GB2355012A - Paramagnetic polymer particles - Google Patents

Abstract

A composite paramagnetic particle comprises a multitude of submicron polymer bead aggregates covalently cross-linked to each other to form larger diameter particles. Distributed throughout the composite paramagnetic particle are vacuous cavities. Each submicron polymer bead has distributed throughout its interior and surface submicron magnetite crystals. Composite particles are made using high energy ultrasound during polymerization of one or more vinyl monomers. In one embodiment, high energy ultrasound is used during an emulsification step and during the early stages of the polymerization process to produce micron sized composite paramagnetic particles. The particles exhibit a high percent magnetite incorporation and water and organic solvent stability.

Description

2355012 PARXMMETIC POLYMER PRACTICE The present invention relates to a

paramagnetic polymer particle and method of making such a particle.

More specifically, the preferred embodiment relates to an ultrasonically generated composite paramagnetic polymer particle including a plurality of paramagnetic polymer bead aggregates.

A typical method for measuring the binding of antigens or ligands to proteins is particle agglutination or precipitation. In such a method a protein bound suspension of beads or particles reacts with some antigen or ligand causing the particles to flocculate or precipitate. Quantitation is accomplished through the use of spectrophotometry to measure the change of light transmission through the medium. Another method which is far more sensitive is the use of radioactivity in the measurement. In such a method, either the ligand or the particle bound protein is radiolabeled and complexed with either unlabeled protein or ligand. The uncomplexed material is then separated from the bound complex by simple filtration and then counted through the process of scintillation. Filtration of insoluble particles incorporating a protein- ligand complex is efficient when 2 large numbers of particles are employed, yet can be difficult when small numbers of particles are used.

In cases where small numbers of particles are used, other insoluble supports are utilized, such as paramagnetic particles. U.S. Patent No. 5, 750,412 discloses exposing antibody-bound paramagnetic particles to a magnetic field in order to separate antibody-bound antigen from unbound antigen in immunoassays. Such materials are attracted to a magnetic field and this attraction is used to separate the bound protein-ligand complex from the surrounding liquid medium. Magnetic separation methods have also been applied successfully in cell sorting. A definite advantage that magnetic separation has over simple filtration is the ability to separate out small numbers of particles from small reaction volumes. Another advantage magnetic separation has over simple filtration is the ease to which one is able to automate the simultaneous washing and separation of hundreds of individual solid phase complexation reactions.

In the pharmaceutical industry, compound libraries for biological testing are routinely synthesized through the use of solid phase organic chemistry. solid phase organic synthesis allows for the quick separation of products from unreacted starting material as well as reagents and sideproducts that are not originally bound to the support. The physical separation of the support from the solubilized 3 components of the reaction mixture has primarily been accomplished by filtration through a glass or polymer filter. Although filtration has been the method of choice in solid phase organic synthesis, it has limitations that warrant the development of newer approaches. One such limitation is the difficulty in automating the simultaneous washing and filtration of hundreds of small scale solidphase reactions.

U.S. Patent No. 5,684,130 discloses the use of magnetic separation in the field of solid-supported organic chemistry has recently been demonstrated in the synthesis of peptides, non-peptides compounds and in the affinity chromatography of compound mixtures using a paramagnetic support in organic solvent. Clearly, paramagnetic supports have many useful applications, including in biology and chemistry which warrant the development of improved paramagnetic supports and/or better methods for their manufacture.

U.S. Patent No. 4,358,388 discloses magnetite crystals encapsulated in polystyrene by the suspension polymerization of styrene in the presence of an organosoluble initiator, a suspending agent, magnetite (Fe304) and an emulsifying agent in water. The mixture was homogenized to give organic phase droplets ranging from 0.03-5 microns in diameter. The homogenized mixture was then polymerized to give spherically shaped magnet ic-po lymer 4 latex particles. The resulting particles consist of a core of polystyrene polymer with magnetite crystals located as inclusions along the outer most periphery of the particles.

U.S. Patent No. 5,091,206 also discloses a process for producing paramagnetic particles with a uniform spherical surface. Magnetite crystals are coated on the surface of pre-made uniformly spherical polystyrene particles (2-6 microns in diameter) to form a non-covalent composite or "seed". A vinyl monomer is then polymerized on the surface of the polymer part ic 1 es-magnetite seeds to produce spherical paramagnetic particles having an average particle diameter of between 4-7 micrometers. The polymerization process involved heating together in a rotating reaction flask small quantities of polymer particles-magnetite seeds, vinyl monomer, water soluble initiator and an anionic surfactant in water. The percent magnetite incorporation of the particles is increased by taking the polymer coated magnetite particles and successively adding more magnetite to them to form new "seeds" which are again polymerized together. This process of successively adding magnetite followed by polymer coating the resulting aggregate layers can enhance the percent magnetite incorporation of the final paramagnetic bead. However, among other disadvantages, in order to achieve relatively high levels of magnetite incorporation repeated polymerization and separation steps are required making this method both very costly and time consuming to perform.

