[0001] This invention relates to the production of improved polyelectrolytes which are useful
in the fractionation of blood and other proteinaceous substances. More particularly,
this invention relates to aggregated water-insoluble, cross-linked polyelectrolyte
polymers having amine-imide functional groups.
[0002] Recently, there has been disclosed the production of certain polyelectrolyte polymers
which are useful for a variety of protein separation systems. Thus, U.S. Patent 3,554,985
describes the preparation of water-insoluble, cross-linked polyelectrolyte polymers
having diloweralkylaminoloweralkylimide functional groups. These polyelectrolytes
have been found to be useful in the fractionation of blood plasma and serum as described
in U.S. Patent 3,555,001 and for the separation of viruses from non-viral proteins
as disclosed in U.S. Patents 3,655,509 and 3,846,543. These polyelectrolyte polymers
also are useful for the immunization of animals against viral diseases as seen from
U.S. Patent 3,651,213 and for the purification of water by the removal of contaminating
bacteria and viruses as set forth in U.S. Patent 3,398,092.
[0003] In U.S. Patent 3,554,985, the water-insoluble, cross- linked polyelectrolytes are
further described as being copolymers of an (a) unsaturated monomer of 2 to 12 carbon
atoms and (b) a monomer selected from the group consisting of (1) a mixture of an
unsaturated polycarboxylic acid or anhydride and an unsaturated polycarboxylic acid
amine-imide, and (2) an unsaturated polycarboxylic acid amine-imide. In a typical
example, the starting copolymer comprises the reaction product of styrene and maleic
anhydride cross-linked with divinyl benzene (Example 1, Column 16) which is subsequently
converted to the amine-imide derivative by reaction with dimethylaminopropylamine
(Example 2, column 16). In other typical examples, a preformed polymer such as a copolymer
of ethylene and maleic anhydride is cross-linked during the reaction with the dialkylaminoalkylamine
by also employing in the reaction a predetermined amount of a difunctional compound
such as ethylenediamine (column 12, lines 27-40).
[0004] Notwithstanding the favorable properties of the foregoing polyelectrolytes for their
intended uses, it has been found in practice that they are difficult to handle in
processing due to certain of the physical and chemical properties of the initially
preformed polymer. Thus, it has been found difficult to filter the preformed polymer
from the mother liquor following the initial polymerization reaction. Thick slurries
of the polymer are formed which are slow to filter and give very dense filter cakes
that do not break up readily. On drying, these products give a hard, lumpy material
that requires excessive grinding.
[0005] While various procedures have been considered for overcoming these problems in processing
of the polymer, a principal ultimate use of the polymer for protein fractionation
following substitution with the functional amine-imide group dictates that the protein
adsorption capacity and protein selectivity of the polymer be not substantially impaired.
[0006] The method of the present invention is one of aggregating and improving the filterability,
drying characteristics and physical form of polymers used in making water-insoluble,
cross-linked polyelectrolytes containing amine-imide functional groups without-substantially
diminishing the protein adsorption capacity of said polyelectrolytes characterized
by heating said polymer in an inert organic solvent or solvent mixture at a temperature
ranging from about 115°C to about 160°C but lower than the softening point of said
polymer for at least about 15 minutes and until said polymer is substantially aggregated,
said polymer comprising a copolymer of (a) unsaturated monomer having from 2 to about
18 carbon atoms and (b) a monomer selected from the group consisting of unsaturated
polycarboxylic acids or anhydrides having from 4 to about 12 Carbon atoms, and said
polymer being thus aggregated prior to the cross- linking and substitution with the
major portion of said amine-imide functional groups.
[0007] The invention also comprises an aggregated, water-insoluble, cross-linked polyelectrolyte
characterized by a copolymer of (a) unsaturated monomer having from 2 to about 18
carbon atoms and (b) a monomer selected from the group consisting of unsaturated polycarboxylic
acid or anhydride having from 4 to about 12 carbon atoms, and in which from about
2 to 100% of the reactive sites in said copolymer are substituted with amine-imide
functional groups, said polyelectrolyte being thus aggregated prior to cross-linking,
and substitution with the major portion of said amine-imide functional groups.
[0008] The invention also comprises the use of a polyelectrolyte according to the invention
in the fractionation of blood or other proteinaceous substances.
[0009] In accordance with the present invention, polyelectrolytes of the general type described
hereinbefore are significantly and substantially improved by an aggregation process
whereby the protein adsorption capacity not only is unimpaired but, surprisingly,
also is improved in certain blood fractionation systems. Briefly stated, the aggregation
process comprises treatment of the preformed copolymer, prior to cross-linking and
the addition of the functional amine-imide group, with refluxing xylene or other such
inert organic solvents. This treatment is carried out at a temperature ranging from
about 115°C to about 160°C but lower than the softening point of the polymer for at
least about 15 minutes and until the polymer is substantiallyaggregated. The product
obtained by this treatment is an aggregated polymer which filters rapidly and in which
the filter cake breaks apart so easily that ball milling is no longer necessary in
most instances. Drying of the filtered material also is faster with the aggregated
polymer than with'the unaggregated polymer. The protein adsorption capacity of the
subsequently prepared cross-linked material containing the amine-imide functional
group is substantially undiminished. In an illustrative preferred example, the albumin
adsorption capacity of the aggregated material has been found to be more than three
timesthat of the unaggregated material. These results are surprising because one would
expect that a more finely divided material having a greater surface area would also
have a correspondingly greater adsorption capacity than an aggregated material.
