U.S. patent application number 10/558541 was filed with the patent office on 2007-06-21 for novel nanocomposites and their application as monolith columns.
This patent application is currently assigned to Waters Investments Limited. Invention is credited to Julia Ding, John E. O'Gara, Daniel Walsh.
Application Number | 20070141325 10/558541 |
Document ID | / |
Family ID | 33490689 |
Filed Date | 2007-06-21 |
United States Patent
Application |
20070141325 |
Kind Code |
A1 |
O'Gara; John E. ; et
al. |
June 21, 2007 |
Novel nanocomposites and their application as monolith columns
Abstract
Novel materials for chromatographic separations, processes for
their preparation, and separation devices containing the
chromatographic materials. In particular, hybrid inorganic/organic
monolith materials comprising a polymerized scaffolding
nanocomposite (PSN), wherein the nanocomposite contains a
scaffolding functionality capable of chemically interacting with a
surface of a second material are described. The hybrid
inorganic/organic materials have enhanced wall adhesion and
increased resistance to shrinkage as compared to prior art monolith
materials. The improved adhesion of the monoliths enable the
preparation of capillary columns with an internal diameter (I.D.)
.gtoreq.50 .mu.m.
Inventors: |
O'Gara; John E.; (Ashland,
MA) ; Ding; Julia; (Madison, WI) ; Walsh;
Daniel; (Danvers, MA) |
Correspondence
Address: |
EDWARDS & ANGELL, LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
Waters Investments Limited
109 Lukens Drive
New Castle
DE
19720
|
Family ID: |
33490689 |
Appl. No.: |
10/558541 |
Filed: |
May 3, 2004 |
PCT Filed: |
May 3, 2004 |
PCT NO: |
PCT/US04/13721 |
371 Date: |
February 15, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60474068 |
May 28, 2003 |
|
|
|
Current U.S.
Class: |
428/332 ;
428/429; 428/447; 528/12; 977/701 |
Current CPC
Class: |
B01J 20/285 20130101;
B01J 2220/82 20130101; B01J 20/26 20130101; B82Y 30/00 20130101;
B05D 7/22 20130101; B01D 15/265 20130101; Y10T 428/26 20150115;
Y10T 428/31663 20150401; C08G 77/20 20130101; C08J 5/005 20130101;
B01D 15/22 20130101; B01D 15/206 20130101; B01J 2220/86 20130101;
Y10T 428/31612 20150401 |
Class at
Publication: |
428/332 ;
428/429; 428/447; 528/012; 977/701 |
International
Class: |
B32B 1/02 20060101
B32B001/02 |
Claims
1. A hybrid inorganic/organic material comprising a polymerized
scaffolding nanocomposite (PSN), wherein the nanocomposite contains
a scaffolding functionality capable of chemically interacting with
a surface of a second material.
2. The hybrid material of claim 1, wherein the second material is a
containment vessel.
3. The hybrid material of claim 1, wherein the scaffolding
functionality is selected from the group consisting of vinyl,
acrylate, methacrylate, acrylamide, methacrylamide, styrene,
divinylbenzene, itaconate, fumarate, alkyne, and combinations
thereof.
4. The hybrid material of claim 1, wherein the surface of the
second material is derivatized with an anchoring functionality.
5. The hybrid material of claim 4, wherein the anchoring
functionality is selected from the group consisting of vinyl,
acrylate, methacrylate, acrylamide, methacrylamide, styrene,
divinylbenzene, itaconate, fumarate, alkyne, azo compounds, and
combinations thereof.
6. The hybrid material of claim 4, wherein the scaffolding
functionality and the anchoring functionality are
copolymerizable.
7. The hybrid material of claim 2, wherein containment vessel is
selected from the group consisting of a capillary column, a glass
lined steel column, a radial compression column, a trap column, a
microfluidic device, a microchip, a sensor, an electronic circuit,
a miniaturized SPE device, and an on-column frit.
8. The hybrid material of claim 2, where the containment vessel is
a fused silica capillary column.
9. The hybrid material of claim 1, wherein the chemical interaction
is formation of a covalent bond.
10. The hybrid material of claim 9, wherein the covalent bond is
formed by polymerization.
11. The hybrid material of claim 10, wherein the polymerization is
initiated with a radical initiator.
12. The hybrid material of claim 11, wherein the radical initiator
is minimally water soluble.
13. The hybrid material of claim 11, wherein the initiator is
selected from the group consisting of
2,2'-azobis(isobutyronitrile),
2,2'-azobis(2-methylpropionamidine)dihydrochloride,
4,4'-azobis(4-cyanovaleric acid), potassium persulfate, and
peracetic acid.
14. The hybrid material of claim 1, wherein the inorganic portion
of the hybrid material is a material selected from the group
consisting of alumina, silica, titanium oxide, zirconium oxide, and
ceramic material.
15. The hybrid material of claim 1, wherein the inorganic portion
of the hybrid material is silica.
16. The hybrid material of claim 1, wherein the PSN is the product
of a reaction of an organosilane and an inorganic silane
monomer.
17. The hybrid material of claim 16, wherein the PSN is the product
of a reaction of a tetraalkoxysilane and an organosilane containing
at least one polymerizable group.
18. The hybrid material of claim 17, wherein said tetraalkoxysilane
has the formula Si(OR.sup.1).sub.4, where R.sup.1 is a
C.sub.1-C.sub.3 alkyl moiety.
19. The hybrid material of claim 17, wherein said organosilane is
an organoalkoxysilane having the formula R.sup.2Si(OR.sup.1).sub.3
or R.sup.6[Si(OR.sup.1).sub.3].sub.m where R.sup.2 is a styryl,
vinyl, an acrylate, methacrylate, acrylamide, methacrylamide,
divinylbenzene, itaconate, fumarate, substituted or unsubstituted
C.sub.1-C.sub.18 alkenylene, alkynylene or arylene, or a
combination thereof; R.sup.1 is a C.sub.1-C.sub.4 alkyl moiety;
R.sup.6 is a substituted or unsubstituted C.sub.1-C.sub.18
alkenylene, alkynylene or arylene moiety bridging two or more
silicon atoms; and m is an integer greater than or equal to
two.
20. The hybrid material of claim 19 wherein R.sup.2 is vinyl,
methacryloxypropyl, methacrylamidepropyl, or styrylethyl and
R.sup.1 is methyl or ethyl; or R.sup.6 is a bridging
N,N-bis(propylene)acrylamide group, m=2, and R.sup.1 is ethyl or
methyl.
21. The hybrid material of claim 17, wherein the organosilane is
minimally water soluble.
22. The hybrid material of claim 17 wherein said tetraalkoxysilane
is selected from the group consisting of tetramethoxysilane and
tetraethoxysilane.
23. The hybrid material of claim 17, wherein the tetraalkoxysilane
is tetramethoxysilane.
24. The hybrid material of claim 17, wherein the polymerizable
group is 3-methacryloxypropyl.
25. The hybrid material of claim 17, wherein the polymerizable
group is styrylethyl.
26. The hybrid material of claim 17, wherein the tetraalkoxysilane
is minimally water soluble.
27. The hybrid material of claim 17, wherein the organosilane is
(3-methacryloxypropyl)trimethoxysilane.
28. The hybrid material of claim 1, wherein said pore structure of
said hybrid material is modified by further including a surfactant
or combination of different surfactants in said reaction, and by
subjecting said material to hydrothermal treatment.
29. The hybrid material of claim 28, wherein said surfactant or
combination of surfactants are nonionic surfactants.
30. The hybrid material of claim 29, wherein the surfactants are
selected from the group consisting of surfactants comprised of
block copolymers of polyethylene glycol and polypropyleneglycol,
surfactants comprised of alkylphenoxypolyethoxyethanol, and
polyethyleneglycol.
31. The hybrid material of claim 29, wherein the surfactant is
Pluronic F38,
32. The hybrid material of claim 28, wherein said surfactant or
combination of surfactants are selected from surfactants with a
hydrophile-lipophile balance ranging from about 0 to 60.
33. The hybrid material of claim 28, wherein said surfactant or
combination of surfactants are selected from surfactants with a
hydrophile-lipophile balance ranging from about 10 to 50.
34. The hybrid material of claim 28, wherein said surfactant or
combination of surfactants are selected from surfactants with a
hydrophile-lipophile balance ranging from about 20 to 40.
35. The hybrid material of claim 28, wherein said surfactant or
combination of surfactants are selected from surfactants with a
hydrophile-lipophile balance ranging from about 30 to 40.
36. The hybrid material of claim 28, wherein said surfactant or
combination of surfactants are selected from surfactants with a
hydrophile-lipophile balance is about 33.
37. The hybrid material of claim 1, wherein said material has been
surface modified by a surface modifier selected from the group
consisting of an organic group surface modifier, a silanol group
surface modifier, a polymeric coating surface modifier, and
combinations thereof.
38. The hybrid material of claim 37, wherein said material has been
surface modified by a polymeric coating surface modifier.
39. The hybrid material of claim 37, wherein said material has been
surface modified by a combination of an organic group surface
modifier and a silanol group surface modifier.
40. The hybrid material of claim 37, wherein said material has been
surface modified by a combination of an organic group surface
modifier and a polymeric coating surface modifier.
41. The hybrid material of claim 37, wherein said material has been
surface modified by a combination of a silanol group surface
modifier and a polymeric coating surface modifier.
42. The hybrid material of claim 37, wherein said material has been
surface modified by a combination of an organic group surface
modifier, a silanol group surface modifier, and a polymeric coating
surface modifier.
43. The hybrid material of claim 37, wherein said material has been
surface modified by a silanol group surface modifier.
44. The hybrid material of claim 37, wherein said material has been
surface modified via formation of an organic covalent bond between
an organic group of the material and a surface modifier.
45. The hybrid material of claim 37, wherein the surface modifier
has the formula Z.sub.a(R').sub.bSi--R, where Z=Cl, Br, I,
C.sub.1-C.sub.5 alkoxy, dialkylamino or trifluoromethanesulfonate;
a and b are each an integer from 0 to 3 provided that a+b=3; R' is
a C.sub.1-C.sub.6 straight, cyclic or branched alkyl group, and R
is a functionalizing group.
46. The hybrid material of claim 45 wherein R' is selected from the
group consisting of methyl, ethyl, propyl, isopropyl, butyl,
t-butyl, sec-butyl, pentyl, isopentyl, hexyl and cyclohexyl.
47. The hybrid material of claim 45 wherein said functionalizing
group R is a C.sub.1-C.sub.30 alkyl group.
48. The hybrid material of claim 45 wherein said functionalizing
group R is a C.sub.1-C.sub.20 alkyl group.
49. The hybrid material of claim 45 wherein said surface modifier
is selected from the group consisting of octyltrichlorosilane,
octadecyltrichlorosilane,
octadecyldimethyl-N,N-dimethylaminosilane,
octyldimethylchlorosilane, and octadecyldimethylchlorosilane.
50. The hybrid material of claim 45, wherein said surface modifier
is octadecyldimethyl-N, N-dimethylaminosilane.
51. The hybrid material of claim 45, wherein said functionalizing
group R is selected from the group consisting of alkyl, alkenyl,
alkynyl, aryl, cyano, amino, diol, nitro, ester, a cation or anion
exchange group, an alkyl group containing an embedded polar
functionality and an aryl group containing an embedded polar
functionality.
52. A hybrid inorganic/organic monolith comprising a polymerized
scaffolding nanocomposite (PSN), wherein the nanocomposite contains
a scaffolding functionality capable of chemically interacting with
a surface of a second material.
53-105. (canceled)
106. A method of preparation of the hybrid inorganic/organic
monolith of claim 52, said method comprising the steps of a)
forming a sol-gel by the reaction of two or more monomers; b)
initiating a polymerization reaction; and c) allowing the monomers
to react through a polymerization sol-gel (PSG) reaction, thereby
preparing the hybrid inorganic/organic monolith.
107. The method of claim 106 further comprising modifying the pore
structure of the material.
108-181. (canceled)
182. A separations device comprising a) a surface capable of
accepting a monolith material comprising a polymerized scaffolding
nanocomposite (PSN) material, said surface comprising an anchoring
functionality and b) a hybrid inorganic/organic monolith comprising
a polymerized scaffolding nanocomposite (PSN), wherein the
nanocomposite contains a scaffolding functionality capable of
chemically interacting with the anchoring functionality on said
surface, and wherein said hybrid organic/inorganic monolith is
anchored to said surface by a chemical interaction between said
scaffolding functionality and anchoring functionality.