Many variables contribute toward the ultimate size, shape and thickness of polymer coatings. The amount and types of surfactant used, the reaction temperature and the stirring speed and/or the agitation method can all have a direct effect on structural properties. For example, power ultrasound has been reported to enhance the uniformity of polymer coverage around titanium dioxide particles under emulsion polymerization conditions and to aid in the formation of nanometer sized iron colloid particles. See J. P. Lorimer et al, 269 Colloid. Polym. Sci. 393-397 (1991); K. S. Suslick et al, 116 J. Am. Chem. Soc. 11960-61 (1996).

There are many drawbacks to prior art paramagnetic polymer particles and methods of making. Current polymer coated paramagnetic particles are relatively expensive to produce. For example, making polymer coated magnetite particles exhibit high levels of magnetite incorporation is both costly and time consuming. Because magnetite is hydrophilic by nature, it is difficult to stabilize in the hydrophobic environment of a monomer droplet. A number of different and sometimes exotic surfactants, co-surfactants and suspending agents are usually required in order to allow the magnetite to be closely associated with the monomer droplets. U.S. Patent No. 5,232,782 discloses metal oxides 6 made more hydrophobic by coating them with an organosilating reagent. However making the magnetite more hydrophobic can produce other problems that can be difficult to predict let alone control. For example, due to an increase in the number of hydrophobic components in the polymer reaction mixture, more surfactant is required to stabilize the magnetite incorporated monomer droplet. However, too much surfactant can force the separation of the hydrophobic magnetite and the monomer droplet itself, resulting in a lowering in the magnetite incorporation. Too little surfactant can result in rapid aggregation of the magnetite itself causing the hydrophobic magnetite to fall out of suspension. It can also cause the coalescence of the monomer droplets into larger droplets containing less magnetite. Another problem is the expense and time required to make very small and uniform magnetite crystals having a diameter of less than 1 micron in size. Lastly, it is difficult to make polymer coated paramagnetic particles be stable both in an aqueous as well as an organic solvent environment.

The present invention seeks to provide an improved paramagnetic polymer particle.

According to an aspect of the present invention there is provided a paramagnetic particle as specified in claim 1 or 2.

7 According to another aspect of the present invention there is provided a method of making paramagnetic particles as specified in claim 9 or 10.

The preferred embodiments can provide a composite paramagnetic particle having high levels of magnetite incorporation; a paramagnetic polymer particle that is stable to both water and organic solvents; a composite paramagnetic polymer particle including a plurality of interconnected beads interspersed by cavities and/or a composite paramagnetic polymer particle including a plurality of interconnected bead aggregates interspersed by cavities.

The preferred embodiments can also provide an easy and inexpensive method of making composite paramagnetic particles, preferably, using ultrasound.

They can also provide a method of making a composite paramagnetic particle 8 using magnetite coated with a hydrophobic compound or a silylating agent, or a method of making magnetite crystals of submicron size suitable for use in making the composite particle of the'present invention.

In a preferred embodiment, the particle of the invention comprises a plurality of interconnected bead aggregates, each aggregate comprising interconnected primary beads. The particles can be made according to the method of the present invention in such quantities as desired.

The bead aggregates are each comprised of submicron primary beads, each primary bead comprising a cross-linked polymer preferably incorporating on the surface and/or inside, inclusions of paramagnetic magnetite crystals. These crystals can also be incorporated within cavities located between proximally attached submicron primary beads. Each submicron primary bead preferably has incorporated at least one metal oxide crystal on the surface or within the cross-linked polymer. However, it is not necessary that every primary bead or bead aggregate contain magnetite as long as the composite paramagnetic particle as 9 a whole contains magnetite. Throughout the composite paramagnetic particle are distributed various sized vacuous cavities.

In making the composite paramagnetic polymer particle, high energy ultrasound is applied during a homogenizktion step and/or the polymerization steps of a reaction mixture including one or more vinyl monomers, a cross-linking agent, submicron magnetite crystals that are either coated with an organosilating agent or uncoated and one or more surfactants in water. The preferred method involves applying high energy ultrasound during both the homogenization and early stages of the polymerization reaction. The vinyl monomers may have reactive side groups. Preferably, the monomers used to form the composite, paramagnetic polymer particle are styrene or styrene type derivatives.

The composite paramagnetic particles may be made by a number of different polymerization methods. A preferred method includes four steps with the first being a homogenization step including the application of high energy ultrasonic energy such as through the use of an ultrasonic probe to an aqueous mixture including one or more vinyl monomers, cross-linking agent, organosilane coated or noncoated magnetite crystals of preferably less than 0.05 microns in diameter, a water soluble or organic soluble initiator and one or more emulsifying agents. When the magnetite crystals are not coated with an organosilane, mineral oil is used as a suspending agent. The second step involves applying high energy bath ultrasound produced by an immersible ultrasonic transducer to the homogenized reaction mixture at an elevated temperature while maintaining constant stirring and under an inert atmosphere for the early stages of the polymerization reaction. The third step involves the continued heating and stirring of the reaction mixture without the presence of high energy bath ultrasound for the duration of the polymerization reaction. The fourth step involves separating out the magnetite encapsulated polymer from the reaction mixture.

