High stability porous zirconium oxide spherules

The present invention provides a stable stationary phase for chromatography which comprises porous ZrO.sub.2 spherules coated with a cross-linked polymer coating wherein said coated spherules have a pore size from about 20-500 .ANG. and a particle diameter range of about 0.5-500 microns, and are stable in basic media to pHs of up to about 14.

BACKGROUND OF THE INVENTION 
A. Inorganic Oxide-Based Chromatographic Supports 
Currently known inorganic chromatography supports comprising particulate 
silica (SiO.sub.2) or alumina (Al.sub.2 O.sub.3) are stable over pH ranges 
of about 1-8 and 3-12, respectively. The solubilization of SiO.sub.2 and 
Al.sub.2 O.sub.3 at pHs outside of these ranges results in deterioration 
of these supports and contamination of the resultant chromatographed and 
separated products with silicon- or aluminum-containing species. Methods 
of improving the alkaline stability of particulate SiO.sub.2 by cladding 
the surface with a more base stable metal oxide such as zirconium oxide 
(ZrO.sub.2) have been disclosed in U.S. Pat. Nos. 4,648,975 and 4,600,646. 
This cladding is disclosed to increase the upper pH limit at which these 
supports, also referred to as packings, can be used to 11 and 9.5, 
respectively. However, these packings still lack adequate stability to 
allow them to be sterilized and cleaned in, for example, 0.1 N aqueous 
sodium hydroxide (NaOH, pH=13). 
Use of porous spherical ZrO.sub.2 particles on a thin layer chromatography 
plate has been disclosed in U.S. Pat. No. 4,138,336, a process for the 
preparation of porous ZrO.sub.2 microspheres is taught in U.S. Pat. No. 
4,010,242, and chromatographic use of these particles is taught in U.S. 
Pat. No. 3,782,075. The microspheres are prepared by a process in which 
colloidal metal oxide particles are mixed with a polymerizable organic 
material and coacervated into spherical particles by initiating 
polymerization of the organic material. This is a time consuming, batch 
process which requires the addition of organic material which is pyrolized 
and hence lost. 
U.S. Pat. No. 3,862,908 discloses microspheres of urania and other metal 
oxides; however, these particles are fired to near full density, have 
reduced surface areas and therefore, would not be attractive for 
chromatographic uses. 
U.S. Pat. No. 3,892,580 discloses a process for preparing porous bodies of 
ZrO.sub.2. This process requires the use of a binder to react with the 
oxide particles during preparation. This binder is subsequently decomposed 
by pyrolysis and therefore lost. The bodies produced by this process are 
not spherical, would pack unevenly, may cause increased column pressure, 
and are therefore not attractive for chromatographic uses. 
U.S. Pat. No. 4,389,385 teaches the preparation of porous gels and ceramic 
materials by dispersing solid particles of an inorganic substance produced 
by a vapor phase condensation method in a liquid to form a sol. The sol 
contains colloidal particles which are aggregates of the primary 
particles. The sol is dried to produce a porous gel of greater than 70% by 
volume porosity. 
B. Reverse Phase High Pressure Liquid Chromatography 
The majority of separations employing high pressure liquid chromatography 
(HPLC) are performed in the so-called reversed-phase mode. In this mode, 
the column-packing material is referred to as stationary phase. The most 
commonly used stationary phases feature a non-polar ligand (e.g., octane 
or octadecane) covalently-bound to a porous silica particle through a 
siloxane bond (Si--O--Si) to render the surface hydrophobic. Although 
these silica-based bonded phases are very useful for a wide range of 
applications in reversed-phase HPLC, their use is strictly limited to the 
pH range of between 2 and 8, due to the hydrolytic instability of both the 
silica support particle and the siloxane bond used to "anchor" the 
non-polar active group. Thus, the production of a pH-stable reversed-phase 
support material must involve the development of both a stable, controlled 
porosity, high surface area support material and a method for rendering 
the surface permanently hydrophobic. 
The eluent, also referred to as the mobile phase, used to elute the various 
components from the stationary phase is relatively polar, e.g., an aqueous 
buffer or a mixture of water and an organic solvent, e.g., aqueous 
alcohol. Its polarity can be changed by increasing the concentration of 
the less polar liquid in the mobile phase, a technique known in the art. 
Thus relative to the use of ZrO.sub.2 -clad silica, a more promising 
approach to developing a highly stable reversed-phase support, involves 
replacing the silica with an alternative inorganic material, such as 
alumina. Although it has been demonstrated that some improvement in pH 
stability is realized by replacing silica with alumina, the dissolution of 
alumina in aqueous solutions at extreme pHs (pH&lt;2 and pH&gt;12), even at room 
temperature, is well known. 
As mentioned previously, in addition to the use of a pH-stable support 
material, the production of a stable, reversed-phase also requires a 
process for modifying the support material which results in a stable, 
hydrophobic surface. Silylation is the most widely used method to 
derivatize silica particles to produce hydrophobic reversed-phase 
supports. The silylation of inorganic bodies other than silica (e.g., 
alumina, titania, zirconia, etc.) has been disclosed in U.S. Pat. No. 
3,956,179. However, it is uncertain whether or not covalent bonds to the 
support surface are actually formed. In any event, the hydrolytic 
instability of the siloxane bond is well known, and it is very likely that 
a Si--O-metal bond will be even more susceptible to aqueous hydrolysis 
because of the increased polarity of the bond. 
An alternate approach to silylation for modifying the surface polarity of 
inorganic bodies is the sorption of a polymer of desired 
polarity/functionality onto an SiO.sub.2 or Al.sub.2 O.sub.3 support 
surface followed by cross-linking of the individual polymer chains to one 
another to impart additional stability to the coating. Reversed-phase 
supports prepared in this fashion exhibit much improved pH stability 
compared to those prepared by silylation. It is important to recognize 
that the formation of a stable, cross-linked polymer layer on the surface 
of the support does not reduce the need for a stable, inorganic support, 
since it may not be possible to cover the entire inorganic surface. 
Although cross-linking of the polymer may keep it in place even as the 
underlying inorganic support dissolves, dissolution of the support will 
undoubtedly lead to a reduction in the mechanical stability of the 
support. In addition, problems related to increasing column back pressure 
are known to accompany the dissolution of the inorganic support and its 
subsequent appearance in the mobile phase and transport through the column 
and the accompanying instrumentation. 
Another problem related to the use of silica-based reversed phase supports 
is the difficulty encountered in the chromatography of amines and other 
basic solutes. This problem results from the presence of acidic silanol 
groups (SiOH) on the silica surface. Basic solutes undergo very strong 
interactions with these silanol groups which may involve cation exchange 
or hydrogen bonding, depending on the pH of the mobile phase. This problem 
is exaggerated by the requirement of working in the pH range 2&lt;pH&lt;8 on 
silica-based columns, since most amines will be protonated in this pH 
range and protonated amines can readily bond to the silica surface. One 
obvious approach to improving the chromatography of amines is at hydrogen 
ion concentrations significantly lower than the ionization constant of the 
amines so that they are unprotonated. For aliphatic amines, this normally 
involves working at a pH greater than 11. However, these pH ranges cannot 
be employed using silica-based columns. 
The presence of the aforementioned acidic silanol groups can also lead to 
irreversible adsorption of many classes of organic molecules onto 
silica-based reversed-phase supports, a problem which is well known to 
those versed in the art. This irreversible adsorption is particularly 
troublesome in the reversed-phase HPLC of proteins. Ultimately, this 
adsorption will result in a change in the properties of the support and 
can lead to its destruction. 
Reversed-phase HPLC is finding increased use in the area of bioprocessing 
because of HPLC's great ability to separate and purify materials. At the 
preparative scale, there are many unique considerations not applicable at 
the analytical scale. One such consideration is the need to sterilize a 
chromatography column prior to its use in the purification of a product 
intended for biological or human use. Another is the desirability of using 
larger particles, typically greater than 20 .mu. in average particle 
diameter. 
C. Ion-Exchange High Pressure Liquid Chromatography 
Ion-exchange chromatography (IEC) has become an important separation 
technique for the purification of biomolecules. Typical supports used in 
IEC are silica, alumina, agarose, polymethacrylate, and 
poly(styrene-divinylbenzene). See H. G. Barth et al., Anal. Chem., 60, 
387R (1988). Agarose is not suitable for high pressure work, while silica 
and alumina have limited pH stability. The matrices of silica and alumina 
must also be derivatized or coated to provide the support with ion 
exchange properties. This often introduces hydrophobic interactions into 
the retention mechanism. The hydrophobic nature of hydrocarbon-based 
supports such as poly(styrene-divinylbenzene) must be masked in order for 
them to be used as IEC supports. The hydrocarbon-based supports are also 
subject to shrinking and swelling whereas inorganic supports are not. 
Zirconium phosphate has been extensively studied as an inorganic ion 
exchanger for the nuclear industry because of its excellent exchange 
capacities, radiation and thermal stability. See A. Clearfield et al., Ion 
Exchange and Solvent Extraction, J. A. Marinsky et al., eds., Marcel 
Decker, New York, (1973) at Chapter 1. However, relatively little work has 
been done using zirconium phosphate as an HPLC support because of its poor 
mechanical properties and the lack of materials with the necessary porous 
structure. Furthermore, zirconium phosphate lacks the mechanical stability 
necessary for high performance chromatographic supports. 
OBJECTS OF THE INVENTION 
It is, therefore, an object of the present invention to produce 
chromatography column support material which resists dissolution and is 
therefore stable in aqueous media over a wide pH range. 
Furthermore, it is an object of the present invention to produce a reverse 
phase support material which possesses a hydrophobic surface and can 
therefore be used for reverse phase chromatographic processes, and which 
may be exposed to solutions having pHs of from about 1 to 14 without 
undergoing significant dissolution. 
Furthermore, it is an object of the present invention to produce a support 
material comprising a non-polar surface which can be used for separation 
by both ion-exchange and reversed-phase processes, wherein the relative 
contribution of these two processes may be controlled by simple adjustment 
of mobile phase conditions. 
Also, it is the object of the present invention to produce a support 
material which can be regenerated by freeing it from "irreversibly 
adsorbed" biological or organic residues by treatment at high pH. 
It is another object of the present invention to provide a support material 
for use in large scale separations, particularly of products generated by 
biotechnology, for example, by fermentation, wherein said support material 
can withstand traditional sterilization techniques involving high pH and 
heat treatment. 
SUMMARY OF THE INVENTION 
The present invention provides a support material adapted for use as the 
stationary phase in high-performance liquid chromatography (HPLC) which 
comprises porous spherules of zirconium oxide (ZrO.sub.2, "zirconia"). 
These spherules display a remarkable physical and chemical stability in 
aqueous media of a pH of about 1 to 14. Preferred ZrO.sub.2 spherules are 
about 0.5-500 .mu., most preferably about 20-500 .mu. in diameter, have 
surface area of about 1-200 m.sup.2 /g, most preferably about 40-150 
m.sup.2 /g; and have pore diameters of from about 20-500 .ANG., most 
preferably about 100-300 .ANG.. 