183. The separations device of claim 182, wherein said device is
selected from the group consisting of chromatographic columns, thin
layer plates, filtration membranes, sample cleanup devices, and
microtiter plates.
184-193. (canceled)
194. The separations device of claim 183, wherein the device is a
fused-silica capillary column.
195. The fused-silica capillary column of claim 194, wherein the
capillary column has an inner diameter (I.D.) greater than 50
.mu.m.
196-245. (canceled)
246. A method of in situ preparation of a hybrid inorganic/organic
monolith in a fused-silica capillary column, said method
comprising: forming an anchoring functionality on an interior
surface of said capillary column; and forming inside said capillary
column a hybrid inorganic/organic monolith comprising a polymerized
scaffolding nanocomposite (PSN), wherein the nanocomposite contains
a scaffolding functionality capable of chemically interacting with
the anchoring functionality on said surface, said monolith being
formed by: a) forming a sol-gel by the reaction of two or more
monomers; b) initiating a polymerization reaction; and c) allowing
the monomers to react through a polymerization sol-gel (PSG)
reaction; whereby said scaffolding functionality and said anchoring
functionality chemically interact to thereby anchor said monolith
to said surface, such that a hybrid inorganic/organic monolith is
prepared in situ in the fused-silica capillary column.
247. The method of claim 246 further comprising modifying the pore
structure of the monolith.
248. The inorganic/organic hybrid monolith of claim 52, produced by
a process comprising the steps of a) forming a sol-gel by the
reaction of two or more monomers; b) initiating a polymerization
reaction; and c) allowing the monomers to react through a
polymerization sol-gel (PSG) reaction.
249. The inorganic/organic hybrid monolith of claim 248, wherein
the process further comprises modifying the pore structure of the
monolith.
250. A method of preparation of a hybrid inorganic/organic material
of claim 1, comprising the steps of a) forming a sol-gel by the
reaction of two or more monomers; b) initiating a polymerization
reaction; and c) allowing the monomers to react through a
polymerization sol-gel (PSG) reaction, thereby preparing the hybrid
inorganic/organic material.
251. The method of claim 250, further comprising modifying the pore
structure of the material.
Description
RELATED APPLICATION
[0001] This application claims the benefit of U.S. provisional
patent application Ser. No. 60/474,068 filed May 28, 2003, the
disclosure of which application is incorporated herein in its
entirety by this reference.
BACKGROUND OF THE INVENTION
[0002] Packing materials for liquid chromatography (LC) are
generally classified into two types: organic materials, e.g.,
polydivinylbenzene; and inorganic materials typified by silica Many
organic materials are chemically stable against strongly alkaline
and strongly acidic mobile phases, allowing flexibility in the
choice of mobile phase pH. However, organic chromatographic
materials generally result in columns with low efficiency, leading
to inadequate separation performance, particularly with low
molecular-weight analytes. Furthermore, many organic
chromatographic materials shrink and swell when the composition of
the mobile phase is changed. In addition, most organic
chromatographic materials do not have the mechanical strength of
typical chromatographic silicas.
[0003] Due in large part to these limitations, silica is the
material most widely used in High Performance Liquid Chromatography
(HPLC). The most common applications employ silica that has been
surface-derivatized with an organic group such as octadecyl
(C.sub.18), octyl (C.sub.8), phenyl, amino, cyano, etc. As
stationary phases for HPLC, these packing materials result in
columns that have high efficiency and do not show evidence of
shrinking or swelling.
[0004] However, a further problem associated with silica particles
and polymer particles is packed bed stability. Chromatography
columns packed with spherical particles can be considered to be
random close packed lattices in which the interstices between the
particles form a continuous network from the column inlet to the
column outlet. This network forms the interstitial volume of the
packed bed, and acts as a conduit for fluid to flow through the
packed column. In order to achieve maximum packed bed stability,
the particles must be tightly packed, and hence, the interstitial
volume is limited in the column. As a result, such tightly packed
columns afford high column backpressures that are not desirable.
Moreover, bed stability problems for these chromatography columns
are still typically observed because of particle
rearrangements.
[0005] A trend in current HPLC development is the miniaturization
of column diameters that is driven by the often limited amount of
samples originating from such areas as the life sciences. For mini-
and microbore columns as well as capillary columns, the trade-off
between particle size and backpressure becomes even more
pronounced. For example, MacNair et al..sup.(1) required
specifically designed hardware that enabled an operating pressure
as high as 500 MPa in order to achieve a HPLC separation of a
tryptic digest in a 25 cm long capillary column packed with 1 .mu.m
silica particles. The pressure is one order of magnitude higher
than the typical 40 MPa limitation of a commercial HPLC system.
[0006] In an attempt to overcome the combined problems of packed
bed stability and high efficiency separations at low backpressures
and high flow rates, several groups have reported the use of
monolith materials in chromatographic separations. Monolith
materials are characterized by a continuous, interconnected pore
structure of large macropores, the size of which can be changed
independent of the skeleton size without causing bed instability.
The presence of large macropores allows liquid to flow directly
through with very little resistance resulting in very low
backpressures even at high flow rates.
[0007] Monolith columns have been designed to disobey the trade-off
rule associated with packed particle beds. Theoretically, they can
exhibit combined properties of low backpressure and high efficiency
that go beyond the limits of packed particle columns in
pressure-driven liquid chromatography. Capillary monolith columns
comprising polymeric, inorganic silica and organic-inorganic hybrid
materials have been studied and reported in the literature..sup.(2,
3) The polymeric monoliths are made primarily via a radical
polymerization of methacrylate or styrene-divinylbenzene(DVB)
monomers and are used under electroosmotic flow in
electrochromatography applications and low pressure pump driven
applications because of their limited mechanical strength under
high pressure.
[0008] Silica monoliths have also been applied in HPLC separations
by Nakanishi et al..sup.(3) and have demonstrated an efficiency
similar to 5 .mu.m particles but with permeability 25-30 times
higher. However, due to the shrinkage of the silica skeleton,
silica capillaries with an I.D. larger than 50 .mu.m showed much
lower efficiency, and in all cases 5-15% of the length of each
capillary end had to be cut off to remove large voids caused by
shrinkage that formed between the monolith and capillary wall
before the capillary could be used.
[0009] In another publication, Nakanishi et al..sup.(4)
demonstrated the possibility of making a capillary column of 200
.mu.m internal diameter (I.D.) from a mixture of tetramethoxysilane
and methyltrimethoxysilane. However, these hybrid-type silica
monoliths capillaries still had large voids caused by shrinkage
that formed between the monolith and capillary wall and required
cutting of 5-15% of the length of each capillary end before use.
The hybrid-type silica monolith also had a three-fold increase in
separation impedance versus the analogous silica monolith column of
50 .mu.m I.D.
[0010] Polymeric capillary monolith columns prepared by a UV
polymerization of (3-methacryloxypropyl)trimethoxysilane were first
reported by Zare et al. in 2001..sup.(5) The elution order of the
Zare column is similar to that of a reversed-phase column where
larger molecular weight or more hydrophobic analytes elute later
than the smaller molecular weight or more hydrophilic analytes,
indicating that the polymerized methylacrylate groups are located
on the surface of the monolith structure. Although Zare's work has
been successfully applied in electrochromatography, poor column
efficiency, poor adhesion between the capillary wall and the
monolith structure, and inhomogeneity of the monolith structure
were observed in pressure driven separations..sup.(6) Moreover, as
a consequence of the utilization of photopolymerization rather than
thermal polymerization, the polyimide coating of the glass
capillary must be removed prior to use. This unprotected fused
silica tubing becomes very fragile and is easily broken. Therefore,
only columns with a limited length can be prepared by this
method.
[0011] Current monolith columns have significant shrinkage,
resulting in poor wall adhesion, and consequently, only columns
with an I.D. of less than 150 .mu.m have been prepared. Therefore,
a need exists for novel materials that overcome the problems that
are associated with known materials. In particular, there is a need
for monolith materials with increased resistance to shrinkage and
enhanced wall adhesion that can be used to prepare chromatographic
columns with an I.D. of 150 .mu.m and greater.
SUMMARY OF THE INVENTION
[0012] The present invention provides novel hybrid
inorganic/organic materials and methods for their preparation. In
particular, the invention provides nanocomposite monolith materials
having increased resistance to shrinkage and novel physical
characteristics. The nanocomposites of the invention have enhanced
capillary wall adhesion as compared to prior art monolith
materials. The improved adhesion of the monoliths of the invention
enables the preparation of capillary columns with an internal
diameter (I.D.) .gtoreq.150 .mu.m.
[0013] Accordingly, in one aspect, the invention provides a hybrid
inorganic/organic material comprising a polymerized scaffolding
nanocomposite (PSN), wherein the nanocomposite contains a
scaffolding functionality capable of chemically interacting with a
surface of a second material.
[0014] In another aspect, the invention provides a hybrid
inorganic/organic monolith comprising a polymerized scaffolding
nanocomposite (PSN), wherein the nanocomposite contains a
scaffolding functionality capable of chemically interacting with a
surface of a second material.
[0015] Additionally, the present invention provides a method of
preparation of a hybrid inorganic/organic monolith comprising a
polymerized scaffolding nanocomposite (PSN), wherein the
nanocomposite contains a scaffolding functionality capable of
chemically interacting with a surface of a second material, said
method comprising the steps of
[0016] a) forming a sol-gel by the reaction of two or more
monomers;
[0017] b) initiating a polymerization reaction; and
[0018] c) allowing the monomers to react through a polymerization
sol-gel (PSG) reaction,
thereby preparing the hybrid inorganic/organic monolith.
[0019] In a related aspect, the invention provides a method of
preparation of a hybrid 15 inorganic/organic monolith comprising a
polymerized scaffolding nanocomposite (PSN), wherein the
nanocomposite contains a scaffolding functionality capable of
chemically interacting with a surface of a second material, said
method comprising the steps of
[0020] a) forming a sol-gel by the reaction of two or more
monomers;
[0021] b) initiating a polymerization reaction;
[0022] c) allowing the monomers to react through a polymerization
sol-gel (PSG) reaction; and
[0023] d) modifying the pore structure of the material,
thereby preparing the hybrid inorganic/organic monolith.
[0024] In yet another aspect, the invention provides a separations
device comprising
[0025] a) a surface capable of accepting a monolith material
comprising a polymerized scaffolding nanocomposite (PSN) material,
said surface comprising an anchoring functionality and
[0026] b) a hybrid inorganic/organic monolith comprising a
polymerized scaffolding nanocomposite (PSN), wherein the
nanocomposite contains a scaffolding functionality capable of
chemically interacting with the anchoring functionality on said
surface, and wherein said hybrid organic/inorganic monolith is
anchored to said surface by a chemical interaction between said
scaffolding functionality and anchoring functionality.
[0027] In a related aspect, the invention provides a fused-silica
capillary column, comprising
[0028] a) a fused-silica capillary column having a cylindrical
interior surface capable of accepting a monolith comprising a
polymerized scaffolding nanocomposite (PSN) material, said interior
surface comprising an anchoring functionality, and
[0029] b) a hybrid inorganic/organic monolith comprising a
polymerized scaffolding nanocomposite (PSN), wherein the
nanocomposite contains a scaffolding functionality capable of
chemically interacting with the anchoring functionality on said
surface, and wherein said hybrid organic/inorganic monolith is
anchored to said surface by a chemical interaction between said
scaffolding functionality and anchoring functionality.
[0030] Another aspect of the invention provides a method of in situ
preparation of a hybrid inorganic/organic monolith in a
fused-silica capillary column, said method comprising: [0031]
forming an anchoring functionality on an interior surface of said
capillary column; and [0032] forming inside said capillary column a
hybrid inorganic/organic monolith comprising a polymerized
scaffolding nanocomposite (PSN), wherein the nanocomposite contains
a scaffolding functionality capable of chemically interacting with
the anchoring functionality on said surface, said monolith being
formed by:
[0033] a) forming a sol-gel by the reaction of two or more
monomers;
[0034] b) initiating a polymerization reaction; and
[0035] c) allowing the monomers to react through a polymerization
sol-gel (PSG) reaction;
whereby said scaffolding functionality and said anchoring
functionality chemically interact to thereby anchor said monolith
to said surface, such that a hybrid inorganic/organic monolith is
prepared in situ in a fused-silica capillary column.
