Method of coating solid particles with a hydrophilic gel

Methods for coating hydrophilic gels onto the surface of solid particles are disclosed which include utilizing hydrophilic solid particles, intimately admixing these hydrophilic solid particles with a hydrophilic gel-forming substance at a temperature above the gelling temperature so as to coat the solid hydrophilic particles, and then separating these coated particles from each other and cooling them to a temperature below the gelling temperature. The use of these coated particles is also disclosed in connection with various separation processes, such as an ion exchanger, in those cases where the gel-forming substance contains charged groups, or providing the particles with suitable adsorbent groups.

FIELD OF THE INVENTION 
The present invention is directed to methods for coating hydrophilic gels 
onto the surface of solid particles. More particularly, the present 
invention is directed to solid particles coated with a layer of 
hydrophilic gel, particularly for use in chromatography. Still more 
particularly, the present invention is directed to solid particles coated 
with hydrophilic gels used as supports for different adsorbent groups. 
BACKGROUND OF THE INVENTION 
There exists a substantial need for various particles which are suitable 
for use as supports for different adsorbent groups in the field of 
chemistry, in general in connection with chromatography, and in particular 
in connection with immuno-absorption therapy. Such supports which have a 
large active surface and which can be rapidly and effectively brought into 
equilibrium with a flowing medium and also offer a low flow resistance 
thereto are especially desirable in this context. A support which presents 
such properties may consist of a solid inner core which has only small 
pores, or preferably none at all, which is coated with a thin layer of a 
gel which acts as a bonding medium for such adsorbent groups. 
Columns which contain solid supports with immuno-adsorbents immobilized 
thereon are found, for example, in U.S. Pat. Nos. 4,180,383 and 4,215,688. 
In at least the above-mentioned '383 patent, there is described a support 
which consists of an inner solid core with one or more outer layers of 
active material. However, the description of how these outer layers is 
produced is quite inadequate in this patent. It is therefore an object of 
the present invention to provide a method for the manufacture of such 
supports and more particularly to solid cores coated with hydrophilic 
gels. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, these and other objects have now 
been realized by applicants' invention of a method for coating a 
hydrophilic gel onto the surface of a solid particle which includes 
providing hydrophilic solid particles, intimately admixing these 
hydrophilic solid particles with a precursor for the hydrophilic gel at an 
admixing temperature which is greater than the temperature at which the 
gel is formed in order to coat each of the hydrophilic solid particles 
with a layer of that precursor, separating the coated hydrophilic solid 
particles from each other, and lowering the admixing temperature to the 
predetermined gelling temperature for that gel in order to produce a layer 
of hydrophilic gel on the hydrophilic solid particles. In accordance with 
a preferred embodiment, the separating of the coated hydrophilic solid 
particles from each other comprises dispersing these particles in a 
hydrophobic solvent. Preferably, the lowering of the admixing temperature 
comprises cooling the hydrophobic solvent, and in a highly preferred 
embodiment, a dispersing agent is added to the hydrophobic solvent in 
order to assist in the dispersion step. 
In accordance with another embodiment of the method of the present 
invention, each of the hydrophilic solid particles, the precursor for the 
hydrophilic gel and the hydrophobic solvent has substantially the same 
density. 
In accordance with another embodiment of the method of the present 
invention, the hydrophilic solid particles and/or the precursor for the 
hydrophilic gel have a density which is greater than the density of the 
hydrophobic solvent, and the hydrophobic solvent has an elevated viscosity 
so as to prevent the coated hydrophilic solid particles from contacting 
each other. 
In accordance with another embodiment of the method of the present 
invention, intimate admixing of the hydrophilic solid particles with the 
precursor for the hydrophilic gel is carried out in the hydrophobic 
solvent. 
In accordance with another embodiment of the method of the present 
invention, intimate admixing of the hydrophilic solid particles with the 
precursor for the hydrophilic gel is carried out prior to dispersing the 
coated hydrophilic solid particles in the hydrophobic solvent. 
