Feb. 5, 174
`
`3,790,492
`M. J. FULWYLER
`METHOD FOR PRODUCTION OF UNIFORM MICROSPHERES
`
`Filed March 11, 1971'
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`INVENTOR.
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`_
`
`Mack J Fu/wy/er ‘
`
`Luminex Ex. 1010
`Luminex/Irori - Page 1
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`

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`3,790,492
`Patented Feb. 5, 1974
`
`1
`
`3,790,492
`METHOD FOR PRODUCTION OF UNIFORM
`MICROSPHERES
`Mack J. Fulwyler, Los Alamos, N. Mex., assignor to the
`United States of America as represented by the United
`States Atomic Energy Commission
`Filed Mar. 11, 1971, Ser. No. 123,362
`Int. Cl. C09k 3/00
`U.S. Cl. 252-301.1 R
`
`1 Claim
`
`ABSTRACT OF THE DISCLOSURE
`Uniform microspheres having any desired diameter
`within the range of about 2 to 40 microns are readily
`produced by introducing a laminar jet of a core liquid
`carrying material in solution or suspension within a lami
`nar flowing sheath liquid, said core liquid jet being pe
`riodically disturbed, allowing the droplets of core liquid
`thus produced to remain suspended within the sheath
`liquid for a time sufficient for the material suspended or
`dissolved in said droplets to form condensed microspheres,
`and removing said microspheres from the sheath liquid.
`The variation in diameter of the microspheres is about
`2%, which is near the accuracy limitation of the measur
`ing instrumentation.
`
`BACKGROUND OF THE INVENTION
`The invention described herein was made in the course
`of, or under, a contract with the U.S. Atomic Energy Com
`mission. It relates to a method and apparatus for prepar
`ing uniform microspheres of various materials through
`dispersal of uniform droplets of a ?rst (core) liquid con
`taining an appropriate material in solution or suspension
`in a second (sheath) liquid immiscible with the core
`liquid and condensation of the material suspended or dis
`solved in the core liquid droplets.
`Small microspheres of very uniform volume have utility
`in a number of ?elds. For example, uniform plastic micro
`spheres having known and controllable physical and op
`tical properties are of great aid in developing instruments
`for biological cell analysis. They are also highly useful
`in the calibration of such instruments. Uniform volume
`microspheres of uranium oxide and plutonium oxide pro
`vide useful reactor fuel materials. Microspheres of radio
`active materials that are uniform in size and volume are
`highly desirable for use in many biological studies, includ
`ing those concerning deposition of various sizes of blood
`borne particles in the lungs, distribution of fetal blood
`?ow, and distribution of cardiac output. An example of
`work of this type is reported in “Preparation of Meta
`bolizable Radioactive Human Serum Albumin Micro
`spheres for Studies of the Circulation,” I. Zolle, B. A.
`Rhodes, and H. N. Wagner, Jr., Internat. J. Appl. Isotopes
`21,155—167 (1970).
`A Well-known technique for forming microspheres in
`volves the use of two liquids which coexist as distinct
`phases with a boundary surface and therefore have sur
`face tension between them. For such a surface tension to
`exist, however, it is essential that the two liquids be im‘
`miscible. Droplets of a ?rst liquid, henceforth called the
`core liquid, in which a material is dissolved or suspended,
`are formed in a moving stream of the second liquid,
`henceforth called the sheath liquid. The liquid of the
`droplets is slightly soluble in the sheath liquid and con
`sequently diffuses out, leaving a spherical particle. Micro
`
`2
`spheres can be produced by this technique over a wide
`range of diameters from a few microns to many hundreds
`of microns. The size of the microspheres depends on the
`size of the droplets formed and on the amount of mate
`rial dissolved or suspended in the droplets.