[0036] In another related aspect, the invention provides a method
of in situ preparation of a hybrid inorganic/organic monolith in a
fused-silica capillary column, said method comprising: [0037]
forming an anchoring functionality on an interior surface of said
capillary column; and [0038] forming inside said capillary column a
hybrid inorganic/organic monolith comprising a polymerized
scaffolding nanocomposite (PSN), wherein the nanocomposite contains
a scaffolding functionality capable of chemically interacting with
the anchoring functionality on said surface, said monolith being
formed by:
[0039] a) forming a sol-gel by the reaction of two or more
monomers;
[0040] b) initiating a polymerization reaction;
[0041] c) allowing the monomers to react through a polymerization
sol-gel (PSG) reaction; and
[0042] d) modifying the pore structure of the monolith,
whereby said scaffolding functionality and said anchoring
functionality chemically interact to thereby anchor said monolith
to said surface, such that a hybrid inorganic/organic monolith is
prepared in situ in a fused-silica capillary column.
[0043] Another aspect of the invention provides an
inorganic/organic hybrid monolith comprising a scaffolding
functionality capable of chemically interacting with a surface of a
second material, produced by the process of
[0044] a) forming a sol-gel by the reaction of two or more
monomers;
[0045] b) initiating a polymerization reaction; and
[0046] c) allowing the monomers to react through a polymerization
sol-gel (PSG) reaction.
[0047] In a related aspect, the invention provides an
inorganic/organic hybrid monolith comprising a scaffolding
functionality capable of chemically interacting with a surface of a
second material, produced by the process of
[0048] a) forming a sol-gel by the reaction of two or more
monomers;
[0049] b) initiating a polymerization reaction;
[0050] c) allowing the monomers to react through a polymerization
sol-gel (PSG) reaction; and
[0051] d) modifying the pore structure of the monolith.
[0052] In yet another related aspect, the invention provides a
method of preparation of a hybrid inorganic/organic material
comprising a polymerized scaffolding nanocomposite (PSN),
comprising the steps of
[0053] a) forming a sol-gel by the reaction of two or more
monomers;
[0054] b) initiating a polymerization reaction; and
[0055] c) allowing the monomers to react through a polymerization
sol-gel (PSG) reaction,
thereby preparing the hybrid inorganic/organic monolith, wherein
the monolith contains a scaffolding functionality capable of
chemically interacting with a surface of a second material.
[0056] In another aspect, the invention provides a method of
preparation of a hybrid inorganic/organic material comprising a
polymerized scaffolding nanocomposite (PSN), comprising the steps
of
[0057] a) forming a sol-gel by the reaction of two or more
monomers;
[0058] b) initiating a polymerization reaction;
[0059] c) allowing the monomers to react through a polymerization
sol-gel (PSG) reaction; and
[0060] d) modifying the pore structure of the material,
thereby preparing the hybrid inorganic/organic material, wherein
the nanocomposite contains a scaffolding functionality capable of
chemically interacting with a surface of a second material.
[0061] Yet another aspect of the invention provides a capillary
column, wherein the interior surface of the capillary column is
derivatized with a polymerizable anchoring functionality.
BRIEF DESCRIPTION OF DRAWINGS
[0062] FIGS. 1A-1D depict a schematic representation of monoliths
made by the polymerization sol gel (PSG) reaction. FIG. 1A depicts
examples of hybrid and inorganic monomers in chemical structure and
schematic form. FIG. 1B is a schematic depiction of hybrid and
inorganic monomer hydrolysis and oligomerization. Figure C is a
simplified depiction of the PSG reaction to form a cross-sectional
block of polymerized scaffolding nanocomposite material. Figure D
is a simplified cross-sectional view of the PSN material, including
expanding views of the PSN material.
DETAILED DESCRIPTION OF THE INVENTION
[0063] The present invention provides novel hybrid
inorganic/organic materials, and methods for their preparation. In
particular, the invention provides nanocomposite monolith materials
having increased resistance to shrinkage and novel physical
characteristics. The nanocomposites of the invention have enhanced
capillary wall adhesion, as compared to prior art monolith
materials. The improved adhesion of the monoliths of the invention
enables the preparation of capillary columns with an internal
diameter (I.D.) .gtoreq.150 .mu.m.
[0064] In an advantageous embodiment, the invention provides novel,
inorganic/organic monolith materials inside a fused silica
capillary column. In accordance with this embodiment, the monoliths
of the invention are prepared in situ inside a surface-modified
fused silica capillary column through simultaneous organic
polymerization and sol-gel reaction of a silane mixture that has an
organosilane monomer containing at least one organic polymerizable
group, such as, e.g., (3-methacryloxyproply)trimethoxysilane.
Definitions
[0065] These and other embodiments of the invention will be
described with reference to following definitions that, for
convenience, are collected here.
[0066] The term "alicyclic group" includes closed ring structures
of three or more carbon atoms. Alicyclic groups include
cycloparaffins or naphthenes that are saturated cyclic
hydrocarbons, cycloolefins which are unsaturated with two or more
double bonds, and cycloacetylenes which have a triple bond. They do
not include aromatic groups. Examples of cycloparaffins include
cyclopropane, cyclohexane, and cyclopentane. Examples of
cycloolefins include cyclopentadiene and cyclooctatetraene.
Alicyclic groups also include fused ring structures and substituted
alicyclic groups such as alkyl substituted alicyclic groups. In the
instance of the alicyclics such substituents can further comprise a
lower alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a
lower alkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl,
--CF.sub.3, --CN, or the like.
[0067] The term "aliphatic group" includes organic compounds
characterized by straight or branched chains, typically having
between 1 and 22 carbon atoms. Aliphatic groups include alkyl
groups, alkenyl groups and alkynyl groups. In complex structures,
the chains can be branched or cross-linked. Alkyl groups include
saturated hydrocarbons having one or more carbon atoms, including
straight-chain alkyl groups and branched-chain alkyl groups. Such
hydrocarbon moieties may be substituted on one or more carbons
with, for example, a halogen, a hydroxyl, a thiol, an amino, an
alkoxy, an alkylcarboxy, an alkylthio, or a nitro group. Unless the
number of carbons is otherwise specified, "lower aliphatic" as used
herein means an aliphatic group, as defined above (e.g., lower
alkyl, lower alkenyl, lower alkynyl), but having from one to six
carbon atoms. Representative of such lower aliphatic groups, e.g.,
lower alkyl groups, are methyl, ethyl, n-propyl, isopropyl,
2-chloropropyl, n-butyl, sec-butyl, 2-aminobutyl, isobutyl,
tert-butyl, 3-thiopentyl, and the like. As used herein, the term
"nitro" means --NO.sub.2; the term "halogen" designates --F, --Cl,
--Br or --I; the term "thiol" means SH; and the term "hydroxyl"
means --H.
[0068] The terms "alkenyl" and "alkynyl" refer to unsaturated
aliphatic groups analogous to alkyls, but which contain at least
one double or triple bond respectively. Suitable alkenyl and
alkynyl groups include groups having 2 to about 12 carbon atoms,
preferably from 1 to about 6 carbon atoms.
[0069] The term "alkoxy" as used herein means an alkyl group, as
defined herein, having an oxygen atom attached thereto.
Representative alkoxy groups include groups having 1 to about 12
carbon atoms, preferably 1 to about 6 carbon atoms, e.g., methoxy,
ethoxy, propoxy, tert-butoxy and the like.
[0070] The term "alkyl" includes saturated aliphatic groups,
including straight-chain alkyl groups, branched-chain alkyl groups,
cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups,
and cycloalkyl substituted alkyl groups. In certain embodiments, a
straight chain or branched chain alkyl has 30 or fewer carbon atoms
in its backbone, e.g., C.sub.1-C.sub.30 for straight chain or
C.sub.3-C.sub.30 for branched chain. In certain embodiments, a
straight chain or branched chain alkyl has 20 or fewer carbon atoms
in its backbone, e.g., C.sub.1-C.sub.20 for straight chain or
C.sub.3-C.sub.20 for branched chain, and more preferably 18 or
fewer. Likewise, preferred cycloalkyls have from 4-10 carbon atoms
in their ring structure, and more preferably have 4-7 carbon atoms
in the ring structure. The term "lower alkyl" refers to alkyl
groups having from 1 to 6 carbons in the chain, and to cycloalkyls
having from 3 to 6 carbons in the ring structure.
[0071] Moreover, the term "alkyl" (including "lower alkyl") as used
throughout the specification and claims includes both
"unsubstituted alkyls" and "substituted alkyls", the latter of
which refers to alkyl moieties having substituents replacing a
hydrogen on one or more carbons of the hydrocarbon backbone. Such
substituents can include, for example, halogen, hydroxyl,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,
phosphinato, cyano, amino (including alkyl amino, dialkylamino,
arylamino, diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfate, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl,
cyano, azido, heterocyclyl, aralkyl, or an aromatic or
heteroaromatic moiety. It will be understood by those skilled in
the art that the moieties substituted on the hydrocarbon chain can
themselves be substituted, if appropriate. Cycloalkyls can be
further substituted, e.g., with the substituents described above.
An "aralkyl" moiety is an alkyl substituted with an aryl, e.g.,
having 1 to 3 separate or fused rings and from 6 to about 18 carbon
ring atoms, e.g., phenylmethyl (benzyl).
[0072] The term "alkylamino" as used herein means an alkyl group,
as defined herein, having an amino group attached thereto.
Exemplary alkylamino groups include groups having 1 to about 12
carbon atoms, preferably from 1 to about 6 carbon atoms.
[0073] The term "alkylthio" refers to an alkyl group, as defined
herein, having a sulfhydryl group attached thereto. Exemplary
alkylthio groups include groups having 1 to about 12 carbon atoms,
preferably from 1 to about 6 carbon atoms. The term "alkylcarboxyl"
as used herein means an alkyl group, as defined above, having a
carboxyl group attached thereto. The term "amino," as used herein,
refers to an unsubstituted or substituted moiety of the formula
--NR.sub.aR.sub.b, in which R.sub.a and R.sub.b are each
independently hydrogen, alkyl, aryl, or heterocyclyl, or R.sub.a
and R.sub.b, taken together with the nitrogen atom to which they
are attached, form a cyclic moiety having from 3 to 8 atoms in the
ring. Thus, the term "amino" includes cyclic amino moieties such as
piperidinyl or pyrrolidinyl groups, unless otherwise stated. An
"amino-substituted amino group" refers to an amino group in which
at least one of R.sub.a and R.sub.b, is further substituted with an
amino group.
[0074] The language "anchoring functionality" is intended to
include functional moieties that promote the ability of the second
material to chemically interact with the PSN material. In certain
embodiments, the anchoring functionality is a polymerizable group,
including, but not limited to vinyl, acrylate, methacrylate,
acrylamide, methacrylamide, styrene, divinylbenzene, itaconate,
fumarate, alkyne, azo compounds, and combinations thereof.
[0075] The term "anchoring" refers to the act of adhesion of one
material to a second material. The extent of adhesion is a direct
result of the chemical interaction of the first material with the
second material, and is intended to include a range of interactions
which extend from flexible interactions to strict
immobilization.
[0076] The term "aromatic group" includes unsaturated cyclic
hydrocarbons containing one or more rings. Aromatic groups include
5- and 6-membered single-ring groups which may include from zero to
four heteroatoms, for example, benzene, pyrrole, furan, thiophene,
imidazole, oxazole, thiazole, triazole, pyrazole, pyridine,
pyrazine, pyridazine and pyrimidine, and the like. The aromatic
ring may be substituted at one or more ring positions with, for
example, a halogen, a lower alkyl, a lower alkenyl, a lower alkoxy,
a lower alkylthio, a lower alkylamino, a lower alkylcarboxyl, a
nitro, a hydroxyl, --CF.sub.3, --CN, or the like.
[0077] The term "aryl" includes 5- and 6-membered single-ring
aromatic groups that may include from zero to four heteroatoms, for
example, unsubstituted or substituted benzene, pyrrole, furan,
thiophene, imidazole, oxazole, thiazole, triazole, pyrazole,
pyridine, pyrazine, pyridazine and pyrimidine, and the like. Aryl
groups also include polycyclic fused aromatic groups such as
naphthyl, quinolyl, indolyl, and the like. The aromatic ring can be
substituted at one or more ring positions with such substituents,
e.g., as described above for alkyl groups. Suitable aryl groups
include unsubstituted and substituted phenyl groups.
