Porous cross-linked polyvinyl alcohol particles, process for producing the same, and separating agent composed of the same

Porous cross-linked polyvinyl alcohol particles which is used for a chromatographic medium is prepared by: PA0 dispersing an aqueous solution of a mixture of polyvinyl alcohol and a salt in an organic solvent; PA0 allowing a gel to form spontaneously from the dispersion; and PA0 reacting the gel with a cross-linking agent.

BACKGROUND OF THE INVENTION 
The present invention relates to porous cross-linked polyvinyl alcohol 
particles that are produced by first dispersing in an organic solvent an 
aqueous solution of polyvinyl alcohol to make spheres of polyvinyl alcohol 
solution, holding the dispersion in such a state that a gel will form 
spontaneously, and then reacting the gel with a cross-linking agent. The 
present invention also relates to a process for producing such porous 
cross-linked polyvinyl alcohol particles, as well as a separating agent 
composed of such particles. More particularly, the present invention 
relates to porous cross-linked polyvinyl alcohol particles that are 
suitable for use as packing materials in chromatography, especially in 
aqueous-phase gel permeation chromatography. 
Water-insoluble crosslinked polyvinyl alcohol particles that are produced 
by reacting polyvinyl alcohol with a cross-linking agent are known but the 
so obtained polyvinyl alcohol is in a gel state. A method has therefore 
been proposed for producing a porous cross-linked polyvinyl alcohol by 
reacting polyvinyl alcohol with a cross-linking agent in water in which a 
salt is dissolved or finely suspended (Unexamined Published Japanese 
patent application No. 90804/81). 
SUMMARY OF THE INVENTION 
An object of the present invention is to provide macro-porous polyvinyl 
alcohol particles having a high exclusion limit of molecular weights that 
are produced by first dispersing in an organic solvent an aqueous solution 
of polyvinyl alcohol to make spheres of polyvinyl alcohol solution, 
holding the dispersion in such a state that a gel will form spontaneously, 
and then reacting the gel with a cross-linking agent. 
Another object of the present invention is to provide a process for 
producing macro-porous polyvinyl alcohol particles having a high exclusion 
limit of molecular weight that are produced by first dispersing in an 
organic solvent an aqueous solution of polyvinyl alcohol to make spheres 
of polyvinyl alcohol solution, holding the dispersion in such a state that 
a gel will form spontaneously, and then reacting the gel with a 
cross-linking agent. 
A further object of the present invention is to provide a packing material 
for use in chromatography that is composed of the macro-porous polyvinyl 
alcohol particles produced by the process described above.

DETAILED DESCRIPTION OF THE INVENTION 
The term "spontaneous gelling" as used herein means the phenomenon in which 
polyvinyl alcohol dissolved in water insolubilized in the absence of any 
cross-linking agent used as a third component. When left to stand, an 
aqueous solution of polyvinyl alcohol increases in viscosity with time 
until a gel forms spontaneously. This phenomenon is believed to occur due 
to the hydrogen bonding between individual vinyl alcohol molecules. The 
higher the concentration of the aqueous polyvinyl alcohol solution and the 
lower the temperature at which it is left to stand, the more rapidly start 
gelation. The exact reason why the particles of the present invention 
which are produced by making use of the spontaneous gelling of an aqueous 
polyvinyl alcohol solution contains macro pores is not clear but most 
probably polyvinyl alcohol which precipitates from the reaction medium and 
undergoes phase separation due to spontaneous gelling to assume a 
heterogeneous and porous structure. The so formed macro pores differ 
essentially from the pores in a network structure that is formed by simply 
reacting polyvinyl alcohol with a cross-linking agent. 
The present invention relates to porous cross-linked polyvinyl alcohol 
particles that are produced by first dispersing in an organic solvent an 
aqueous solution of a mixture of polyvinyl alcohol and a salt, holding the 
dispersion in such a state that a gel will form spontaneously, and then 
reacting the gel with a cross-linking agent. The present invention also 
relates to a process for producing such porous cross-linked polyvinyl 
alcohol particles, as well as a separating agent composed of such 
particles. 
The polyvinyl alcohol that is suitable for use as the starting material in 
the present invention has a degree of polymerization in the range of from 
several tens to several thousands, preferably from 200 to 2,000, and a 
saponification degree of at least 90 mol%, preferably at least 95 mol%. 