`Clinton et al. in U.S. Pat. 3,290,122, issued Dec. 6,
`1966, disclose the application of this technique to the
`preparation of oxide gel microspheres from sols. They use
`a two-?uid nozzle in which droplets of the core liquid
`are produced by concurrently introducing a ?ne stream of
`the core liquid and a surrounding stream of the sheath
`liquid into a droplet forming zone through a central aper
`ture and a concentric annular aperture, with the flow rate
`of the sheath liquid being substantially greater than that
`of the core liquid. The literature indicates that to produce
`uniformly sized droplets with a two-?uid nozzle, the
`sheath liquid ?ow must be laminar, and the core liquid
`should be injected in such a manner as to minimize tur
`bulence.
`Haas et al. in U.S. Pat. 3,331,898, issued July 18, 1967,
`disclose a method of preparing gel microsphere wherein
`the core liquid is formed into droplets by passing the core
`liquid stream through small ori?ces into the sheath liquid
`stream at an angle to the direction of ?ow of the sheath
`liquid. Droplets of core liquid are produced by the high
`shearing force that results.
`A signi?cant problem with gel microspheres produced
`by the methods disclosed in U.S. Pats. 3,290,122 and
`3,331,898 is that they are not uniform in size. The ex
`amples disclosed within these two patents show a Wide
`spread in diameters of the microspheres formed. For many
`applications, such variation in size cannot be tolerated.
`Wymer, “Laboratory and Engineering Studies of Sol-Gel
`Processes at Oak Ridge National Laboratory,” ORNL
`TM~2205, pp. 33-39 (1968), discloses that droplets
`formed from capillaries vibrated transversely by an elec
`trodynamic device are more uniformly sized than those
`from a two-?uid nozzle or a shear disperser. However,
`even these droplets produce microspheres having an unde~
`sirably wide range of sizes.
`In addition, the disclosure of Wymer is limited to the
`production of microspheres having an average diameter
`in excess of 250 microns. He does not disclose the pro
`duction of very small microspheres, i.e., those having di
`ameters less than 50 microns. It is well known in the art
`that techniques useful for the production of fairly large
`microspheres, that is, those having diameters in excess
`of several hundred microns are not generally applicable
`to the production of very small microspheres.
`SUMMARY OF THE INVENTION
`I have now found that by using a two-?uid nozzle in
`which a laminar jet of a core liquid carrying material in
`solution or suspension is introduced within a laminar flow
`ing sheath liquid and periodically disturbed, very uni
`formly sized droplets and hence very uniformly sized gel
`microspheres can be readily produced. The laminar sheath
`?ow maintains a uniform separation of the droplets, thus
`preventing droplet coalescence to give larger droplets and
`therefore larger microspheres. The periodic disturbance
`may be introduced into the core liquid jet either axially
`or transversely; however, it is essential that it have a uni
`form frequency. The optimum frequency is determined by
`the diameter and velocity of the jet. Because of the reso
`nant nature of the system, under a given set of conditions
`several suitable frequencies are usually available.
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`3,790,492
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`Moreover, the method and apparatus that I have devel
`across nozzle 7 to vary periodically. Although FIG. 1
`oped are not limited to the production of gel microspheres
`shows the periodic disturbance applied axially to the core
`but are useful in the production of a wide variety of very
`liquid jet, there is no requirement that the disturbance be
`small uniformly sized microspheres, i.e., those having
`introduced axially. A transverse periodic disturbance will
`diameters ranging vfrom several microns to about 40 mi
`produce essentially the same results.
`crons. For example, my invention is easily directed toward
`The size of the droplets 12 formed is dependent on the
`the production of plastic microspheres useful in the cali
`size of nozzle 7 and on the rate of ?ow through nozzle 7.
`bration of instrumentation such as Coulter cell counters.
`Th size of the microspheres that are produced from the
`To my knowledge, there is no other process that can
`droplets in turn depends on the droplet size and the
`readily produce uniformly sized plastic microspheres in
`amount of material held in solution or suspension within
`the 4 to 30 microns range. I have found that the variation
`the droplets. The volume and thereby the diameter of a
`in diameter of the microspheres produced by the method
`microsphere produced from a droplet of a known size is
`and apparatus of my invention is about 2%, which is near
`easily controlled by varying the concentration of solute or
`the accuracy limitation of the measuring instrumentation.