[0078] The term "aryloxy" as used herein means an aryl group, as
defined above, having an oxygen atom attached thereto. The term
"aralkoxy" as used herein means an aralkyl group, as defined above,
having an oxygen atom attached thereto. Suitable aralkoxy groups
have 1 to 3 separate or fused rings and from 6 to about 18 carbon
ring atoms, e.g., O-benzyl.
[0079] The language "azo compounds" is intended to include azo
containing groups as surface functionalities. These azo groups on
the surface can initiate the polymerization from the surface into
the bulk nanaocomposite. This approach is achieved using silanes
that contain azo groups in them and that can be bonded onto the
walls of the glass capillaries..sup.9,10
[0080] The language "chemical interaction" is intended to include,
but is not limited to hydrophobic/hydrophilic, ionic (e.g.,
coulombic attraction/repulsion, ion-dipole, charge-transfer),
chemical bonding, Van der Waals, and hydrogen bonding. The term
chemical interaction is meant to be distinguished from physical
interactions, such as physical friction between surfaces.
[0081] The language "chemical bonding" is intended to include the
formation of a covalent bond, e.g., organic covalent bond or
inorganic covalent bond. Organic covalent bonds are defined to
involve the formation of a covalent bond between the common
elements of organic chemistry including but not limited to
hydrogen, boron, carbon, nitrogen, oxygen, silicon, phosphorus,
sulfur, and the halogens. In addition, carbon-silicon and
carbon-oxygen-silicon bonds are defined as organic covalent bonds,
whereas silicon-oxygen-silicon bonds that are not defined as
organic covalent bonds, i.e., silicon-oxygen-silicon bonds are
defined as inorganic covalent bonds.
[0082] The language "capillary column" refers to a chromatographic
column with internal diameters (I.D.) in the range of 50-2000
.mu.m.
[0083] The terms "coalescing" and "coalesced," as used in the
language "coalesced particles" are intended to describe a material
in which several individual components have become coherent to
result in one new component by an appropriate chemical or physical
process, e.g., heating. The term coalesced is meant to be
distinguished from a collection of individual particles in close
physical proximity, e.g., in a bed formation, in which the end
product comprises individual particles.
[0084] The language "dense material" is intended to include hybrid
materials, which upon view of an axial cross-section of the
material's surface are comprised of the hybrid material and
macropores with an average diameter of less than 0.5 .mu.m that
separate the hybrid material.
[0085] The terms "derivatized" or "derivatization" are intended to
include to the property or characteristic of anchoring or coating
an agent of alternate functionality onto a second material by
conversion of the functionality of the receiving surface of the
second material to the alternate functionality, e.g., by coating or
chemical bonding, e.g., polymerization.
[0086] The term "functionalizing group" includes organic groups
which impart a certain chromatographic functionality to a
chromatographic stationary phase, including, e.g., octadecyl
(C.sub.18) or phenyl. Such functionalizing groups are present in,
for example, surface modifiers such as disclosed herein which are
attached to the base material, e.g., via derivatization or coating
and later crosslinking, imparting the chemical character of the
surface modifier to the base material. In one embodiment, such
surface modifiers have the formula Z.sub.a(R').sub.bSi--R, where
Z=Cl, Br, I, C.sub.1-C.sub.5 alkoxy, dialkylamino or
trifluoromethanesulfonate; a and b are each an integer from 0 to 3
provided that a+b=3; R' is a C.sub.1-C.sub.6 straight, cyclic or
branched alkyl group, and R is a functionalizing group. R' may be,
e.g., methyl, ethyl, propyl, isopropyl, butyl, t-butyl, sec-butyl,
pentyl, isopentyl, hexyl or cyclohexyl; preferably, R' is
methyl.
[0087] The functionalizing group R may include alkyl, alkenyl,
alkynyl, aryl, cyano, amino, diol, nitro, ester, a cation or anion
exchange group, embedded polar functionalities, or an alkyl or aryl
group containing an embedded polar functionality. Examples of
suitable R functionalizing groups include C.sub.1-C.sub.30 alkyl,
including C.sub.1-C.sub.20, such as octyl (C.sub.8), octadecyl
(C.sub.18), and triacontyl (C.sub.30); alkaryl, e.g.,
C.sub.1-C.sub.4-phenyl; cyanoalkyl groups, e.g., cyanopropyl; diol
groups, e.g., propyldiol; amino groups, e.g., aminopropyl; and
alkyl or aryl groups with embedded polar functionalities, e.g.,
carbamate functionalities such as disclosed in U. S. Pat. No.
5,374,755, the text of which is incorporated herein by reference.
Such groups include those of the general formula ##STR1## wherein
l, m, o, r, and s are 0 or 1, n is 0, 1, 2 or 3 p is 0, 1, 2, 3 or
4 and q is an integer from 0 to 19; R.sub.3 is selected from the
group consisting of hydrogen, alkyl, cyano and phenyl; and Z, R', a
and b are defined as above. Preferably, the carbamate functionality
has the general structure indicated below: ##STR2## wherein R.sup.5
may be, e.g., cyanoalkyl, t-butyl, butyl, octyl, dodecyl,
tetradecyl, octadecyl, or benzyl. Advantageously, R.sup.5 is octyl,
dodecyl, or octadecyl.
[0088] The term "heterocyclic group" includes closed ring
structures in which one or more of the atoms in the ring is an
element other than carbon, for example, nitrogen, sulfur, or
oxygen. Heterocyclic groups can be saturated or unsaturated and
heterocyclic groups such as pyrrole and furan can have aromatic
character. They include fused ring structures such as quinoline and
isoquinoline. Other examples of heterocyclic groups include
pyridine and purine. Heterocyclic groups can also be substituted at
one or more constituent atoms with, for example, a halogen, a lower
alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a lower
alkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl, --CF.sub.3,
--CN, or the like. Suitable heteroaromatic and heteroalicyclic
groups generally will have 1 to 3 separate or fused rings with 3 to
about 8 members per ring and one or more N, O or S atoms, e.g.
coumarinyl, quinolinyl, pyridyl, pyrazinyl, pyrimidyl, furyl,
pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl,
benzofuranyl, benzothiazolyl, tetrahydrofuranyl, tetrahydropyranyl,
piperidinyl, morpholino and pyrrolidinyl.
[0089] The term "hybrid", i.e., as used in the language
"inorganic/organic hybrid material" or "inorganic/organic hybrid
monolith" includes inorganic-based structures wherein an organic
functionality is integral to both the internal or "skeletal"
inorganic structure. The inorganic portion of the hybrid material
may be selected from, but are not limited to alumina, silica,
titanium oxide, zirconium oxide, and ceramic material. In one
embodiment the inorganic portion of the hybrid material is
silica.
[0090] The language "Hydrophile-Lipophile Balance (HLB)," is art
recognized and is intended to characterize the solubility of
surfactants. The number provides a guide to the skilled artisan in
deciding which surfactant to use in a given emulsion. Surfactants
with an HLB number of <10 (or depending on your reference, 3-6)
have an affinity for oil over water, and as a result water droplets
tend to form in an oil phase. The converse is true for surfactants
with HLB numbers >10 (or 8-13), which have an affinity for water
over oil, and as a result oil droplets tend to form in a water
phase. In certain embodiments the HLB number typically ranges from
0 to about 15, but can go as high as 60. The HLB number may be
derived by a variety of methods, providing a relative, rather than
an exact value.
[0091] The term "material" as it is used herein, is intended to
include three-dimensional copolymers that have been formed in a
single piece. In one embodiment, the material of the invention is
prepared by casting precursors into a mold of a desired shape.
Materials of the invention include, but are not limited to
polymeric materials such as monoliths or non-monolith materials,
e.g., particles, copolymers with low porosity, or porous copolymers
that do not have interconnected pore structure.
[0092] The term "macropore" is intended to include pores of a
material, or channels through the material, which allow liquid to
flow directly through the material with reduced resistance at
chromatographically-useful flow rates. For example, macropores of
the present invention are intended to include, but are not limited
to pores with a pore diameter larger than about 0.05 .mu.m, pores
with a pore diameter ranging from about 0.1 .mu.m to about 10
.mu.m, pores with a pore diameter ranging from about 0.5 .mu.m to
about 5 .mu.m, and pores with a pore diameter ranging from about
0.8 .mu.m to about 4 .mu.m. It should be understood that the
selection of macropore size in the materials of the invention for
use in chromatography may be analyzed based on, at least, column
backpressure and efficiency. If the macropore size is too small,
the column backpressure will become too great for chromatographic
use, while if the macropore diameter is too large, the column will
lose chromatographic efficiency.
[0093] The language "minimally water soluble," is intended to
include the ability of the compound to remain at least soluble
enough to allow the intended reaction to move forward. In certain
embodiments, the language "minimally soluble" is intended to
include a range of solubility between sparingly soluble and fully
soluble. It should be understood that the solubility includes
solubility induced by using, for example, heat or additional
reagents. In certain embodiments, 2,2'-azobis(isobutyronitrile)
would be understood by one skilled the art to be minimally water
soluble.
[0094] The term "monolith" refers to a porous, three-dimensional
material having a continuous interconnected pore structure in a
single piece. The term monolith is meant to be distinguished from a
collection of individual particles packed into a bed formation, in
which the end product comprises individual particles. The term
monolith is also meant to be distinguished from three-dimensional
polymers with low porosity or porous three-dimensional polymers
that do not have interconnected pore structure. In one embodiment,
a monolith of the invention is prepared by casting precursors into
a mold of a desired shape. In another embodiment, the monolith
comprises coalesced particles. In yet another embodiment, the
monolith material may be used for chromatography, i.e., the
monolith is a chromatographically useful material, e.g., a
chromatographic column.
[0095] The term "nanophase" refers to organic or inorganic phase
aggregations that have formed based on phase separations at the
molecular level. In particular embodiments of the invention, phase
separation at the molecular level is generated upon the
simultaneous reaction of hybrid and inorganic monomers through a
polymerization sol-gel reaction. FIG. 1 is a graphical depiction of
the polymerization sol-gel reaction of the invention that leads to
the production of organic and inorganic nanophases.
[0096] The language "polymerized scaffolding nanocomposite (PSN)"
refers to hybrid composite materials that comprise a polymerized
organic nanophase surrounded by an inorganic nanophase, e.g.,
polymerized siloxane, wherein the nanophases form independently
through independent reactions, e.g., a polymerization and a sol gel
reaction, and wherein the organic and inorganic nanophases
interconnect by chemical bond formation through a hybrid monomer,
e.g., by condensation of the inorganic phase, e.g., of the hybrid
monomer to an inorganic monomer; and polymerization of the organic
phase, e.g., radical polymerization, such that a hybrid material
forms with a plurality of surface scaffolding functionalities.
[0097] The language "polymerization sol-gel (PSG) reaction" refers
to the approach of preparing hybrid materials using simultaneous
polymerization reactions and sol-gel reactions, wherein the organic
and inorganic nanophases interconnect by chemical bond formation
through an interaction with a hybrid monomer, e.g., by condensation
of the inorganic phase, e.g., of the hybrid monomer to an inorganic
monomer; and polymerization of the organic phase, e.g., radical
polymerization, such that a hybrid material forms. In a particular
embodiment, the materials are made from a mixture of a
tetraalkoxysilane, e.g., tetramethoxysilane, and a polymerizable
organosilane, e.g., (3-methacryloxypropyl)trimethoxysilane, wherein
the organosilane is minimally water soluble, e.g., sufficient to
produce a polymerized scaffolding nanocomposite (i.e., the
polymerizable group will have an affect on this solubility). For
example, the starting silanes have limited solubility in water. As
the SiOR bonds are hydrolyzed to SiOH, the SiOH monomer that forms
is substantially soluble in water.
[0098] The term "polymerization" refers to any process that results
from the bonding or coupling of smaller molecules to form a larger
molecule. One skilled in the art would recognize that the term
"polymerization" is intended to include a range of different
degrees of polymerization, i.e., ranging from a single reaction,
e.g., two molecules reacting to form a dimer, to a plurality of
reactions, e.g., a large number of molecules reacting to form a
macromolecule.
[0099] The language "scaffolding functionality" refers to
functional moieties, such as organic functionalities, e.g., of
organosilanes, that are capable of chemically interacting with a
surface of a second material, e.g., a containment vessel, thus
allowing the PSN material to anchor to the second material, and
thereby become immobilized relative thereto. In certain embodiments
of the invention, the functional moiety is a "polymerizable group"
including, but not limited to vinyl, acrylate, methacrylate, e.g.,
3-methacryloxypropyl, acrylamide, methacrylamide, styrene, e.g.,
styrylethyl, divinylbenzene, itaconate, fumarate, alkyne, and
combinations thereof. The ordinarily skilled artisan would
understand that the scaffolding functionality would be consumed
during a polymerization reaction to varying extents depending on
the reaction conditions chosen, and that the above listed
functionalities are provided in their unreacted or monomeric
state.