An embodiment of the present invention is described below, by way of example only, with reference to the accompanying drawings, in which:

Figure 1 is an environmental scanning electron micrograph of the surface of an embodiment of composite paramagnetic particle at a magnification of 400OX; Figure 2 is a transmission electron micrograph of a cross-section of an embodiment of several composite paramagnetic particles at a magnification of 400OX; Figure 3 is transmission electron micrograph of a cross-section of an embodiment of composite paramagnetic particle at a magnification of 22, 50OX; 11 FIGURE 4 is transmission electron micrograph of prior art paramagnetic particles made without the introduction of ultrasound either during the homogenization or during the polymerization process at a magnification of 22,50OX; and

FIGURE 5 is an environmental scanning electron micrograph of the surface of two composite paramagnetic particles at a magnification of 10,OOOX.

In one embodiment, a composite paramagnetic particle comprises a plurality of submicron primary beads, preferably arranged as covalent bead aggregates, the aggregates covalently attached to each other and composed of cross-linked polymer which may be functionalized. Embedded in many of the polymer beads are magnetite crystals. Throughout the composite particle are located vacuous cavities that are devoid of both submicron primary beads and magnetite crystals. The particle is stable in both water and organic solvents including acetone, methanol, ethanol and hexane. With higher levels of crosslinking the composite paramagnetic particles become more stable to polar organic solvents such as methylene chloride and tetrahydrofuran at elevated temperatures and under high energy sonication.

12 The surface of an embodiment of composite paramagnetic particie is shown in the environmental scanning electron micrograph of FIGURE 1. The surface of the particle has a multitude of submicron, spherically shaped beads that are covalently bonded together. A cross-section of several different sized composite paramagnetic particles of the invention are shown in the scanning electron micrograph of FIGURE 2. An enlarged cross-sectional view of one of these composite paramagnetic particles is shown in FIGURE 3. Vacuous cavities 301 of various sizes are distributed throughout the composite paramagnetic particle as shown in FIGURE 3. The individual submicron beads 303 that make up the composite paramagnetic particle can be seen as rounded dark images.

The black dots interspersed throughout the composite paramagnetic particle are believed to be magnetite crystals either as individual crystals or in the form of crystal aggregates. While not limiting, -_ the particle shown in FIGURE 3 has a diameter of approximately 6 microns, whereas the submicron beads 303 are approximately 0.1 to 0.13 microns in diameter.

The magnetite crystals can be made by several methods using a combination of iron(II) and iron(III) metal salts. A preferred method involves the addition by dropping funnel of an excess of sodium hydroxide solution to a heated, sonicated and stirred reaction mixture made up of 13 equal molar quantities of iron(II)sulfate and iron (III) sulfate dissolved in deionized water. The magnetite crystals that are formed are then magnetically separated and washed repeatedly until neutral to pH. The magnetite crystals formed are preferably under 1 micron in size. It is observed that in the absence of the application of ultrasonic energy to the reaction mixture one obtains a larger percentage of magnetite crystals that are larger than 1-2 microns in diameter. After washing and magnetically separating the magnetite crystals they are dried. The dried crystals are then crushed using a mortar and pestle to give crystals with a diameter preferably equal to or less than 0.06 microns as observed using transmission electron microscopy.

The quantity of magnetite crystals used during the polymerization process may range from 1-60% by weight based upon the weight of the total monomer(s) added during the homogenization step. The magnetite crystals may be precoated with a hydrophobic coating before use in the homogenization step such as with silica oil or mineral oil or some hydrophobic silylating agent such as phenyltriethoxysilane or a polymerizable vinyl containing triethoxyvinylsilane such as methacryloxypropyltrimethoxys, i lane available commercially from Dow Corning. It is preferred that the magnetite crystals be precoated with some hydrophobic coating before use in the homogenization step.

14 It is preferred that the amount of magnetite crystals used be between 10- 50% by weight of total monomer added during the homogenization step.

The monomers which are suitable for making the preferred composite paramagnetic particle are vinyl monomers such as styrene and substituted styrenes, acrylates and methacrylates. Inclusion of a desired percentage of functionalized monomers eliminates the step of providing functionalized groups after the polymerization process. The cross-linking agents may be composed of any divinyl monomer such as divinyl benzene. An amount of divinyl monomer is added so that the final polymer is cross-linked in the range of 1 to 50%. It is preferred that the level of crosslinking in the composite paramagnetic particle be in the range of between 4-20%.