`suspended material in the droplets. For example, to
`double the diameter of the microspheres produced from
`droplets of a known size, it is necessary to increase the
`concentration of solute or suspended material within the
`droplets by a factor of 2a or 8.
`The rate of droplet formation and hence of micro
`spheres formation is dependent on the frequency of dis
`turbance of the core liquid, as [for example, by means of
`oscillator 4, and upon the flow rates of the core liquid
`and the sheath liquid in region 21 of FIG. 1.
`On FIGS. 2 and 3 the horizontal axis represents particle
`volume on a linear scale and the vertical axis represents
`the number of particles per volume increment. On FIG. 2
`th horizontal axis also represents particle ?uorescence on
`a linear scale. The curves of FIGS. 2 and 3 were obtained
`with a modi?ed Coulter counter used as the measuring
`device. Modal value of these volume distributions is de
`termined by an optical measurement of microsphere diam
`eter. It will be apparent that a measurement of the
`volume of these microspheres gives a sensitive indication
`of the diameter of the microspheres.
`The coe?icient of variation for the volume distribution
`is determined according to the following equation.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is a schematic illustrating an apparatus useful in
`the practice of the invention.
`FIG. 2 illustrates the degree of uniformity of volume
`and of ?uorescence of representative plastic microspheres
`produced according to the process of this invention.
`FIG. 3 illustrates the degree of uniformity of volume of
`representative zirconia microspheres produced according
`to the process of this invention.
`As shown in FIG. 1, core liquid 16 stored in reservoir
`1 at a desired pressure ?ows through a suitable line 2 to
`vibration chamber 3 where vibrational energy is intro
`duced at a desired frequency by means of an oscillator 4.
`The core liquid then flows through line 5 into injection
`tube 6 and emerges from nozzle 7 as a jet 11. Injection
`tube 6, which is centered coaxially in ?ow chamber 10,
`extends vertically from ?ow chamber 10 through end
`closure 18 and only partially through the length of ?ow
`chamber 10. Sheath liquid 17 stored at a desired pressure
`in reservoir 8 flows through line 9 to ?ow chamber 10,
`around injection tube 6 and core liquid jet 11 and emerges
`from nozzle 19 to be collected in a stirred beaker 14.
`Neither the location of sheath liquid inlet 20 nor the shape
`of ?ow chamber 10 are critical except as they may cause
`the flow of sheath liquid through region 21 to be non~
`laminar. It is essential that sheath liquid ?ow in region 21
`where the sheath liquid surrounds core liquid jet 11 and
`newly formed core liquid droplets 12 be laminar.
`Because of the vibrational energy imparted to the core
`liquid ?ow at vibration chamber 3, the pressure at nozzle 7
`varies periodically around the pressure maintained in core
`liquid reservoir 1. As a consequence, the velocity of core
`liquid jet 11 varies periodically, producing minute disturb
`ances or bunching on jet 11. By producing these ditsurb
`ances at a proper frequency, determined by the jet diam
`eter and velocity, they are made to grow in amplitude by
`surface tension until jet 11 is broken into evenly spaced,
`uniformly sized droplets 12. Because both the sheath
`liquid flow and core liquid ?ow are laminar, droplets 12
`once produced maintain substantially constant spacing as
`they emerge from nozzle 19 and ?ow 13 to beaker 14
`where they are dispersed. Maintenance of such uniform
`spacing between droplets is essential if collisions between
`droplets and hence coalescence to give droplets with
`volumes 2, 3, or 4 times that of single droplets is to be
`minimized before the droplets are dispersed in beaker 14.
`Collision and coalescence of droplets is a major reason for
`the formation of nonuniform microspheres.