[0100] The language "second material" is intended to include any
second material with a surface capable of receiving the PSN
material.
[0101] The language "surface modifiers" is intended to include
functionalizing groups that impart a certain chromatographic
functionality to a chromatographic stationary phase. Surface
modifiers such as disclosed herein are attached to the base
material, e.g., via derivatization or coating and later
crosslinking, imparting the chemical character of the surface
modifier to the base material.
[0102] The language "wall adhesion" refers to a property of a
material, e.g., a PSN material, in which the material possesses
sufficient chemical interaction between the material and a second
material (e.g., the inside wall of a capillary column) such that
the chemical interactions are retained upon subjection of the
material to additional factors, e.g., during use of the material in
chromatographic separations. In certain embodiments, the PSN
material possesses sufficient wall adhesion so as to minimize or
preclude flow paths between the PSN material and the second
material, e.g., chromatographic column, where said flow paths would
be deleterious to separation efficacy in the chromatographic
column.
I. Compositions of Matter
[0103] In one aspect, the invention provides a hybrid
inorganic/organic material comprising a polymerized scaffolding
nanocomposite (PSN), wherein the nanocomposite contains a
scaffolding functionality capable of chemically interacting with a
surface of a second material. The hybrid material includes
inorganic-based structures wherein an organic functionality is
integral to both the internal or "skeletal" inorganic structure,
wherein the inorganic portion is selected from, but not limited to
silica, alumina, zirconium oxide, titanium oxide, ceramics, tin
oxide, and combinations thereof.
[0104] In certain embodiments, the organic functionality of the
hybrid material is derived, at least in part, from a hybrid
monomer, e.g., an organosilane, e.g., an organoalkoxysilane, which
comprises both an inorganic and organic component, i.e., relating
to the presence of both a silicon-oxygen bond and a carbon-silicon
bond in a single monomeric unit. In certain embodiments, the
organosilane is an organoalkoxysilane having the formula
R.sup.2Si(OR.sup.1).sub.3 or R.sup.6[Si(OR.sup.1).sub.3].sub.m
where R.sup.2 is a styryl, vinyl, an acrylate, methacrylate,
acrylamide, methacrylamide, divinylbenzene, itaconate, fumarate,
substituted or unsubstituted C.sub.1-C.sub.18 alkenylene,
alkynylene or arylene, or a combination thereof; R.sup.1 is a
C.sub.1-C.sub.4 alkyl moiety; R.sup.6 is a substituted or
unsubstituted C.sub.1-C.sub.18 alkenylene, alkynylene or arylene
moiety bridging two or more silicon atoms; and m is an integer
greater than or equal to two. In certain embodiments, R.sup.2 is
vinyl, methacryloxypropyl, methacrylamidepropyl,or styrylethyl and
R.sup.1 is methyl or ethyl; or R.sup.6 is a bridging
N,N-bis(propylene) acrylamide group, m=2, and R.sup.1 is ethyl or
methyl. In a specific embodiment of the invention, the organosilane
is (3-methacryloxypropyl)trimethoxysilane. In certain embodiments,
the organosilane is minimally water soluble, e.g., sufficient to
produce a polymerized scaffolding nanocomposite.
[0105] In one embodiment, the PSN is the product of a reaction of a
hybrid organic/inorganic monomer, e.g., an organosilane, and an
inorganic monomer, e.g., a silane monomer. In a specific
embodiment, the PSN is the product of a reaction of a
tetraalkoxysilane and an organosilane containing at least one
polymerizable group, e.g., 3-methacryloxypropyl or styrylethyl.
[0106] In one embodiment, the tetraalkoxysilane has the formula
Si(OR.sup.1).sub.4, where R.sup.1 is a C.sub.1-C.sub.3 alkyl
moiety, e.g., tetramethoxysilane or tetraethoxysilane. In certain
embodiments, the organosilane and/or tetraalkoxysilane are
minimally water soluble, e.g., sufficient to produce a polymerized
scaffolding nanocomposite. In a particular embodiment, the
(3-methacryloxypropyl)trimethoxysilane and the tetramethoxysilane
are present in a volume to volume ratio of 1 to 4.
[0107] The hybrid inorganic/organic material may be cast in any
shape by techniques well known in the art, such as molding, so long
as the properties of the material are not substantially affected.
In one embodiment of the invention the hybrid inorganic/organic
material is a porous hybrid inorganic/organic monolith, e.g., a
capillary monolith. The monoliths of the present invention have
improved properties including, but not limited to enhanced wall
adhesion, i.e., capillary wall adhesion, and increased resistance
to shrinkage and improved mechanical stability in comparison to
capillary monoliths known in the art.
[0108] In certain embodiments of the invention, the polymerizable
organo groups are embedded inside the silica skeleton, which yields
monoliths that have high surface silanol concentrations, i.e., a
large population of chromatographically accessible silanols, and
with essentially no chromatographically accessible or useful organo
groups. The monolith skeletons of these monoliths are composed of
an organic-inorganic nanocomposite, wherein the organic moiety is
embedded inside the inorganic silica network: This approach offers
monoliths with an increased resistance to shrinkage as well as good
resistance to shrinking and swelling upon exposure to organic
solvent. In certain embodiments, the shrinkage of the silica
skeleton/scaffold will be minimized due to the high organic
content. Thus, monolith columns with IDs of greater than 50 .mu.m,
e.g., 1-2 mm, can be prepared in-situ using this method of
reaction.
[0109] In an alternate embodiment, the polymerized organic phase is
chromatographically accessible and changes the hydrophobicity of
the porous surface, resulting in novel selectivities for
chromatographic separations. In certain embodiments, the extent to
which the organic phase is chromatographically accessible depends
on the extent to which the scaffolding functionality is consumed
upon anchoring of the PSN material to the interior surface of the
chromatographic column. In particular embodiments, unpolymerized
scaffolding functionality may directly, e.g., methacrylate groups,
or indirectly, e.g., hydrolyzed methacrylate groups, provide the
chromatographically accessible organic phase. This polymerization
reaction may be regulated to produce desired chromatographic
selectivities.
[0110] In accordance with the invention, the PSN contains a
scaffolding functionality that is capable of chemically interacting
with a surface of a second material, e.g., a containment vessel,
thus allowing the PSN material to anchor to the second material. In
certain embodiments of the invention, the scaffolding functionality
is a "polymerizable group" including, but not limited to vinyl,
acrylate, methacrylate, e.g., 3-methacryloxypropyl, acrylamide,
methacrylamide, styrene, e.g., styrylethyl, divinylbenzene,
itaconate, fumarate, alkyne, and combinations thereof.
[0111] The improved properties afforded by the invention, e.g.,
enhanced wall adhesion and/or increased resistance to shrinkage,
are the result of the scaffolding functionality that anchors the
polymer to the wall of the containment vessel. The ordinarily
skilled artisan would understand that the scaffolding functionality
would be substantially consumed during a polymerization reaction,
and that the above-listed functionalities are provided in their
unreacted or monomeric state.
[0112] The second material, in accordance with the invention, is
any material having a surface that is capable of receiving the PSN
material. In certain embodiments, the second material is a
containment vessel, which provides supportive enclosure to the PSN
material. The second material, e.g., containment vessel, may be
used as the primary reaction vessel for preparation of the PSN
material, or the PSN material may be prepared independently of the
second material and subsequently transferred to the second
material, e.g., a secondary containment vessel, such that the PSN
material is capable of interacting with the second material.
[0113] In one embodiment, the containment vessel is used as the
reaction vessel, whereby chemical interactions, such as chemical
bonding, e.g., covalent bonding, occur between the forming PSN
material and the second material during the polymerization of the
PSN material. In an alternative embodiment, the PSN material is
subsequently transferred to the second material, such as a
secondary containment vessel, wherein chemical interactions, such
as chemical bonding, e.g., covalent bonding, occur through further
chemical processing, e.g., addition of a crosslinking agent,
photoinitiation of a radical polymerization reaction, or addition
of a hydrosilylation catalyst for a hydrosilylation reaction. The
containment vessels that may be utilized for PSN materials of the
present invention include, but are not limited to a chromatographic
column, e.g., capillary column, e.g., a fused silica capillary
column, a glass lined steel column, a radial compression column, a
trap column, a microfluidic device, a microchip, a sensor, an
electronic circuit, a solid phase extraction (SPE) device, e.g., a
miniaturized SPE device, and an on-column frit.
[0114] In certain embodiments, the second material in itself has
functionality suitable for interaction with the nanocomposite. In
other embodiments, the surface of the second material is
derivatized with an anchoring functionality or treated to provide
the anchoring functionality.
[0115] The anchoring functionality and the scaffolding
functionality may be copolymerizable, such that a covalent bond is
formed as a result of copolymerization of the scaffolding and
anchoring functionalities. In one embodiment, this polymerization
may be initiated by a radical initiator, including, but not limited
to 2,2'-azobis(isobutyronitrile),
2,2'-azobis(2-methylpropionamidine)dihydrochloride,
4,4'-azobis(4-cyanovaleric acid), potassium persulfate, and
peracetic acid. In addition, the radical initiator is at least
minimally soluble, e.g., minimally water soluble.
[0116] In certain embodiments, the present invention involves the
interaction of silanol functionalities, e.g., chemical bonding
between a silanol of second surface and a silanol of the PSN
material (i.e., a glass capillary surface containing silanols that
react with the Si--OH groups of a silica sol to form a bridging
siloxane between a gelling monolith and the capillary wall).
However, it should be understood that in certain embodiments where
the chemical interaction is the interaction of silanol
functionalities, this interaction is in combination with other
chemical interaction(s), e.g., chemical bonding, through
polymerization, e.g., forming an organic chemical bond. In certain
other embodiments the chemical interaction is not the interaction
of silanol functionalities.
[0117] In another aspect, the invention provides a PSN that is a
composite material prepared by the polymerization sol-gel reaction
described herein below. For example, in one embodiment, the
preparation of a PSN material comprises the steps of
[0118] a) forming a sol-gel by the reaction of two or more
monomers;
[0119] b) initiating a polymerization reaction; and
[0120] c) allowing the monomers to react through a polymerization
sol-gel (PSG) reaction.
[0121] In certain embodiments, the preparation of the PSN may
further comprise the step of (d) modifying the pore structure of
the material. The porous inorganic/organic hybrid materials of the
invention may be used as prepared by the process noted above and
further described in Section II below, without further
modification.
[0122] Alternatively, the hybrid materials of the invention may be
further modified by one or more processing treatments, e.g., by:
chemical processing treatment, such as derivatization of surface
functionalities of the thus-prepared material; incorporating
additives to the PSG reaction that affect the physical properties,
e.g., porogens, such as surfactants or solvents, e.g., toluene;
physical processing treatments, such as hydrothermal treatment; or
a combination of several processing treatments.
Chemical Processing
[0123] A. Incorporation of Additives
[0124] In one embodiment, the pore structure of the materials of
the invention, e.g., monolith materials, is modified by further
including a surfactant or combination of different surfactants in
the PSG reaction. In a specific embodiment, the surfactant or
combination of surfactants includes at least one nonionic
surfactant. Exemplary nonionic surfactants include but are not
limited to surfactants comprised of block copolymers of
polyethylene glycol and polypropyleneglycol, surfactants comprised
of alkylphenoxypolyethoxyethanol, and polyethyleneglycol, e.g.,
Pluronic F38. In particular embodiments, the surfactant or
combination of surfactants are selected from surfactants with a
hydrophile-lipophile balance ranging from about 0 to 60, e.g.,
about 10 to 50, e.g., about 20 to 40, e.g., about 30 to 40, e.g.,
about 33.
[0125] The surfactants are believed to enhance the concentration of
water and the acid/base catalyst on the surface of the material
during the polymerization sol-gel reaction. Use of surfactants to
modulate the surface structure of the material stabilizes the
polymer material that is forming throughout the reaction, and
minimizes or suppresses inhomogeneous morphology.
[0126] B. Surface Modification
[0127] The methods of preparation may further comprise surface
modifying the materials of the invention, e.g., the monolith
materials of the invention. The hybrid materials of the invention
possess both organic groups and silanol groups, which may be
additionally substituted or derivatized with a surface
modifier.
