U.S. patent application number 10/911389 was filed with the patent office on 2005-06-23 for water-wettable chromatographic media for solid phase extraction.
This patent application is currently assigned to Waters Investments Ltd.. Invention is credited to Bouvier, Edouard S. P., McDonald, Patrick D., Meirowitz, Randy E..
Application Number | 20050133424 10/911389 |
Document ID | / |
Family ID | 24544907 |
Filed Date | 2005-06-23 |
United States Patent
Application |
20050133424 |
Kind Code |
A1 |
Bouvier, Edouard S. P. ; et
al. |
June 23, 2005 |
Water-wettable chromatographic media for solid phase extraction
Abstract
A method for removing an organic solute from a solution
comprises contacting the solution with a polymer formed by
copolymerizing one or more hydrophobic monomers and one or more
hydrophilic monomers, whereby the solute is adsorbed onto the
polymer. The solution can comprise a polar solvent such as a polar
organic solvent or water or an aqueous buffer. The hydrophobic
monomer can be, for example, divinylbenzene. The hydrophilic
monomer can be, for example, a heterocyclic monomer, such as a
vinylpyridine or N-vinylpyrrolidone.
Inventors: |
Bouvier, Edouard S. P.;
(Stow, MA) ; Meirowitz, Randy E.; (Somerville,
MA) ; McDonald, Patrick D.; (Holliston, MA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
Waters Investments Ltd.
Milford
MA
|
Family ID: |
24544907 |
Appl. No.: |
10/911389 |
Filed: |
August 4, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10911389 |
Aug 4, 2004 |
|
|
|
10615820 |
Jul 9, 2003 |
|
|
|
6773583 |
|
|
|
|
10615820 |
Jul 9, 2003 |
|
|
|
10235608 |
Sep 5, 2002 |
|
|
|
6726842 |
|
|
|
|
10235608 |
Sep 5, 2002 |
|
|
|
09874763 |
Jun 5, 2001 |
|
|
|
6468422 |
|
|
|
|
09874763 |
Jun 5, 2001 |
|
|
|
09643094 |
Aug 21, 2000 |
|
|
|
6254780 |
|
|
|
|
09643094 |
Aug 21, 2000 |
|
|
|
09374945 |
Aug 16, 1999 |
|
|
|
6106721 |
|
|
|
|
09374945 |
Aug 16, 1999 |
|
|
|
09216047 |
Dec 18, 1998 |
|
|
|
5976367 |
|
|
|
|
09216047 |
Dec 18, 1998 |
|
|
|
08634710 |
Apr 18, 1996 |
|
|
|
5882521 |
|
|
|
|
Current U.S.
Class: |
210/198.2 ;
210/511; 422/400; 422/70 |
Current CPC
Class: |
B01J 2220/54 20130101;
B01J 2220/62 20130101; B01D 15/00 20130101; G01N 30/482 20130101;
B01D 15/08 20130101; B01J 2220/64 20130101; B01J 20/285
20130101 |
Class at
Publication: |
210/198.2 ;
422/070; 422/101; 210/511 |
International
Class: |
B01D 015/08 |
Claims
What is claimed is:
1. A solid phase extraction cartridge comprising a water-wettable
polymer packed inside an open-ended container, said polymer being
formed by copolymerizing at least one hydrophobic monomer and at
least one hydrophilic monomer having a hydrophobic to hydrophilic
monomer ratio sufficient for the polymer to be water-wettable while
being effective to retain organic solutes thereon and wherein said
polymer is capable of adsorbing a less polar solute more strongly
than a more polar solute and wherein said solutes are capable of
being desorbed from said polymer in order of decreasing polarity by
washing said polymer with a sequence of solvents of decreasing
polarity.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/615,820, filed Jul. 9, 2003, (now allowed), which is a
divisional of U.S. application Ser. No. 10/235,608, filed Sep. 5,
2002 (now U.S. Pat. No. 6,726,842; issue date Apr. 27, 2004), which
is a continuation of U.S. application Ser. No. 09/874,763, filed
Jun. 5, 2001 (now U.S. Pat. No. 6,468,422; issue date Oct. 22,
2002), which is a continuation of U.S. application Ser. No.