`It is therefore critical to this invention that a laminar
`core liquid jet 11 be periodically disturbed when it enters
`into a laminar ?ow of sheath liquid as in region 21 of flow
`chamber 10. The periodic disturbance may take the form
`of axial velocity modulation of the core liquid. One man
`ner in which such axial velocity modulation can be readily
`achieved is shown by vibration chamber 3 and oscillator
`4 of FIG. 1. However, the vibrational energy can be intro~
`duced in a manner other than that shown in FIG. 1. Alter
`natively, the entire injection tube 6 can be vibrated along
`its long axis, or vibrational energy can be introduced into
`the sheath liquid ?ow, causing the pressure difference
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`Full width at half maximum
`1
`Modal value
`X 100X—_C'V'
`2.35
`
`The last factor in the equation represents approximately
`the relationship between resolution and coef?cient of vari
`ation for Gausian distribution.
`
`DESCRIPTION OF THE PREFERRED
`EMBODIMENTS
`
`The apparatus of FIG. 1 can be used to produce uni
`form microspheres having any desired diameter within the
`range of about 2 to 40 microns although these limits have
`been challenged. The inside diameter of nozzle 7 is 50
`microns while that of nozzle 19 is 1 mm. The ID. of
`?ow chamber 22 is 3 mm. Typically, the core liquid is held
`in reservoir 1 at a pressure of about 50 p.s.i., while the
`sheath liquid is held in reservoir 8 at a pressure of about
`30 p.s.i. Under these conditions the flow rates of core and
`sheath liquid through region 21 are about 0.9 cc./min.
`and about 110 cc./min., respectively. Note that the diow
`rate through an ori?ce of given size under a set pressure
`ditference is dependent on the viscosity of the liquid. The
`oscillator 4 normally operates at a frequency of about 20
`kHz. This results in droplets and therefore microspheres
`being formed at a rate of about 20,000 per second.
`
`EXAMPLE I
`
`The apparatus of FIG. 1 was used to produce uniform
`thoria microspheres in the following manner. A core
`liquid consisting of an aqueous solution containing 4.15
`mg./cc. of thoria sol was placed in reservoir 1 at a pres
`sure of 50 p.s.i. A sheath liquid consisting of 2-ethyl-l
`hexanol containing 0.2% by volume of ammonium hy
`droxide, 0.005% by volume of Ethomeen S/ 15, and
`0.05% by volume of Triton X-100 was placed in res
`ervoir 8 at a pressure of 30‘ p.s.i. Ethomeen 8/15 is a
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`surfactant which consists of an ethylene oxide condensa
`tion product of the primary fatty tertiary aminess. Triton
`X-100 is a surfactant product of Rohm & HaasCompany
`which consists of alkylphenoxy polyethoxy ethanol with
`9 or 10 oxyethylene groups. At these respective pressures,
`the ?ow rate of the core liquid was approximately 0.9
`cc. /min. while that of the sheath liquid was approximately
`110 cc./min. Once flow of core liquid and sheath liquid
`had commenced through injection tube 6 and flow cham
`ber 22, respectively, oscillator 4 was operated at a frequen~
`cy of about 19.2 kHz. This imparted a periodic disturb
`ance to the core liquid and produced 19,200 droplets per
`second from the core liquid jet emerging from nozzle 7.
`Nozzle 7 had an I.D. of 50 microns. These droplets were
`held in a spaced relationship to each other by the laminar
`flow of sheath liquid through region 21 of the apparatus
`of FIG. 1. The droplets and their surrounding sheath
`liquid were allowed to ?ow into a beaker which was
`magnetically stirred. The water in the droplets is slightly
`soluble in the 2-ethylhexanol so that it diffuses out, leav
`ing gelled microspheres. The droplets are left in the sheath
`liquid for a time su?icient for the microspheres to gel.
`In this particular example, the droplets were left in the
`2-ethylhexanol for approximately 20 minutes. The gelled
`microspheres thus produced had diameters of 14 microns.