Such polyvinyl alcohol is dissolved in water for a concentration of from 2 
to 30%, preferably from 5 to 15% and placed under such a condition that a 
gel will form spontaneously. However, with the aqueous solution of 
polyvinyl alcohol alone, it will take so prolonged time for gelling 
spontaneously that in order to speed up the occurrence of spontaneous 
gelling, a salt is added to the aqueous polyvinyl alcohol solution. A 
suitable salt is sodium chloride, sodium sulfate or any other salt capable 
of coagulating and precipitating polyvinyl alcohol. 
The amount in which the salt is added will vary with the specific type of 
salt used but it is usually added in an amount within the range of from 3% 
to the concentration where polyvinyl alcohol will not precipitate before 
spontaneous gelling. If sodium chloride is used as the salt, the amount of 
its addition will vary with the degree of polymerization and 
saponification of polyvinyl alcohol and its concentration. If, for 
example, an aqueous solution of 10% polyvinyl alcohol having a 
polymerization degree of 500 and a saponification degree of at least 98 
mol% is used, sodium chloride is to be added in an amount of from 1 to 12% 
preferably from 3 to 10%. If the same polyvinyl alcohol is used as a 5% 
aqueous solution, sodium chloride is to be added in an amount of 1 to 15%, 
preferably 5 to 13%. Further acceleration of spontaneous gelling can be 
achieved by adding an alkali, such as sodium hydroxide, etc. to the 
aqueous polyvinyl alcohol solution to which the salt has been added. 
According to the present invention, the salt-added aqueous polyvinyl 
alcohol solution is subjected to spontaneous gelling after it has been 
dispersed in particles in an organic solvent. Useful organic solvents 
include aromatic hydrocarbons such as toluene, benzene, chlorobenzene, 
dichlorobenzene, etc. and halogenated derivatives thereof; and aliphatic 
and alicyclic hydrocarbons such as n-heptane, n-hexane, liquid paraffin, 
cyclohexane, dichloromethane, dichloroethane, etc. and halogenated 
derivatives thereof. These organic solvents are typically used dependently 
but may be used in admixture. 
The organic solvents are used in amounts which are at least twice, 
preferably from three to six times, the volume of the aqueous polyvinyl 
alcohol solution. The organic solvents preferably contain dispersion 
stabilizers such as oil-soluble cellulose compounds (e.g., ethyl 
cellulose, cellulose acetate butyrate, and ethyl hydroxyethyl cellulose) 
and other oil-soluble compounds as exemplified by gum arabic, sorbitan 
sesquioleate, sorbitan monooleate, and sorbitan monostearate. These 
dispersion stabilizers are typically used in amounts of from 0.05 to 10%, 
preferably from 0.1 to 5%, of the organic solvent. 
After being dispersed in particles in the organic solvent, the aqueous 
polyvinyl alcohol solution is subjected to spontaneous gelling at a 
temperature of from 0.degree. to 50.degree. C., preferably from 3.degree. 
to 30.degree. C., for a period of from 2 to 200 hours, preferably from 5 
to 100 hours. 
After a gel forms spontaneously under these conditions, it is subjected to 
reaction with a cross-linking agent. Useful cross-linking agents are those 
which are capable of forming cross-linked portions with 2 or more carbon 
atoms between vinyl alcohol molecules, which include dialdehyde compounds 
such as glyoxal, glutaraldehyde and terephthalaldehyde; diepoxy compounds 
such as 1,2,3,4-diepoxybutane; glycidyl ether compounds such as ethylene 
glycol diglycidyl ether and 1,4-butanediol diglycidyl ether; and 
epihalohydrin compounds such as epichlorohydrin and epibromohydrin. 
Advantageous examples are dialdehyde compounds which form cross-linking 
with acetal bonding between vinyl alcohol molecules. The cross-linking 
agent is typically used in an amount of at least 4 mol% of the total 
hydroxyl groups in the polyvinyl alcohol. 
Cross-linking reaction is carried out by adding the above-mentioned 
cross-linking agents to the gel that has formed spontaneously. 
Alternatively, the spherical particles resulting from the spontaneous 
gelling may be filtered to separate from organic solvent and the particle 
retained on the filter are subsequently washed with acetone or methanol 
and subjected to cross-linking reaction in water. 