09/643,094 (U.S. Pat. No. 6,254,780; issue date Jul. 3, 2001),
filed Aug. 21, 2000, which is a continuation of U.S. application
Ser. No. 09/374,945, filed Aug. 16, 1999 (U.S. Pat. No. 6,106,721;
issue date Aug. 22, 2000), which is a continuation of U.S.
application Ser. No. 09/216,047, filed Dec. 18, 1998 (U.S. Pat. No.
5,976,367; issue date Nov. 2, 1999), which is a divisional of U.S.
application Ser. No. 08/634,710 filed Apr. 18, 1996 (U.S. Pat. No.
5,882,521; issue date Mar. 16, 1999). The teachings of each of
these referenced applications are expressly incorporated herein by
reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] Solid phase extraction is a chromatographic technique of
frequent use in the preparation of samples for quantitative
analysis, for example, via high performance liquid chromatography
(HPLC) or gas chromatography (GC) (McDonald and Bouvier, eds. Solid
Phase Extraction Applications Guide and Bibliography, sixth
edition, Milford, Mass.: Waters (1995)). Solid phase extraction can
be used to separate a component of interest in a complex solution
from potentially interfering matrix elements and to concentrate the
analyte to levels amenable to detection and measurement. Thus,
solid phase extraction is of use in the analysis of environmental
samples, where, for example, various soluble components of soils
may interfere with the analysis of trace organic materials. Solid
phase extraction is also of importance in the analysis of
pharmaceutical agents or metabolites in blood plasma, which
requires the prior removal of plasma proteins and other matrix
constituents which may interfere with the analysis.
[0003] Solid phase extraction of an aqueous solution is typically
performed by passing the solution through a single-use cartridge
containing a chromatographic sorbent. The most commonly used
sorbents consist of porous silica particles that have been
functionalized on their surface with hydrophobic octyl (C.sub.8)
and octadecyl (C.sub.18) functional groups. Prior to use, such
sorbents must be wetted with a water-miscible polar organic solvent
to solvate the alkyl chains. This increases the contact of these
chains with the aqueous phase, increasing the sorbent surface area
available to solutes and, therefore, retention of solutes. Such
sorbents which are not pre-wetted or have dried out display poor
solute retention, and, thus, inadequate separation of solution
components.
[0004] The requirement that the sorbent remain wetted during the
extraction procedure complicates solid phase extractions and
substantially slows sample analysis. For example, solid phase
extraction cartridges, in general, have differing flow rates and
must be monitored individually to prevent drying out when used on a
vacuum manifold, the current state of the art for processing
multiple samples. This further complicates the development of
instruments for automated solid phase extraction, which often
incorporate elaborate safeguards to prevent drying out of the
sorbent.
[0005] Thus, there is need for a solid phase extraction method
which utilizes a sorbent that does not require wetting with an
organic solvent or that stays wetted even if the bulk of the
wetting solvent is removed during use on a vacuum manifold. Such a
method would enable more rapid sample preparation for quantitative
analysis, particularly for multiple samples, and allow the
development of less expensive and simpler methods for automated
solid phase extraction.
SUMMARY OF THE INVENTION
[0006] The present invention relates to a method for removing an
organic solute from a solution. The method comprises contacting the
solution with a water-wettable polymer formed by copolymerizing one
or more hydrophobic monomers and one or more hydrophilic monomers,
whereby the solute is adsorbed onto the polymer. The solution can
comprise a polar solvent such as a polar organic solvent, a
water/organic mixture or, preferably, water or an aqueous solution,
such as an aqueous buffer, acid, base or salt solution.
[0007] The hydrophobic monomer can comprise a hydrophobic moiety.
Suitable hydrophobic moieties include, but are not limited to
phenyl, phenylene and C.sub.2-C.sub.18-alkyl groups. Suitable
hydrophobic monomers include divinylbenzene and styrene.
[0008] The hydrophilic monomer can comprise a hydrophilic moiety.