`They were then dried at 180° C. and calcined at 950°
`C. The thoria microspheres produced on calcining had
`diameters of 8.3 microns. The volume distribution of
`these microspheres was similar to that shown in FIG. 3
`for zirconia microspheres produced in a similar manner.
`
`EXAMPLE II
`
`The apparatus of FIG. 1 was used to produce uniform
`zirconia microspheres in the following manner. A core
`liquid consisting of an aqueous solution containing 5.3
`mg./cc. of zirconia sol was placed in reservoir 1 at a
`pressure of 49 p.s.i. A sheath liquid consisting of 2-ethyl
`l-hexanol containing 0.1% by volume of ammonium hy
`droxide and 0.05% by volume of Triton X~100 was
`placed in reservoir 8 at a pressure of 34 p.s.i. Triton X
`100 is a surfactant which consists of alkylphenoxy poly
`ethoxy ethanol with 9 or 10 oxyethylene groups. At these
`respective pressures, the ?ow rate of the core liquid was
`approximately 0.6 cc./min. while that of the sheath
`liquid was approximately 100 cc./min. Once ?ow of core
`liquid and sheath liquid had commenced through injec
`tion tube 6 and ?ow chamber 22, respectively, oscillator
`4 was operated at a frequency of about 20.6 kHz. This
`imparted a periodic disturbance to the core liquid and
`produced 20,600 droplets per second from the core liquid
`jet emerging from nozzle 7. Nozzle 7 had an ID. of 50
`microns. These droplets were held in spaced relationship
`to each other by the laminar ?ow of sheath liquid through
`region 21 of the apparatus of FIG. 1. The droplets and
`their surrounding sheath liquid were allowed to ?ow into
`a beaker which was magnetically stirred. The water in
`the droplets diffused into the Z-ethylhexanol in the man
`ner described in Example I. The droplets were left in the
`Z-ethylhexanol for approximately 20 minutes. The gelled
`microspheres thus produced had diameters of 18 microns.
`They were then removed, dried, and calcined at 950° C.
`The zirconia microspheres produced on calcining had
`diameters of 10 microns. The coe?icient of variation in
`volume of the microspheres, as calculated from the curve
`shown in FIG. 3, was 3.7%.
`
`EXAMPLE III
`
`The apparatus of FIG. 1 was used to produce uniform
`polystyrene microspheres containing a ?uoroescent dye in
`the following manner. A core liquid consisting of a 25%
`dichloroethane-75% dichloromethane solution containing
`0.0533% by wt./vol. of polystyrene and 1.5><10—5 g./l.
`of solvent of a ?uorescent dye was placed in reservoir 1
`at a pressure of 6 p.s.i. The dye was Maxilon Brilliant
`
`3,790,492
`6
`Flavine 10GFF (63040) manufactured by Geigy Com
`pany. A sheath liquid consisting of water containing
`0.15% by volume of Liquinox was placed in reservoir 8
`at a pressure of 4.2 p.s.i. Liquinox is a surfactant pro
`duced by Alconox, Inc. At these respective pressures, the
`?ow rate of the core liquid was approximately 0.17 cc./
`min. while that of the sheath liquid was approximately
`60 cc./min. Once ?ow of core liquid and sheath liquid
`had commenced through injection tube 6 and ?ow cham~
`ber 22, respectively, oscillator 4 was operated at a frequen
`cy of about 6,300 cycles/ second. This imparted a periodic
`disturbance to the core liquid and produced 6,300 droplets
`per second from the core liquid jet emerging from nozzle
`7. Nozzle 7 had an I.D. of 31 microns. These droplets
`were held in a spaced relationship to each other by the
`laminar ?ow of sheath liquid through region 21 of the
`apparatus of FIG. 1. The droplets and their surrounding
`sheath liquid were allowed to ?ow into a beaker which
`was magnetically stirred. The solvent in the droplets is
`slightly soluble in the water of the sheath liquid so that
`it diffuses out, leaving solid microspheres of polystyrene.