The ZrO.sub.2 spherules of the invention can be prepared by a process 
consisting essentially of (a) dispersing an aqueous sol containing 
colloidal ZrO.sub.2 particles in a medium which extracts the water from 
the dispersed sol to afford gelled ZrO.sub.2 spherules; (b) recovering the 
gelled spherules from the medium; and (c) heating the gelled spherules to 
yield solid porous ZrO.sub.2 spherules. This process yields porous 
particles of ZrO.sub.2 which are essentially spherical. When formed into a 
bed, the spherules provide improved mobile phase flow characteristics over 
those exhibited by irregularly-shaped, jagged-edged or angular particles. 
In a preferred embodiment of this process, the colloidal ZrO.sub.2 sol is 
centrifuged, the supernatant liquid decanted and the residue re-dispersed 
in an about equal volume of water. This procedure is preferably repeated a 
plurality of times (2-5.times.). The re-dispersed ZrO.sub.2 yields 
spherules having a larger pore diameter and an increased pore volume, when 
they are formed in accord with the present method. 
These particulate spherules can be formed into a bed, and employed as the 
stationary phase in separations performed via chromatography. Therefore, 
the spherules can be used as the stationary phase in conventional 
chromatography columns which have an axial flow path, with or without 
rigid walls. For example, the ZrO.sub.2 spherules can be packed into a 
column such as a HPLC column, where the packing functions as the 
stationary phase during HPLC separations which are accomplished by ion 
exchange and size exclusion processes. The spherules can also be used in 
columns which have a radial flow path or to form a fluidized bed, with 
single or multiple stage absorbers. The bed can also be formed of a mass 
of spherules which are contained in an immobilized enzyme reactor or other 
type of bioreactor. 
The majority of HPLC methodologies involve use of the reverse phase mode, 
wherein the column-packing material (stationary phase) is non-polar, and 
the mobile phase is polar. Therefore, the present invention also provides 
a support material comprising porous ZrO.sub.2 spherules coated with a 
hydrophobic polymeric layer. The coated spherules are prepared by 
adsorbing a polymerizable monomer or oligomer onto the surface of the 
spherules and subsequently cross-linking it, e.g., by reaction of the 
adsorbed material with a free radical initiator or by irradiation. The 
polymeric coating renders the ZrO.sub.2 particles hydrophobic without 
substantially altering any of their desirable physical and mechanical 
properties. Likewise, the ZrO.sub.2 spherules can be coated with a 
hydrophilic, cross-linked polymer to form an ion-exchange support 
material. 
The coated spherules can also be combined with a suitable binder and used 
to coat a glass or plastic substrate to form plates for thin-layer 
chromatography. 
Therefore, another preferred embodiment of the present invention is 
directed to a chromatographic support material comprising porous ZrO.sub.2 
spherules having a cross-linked polymeric coating thereon, wherein said 
coated spherules are hydrophobic, have a pore size from about 20-500 .ANG. 
and an average diameter of about 0.5-500 .mu.. 
As a result of the support material's remarkable stability over a wide pH 
range, it is useful for the chromatographic separation of compounds at 
their optimal pHs. For example, the coated material prepared in this 
fashion can be used for the separation of amines at a variety of pHs and 
mobile phase conditions such that the separation occurs either by a 
reversed-phase retention mode, a cation-exchange mode, or some combination 
of the two. For example, at high pH (pH=12), the amines are unprotonated 
so that separation occurs entirely by a reversed-phase mode. At low pH in 
the presence of a low ionic strength phosphate buffer and with an organic 
solvent-rich mobile phase, the separation occurs via a cation-exchange 
mode. By adjustment of mobile-phase conditions, selectivity can thus be 
significantly adjusted. 
The ZrO.sub.2 spherules of the present invention can also be employed to 
immobilize bioactive materials for a variety of purposes, including 
catalysis, analysis, affinity chromatography and synthetic 
transformations. Bioactive materials can be strongly sorbed onto the 
exterior and interior surfaces of both the uncoated and the polymer-coated 
ZrO.sub.2 spherules, while retaining a large percentage of their initial 
bioactivity. Useful biomaterials include proteins such as enzymes and 
antibodies. 
In addition, "irreversibly adsorbed" organic or biological residues can be 
removed from fouled columns packed with coated or uncoated spherules by 
flushing the column with a mobile phase at high pH or by injecting pulses 
of the high pH mobile phase. The term "irreversible adsorption" refers to 
the very strong tendency which surface-adsorbed proteins, biopolymers and 
the like exhibit to remain sorbed under normal elution conditions, until 
the mobile phase conditions are changed sufficiently to desorb them. 
Therefore, coated or uncoated ZrO.sub.2 spherules can be prepared which 
comprise a biologically active material such as an enzyme or a protein 
such as an immunoglobulin. Upon depletion of the biological activity, the 
enzyme or other protein can be removed from the spherules by exposing them 
to an aqueous medium at high pH, e.g., by washing them with a solution of 
an alkali metal hydroxide. The spherules, stripped of the biological 
materials, can then be treated with a buffer to return them to a 
physiological pH, and subsequently reloaded with the same, or a different 
bioactive material. 
The ZrO.sub.2 spherules may also be exposed in situ to traditional 
sterilization conditions, for example, by exposing the packing or the 
packed column to heat and high pH, without significant degradation. 
In a further preferred embodiment of the invention, the surface of the 
coated or uncoated ZrO.sub.2 spherules is deactivated or modified by 
treatment with an effective amount of an inorganic phosphate, such as 
phosphoric acid or an alkali metal phosphate salt, or with an 
organophosphonate, prior to or following application of the hydrophobic 
polymer coating. The treatment conditions can be varied so as to either 
reversibly adsorb phosphate, which may be phosphate ion, onto the 
ZrO.sub.2 surface, or to bind the phosphate onto and/or into the ZrO.sub.2 
surface, for example, as zirconium phosphate. These treatments render the 
particles effective to separate negatively charged molecules such as 
sulfonates, carboxylates, and other oxyanions. It is also believed that 
the organophosphonate becomes incorporated into the organic matrix of the 
polymeric coating.

DETAILED DESCRIPTION OF THE INVENTION 
I. Zirconium Oxide 
In the practice of this invention, a portion, or preferably a majority of 
the initial zirconium oxide (ZrO.sub.2) used to form the present spherules 
is in the sol state; a colloidal dispersion of ZrO.sub.2 particles in 
water. 
Colloidal dispersions of zirconium oxide suitable for use as the ZrO.sub.2 
source used to prepare the present spherules are manufactured by Nyacol 
Inc., Ashland, Mass. These dispersions contain about 20 wt-% ZrO.sub.2, 
wherein the ZrO.sub.2 particles vary in average diameter, e.g., from about 
10-250 nm. For example, Nyacol.TM. Zr 95/20 is an aqueous dispersion 
containing 20 wt-% ZrO.sub.2 of colloidal ZrO.sub.2 particles, the 
majority of which are about 95 nm in diameter. 
Non-colloidal ZrO.sub.2 sources may be included along with the colloidal 
ZrO.sub.2 particles used to prepare these spherules. Thus, chloride, 
nitrate, sulphate, acetate, formate or other inorganic or organic salts of 
zirconium such as the oxysalts and alkoxides may be included with the 
ZrO.sub.2 sol and the mixture used to make spherules. In preferred 
mixtures, colloidal ZrO.sub.2 particles make up a major part of the total 
ZrO.sub.2 present. 
Organic compounds may also be included with the ZrO.sub.2 precursors used 
to prepare the spherules. These organic materials are fugitives which are 
removed during the firing of the spherules. In particular, water-soluble 
polymers such as polyvinylpyrrolidone, polyethylene glycol, polyethylene 
oxide, and the like, or latex particles may be included in the liquid 
mixture used to prepare the spherules. These fugitives may be added to 
alter the rheology of the precursor solution or the pore structure of the 
resulting fired spherule. 
It is also within the scope of the present invention to include precursors 
for other metal oxides with the ZrO.sub.2 precursors so as to stabilize a 
particular crystalline phase of ZrO.sub.2 or to retard grain growth in the 
fired spherules. Thus, salts or sols of metals such as yttrium, magnesium, 
calcium, cerium, aluminum, and the like may be included in levels of from 
approximately 0-20 mole-%. ZrO.sub.2 spherules fired in air or in 
oxidizing atmospheres which do not contain other oxide additives display 
either monoclinic, tetragonal or pseudocubic crystal structures when 
cooled to room temperature. Higher firing temperatures and longer firing 
times favor the presence of the monoclinic phase. The inclusion of other 
metal oxides allows the preparation of spherules which possess either 
monoclinic, tetragonal, or cubic crystalline structures. 
II. Preparation of ZrO.sub.2 Spherules 
To prepare the spherical ZrO.sub.2 particles, or "spherules" of the present 
invention, an aqueous sol containing a colloidal dispersion of ZrO.sub.2 
particles is dispersed in a medium which can extract water from the 
dispersed sol in the form of droplets. Removal of all or a portion of the 
water results in gelled solid spherules which consist of aggregated sol 
particles. One medium which may be used is 2-ethyl-1-hexanol as disclosed 
in U.S. Pat. No. 4,138,336. A preferred medium for safety reasons and ease 
of processing is peanut oil, which is preferably used at a temperature of 
about 80-100.degree. C. The most preferred medium is a mixture of peanut 
oil and oleyl alcohol which are combined in a ratio of about 1:1, and used 
at a temperature of about 80-100.degree. C. Oleyl alcohol possesses a 
higher extraction capacity than peanut oil and mixtures of the two allow 
the extraction capacity of the medium to be controlled. Depending upon the 
ratio of sol to forming medium extraction times of from about 1-60 minutes 
can be used to fully gel the ZrO.sub.2 particles. The gelled spherules may 
be conveniently separated from the extracting medium, e.g., by filtration. 
The spherules of the present invention may also be prepared by spray drying 
a suitable zirconium precursor, as disclosed in U.S. Pat. No. 4,138,336. 
It is difficult to prepare spherical particles larger than about 45 .mu. 
in diameter when using the spray drying process, however. 
Once the ZrO.sub.2 particles are condensed into spherules by one of the 
above processes, thermal treatment at firing temperatures of from about 
100-1500.degree. C., preferably about 400-1100.degree. C., is performed. 
The resulting fired spherules may be from about 0.5-500 .mu. in diameter 
and can possess a surface area of 1-200 m.sup.2 /g and pore diameters of 
from about 20-500 .ANG.. These particles have high mechanical strength and 
exceptional stability to aqueous solutions of pHs of about 1-14. 
The particles may be packed into a HPLC column and used to perform HPLC 
chromatographic separations by ion exchange and size exclusion mechanisms. 
For a general discussion of HPLC techniques and apparatuses, see 
Remington's Pharmaceutical Sciences, A. Osol, ed., Mack Publishing Col, 
Easton, PA (16th ed. 1980), at pages 575-576, the disclosure of which is 
incorporated by reference herein. 
III. Polymer-Coated ZrO.sub.2 Spherules 
The majority of HPLC methodology employs the so-called "reverse phase" 
mode, i.e., the column-packing material (stationary phase) is non-polar 
and the eluent (mobile phase) is polar. Therefore, it is preferred to coat 
the surface of the ZrO.sub.2 spherules with a hydrophobic coating, which 
is also preferably stable to aqueous solutions having a pH of about 1-14. 
