The present invention is concerned with the field of chromatographic
separation and relates in particular to a novel type of anion exchangers containing
two positive charges at a distance of two atoms from each other.
Ion exchange chromatography - a technique where a sample is made
to pass through a matrix containing immobilized charged groups which will bind
sample components of the opposite charge - is used interalia for
the separation of biomolecules such as for example proteins, peptides and nucleic
acids. Although this is one of the oldest separation methods it continues to be
one of the basic techniques for modern biochemical separation procedures. As far
as anion exchangers are concerned, i.e. ion exchangers containing positively charged
groups, the substances employed as such exchangers are in the first place amines
attached to a solid phase of some kind or other in order to thus form either the
charged groups themselves or groups chargeable in some particular environment.
Primary, secondary and tertiary amine functions are classed as weak anion exchanger
groups whereas quaternary amine functions are classed as strong anion exchanger
groups. However, the use of these terms "weak" and "strong" does not reflect any
qualitative assessment of the function of the ion exchanger; rather, it refers
to the fact that a "strong" ion exchanger is charged over a broader pH range. In
some practical applications, a weak ion exchanger is to be preferred over a strong
one, and vice versa. As examples of anion exchangers that have been available
commercially, for many years, may be mentioned DEAE Sephadex® and QAE Sephadex®
(Pharmacia LKB Biotechnology AB, Uppsala, Sweden) containing diethylaminoethyl
and quaternary aminoethyl groups, respectively, as their functional groups. Ion
exchangers containing amino groups are disclosed also in a great number of publications,
see for instance WO 89/04203, EP-A-167 488 and EP-A-295 495.
In all forms of chromatographic separation techniques it is imperative
that one should achieve the best possible separation/resolution of the sample components
into either individual components or individual groups of components. Resolution
is a function of i.a. the efficiency and selectivity of the column employed. These
factors are determined in the first place by the properties of the separation
matrix, in combination with the geometry of the column; so these factors constitute
fixed parameters of the system. Other factors affecting resolution are for instance
sample loading, flow rate, temperature, pH, gradients etc. which thus have to
be optimized for any given column in each given separation situation. A trend during
recent years has been that efforts should be made towards obtaining an increased
efficiency and concomitantly an increased resolution by means of employing a column
packing material of lesser particle size. On the other hand, it seems that very
little work has been devoted to efforts aimed at improving the selectivity of the
ion exchangers. One of the factors affecting selectivity is the structure of the
charged group that has been attached to the matrix.
Another important property of ion exchangers is their ion exchange
capacity; in case the mobile phase ions are small monovalent ions, this capacity
is equal to the number of charges in the matrix. A small ion is capable of penetrating
through the surface layer formed by the charged substituents and reaches for example
also charges lying deeply in the interior of narrow pores. But when it comes to
binding of biomolecules, the situation is different. What determines the amount
of a protein that can be bound to an ion exchanger is not only the number of charges
on the gel but also the manner in which the charges are exposed on the matrix
surface, implying that the porosity of the matrix and the structure of the ion-exchanging
group are of decisive importance. Of course, also the charge properties of the
protein will have an influence on the degree of binding.
In WO 91/00145 (which application was not published or made publicly
available prior to the filing date of the present application) it is disclosed
that the selectivity and capacity of strong anion exchangers may be improved considerably
by means of introducing functional groups with charges arranged pairwise, these
charges being located in a special structure in which they are exposed in an optimum
manner to the ambient medium due to minimisation of charge shielding. The functional
groups specifically described in WO 91/00145 are 4-methyl-1,4-diazabicyclo[2.2.2]octan-1-yl
(1), 1,4,4-trimethylpiperazinium-1-yl (2), 1,4-dimethyl-1,4-diazabicyclo[2.2.2]octan-2-yl
(3), and 1,1,4,4,4-pentamethyl-1,4-diazabutan-1-yl (4) having the respective structures:
the characteristic feature being that the two charged nitrogen atoms are located
at a distance of two atoms from each other. In structures (1), (2) and (3) the
nitrogen atoms moreover form part of a cyclic structure. It is further stated
in the specification that by choosing structures (1) and (3) on ion exchangers
one will minimize steric hindrance thwarting interaction with sample molecules
in a solution containing the ion exchanger.
In accordance with the present invention the above basic concept
has been developed to provide a diversity of other functional groups for improving
the selectivity and capacity of strong anion exchangers.