A polymerization initiator is preferably added at about 1-10% by weight based upon the weight of the total monomer(s) added during the homogenization step. The initiator may be either organic solvent soluble or water soluble. When an organically soluble initiator is used the preferred initiators are alkyl and aryl peroxides. Preferably, lauroyl peroxide is used. When a water soluble initiator is used the preferred initiator is potassium persulfate. However, other means of initiation may be used such as ultrasonically induced radical formation. It has been observed that when a water soluble initiator is used a greater percentage of magnetite is found incorporated in the composite paramagnetic particle as compared to when an organic soluble initiator is used.

one or more dispersing agents are added preferably at about 1-15% each by weight based upon the weight of the total monomer(s) added during the homogenization step. The dispersing agents may be anionic, cationic or neutral in nature. In a preferred embodiment both a charged and a neutral emulsifying agent -are used at concentrations of 1-15% each by weight of the total monomer(s) present during the homogenization step. The preferred charged emulsifying agent is the anionic surfactant sodium lauryl sulfate. Examples of non-ionic dispersants are polyvinyl alcohol, cellulose based compounds and alkylphenols. The preferred non-ionic dispersants are both branched and unbranched phosphated, ethoxylated nonylphenol.

In another aspect of the present invention, the application of ultrasound is important to the successful production of composite paramagnetic particle of the invention. If ultrasound is not used in the process one obtains particles that are substantially different from those made with the use of high energy ultrasound. A crosssection of several paramagnetic particles made without the aid of ultrasound are shown in the scanning electron micrograph, FIGURE 4. The paramagnetic particles 405 incorporate magnetite crystals 407 primarily as inclusions 16 on the surface of each individual spherical particle 405. By way of comparison, spherical particles 405 contain neither magnetite crystals nor vacuous cavities within the core of the particle 405 themselves nor are they composed of a multitude of smaller submicron polymer particles.

Ultrasound can be applied during either the homogenization stage and/or the polymerization stage of the method of the invention. The preferred application of ultrasound is during both the homogenization stage and during the early stages of the polymerization process. Various methods for applying ultrasonic energy are possible. Examples of sonicating systems are high energy probe sonicators, immersible sonicators and sonicating cup horns. It is preferred that a high energy probe sonicator be used during the homogenization step and a high energy, immersible sonicator be used during the early stages of the polymerization process.

In a preferred embodiment of the invention, noncoated magnetite crystals are mixed with mineral oil and added to a solution composed of styrene monomer, divinyl benzene monomer and an organic soluble polymerization initiator to form a dispersed phase. In another embodiment of the invention, when organosilane coated magnetite is used no mineral oil is required. Instead, the organosilane coated magnetite is mixed directly with the styrene monomer, 17 divinyl benzene monomer and the organic soluble polymerization initiator to form the dispersed phase. A continuous fluid phase is formed from degassed, deionized water and dispersant agents. When the initiator is water soluble, it is dissolved in the continuous fluid phase rather than the dispersed phase.

The dispersed phase is added to the continuous f luid phase in a ratio of about 1: 15 to about 1: 3 dispersed to continuous phase (vol:vol) and preferably at a ratio of about 1: 7 to about 1: 5. The continuous fluid phasedispersed phase is homogenized using a high energy sonicating probe for about 1-5 minutes and at a power level of about 40-100%. Preferably the fluid phase-dispersed phase is sonicated f or about 1-2 minutes at a power of about 50-80%. The homogenized mixture is then simultaneously sonicated, heated and stirred for about 5-120 minutes. It is preferred that the homogenized mixture be bath sonicated for no more than 30 minutes while heated and stirred under closed nitrogen atmosphere. Heating and stirring of the mixture is continued under a closed atmosphere for the duration of the polymerization reaction. It is preferred that the heating and stirring continue for a total of about 18- 24 hours beyond the bath sonicating phase. The crude polymer product is then washed and either filtered or magnetically separated to give the desired composite, paramagnetic particles. The final particle size preferably 18 ranges from about 0.75 to 300 microns. Particles equal to or under 5 microns are preferred.

Although the overall composition of the paramagnetic particles maintains constant, it has been observed that factors such as the stirring speed as well as the shape of the stir blade itself can have a profound effect on the size distribution of the final composite paramagnetic particle product. With faster stirring speed one obtains composite paramagnetic particles having a smaller average particle size. It is pref erred that a rate raging between about 350-900 rpm be maintained throughout the polymerization reaction. In order to obtain the largest amount of composite paramagnetic particles having an average particle size of 5 micron or below, it is preferred to have a stirring speed during the polymerization reaction of about 700-900 rpm.

For purposes of example only, the particle finds use as a separation support system which can be resuspended in solution, then magnetically separated by applying a magnetic field, and subsequently resuspended. Such a separation system is useful, for example, in immunodiagnostics, affinity chromatography and receptorligand binding assays as well as in the solid phase synthesis of organic molecules. In addition, it may also find uses as a building block for the synthesis of other paramagnetic materials.