The cross-linking reaction that follows the spontaneous gelling is carried 
out at a temperature of from 0.degree. to 100.degree. C., preferably from 
30.degree. to 80.degree. C., for a period of from 1 to 20 hours, 
preferably from 2 to 8 hours. The cross-linking reaction may be catalyzed 
to an acid such as hydrochloric acid or sulfuric acid or an alkali such as 
sodium hydroxide or sodium potassium, the choice of which will depend on 
the specific type of cross-linking agent used. If a dialdehyde compound is 
used as the cross-linking agent, an acid is used as the catalyst. The acid 
is preferably added in such an amount that its concentration in the 
aqueous solution will be at least 0.5N. If the cross-linking agent is an 
epihalohydrin compound, an alkali is used as the catalyst. The amount of 
alkali agent is preferably added so as to be at least 2N of concentration 
in the aqueous solution. 
The cross-linking reaction, including the case where it is effected in 
water is preferably carried out in the presence of a salt such as sodium 
sulfate, sodium chloride, etc. The amount in which the salt is added will 
vary with the specific type of salt used but it is usually added in an 
amount of at least 6%, or the concentration at which the aqueous polyvinyl 
alcohol solution undergoes coagulation and precipitation during 
spontaneous gelling. For instance, sodium chloride is added in an amount 
of at least 20%, and sodium sulfate of at least 6%. 
The cross-linked polyvinyl alcohol particles so prepared are separated from 
the reaction medium by filtration. Thereafter, the recovered particles are 
washed with water-miscible organic solvents such as acetone, methanol, 
etc. followed by thoroughly washing with water. For complete removal of 
the organic solvent, the separated cross-linked linked polyvinyl alcohol 
particles are preferably heated in water so that the organic solvent is 
eliminated by azeotropic distillation. 
The porous cross-linked polyvinyl alcohol particles produced by the method 
of the present invention are useful as packing materials in 
chromatography, especially in aqueous-phase gel-permeation chromatography. 
If a water-soluble polymer such as dextran, polyethylene glycol, etc. that 
has high comparability with polyvinyl alcohol is added to the starting 
aqueous solution of polyvinyl alcohol, macro-porous cross-linked polyvinyl 
alcohol particles having an even higher exclusion limit of molecular 
weights can be attained. If desired, packing materials for use in various 
modes of chromatography such as ion-exchange chromatography, hydrophobic 
chromatography, affinity chromatography, and the like, can be prepared by 
introducing a various functional groups or ligands to hydroxyl groups in 
the polyvinyl alcohol. 
When used as separating agents in the various fields of application 
mentioned above, the porous cross-linked polyvinyl alcohol particles of 
the present invention are usually adjusted to have an average particle 
size of from 20 to 1,000 .mu.m, preferably from 50 to 200 .mu.m, a water 
content of from 50 to 95%, preferably from 60 to 90%, and a degree of 
swelling of from 3 to 20 ml/g, preferably from 5 to 15 ml/g, of a dry 
resin. Among these physical characteristics, the average particle size can 
be readily imparted by proper selection of the conditions for dispersing 
polyvinyl alcohol in particles in an organic solvent, such as amount and 
type of each of the organic solvent and dispersion stabilizer used, and 
the desired water content and the degree of swelling can be readily 
attained by proper selection of the conditions for spontaneous gelling 
such as the molecular weight and amount of polyvinyl alcohol used and the 
gelling temperature. 
The separating agents composed of the porous cross-linked polyvinyl alcohol 
particles of the present invention are highly hydrophilic and hence have 
the advantage of showing less non-specific adsorption of proteins. Because 
of this feature, the agents are useful for the purpose of separating a 
variety of proteins such as globulin, ferritin, albumin, myoglobin, etc. 
In addition, these separating agents exhibit superior mechanical strength 
due to the cross-linked structure of the polyvinyl alcohol particles of 
which they are composed. 
The following examples are provided for the purpose of further illustrating 
the present invention but it should be understood that various 
modifications of these examples are possible without departing from the 
scope and spirit of the invention. In the examples, the water content and 
the degree of swelling, both being an index of porosity, were measured by 
the following methods. 