The present invention thus relates to an anion exchanger for chromatographic
separation, said anion exchanger having the structure
P - S - A
where P is an insoluble support, preferably in the form of discrete particles such
as e.g. spherical particles of the type as known within this technical field, having
a particle diameter of for example 1 to 500 µm.
S is a so-called spacer, idest a molecule chain that,
preferably, does not interact with the sample molecules in any manner interfering
with the separation procedure, but does promote exposure of the charged structure
on the particle surface. If it is desired that for instance a certain amount of
hydrophobic groupings are employed in the separation procedure, the spacer can
of course be made hydrophobic in a known per se manner.
A is the charged ligand which in accordance with the present invention
comprises two positively charged atoms, each selected from nitrogen and sulphur,
at a distance of two atoms from each other, with the proviso that this functional
ligand A can not be 4-methyl-1,4-diazabicyclo[2.2.2]octan-1-yl, 1,4,4-trimethylpiperazinium-1-yl,
1,4-dimethyl-1,4-diazabicyclo[2.2.2]octan-2-yl or 1,1,4,4,4-pentamethyl-1,4-diazabutan-1-yl.
Preferably, the two atoms separating the positively charged atoms
are carbon atoms.
The charged atoms may both be nitrogen atoms, or one may be sulphur
and the other nitrogen, or both may be sulphur atoms. In case both the charged
atoms are nitrogen atoms, the ligand comprises two quaternary amino groups.
The spacer S may be bound either to one of the charged atoms or to
one of the two atoms separating the charged atoms.
A preferred group of ligands A within the above given definition
for A are represented by the following formulae (IA) and (IB) (the use of two formulae
reflecting the different positions of the free valence for the binding of the
ligand to the spacer S):
wherein
Y1 and Y2 independently of each other are nitrogen or
sulphur,
R1, R2, R3, R4, R5 and
R6 independently of each other are represented by the formula (II):
R7, R8, R9 and R10 independently
of each other are hydrogen or C1-4alkyl, C1-4alkoxyalkyl
or hydroxy-C1-4alkyl;
R11 is hydrogen, C1-6alkyl, C1-6alkoxyalkyl
or hydroxyC1-6alkyl;
R12 is hydrogen, hydroxy or C1-4alkyl;
l is 0 or 1;
m is 0 or 1;
n is 1 to 5;
x is 0 or 1;
y is 0 or 1;
z is 0 or 1;
with the provisos that:
l is 0 when Y2 is sulphur;
m is 0 when Y1 is sulphur;
z is 0 when R12 is hydroxy;
R2 together with R4 and/or R3 together with
R6 may form an ethylene group in which one or both carbon atoms may
be mono- or disubstituted by C1-4alkyl, C1-4alkoxy, C1-4alkoxyalkyl
or hydroxy-C1-4alkyl.
The term alkyl as used herein (separately or in combinations) is
meant to comprise both straight and branched groups.
In one subgroup of ligands of formulae IA or IB, each substituent
R1 to R6 independently has R12 coupled to a group
selected from: -[CH2-CH2-O]n-; -[CH2]n-;
and
n is preferably 1 to 3, particularly 1 or 2, and more preferably
1.
A preferred group of ligands A consists of those wherein R1
to R6 (independently of each other) are alkyl, alkoxyalkyl or hydroxyalkyl
having one to three carbon atoms (such as e.g. ethyl, methoxyethyl or hydroxyethyl,
respectively).
In another preferred subgroup of ligands of formulae IA or IB at
least one of the substituent pairs R2,R4 and R3,R6
forms an ethylene group which may optionally be substituted, preferably by alkyl,
alkoxy or hydroxyalkyl of one or two carbon atoms.
R7 to R10 are preferably (independently of each
other) hydrogen or alkyl of one or two carbon atoms, and more preferably hydrogen.
R11 is preferably alkyl having no more than five carbon
atoms, more preferably no more than three carbon atoms.
R12 is preferably hydrogen, hydroxy or methyl.
As regards choosing the supportive matrix for binding of the functional
group, this is an item which does not form part of the invention; a person skilled
in the art can apply the inventive concept to the large number of supportive matrices
that have been described for use in chromatographic separation procedures, and
from among these he may choose one having desirable properties in respect of the
other separation parameters. Examples of such matrices are interalia gels of polysaccharides as for instance dextran, starch, cellulose
and agarose, optionally after crosslinking for the purpose of increasing the rigidity
of the material and thus improving its compressive and flow properties. Other
examples are supportive matrices based on polystyrene-divinylbenzene, silica and
acrylates.