In accordance with another embodiment of the method of the present 
invention, separating of the coated hydrophilic solvent particles from 
each other includes a mechanical separation step. In a preferred 
embodiment, this step includes passing the coated hydrophilic solid 
particles through a screen having a plurality of openings of a size which 
permit only one of the coated hydrophilic solid particles to pass 
therethrough at a time. Preferably, the lowering of the admixing 
temperature in this embodiment comprises passing the coated hydrophilic 
solid particles into a cooling medium, which can be a cold gas, air, or a 
liquid solvent medium. In an embodiment in which the solvent medium is a 
hydrophobic solvent the method includes passing the coated hydrophilic 
solid particles through the hydrophobic solvent in a predetermined 
direction, and decreasing the temperature of the hydrophobic solvent along 
that predetermined direction so that the temperature of the hydrophobic 
solvent initially contacted by the coated hydrophilic solid particles is 
greater than the predetermined gelling temperature for the gel and the 
temperature of the hydrophobic solvent at a predetermined distance along 
the predetermined direction comprises the predetermined gelling 
temperature for the gel. 
In accordance with the method of the present invention, it is therefore 
possible to coat solid particles with a hydrophilic gel by utilizing a 
method which is based at least in part on the generally known principle 
that hydrophilic substances and hydrophilic particles are drawn towards 
one another, while at the same time there is a negative or rejection 
effect existing with respect to hydrophobic substances. Utilization is 
made of this principle in the method of the present invention by selecting 
as the solid particles hydrophilic particles, which are then mixed with a 
gel-forming substance at a temperature above the gelling temperature of 
that substance so that the gel-forming substance is made to cover each 
individual particle, and whereupon the particles are thus separated from 
each other and cooled to a temperature below the gelling temperature. As 
can be seen from the present disclosure, mixing as well as separations may 
take place in a number of different ways. 
As is thus set forth in a preferred embodiment of the present invention, 
the separation step takes place by dispersing the coated particles in a 
hydrophobic solvent which is subsequently cooled. This can be carried out, 
for example, with vigorous and effective stirring which initially 
separates particles which might have a tendency to cohere with each other, 
and then keeping these particles separate from each other. It has thus 
been found that the hydrophilic gel-forming substance is drawn toward the 
individual hydrophilic particles and deposits as a thin layer around these 
particles. Thus, on cooling the gel-forming substance naturally 
solidifies, whereafter the coated particles can be readily separated from 
the hydrophobic solvent. 
The dispersion step is greatly facilitated by the use of an additional 
dispersing agent, such as a soap with a hydrophilic end and a hydrophobic 
end. Because of the nature of these structures, these agents automatically 
settle in the boundary layer between the hydrophilic gel and the 
hydrophobic solvent, again assisting in the dispersion step. 
The dispersion step is further facilitated if the particles, the 
gel-forming substance and the hydrophobic solvent are selected so that 
they have substantially identical densities. It is also possible, however, 
to use particles and/or gel-forming substances which have a different 
density, preferably a higher density, than that of the hydrophobic 
solvent. In that case, however, it becomes necessary to select a 
hydrophobic solvent which has a high viscosity, so that the particles with 
the gel-forming substance coated thereon are prevented from rising or 
sinking too rapidly, and they are also prevented from making contact with 
one another, which can lead to clotting or adherence together. 
It is preferable to mix the gel-forming substance with the solid particles 
separately before dispersion in the hydrophobic solvent. However, this can 
also be accomplished in the solvent itself. 