[0010] The properties of the aggregated polyelectrolyte in which grinding is unnecessary
for obtaining suitable handling characteristics differ markedly from those of a ground,
unaggregated polyelectrolyte. These differing products have non-equivalent particle
structures. It has been found that the protein adsorption characteristics of these
products involve both their chemical and physical properties. The desired aggregated
polyelectrolyte is prepared with due consideration of difference in the molecular
structure of the external shell and the internal core of the particles. When the particles
are reduced by grinding, the shell-core relationships are changed. For this reason,
(1) size reduction of polyelectrolyte particles is preferably avoided and (2) the
desired structures for polyelectrolyte particles_are achieved by synthesis sequences
which develop the surface characteristics and basic core structure preferred for selective
adsorption and elution of specific proteins. Thus, aggregation relates to the overall
particle structure and its requirements for protein fractionation as well as providing
important process advantages.
[0011] In the present invention, several embodiments of the aggregated polymer are contemplated
by the inventors. The following is a description of preferred embodiments taken in
connection with the accompanying drawings in which:
FIG. 1 is a photomicrograph (200 X) of an aggregated water-insoluble cross-linked
polymer of this invention.
FIG. 2 is a photomicrograph (200 X) of another batch of the polymer of FIG. 1 but
in unaggregated form.
[0012] In general, the initial polymers which are aggregated in accordance with this invention
include those disclosed in the aforementioned U.S. Patents 3,554,985 and 3,555,001,
said patents being incorporated herein by reference. Preferably, the initial polymer
comprises a copolymer of (a) unsaturated monomer-.having from 2 to about 18 carbon
atoms and (b) a monomer selected from the group consisting of unsaturated polycarboxylic
acids and anhydrides having from 4 to about 12 carbon atoms. Following the aggregation
process, the aggregated polymer is cross-linked and substituted with an appropriate
amine-imide group.
[0013] Suitable amine-imide groups include not only those specifically described in U.S.
Patents 3,554,985 and 3,555,001, but also cyclic amine-imide groups as defined hereinbelow.
[0014] It is critical to the present invention that the desired aggregation process be carried
out prior to the cross-linking and substitution with the major proportion of amine-imide
groups. It has been found that when the cross-linking and/or the substitution with
excessive amounts of the functional amine-imide group is carried out prior to the
attempted aggregation process, the desired aggregation is not achieved and the advantages
of the invention are not obtained. These results are surprising inasmuch as they are
contrary to the expectation that the presence of the functional group would tend to
make the polymer softer and thereby more readily susceptible to aggregation by simple
particle fusion By the term "major proportion" is meant more than 50% of said groups.
[0015] Although the inventors are not bound by theory, the importance of carrying out the
aggregation process prior to cross-linking and/or the addition of the major portion
of the functional group may be due in part to a bridging reaction to form acyclic
anhydride groups between carboxyl groups on the backbones of different polymer molecules
on adjacent particle surfaces. This bridging differs from the usual anhydride formation
by adjacent carboxyl groups on a backbone of a given polymer molecule. These differences
can be illustrated structurally as follows:

[0016] The anhydride copolymers normally contain up to 2% moisture, and a portion of this
reacts with anhydride groups to form carboxylic acid groups while the remainder is
assumed to be present as free water. The latter is rather easily lost on drying, while
the former is released by reforming either cyclic or acyclic anhydride groups. This
is a slower process; however, it occurs readily under conditions that favor aggregation,
e.g., refluxing xylene.
[0017] For purposes of convenience, the preferred initial polymers which are subjected to
the aggregation process as defined herein will be referred to as EMA-type polymers
(ethylene/maleic anhydride or acid). The EMA-type polymers have been described previously
in U.S. Patents 3,554,985 and 3,555,001 and are further illustrated by the general
examples in the following section I:
I
[0018] The polycarboxylic acid polymers can be of the non-vicinal-type including those containing
monomer units, such as acrylic acid, acrylic anhydride, methacrylic acid, crotonic
acid or their respective derivatives, including partial salts, amides and esters or
of the vicinal type, including maleic, itaconic, citraconic, α-dimethyl maleic, α-butyl
maleic, α-phenyl maleic, fumaric, aconitic, α-chloromaleic, α-bromomaleic, and α-cyanomaleic
acids including their salts, amides and esters. Anhydrides of the foregoing acids
are also advantageously employed.