In one embodiment the hydrophilic moiety is a saturated,
unsaturated or aromatic heterocyclic group, such as a pyrrolidonyl
group or a pyridyl group. In another embodiment, the hydrophilic
moiety is an ether group. Suitable hydrophilic monomers are, for
example, N-vinylpyrrolidone, 2-vinylpyridine, 3-vinylpyridine,
4-vinylpyridine and ethylene oxide.
[0009] In one embodiment of the method, the polymer is a
poly(divinylbenzene-co-N-vinylpyrrolidone) copolymer which
comprises greater than about 12 mole percent N-vinylpyrrolidone. In
a preferred embodiment, the copolymer comprises from about 15 mole
percent to about 30 mole percent N-vinylpyrrolidone.
[0010] The present invention further includes a method for forming
a solution, containing a solute, which is suitable for quantitative
analysis. In one embodiment, the method comprises contacting a
first solution including the solute with a water-wettable polymer
formed by copolymerizing at least one hydrophobic monomer and at
least one hydrophilic monomer, whereby the solute is adsorbed onto
the polymer. This is followed by washing the polymer with a
suitable solvent or mixture of solvents, so that the solute is
desorbed from the polymer, thereby forming a second solution
including the solute. This second solution is suitable for
quantitative analysis.
[0011] In another embodiment, the invention provides a method for
forming a solution comprising a polar organic solute which is
suitable for quantitative analysis. The method comprises contacting
a solution which includes the polar organic solute and at least one
additional solute of lesser polarity with a water-wettable polymer
formed by copolymerizing at least one hydrophobic monomer and at
least one hydrophilic monomer, whereby the additional solute is
adsorbed onto the polymer and the polar solute remains in the
aqueous phase. The resulting aqueous phase is, thus, a solution of
the polar organic solute which is suitable for quantitative
analysis.
[0012] The present invention further includes a solid phase
extraction cartridge comprising an open-ended container and a
polymer packed within the container. The solid phase extraction
cartridge can, optionally, further comprise a porous retaining
means, such as a frit. The polymer is formed by copolymerizing at
least one hydrophobic monomer and at least one hydrophilic monomer.
Suitable polymers include
poly(divinylbenzene-co-N-vinylpyrrolidone) copolymers which
comprise about 12 mole percent or more, preferably from about 15
mole percent to about 30 mole percent, N-vinylpyrrolidone. The
solid phase extraction cartridge preferably comprises from about
0.025 g to about 1 g of the polymer.
[0013] The present invention enables the solid phase extraction of
one or more solutes from an aqueous solution, without prior wetting
of the sorbent with an organic solvent. The method is versatile
with respect to solute identity, resulting in extraction of a broad
range of solutes of varying polarity. A particular advantage of the
method is that the sorbent can dry out during the extraction
procedure without diminishing the ability of the sorbent polymer to
retain solutes. Thus, the present invention provides a simpler
method for the preparation of analytical samples, decreasing sample
preparation time and increasing sample throughput. The present
method is, thus, also more amenable to automation than currently
used methods.
BRIEF DESCRIPTION OF THE DRAWING
[0014] The FIGURE is a schematic, in cross-section, of one
embodiment of the solid phase extraction cartridge of the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0015] The present invention provides a method for solid phase
extraction of aqueous or buffered aqueous solutions which does not
require that the sorbent be wetted with an excess of organic
solvent prior to and during the solid phase extraction process. The
invention is based on the discovery that polymers or resins
comprising both a hydrophilic monomer and a hydrophobic monomer in
a suitable ratio can be wetted by water while maintaining
surprisingly effective retention of organic solutes with a wide
range of chromatographic polarities.
[0016] As described in the Exemplification, a relatively small
increase in the N-vinylpyrrolidone content of a
poly(divinylbenzene-co-N-vinylpyrroli- done) copolymer resulted in
a dramatic improvement in retention of polar organic solutes under
conditions in which the pre-wetted polymer was dried under reduced
pressure for several minutes. For example, under these conditions,
recovery of acetaminophen from such a copolymer comprising 9 mole
percent N-vinylpyrrolidone was 10.4%. Increasing the mole percent
N-vinylpyrrolidone in the copolymer to 13 resulted in a 92%
recovery of acetaminophen. Similar results were observed for
procainamide, ranitidine, and caffeine. For relatively nonpolar
solutes the difference in recovery between the two copolymers was
less dramatic.