19 The following Examples are provided to illustrate the presently preferred embodiments and not to limit their scope.

Synthesis and.Isolation of Magnetite Crystals Example-I

To a 500 ml round bottom flask is added a solution of 30.09 grams (0.108 moles) of iron(II)sulfate heptahydrate dissolved in 120 ml of deionized water and a solution of 54.12 grams (0.110 moles) of iron (III) sulfate pentahydrate dissolved in 120 ml of deionized water. The stirred solutions was then heated at 850C and sonicated using a Heat Systems Inc. 20 KHz sonicating probe, model XL2020 (Farmingdale, New York) at 25% power following the program of 1 minute "on" and 1 minute "off". To the stirred, heated and sonicated reaction mixture was slowly added by dropping funnel a solution of 60 grams of NaOH in 250 ml of deionized water over a period of 1 hour. After the addition of the sodium hydroxide solution the heated and stirred reaction was sonicated for an additional 40 minutes and then the reaction was allowed to cool to room temperature to give magnetite crystals as a fine black suspension. The reaction mixture was transferred to a 1 liter beaker. A series of neodymium permanent magnets embedded in a flat plastic sheet was then placed underneath the beaker. The magnetite suspension was allowed to sit at room temperature until all of the magnetite crystals magnetically separated to the bottom of the beaker. The water was then aspirated off and the resulting magnetite crystals washed with deionized water. The magnetite crystals were magnetically separated again and then washed. The water washes and magnetic separations continued until the washes were neutral to pH. The magnetite crystals were then dried under reduced pressure to give 24.9 grams of magnetite crystals as a black solid. The solid was then lightly crushed with a mortar and pestle to give magnetite crystals (Fe304) as a fine powder that by transmission electron microscopy consisted of individual crystals of magnetite of equal to or under 0.06 microns in size.

Synthesis and Isolation of Ultrasonically Generated Composite Paramagnetic Particles Example 2 To a 500 ml three neck flask is added a solution consisting of 1 gram of sodium dodecyl sulfate and 1 gram of ethoxylated, phosphated, nonylphenol (Servoxyl VPNZ 20/100 obtained from SERVO DELDEN BV, The Netherlands) dissolved in 200 ml of deionized and degassed water to form the continuous phase. To a beaker containing three grams of magnetite crystals (Fe304) is added 2.5 grams of mineral oil and manually mixed. To the mixture is then added 30 grams of styrene, 1.5 grams of divinyl benzene and 3 grams of 21 lauroyl peroxide to form the dispersed phase. The dispersed phase is then transferred to the 500 ml three neck round bottom f lask and the combined continuous-dispersed phases are sonicated using a Heat Systems Inc. 20 KHz, 550 watts sonicating probe, model XL2020 (Farmingdale, New York) at 50% power for 1 minute. The round bottom flask is then transferred to a temperature controlled sonicating tank and the homogenized mixture is then simultaneously heated at 500C, stirred using an overhead stirrer at 350 rpm and sonicated using a 40 KHz, 600 watts, immersible sonicator (CAE Blackstone, Jamestown, New York) at 55% power for 30 minutes under a nitrogen atmosphere. After 30 minutes the sonication was stopped and the reaction mixture was stirred and heated under a closed nitrogen atmosphere for an additional 18 hours at 500C. The reaction mixture was then filtered through a 0.5 mm metal mesh screen into a 1 liter beaker. To the beaker is added 400 ml of methylene chloride and 400 ml of methanol and manually stirred. To the bottom of the beaker was placed a plate incorporating a series of embedded neodymium permanent magnets (Master Magnetics, Castle Rock, Colorado). The top milky white, aqueous layer was then aspirated off. To the remaining organic layer was added another 400 ml of methanol to give a light brown suspension. The brown suspension was then filtered, washed with methanol and dried under reduced pressure to give 18.4 grams of composite, paramagnetic particles. Elemental 22 analysis of the product gave 84.25% carbon, 7.25% hydrogen and 4.37% iron. Electron microscopy of a sampling of the composite, paramagnetic, particles can be seen in FIGURES 1-3.

ExaCmiple 3 This example demonstrates the incorporation of high levels of cross- linking and functionalization into a composite paramagnetic support. To a 500 ml three neck flask is added a solution consisting of 1 gram of sodium dodecyl sulfate, 1 gram of ethoxylated, phosphated, nonylphenol (Servoxyl VPNZ 20/100 obtained from SERVO DELDEN BV, TheNetherlands) dissolved in 200 ml of deionized and degassed water to form the same continuous phase as experiment 2. To a beaker containing three grams of magnetite crystals (Fe304) is added 2.5 grams of mineral oil and manually mixed. To the mixture is then added 24.1 grams of styrene, 3.1 grams of divinyl benzene, 4.2 grams of chloromethyl styrene and 3 grams of lauroyl peroxide to form the dispersed phase. The dispersed phase is then transferred to the 500 ml three neck round bottom flask and the combined continuous-dispersed phases are sonicated using a Heat Systems Inc. 20 KHz, 550 watts sonicating probe, model XL2020 (Farmingdale, New York) at 50% power for 1 minute. The round bottom flask is then transferred to a temperature controlled sonicating tank and the homogenized 23 mixture is then simultaneously heated at 500C, stirred using an overhead stirrer at 350 rpm and sonicated using a 40 KHz, 600 watts, immersible sonicator (CAE Blackstone, Jamestown, New York) at 55% power for 30 minutes under a nitrogen atmosphere. After 30 'minutes the sonication was stopped and the reaction- mixture was stirred and heated under a-closed nitrogen atmosphere for an additional 18 hours at 500C. The reaction mixture was then filtered through a 0.5 min metal mesh screen into a I liter beaker. To the beaker is added 400 mL of methylene chloride and 400 ml of methanol and manually stirred. To the bottom of the beaker was placed a plate incorporating a series of embedded neodymium permanent magnets (Master Magnetics, Castle Rock, Colorado). The top milky white, aqueous layer was then aspirated off. To the remaining organic layer was added another 400 ml of methanol to give a light brown suspension. The brown suspension was then filtered, washed with methanol and dried under reduced pressure to give 3.0 grains of composite paramagnetic particles. Elemental analysis of the product gave 6.34% iron and 2.55% chlorine (which corresponds to 0.71 mmoles chloromethyl /gram of composite paramagnetic support).