As discussed above, the separation step can be carried out through the use 
of vigorous stirring of the coated particles in a solvent. As an 
alternative, however, the separation step can be effected by forcing the 
particles mixed with the gel-forming substance through a sieve or the like 
which has a mesh size such that only one coated particle at a time can 
pass through the respective holes therein. Such a sifting procedure also 
makes it clear that the coating on the individual particles becomes very 
even. Furthermore, in such a process from the sieve itself the separated 
particles can then be made to drop freely down into a cooling agent and/or 
solvent. The simplest and least expensive system that can be used in such 
a case is that of a cooling agent which consists of water which has a 
temperature below the gelling temperature. It has also been found, 
however, that a somewhat more uniform coating can be achieved if a 
hydrophobic solvent is instead utilized, in the same manner as set forth 
above. The surface temperature of this solvent can be made to exceed the 
gelling temperature, and at the same time the bottom temperature can be 
kept below that temperature. In this manner a uniform distribution of the 
gel-forming layer can be attained as the particles sink down to the region 
where the gel itself begins to solidify. 
As the particular material for the solid particles a hydrophilic glass is 
preferably used. However, other materials can also be used, such as 
polyvinyl chloride (PVC), polyamide, polycarbonate, etc. In such a case, 
however, the particles generally need to be pretreated so as to first 
achieve a hydrophilic surface. 
The particle size for the solid particles which is selected is preferably 
between about 0.001 and 5 mm, and most preferably between about 0.15 and 1 
mm. 
As a suitable gel material agarose may be utilized. For this material it 
has been found particularly appropriate to select a thickness for the gel 
layer of between about 0.0001 and 1.0 mm, and most preferably between 
about 0.001 and 0.004 mm. Alternatively, a gel can be used such as agar, 
cappa carrageenan, starch, and chitosan. 
Subsequent to the gelling step, the coated particles can be separated from 
the cooling agent and/or solvent which has been utilized. The gel may then 
be washed and possibly strengthened, such as through cross-linking. 
The gel-coated solid particles which are so obtained may either be used as 
such in various separating processes, for example as an ion exchanger, in 
the case where the gel-forming substance contains charged groups. As an 
alternative, the gel-coated particles may then be provided with suitable 
adsorbent groups, such as ion exchange groups, hydrophobic groups, or 
groups with biospecificity. Examples of such groups with biospecificity 
are enzyme inhibitors, enzymes, antibodies, and protein A from 
staphylococcus Aureus. 
The present invention also relates to coated particles, which are 
manufactured in accordance with the method set forth above. 
The present invention may be further understood with reference to the 
following working examples thereof.

EXAMPLE 1 
Agarose covering of glass beads in paraffin oil 
Materials utilized: 
150 ml of paraffin oil; 
1.2 grams of sorbitan sesquioleate; 
6.0 grams of glass beads, having a diameter of approximately 0.2 mm; and 
3.0 ml of 3% agarose, having a gelling temperature below about 30.degree. 
C. 
The procedure employed with these materials included heating of the 
paraffin oil with the above-identified sesquioleate to a temperature of 
between 40.degree. and 45.degree. C. 
The agarose was then melted in the water, which was heated to the boiling 
point, and was then mixed with the glass beads at 45.degree. C., whereupon 
the mixture was added to the oil, with stirring at about 750 rpm. 
Stirring was continued at a temperature of between 40.degree. and 
45.degree. C. for five minutes. Thereafter, the mixture was cooled for 
five minutes in ice water, with continued stirring, and this was continued 
for a further period of ten minutes, but now essentially at room 
temperature. 
Finally, the coated glass beads were separated from the paraffin oil and 
washed on a coarse filter (0.1 mm) with small portions of ether and water. 
The result obtained comprised glass beads coated with a gel layer having a 
thickness of between about 5 and 15.mu.. 
EXAMPLE 2 
Agarose covering of dextran (Sephadex.RTM. G 25 coarse) in n-butanol 
Materials utilized: 
150 ml of n-butanol; 
2 grams of Sephadex.RTM. G 25, coarse; and 
10 ml of 0.5 agarose, having a gelling temperature below about 30.degree. 
C. 
The procedure employed with these materials included heating of the 
n-butanol in a water bath to approximately 50.degree. C. The dextran was 
then measured and allowed to swell in about 20 ml of distilled water. 