Measuring the water content and the degree of swelling 
A thoroughly water-washed resin is put into a 10-ml messcylinder and 
exactly 10 ml of the resin is metered with light tapping on the bottom of 
the cylinder. The resin is dehydrated with a centrifuge at 350 G for 5 
minutes and its weight (a.sub.1 g) is measured precisely. The resin is 
then vacuum-deried at 60.degree. C. and at 10 mm Hg for 24 hours and its 
weight (a.sub.2 g) measured again precisely. The water content and the 
degree of swelling of the resin can be calculated by the following 
formulae: 
##EQU1## 
EXAMPLE 1 
A three-necked flask (0.5 l) equipped with a stirrer and a reflux condenser 
was charged with 250 ml of dichloroethane in which 0.5 g of cellulose 
acetate butyrate (CAB 381-20 of Eastman Kodak Company) was dissolved. 
In a separate step, a three-necked flask (200 ml) equipped with a stirrer 
and a reflux condenser was charged with 5 g of sodium chloride and 50 ml 
of water. After a solution was formed by agitation, 5 g of a polyvinyl 
alcohol powder (Gosenol NLO5 of the Nippon Synthetic Chemical Industry 
Co., Ltd.) was added to the solution and the resulting dispersion was 
heated at 98.degree. C. for 1 hour to make a solution. After being cooled 
to room temperature, the solution was dispersed in the previously prepared 
organic solvent. The dispersion was left at room temperature for 97 hours 
with agitation until a gel formed spontaneously. To the gel were added 50 
ml of 20% aqueous NaCl solution and 12 ml of a cross-linking agent (25% 
glutaraldehyde), and 1 ml of 1N HCl was added to the stirred mixture. The 
resulting mixture was heated at 65.degree. C. for 2 hours with agitation. 
Following addition of 10 ml of 5N HCl, the mixture was further heated at 
65.degree. C. for 2 hours to complete the cross-linking reaction. 
The reaction mixture was cooled to room temperature, filtered, and washed 
first with methanol, then with water. The washed product was transferred 
into a three-necked flask (1 l) equipped with a stirrer. Following 
addition of 700 ml of water, the contents of the flask were heated at 
95.degree. C. for 1 hour to have the residual dichloroethane distilled off 
the cross-linked polyvinyl alcohol particles, which were then recovered 
from the flask and thoroughly washed with water. 
The finally attained particles of cross-linked polyvinyl alcohol were 
milky-white color and spherical; the water content and the degree of 
swelling of these particles are shown in Table 1. 
Measurement for pressure loss 
A glass column (i.d., 8 mm) having water jacket was packed with a 
separating agent that was composed of the particles prepared in Example 1. 
The particles had been classified to a size range of 63 to 149 .mu.m. The 
packed layer was 50 cm high. Distilled water was supplied from the top of 
the column at a constant flow rate, with the column being held at 
25.degree. C. by circulating water through the jacket. When the packed 
layer became stable and the reading of the pressure gage on top of the 
column levelled off, the latter was recorded. The same procedures were 
followed with the column left empty (i.e., unpacked with the separating 
agent). The reading of the pressure gage in this case was substracted from 
the value for the packed column so as to determine the pressure loss 
(.DELTA.p in kg/cm.sup.2 /50 cm-bed). 
The results of pressure loss (.DELTA.p) measurement for varying values of 
flow rate (LV: linear velocity; unit: m/hr) are shown in FIG. 2 by line 
(1). Within the measured range of flow rates (LV.ltoreq.10 m/hr), a linear 
relationship was established between .DELTA.p and LV with the separating 
agent of the present invention, and passage at a flow rate of as high as 
LV=10 m/hr was possible without causing deformation or disintegration of 
the carrier particles. 
The same experiment was conducted on a cross-linked agarose gel and the 
resulting .DELTA.p vs LV profile is shown in FIG. 1 by curve (2). When the 
cross-linked agarose gel was used as a medium, there occurred a marked 
increase in .DELTA.p when the flow rate exceeded LV=1 m/hr and much 
difficulty was encountered in passage through the column at LV.gtoreq.2.5 
m/hr. 
EXAMPLE 2 
Cross-linked polyvinyl alcohol particles were prepared by repeating the 
procedures of Example 1 except that the amount of polyvinyl alcohol used 
and the duration for which the aqueous solution of polyvinyl alcohol was 
left to cause spontaneous gelling were changed to the values shown in 
Table 1. The water content and the degree of swelling of the particles 
obtained are also shown in Table 1. 