24 SynthQsis of Polymeric Paramacfnetic Partigles Without Use of Ultrasound Example 4 This example is provided to demonstrate the differences obtained in the overall composition of a polymeric paramignetic particle product when ultrasound is not used in making the particle. The procedure used in this example is exactly the same as that of Example 2 except that a conventional mechanical homogenizer is used during the homogenization step and no ultrasound is used during the poly-merization step. To a 500 ml three neck flask is added a solution consisting of 1 gram of sodium dodecyl sulfate, 1 gram of ethoxylated, phosphated, nonylphenol (Servoxyl VPNZ 20/100 obtained from SERVO DELDEN BV, The Netherlands) dissolved in 200 ml of deionized and degassed water to form the same continuous phase as experiment I.. To a beaker containing three grams of magnetite crystals (Fe304) is added 2.5 grams of mineral oil and manually mixed. To the mixture is then added the same quantities of styrene, divinyl benzene and lauroyl peroxide as Example 2 to form the dispersed phase. The dispersed phase is then transferred to the 500 ml three neck round bottom flask and the combined continuous -dispersed phases are homogenized for 2 minutes using a Virtis brand homogenizer (The Virtis Company, Gardiner, New York) incorporating a 20 mm diameter homogenizer shaft. The round bottom flask is then heated at OC while stirring using an overhead stirrer at 350 rpm under a closed nitrogen atmosphere f or a total of 18k hours. The reaction mixture was then worked up in the same manner as that of Experiment 2 to give 13.0 grams of polymer, paramagnetic beads. Elemental analysis of the product gave carbon, 7.78% hydrogen and 0.80!.-' iron. Electron microscopy of a sampling of the polymeric, paramagnetic particles can be seen in FIGURE 4.

Ultrasonically Induced Composite Paramagnetic Particles With High Levels of Magnetite Incorporation Example 5 This example is provided to demonstrate that one can optimize upward the percent incorporation of magnetite crystals in the composite paramagnetic particles of the invention by moderately modifying some of the reaction conditions. In this case, a water soluble initiator is used in place of an organic soluble initiator and the quantities of magnetite and dispersants used in the beginning of the process are increased. To a 500 ml three neck flask is added a solution consisting of 2 gram of sodium dodecyl sulfate, 2 gram of ethoxylated, phosphated, nonylphenol (Servoxyl VPNZ 20/100 obtained from SERVO DELDEN BV, The Netherlands) and 2 grams of potassium persulfate dissolved in 200 ml of deionized and degassed water to form the continuous phase. To a beaker containing six grams of 26 magnetite crystals (Fe.04) is added 4 grains of mineral oil and manually mixed. To the mixture is then added 28.3 grams of styrene and 3.1 grams of divinyl benzene to form the dispersed phase. The dispersed phase is then transferred to the 500 ml three neck round bottom flask and the combined continuous -dispersed phases are sonicated using a Heat Systems Inc. 20 KHz, 550 watts sonicating probe, model XL2020 (Farmingdale, New York) at 70% power for 1 minute. The round bottom flask is then transferred to a temperature controlled sonicating tank and the homogenized mixture is then simultaneously heated at 580C, stirred using an overhead stirrer at'. 430 rpm and sonicated using a 40 KHz, 600 watts, immersible sonicator (CAE Blackstone, Jamestown, New York) at 55% power for 30 minutes under a nitrogen atmosphere. After 30 minutes the sonication was stopped and the reaction mixture was stirred and heated under a closed nitrogen atmosphere for an additional 19 hours at 58-600C. To the reaction mixture which was allowed to cool to room temperature was added 200 mL of deionized water. The reaction mixture was then filtered through a 0.5 mm metal mesh screen into a 1 liter flask. To the flask is added 400 ml of methylene chloride and manually stirred. To the bottom of the flask was placed a plate incorporating a series of embedded neodymium permanent magnets (Master Magnetics, Castle Rock, Colorado). The top milky white, aqueous layer was then aspirated off. To the remaining 27 organic layer was added another 100 ml of methylene chloride and organic was aspirated off leaving behind the magnetically separated solid. The dark brown solid was washed with another 200 ml of methylene chloride, magnetically separated and the organic solvent aspirated off and the resulting brown solid placed under vacuum to give 4.0 grams of a dark brown powder. Elemental analysis of the product gave 21.59% iron. Electron microscopy of a sampling of the composite paramagnetic particles can be seen in FIGURE 5.