Synthesis of ion exchangers according to the invention is carried
out either by introducing reactive groups into the matrix chosen, said groups being
reacted with either the ion exchanging group or a derivative thereof, or by causing
a reactive derivative of the ion exchanging group to directly react with the matrix.
Coupling is performed with the aid of a so-called spacer, to be bound
at one of its ends to the matrix and at the other end to the reagent that will
produce one of the aforesaid ligand structures A. Such coupling of the spacer
to the gel on one hand, and to the reagent on the other hand, is carried out by
means of any of the numerous methods developed for couplings in this type of technological
contexts, especially in the fields of affinity chromatography; examples of such
methods are CNBr, epoxide, cyanate, hydrazide and sulfonyl coupling, to mention
just a few out of a large number. The use of spacers or exposing functional groups
on a matrix is likewise a very well-known method within this field of technology
and does not form a part of the invention.
The invention is illustrated by way of the following non-limitative
examples dealing with both the synthesis of ion exchangers and the use of these
ion exchangers in chromatograpich separation procedures.
WORKING EXAMPLESI. SYNTHESIS OF ION EXCHANGER ON POLYSTYRENE/DIVINYLBENZENE MATRIX.Example 1A) Allylation
Hydroxylated polystyrene/divinylbenzene gel (100 ml) was washed with
76 ml of 3 M aqueous sodium hydroxide solution containing 0.38 g of sodium borohydride.
The gel was added to 40 ml of the alkali solution, and 115.5 g of allyl glycidyl
ether were added. The reaction mixture was then stirred overnight at 45oC,
whereupon the gel was filtered and washed with ethanol and then with water.
B) Bromination and coupling of DABCO.Ion exchanger no. 1 (comparative)
The allylated gel (50 ml) from step A) above was added to 30 ml of
0.014 M sodium acetate solution. Bromine water was added, with stirring, until
a lasting yellow color appeared. Excess bromine was removed with sodium formate.
12.1 g of DABCO (1,4-diazabicyclo[2.2.2]octane) were added, and the pH was adjusted
to 10.5 by the addition of 45% aqueous sodium hydroxide solution. The reaction
mixture was then stirred overnight at 45oC, whereupon the gel was filtered
and washed with water, ethanol and finally water.
Example 2Methylation of DABCO-coupled gel.Ion exchanger no. 2 (comparative; WO 91/00145)
7 ml of the DABCO-coupled gel from Example 1 were washed 3 times
with ethanol and then 3 times with acetonitrile. The gel was then subjected
to suction on a glass filter until it was dry. Thereafter the gel was added to
14 ml of acetonitrile. 0.43 g of methyl iodide was added at 30oC. The
reaction mixture was stirred overnight at 30oC, whereupon the gel was
filtered and washed with water, ethanol, water, 1 M aqueous sodium chloride-solution
and water.
Example 3Ethylation of DABCO-coupled gel.Ion Exchanger no. 3
5 ml of the DABCO-coupled gel from Example 1 were alkylated with
ethyl iodide (0.34 g) by using the reaction conditions described for the synthesis
of ion exchanger no. 2 in Example 2.
Example 4Propylenoxide alkylation of DABCO-coupled gel.Ion Exchanger no. 4
7 ml of the DABCO-coupled gel from Example 1 were added to 14 ml
of water, and the pH was adjusted to 5.7 with 2 M hydrochloric acid solution. Then
7 g of propylene oxide were added, and the reaction mixture was stirred overnight
at 45oC. The gel was filtered and washed with water, ethanol, water,
1 M aqueous sodium chloride solution and water.
Example 5Allylglycidyl ether alkylation of DABCO-coupled gel and hydroxylation.Ion exchanger no. 5
5 ml of the DABCO-coupled gel from Example 1 were added to 10 ml
of water, and the pH was adjusted to 5.7 with 2 M hydrochloric acid solution. Then
10 g of allylglycidyl ether were added and the reaction mixture was stirred overnight
at 50oC. The gel was filtered and washed with ethanol, water, 1 M aqueous
sodium chloride solution and water. The allylglycidyl ether alkylated gel was
hydroxylated by adding the gel to 6 ml of 0.014 M aqueous sodium acetate solution.