[0017] The ability of poly(divinylbenzene-co-N-vinylpyrrolidone)
copolymers comprising between 13 mole percent and 22 mole percent
N-vinylpyrrolidone to retain organic solutes was also compared with
that of octadecyl (C.sub.18)-bonded silica gel. As discussed in the
Exemplification, the C.sub.18-bonded silica sorbent showed poor
retention of polar organic solutes when the sorbent was pre-wetted
with an organic solvent and then dried under reduced pressure prior
to extraction. For example, this sorbent showed a 2.8% recovery of
m-toluamide under these conditions. In contrast, the
poly(divinylbenzene-co-N-vinylpyrrolidone) copolymer comprising 13
mole percent N-vinylpyrrolidone displayed a 96.3% recovery of
m-toluamide under similar conditions. Overall, the results
demonstrate that a balance between the mutually exclusive
properties of water-wettability and retention of organic solutes
can be achieved in a copolymer which has a suitable ratio of
hydrophilic monomers and hydrophobic monomers.
[0018] In one embodiment, the invention is a method for removing a
solute from a solution. The method comprises the step of contacting
the solution with a water-wettable polymer formed by copolymerizing
at least one hydrophobic monomer and at least one hydrophilic
monomer, whereby the solute is adsorbed onto the polymer. The
solution can comprise water, or a mixture of water and a
water-miscible polar organic solvent such as methanol, ethanol,
N,N-dimethylformamide, dimethylsulfoxide or acetonitrile. The
solution can also comprise a mixture of water or an aqueous buffer
and a polar, water-miscible organic solvent. In a particularly
preferred embodiment, the solution is an acidic, basic or neutral
aqueous or predominately aqueous, i.e., greater than about 50%
water by volume, solution. The solute is preferably an organic
compound.
[0019] The solution can be contacted with the polymer in any
fashion which permits intimate contact of the polymer and the
solution, such as a batch or chromatographic process. For example,
the solution can be forced through a porous polymer column, disk or
plug, or the solution can be stirred with the polymer, such as in a
batch-stirred reactor. The solution can also be added to a
polymer-containing well of a microtiter plate. The polymer can take
the form of, for example, beads or pellets. The solution is
contacted with the polymer for a time period sufficient for the
solute of interest to substantially adsorb onto the polymer. This
is typically the time necessary for the solute to equilibrate
between the polymer surface and the solution. The adsorption or
partition of the solute onto the polymer can be partial or
complete.
[0020] A preferred polymer for use in the present method is
water-wettable and has the ability to retain a variety of solutes
of varying polarity. The term "water-wettable", as used herein,
describes a material which is solvated, partially or completely, by
water. The material, thus, engages in energetically favorable or
attractive interactions with water molecules. These interactions
increase the amount of surface area of the material which, upon
contact with water, is accessible to water molecules, and, hence,
to solutes present in aqueous solution.
[0021] The term "monomer", as used herein, refers to both a
molecule comprising one or more polymerizable functional groups
prior to polymerization, and a repeating unit of a polymer. A
polymer can comprise two or more different monomers, in which case
it can also be referred to as a copolymer. The "mole percent" of a
given monomer which a copolymer comprises is the mole fraction,
expressed as a percent, of the monomer of interest relative to the
total moles of the various (two or more) monomers which compose the
copolymer.
[0022] In one embodiment of the method, the solution is contacted
with the polymer in dry form. In another embodiment the polymer is
wetted prior to contacting the solution with the polymer, for
example, by treating the polymer with a polar organic solvent,
followed by water or an aqueous buffer.
[0023] The hydrophilic monomer can comprise hydrophilic group. In
one embodiment, the hydrophilic group is a heterocyclic group, for
example, a saturated, unsaturated or aromatic heterocyclic group.