EXAMPLE 3 
Two and a half gram of polyvinyl alcohol was dissolved in aqueous sodium 
chloride solution under the same conditions as employed in Example 1. 
Three milliliters of 5N NaOH was added to the solution and the mixture was 
stirred. After it was cooled to room temperature, the mixture was 
dispersed in the same organic solvent as used in Example 1 and the 
resulting dispersion was left at room temperature for 19 hours under 
agitation until a gel formed spontaneously. To the gel, 50 ml of 20% 
aqueous NaCl solution was added and the mixture was agitated for 5 hours. 
The mixture was filtered, washed first with methanol, then with water, and 
transferred into a three-necked flask (200 ml) equipped with a stirrer. 
Following addition of NaCl (20 g) and water (100 ml), 6 ml of an aqueous 
solution of 25% glutaraldehyde was charged into the flask under agitation. 
Thereafter, 1 ml of 1N HCl was added and the contents of the flask were 
heated at 65.degree. C. for 2 hours with agitation. Subsequently, 10 ml of 
5N HCl was added and the mixture was heated at 65.degree. C. for 2 hours 
until the cross-linking reaction was completed. After it was cooled to 
room temperature, the reaction mixture was filtered and thoroughly washed 
with water. 
The finally attained particles of cross-linked polyvinyl alcohol was 
milky-white and spherical; the water content and the degree of swelling of 
these particles are shown in Table 1. 
EXAMPLE 4 
Cross-linked polyvinyl alcohol particles were prepared by repeating the 
procedures of Example 3 except that the type and amount of polyvinyl 
alcohol used and the amount of 5N NaOH added were changed to those shown 
in Table 1. The water content and the degree of swelling of the particles 
obtained are also shown in Table 1. 
EXAMPLE 5 
A polyvinyl alcohol solution was prepared as in Example 3 except that the 
type and amount of polyvinyl alcohol used were changed to those shown in 
Table 1. This solution was dispersed in an organic solvent in which 1.13 g 
of ethyl cellulose (EC-T100 of Hercules Incorporated) was dissolved in 125 
ml of toluene. Subsequent procedures were the same as in Example 3 except 
that the amount of aqueous solution of 25% glutaraldehyde was changed to 
the value shown in Table 1. The water content and the degree of swelling 
of the cross-linked polyvinyl alcohol particles obtained are also shown in 
Table 1. 
COMATIVE EXAMPLE 1 
Polyvinyl alcohol was dissolved in aqueous sodium chloride solution under 
the same conditions as employed in Example 1. The solution was cooled to 
0.degree. C. and 6 ml of an aqueous solution of 25% glutaraldehyde and 1 
ml of 1N HCl were added to the solution, followed by rapid agitation. The 
mixture was dispersed at room temperature in the same organic solvent as 
used in Example 1, and the dispersion was agitated at room temperature for 
15 minutes. Immediately thereafter, cross-linking reaction and washing 
were conducted as in Example 1. 
The water content and the degree of swelling of the finally obtained 
polyvinyl alcohol particles are shown in Table 1. 
COMATIVE EXAMPLE 2 
Cross-linked polyvinyl alcohol particles were prepared as in Comparative 
Example 1 except that the amount of aqueous solution of 25% glutaraldehyde 
was changed to the value shown in Table 1. The water content and the 
degree of swelling of the particles are shown in Table 1. 
TABLE 1 
__________________________________________________________________________ 
Comparative 
Example Example 
Conditions and Results 
1 2 3 4 5 1 2 
__________________________________________________________________________ 
Polyvinyl alcohol* 
NL-05 
NL-05 
NL-05 
NL-18 
NH-18 
NL-05 
NL-05 
Amount of polyvinyl 
5 7.5 2.5 1.5 5 5 5 
alcohol added (g) 
Amount of 5 N NaOH 
-- -- 3 5 3 -- -- 
added (ml) 
Time permitted to stand 
97 25 19 19 19 -- -- 
for spontaneous gelling (hr) 
Amount of aqueous solution 
12 12 6 6 1 6 10 
of 25% glutaraldehyde added (ml) 
Water content (%) 
85.2 
80.6 
88.7 
90.0 
79.6 
87.0 
89.7 
Degree of swelling (ml/g) 
12.5 
8.8 15.0 
21.0 
11.8 
12.7 
15.0 
__________________________________________________________________________ 
*GOSENOL Series of The Japan Synthetic Chemical Industry Co., Ltd. 