Bromine water was added, with stirring, until a stable yellow color was obtained.
Excess bromine was removed with sodium formate. The pH was then adjusted to 12
by the addition of 4 M sodium hydroxide. The reaction mixture was stirred overnight
at 45oC, whereupon the gel was filtered and washed with water, ethanol
and water.
Example 6A) Bromination and coupling of thiomorpholine.
10 ml of the allylated gel from Example 1 were added to 6 ml of 0.014
M aqueous sodium acetate solution. Bromine water was added, with stirring, until
a stable yellow color was obtained. Excess bromine was removed with sodium formate.
The pH was then adjusted to 9 by the addition of 4 M aqueous sodium hydroxide solution
and 2.5 g of thiomorpholine were added. The reaction mixture was then stirred
overnight at 45oC, whereupon the gel was filtered and washed with water,
ethanol and water.
B) Methylation of the thiomorpholine-coupled gel.Ion exchanger no. 6
4 ml of the thiomorpholine-coupled gel from step A) above were washed
3 times with ethanol and 3 times with acetonitrile. It was then subjected to suction
on a glass filter until the gel was dry. Thereafter the gel was added to 8 ml
of acetonitrile and 1.5 g of methyl iodide were added at 30oC. The reaction
mixture was stirred overnight at 30oC. The gel was then filtered and
washed with water, ethanol, water, 1 M aqueous sodium chloride solution and water.
Example 7A) Azasulfenylation on allylated gel.
20 ml of the allylated gel from Example 1 were washed 3 times with
nitromethane. The gel was then added to 60 ml of nitromethane and 2.1 g of dimethyl(methylthio)sulfonium
fluoroborate were added at 2 oC. The reaction mixture was stirred for
2 hours at 2 oC, then overnight at room temperature, whereupon the gel
was filtered and washed with nitromethane.
B) Methylation of azasulfenylated gel.Ion exchanger no. 7
5 ml of the azasulfenylated gel from step A) above were washed 3
times with acetonitrile. The gel was then subjected to suction on a glass filter
until it was dry. Thereafter the gel was added to 10 ml of acetonitrile, and 0.77
g of methyl iodide was added at 30 oC. The reaction mixture was then
stirred overnight at 30oC, whereupon the gel was filtered and washed
with water, ethanol, water, 2 M aqueous sodium hydroxide solution and water.
Example 8Substitution with amine on azasulfenylated gel followed by methylation.Ion exchanger no. 8
5 ml of the azasulfenylated gel from Example 8 were added to a solution
containing 20 ml of nitromethane and 1.09 g of triethylamine. The reaction mixture
was stirred for 4 days at room temperature. The gel was then filtered and alkylated
with methyl iodide (0.77 g) by using the reaction conditions described for the
synthesis of ion exchanger no. 7 in Example 7.
II. CHARACTERIZATION OF THE SELECTIVITY OF THE ION EXCHANGERS
With a test mixture consisting of
transferrin
10 mg/ml
ovalbumin
20 mg/ml
β-lactoglobulin
20 mg/ml
the selectivity of the ion exchangers 1 - 6 produced above were characterized by
means of determining the difference in elution volume divided by the void volume
V0, (Ve,m -Ve,n)/V0; m and n represent
sequential numerals assigned to peaks of the chromatograms
Column:
HR 5/5 (Pharmacia LKB Biotechnology AB); volume 1.0 ml
Sample loading:
0.5 mg protein/ml gel
Buffer A:
20 mM piperazine pH 6.0
Buffer B:
Buffer A +0.6 M NaCl
Flow rate:
150 cm/h
Gradient:
0-100% buffer B/20 column volumes (20 ml)
The test mixture listed above produces 4 peaks when eluted with a
strong anion exchanger at pH 6.0. The mixture contains proteins of different sizes;
and in reality the V0
value is a different one for each of the different
proteins, in as much as these would have been eluted at different volumes even
if none of them were retarded by ion exchange interactions. This is a gel filtration
phenomenon. However, all the proteins would have been eluted within one column
volume. In the test series, V0 has not been corrected in respect of
this effect because in comparisons made on the same supportive matrix the V0
error is the same for each protein on the different ion exchangers.