Hydrophilic polymer coatings can also be applied and cross-linked for 
modification of the ZrO.sub.2 spherules to form an ion exchange support or 
a steric exclusion support. These hydrophilic polymer coatings are formed 
from monomers or oligomers which comprise polar groups such as sulfonic 
acids, carboxylic acids, amino groups, hydroxyl groups, amido groups or 
quaternary ammonium groups. A preferred method to prepare such a coating 
comprises sorbing a polymerizable monomer or oligomer onto the surface of 
the spherules, and cross-linking the monomer or oligomer. See G. Shomberg, 
LC-GC, 6, 36 (1988). 
A. Polymerizable Monomers or Oligomers 
A wide variety of cross-linkable organic materials, which may be monomers, 
oligomers or polymers, can be employed to coat the porous ZrO.sub.2 
spherules. For example, such materials include polybutadiene, polystyrene, 
polyacrylates, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), 
polyorganosiloxanes, polyethylene, poly(t-butyl)styrene, polyisoprene, 
polyethyleneimine, polyaspartic acid and multifunctional silanes. 
A preferred material for the preparation of a reversed phase support 
material is an oligomer of polybutadiene. A preferred material for 
modification of the ZrO.sub.2 spherules to form a cation ion exchange 
support is polyaspartic acid. A preferred material for construction of a 
support suitable for aqueous steric exclusion chromatography is a tri- or 
di-alkoxy-,gamma-glycidoxy silane. 
B. Cross-linking Agents 
Any of the common free radical sources including organic peroxides such as 
dicumyl peroxide, benzoyl peroxide or diazo compounds such as 
2,2'-azobisisobutyronitrile (AIBN) may be employed as cross-linking agents 
in the practice of the present invention. Useful commercially available 
peroxyesters include the alkylesters of peroxycarboxylic acids, the 
alkylesters of monoperoxydicarboxylic acids, the dialkylesters of 
diperoxydicarboxylic acids, the alkylesters of monoperoxycarbonic acids 
and the alkylene diesters of peroxycarboxylic acids. These peroxyesters 
include t-butyl peroctoate, t-butyl perbenzoate, t-butyl 
peroxyneodecanoate and t-butyl peroxymaleic acid. These compounds are 
commercially available from Pennwalt Chemicals, Buffalo, N.Y. The amount 
of any free radical initiator required to catalyze the polymerization 
reaction will vary depending upon the molecular weight of the initiator 
and its thermal stability. Oligomers may also be polymerized by thermal 
treatment, by irradiation with UV light or gamma rays or by exposure to 
high energy electrons. 
C. Coating/Cross-linking Process 
Zirconium oxide may be modified in different ways to achieve materials with 
a light, intermediate or heavy carbon load. Preferably, the ZrO.sub.2 
spherules are first surface-hydrated and then dried in vacuo. Depending on 
the load desired, the dried ZrO.sub.2 spherules are added to 15-50 ml of a 
pentane solution containing from 5-250 mg of an oligomer, such as 
polybutadiene, per gram of ZrO.sub.2 spherules. The resultant slurry is 
placed in an ultrasonic bath and a vacuum applied in order to degas the 
particles and to insure that the oligomer solution has infiltrated 
substantially all of the pores. A free radical initiator, such as dicumyl 
peroxide, is then added at a level of 2-20% (w.w/) relative to the amount 
of polymer used. Solvent is then removed either by evaporation or by 
filtration, again depending on the desired carbon load. The treated 
ZrO.sub.2 spherules are then heated to about 60-70.degree. C. under vacuum 
(10-20 mm Hg) for 12 hrs to remove any remaining solvent. The 
cross-linking reaction is then carried out by heating the coated ZrO.sub.2 
spherules in a tube furnace at 175-200.degree. C. for 2-4 hours under a 
flow of nitrogen. 
The resultant coated spherules can then be packed into 5 cm.times.0.46 cm 
HPLC columns by dry packing or stirred upward slurry packing, depending on 
their particle size. 
Mixed-mode chromatography of amines can be performed in aqueous/organic 
mobile phases at various pHs containing different amounts of organic 
solvent, phosphate buffer and neutral salt for ionic strength adjustment. 
A column "fouled" by repeated injections of large amounts of material, to 
the point that a marked change in characteristics is observed, can be 
stripped of irreversibly adsorbed material. The original column 
performance can be restored by pulsing the column with 100 .mu.l injection 
of 1 M NaOH or by flushing the column for about 0.5-10 hrs with aqueous 
alkali metal hydroxide, i.e., with a 0.1 M NaOH solution. 
The stability of the polymer-coated ZrO.sub.2 spherules or uncoated 
ZrO.sub.2 spherules to sterilizing conditions can be demonstrated by 
heating a previously characterized column to 100.degree. C. while pumping 
a 1 M NaOH solution through it for 1-4 hrs. Recharacterization of the 
column demonstrates that no significant change in column properties or 
decreased retention of a non-polar substance has taken place. 
IV. Bioactive Materials 
A wide variety of bioactive materials can be bound to the uncoated or 
polymer-coated spherules by presently-available techniques so that their 
bioactivity is retained and prolonged, or "stabilized" with respect to the 
unbound bioactive material. For example, antibodies or enzymes can be 
bound to the uncoated spherules in high concentrations by agitating an 
aqueous mixture of degassed spherules and antibody in a buffer, e g., for 
about 0.1-5 hrs under ambient conditions. For a review of other 
noncovalent and covalent enzyme-binding methodologies, see R. A. Messing 
(U.S. Pat. No. 3,850,751), the disclosure of which is incorporated by 
reference herein. 
Enzymes capable of being bound and stabilized as described herein include a 
wide variety of enzymes which may be classified under six general groups: 
hydrolytic enzymes, redox enzymes, transferase enzymes, lyases, isomerases 
and ligases. The first group, hydrolase enzymes include proteolytic 
enzymes which hydrolyze proteins, e.g., papain, ficin, pepsin, trypsin, 
chymotrypsin, bromelin, keratinase, carbohydrases which hydrolyze 
carbohydrates, e.g., cellulase, glucuronidase, amylase, maltase, 
pectinase, chitinase; esterases which hydrolyze esters; e.g., lipase, 
cholinesterase, lecithinase, phosphatase; nucleases which hydrolyze 
nucleic acid, e.g., ribonuclease, deoxyribonuclease; and amidases which 
hydrolyze amines, e.g., arginase, asparaginase, glutaminase, and urease. 
The second group are redox enzymes that catalyze oxidation or reduction 
reactions. These include glucose oxidase, catalase, peroxidase, 
lipoxidase, and cytochromes. The third group are transferase enzymes that 
transfer groups from one molecule to another. Examples of these are 
glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, 
transmethylase, phosphopyruvic transphosphorylase and dehydrogenase. The 
fourth group are lyase enzymes that catalyze the cleavage of C--C, C--O, 
C--N and other bonds by elimination, leaving double bonds, or conversely, 
adding groups to double bonds. Examples of these are pyruvate 
decarboxylase, amino acid decarboxylases, aldolase, fumarate hydratases, 
aconitate hydratases and ammonia lyase. The fifth group are isomerase 
enzymes that catalyze the dehydrogenation and epimerization of amino acids 
and sugars. An example of an isomerase is phosphoglucomutase. The sixth 
group are ligase enzymes that catalyze the synthetic linking of two 
molecules, simultaneously with the breakdown of ATP. Examples of these are 
aminoacyl-tRNA synthetases and biotinyl-dependent carboxylases. 
Other proteins capable of being bound and stabilized as described herein 
include Con-A, Protein-A, acid glycoproteins, plasma immunoglobulins, 
monoclonal antibodies, bioactive polypeptides such as serum proteins and 
immunomodulators, e.g., lymphokines and the like. Other examples of 
proteins which are bound by the present spherules are provided in the 
working example hereinbelow. 
V. Phosphate Modification 
The surface of uncoated or polymer-coated ZrO.sub.2 spherules can be easily 
and dramatically modified in a chromatographically-beneficial way by 
treatment with aqueous inorganic phosphate solutions. The combination of 
polymer coating and phosphate treatment produces a mixed mode stationary 
phase exhibiting both cation-exchange and reversed-phase properties. This 
allows one to adjust the selectivity of the present support material with 
respect to a group of basic solutes by appropriate adjustment of mobile 
phase pH, ionic strength, and reversed-phase eluting strength (i.e., 
volume fraction of the adjuvant organic solvent). 
Useful aqueous inorganic phosphate solutions include about 0.01-1.0 M 
solutions of phosphoric acid (H.sub.3 PO.sub.4) or of alkali metal 
phosphate salts, e.g., orthophosphates, pyrophosphates, metaphosphates, 
tripolyphosphates and the like. 
Although phosphate ions can be adsorbed onto the ZrO.sub.2 surface by 
exposure to dilute (0.01-0.05 M) aqueous solutions of various inorganic 
phosphates for relatively short periods of time (e.g., 1-3 hours) at 
ambient temperatures (20.degree.-30.degree. C.), the phosphate is slowly 
removed from the surface under conditions of high pH. Therefore, it is 
preferred to treat the surface of the ZrO.sub.2 spherules with relatively 
concentrated (0.05-1.0 M) aqueous solutions of inorganic phosphates for 
longer periods of time (three or more hours) and/or at elevated 
temperatures (e.g., 90.degree.-110.degree. C.), so that the phosphate ions 
react with and become incorporated into an outer layer of the spherule, 
for example, as, e.g., zirconium phosphate. Preferably, the treated 
spherules will comprise about 0.5-15.0 wt-% phosphate. 
This phosphate incorporated into the structure as zirconium phosphate is 
less readily removed by hydrolysis reactions than the surface adsorbed 
phosphate ions are by exchange processes. Both of these types of phosphate 
groups will nevertheless be gradually lost upon exposure to conditions of 
high pH (&gt;10) in flowing mobile phases. This loss of phosphate can be 
reduced by keeping phosphate present in the mobile phase. Additionally, it 
is also possible to recondition a column which has lost phosphate by 
exposing it to phosphating conditions. 
It is important to note that the underlying ZrO.sub.2 spherules remain 
stable. It is therefore possible to perform an ion exchange separation 
with a column packed with phosphate-coated spherules, clean the column by 
flushing with strong base, and if necessary expose the column to 
phosphating conditions prior to the next separation operation. These 
cycles may be repeated indefinitely. 
For purposes of calculating wt-% phosphate in the treated spherules, it 
will be assumed that each phosphate ion incorporated into the ZrO.sub.2 
spherule possesses four oxygen atoms. The weight percentage of phosphate 
can thus be calculated from a knowledge of the weight percentage of 
phosphorus in the spherule by the following formula: 
##EQU1## 
The weight percentage of phosphorus in the spherules can be measured by 
inductively coupled plasma spectroscopy (ICP). The amount of phosphorous 
incorporated in the spherules for a given exposure condition is directly 
related to the specific surface area of the ZrO.sub.2 spherule. 