[0019] Co-monomers suitable for use with the above polycarboxylic acid monomers include
α-olefins, such as ethylene, 2-methyl-pentene-l, propylene, isobutylene, 1- or 2-butene,
1-hexene, 1-octene, 1-decene, 1-dodecene, 1-octadecene, and other vinyl monomers,
such as styrene, α-methyl styrene, vinyltoluene, vinyl acetate, vinyl chloride, vinyl
formate, vinyl alkyl ethers, e.g., methyl-vinyl-ether, alkyl acrylates, alkyl methacrylates,
acrylamides and alkylacrylamides, or mixtures of these monomers. Reactivity of some
functional groups in the copolymers resulting from some of these monomers permits
formation of other useful functional groups in the formed copolymer, including hydroxy,
lactone, amine and lactam groups.
[0020] Any of the said carboxylic acids or derivatives may be copolymerized with any of
the other monomers described above, and any other monomer which forms a copolymer
with unsaturated carboxylic acids or derivatives. Although these copolymers can be
prepared by direct polymerization of the various monomers, frequently they are more
easily prepared by an after-reaction modification of an existing copolymer. Copolymers
are conveniently identified in terms of their monomeric constituents. The names so
applied refer to the molecular structure and are not limited to the polymers prepared
by the copolymerization of the specified monomers.
[0021] Representative EMA-type carboxylic acid or anhydride-olefin polymers, especially
maleic acid or anhydride-olefin polymers of the foregoing type are known: for example,
from U.S. Patents 2,378,629; 2,396,785, 3,157,595; and 3,340,680. Generally, the copolymers
are prepared by reacting ethylene or other unsaturated monomer, or mixtures thereof,
with the acid anhydride in the presence of a peroxide catalyst in an aliphatic or
aromatic hydrocarbon solvent for the monomers but nonsolvent for the interpolymer
formed. Suitable solvents include benzene, toluene, xylene, chlorinated benzene and
the like. While benzoyl peroxide is usually the preferred catalyst, other peroxides
such as acetyl peroxide, butyryl peroxide, ditertiary butyl peroxide, lauroyl peroxide
and the like, or any of the numerous azo catalysts, are satisfactory since they are
soluble in organic solvents. The copolymer preferably contains substantially equimolar
quantities of the olefin residue and the anhydride residue. Generally, it will have
a degree of polymerization of about 8 to 100,000, preferably about 100 to 5,000, and
a molecular weight of about 1,000 to 1,000,000, preferably about 10,000 to 500,000.
The properties of the polymer, such as molecular weight, for example, are regulated
by suitable choice of the catalyst and control of one or more of the variables such
as ratio of reactants, temperature, and catalyst concentration or the addition of
regulating chain transfer agents, such as diisopropyl benzene, propionic acid, alkyl
aldehydes, and the like. "Numerous of these polymers are commercially available.
[0022] The aggregation of the foregoing EMA-type polymers and other such polymers as defined
herein is carried out by stirring the polymer as a suspension in refluxing or heated
organic solvent which is inert to the polymer. This refluxing or heating is carried
out at a temperature ranging from about 115°C to about 160°C but lower than the softening
point of the polymer. A preferred solvent is xylene. Other solvents which can be used
are, for example, ethylbenzene, mono- and dichlorobenzene and cumene. Solvents such
as benzene and toluene having boiling points below about 115°C are not practical for
purposes of this invention. However, dioxane which boils at 101.5 can yield appreciable
polymer coagulation probably due to stronger solvent action on EMA. It has been found
that treatment of the polymer in boiling solvent at temperatures below about 115°C
does not provide any significant aggregated product as desired herein when using weak
solvents such as hydrocarbons. Aggregation can occur in high boiling solvents at temperatures
below the boiling point, but temperatures above the melting point of the polymer are
unsuitable in view of the detrimental effect they can have upon the polymer structure
and on subsequent use in the polyelectrolyte adsorption of proteins.
[0023] Heating of the polymer in the refluxing solvent for at least about 15 minutes is
desired, and good results have been obtained by heating up to about one hour. Heating
for prolonged periods of time substantially in excess of about one hour is unnecessary,
but the aggregates remain stable in weak solvents even during such prolonged heat
treatment up to 7 hours. The stronger solvents such as chlorobenzene, dichlorobenzene,
dioxane and N,N-dimethylamine are less preferred solvents because of excessive coagulation
that occurs on extended aggregation times.
[0024] Following the aggregation process, the aggregated polymer is cross-linked and substituted
with the desired amine-imide groups in whatever sequence optimizes the properties
being sought.by tailoring the distribution of specific groups within the particles.