`The droplets were left in the water for approximately 20
`minutes. The polystyrene microspheres produced had
`diameters of 10 microns. The coe?icient of variation in
`volume of the microspheres, as calculated from the vol
`ume curve of FIG. 2, was 2.9%. The coefficient of varia—
`tion in ?uorescence, as calculated from the ?uorescence
`curve of FIG. 2, was 4%.
`Examples I and II disclose the formation of small very
`uniform microspheres of thoria and zirconia from a start
`ing material comprising an aqueous sol of the oxide. It
`is well known in the art that numerous metal oxides form
`such sols and can be reduced to gels by appropriate dehy
`dration techniques. In particular, Clinton et al. in U.S. Pat.
`3,290,122 disclose the formation of microspheres by the
`“sol-gel” process from actinide metal oxides. On the basis
`of that patent and various other disclosures in the liter
`ature, one of reasonable skill in the art will realize that
`my method of forming small uniform microspheres is
`not limited to formation of thoria and zirconia micro
`spheres but is applicable to the production of a wide
`variety of metal oxide and mixed metal oxide gel micro
`spheres in accordance with sol-gel techniques. For ex
`ample, such techniques are described in some detail in the
`International Atomic Energy Agency document “Sol-Gel
`Processes for Ceramic Nuclear Fuels” (Vienna, 1968).
`US. Pat. 3,290,122 is hereby incorporated into and made
`a part of this application by reference.
`In US. vPat. 3,422,167, issued Jan. 14, 1969, Bowman
`et al. disclose metal oxide gel microspheres of alumina,
`zirconia, hafnia, europia, thoria, urania, plutonia, and
`mixtures thereof formed by jetting a corresponding metal
`oxide sol into a freezing medium, freezing, subsequently
`dehydrating by vacuum distillation after removal of the
`freezing medium, and calcining into ?red product. US.
`Pat. 3,422,167 is hereby incorporated into and made a
`part of this application by reference. In US. Pat. 3,551,
`533, issued Dec. 29, 1970, Monforte et al. disclose the
`formation of uniform spheres of a wide variety of mate
`rials by freeze-drying an atomized solution. Again, it will
`be readily understood that my method of forming small
`uniform microspheres is easily adapted to such freeze
`dry techniques by using a sheath liquid that will serve
`as an appropriate freezing medium.
`What is claimed is:
`1. A method for producing uniformly sized particles
`which comprises introducing through a two-fluid nozzle a
`laminar jet of a stable hydrous oxide sol core liquid into
`a laminar ?ow sheath liquid consisting of an organic liquid
`having a water solubility of 0.3 to 10 volume percent
`and a solubility in water of less than one volume percent
`and containing a surfactant, said core liquid jet being
`periodically disturbed prior to its introduction into the
`laminar ?ow sheath liquid, to form uniformly sized drop
`lets of core liquid in uniform space relation within said
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`7
`laminar ?ow sheath liquid, allowing the droplets of core
`liquid thus produced to remain within the sheath liquid
`for a time suf?cient for the material to form condensed
`spheres between 2 and 40 microns in diameter and then
`’ removing said spheres from the sheath liquid.
`
`3,790,492
`
`8
`3,352,950 11/1967 ‘Helton et al _________ __ 264—.5
`3,463,842
`8/1969 Flack et a1, ____ __ 252_301_1 s
`3,617,534 11/1971 Flack et a1, ____ __ 252__301,1S
`
`5 STEPHEN J. LECHERT, JR., Primary Examiner
`
`References Cited
`UNITED STATES PATENTS
`
`264——-5
`
`U.S. Cl. X.R.
`
`l
`
`3,331,898
`3,340,567
`
`7/1967 Haaset a1 ___________ __ 264—.5 10
`9/1967 Flack et a1. ______ __ 264—.5X
`
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