For example, treatment of the ZrO.sub.2 spherules having a specific surface 
area of about 117 m.sup.2 /g for about 1-4 hours at about 25.degree. C. 
with an excess of an aqueous solution of phosphoric acid with a 
concentration from about 0.01-1.0 molal yields particles containing about 
2.0-5.0 wt-% phosphate. Treatment of ZrO.sub.2 spherules for about 1-4 
hours at about 100.degree. C. with an excess of about 0.01-1.0 molal 
H.sub.3 PO.sub.4. yields spherules containing about 2.0-12.0 wt-% 
phosphate. 
Although not intending to be bound by any particular theory of action, it 
is believed that these more rigorous treatment conditions, including 
temperatures of about 90.degree.-110.degree. C., cause the phosphate ions 
to chemically react with and be incorporated into the ZrO.sub.2 spherules. 
Thus, the outer surfaces (both external and internal) of the spherules are 
at least partially converted to zirconium phosphate. The thickness of this 
zirconium phosphate layer is governed by the reaction conditions employed. 
Higher phosphate concentrations, higher temperatures and longer reaction 
times lead to the formation of thicker layers. These particles exhibit 
desirable cation exchange properties, while retaining the high mechanical 
and pH stability exhibited by untreated particles. As discussed above, 
while less stable at elevated pHs and temperatures than the underlying 
ZrO.sub.2 particles, the phosphate coatings possess useful stabilities and 
can be readily regenerated by exposure to solution sources of inorganic 
phosphate. 
VI. Modification with Organophosphorus Compounds 
For some applications, it is desirable to further deactivate or modify the 
surface of the uncoated or polymer-coated ZrO.sub.2 spherules. This can be 
accomplished by treating the uncoated ZrO.sub.2 spherules with an 
organophosphorus compound in a suitable solvent for the organophosphorus 
compound. Preferred organophosphorus compounds include the saturated or 
unsaturated organophosphonic acids and the water-soluble salts thereof, 
e.g. the alkali metal salts. Useful organophosphorus compounds include 
organophosphonates such as allylphosphonates, octyl phosphonates, diallyl 
phosphorates, allylphosphonic acid, phenyl phosphonic acid, naphthyl 
phosphonic acid, phenyl phosphinic acid, phenylphosphoric acid, and the 
salts thereof. 
Useful solvents for the organophosphorus compound include aqueous alcohol, 
e.g., a solution of water and a (C.sub.1 -C.sub.5) alkanol. The ZrO.sub.2 
spherules are preferably coated by agitating the spherules in a solution 
of the organophosphorus compound so that the weight ratio of the 
organophosphorus compound to spherules is about 0.25-1:1. The treated 
particles are then separated from the treating solution, and dried. The 
cross-linked polymeric coating then can be applied as disclosed 
hereinabove. 
The invention will be further described by reference to the following 
detailed examples. 
EXAMPLE 1 
Preparation of ZrO.sub.2 Spherules 
Peanut oil (3 liters) was placed in a 4 liter beaker and heated to 
90.degree. C. A mechanical agitator was inserted and the peanut oil was 
vigorously stirred. One hundred grams of Nyacol.TM. Zr 95/20, a colloidal 
ZrO.sub.2 manufactured by Nyacol, Inc. and containing 20 wt-% of 
ZrO.sub.2, primarily as about 95 nm particles, was sprayed into the peanut 
oil through an aerosol atomizer. After approximately 30 minutes, the batch 
was filtered through a No. 54 Whatman filter. Approximately 17 g of solids 
were recovered, which were predominately spherules having a diameter of 
&lt;30 .mu.. 
EXAMPLE 2 
Preparation of ZrO.sub.2 Spherules 
Peanut oil (600 g) and 600 g of oleyl alcohol were mixed and heated to 
about 90.degree. C. Under vigorous agitation, 100 g of Nyacol.TM. Zr 95/20 
was sprayed into the peanut oil/oleyl alcohol mixture as described in 
Example 1. After 30 minutes, the batch was filtered and the particles 
collected. The particles were predominately (ca. 70%) spherules having a 
diameter of &lt;50 .mu.. 
Spherules prepared as described in Examples 1 and 2 were thermally treated 
at a series of temperatures and the surface area, average pore diameter 
and pore volume were measured by nitrogen adsorption isotherm on a 
Quantasorb surface area analyzer. These results are summarized in Table I, 
below. 
TABLE I 
______________________________________ 
Firing Surface Average Pore 
Pore 
Temp (.degree.C.)* 
Area (m.sup.2 /g) 
Diameter (.ANG.) 
Volume (%) 
______________________________________ 
400 142 42 47 
500 92 71 50 
600 34 110 36 
800 17 205 34 
900 14 220 31 
______________________________________ 
*6 hrs 
The data summarized on Table I show that it is possible to increase the 
average pore diameter by increasing the firing temperature from 
400.degree. to 900.degree. C. The surface area and pore volume decrease 
with increasing firing temperature. Chromatographic activity of the 
ZrO.sub.2 spherules is determined by the parameters of the surface area, 
average pore diameter and pore volume. Accordingly, the appropriate firing 
temperature is selected. 
EXAMPLE 3 
Preparation of ZrO.sub.2 Spherules 
The procedure of Example 2 was used to prepare spherules using Nyacol.TM. 
Zr 50/20 (50 nm ZrO.sub.2 colloidal size) as the ZrO.sub.2 source. 
EXAMPLE 4 
Preparation of ZrO.sub.2 Spherules 
The procedure of Example 2 was used to prepare spherules using Nyacol.TM. 
Zr 150/20 (150 nm ZrO.sub.2 colloid size) as the ZrO.sub.2 source. 
Table II summarizes the surface area, average pore diameter and pore volume 
of spherules prepared as per Examples 2-4 and fired at 600.degree. C. for 
6 hrs. 
TABLE II 
______________________________________ 
ZrO.sub.2 
Colloid Pore 
ZrO.sub.2 
Size Surface Average Pore 
Volume 
Source* (nm) Area (m.sup.2 /g) 
Diameter (.ANG.) 
(%) 
______________________________________ 
Zr 50/20 
50 33 92 31 
Zr 95/20 
95 34 110 36 
Zr 150/20 
150 40 147 45 
______________________________________ 
*Nyacol .TM. series. 
The data summarized in Table II show that it is possible to control the 
average pore diameter of the fired spherules by appropriate selection of 
the colloid size of the ZrO.sub.2 source. Larger colloids produce fired 
spherules with larger pore diameters and pore volumes. 
EXAMPLE 5 
Preparation of ZrO.sub.2 Spherules 
Preparation A 
A 4500 g sample of Nyacol.TM. Zr 100/20, which contained 20 wt-% ZrO.sub.2 
primarily as about 100 nm particles, was concentrated on a rotary 
evaporator until its concentration was 35% ZrO.sub.2 by weight. This sol 
was then spray dried on a spray drier manufactured by Nyro Incorporated. 
About 900 g of dried solids were obtained. When examined under an optical 
microscope, the solids were observed to be spherules from about 0.5 to 30 
.mu. in diameter. The dried spherules were fired by heating them in a 
furnace to a temperature of 600.degree. C. over 6 hours, with additional 
heating applied at a constant temperature of 600.degree. C. for 6 more 
hours. Nitrogen adsorption measurements on the fired ZrO.sub.2 spherules 
indicated that their average surface area was 48.1 m.sup.2 /g and their 
average pore diameter was 116 .ANG.. The spherules were air classified, 
and the fraction ranging in size from approximately 5-10 .mu. was 
subsequently used for chromatography experiments. 
Preparation B 
To prepare spherules with larger diameter pores than those of Preparation 
A, the procedure described below was followed. 1200 g of Nyacol.TM. Zr 
100/20 colloidal ZrO.sub.2 were centrifuged on a laboratory centrifuge at 
5000 rpm for 55 minutes. The supernatant was discarded and the sediment 
was re-dispersed in distilled water. The centrifuged sol was placed on a 
rotary evaporator and concentrated until it contained 35% by weight of 
ZrO.sub.2. Following spray drying of the sol under conditions similar to 
those described in Preparation A, about 300 g of dried solids were 
obtained. When examined under an optical microscope, the solids were 
observed to be spherules ranging in size from about 1 to 30 .mu. in 
diameter. Many of the spherules (&gt;50%) were observed to possess cracks, 
especially those spherules of larger size. 
A portion of the fired spherules was then placed in a furnace and heated to 
a temperature of 1100.degree. C. over 9 hours, with additional heating at 
a constant temperature of 1100.degree. C. for 6 more hours. The surface 
area of the fired spherules was determined to be 16.1 m.sup.2 /g, and the 
average pore diameter was 408 .ANG., as measured by mercury porosimetry. 
This technique is a preferred method for measuring the size of pores 
greater than about 250 .ANG. in diameter. The fired spherules were 
unchanged in appearance from the dried spherules. They were nearly all 
intact, but many (&gt;50%) were cracked. 
A portion of the fired spherules was classified by size fraction as 
described in Preparation A. Examination of the classified fractions 
indicated that a portion of the spherules had fractured during the 
classification procedure. Many intact spherules remained, but a portion of 
each fraction contained irregularly shaped particles which appeared to 
have been produced by the fracturing of the spherules during the 
classification process. 
Preparation C 
To prevent the cracking observed in the spherules prepared according to 
Preparation B, spherules were also prepared as follows: 1250 g of 
Nyacol.TM. Zr 100/20 colloidal ZrO.sub.2 were placed in a laboratory 
centrifuge and spun at 5000 rpm for 55 minutes. The supernatant was 
discarded and the sediment was re-dispersed in distilled water. This 
centrifuged sol was placed on a rotary evaporator and concentrated until 
the concentration of ZrO.sub.2 in the sol was 32 wt%. To 513 g of this sol 
were added 34.6 g of a solution of zirconyl acetate containing 25% by 
weight ZrO.sub.2 equivalent (Harshaw, Inc., Cleveland, Ohio), and 61 g of 
a solution containing 50 wt% PVP K30, a polyvinylpyrrolidone polymer (GAF 
Corporation, Texas City, Tex.) were added to the concentrated sol. The 
resulting mixture was then agitated rapidly into a 50/50 mixture of peanut 
oil and oleyl alcohol which had been heated to a temperature of 90.degree. 
C. The resulting mixture contained gelled spherules of about 1 to 30 .mu. 
in diameter, which were observed under an optical microscope to be intact 
and crack-free. 
The spherules were then fired to a temperature of 900.degree. C. over 7 
hours and 20 minutes, with heating at a constant temperature of 
900.degree. C. for an additional 6 hours. After firing, the resulting 
spherules were from about 1 to 25 .mu. in diameter, and were observed 
under an optical microscope to be intact and crack-free. The surface area 
and average pore diameter of these microspheres were measured by mercury 
porosimetry to be 28 m.sup.2 /g and 415 .ANG., respectively. A portion of 
these spherules was classified into 5-10 .mu. and 10-20 .mu. fractions by 
sieving. Following classification, the classified spherules remained 
uncracked and intact. 