These groups are essentially basic groups which can be aliphatic straight chain groups
or can be alicyclic or aromatic groups. The aliphatic straight chain groups preferably
are diloweralkylaminoloweralkylimide groups or lower- alkyliminodi(loweralkylimide)
linkages as described previously in U.S. Patents 3,554,985 and 3,555,001. Such products
are further illustrated by the general examples in the following section II:
II
[0025] The initial copolymers of anhydrides and another monomer can be converted to carboxyl-containing
copolymers by reaction with water, and to ammonium, alkali and alkaline earth metal
and alkylamine salts thereof by reaction with alkali metal compounds, alkaline earth
metal compounds, amines or ammonia. Other suitable derivatives of the above polymers
include the alkyl or other esters, alkyl amides, dialkyl amides, phenylalkyl amides
or phenyl amides prepared by reacting carboxyl groups on the polymer chain with the
selected amines or alkyl or phenylalkyl alcohol, as well as amino esters, amino amides,
hydroxy amides and hydroxy esters, wherein the functional groups are separated by
alkylene, phenyl, phenylalkyl, phenyl- alkylphenyl, or alkylphenylalkyl or other aryl
groups. Moieties bearing amine or amine salts including quaternary salt groups are
conveniently formed by reaction of the carboxyls of their anhydride precur- ―_ sors
where applicable with polyfunctional amines such as dimethylaminopropylamine at higher
temperatures forming an imide linkage with vicinal carboxyls. Such pendant free amine
groups can then be converted, if desired, to their simple or quaternary salts.
[0026] Partial imides of a starting carboxyl or carboxylic acid anhydride containing polymer,
e.g., EMA, are produced by:
(A) Heating a limiting amount of a secondary or tertiary aminoloweralkylamine with
the anhydride or carboxyl-containing form of the polymer in a suitable solvent (e.g.,
xylene) at a temperature of about 140-150°C until water is no longer given off. Such
a reaction simultaneously results in formation of imide groups in proportion to the
amount of amine added and in the reformation of anhydride groups for the remainder
of the polymer units. In this manner, imide-polymer products are formed which typically
possess 2-100% imide linkages, the remaining carboxyl groups, when present, being
in the anhydride form.
(B) Alternatively, a partial amide polymer product can be converted to the partial
imide polymer product by heating a partial amide-polymer product in vacuo at 140-150°C
until water is no longer given off. Such an imide polymer product likewise possesses
comparable proportions of imide and anhydride groups depending upon the number of
amide groups originally contained in the starting partial amide-polymer product.
[0027] Partial secondary or tertiary aminoloweralkyl- amides of the starting carboxyl or
carboxylic acid anhydride-containing polymer, e.g., E.M.A., are obtained by contacting
the polymer with a limiting amount of the selected amine in suspension in a solvent
such as benzene or hexane, resulting in formation of a partial amide-acid anhydride
derivative of the polymer, or a corresponding amide-carboxylate derivative thereof.
[0028] The number of amide groups is dependent upon the quantity of the amine used as compared
with the quantity of polymer employed. Such amide-polymer products typically comprise
2-100% amide groups, with remaining carboxyl groups being present as acid or anhydride
groups.
[0029] Suitable blocking and unblocking of the amine moiety of the reactant employed in
preparing amines or imides may be effected when required. Residual, non- modified
polymer units may optionally be converted to neutral groups or units by attachment
to the polymer molecule of compounds including alkylamines, aminoalcohols and alcohols.
[0030] Alternatively, additional cationic character can be provided in the polymer through
incorporation of monomers which impart a basic or cationic character such as C-vinyl
pyridines, vinyl amine, the several amino-substituted vinyl benzenes (or toluenes
and the like), amine-bearing acrylates (or methacrylates and the like), vinyl imidazole
and similar such monomers.
[0031] Thus, in any event, the polymer product will have residual active or reactive groups
which can be of various types, including mixtures, but these residual active or reactive
groups or residual "reactive sites" in the polymer will in one way or another comprise
a certain percentage which are of a basic nature, so as to impart the requisite basic
nature to the polymer product.
[0032] Especially preferred polymers subject to the previously referred to requirements
are selected from the group consisting of ethylene/maleic acid or anhydride copolymers,
styrene/maleic acid or anhydride copolymers, and isobutylene/maleic acid or anhydride
copolymers.
[0033] As will be apparent from the foregoing, the essential basic groups of the polycationic
or polyampholytic polyelectrolyte (PE) employed are of an imide nature involving diloweralkylaminoloweralkylimide
groupings, e.g., as produced by reacting a diloweralkylaminoloweralkylamine with the
carboxyl groups of a pre-formed polymer or by polymerizing an unsaturated olefin with
an unsaturated anhydride or acid-having such pre-formed imide groups in at least a
portion of the unsaturated polycarboxylic acid reactant. According to the invention,
such groups are preferred for purposes of the invention.
[0034] Alternatively, whether such pre-formed groups are or are not present, imide groups
can be provided by cross- linking the polymer with a loweralkyliminobis(loweralkyl-
amine) which in the process of cross-linking by reaction between the terminal amine
groups of the cross-linker and carboxyl groups in the polymer chain is productive
of imido groups at both ends of the cross-linking chain with formation of the desired
loweralkyliminobis(loweralkyl- imide) linkages. Other groups, such as diloweralkylaminoloweralkylimide
groups, from which the desired imide groups can be obtained by heating at elevated
temperatures, can also be present. Also, diloweralkyl- aminoloweralkyl ester groups
can be present, as well as other groups, so long as the
imide groups of the prescribed type are also present in the polyelectrolyte molecule
as well as the residual acid groups of the starting unsaturated acid or anhydride
when the polyelectrolyte is a polyampholyte. As will be recognized, both the acid
groups and the imide groups need not necessarily be present in the polyelectrolyte
as such, but can be present in the form of their simple derivatives, e.g., salts,
as already indicated.