[0128] In one embodiment of the invention, surface organic groups
of the porous inorganic/organic hybrid material are derivatized or
modified in a subsequent step via formation of an organic covalent
bond between the modifying reagent and organic groups of the
material, wherein the material retains sufficient scaffolding
functionality such that the material is able to interact with a
second material. Alternatively, the surface silanol groups of the
hybrid silica are derivatized into siloxane organic groups, such as
by reacting with an organotrihalosilane, e.g.,
octadecyltrichlorosilane, or a halopolyorganosilane, e.g.,
octadecyldimethylchlorosilane. Alternatively, the surface organic
and silanol groups of the hybrid silica are both derivatized.
[0129] In a particular embodiment, the silanol groups are surface
modified with compounds having the formula Z.sub.a(R').sub.bSi--R,
where Z=Cl, Br, I, C.sub.1-C.sub.5 alkoxy, dialkylamino or
trifluoromethanesulfonate; a and b are each an integer from 0 to 3
provided that a+b=3; R' is a C.sub.1-C.sub.6 straight, cyclic or
branched alkyl group, and R is a functionalizing group. R' may be,
e.g., methyl, ethyl, propyl, isopropyl, butyl, t-butyl, sec-butyl,
pentyl, isopentyl, hexyl or cyclohexyl; preferably, R' is methyl.
In additional embodiments, the organic groups may be similarly
functionalized.
[0130] In one embodiment, the organic groups of the hybrid
materials react to form an organic covalent bond with a surface
modifier. The modifiers can also form an organic covalent bond to
the material's organic group via a number of mechanisms well known
in organic and polymer chemistry including but not limited to
nucleophilic, electrophilic, cycloaddition, free-radical, carbene,
nitrene, and carbocation reactions.
[0131] In one embodiment, the surface modifier may be an
organotrihalosilane, such as octyltrichlorosilane or
octadecyltrichlorosilane. In an additional embodiment, the surface
modifier may be a halopolyorganosilane, such as
octyldimethylchlorosilane or octadecyldimethylchlorosilane. In
certain other embodiments the surface modifier is
octadecyldimethyl-N,N-dimethylaminosilane.
[0132] In another embodiment, the surface of the hybrid silica
material may also be surface modified by coating with a polymer.
Polymer coatings are known in the literature and may be provided
generally by polymerization or polycondensation of physisorbed
monomers onto the surface without chemical bonding of the polymer
layer to the support (type I), polymerization or polycondensation
of physisorbed monomers onto the surface with chemical bonding of
the polymer layer to the support (type II), immobilization of
physisorbed prepolymers to the support (type III), and
chemisorption of presynthesized polymers onto the surface of the
support (type IV).sup.(11). As noted above, coating the hybrid
material with a polymer may be used in conjunction with various
surface modifications described in the invention.
[0133] Moreover, the surface of the materials of the invention may
contain residual organic groups, which can be derivatized by
reacting with a reagent that is reactive towards the organic group.
For example, vinyl groups, e.g., unreacted methacrylate groups, on
the particle can be reacted with a variety of olefin reactive
reagents such as bromine (Br.sub.2), hydrogen (H.sub.2), free
radicals, propagating polymer radical centers, dienes, and the
like. In another example, hydroxyl groups on the material can be
reacted with a variety of alcohol reactive reagents such as
isocyanates, carboxylic acids, carboxylic acid chlorides, and
reactive organosilanes as described below. Reactions of this type
are well known in the literature, see, e.g., March, J. "Advanced
Organic Chemistry," 3.sup.rd Edition, Wiley, New York, 1985; Odian,
G. "The Principles of Polymerization," 2.sup.nd Edition, Wiley, New
York, 1981; the texts of which are incorporated herein by
reference. Moreover, regulation of the extent of the (methacrylate)
polymerization reaction may be used to supply more or less organic
groups for derivatization with reagents that are reactive to the
organic functionalities.
[0134] In addition, the surface of the materials of the invention
contain silanol groups, which can be derivatized by reacting with a
reactive organosilane. The surface derivatization of the hybrid
material may be conducted according to standard methods, for
example by reaction with octadecyltrichlorosilane or
octadecyldimethylchlorosilane in an organic solvent at elevated
temperature. An organic solvent such as toluene or methylene
chloride is typically used for this reaction. An organic base such
as pyridine or imidazole is added to the reaction mixture to
catalyze the reaction. The product of this reaction is then washed
with one or more solvents such as methanol, water, toluene,
methylene chloride, and/or acetone and can be sometimes further
dried at about 80.degree. C. to 100.degree. C. under reduced
pressure for about 16 h. The resultant hybrid material can be
further reacted with a short-chain silane such as
trimethylchlorosilane to endcap any remaining silanol groups, by
using a similar procedure described above.
[0135] C. Hydrothermal Treatment
[0136] In addition, materials of the invention, e.g., the monolith
materials of the invention, may be subjected to hydrothermal
treatment to improve the material's pore structure, e.g., by
further condensation of the siloxane polymer network formed during
the PSG reaction, and dissolution and redeposition of silicic acid
silicates from and to the surface of the pore structure. In
particular, this treatment would be applicable after the
polymerization achieved a solid state. In one embodiment, the
hydrothermal treatment is in a basic solution at an elevated or
high temperature, e.g., 120.degree. C. (i.e., the hydrothermal
treatment is run in the capillary). The range of pH of the
hydrothermal treatment is about 6-12, e.g., about 8-11, e.g., about
9-10. The monolith material is then rinsed with water followed by a
solvent exchange, e.g., with methanol; ethanol; acetonitrile; or
tetrahydrofuran followed by room temperature, e.g. about
20-25.degree. C., drying. The material is then dried at about
25-120.degree. C., and preferably at about 70.degree. C., under
vacuum overnight.
[0137] In one embodiment, the condensation is base catalyzed, e.g.,
by ammonia. In certain embodiments, the ammonia is generated by the
high temperature decomposition of urea or a suitable organic
amide.
[0138] The surface of the hydrothermally treated hybrid material
may be modified in a similar fashion to that of the hybrid material
that is not modified by hydrothermal treatment as described above.
For example, in a subsequent step, the surface organic groups of
the hybrid material may be optionally modified via formation of a
covalent bond between the monolith material's organic and/or
silanol group and the modifying reagent, and optionally including
coating with a polymer, i.e., also as is described above.
II. Methods of Preparation
[0139] In a related aspect, the invention provides a method of
preparation of a hybrid inorganic/organic material comprising a
polymerized scaffolding nanocomposite (PSN), comprising the steps
of
[0140] a) forming a sol-gel by the reaction of two or more
monomers;
[0141] b) initiating a polymerization reaction; and
[0142] c) allowing the monomers to react through a polymerization
sol-gel (PSG) reaction, thereby preparing the hybrid
inorganic/organic monolith, wherein the monolith contains a
scaffolding functionality capable of chemically interacting with a
surface of a second material. In certain embodiments, the methods
further comprise the step of (d) modifying the pore structure of
the material. As described above, the resulting PSN material may be
subsequently transferred to the second material, such as a
secondary containment vessel, wherein chemical interactions, such
as chemical bonding, e.g., covalent bonding, occur through further
chemical processing, e.g., addition of a crosslinking agent,
photoinitiation of a radical polymerization reaction, or addition
of a hydrosilylation catalyst for a hydrosilylation reaction.
[0143] Alternatively, the invention provides a method for in situ
preparation of a hybrid inorganic/organic monolith in a
chromatographic column, e.g., a capillary column, e.g., a
fused-silica capillary column. The method comprises: [0144] forming
an anchoring functionality on an interior surface of said
chromatographic column; and [0145] forming inside the
chromatographic column a hybrid inorganic/organic monolith
comprising a polymerized scaffolding nanocomposite (PSN), wherein
the nanocomposite contains a scaffolding functionality capable of
chemically interacting with the anchoring functionality on the
surface, the monolith being formed by:
[0146] a) forming a sol-gel by the reaction of two or more
monomers;
[0147] b) initiating a polymerization reaction; and
[0148] c) allowing the monomers to react through a polymerization
sol-gel (PSG) reaction; whereby the scaffolding functionality and
the anchoring functionality chemically interact to thereby anchor
the monolith to the surface, such that a hybrid inorganic/organic
monolith is prepared in situ in the chromatographic column. In
certain embodiments, the in situ process involves covalent bond
formation between the PSN material and the surface of the
chromatographic column, i.e., consumption of a plurality of
scaffolding functionalities in covalent bond formation to the
second material.
[0149] In accordance with these aspects of the invention, the
hybrid materials are prepared using a polymerization sol-gel (PSG)
reaction. More particularly, the hybrid materials are prepared
using simultaneous polymerization reactions and sol-gel reactions,
wherein the organic and inorganic nanophases interconnect by
chemical bond formation through an interaction with a hybrid
monomer, e.g., by condensation of the inorganic phase, e.g., of the
hybrid monomer to an inorganic monomer; and polymerization of the
organic phase, e.g., radical polymerization, such that a hybrid
material forms. In a particular embodiment, the materials are made
from a mixture of a tetraalkoxysilane, e.g., tetramethoxysilane,
and a polymerizable organosilane, e.g.,
(3-methacryloxypropyl)trimethoxysilane, wherein the organosilane is
minimally water soluble, e.g., sufficient to produce a polymerized
scaffolding nanocomposite (i.e., the polymerizable group will have
an affect on this solubility).
[0150] In one exemplary embodiment, the PSG reaction begins with
hydrolysis, e.g., acid catalyzed hydrolysis, e.g., by acetic acid,
and slow condensation of a hybrid monomer and an inorganic monomer,
e.g., a mixture of tetramethoxysilane and a polymerizable
organosilane, such as (3-methacryloxypropyl)trimethoxysilane, and
proceeds at low temperature (e.g., a temperature ranging from about
0.degree. C. to about room temperature, e.g. about 20-25.degree.
C.) and low pH (e.g., about 2-3), forming a mixture of low
molecular weight oligomers. In a subsequent step, a polymerization
reaction is initiated, e.g., radically initiated. In certain
embodiments, the radical initiation occurs at an increased
temperature (e.g., above room temperature, e.g., about
60-65.degree. C.). The oligomers formed are then simultaneously
polymerized, e.g., via radical chain polymerization, and further
condensed to form a polymerized sol-gel (illustrated in FIG. 1).
Moreover, the polymerizable group (depicted in FIG. 1A, in both
chemical structure and schematic form) may react with additional
polymerizable groups or terminate in a reaction with the surface of
a second material, e.g., with an anchoring functionality on the
inner surface of a chromatographic column. A simplified example of
the product of the polymerization sol-gel reaction is shown in a
cross-sectional block of an interior portion of the PSN material in
FIGS. 1C and 1D, i.e., polymerization to the second material is not
depicted. However, the depictions of FIG. 1 are not intended to
limit the scope of the invention.
[0151] The PSG reaction is performed at a temperature sufficient to
achieve simultaneous polymerization and sol gel reactions, e.g., at
65.degree. C., and is performed for an amount of time sufficient to
prepare the hybrid inorganic/organic material, e.g., the PSN
material. In certain embodiments, the polymerization is initiated
with a radical initiator, e.g., a radical initiator that is
minimally water soluble, e.g., 2,2'-azobis(isobutyronitrile),
2,2'-azobis(2-methylpropionamidine) dihydrochloride,
4,4'-azobis(4-cyanovaleric acid), potassium persulfate, or
peracetic acid. In certain embodiments of the invention, the
sol-gel reaction occurs in the presence of urea.
III. Devices
[0152] The porous inorganic/organic hybrid materials of the current
invention have a wide variety of end uses in the separation
sciences, such as materials for chromatographic columns, thin layer
chromatographic (TLC) plates, filtration membranes, microtiter
plates, scavenger supports, solid phase organic synthesis supports,
Capillary-LC columns, radial compression columns, trap columns,
microfluidic devices, microchips, sensors, electronic circuits,
miniaturized solid phase extraction (SPE) devices and on-column
frits, and the like, having a stationary phase that includes porous
inorganic/organic hybrid materials, e.g., monolith materials, of
the present invention. The stationary phase may be introduced into
the device by coating, impregnation, cladding, wrapping, or other
art-recognized techniques consistent with the methods of
preparation of the present invention, etc., depending on the
requirements of the particular device.
