms selected from chlorine and bromine.

33. A method as defined in claim 14, wherein said catechol compounds are selected from the group consisting of catechol monosubstituted on the benzene ring by a substi-tuent selected from NO and NO2.

34. A method as defined in claim 13, wherein said catechol compounds are selected from the group consisting of catechol substituted on the benzene ring by one or two electrophilic substituents.

35. A method as defined in claim 34, wherein said solution has a pH of about 4 to about 6.

36. A method as defined in anyone of claims 34 and 35, wherein said chelating agent is a tricarboxylic acid and said organic acid is a tricarboxylic acid.

37. A method as defined in claim 35, wherein said chelating agent and said organic acid are selected from the group consisting or citric acid, iso-citric acid, cis aconitic acid and oxalosuccinic acid.