For comparison also the corresponding values obtained with the prior
art ion exchanger Mono Q (Pharmacia LKB Biotechnology AB), these ion exchangers
having the functional group (ion exchanger no. 0):
Results from separations performed on polystyrene/divinylbenzene-based ion
exchangers according to Example 1 Ligand no. Conc of ligand on gel mmol/ml gel Capacity for Cl― mmol/ml (Ve,m―Ve,n)/V0m=2 m=3 m=4 m=4 n=1 n=2 n=3 n=1 00.280.282.221.341.364.92 10.120.121.930.771.263.96 20.120.243.962.592.298.84 30.120.152.401.341.465.20 40.120.233.632.301.967.89 50.120.163.431.481.596.50 60.060.123.801.411.436.64
In conclusion, the ion exchangers no. 2 - 6 (nos. 3 - 6 being within
the scope of the present invention) have a better selectivity than the conventional
type of anion exchanger no. 0.
Anspruch[de]
Ionenaustauscher zur chromatographischen Trennung mit der Struktur
P - S - A,
wobei P ein unlöslicher Träger ist, S ein Abstandhalter ist und A ein funktioneller
Ligand ist, dadurch gekennzeichnet, daß der funktionelle Ligand A zwei positiv
geladene Atome umfaßt, jeweils ausgewählt aus Stickstoff und Schwefel, in einem
Abstand von zwei Atomen voneinander mit der Maßgabe, daß jedes der geladenen Atome
ein quaternärer Aminostickstoff ist, wenn die beiden geladenen Atome Stickstoffatome
sind, und daß A nicht 4-Methyl-1,4-diazabicyclo-[2,2,2]octan-1-yl, 1,4,4-Trimethylpiperazinium-1-yl,
1,4-Dimethyl-1,4-diazabicyclo[2,2,2]octan-2-yl und 1,1,4,4,4-Pentamethyl-1,4-diazabutan-1-yl
sein kann.
Ionenaustauscher nach Anspruch 1, dadurch gekennzeichnet, daß die beiden Atome,
die die positiv geladenen Atome trennen, Kohlenstoffatome sind.
Ionenaustauscher nach Anspruch 1 oder 2, dadurch gekennzeichnet, daß die positiv
geladenen Atome Stickstoffatome sind.
Ionenaustauscher nach Anspruch 1 oder 2, dadurch gekennzeichnet, daß eines
der positiv geladenen Atome ein Stickstoffatom und das andere ein Schwefelatom
ist.
Ionenaustauscher nach einem der Ansprüche 1 bis 4, dadurch gekennzeichnet,
daß der funktionelle Ligand A angegeben wird durch die Strukturformeln (IA) oder
(IB):
wobei Y1 und Y2 unabhängig voneinander Stickstoff oder Schwefel
sind,
R1, R2, R3, R4, R5 und
R6 unabhängig voneinander angegeben sind durch die Formel (II):
R7, R8, R9 und R10 unabhängig voneinander
Wasserstoff, C1-C4-Alkyl, C1-C4-Alkoxyalkyl
oder Hydroxy-C1-C4-alkyl sind,
R11 Wasserstoff, C1-C6-Alkyl, C1-C6-Alkoxyalkyl
oder Hydrox-C1-C6-alkyl ist,
R12 Wasserstoff, Hydroxy oder C1-C4-Alkyl ist,
l 0 oder 1 ist,
m 0 oder 1 ist,
n 1 bis 5 ist,
x 0 oder 1 ist, y 0 oder 1 ist, z 0 oder 1 ist, mit der Maßgabe, daß
l 0 ist, wenn Y2 Schwefel ist,
m 0 ist, wenn Y1 Schwefel ist,
z 0 ist, wenn R12 Hydroxy ist,
R2 zusammen mit R4 und/oder R3 zusammen mit
R6 eine Ethylengruppe bilden können, in der eines oder beide Kohlenstoffatome
mono- oder disubstituiert sein kann/können durch C1-C4-Alkyl,
C1-C4-Alkoxy, C1-C4-Alkoxyalkyl oder
Hydroxy-C1-C4-alkyl.
Ionenaustauscher nach Anspruch 5, dadurch gekennzeichnet, daß R1
bis R6 eine Gruppe umfassen, unabhängig ausgewählt aus: -[CH2-CH2-O]n-;
-[CH2]n-;
und
wobei n wie in Anspruch 5 definiert ist.