[0153] Thus, in another aspect, the invention provides a
separations device comprising
[0154] a) a surface capable of accepting a monolith material
comprising a polymerized scaffolding nanocomposite (PSN) material,
said surface comprising an anchoring functionality and
[0155] b) a hybrid inorganic/organic monolith comprising a
polymerized scaffolding nanocomposite (PSN), wherein the
nanocomposite contains a scaffolding functionality capable of
chemically interacting with the anchoring functionality on said
surface, and wherein said hybrid organic/inorganic monolith is
anchored to said surface by a chemical interaction between said
scaffolding functionality and anchoring functionality. In certain
embodiments, the devices in accordance with the invention include
chromatographic columns, e.g., a glass lined steel column,
capillary column, thin layer plates, filtration membranes, sample
cleanup devices, and microtiter plates.
[0156] In certain embodiments, the separations device is a
fused-inorganic capillary column, wherein the inorganic portion may
be selected from, but is not limited, to silica, alumina, zirconium
oxide, titanium oxide, ceramics, tin oxide, and combinations
thereof.
[0157] In an particularly advantageous embodiment, the invention
provides a chromatographic column, e.g., a capillary column, e.g.,
a fused-silica capillary column, comprising
[0158] a) a chromatographic column having a cylindrical interior
surface capable of accepting a monolith comprising a polymerized
scaffolding nanocomposite (PSN) material, the interior surface
comprising an anchoring functionality, and
[0159] b) a hybrid inorganic/organic monolith comprising a
polymerized scaffolding nanocomposite (PSN), wherein the
nanocomposite contains a scaffolding functionality capable of
chemically interacting with the anchoring functionality on the
surface, and wherein the hybrid organic/inorganic monolith is
anchored to the surface by a chemical interaction between the
scaffolding functionality and anchoring functionality.
[0160] In certain embodiments, the device is a chromatographic
column, e.g., a capillary column, e.g., a fused-silica capillary
column, wherein the interior surface of the capillary column is
derivatized with a polymerizable anchoring functionality. In
certain embodiments of the invention the capillary column has an
inner diameter (I.D.) greater than about 50 .mu.m, e.g., greater
than about 100 .mu.m, e.g., greater than about 150 .mu.m, e.g.,
greater than about 200 .mu.m, e.g., greater than about 250 .mu.m,
e.g., greater than about 500 .mu.m, e.g., greater than about 1.0
mm, e.g., greater than about 2 mm, e.g., greater than about 3 mm.
In a particularly advantageous embodiment, the chromatographic
device is a chromatographic column, such as commonly used in
HPLC.
EXAMPLES
[0161] The present invention may be further illustrated by the
following non-limiting examples describing the preparation of
porous inorganic/organic hybrid monolith materials, and their
use.
Materials
[0162] All reagents were used as received unless otherwise noted.
Those skilled in the art will recognize that equivalents of the
following supplies and suppliers exist, and as such the suppliers
listed below are not to be construed as limiting.
[0163] Gelest Inc., Morrisville, Pa.:
(3-Methacryloxypropyl)trimethoxysilane (MAPTMOS),
tetramethoxysilane (TMOS), and
octadecyldimethyl(dimethylamino)silane; BASF Corp., Mount Olive,
N.J.: Pluronic.RTM. F38; Sigma-Aldrich Chemical Co., Milwaukee,
Wis.: thiourea, acenaphthene, naphthalene, sodium hydroxide;
DuPont, Wilmington, Del., Vazo.RTM. 64 and Vazo.RTM. 44; J. T.
Baker, Phillipsburgh, N.J.: urea, methylene chloride, methanol,
acetonitrile, acetone, toluene, pyridine, hydrochloride acid and
glacial acetic acid. All solvents were HPLC grade. Water was used
directly from a Millipore Milli-Q (Millipore Corp., Bedford,
Mass.). Fused silica capillary columns (Cat # FS-115, 150 .mu.m
I.D., Cat # FS-150, 50 .mu.m I.D., or equivalents) and all the
end-fittings and unions (Cat #P-760) were from Upchurch Scientific
(Oak Harbor, Wash.) and from Polymicro Technologies, Phoenix, Ariz.
(Cat # TSP 530660, 520 .mu.m I.D., or equivalents). Compression
screws were from Waters Corp. (Milford, Mass.,).
Example 1
[0164] A Harvard Apparatus Model `33` Dual Syringe Pump(Harvard
Apparatus Inc., Hollistion, Mass., or equivalent) and 10 mL
Pharmaseal.RTM. syringe (American Pharmaseals Laboratory, Glendale,
Calif., or equivalent) were used in all capillary filling and
purging steps.
[0165] A fused silica capillary column (ca. 2 m in length) was
treated by the following five steps: (1) the column was purged with
1N NaOH at a flow rate of 50 .mu.L/min for at least 5 minutes,
sealed via compression fittings and heated to 90.degree. C. for 17
hours; (2) the column was purged with 1N HCl water solution at a
flow rate of 50 .mu.L/min for at least 5 minutes; (3) the column
was rinsed with water, acetone, and then toluene at a flow rate of
50 .mu.L/min for 10 minutes each (total volume .about.500 .mu.L,
>10 times column volume); (4) the column was filled with a
mixture of 0.5 mL (3-methacryloxypropyl)trimethoxysilane/0.5 mL
pyridine/10 mL toluene at a flow rate of 50 .mu.L/min for 5
minutes, sealed at both ends of the column and the column was then
heated in a 90.degree. C. oven for 21 hours; and (5) the column was
washed with toluene, acetone and water for 10 minutes each at a
flow of 50 .mu.L/min.
[0166] After surface derivatization, sections of the 2 meter
capillary are cut to make multiple, shorter capillary monoliths
that may be used in further experiments.
Example 2
[0167] Pluronic.RTM. F38 (Example 2a--0.622 g, Example 2b--0.596 g)
and urea (Example 2a--0.517 g, Example 2b--0.515 g) were added to 5
mL of a 15 mM acetic acid solution at room temperature in a glass
vial. The solutions were deoxygenated by nitrogen gas purging for
at least 2 minutes before Vazo.RTM. 64 (1-2 mg) was added, and then
the solution was cooled to 0.degree. C. In a separate glass vial, a
2 mL silane mixture (4/1 TMOS:MAPTMOS v/v) was prepared and then
cooled to 0.degree. C. Next, the silane mixture was added slowly to
the acetic acid solution. The combined solutions were stirred at
0.degree. C. for 1 h and then at room temperature (rt) for 1.75
h.
[0168] The resulting solutions were delivered into two separate 50
.mu.m (I.D.).times.40 mm (L) capillary columns that were surface
treated as described in Example 1 with the exception that step (1)
was run for 2 hours. The columns were then sealed at both ends with
two compression screws and were heated at 45.degree. C. in an oven
for 18.5 h.
[0169] Monolith morphology of the cross-sections of each column was
observed by Scanning Electron Microscopy (SEM) (JEOL, Peabody,
Mass. or equivalent) and showed the formation of monolith materials
with a majority of macropores with diameters greater than 0.5 .mu.m
and the absence of shrinkage away from the capillary wall.
Example 3
[0170] As described in Example 2, Pluronic.RTM. F38 and urea were
added to 5 mL of an acetic acid solution at room temperature. The
solutions were deoxygenated by nitrogen gas purging for at least 2
minutes before Vazo.RTM. 64 was added. The stirred solutions were
cooled to 0.degree. C. for a specific time, and 2 mL of a 0.degree.
C. silane mixture (4/1 TMOS:MAPTOS v/v) was added slowly to the
acetic acid solution. The combined solutions were stirred at
0.degree. C. for a prescribed time and then at rt for an additional
time period.
[0171] The resulting solutions were delivered into separate 150
.mu.m (I.D.).times.40 mm (L) capillary columns that had been
surface treated as described in Example 1. The columns were then
sealed at both ends with two compression screws and were heated at
65.degree. C. for a prescribed time and then at an elevated
temperature (Example 3a,b 120.degree. C.; Example 3c-j 105.degree.
C.; Example 3k-o 110.degree. C.; Example 3p-t 125.degree. C.) for
an additional time period.
[0172] Monolith morphology of the cross-sections of each column was
observed by SEM as described in Example 2. Depending on the
reaction conditions, dense monolith materials (a majority of
macropores with diameters less than 0.5 .mu.m) to non-dense
monolith materials (a majority of macropores with diameters greater
than 0.5 .mu.m) were observed, and varying levels of wall adhesion
were achieved. Specific amounts of reagents used to prepare these
products and characterization data are listed in Table 1.
[0173] Example 3k and 3t were cut to 20 cm lengths and the
surfactant was removed from the monolith structure by prolonged
methanol purging using an Eldex Micropro Syringe Pump (Eldex, Napa,
Calif., or equivalent). The column was purged with methanol for 5
days at a 1.0 .mu.L/min flow rate, at which time no surfactant
could be detected in the mobile phase as measured by FTIR
spectroscopy (Polaris, ThermoMattson, Madison, Wis., or
equivalent).
Surface Modification:
[0174] After chromatographic evaluation as described in Example 9
(see Table 4), Example 3t was purged with methylene chloride at 1.0
.mu.L/min for 16 h and then purged with a solution of
octadecyldimethyl(dimethylamino)silane in methylene chloride (1/4
v/v) at 1 .mu.L/min for 3 h. After surface modification, the column
was purged with methylene chloride at 1 .mu.L/min flow rate for 16
hours, which was then exchanged with acetonitrile by purging with
greater that 3 column volumes. The resulting surface modified
monolith is identified as Example 3u, which was in turn evaluated
as described in Example 9 (see Table 4).
Example 4
[0175] As described in Example 2, Pluronic.RTM. F38 and urea were
added to 5 mL of an acetic acid solution at room temperature. The
solutions were deoxygenated with N.sub.2 purge for at least 2
minutes before Vazo.RTM. 64 was added. The stirred solutions were
cooled to 0.degree. C. for a specific time, and 2 mL of a 0.degree.
C. silane mixture (4/1 TMOS:MAPTMOS v/v) was added slowly to the
acetic acid solution. The combined solutions were stirred at rt for
prescribed time.
[0176] The resulting solutions were delivered into separate 150
.mu.m (I.D.).times.40 mm (L) capillary columns that were surface
treated as described in Example 1. The columns were then sealed at
both ends with two compression screws and were heated at 65.degree.
C. for a prescribed time and then at an elevated temperature (4a-e
120.degree. C.; 4f-n 110.degree. C.) for an additional time
period.
[0177] Monolith morphology on the cross-section of the column was
observed by SEM as described in Example 2. Depending on the
reaction conditions, dense monolith materials (a majority of
macropores with diameters less than 0.5 .mu.m) to non-dense
monolith materials (a majority of macropores with diameters greater
than 0.5 .mu.m) were observed, and varying levels of wall adhesion
were achieved. Specific amounts of reagents used to prepare these
products and characterization data are listed in Table 2.