[0035] Alicyclic or aromatic groups which can be substituted on the aggregated EMA-type
polymers are for example, aminoloweralkyl-pyridine, piperidine, piperazine, picoline,
pyrrolidine, morpholine and imidazole. These groups can be substituted on the aggregated
polymer in a manner analogous to the aliphatic chain amines but by using, instead,
cyclic amines such as, for example:
2-aminopyridine
2-amino-4-methylpyridine
2-amir.o-6-methylpyridine
2-(2-aminoethyl)-pyridine
4-(aminoethyl)-piperidine
3-amino-N-ethylpiperidine
N-(2-aminoethyl)-piperidine
N-(2-aminoethyl)-piperazine
3-picolylamine
4-picolylamine
2-(aminomethyl)-i-ethylpyrrolidine
N-(3-aminopropyl)-2-pyrrolidine
N-(2-aminoethyl)-mQrpholine
N-(3-aminopropyl)-morpholine
4-imidazole
[0036] The following specific examples will further illustrate the production and use of
the aggregated polyelectrolyte polymers of this invention although it will be understood
that the invention is not limited to these specific examples. The results obtained
in several examples are set forth-in convenient tabular form following the respective
examples. In these examples, abbreviations of several materials are defined as follows:
MIBPA = methyl-imino-bis-propylamine
DMAPA = dimethylaminopropylamine
DEAEA = diethylaminoethylamine
HOEtA = monoethanolamine
HMDA = hexamethylenediamine
[0037] Figure 1 of the drawings shows a photomicrograph of the aggregated polymer prepared
in Example 3 at a magnification of 200 X. A similar polymer was prepared as in this
example but without the aggregation process. Figure 2 of the drawings shows a photomicrograph
of this unaggregated polymer prepared in Example 9 also at a magnification of 200
X. The striking differences in physical structure are readily apparent from these
comparative photomicrographs.
[0038] The polymer aggregates are a multiplicity of small individual particles held together
in clusters without fusion. In general, the major portion of the unaggregated polymer
has a particle size ranging from about 0.1 microns to about 10 microns whereas the
major portion of the aggregated polymer has a particle size ranging from about 50
to about 200 microns.
EXAMPLE 1
[0039] A 5-liter reaction flask, equipped with reflux condenser, Dean-Stark water take-off,
stirrer, reagent addition vessel, thermometer and nitrogen-purge attachments is charged
with 193.05 g. ethylene/maleic anhydride copolymer (EMA) (1.5 moles, anhydride basis)
aha ml. xylene. The charge is stirred at 200 r.p.m. with a 6.5 inch blade-type stirrer
while it is heated to the reflux temperature. This reflux temperature will vary from
135 to 139°C depending on the water content of the EMA and upon whether this water
is azeotropically removed during the ensuing reflux period. In the present example
the slurry was maintained at total reflux for 60 minutes under total reflux return
at a temperature of 135°C. After 1 hour the reactor was cooled to 125°C under nitrogen
and a solution mixture of 10.89 g. MIBPA (0.075 mole) and 1.5 ml. water was added.
The mixture was heated to reflux (134°C) and maintained at reflux for 1 hour while
continuously removing water azeotrop (final temperature was 137°C). The reaction mixture
was again lowered to 125°C under nitrogen and a solution mixture of 153.3 g. DMAPA
(1.5 moles) and 4.5 ml. water was added. The slurry was then heated to 133°C and held
at this temperature (1-10 min.) until refluxing began as a conse-
quence of water being formed during the chemical reaction. Stirring and refluxing of
the reacting slurry was continued until water removal by azeotropic distillation was
complete. The final temperature was 139°C.
Product work-up
[0040] For work-up as the free amine form, the above slurry was filtered hot and the product
cake was reslurried in 2700 ml of a 3:1 mixture of xylene and ethanol, stirred at
reflux temperature for one hour and then filtered hot. This was repeated a second
time for a two hour period and a third time for a three hour refluxing period, in
each case followed by hot filtration. The resulting extracted product cake was then
reslurried in 2700 ml. hexane for 1 hour at room temperature and filtered. The hexane
extraction was repeated a total of four times. The final product was airdried for
30 minutes and finally dried in a vacuum oven at 55°C...
[0041] For work-up as the hydrochloride salt form, the final reaction slurry was filtered
hot and the product cake reslurried at reflux in 3:1 xylene-alcohol three times, as
above, followed by two 1-hour room temperature extractions with 2700 ml. acetone.
The filtered product was converted to the hydrochloride by reslurrying in either 2700
ml. alcohol or acetone and gradually adding with stirring (over 10 min.) 112 ml. conc.