Example 6

This example is provided to demonstrate that one can optimize upward the percent incorporation of magnetite crystals in the composite paramagnetic particles of the invention by utilizing organosilane coated magnetite. 20 grams of magnetite synthesized following the procedure in Example 1 is added to a 250 ml beaker. To the beaker is then added 30 gram of a 10% aqueous solution of methacryloxypropyltrimethoxysi lane obtained from Dow Corning. To the mixture is then added 50 ml of deionized water and 10 ml of methanol and mixture stirred at 65-750C for 30 minutes then the solvent removed under reduced pressure at 60-700C and then placed under pump vacuum for 24 hours to give a black solid which was then manually crushed to a fine black powder that by transmission electron 28 microscopy consisted of individual crystals of magnetite of equal to or under 0. 06 microns in size. Three grams of the f ine black powder is then added to a 10 ml beaker and added 3 grams of styrene, 0. 18 grams of divinyl benzene and 0. 06 grams of lauroyl peroxide to form the dispersed phase. To a 500 ml three neck flask is added a solution consisting of 0.2 gram of sodium dodecyl sulfate, 0.2 gram of ethoxylated, phosphated, nonylphenol (Servoxyl VPNZ 20/100 obtained from SERVO DELDEN BV, The Netherlands) dissolved in 250 ml of deionized and degassed water to form the same continuous phase as in Example 2. The dispersed phase is then transferred to the 500 ml three neck round bottom flask and the combined continuous-dispersed phases is then first mechanically mixed and then homogenized using a Heat Systems Inc. 20 KHz, 550 watts sonicating probe, model XL2020 (Farmingdale, New York) at 50% power for 2. minute. The round bottom flask is then transferred to a temperature controlled sonicating tank and the homogenized mixture is then simultaneously heated at 600C, stirred using an overhead stirrer at 350 rpm and sonicated using a 40 KHz, 600 watts, immersible sonicator (CAE Blackstone, Jamestown, New York) at 55% power for 30 minutes under a nitrogen atmosphere. After 30 minutes the sonication was stopped and the reaction mixture was stirred and heated under a closed nitrogen atmosphere for an additional 18 hours at 600C. The reaction mixture was then filtered through a 0.5 mm metal 29 mesh screen into a I liter beaker. To the beaker is added 400 ml of methanol and manually stirred for 2-3 minutes. To the bottom of the beaker is placed a plate incorporating a series of embedded neodymium permanent magnets (Master Magnetics, Castle Rock, Colorado) and the particles allowed to magnetically separate over 12 hours. At the end of 12 hours, the top layer was then aspirated off. The brown bottom layer was then filtered, washed with methanol and dried under reduced pressure to give 2.1 grams of composite, paramagnetic particles. Elemental analysis of the product gave 29.94% iron.

Ultrasonically Generated Composite Paramagnetic Particles Exhibiting Smaller Average Particle Size Example 7 This example is provided to demonstrate that one can reduce the average particle size of the composite, paramagnetic particles by increasing the stirring speed. To a 500 ml three neck flask is added a solution consisting of 0.4 gram of sodium dodecyl sulfate and 0.4 gram of ethoxylated, phosphated, nonylphenol (Servoxyl VPNZ 20/100 obtained from SERVO DELDEN BV, The Netherlands) dissolved in 350 ml of deionized and degassed water to form the continuous phase. To a beaker containing 6 grams of methacryloxypropyltrimethoxysilane coated magnetite crystals is added 20 grams of styrene, 2.5 grams of divinyl benzene and 0.4 grams of lauroyl peroxide to form the dispersed phase. The dispersed phase is then transferred to the 500 ml four neck round bottom flask and the combined continuousdispersed phases are sonicated using a Heat systems Inc. 20 KHz, 550 watts sonicating probe, model XL2020 (Farmingdale, New York) at 50% power for 1 minute. The round bottom flask is then transferred to a temperature controlled sonicating tank and the homogenized mixture is then simultaneously heated at 650C, stirred using an overhead stirrer at 800 rpm and sonicated using a 40 KHz, 600 watts, immersible sonicator (CAE Blackstone, Jamestown, New York) at 80% power for 30 minutes under a nitrogen atmosphere. After 30 minutes the sonication was stopped and the reaction mixture was stirred and heated under a closed nitrogen atmosphere for an additional 24 hours at 650C. The reaction mixture was then filtered through a 0.5 mm metal mesh screen into a 1 liter beaker. To the beaker is added 400 ml of methanol and manually stirred for 2-3 minutes. To the bottom of the beaker is placed a plate incorporating a series of embedded neodynium permanent magnets (Master Magnetics, Castle Rock, Colorado) and the particles allowed to magnetically separate over 12 hours. At the end of 12 hours, -the top layer was then aspirated off. The brown bottom layer was then filtered, washed with methanol and dried under reduced pressure to give 5.2 grams of composite, paramagnetic particles. Elemental analysis of the product gave 5.12% 31 iron. Transmission electron microscopy and scanning electron microscopy of a sampling of the composite paramagnetic particles indicate the majority of the particles to be on average 2-4 microns in diameter.