EXAMPLE 6 
Preparation of ZrO.sub.2 Spherules with Single Centrifugation 
Nyacol.TM. Zr 95/20. colloidal ZrO.sub.2 was placed in a laboratory 
centrifuge and sedimented. The supernatant was decanted and discarded. The 
sedimented ZrO.sub.2 was re-dispersed in an equal volume of distilled 
water. Spherules were prepared from this centrifuged sol following the 
procedures of Example 2. 
EXAMPLE 7 
Preparation of ZrO.sub.2 Spherules with Double Centrifugation 
The centrifugation procedure of Example 6 was performed and the 
re-dispersed sol was subsequently re-centrifuged to sediment, the 
supernatant decanted and the ZrO.sub.2 re-dispersed. Spherules were 
prepared from this doubly centrifuged sol following the procedure of 
Example 2. 
Example 8 
Preparation of ZrO.sub.2 Spherules with Triple Centrifugation 
The double centrifugation procedure used in Example 7 was performed and the 
re-dispersed sol was subsequently re-centrifuged to sediment, the 
supernatant decanted and the ZrO.sub.2 re-dispersed. Spherules were 
prepared from this triply centrifuged sol following the procedures of 
Example 2. 
Table III summarizes the surface area, pore diameter and pore volume of 
spherules prepared as per Examples 2, 6, 7 and 8, and heated to 
600.degree. C. for 6 hrs. 
TABLE III 
______________________________________ 
ZrO.sub.2 Surface Average Pore 
Pore 
Source* Area (m.sup.2 /g) 
Diameter (.ANG.) 
Volume (%) 
______________________________________ 
Zr 95/20 34 110 36 
Zr 95/20 cent. 
(1x) 50 162 55 
Zr 95/20 cent. 
(2x) 52 235 62 
Zr 95/20 cent. 
(3x) 46 250 62 
______________________________________ 
*Nyacol .TM. Zr series. 
Centrifugation, removal of the supernatant, and re-dispersion of the 
colloidal ZrO.sub.2 starting material results in increases in the average 
pore diameter, pore volume and surface area of fired spherules. This 
increase is believed to result from the removal of small (ca. 5-10 nm) 
colloidal ZrO.sub.2 particles which are known to be present in the 
Nyacol.TM. Zr series sols as a minor component. Many of these smaller 
ZrO.sub.2 particles remain suspended during centrifugation and are removed 
when the supernatant is discarded prior to redispersion of the larger 
sedimented ZrO.sub.2 particles. If present, these small ZrO.sub.2 
particles are believed to increase the packing density of the spherules by 
filling the interstices between larger ZrO.sub.2 particles and therefore 
decreasing the average pore diameter, pore volume and surface area of the 
fired spherules. 
It is also possible that sedimentation by centrifugation may result in 
agglomeration of the colloidal ZrO.sub.2 particles into aggregates which 
pack together in a more open structure (effectively behaving as larger 
particles) than unaggregated particles. 
Regardless of mechanism, the centrifugation treatments described in 
Examples 6-8 provide a method of preparing spherules with increased 
average pore diameter, pore volume and surface area relative to spherules 
prepared from untreated colloidal ZrO.sub.2 sols. 
The following example demonstrates the use of the unmodified ZrO.sub.2 
spherules prepared as described above in the chromatographic separation of 
proteins. 
EXAMPLE 9 
Protein Separation 
ZrO.sub.2 spherules prepared as described in Example 2 were heated to 
600.degree. C. for 6 hrs. The spherules were classified and the 5-10 .mu. 
fraction was used. The surface area of the spherules was 55 m.sup.2 /g and 
the average pore diameter was 146 .ANG.. The ZrO.sub.2 spherules were 
slurried in methanol and packed into a 30.times.0.46 cm stainless steel 
column at a constant pressure of 4,000 p.s.i. to rapidly compress the 
ZrO.sub.2 /methanol slurry to yield a uniform packing. After packing, the 
flow was maintained at 1 ml/min. at 1,000 p.s.i. The column was washed 
with 150 ml of 100 mM sodium phosphate, pH 7.0. All subsequent 
chromatography was performed in phosphate buffer. The column was stored in 
20% methanol/water. 
Protein solutions (2 mg/ml) were prepared in the same phosphate buffer: 20 
.mu.l samples of bacitracin, 1.4 KDa; ovalbumin, 45 KDa; and bovine serum 
albumin (67 KDa) samples were injected and eluted with 30 ml of buffer. 
All chromatographic runs were performed with a Spectra Physics Model 
8700XR HPLC system with their Model 757 variable wavelength detector set 
at 280 nm. Elution profiles, peak areas and elution volumes were recorded 
on a Model 4290 integrator/recorder. The proteins eluted as shown in Table 
IV, below, consistent with results expected in exclusion chromatography. 
TABLE IV 
______________________________________ 
Protein Elution Volume (ml) 
______________________________________ 
Bacitracin 3.35 
Ovalbumin 2.51 
Bovine Serum Albumin 
2.38 
______________________________________ 
EXAMPLE 10 
Protein Separation 
ZrO.sub.2 spherules prepared as described in Example 2 to 600.degree. C. 
for 6 hrs. Particles in the 30-50 .mu. diameter range having a surface 
area of 30 m.sup.2 /g and an average pore diameter of 100 .ANG. were used. 
The spherules were hand-packed into a 5 cm.times.0.21 cm column via a 
methanol slurry. After packing, the column was washed for 12 hrs at 0.2 
ml/min. with pH 7.0, 50 mM phosphate buffer. All subsequent chromatography 
was done on an IBM 9533 LC at a flow rate of 1 ml/min. and used a pH 
gradient of 50 mM H.sub.3 PO.sub.4 at pH 2.0 to 50 mM Na.sub.2 HPO.sub.4 
at pH 10 over a time of 10 min., followed by an additional 10 min. of 
isocratic operation at pH 10 with 50 mM Na.sub.2 HPO.sub.4. Bovine serum 
albumin (BSA) and myoglobin were separated by adsorption and ion exchange 
chromatography, yielding retention times of 13.3 min. (BSA) and 17.8 min. 
(myoglobin). 
EXAMPLE 11 
Anion Exchange Chromatography 
A stationary phase suitable for anion exchange chromatography was prepared 
by adsorption of polyethyleneimine [Polysciences, Inc., Warrington, 
Pa.]and subsequent cross-linking with 1,4-butanediol digylcidyl ether 
(95%, Aldrich Chemical Co., Milwaukee, Wis.); by the method of Regnier et 
al., J. Chromatog., 185, 375 (1979); 318, 157 (1985); 359, 121 (1986). 
The anion exchange capacity for adsorption of picric acid was determined to 
be 230 .mu.moles/g of modified ZrO.sub.2. This substrate was used to 
separate ovalbumin from BSA. The column was operated with a gradient of 10 
mM Tris buffer at pH 7.5 to 10 mM Tris at pH 7.5 with 0.5 M NaCl over 20 
min., followed by an additional 10 min. of isocratic operation at pH 7.5 
with 0.5 M NaCl. The flow rate was 1 ml/min. The retention times were 9.75 
(ovalbumin) and 22.8 min. (BSA). 
The following example demonstrates the use of the ZrO.sub.2 spherules to 
immobilize proteins. 
EXAMPLE 12 
Protein Immobilization 
ZrO.sub.2 spherules with a diameter of approximately 30 .mu. and a surface 
area of 50 m.sup.2 /g and an average pore diameter of 124 A were used. 
Mouse antihuman IgE antibody was purified and radioiodinated (I.sup.125) 
by the method of S. M. Burchiel et al., J. Immunol. Meth., 69, 33 (1984); 
K. L. Holmes et al., PNAS USA, 82, 7706 (1985), and diluted with 
unlabelled antibody to yield a specific radioactivity of 5,000 cpm/.mu.g. 
A portion of 250 .mu.l of antibody (250 .mu.g/ml in 5 mM Tris, pH 8.0) was 
added to tubes containing 10 mg of spherules. The mixture was briefly 
evacuated, then rocked at ambient temperature for the appropriate time, 
5-120 min., with three replicates for each time point. The tubes were 
centrifuged and rinsed twice with 1 ml of buffer. The spherules were 
transferred to a fresh tube along with 2 ml of buffer, the buffer removed 
and the radioactivity of each tube was determined in a Packard Model 5230 
gamma scintillation counter. The amount of bound protein in ng, converted 
from cpm, is shown in Table V. 
TABLE V 
______________________________________ 
Time (Min.) Antibody Bound/mg Spherules 
______________________________________ 
10 54 ng 
20 66 ng 
30 72 ng 
60 69 ng 
120 62 ng 
______________________________________ 
EXAMPLE 13 
Extent of Binding of Monoclonal Antibodies 
Using the same materials and techniques described in Example 12, the extent 
of binding of mouse antihuman IgE antibody in 2 hr incubations as a 
function of its concentration (1-250 .mu.g/ml) was determined. The 
averages of three replicates show a saturation (Table VI). 
Double-reciprocal analysis of these data extrapolate to 100 .mu.g antibody 
bound per g spherule at saturation. 
TABLE VI 
______________________________________ 
Conc. Protein (.mu.g/ml) 
Antibody Bound/mg Spherules 
______________________________________ 
1 1.5 ng 
5 7.5 ng 
10 14.0 ng 
50 38.0 ng 
250 62.0 ng 
______________________________________ 
EXAMPLE 14 
A. Trypsin Immobilization 
Solutions (2 mg/ml) of trypsin, a 24 KDa proteolytic enzyme and bovine 
serum albumin (BSA), a 67 KDa protein, were bound to 70 mg of the 
ZrO.sub.2 spherules (average pore diameter 100 A, surface area of 30 
m.sup.2 /g) in 5 mM tris, pH 8.0 by agitating the degassed spherules in 
1.0 ml of buffer for 17.5 hrs. Trypsin (15.3 mg) and 0.2 mg (BSA) bound 
per g of spherule, a proportion which might be expected from their 
relative sizes and the size of the pores. 
Trypsin was assayed using the thioesterase assay disclosed by P. L. Coleman 
et al., Meth. Enzymol., 80, 408 (1981). The bound spherules were suspended 
in 1 ml of buffer and a 5 .mu.l aliquot was added to a tube containing 1.0 
ml of substrate. After 2.5 min. of continuous shaking, a citrate-soybean 
trypsin inhibitor (STI) solution was added to quench the reaction. It was 
rapidly centrifuged and the supernatant removed for determination of the 
absorbance (A) at 412 nm. Assays were performed with the trypsin inhibitor 
in the substrate solution to determine whether it was able to inhibit the 
bound trypsin. The results of these assays are summarized on Table VII, 
below. 
TABLE VII 
______________________________________ 
Trypsin Activity 
(A at 412 nm) 
Sample -STI +STI 
______________________________________ 
Trypsin spherules 2.36 1.79 
BSA spherules 0.10 0.13 
Trypsin supernatant 
0.19 0.12 
______________________________________ 
The results shown in Table VII indicate that about 75% of the bound 
activity is unavailable to STI, even though STI is smaller than trypsin. 
In addition, only 4% of the activity is attributable to unbound trypsin, a 
surprisingly low value given the inefficient batch washing method which 
was used. 