(12N) hydrochloric acid and stirring at room temperature for two hours. The filtered
product was subsequently washed (slurry with stirring) three consecutive times with
10 liters of water (deionized) for 2 hours each time and finally filtered. The filtered
salt cake was reslurried four times in 2700 ml. acetone (1 hour each time) to remove
the water, filtered, air dried for 30 minutes and vacuum oven dried at 55°C.
[0042] The final dried product, either as free amine or as salt, was screened without grinding
with 95% of the product going through a 100 mesh screen before bottling for use.
EXAMPLE 2
[0043] The aggregated diethylaminoethyl derivative was prepared using the identical procedure
of Example 1 except that 174.32 g. DEAEA (1.5 mole) was substituted for the DMAPA
in Example 1. The final product was obtained as the free amine form using the work-up
procedure of Example 1 wherein the reaction product was consecutively extracted with
three 3:1 xylene-alcohol extractions followed by four hexane extractions. The product
was sieved unground through a 100 mesh screen to yield 229 g. of material finer than
100 mesh and 13.0 g. coarser than 100 mesh.
EXAMPLE 3
[0044] This example utilized the same equipment, the same aggregation procedure and the
same initial charge (EMA and xylene) as described in Example 1. After the aggregation
period (1 hour reflux) the slurry temperature was lowered to 125°C and 10.89 g. (0.075
moles) MIBPA was added. The slurry was stirred at 120-125
0C for one hour without reflux. After one hour 7.66 g. (0.075 moles) DMAPA was added
and the slurry was again stirred at 120-125°C. for one hour without reflux. After
this period the slurry was heated to reflux and the total water of the condensation
reaction was removed by distillation as the azeotrope. The final temperature was 139°C.
The reaction mixture was then cooled to 120°C, 87.05 g. of HOEtA was added, and the
slurry maintained at 120° for 1 hour. The temperature was then raised to reflux and
the water from this final condensation reaction completely removed over a 6 hour period
by distillation as the azeotrope. The final temperature was 140°C. The product was
worked up as the free amine as described in Example 1 for free amine work-up procedure.
230 g. of product was obtained which passed through a 100 mesh screen unground; 17
g. of product was retained on the screen.
EXAMPLE 4
[0045] In order to improve the dispersion characteristics of the product of Example 3 the
order of addition of MIBPA and DMAPA was reversed following the aggregation step.
[0046] The same amounts of amines and other raw materials of Example 3 were used. The procedure
was identical through the aggregation step. After cooling the aggregated slurry to
125°C., 7.66 g. of DMAPA was added and the slurry was held at 120-125°C for one hour.
Then 10.89 g. MIBPA was added and the slurry was again held at 120-125°C. for one
hour. From this point on the procedure was exactly the same as described in Example
3. The final product was worked-up as the free amine form.
EXAMPLE 5
[0047] The identical procedure of Example 4 was repeated except that the final product was
worked-up as the hydrochloric acid salt by the procedure described in Example 1. For
this purpose only 14 ml. concentrated hydrochloric acid (12N) was used instead of
the 112 ml. used in Example 1. After drying, 240 grams of product was obtained.
EXAMPLE 6
[0048] The procedure of Example 3 was repeated except that water of the condensation reaction
was removed by azeotropic distillation after each of the amine reactions and holding
times, i.e., after MIBPA, after DMAPA and after HOEtA reaction instead of as in Example
3. The product was obtained as the hydrochloride salt in 240 g. yield.
EXAMPLE 7
[0049] Many preparative examples were made in which the composition was varied with respect
to the various amines used as crosslinker or functional moiety both in type and concentration.
These aggregated compositions are summarized in the following table:

EXAMPLE 8
[0050] The same equipment and the same initial charge of EMA and xylene was used as in Example
1. Aggregation, as obtained in Example 1, was precluded by one of two methods: (a)
heat slurry of EMA at 200 r.p.m. to 90°C and add 10.89 g MIBPA plus 1.5 ml. water,
continue stirring at 90°C for one hour, raise temperature to reflux (136°C) and take
off total water of reaction in the Dean-Stark trap by continued reflux (final temperature
139°C); or (b) heat slurry of EMA at 200 r.p.m. to 125°C and add the MIBPA and water
and immediately raise to reflux temperature of 136°C and continue refluxing until
all water of reaction has been removed by azeotropic distillation at a final temperature
of 139°C. After either procedure (a) or (b), above, the flask contents temperature
was lowered to 125°C. and 153.3 g. DMAPA plus 4.5 ml water was added. The slurry was
heated to 133°C until refluxing began and refluxing was maintained until all water
of reaction was removed by azeotropic distillation to a final temperature of 139-140°C.
The final slurry was filtered hot (over 100
0C). Filtering time at this point required from 30 to 60 minutes in contrast to filtering
times of less than 5 minutes for aggregated products prepared by procedures of Examples
1 through 7. The filtered product was worked up as either the free amine or as the
hydrochloride salt by procedures described in Example 1. Again, during work-up, filtering
times were long (30 minutes to 2 hours) as contrasted to work-up filtering times associated
with aggregated products of Examples 1 through 7 where these times varied from 5 to
10 minutes. Finally, non-aggregated products, prepared by this procedure and others
to follow, dried poorly and had to be ground or ball-milled prior to sieving through
a 100 mesh screen in contrast to aggregated products from Examples 1 through 7 which
required no grinding or ball-milling prior to screening through 100 mesh screens after
drying.