As will be apparent to one skilled in the art, various modifications can be made within the scope of the aforesaid description. Such modifications being within the ability of one skilled in the art form a part of the present invention and are embraced by the appended claims.

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Claims (16)

1. A paramagnetic particle comprising; a plurality of interconnected bead aggregates separated by vacuous cavities, wherein a portion of said);)ead aggregates are covalently interconnected; each said bead aggregate comprises two or more primary beads attached to each other and at least two of said two or more primary beads are covalently attached to each other; each said primary bead comprising a polymer of at least one vinyl monomer; a plurality of magnetite crystals dispersed within said primary beads, on the surface of said primary beads and between said primary beads.

2. A paramagnetic particle as in claim 1 wherein the diameter of said composite particle is at least ten times the diameter of each of said primary beads.

3. A paramagnetic particle as in claim 2, wherein the size of ea.ch said primary bead is less than 1 micrometer.

4. A paramagnetic particle as in claim 1, 2 or 3, wherein said polymer has a level of.--ross-1 inking of greater than or equal to 1%.

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5. A paramagnetic particle as in any preceding claim, wherein the diameter of said magnetite crystals is less than about 0.1 micrometers.

6. A paramagnetic particle as in any preceding claim, wherein the size. of said composite particle is between from about 0.5 to about 1000 micrometers.

7. A paramagnetic particle as in any preceding claim, wherein said at least one vinyl monomer is selected from the group consisting of styrene, chloromethyl styrene, divinyl benzene and divinyl benzene derivatives.

8. A paramagnetic particle comprising: a plurality of interconnected submicron primary beads separated by vacuous cavities, wherein a portion of said primary beads are covalently interconnected; each said primary bead comprising a polymer of at least one vinyl monomer; a plurality of magnetite crystals dispersed within said primary beads, on the surface of said primary beads and between said primary beads.

9. A method of making composite paramagnetic particles having a plurality of interconnected primary beads, said method comprising the steps of: preparing a dispersed phase comprising water and at least one dispersing agent; 34 preparing a dispersed phase comprising in combination an organic soluble initiator, magnetite crystals with an average size of under I micrometre and at least one vinyl monomer selected from the group consisting of styrene, chloromethyl styrene, styrene de-rivatives, divinyl benzene and divinyl benzene derivatives; mixing said dispersed phase with said continuous fluid phase; homogenizing said dispersed-continuous phases using ultrasound to form an emulsion; polymerizing said at least one vinyl monomer in the presence of ultrasound.

10. A method of making composite paramagnetic particles having a plurality of interconnected primary beads, said method comprising the steps of: preparing a continuous fluid phase comprising in combination water, at least one dispersing agent and a water-soluble initiator; preparing a dispersed phase comprising in combination magnetite crystals with an average size of under 1 micrometre, at least one vinyl monomer selected from the group consisting of styrene, chloromethyl styrene, styrene derivatives, divinyl benzene and divinyl benzene derivatives; mixing said dispersed phase with said continuous fluid phase; homogenizing said dispersed-continuous phases using ultrasound to form an emulsion; polymerizing said vinyl monomer in the presence of ultrasound.

11. A method as in claim 9 or 10, wherein said magnetite crystals are precoated with a hydrophobic compovnd selected from the group consisting Qf hydrocarbon oil, silica oil and derivatives thereof.

12. A method as in claim 9 or 10, wherein said magnetite crystals are precoated with a silylating agent selected from the group consisting of triethoxyphenylsilane, trimethoxyphenylsilane, trial koxyvinylsilane and derivatives thereof.

13. A method as in claim 9 or 10, wherein said magnetite crystals are protected with methacryloxypropyltrimethoxysilane.

14. A method as in claim 9 or 10, wherein said magnetite crystals are made by a method comprising the steps of: making an equimolar solution of iron (II) and iron (III) salts in deionized water; applying ultrasound to said solution while heating; adding a molar excess of an alkaline solution to said heated and sonicated iron (II)/iron (III) salt solution to form magnetite crystals in the form of a suspension; washing and magnetically separating said magnetite crystals; 36 drying said crystals; crushing said dry crystals into a powder.

15. A paramagnetic particle substantially as hereinbefore described with reference to and as illustrat,ed in the accompanying drawings.

16. A method of making composite paramagnetic particles substantially as hereinbefore described with reference to and as illustrated in the accompanying drawings.