Calculations based on these observations demonstrated several unexpected 
results. For example, 15 mg of trypsin/g ZrO.sub.2 corresponds to 51 mg/ml 
using 3.3 g/ml as the density of the spherules. This corresponds to a 
trypsin concentration of 2 mM in the column. A check on this may be made 
by estimating the expected absorbance at 412 nM for the assay. In these 
assays, the spherule-bound enzyme was 0.21 .mu.M, the kcat for the 
substrate is 75/sec [G. D. J. Green et al., Anal. Biochem., 93, 223 
(1979)] and the extinction coefficient is 14,100, yielding an estimated 
3.3 absorbance change, which compares favorably with the 2.4 observed. 
Since chromogen was present in amount sufficient to give only 2.8 .ANG. at 
412 nm, it is safe to assume that nearly all of the bound trypsin is 
active. Thus, an extraordinary amount of protein is bound and retains its 
enzymic activity. 
B. Chymotryosinogen-Chymopapain-BSA Immobilization 
The procedure of Example 8 (triple centrifugation) was employed to prepare 
ZrO.sub.2 spherules having 240 .ANG. pores and a surface area of 27 
m.sup.2 /g. Small columns were poured, each containing about 1.0 g of 
spherules, and were equilibrated with either 20 mM tris-chloride buffer 
(pH 8.0) or 50 mM sodium acetate buffer (pH 4.5). Chymotrypsinogen (24.5 
kDa) and chymopapain (32 kDa) were dissolved in the tris buffer and BSA 
was dissolved in the acetate buffer. Protein-containing solution was 
continuously added to the column until the 280 nm absorbance of the eluate 
equalled that of the starting solution. Unbound protein was rinsed from 
the column, and the amount of bound protein was calculated from the 
difference between that added and that recovered in the eluate. 
Chymotrypsinogen and chymopapain bound at 76.9 mg and 24.5 mg of protein/g 
of spherules at pH 8.0, respectively, and 64 mg of BSA bound per gram of 
spherules at pH 7.5. Converting these values into binding densities per ml 
of column volume yields 254, 81 and 211 mg.m/1 of protein, respectively. 
The fact that at acidic pH, albumin binds to a greater extent than does the 
smaller chymopapain, and almost to the extent as the event smaller 
chymotrypsinogen suggests that the latter enzymes would bind to even 
greater densities at lower pH, i.e., below their pIs. 
EXAMPLE 15 
Phosphoric Acid Treatment of ZrO.sub.2 Particles 
The following experiment was performed in order to determine the extent of 
reaction between phosphoric acid and ZrO.sub.2 particles as a function of 
concentration, temperature and time. The ZrO.sub.2 particles were prepared 
by a procedure similar to that described in Example 5, Preparation A, with 
the exception that they were fired to a temperature of 400.degree. C. 
rather than 600.degree. C. The resulting particles were about 100-400 .mu. 
in size, and had a surface area of about 117 m.sup.2 /g. Since no 
chromatographic evaluation of the particles prepared in this experiment 
was planned, these irregularly shaped particles were used, rather than 
spherules. Since the particles were prepared using the same raw materials 
and process as the spherules of Example 5A, the particles possess the same 
pore structure as the spherules of Example 5A. 
Sixteen samples of particles were treated according to the combinations of 
concentration, time, and temperature shown in Table VIII, below. For each 
treatment condition, a 5.0 g portion of the particles was placed in a 
filter flask and 200 g of phosphoric acid solution at the concentrations 
indicated in Table VIII was added to the flask. The flask was then 
evacuated to remove air from the pores and to allow the acid solutions to 
wet the pores. The vacuum was then released and the flask maintained at 
the temperature indicated in Table VI for the indicated time period. A 
total of 16 samples were treated; eight of the samples at 25.degree. C., 
and the other eight at 100.degree. C. Within each group of eight samples, 
half of the samples were treated for one hour at the temperature 
indicated; the other half were treated for four hours. 
Following the phosphoric acid treatment, all samples were collected on a 
filter paper and the particles washed thoroughly with distilled water. 
Following drying for 24 hours at 80.degree. C. and examination under an 
optical microscope, the particles were observed to be intact and 
crack-free. The surface area of each sample of particles was determined by 
nitrogen adsorption. The wt-% phosphate in each sample of treated 
particles was determined by dissolving a portion of each sample of the 
particles in hydrofluoric acid (HF) and analyzing the solutions by 
Inductively Coupled Plasma Spectroscopy (ICP). From the wt-% of phosphorus 
obtained from ICP, the wt-% of phosphate was calculated by assuming all of 
the phosphorus to be in the form of PO.sub.4 ions, as described above. The 
results of these analyses are shown in Table VIII, below. 
TABLE VIII 
______________________________________ 
Results of Phosphoric Acid Treatment 
Sample 
H.sub.3 PO.sub.4 Conc. 
Temp. Time S.A. PO.sub.4 
P/Zr 
# (moles/kg) (C.) (hrs) m.sup.2 /g 
wt % (molar) 
______________________________________ 
1 0.00 25 1 116 0.27 0.004 
2 0.01 25 1 115 2.44 0.032 
3 0.10 25 1 104 4.28 0.057 
4 1.00 25 1 110 4.77 0.063 
5 0.00 25 4 113 0.27 0.004 
6 0.01 25 4 116 2.63 0.034 
7 0.10 25 4 121 4.65 0.061 
8 1.00 25 4 117 4.77 0.065 
9 0.00 100 1 124 0.27 0.004 
10 0.01 100 1 124 2.48 0.032 
11 0.10 100 1 114 5.20 0.070 
12 1.00 100 1 109 7.47 0.101 
13 0.00 100 4 105 0.31 0.004 
14 0.01 100 4 110 3.46 0.045 
15 0.10 100 4 117 6.82 0.089 
16 1.00 100 4 111 10.25 0.149 
______________________________________ 
The results shown in Table VIII indicate that the surface area (S.A.) of 
the particles was not greatly or systematically affected by the treatments 
described. The results also indicate that the amount of phosphate 
incorporated in the particles increased for a given temperature and time 
with increasing H.sub.3 PO.sub.4 concentration. For a given H.sub.3 
PO.sub.4 concentration, the amount of phosphate (as calculated from 
phosphorous wt-%) incorporated into the particles increased with 
increasing treatment temperature and time. On samples treated at 
25.degree. C., however, the phosphorous content of the particles was only 
slightly greater after a treatment time of four hours than after a 
treatment time of one hour for a given H.sub.3 PO.sub.4 concentration. 
EXAMPLE 16 
Mechanical and Physical Characterization of Phosphoric Acid Treated 
Spherules 
The following experiment was performed in order to determine the effect of 
treating ZrO.sub.2 spherules with an inorganic phosphate after rigorous 
pretreatment of the spherules with acid and base solutions. 
ZrO.sub.2 spherules were prepared according to Example 5, Preparation B, 
above. Fifteen g of the spherules were slurried in 200 ml of 0.5 M HCl and 
thoroughly degassed by sonication and application of a vacuum. After one 
hour, during which the spherules were re-suspended three times by shaking, 
the HCl was decanted and the spherules rinsed five times with freshly 
boiled and cooled deionized water. This procedure was then repeated 
substituting 0.5 M NaOH for the 0.5 M HCl. The rinsed spherules were 
placed in a 250 ml round bottomed flask, to which 200 ml of an aqueous 
solution of 0.10 M phosphoric acid in 1.0 M KCl was added. The slurry was 
refluxed at about 100.degree. C. for four hours. The flask was swirled 
several times during this period to insure that the particles remained 
suspended. After four hours of refluxing, the supernatant was decanted and 
the particles thoroughly rinsed with freshly boiled and cooled deionized 
water. 
The surface areas of the untreated and H.sub.3 PO.sub.4 treated spherules 
were measured to be 12.4 and 14.7 m.sup.2 /g, respectively. 
In order to test their mechanical stability, the H.sub.3 PO.sub.4 treated 
spherules were packed into a 50.times.4.6 mm i.d. HPLC column from a 
slurry of isopropanol, using an upward slurry packing technique at 4500 
p.s.i. The spherules did not appear to have suffered any loss of 
mechanical stability due to the phosphating process, as evidenced by the 
fact that no fines developed during the packing procedure to clog the 
column frit. During almost daily use of the column over a three-month 
period, the column back pressure remained at about 200-300 p.s.i., 
providing further evidence of the stability of the phosphate treated 
spherules. 
All chromatographic studies in these experiments were performed using an 
IBM Instruments 9533 Ternary Chromatograph with an IBM Instruments 9522 UV 
absorbance detector. Data were acquired using an IBM Instruments Series 
9000 laboratory computer with the Chromatography Applications Program 
(CAP) software or a Hewlett-Packard 3393A Integrator. All proteins to be 
chromatographically separated were obtained from Sigma Chemical Co. (St. 
Louis, Missouri) and were used without further purification. 
The loading capacity of the HPLC column packed with H.sub.3 PO.sub.4 
treated spherules was investigated chromatographically using lysozyme at 
three different injection concentrations. The spherules were prepared 
according to Example 5, Preparation A and treated with H.sub.3 PO.sub.4 as 
described above (100.degree. C. for four hours), and had an average 
diameter of 5 .mu. and average pore size of 100 .ANG.. In each of the 
three chromatographic studies, lysozyme was eluted using a 30 minute 
linear gradient from 50 mM potassium phosphate at pH 7.00 to 0.5 M 
potassium phosphate at pH 7.00, with a flow rate of 1 ml/min. The area to 
height ratio was used as an indication of column performance. Table IX 
below lists the results of these studies. 
TABLE IX 
______________________________________ 
Column Loading Studies 
Amount of Lysozyme 
Area/Height Ratio 
______________________________________ 
3 .mu.g 14.53 
150 .mu.g 15.20 
1.5 mg 29.74 
______________________________________ 
As shown by the data of Table IX, the area/height ratio at 1.5 mg of 
lysozyme is nearly twice that at 150 .mu.g of lysozyme, indicating the 
column capacity lies between these two loading amounts. 
Additional studies were performed to determine whether lysozyme retained 
any significant enzymatic activity after being retained on the column 
packed with the H.sub.3 PO.sub.4 treated ZrO.sub.2 spherules. The lysozyme 
activity assay was performed according to procedures developed by the 
Technical Assistance Department of Sigma Chemical Company, St. Louis, Mo. 
Total protein was determined by the BCA total protein assay, according to 
Smith et al., Anal. Biochem., 150, 76-85 (1985). A reagent kit available 
from Pierce Chemical Company, Rockford, Ill., was utilized in the assay. 
Lysozyme was used for the calibration standards. The assay results showed 
that the specific enzymatic activity of the lysozyme was retained. 
EXAMPLE 17 
Protein Separation 
Inorganic phosphate-treated ZrO.sub.2 spherules were tested for their 
ability to separate large biomolecules such as proteins. Spherules having 
an average diameter of 20 .mu. and an average pore diameter of 408 .ANG. 
were prepared according to Example 5, Preparation B, and were treated with 
H.sub.3 PO.sub.4 following the procedure and conditions described in 
Example 16. The treated spherules were packed into a 50.times.4.6 mm HPLC 
column. A test mixture of cytochrome-c, ribonuclease A, and lysozyme was 
chromatographically separated using a 30 minute linear gradient from 0.05 
M potassium phosphate at pH 7.00 to 0.5 M potassium phosphate at pH 7.00. 