EXAMPLE 9
[0051] This example utilized the same equipment and the same EMA and xylene charge as in
Example 8. The slurry was heated to 90-95°C and 10.89 g. (0.075 mole) MIBPA was added
and stirred at 95°C for 1 hour. Then 7.66 g (0.075 mole) DMAPA was added and stirred
at 95°C for 1 hour. The slurry was heated to reflux (134°C) and water of reaction
was completely removed by azeotropic distillation to a final temperature of 139°C.
The slurry was then cooled to 95°C and 87.05 g of hydroxyethylamine was added and
the slurry stirred at 95°C for 1 hour. The slurry temperature was then raised to 134°C
and the total water of reaction was completely removed by azeotropic distillation
to a final temperature of 139 to 140°C. The final slurry was filtered hot (30 minutes)
and worked up as the free amine by the procedure of Example 1, dried, ground by extensive
ball milling and screened through a 100 mesh screen. The recovered yields over 12
runs varied from 219 to 244 grams depending on the ball-milling efficiency prior to
screening.
EXAMPLE 10
[0052] This procedure was the same as Example 8 except that the water of reaction was not
removed after addition of MIBPA but was allowed to remain in the reaction slurry until
after the DMAPA addition and then the total water of reaction from both amine reactions
was removed in a single final azeotropic distillation. Final slurry temperature was
140°C. The product was worked-up as the free amine.
EXAMPLE 11
[0053] This procedure was identical to that of Example 1 except that the water of reaction
was not removed after addition of MIBPA, following aggregation, but was allowed to
remain in the reaction mixture slurry until after the DMAPA addition and then the
total water of reaction from both amine reactions was removed in a single final azeotropic
distillation. The final slurry was filtered hot in less than 5 minutes and the product
was worked up as the free amine by the procedure of Example 1.
EXAMPLE 12
[0054] Effects of time and stirring rate during aggregation were considered with respect
to final hot slurry filtration rates.
[0055] A series of comparable runs were made using the procedure of Example 1 wherein the
aggregation time and stirring speed were varied. The products were all finished as
the free amine following the Example 1 procedures. The results are shown in the following
table as compared with a non-aggregated product prepared by Example 8.

EXAMPLE 13
[0056] In order to more completely characterize the solvent and temperature requirements
for aggregation, a series of tests were carried out on EMA as received raw material
in a variety of solvents and over a range of temperatures and stirrer speeds. It was
found that variation in stirrer speeds from 150 to 400 r.p.m. affected only the formed
aggregate size. Of more importance was the nature of the solvent and the temperature
range at which aggregation occurred.
[0057] For this example, a one-liter flask was used and the charge was 700 ml solvent and
50 g. of EMA. At various times and temperatures a 20 ml aliquot of the slurry was
removed and placed in vials. After cooling, the vials were shaken and the time for
settling of the polymer from the solvent was measured with a stop watch as an indication
of aggregate development.
[0058] The results are recorded below.

Example 13 (continued)

Example 13 (continued)
[0059]

EXAMPLE 14
[0060] A measure of particle size in dispersion, whether aggregated or non-aggregated, is
the swelling index defined as the grams of aqueous or other dispersant which is absorbed
at equilibrium per gram of polymer derivative. A suitable sized sample is dispersed
in excess dispersant and adjusted to pH 4 or any other desired pH value.
[0061] The dispersion is allowed to reach equilibrium over a 1 hour period and is then centrifuged
at 750 x g. for thirty minutes in a preweighed centrifuge bottle. The supernate is
decanted and the weight of centrifuged swollen gel is determined. All of the values
are reported using 0.04 Molar saline as the dispersant and a pH of 4.0. The swelling
index number is thus the weight of 0.04M saline absorbed by one gram of polymer.
[0062] Swelling is known to be inversely proportional to the crosslink density for crosslinked
insoluble resins. For a series of derivatives with increasing MIBPA, the swelling
decreases as expected, all other parameters being equal.
[0063] However for the present aggregated materials it was found that aggregate swelling
at any constant MIBPA concentration, in equal crosslink density, could be varied easily,
and differed markedly from non-aggregated derivatives, by varying the amount of water
added during the aggregated polyelectrolyte preparation and by varying the removal
steps of water from the reaction.
[0064] Several preparations of aggregated and non-aggregated polymer with the MIBPA and
DMAPA charge composition of Examples 1, 8, 10 and 11 but with varied amounts of water
added with either MIBPA or DMAPA are shown in the following table with the resultant
variation in product swelling.

EXAMPLE 15
[0065] As the swelling varied with varying amounts of water added with MIBPA and DMAPA and
with the method of water removal, so did the protein adsorption capacity of the product
resin.