APPLICATION EXAMPLE 1 
Four glass columns (10 mm.sup..phi. .times.500 mm.sup.H) were filled with 
the samples of the cross-linked polyvinyl alcohol particles that were 
prepared in Examples 1 and 3 and Comparative Examples 1 and 2. After 
thoroughly washing with water, calibration curves were obtained and the 
performance of the particles as packing materials for use in gel 
permeation chromatography were evaluated by the following procedures. 
Each of the columns was charged with 100 .mu.l of an aqueous solution (5 
w/v%) of dextran with a known molecular weight and 300 .mu.l of an aqueous 
solution (2 w/v%) of polyethylene glycol with a known molecular weight; 
thereafter, distilled water was passed through the column at a flow rate 
of 0.4 ml/min to elute the dextran and polyethylene glycol; the dextran 
and polyethylene glycol in the effluent were detected with a differential 
refractometer and the eluting position (the position of the peak maximum) 
was determined for each sample. The obtained calibration curves are shown 
in FIG. 2. 
APPLICATION EXAMPLE 2 
The cross-linked polyvinyl alcohol particles prepared in Example 5 were 
sieved to obtain particle sizes within the range of 63 to 105 .mu.m. The 
classified particles were filled into a glass column (i.d., 8.2 mm; 
height, 500 mm) and thoroughly equilibrated with a 50 mM phosphate buffer 
solution (pH, 7.40) containing 150 mM NaCl. A calibration curve for 
molecular weight distribution of proteins was obtained and the performance 
of the particles for use as a packing material in gel permeation 
chromatography was evaluated by the following procedures. 
Six proteins (thyroglobulin, ferritin, bovine serum .gamma.-globulin, 
bovine serum albumin, ovalbumin and myoglobin) having different molecular 
weights were dissolved in a 50 mM phosphate buffer (pH, 7.40) containing 
150 mM NaCl so as to form solutions each having a concentration of 0.1%. A 
portion (150 .mu.l) of each of these solutions was charged into the 
above-described column and the respective proteins were eluted by allowing 
a 50 mM phosphate buffer solution containing 150 mM NaCl to pass through 
the column at a flow rate of 0.2 ml/min. Protein detection was conducted 
with a UV monitor (280 nm). 
Based on the eluting positions of respective proteins, their Kav values 
were calculated by the following equation and plotted against the 
molecular weight to obtain a calibration curve: 
##EQU2## 
where Vt: the total volume of separating agent (ml); 
Ve: the elution volume (ml); 
Vo: the void volume (ml) measured on Blue Dextran with an approximate 
molecular weight of 2.times.10.sup.6. 
The constructed calibration curve is shown in FIG. 3. Its linearity shows 
that the separating agent composed of the cross-linked polyvinyl alcohol 
particles prepared in accordance with the present invention is suitable 
for use as a packing material in gel permeation chromatography. The 
molecular weights and isoelectric points of the eluted proteins are noted 
below: 
______________________________________ 
No. in Molecular weight 
Isoelectric 
FIG. 3 Protein (.times. 10.sup.3) 
point 
______________________________________ 
1 thyroglobulin 
667 4.5 
2 ferritin 440 4-5 
3 bovine serum 
.gamma.-globulin 
159 5.8-7.3 
4 bovine serum 
albumin 66.3 4.6-4.8 
5 ovalbumin 45 4.6 
6 myoglobin 17.8 8.1-8.2 
______________________________________ 
The cross-linked polyvinyl alcohol particles prepared in accordance with 
the present invention are macroporous and have a high exclusion limit of 
molecular weights. In spite of their high water content, the particles 
have high mechanical strength and are specifically useful as a packing 
material in aqueous-phase gel permeation chromatography of high-molecular 
weight substances such as proteins. 
While the invention has been described in detail and with reference to 
specific embodiment thereof, it will be apparent to one skilled in the art 
that various changes and modifications can be made therein without 
departing from the spirit and scope thereof.