Ionenaustauscher nach Anspruch 5 oder 6, dadurch gekennzeichnet, daß
n 1 oder 2 ist,
R7 bis R10 unabhängig voneinander Wasserstoff oder C1-C2-Alkyl
sind,
R11 C1-C3-Alkyl ist,
R12 Wasserstoff, Hydroxy oder Methyl ist.
Ionenaustauscher nach einem der Ansprüche 5 bis 7, dadurch gekennzeichnet,
daß R1 bis R6 unabhängig voneinander C1-C3-Alkyl,
C1-C3-Alkoxyalkyl oder Hydroxy-C1-C3-alkyl
sind.
Ionenaustauscher nach einem der Ansprüche 5 bis 8, dadurch gekennzeichnet,
daß mindestens eines der Substituentenpaare R2, R4 und R3,
R6 eine gegebenenfalls substituierte Alkylengruppe bildet.
Ionenaustauscher nach einem der Ansprüche 1 bis 9, dadurch gekennzeichnet,
daß der Träger eine teilchenförmige vernetzte Agarose-oder Polystyrol/Divinylbenzol-Matrix
ist.
Anspruch[en]
An ion exchanger for chromatographic separation, having the structure
P - S - A,
wherein P is an insoluble support, S is a spacer, and A is a functional ligand, characterized in that said functional ligand A comprises two positively
charged atoms, each selected from nitrogen and sulphur, at a distance of two atoms
from each other, with the provisos that each of the charged atoms is a quaternary
amino nitrogen in case the two charged atoms are nitrogens and that A can not
be 4-methyl-1,4-diazabicyclo-[2.2.2]octan-1-yl, 1,4,4-trimethylpiperazinium-1-yl,
1,4-dimethyl-1,4-diazabicyclo[2.2.2]octan-2-yl and 1,1,4,4,4-pentamethyl-1,4-diazabutan-1-yl.
An ion exchanger according to claim 1, characterized
in that the two atoms
separating said positively charged atoms are carbon atoms.
An ion exchanger according to claim 1 or 2, characterized in that said
positively charged atoms are nitrogen atoms.
An ion exchanger according to claim 1 or 2, characterized in that one
of said positively charged atoms is a nitrogen atom and the other is a sulphur
atom.
An ion exchanger according to any one of claims 1 to 4, characterized
in that said functional ligand A is represented by the structural formulae (IA)
or (IB):
wherein
Y1 and Y2 independently of each other are nitrogen or
sulphur,
R1, R2, R3, R4, R5 and
R6 independently of each other are represented by the formula (II):
R7, R8, R9 and R10 independently
of each other are hydrogen, C1-4alkyl, C1-4alkoxyalkyl or
hydroxy-C1-4alkyl;
R11 is hydrogen, C1-6alkyl, C1-6alkoxyalkyl
or hydroxy-C1-6alkyl;
R12 is hydrogen, hydroxy or C1-4alkyl;
l is 0 or 1;
m is 0 or 1;
n is 1 to 5;
x is 0 or 1;
y is 0 or 1;
z is 0 or 1;
with the provisos that:
l is 0 when Y2 is sulphur;
m is 0 when Y1 is sulphur;
z is 0 when R12 is hydroxy;
R2 together with R4 and/or R3 together with
R6 may form an ethylene group in which one or both carbon atoms may
be mono- or disubstituted by C1-4alkyl, C1-4alkoxy, C1-4alkoxyalkyl
or hydroxy-C1-4alkyl.
An ion exchanger according to claim 5, characterized
in that R1
to R6 comprise a group independently selected from: -[CH2-CH2-O]n-;
-[CH2]n-;
and
wherein n is as defined in claim 5.
An ion exchanger according to claim 5 or 6, characterized in that
n is 1 or 2;
R7 to R10 independently of each other are hydrogen or
C1-2alkyl;
R11 is C1-3alkyl;
R12 is hydrogen, hydroxy or methyl.
An ion exchanger according to any one of claims 5 to 7, characterized
in that R1 to R6 independently of each other are C1-3alkyl,
C1-3alkoxyalkyl or hydroxy-C1-3alkyl.
An ion exchanger according to any one of claims 5 to 8, characterized
in that at least one of the substituent pairs R2, R4 and
R3, R6 forms an optionally substituted ethylene group.