Excess water was removed by suction through a glass filter funnel. The 
agarose was then heated in the water until it had melted, and it was then 
mixed with the dextran at approximately 40.degree. C. The mixture was then 
added to the n-butanol, and stirred for five minutes at 40.degree. C. 
The water bath was then removed, and the n-butanol was allowed to assume 
room temperature, while stirring was continued. Finally, the gel was 
washed on a glass filter funnel with 50% acetone and distilled water. 
The result obtained comprised dextran particles coated with a gel layer 
having a thickness of between about 15 and 30.mu.. 
EXAMPLE 3 
Coating of agarose on glass beads of 0.5 mm diameter. 
In this example, 0.5 mm glass beads were boiled under reflex with 5% 
HNO.sub.3 for a period of 10 minutes, and then washed with distilled 
water. They were then dried overnight at a temperature of 170.degree. C. 
3% agarose (Sea Plaque.RTM. from FMC Corp.) having a gelling temperature of 
below 30.degree. C. was then placed in a heating chamber, at a temperature 
of 41.degree. C. 
40 ml of the glass beads and 4 ml of the agarose were then mixed in a 200 
ml round-bottom flask with a ground-glass stopper. Everything had been 
preheated to 41.degree. C. Mixing was then carried out for three minutes 
by shaking the flask vigorously by hand. The flask was then maintained at 
41.degree. C. with continued shaking for a period of 10 minutes, whereupon 
the shaking was stopped, and the glass beads were poured out onto a 
strainer with 0.8 mm holes. The strainer had also been heated to 
41.degree. C. The straining step was then also thus carried out at this 
temperature. The glass beads with the adsorbed agarose were then forced 
through the strainer, with the help of a brush. They were then allowed to 
fall down directly into a 5 liter beaker, which was half filled with 
ice-cold water. 
The results obtained comprised glass beads covered by solidified agarose 
which collected at the bottom of the beaker. The gel layer had a thickness 
of between about 4 and 10.mu., and a relatively even coating was obtained. 
EXAMPLE 4 
Cross-linkage of agarose-coated glass beads 
Materials utilized: 
6.0 grams of agarose-coated glass beads (0.2 mm in diameter); 
6.0 ml of 1 molar NaOH; 
30 mg of NaBH.sub.4 ; and 
0.6 ml of epichlorhydrin. 
The procedure employed with these materials included washing the 
agarose-coated glass beads with distilled water, and then weighing out a 
quantity of 6.0 grams. This measured quantity was then suspended in a 
small amount of distilled water, and the NaOH with NaBH.sub.4 were added, 
followed by the epichlorhydrin. The mixture was then contained in a tube, 
which was rocked very gently overnight, with one turn approximately every 
twenty minutes. 
The gel was then washed with distilled water until all of the NaOH, and any 
epichlorhydrin which might remain therein, had been removed. 
Neutralization with acetic acid was then carried out. 
The result obtained comprised a substantially strengthened gel layer. 
EXAMPLE 5 
Coating of agarose on polyamide particles 
In this example, polyamide-6 particles, having a diameter of 0.5 mm, were 
washed with a detergent (Duponol R A.RTM.) in an alkaline medium. The 
polyamide was subsequently weakly hydrolyzed with 3.65 molar HCL at an 
elevated temperature. The polyamide was then washed with distilled water 
and dried. 900 mg of agarose (Sea Kem ME.RTM. from FMC Corp.) was then 
suspended in 30 ml of distilled water, and boiled under reflex for 10 
minutes. 
A mixture of 150 ml of toluene, 50 ml of carbon tetrachloride and 0.3 grams 
of sorbitan sesquiolate were then heated to 50.degree. C. 
40 ml polyamide granules were then added to the agarose. Alternatively, the 
polyamide granules could be poured into the organic solvent first, and the 
agarose then added to the suspension of polyamide in toluene-carbon 
tetrachloride. 