TABLE-US-00001 TABLE 1 Pluronic Time Time Time Time Wall HOAc F38
Urea Vazo 64 0.degree. C. rt 65.degree. C. elevated Dense Adhesion
Product (mM) (g) (g) (mg) (min) (h) (h) temp (h) (Y/N) (Y/N) 3a 15
0.715 0.508 5 30 60 15 8 Y N 3b 30 0.708 0.503 5 30 1 18 7 Y Y 3c
15 0.709 0.502 5 0 3 17 5 Y Y 3d 30 0.698 0.501 5 0 3 17 5 Y N 3e
15 0.797 0.509 3 0 3 17 5 Y N 3f 30 0.793 0.511 3 0 3 17 5 Y N 3g
15 0.695 0.511 5 0 3 17 5 Y N 3h 15 0.793 0.504 5 0 3 17 5 Y N 3i
15 0.593 0.511 4 0 4 5 72 N Y 3j 15 0.558 0.496 4 0 4 5 72 N Y 3k
30 0.561 0.502 0.5 0 3 2 18 N Y 3l 30 0.595 0.498 1 0 3 2 18 Y N 3m
30 0.659 0.496 1 0 3 2 18 Y N 3n 30 0.703 0.497 1 0 3 2 18 Y N 3o
30 0.790 0.496 1 0 3 2 18 Y N 3p 30 0.560 0.499 1 0 3 2 16 N Y 3q
30 0.533 0.508 3 0 3 2 16 N N 3r 30 0.508 0.508 6 0 3 2 16 N Y 3s
30 0.470 0.513 5 0 3 2 16 N N 3t 30 0.415 0.501 2 0 3 2 16 N Y
[0178] TABLE-US-00002 TABLE 2 Pluronic Time Time Time Wall HOAc F38
Urea Vazo 64 rt 65.degree. C. elevated Dense Adhesion Product (mM)
(g) (g) (mg) (h) (h) temp (h) (Y/N) (Y/N) 4a 50 0.605 0.484 1 2 4
17 N Y 4b 50 0.505 0.512 2 2 4 17 N Y 4c 50 0.401 0.518 2 2 4 17 N
Y 4d 50 0.704 0.500 2 2 4 17 N N 4e 50 0.358 0.495 3 2 4 17 N Y 4f
50 0.403 0.502 1 2 20 8 N Y 4g 70 0.418 0.500 1 2 20 8 N Y 4h 100
0.400 0.492 1 2 20 8 N N 4I 50 0.404 0.493 1 4 3 17 N N 4j 70 0.419
0.492 1 4 3 17 N N
[0179] TABLE-US-00003 TABLE 3 Pluronic Time Time Time Wall HOAc F38
Urea Vazo 44 rt 60.degree. C. 100.degree. C. Dense Adhesion Product
(mM) (g) (g) (mg) (h) (h) (h) (Y/N) (Y/N) 8a 50 0.694 0.486 20 2 17
72 Y N 8b 50 0.653 0.501 20 2 17 72 N Y 8c 50 0.649 0.546 2 1 2 17
Y N 8d 50 0.659 0.514 2 1 2 17 N Y 8e 50 0.640 0.530 2 1 2 17 Y N
8f 50 0.628 0.556 2 1 2 17 Y N 8g 50 0.674 0.553 2 1 2 17 N Y
Example 5
[0180] Pluronic.RTM. F38 (0.424 g) and 0.503 g urea were added to 5
mL of a 15 mM acetic acid solution at room temperature in a glass
vial. The solutions were deoxygenated by nitrogen gas purging for
at least 2 minutes before 3 mg of Vazo.RTM. 64 was added, and then
the solution was cooled to 0.degree. C. In a separate glass vial, a
2 mL silane mixture (4/1 TMOS:MAPTMOS v/v) was prepared and then
cooled to 0.degree. C. Next, the silane mixture was added slowly to
the acetic acid solution. The combined solutions were stirred at
0.degree. C. for 1 h and then at room temperature (rt) for 1.75
h.
[0181] The resulting solution was delivered into a fused silica
capillary with a length of 40 cm (Examples 5a-b) or 120 cm
(Examples 5c-d), and an inner diameter of 150 .mu.m (Examples 5a-c)
or 520 .mu.m (Example 5d), which was pretreated as described in
Example 1, at a flow rate of 50 .mu.L/min for 5-7 minutes.
[0182] The column was then sealed at both ends using compression
screws (Examples 5a-b) or Upchurch P760 compression fittings and a
single Upchurch P760 union (Examples 5c-d). The sealed column was
held at room temperature for two hours after the mixed silanes were
added to the acetic acid mixture. The column was then heated in a
65.degree. C. oven for 2 hours, and transferred to a 125.degree. C.
oven and held overnight (20 hours).
[0183] The column was then slowly cooled to room temperature. The
surfactant was removed from the structure by prolonged methanol
purging using an Eldex Micropro Syringe Pump (Eldex, Napa, Calif.).
The column was purged with methanol for 6 hours at a 0.5 .mu.L/min
flow rate, then for 20 hours at 1 .mu.L/min flow rate.
[0184] Monolith morphology on the cross-section of the column was
observed by SEM as described in Example 2. Non-dense monolith
materials (a majority of macropores with diameters greater than 0.5
.mu.m) were observed, and wall adhesion was achieved.
Example 6
[0185] The monolith surfaces of columns from Examples 5a-d were
surface modified using the following procedure producing surface
modified monoliths defined analogously as Examples 6a-d.
[0186] A 0.32 M solution of octadecyldimethyl(dimethylamino)silane
was prepared in toluene or tetrahydrofuran. The column was purged
with this solution using an Eldex Micropro Syringe Pump at a flow
rate of 1 .mu.L/min for several column volumes and then heated from
25.degree. C. to 50-55.degree. C. and maintained at 50-55.degree.
C. for 19 hours. The column was heated using a Hot Pocket column
heater (Thermo Hypersil-Keystone, Bellefonte, Pa., or equivalent).
Upon cooling, methanol was purged through the column at 1 .mu.L/min
flow rate for at least 5 hours at 35.degree. C.
Example 7
[0187] A 1.0 mm I.D..times.150 mm L borosilicate glass lined steel
column (SGE Inc., Austin, Tex., or equivalent) was surface treated
as described in Example 1, with the exception that 6 mL aliquots of
solvent was used in step (5). Pluronic.RTM. F38 (1.272 g) and urea
(1.506 g) were weighed into a glass vial and dissolved into 15.0 mL
of a 32 mM acetic acid solution. The mixture was deoxygenated by
nitrogen gas purging for 5 minutes before 9 mg of Vazo.RTM. 64 were
added. The mixture was stirred for 5 minutes at room temperature,
and then chilled to 0.degree. C. for 23 min.
[0188] Next, 6 mL of silane mixture (4/1 TMOS:MAPTMOS v/v) was
slowly added into the above mixture at 0.degree. C. After silane
addition, the mixture was stirred at 0.degree. C. for 1.5 h. The
resulting mixture was delivered into the glass lined steel column.
The column was then sealed at both ends using two Waters stainless
steel compression plugs. The sealed column was held at room
temperature for two hours after the mixed silanes were added to the
acetic acid mixture. The column was heated in a 65.degree. C. oven
for 2 hours, and then transferred to a 125.degree. C. oven and held
for 18 hours. The column was then slowly cooled to room
temperature.
[0189] The surfactant was removed from the monolith structure by
prolonged methanol purging using a HPLC pump (Model 515, Waters
Corp, or equivalent). After methanol purging the column
end-fittings and frits were removed. Inspection of the monolith
structure yielded no evidence of monolith shrinkage, as observed by
optical microscope (Model C-P-S, Nikon, Tokyo, Japan, or
equivalent).
Example 8
[0190] Solutions of surfactant, urea, and silane in acetic acid
were made as described in Example 4, except Vazo.RTM. 44 was
substituted for Vazo.RTM. 64. The resulting solutions were
delivered into separate 150 .mu.m I.D..times.40 mm L capillary
columns that were surface treated as described in Example 1. The
columns were sealed, heated, and analyzed as described in Example
4, where the elevated temperature was 110.degree. C. The specific
amounts of reagents used to prepare these products and the
resulting characterization data are listed in Table 3.
Example 9
[0191] Pressure data on monolith columns were collected on
non-surface modified and surface modified monoliths using the
pressure transducer of an Eldex MicroPro Syringe Pump (Eldex, Napa,
Calif., or equivalent). An equivalent particle diameter (apparent
particle size) was obtained based on the Kozeny-Carman equation. An
interstitial porosity of 0.4 of the particle packed column that
would give the same permeability as the monolith bed was assumed.
Solvent viscosities of solvent mixtures were obtained based on the
data of Colin et. al. [H. Colin, J. C. Diez-Masa, G. Guiochon, T.
Czajkowska, 1. Miedziak, J. Chromatogr., 167 (1978) 41-65]. Solvent
viscosities of neat solvents were obtained from Handbook of
Chemistry and Physics on CD-ROM (version 2002, Editor-in-chief D.
R. Lide, Chapman & Hall/CRC).
[0192] Results of the determination of the equivalent particle size
are summarized in Table 4. TABLE-US-00004 TABLE 4 Equivalent
Particle Size [.mu.m] in Selected Solvents Acetonitrile-
Acetonitrile- Pro- Meth- Methylene Acetoni- water water duct anol
Chloride trile 55:45 (v/v) 40:60 (v/v) 3t 3 4 -- 4 -- 3u -- 3 4 3 3
5b 4 -- -- -- -- 6b -- -- -- -- 4 5c 21 -- -- -- -- 6c -- -- -- --
17 5d -- -- -- 26 -- 6d -- -- -- -- 26
Example 10
[0193] The following example demonstrates the porosity of the
hybrid inorganic/organic materials and further characterizes the
materials using chromatographic criteria, i.e., efficiency and
tailing factor.
Experimental Conditions:
[0194] The experimental data was acquired using an Eldex MicroPro
Syringe Pump (Eldex, Napa, Calif., or equivalent), a Valco internal
sample injector (Valco Instruments Co. Inc., Houston, Tex., or
equivalent) and a Waters 2487 Dual .lamda. absorbance detector
equipped with a 250 nL capillary flow cell (Waters Corp., or
equivalent). Data acquisition and determination of chromatography
parameters was performed using Millenium 32 software (Waters Corp.,
or equivalent). Acetonitrile and water was mixed in 40/60 volume
ratio. Thiourea, naphthalene and acenaphthene were used as
analytes. The pump was operated at volumetric flow rates between
0.25 and 4.0 .mu.L/min. Thiourea, naphthalene and acenaphthene were
dissolved in acetonitrile-water 55:45 (v/v) mixture at 20, 100 and
400 .mu.g/ml respectively. The injection volume was 20 nL. UV
detection was carried out at 254 nm. The experiments were performed
at ambient temperature (24.degree. C.).
Experimental Protocol:
[0195] The columns were directly connected to the injector and to
the inlet of capillary flow cell except 3u which was connected with
50 .mu.m I.D. fused silica capillaries. Results of chromatography
evaluation are summarized in Table 5. Total porosity was calculated
by dividing the elution volume of non-retained compound (thiourea)
by the total column volume. TABLE-US-00005 TABLE 5 Flow USP Column
Rate Test Capacity N/meter Tailing Total No. [.mu.L/min] Solute
Factor (half width) Factor Porosity 3u 0.25 Naphthalene 3.76 12540
1.27 0.96 0.5 Naphthalene 3.47 11890 1.40 0.75 Naphthalene 3.38 980
1.42 0.25 Acenaphthene 7.79 17550 1.21 0.5 Acenaphthene 7.24 14450
1.32 0.75 Acenaphthene 7.06 12660 1.33 6b 0.25 Naphthalene 4.55
42123 1.46 0.97 0.5 Naphthalene 4.69 45205 1.42 0.75 Naphthalene
4.61 41095 1.36 1.0 Naphthalene 4.62 36986 1.34 0.25 Acenaphthene
9.88 49315 1.20 0.5 Acenaphthene 10.18 52053 1.29 0.75 Acenaphthene
9.88 45204 1.27 1.0 Acenaphthene 9.91 39041 1.25 6c 0.25
Naphthalene 5.2 40650 1.24 0.92 0.5 Naphthalene 5.2 48260 1.28 1.0
Naphthalene 4.9 43700 1.24 2.0 Naphthalene 5.0 30270 1.17 4.0
Naphthalene 5.2 19207 1.14 0.25 Acenaphthene 11.44 56267 1.26 0.5
Acenaphthene 11.4 63878 1.28 1.0 Acenaphthene 11.0 45397 1.23 2.0
Acenaphthene 11.0 29550 1.16 4.0 Acenaphthene 11.4 19593 1.14
References .sup.1MacNair J. E., etc. Anal Chem. 1999, 71, 700
.sup.2Svec F., etc. Review J. Chromatography A, 2000, 887, 3-29
.sup.3Nakanishi K., Tanaka N. etc., J. High Resol. Chromatogr.
1998, Vol. 21, No. 8 p477-479; Nakanishi K., Tanaka N. etc., J
Chromatography A 2002, 960, 85-96 .sup.4Nakanishi K., Tanaka N.
etc., J. Chromatography A 2002, 961, 53-63 .sup.5Zare R. N. etc.,
Anal. Chem. 2001, 73, 3921-3926 .sup.6Zare R. N. etc. J.
Chromatography A, 2002, 961, 45-51. .sup.7Zare R. N. etc. US Patent
Application 2002/0079257 .sup.8Nakanishi K. etc. WO 99/50654
.sup.9O. Prucker and J. Ruhe, Macromolecules 1998, 31, 592-601
.sup.10O. Prucker and J. Ruhe, Macromolecules 1998, 31, 602-613
.sup.11Hanson et al., J. Chromat. A656 (1993) 369-380
Incorporation By Reference
[0196] The entire contents of all patents, published patent
applications and other references cited herein are hereby expressly
incorporated herein in their entireties by reference.
Equivalents
[0197] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures described herein. Such
equivalents were considered to be within the scope of this
invention and are covered by the following claims. The contents of
all references, issued patents, and published patent applications
cited throughout this application are hereby incorporated by
reference.
* * * * *