[0066] Protein adsorption capacity was measured by the following method. 40 mg. human albumin
and 10 mg- of polymer product in the amine or salt form were dispersed in 1.0 ml.
of 0.04 molar saline and the pH adjusted to 7.0. The slurry was shaken for 30 minutes
while keeping the pH at 7.0. After the 30 minute adsorption period the resin-albumin
complex was centrifuged while saving the supernate. The solids were washed 3 times
with 1.0 ml. water (5-min. shaking, centrifuging) and the combined supernates were
assayed for protein by the method of Miller-Lowry, Analytical Chemistry, 31, 964 (1959).
The albumin capacity values are given in terms of mg. albumin adsorbed per mg. polymer
product.
[0067] The preparations listed in Example 14 are summarized in the following table wherein
their albumin capacity is given as a result of preparation variation.

EXAMPLE 16
[0068] Aggregated products of the type described not only have improved filtration characteristics
during synthesis processes but have been found to give high flow characteristics during
processes of plasma fractionation. Thus, non-aggregated polymers used for plasma fractionation
by adsorbing desired proteins from plasma solutions had to be separated from the mother
liquor by centrifugation processes because'of immediate clogging of filter papers
and cloths. The present aggregated products, with their improved and stable filtration
character, were able to be used in plasma fractionation processes and separated from
mother liquors by conventional vacuum filtration processes with fast filter times
and little or no filter plugging in the presence of proteinaceous material, thus avoiding
the need for expensive centrifugation-type apparatus.
[0069] The fast-flow properties of the described polyelectrolytes were further demonstrated
in packed columns with only gravity flow. For this purpose glass columns were prepared,
2.2 cm I.D. by 30 cm in length, properly stoppered to hold resin with a small fiber
glass plug on top of the bottom outlet. The resins as described in the following table
were equilibrated overnight in 0.04 molar saline at pH 4 and the column filled or
packed to a height of 8.5-10 cm. All resins used contained 5 mole percent MIBPA, thus
keeping the crosslink density factor constant. Swelling differences result from variations
in water added and water removal steps as noted.
[0070] Flow rates of 0.04 molar saline were measured for both aggregated and non-aggregated
polyelectrolyte types. The expressed value of "relative flow rate" is given in cubic
cm. per hour per unit volume of resin bed in the column under gravity flow and maintaining
a 20 cm. saline head on top of the resin during the test.

EXAMPLE 17
[0071] Several preparations of aggregated polyelectrolytes were prepared using the procedure
of Example 4 except that the third amine used to react with all anhydrides, except
those reacted with MIBPA and DMAPA, was varied, i.e., other than HOEtA as in Example
4. The compositions and other variations, if any, of these aggregated products are
listed in the following table.

All of these polyelectrolyte preparations possessed fast filtering properties characteristic
of aggregated resins. All were aggregated for a period of 1 hour.
EXAMPLE 18
[0072] The DMAPA used in several of the previous Examples illustrates an example of a dialkylaminoalkylimide
substituent on the polyelectrolyte. Another such substituent was the DEAEA (diethylaminoethylamine)
used in Examples 2 and 7 (run 3). These two amines were used to represent aggregated
dialkylaminoalkylimide polyelectrolyte substitution with 5 mole percent MIBPA in a
5/90 composition and with 5 mole percent HMDA and 85 mole percent HOEtA in a 5/5/85
composition.
[0073] Other non-aggregated dialkylaminoalkylimide preparations were made using the Example
9 procedure for the 5/5/85 compositions and the Example 8 procedure for the 5/90 compositions
except that in no case was water added with any of the amines. The amines used were
dimethylaminoethylamine, diethylaminoethylamine, diethylaminopropylamine, dimethylaminopropylamine,
di-n-butylaminopropylamine, di-hydroxyethylamino- propylamine and 2-amino-5-diethylaminopentane.
N-phenylethylenediamine was also used to prepare the above non-aggregated resins.
[0074] Similar non-aggregated compositions, using procedures of Examples 8 and 9, were prepared
using various heterocyclic amines to give heterocyclic aminoalkylimide substituents.
These amines included 2-(aminomethyl)-ethylpyrrolidine, 3-amino-N-ethylpiperidine,
N-(2-aminoethyl)-piperidine, N-(3-aminopropyl)-2-pyrrolidone, N-(2-aminoethyl)-morpholine,
N-(3-aminopropyl)-morpholine, N-(2-aminoethyl)-piperazine and 2-(2-aminoethyl)-pyridine.
[0075] Two of these heterocyclic type amines were used to prepare aggregated polyelectrclyte
compositions of the 5/5/85 type using the reactants listed in the following table.
The procedure in Example 4 was followed. In all cases the products were obtained as
hydrochloride salts (according to the Example 5 procedure) and possessed fast filtration
properties characteristic of aggregated derivatives in contrast to extremely slow
filtration properties for the comparative non-aggregated preparations described above.

[0076] Various other examples will be apparent to the person skilled in the art after reading
the foregoing disclosure without departing from the spirit and scope of the invention
and it is intended that all such examples be included within the scope of the appended
claims.