The agarose with polyamide was then poured into the organic solvent and 
stirred at a stirring rate of 1500 rpm. This stirring was then allowed to 
continue for five minutes, whereafter the suspension was cooled with 
continued stirring. The cooling was performed by placing the beaker in an 
ice bath. When the solution had cooled down to room temperature the excess 
solution was filtered off through a glass filter. 
The gel was finally washed on the glass filter with toluene, diethyl and 
water. 
Upon microscopic examination of the gel it was evident that each polyamide 
particle had acquired a thin layer of agarose having a thickness of 
between about 15 and 30.mu.. 
As can be seen from the above examples, where organic solvent and water are 
utilized it is preferable that they have a low mutual solubility, in which 
case they would tend to form two phases. 
In the case where the various substances utilized in this operation have 
the same density, and a water soluble gel is utilized, the density of this 
gel-forming substance in the liquid state can be adjusted through the 
addition of a salt. Therefore, the density may be adjusted, for example, 
with the aid of potassium iodide in the range of from about 1.0 grams per 
cm.sup.3 to 1.7 grams per cm.sup.3 for the gelling water phase. 
In order to adjust the density of the hydrophobic phase, mixtures, for 
example, of toluene and carbon tetrachloride can be used, in the range of 
from about 0.9 to 1.6 grams per cm.sup.3. If even greater densities are 
required, bromoform can be utilized instead of carbon tetrachloride. Most 
plastic materials, including polyamides, polycarbonates, polystyrenes, 
PVC, acrylates, etc. have a density within the range of between about 1 
gram per cm.sup.3 to 1.6 grams per cm.sup.3. Inorganic materials, such as 
glass or coal, for example, on the other hand, have considerably higher 
densities. 
Many plastic materials are hydrophobic by their very nature. However, most 
plastics can be rendered hydrophilic, at least on their surface, by means 
of modification of that surface so that the surface layer becomes 
hydrophilic. Such modification can be carried out in a number of different 
ways. Most often, however, the material is subjected to strong acids, 
oxidizing agents, radiation, or strong bases in an initial step. This 
achievement is sometimes sufficient to render the surface hydrophilic. In 
other cases, however, simultaneous with this aggressive treatment, some 
substance may be present which helps bond to the surface and render it 
hydrophilic. 
The gel layer can be alternatively strengthened by chemical cross-linkages 
which can be obtained by bonding together bifunctional reagents with the 
matrix. The majority of hydrophilic gels contain free hydroxyl groups. 
Cross-linkage between free hydroxyl groups can be established, for 
example, by means of: 
Epichlorhydrin: 
##STR1## 
2-3 dibromopropanol: Br--ChH.sub.2 --CHBr--CH.sub.2 OH Diphenyl sulphone: 
CH.sub.2 .dbd.CH--SO.sub.2 --CH.dbd.CH.sub.2 
An alternative method for providing such cross-linking would be to 
manufacture the gel in a procedure which itself contains a quantity of 
reactive groups. These reactive groups can be obtained, for example, by 
oxidizing the hydroxyl groups to aldehyde groups. A variation of this 
procedure is to utilize gel-forming substances which from the outset 
contain a number of active groups. An example of this is glyoxyl agarose 
which contains free aldehyde groups. 
After the glyoxyl agarose containing free aldehyde groups has been made to 
solidify, the gel can then simply be cross-linked by the addition of a 
diamino compound H.sub.2 N--CH.sub.2 --R--CH.sub.2 --NH.sub.2, wherein R 
is (CH.sub.2)n and where n is a number from 0 to 10, whereafter the Schiff 
bases formed are reduced, for example, with sodium borohydride NaBH.sub.4. 
Although the invention herein has been described with reference to 
particular embodiments, it is to be understood that these embodiments are 
merely illustrative of the principles and applications of the present 
invention. It is therefore to be understood that numerous modifications 
may be made to the illustrative embodiments and that other arrangements 
may be devised without departing from the spirit and scope of the present 
invention as defined by the appended claims.