An ion exchanger according to any one of claims 1 to 9, characterized
in that said support is a particulate cross-linked agarose or polystyrene-divinylbenzene
matrix.
Anspruch[fr]
Echangeur d'ions pour séparation chromatographique, ayant la structure
P - S - A,
dans laquelle P est un support insoluble, S est un groupe espaceur, et A est un
ligand fonctionnel,
caractérisé en ce que ledit ligand fonctionnel A comprend deux atomes chargés positivement,
chacun étant choisi parmi l'azote et le soufre, à une distance de deux atomes
l'un de l'autre,
aux conditions que chacun des atomes chargés soit un azote d'amine quaternaire
au cas où les deux atomes chargés sont des azotes, et que A ne puisse pas être
le 4-méthyl-1,4-diazabicyclo-[2.2.2]octan-1-yle, le 1,4,4-triméthylpipérazinium-1-yle,
le 1,4-diméthyl-1,4-diazabicyclo[2.2.2]octan-2-yle et le 1,1,4,4,4-penta-méthyl-1,4-diazabutan-1-yle.
Echangeur d'ions selon la revendication 1, caractérisé en ce que les deux atomes
séparant lesdits atomes chargés positivement sont des atomes de carbone.
Echangeur d'ions selon la revendication 1 ou 2, caractérisé en ce que lesdits
atomes chargés positivement sont des atomes d'azote.
Echangeur d'ions selon la revendication 1 ou 2, caractérisé en ce que l'un
desdits atomes chargés positivement est un atome d'azote et l'autre est un atome
de soufre.
Echangeur d'ions selon l'une quelconque des revendications 1 à 4, caractérisé
en ce que ledit ligand fonctionnel A est représenté par la formule structurale
(IA) ou (IB):
dans lesquelles
Y1 et Y2, indépendamment l'un de l'autre, sont l'azote
ou le soufre,
R1, R2, R3, R4, R5 et
R6, indépendamment les uns des autres, sont représentés par la formule
(II):
R7, R8, R9 et R10, indépendamment
les uns des autres, sont l'hydrogène, un alkyle en C1-4, un alcoxyalkyle
en C1-4 ou un hydroxy(alkyle en C1-4);
R11 est l'hydrogène, un alkyle en C1-6, un alcoxyalkyle
en C1-6 ou un hydroxy(alkyle en C1-6);
R12 est l'hydrogène, un hydroxy ou un alkyle en C1-4;
l vaut 0 ou 1;
m vaut 0 ou 1;
n vaut 1 à 5;
x vaut 0 ou 1;
y vaut 0 ou 1;
z vaut 0 ou 1;
aux conditions que:
l vaille 0 lorsque Y2 est le soufre;
m vaille 0 lorsque Y1 est le soufre;
z vaille 0 lorsque R12 est un hydroxy;
R2 conjointement à R4 et/ou R3 conjointement
à R6 peuvent former un groupe éthylène dans lequel l'un des atomes
de carbone ou les deux peuvent être mono- ou disubstitués par un alkyle en C1-4,
un alcoxy en C1-4, un alcoxyalkyle en C1-4 ou un hydroxy(alkyle
en C1-4).
Echangeur d'ions selon la revendication 5, caractérisé en ce que R1
à R6 comprennent un groupe choisi indépendamment parmi: -[CH2-CH2-O]n-;
-[CH2]n-;
et
dans lequel n est comme défini à la revendication 5.
Echangeur d'ions selon la revendication 5 ou 6, caractérisé en ce que
n vaut 1 ou 2;
R7 à R10, indépendamment les uns des autres, sont l'hydrogène
ou un alkyle en C1-2;
R11 est un alkyle en C1-3;
R12 est l'hydrogène, un hydroxy ou un méthyle.
Echangeur d'ions selon l'une quelconque des revendications 5 à 7, caractérisé
en ce que R1 à R6, indépendamment les uns des autres, sont
un alkyle en C1-3, un alcoxyalkyle en C1-3 ou un hydroxy(alkyle
en C1-3).
Echangeur d'ions selon l'une quelconque des revendications 5 à 8, caractérisé
en ce que au moins l'un des couples de substituants R2, R4
et R3, R6, forme un groupe éthylène éventuellement substitué.
Echangeur d'ions selon l'une quelconque des revendications 1 à 9, caractérisé
en ce que ledit support est un agarose réticulé particulaire ou une matrice de
polystyrène-divinylbenzène.