The results are depicted in FIG. 1. Bovine serum albumin (BSA) was 
unretained at the initial pH and phosphate concentration. The chromatogram 
of FIG. 1 indicates that the inorganic phosphate-treated ZrO.sub.2 
spherules are a useful support for protein separation. 
EXAMPLE 18 
Effect on Selectivity by Addition of Phosphate to Mobile Phase 
Bovine Serum Albumin (BSA) is irreversibly retained in a 10 mM 
2-[N-Morpholino]ethanesulfonic acid (MES) pH 6.00 buffered mobile phase, 
yet it is unretained upon addition of 1 mM phosphate to the mobile phase. 
In this experiment, the separation of a protein mixture of myoglobin, 
lysozyme, ribonuclease A and cytochrome C was attempted using a 
50.times.4.6 mm chromatographic column packed with ZrO.sub.2 spherules 
having an average pore diameter of 408 .ANG., prepared according to 
Example 5, Preparation B. A 10 mM MES pH 6.00 buffered mobile phase and a 
15 minute linear KCl elution gradient (0.00 M KCl to 0.80 M KCl) were 
utilized. No phosphate was present in the mobile phase. These conditions 
resulted in an almost total loss of selectivity. 
The experiment was then repeated by adding 1.0 mM potassium phosphate to 
the mobile phase. The 50.times.4.6 mm column was packed with 20 .mu. 
spherule diameter, 408 .ANG. average pore diameter ZrO.sub.2 spherules, 
prepared according to Example 5, Preparation B, and treated with H.sub.3 
PO.sub.4 according to the procedure and conditions of Example 16. The 
results depicted in FIG. 2 indicate that selectivity was mostly restored 
by the addition of the phosphate to the mobile phase, and demonstrate that 
phosphate ions played a critical role in the elution process of the 
proteins. It is possible that phosphate adsorbs onto the stationary phase 
from the mobile phase, modifying the phase and its retention properties. 
EXAMPLE 19 
Comparison of Inorganic Phosphate Treated ZrO.sub.2 to Hydroxyapatite 
Supports 
High performance hydroxyapatite (Ca.sub.10 (PO.sub.4).sub.6 (OH).sub.2) 
supports for HPLC separations of proteins have become commercially 
available. They are limited in several respects, however. The pressure 
limit of columns packed with the hydroxyapatite supports is at most 3000 
psi (Regis Chemical Co., Morton Grove, Ill.), and the pH range operating 
range of the columns is generally from 5.5 to 10.5. Furthermore, all 
manufacturers note the need for guard precolumns with these supports to 
prevent degradation of the main column. The guard columns must also be 
periodically replaced at considerable cost. 
In the present studies, columns packed with inorganic phosphate treated 
ZrO.sub.2 according to Example 5, Preparation B, and Example 16 (procedure 
of second paragraph: HCL wash, NaOH wash, and phosphoric acid treatment) 
have been operated at pressures of up to 6000 p.s.i. Furthermore, the pH 
operating range limits of these columns are considerably wider than those 
of the hydroxyapatite supports. Additionally, the phosphate treated 
ZrO.sub.2 column was used extensively without a guard column. When fouled, 
a sodium hydroxide rinse could be used to clean the column. 
EXAMPLE 20 
Separation of Monoclonal Antibodies 
The purpose of this experiment was to determine whether the inorganic 
phosphate treated ZrO.sub.2 spherules could be used to separate IgG 
monoclonal antibodies from a broth containing large amounts of albumin and 
some transferrin. ZrO.sub.2 spherules prepared according to Example 5, 
Preparation B and treated with H.sub.3 PO.sub.4 according to the 
procedures and conditions of Example 16, were used as the chromatographic 
10 support for a 50.times.4.6 mm column operated at a 30 minute linear 
gradient from 0.05 M potassium phosphate at pH 6.0 to 0.5 potassium 
phosphate at pH 6.0. The mobile phase was a broth of the IgG monoclonal 
antibodies, bovine serum albumin (BSA) and transferrin. As shown in FIG. 
3, antibodies were retained by the column. In this manner, it is believed 
that the inorganic phosphate treated ZrO.sub.2 can be used in an initial 
cleanup step to retain antibodies, while the albumin and transferrin are 
passed through the column unretained. 
EXAMPLE 21 
Polymer Adsorption/Cross-linking 
Preparation A - Heavily loaded ZrO.sub.2 
A solution of 0.55 g of polybutadiene (Aldrich Chiemical Co., Milwaukee, 
Wis., m.w. 4500, Cat. No. 20-050-6) in 50 ml of pentane was added to 3.5 g 
of ZrO.sub.2 spherules prepared as described in Example 2 (fired at 
600.degree. C. for 6 hrs; particle size =20-45 microns) which had been 
boiled in CO.sub.2 -free water to fully hydrate the surface and then dried 
at 25.degree. C. The slurry was placed in an ultrasonic bath and a water 
aspirator vacuum applied. Dicumyl peroxide (DCP) (0.01 g) was then added 
and the slurry was again placed in an ultrasonic bath and a vacuum 
applied. The pentane was removed in vacuo and the material dried at 
70.degree. C. under vacuum. The material was then heated in a tube furnace 
to 200.degree. C. for 2 hrs and then washed successively with pentane, 
toluene, methylene chloride, tetrahydrofuran, methanol and 0.1 M sodium 
hydroxide. Elemental analysis of the coated spherules showed a carbon load 
of 7.7%. A duplicate sample was prepared in an identical fashion and had a 
carbon load of 7.5%. Because of the extremely heavy load of polybutadiene, 
the specific surface area of the porous spherules, as determined by a BET 
measurement, decreased from 50.4 to 4 m.sup.2 /gm. 
Preparation B--Lightly loaded ZrO.sub.2 
35 ml of a solution of 0.09 g of polybutadiene in pentane was added to 3.5 
g of ZrO.sub.2 spherules and the resultant slurry was placed in an 
ultrasonic bath and a water aspirator vacuum applied. Pentane (10 ml) 
containing 0.002 g of DCP was then added and the slurry was again placed 
in an ultrasonic bath and a vacuum applied. The slurry was then shaken for 
one hr and the supernatant removed by filtration. The material was then 
washed as described in Preparation A. Elemental analysis of the coated 
spherules showed 0.84% carbon, while the BET results showed a specific 
surface area of 38.7 m.sup.2 /gm. The decrease in specific surface area 
from 50.4 to 38.7 m.sup.2 /gm is similar to the reduction in surface area 
which occurs upon silylation of typical inorganic supports. 
Preparation C--Intermediate load 
A solution of 0.27 g of PBD in 50 ml pentane was added to 3.0 g of 
ZrO.sub.2 spherules (mean particle diameter 3.5 microns). The slurry was 
placed in an ultrasonic bath and a vacuum applied. 5.2 mg of DCP in 10 ml 
of pentane were then added. The methodology of Preparation A was then 
followed. Elemental analysis showed 2.7% carbon. 
It is clear from the results of carbon analysis that carbon had been 
deposited on the surface of the ZrO.sub.2 spherules. FIG. 4 further 
demonstrates the reversed-phase nature of the polymer-modified ZrO.sub.2 
spherules as exhibited by a 5 cm.times.0.46 cm column packed using 
Preparation C. The linearity of the log k' (capacity factor) vs. carbon 
number plot for the members of a homologous series of alkylphenones is 
clearly indicative of a reversed-phase retention mechanism. 
EXAMPLE 22 
Alteration of Selectivity 
A mixed cation-exchange/reversed phase support was prepared by treating a 
material prepared as described in Example 21, Preparation C with a 100 mM 
aqueous H.sub.3 PO.sub.4 solution at pH 3 for about one hour at 25.degree. 
C. The retention data given in Table X show distinct changes in 
selectivity as a function of pH, volume fraction organic solvent and 
mobile phase ionic strength. 
TABLE X 
______________________________________ 
Selectivity Factor* 
Solute A B C D E 
______________________________________ 
Butyl Benzene 
5.02 5.18 4.86 6.86 6.94 
Lidocaine 0.28 0.074 0.1 0.44 0.32 
Quinine 2.9 0.39 0.22 5.17 0.6 
Nortriptyline 
68.0 2.61 2.07 99.2 3.38 
Amitriptyline 
15.6 3.29 3.45 33.7 5.69 
______________________________________ 
*Selectivity Factor = [k' (solute)]/[k' 
Conditions and capacity factors of toluene are given below: 
A = 60% MeOH/40% 10 mM PO4 at pH 7; k' (toluene) = 0.57 
B = 60% meOH/40% 10 mM PO4 at pH 7 with 0.5M NaCl k' (toluene) = 0.54 
C = 60% MeOH/40% 10 mM PO4 at pH 12; k' (toluene) = 0.58 
D = 50% MeOH/50% 10 mM PO4 at pH 7; k' (toluene) = 1.2 
E = 50% MeOH/50% 10 mM PO4 at pH 12; k' (toluene) = 1.2 
Separations at high pH (above the pKa of the amines) are dominated by a 
reversed-phase retention mechanism as are separations at lower pH in high 
ionic strength mobile phase. Conversely, separations at low pH in a low 
ionic strength environment are controlled primarily by cation-exchange 
processes. In addition to the ability to alter selectivity in several 
ways, the subject material also exhibits dramatic improvement in terms of 
the peak symmetry of amine solutes relative to silica. 
EXAMPLE 23 
pH Stability Testing 
The pH stability of the material of Example 21, Preparation A, was 
demonstrated in chromatographic experiments at high pH and elevated 
temperature by monitoring the retention of test solutes and by measurement 
of the amount of carbon on the support before and after prolonged exposure 
to high pH. These experiments were carried out under the following 
chromatographic conditions: Mobile Phase A: 0.1 M CO.sub.2 -free NaOH; 
Mobile Phase B: Methanol; Flow Rate: 1 ml/min.; Oven Temp: 50.degree. C. 
The retention of two test solutes in a mobile phase of 50% B/50% A as a 
function of the number of column volumes of mobile phase flushed through 
the column is shown in FIG. 5. Note that the initial decrease in retention 
reflects the equilibration of the column to the elevated temperature and 
not a loss in bonded phase. The evaluation was repeated on the lightly 
loaded material (Preparation B); the retention data on this material is 
shown in FIG. 6. Once again, there is an initial decrease in retention 
associated with column equilibration. There is also a slight decrease in 
retention at approximately 15 hours which accompanied a change in the lot 
of mobile phase; this change does not reflect a significant drop in carbon 
load. 
It is believed that the above evaluations represent the most challenging 
test of pH stability which has been reported for any reversed-phase 
material and it is also believed that the data clearly show that the 
spherules of Example 22, Preparations A and B, are essentially stable 
under these conditions. 
EXAMPLE 24 
Allylphosphonate Treatment 
ZrO.sub.2 spherules prepared by the procedure of Example 2 (3.4 g, surface 
area: 60 m.sup.2 /g; pore diameter: 95.ANG.) were treated with a solution 
of 1.6 g of allylphosphonic acid in 50 ml of 95/5 (v/v) methanol/water. 
After "ultrasonicating" under vacuum and shaking for one hr, the 
supernatant was removed by filtration and the phosphonate-treated 
ZrO.sub.2 was dried at 70.degree. C. for 12 hrs. The material was then 
modified with PBD according to Example 21, Preparation C. In this manner, 
the residual ZrO.sub.2 surface was deactivated as is clearly shown by the 
data in Table XI, below. Note that carboxylic acids are not eluted on the 
non-phosphonated ZrO.sub.2 material but are eluted on the phosphonated 
material. 
TABLE XI 
______________________________________ 
Solute k' (untreated) 
k' (treated) 
______________________________________ 
Toluene 0.46 0.49 
Benzoic Acid not eluted 6.1 
______________________________________ 
EXAMPLE 25 
Regeneration of Column Retentron Characteristics 
Several 100 .mu.l injections of cytochrome C were made on a column packed 
with material prepared as described in Example 21, Preparation C. The 
retention of cytochrome C on this material decreased due to "irreversible" 
adsorption of protein upon each injection. 
The column was then "pulsed" with 5, 100 .mu.l injections of 1 M NaOH in 
order to strip the "irreversibly adsorbed" cytochrome C. The effect of the 
pulses is to strip the column of adsorbed protein such that the original 
retention characteristics can be regenerated. 
EXAMPLE 26 
Exposure to Sterilizing Conditions 
A. Polybutadiene-Coated Spherules 
The ability of the PBD-coated spherules to withstand sterilizing conditions 
was demonstrated by evaluation of the chromatographic characteristics of a 
sample of the spherules prepared as described in Example 21, Preparation 
C, before and after exposure of the sample to a mobile phase of 1 M NaOH 
at 100.degree. C. for 1 hr. As indicated by the data in Table XII below, 
there was no decrease in retention of nonpolar substances upon challenging 
the packing in this fashion. 
TABLE XII 
______________________________________ 
k' k' 
Solute Before Treatment 
After Treatment 
______________________________________ 
Benzene 1.36 1.47 
Toluene 2.68 3.01 
Ethyl Benzene 
4.83 5.57 
Propyl Benzene 
9.21 10.86 
______________________________________ 
A second column (ES Industries, Marlton, N.J.), packed with an alumina 
support modified by the method of G. Shomberg, LC-GC, 6, 36 (1988), was 
challenged with a mobile phase of lM NaOH, which was collected in two 
fractions. The first corresponded to an elution time of 1 hr and the 
second to an additional elution of 2.25 hrs. 
The eluents were analyzed via an inductively coupled plasma spectrometer. 
The concentration of alumina in the eluent from the second column 
corresponded to the dissolution of a total of 10% of the mass of the 
alumina in the column. 
In marked contrast, zirconium was absent in the eluent of the zirconium 
colum at a level of detectability of 0.03 .mu.g/ml. Even if Zr was present 
at the detection limit, this would correspond to loss of less than 0.001% 
of the mass of ZrO.sub.2 on the test column. 
B. Polystrene-, Poly(t-butyl)styrene- and Polyisoprene-Coated Spherules 
The ability of three additional polymer-coated phases to withstand the 
sterilizing conditions described above was also evaluated. The additional 
polymer coatings tested were polystyrene ("PS", 3850 m.w.), 
poly(t-butyl)styrene ("PTBS", 3930 m.w.), and polyisoprene ("PI", 3000 
m.w.). The spherules coated with these additional polymers were prepared 
as follows: 
1. Preparation of Polystyrene-Coated Spherules 
Porous ZrO.sub.2 spherules having a diameter from about 1 to 10 .mu., a 
surface area of 47.7 m.sup.2 /g, and an average pore diameter of about 118 
.ANG. were coated with polystyrene, a hydrophobic, aromatic polymer, as 
follows: 0.45 g of the oligimeric polystyrene (m.w. 3850) were dissolved 
in 200 ml of toluene. To this solution were added 10 ml of toluene in 
which 0.01 g of dicumyl peroxide initiator had been dissolved. The 
resulting solution was placed in a 1000 ml round bottom flask. 15 0 g of 
the ZrO.sub.2 spherules were added to the flask before placement of the 
flask on a rotary evaporator and rotation under a pressure of 15 in. of Hg 
for about 15 mins. The pressure was then reduced to 26-28 in. of Hg, until 
the toluene was removed. Some agglomerating of the resulting spherules was 
observed. 
After release of the vacuum, toluene was added to the flask to redissolve 
the oligimer and initiator. An additional 1.0 g of ZrO.sub.2 spherules 
were added to the flask, and the toluene was removed under a vacuum of 
26-28 in. of Hg. The resulting oligimer-coated spherules were not 
agglomerated, and the resulting batch flowed easily when poured. 
The flask containing the spherules was then placed in a vacuum oven and was 
heated to a temperature of 170.degree. C. under a vacuum of about 29-30 
in. Hg for 4 hours, in order to cross-link the oligimeric polystyrene. 
After curing, analysis of a sample of these coated spherules indicated 
that the carbon and hydrogen content of the spherules were 1.7 wt-% and 
0.3 wt-%, respectively. The polystyrene-coated spherules were then 
Soxhlet-extracted with toluene for 4 hours Surface area analysis of the 
coated spherules indicated that their average surface area was 39.5 
m.sup.2 /g, and the average pore diameter about 100 .ANG.. The spherules 
were then classified on a Gilson Inc. sonic siever; spherules having 
diameters between 5 and 10 .mu. were used to pack an HPLC column. 
2. Preparation of Polyisoprene-Coated Spherules 
A second sample of ZrO.sub.2 spherules, having a diameter of 1-10 .mu. and 
a surface area of 28 m.sup.2 /g, were coated with polyisoprene, an 
aliphatic, hydrophobic polymer. 0.2 g of the oligimeric polyisoprene (m.w. 
3000) and 0.005 g of dicumyl peroxide initiator were dissolved in 300 ml 
of heptane. The resulting solution was placed in a 1000 ml round bottom 
flask. 10 g of ZrO.sub.2 spherules were added to the flask, which was then 
placed on a rotary evaporator and rotated under a pressure of about 15 
inches of Hg for about 15 minutes until the heptane was removed. The flask 
containing the spherules was then exposed to a vacuum of 29-30 in. of Hg 
for 2 hours in order to cross-link the polyisoprene. The spherules were 
then extracted with heptane for 4 hours in a Soxhlet extractor. 
3. Preparation of Poly(t-butyl)styrene-Coated Spherules 
A third sample of ZrO.sub.2 spherules having a diameter from about 1 to 10 
.mu., a surface area of 47.7 m.sup.2 /g, and an average pore diameter of 
about 118 A were coated with poly(t-butyl)styrene, a hydrophobic polymer 
with both aliphatic and aromatic character. 0.4 g of the 
poly(t-butyl)styrene (3930 m.w.) was dissolved in 200 ml of toluene. To 
this was added a solution of 0.01 g of dicumyl peroxide dissolved in 10 ml 
of toluene. The resulting solution was placed in a 1000 ml round bottom 
flask, and 20.0 g of the ZrO.sub.2 spherules were added. The flask was 
then placed on a rotary evaporator and rotated under a pressure of about 
15 inches of Hg for about 15 minutes. The pressure was then reduced to 
about 26-28 inches of Hg with removal of the toluene over about 1 hour. 
The coated spherules were observed to be slightly agglomerated. 
150 ml of toluene were then added to the flask, in order to redissolve the 
poly(t-butyl)styrene and the initiator. An additional 4.2 g of ZrO.sub.2 
spherules were added to the flask, and the toluene was removed on the 
rotary evaporator over about an hour under the previously described 
conditions. The coated spherules flowed easily and were not agglomerated. 
The spherules were next placed in a ceramic tray and cross-linked in a 
vacuum oven at 170.degree. C. for 4 hours. Analysis of the carbon and 
hydrogen content of the poly(t-butyl)styrene coated spherules indicated 
that these were 1.4 wt-% and 0.3 wt-%, respectively. Surface area analysis 
of the coated spherules indicated that the surface area was 33.5 m.sup.2 
/g. 
4. Exposure to Sterilizing Conditions 
After coating and cross-linking as noted, a portion of each of the three 
samples of polymer-coated spherules were used to pack HPLC columns without 
further modification. The HPLC columns were prepared by packing each 5 
cm.times.4.6 mm id 316 stainless steel column blank with a sample of 
spherules coated with polystyrene, poly(t-butyl)styrene, or polyisoprene. 
Each HPLC column was equipped with 1/4 inch 316 SS Parker-Hanifan end 
fittings and 1/4 inch.times.1/32 inch 2 .mu.m titanium frits. The coated 
spherules were packed at 6000 p.s.i. from a methanolic slurry, using a 
downward slurry packing technique. 
The stabilities of the polymer-coated zirconia materials were then 
evaluated under "sterilizing" conditions by exposing the columns to a 
mobile phase of 1.0 M NaOH for 3.25 hours, at a flow rate of 1.0 ml/min, 
while the column was held at a temperature of 100.degree. C. The effluents 
of all three columns were collected, and half of each effluent was 
filtered through 0.45 .mu.m Teflon.TM. filter. Both fractions of each 
effluent were then evaluated by inductively coupled plasma spectroscopy 
(ICP), in order to determine levels of zirconia and other metals present 
in each sample. The limit of detection for the ICP analysis was 0.03 
.mu.g/ml for zirconium. 
The results of the ICP analysis of the three additional polymer-coated 
zirconium samples are summarized in Table XIII below, which indicates that 
no detectable amount of zirconium was present in the effluent from any of 
the samples. Nor was any zirconium detected in the sodium hydroxide blank 
solution. Upon opening each of the columns following the alkaline 
treatment, no voids were observed in the packing. 
TABLE XIII 
______________________________________ 
Aluminum, Silicon, and Zirconium Levels in the 
Effluent of Polymer Coated ZrO.sub.2 Columns Exposed to 
Sterilizing Conditions.sup.a 
Column Al (.mu.g/ml) 
Si (.mu.g/ml) 
Zr (.mu.g/ml) 
______________________________________ 
PS/ZrO.sub.2 
&lt;0.13.sup.b 
0.99 &lt;0.03.sup.b 
PTBS/ZrO.sub.2 
&lt;0.13.sup.b 
2.3 &lt;0.03.sup.b 
PI/ZrO.sub.2 
&lt;0.13.sup.1 
1.1 &lt;0.03.sup.b 
NaOH blank &lt;0.13.sup.b 
0.2 &lt;0.03.sup.b 
______________________________________ 
.sup.a 1.0M NaOH at 100.degree. C. for 3.25 hours 
.sup.b indicates limit of detection under these conditions 
The data of Table XIII demonstrate that the application of other 
hydrophobic polymer coatings to the zirconia spherules resulted in phases 
which behaved similarly to the PBD/ZrO.sub.2 described above. No 
measurable zirconia was leached from these materials during sterilizing 
sodium hydroxide treatment, in contrast with the significant loss of 
alumina from the alumina support, described above. 
The invention has been described with reference to various specific and 
preferred embodiments and techniques. However, it should be understood 
that many variations and modifications may be made while remaining within 
the spirit and scope of the invention.