Suitable examples include nitrogen-containing heterocyclic groups
such as pyrrolidonyl and pyridyl groups. In another embodiment, the
hydrophilic moiety is an ether group. The hydrophilic monomer can
be, for example, N-vinylpyrrolidone, 2-vinylpyridine,
3-vinylpyridine, a hydrophobic moiety, 4-vinylpyridine or ethylene
oxide.
[0024] The hydrophobic monomer can comprise, for example, an
aromatic carbocyclic group, such as a phenyl or phenylene group, or
an alkyl group, such as a straight chain or branched
C.sub.2-C.sub.18-alkyl group. Suitable hydrophobic monomers
include, but are not limited to, styrene and divinylbenzene.
[0025] In a preferred embodiment, the polymer to be contacted with
the solution is a poly(divinylbenzene-co-N-vinylpyrrolidone)
copolymer. The polymer can comprise about 12 mole percent or more
N-vinylpyrrolidone. In a particularly preferred embodiment, the
polymer comprises from about 15 mole percent to about 30 mole
percent N-vinylpyrrolidone.
[0026] The polymer can be in the form of, for example, beads having
a diameter in the range from about 5 to about 500 .mu.m, preferably
from about 20 to about 200 .mu.m. The copolymer, preferably, has a
specific surface area in the range from about 200 to about 800
square meters per gram and pores having a diameter ranging from
about 0.5 nm to about 100 nm.
[0027] The solution comprising the solute can, optionally, further
contain one or more additional solutes. In one embodiment, the
solution is an aqueous solution which includes a complex variety of
solutes. Solutions of this type include blood plasma, urine,
cerebrospinal fluid, synovial fluid and other biological fluids,
including extracts of tissues, such as liver tissue, muscle tissue,
brain tissue and heart tissue. Such extracts can be aqueous
extracts or organic extracts which have been dried and subsequently
reconstituted in water or in a water/organic mixture.
[0028] The solution can also be ground water, surface water,
drinking water or an aqueous or organic extract of an environmental
sample, such as a soil sample. The solution can further be a food
substance, such as a fruit or vegetable juice or milk or an aqueous
or aqueous/organic extract of a food substance, such as a fruit,
vegetable, cereal, or meat.
[0029] The solute can be any organic compound of polarity suitable
for adsorption onto the polymer. Such solutes can include, for
example, drugs, pesticides, herbicides, toxins and environmental
pollutants resulting from the combustion of fossil fuels or other
industrial activity, such as metal-organic compounds comprising a
heavy metal such as mercury, lead or cadmium. The solutes can also
be metabolites or degradation products of the foregoing materials.
The solutes can also include biomolecules, such as proteins,
peptides, hormones, polynucleotides, vitamins, cofactors,
metabolites, lipids and carbohydrates.
[0030] In one embodiment of the method, the polymer is packed as
particles within an open-ended container to form a solid phase
extraction cartridge. The container can be, for example, a
cylindrical container or column which is open at both ends so that
the solution can enter the container through one end, contact the
polymer within the container, and exit the container through the
other end. The polymer can be packed within the container as small
particles, such as beads having a diameter between about 5 .mu.m
and about 500 .mu.m, preferably between about 20 .mu.m and about
200 .mu.m. The polymer particles can also be packed in the
container enmeshed in a porous membrane.
[0031] The container can be formed of any material which is
compatible, within the time frame of the extraction process, with
the solutions and solvents to be used in the procedure. Such
materials include glass and various plastics, such as high density
polyethylene and polypropylene. In one embodiment, the container is
cylindrical through most of its length and has a narrow tip at one
end. One example of such a container is a syringe barrel.
[0032] The solid phase extraction cartridge can further comprise a
porous retaining means, such as a filter element, or frit, at one
or both ends of the cartridge adjacent to the polymer to retain the
polymer within the cartridge and to remove undissolved solid
materials from the solution as it flows into the cartridge, while
still permitting solution flow into and out of the cartridge. Such
a filter can be formed from, for example, fritted glass or a porous
polymer, such as a porous high density polyethylene.
[0033] The amount of polymer within the container is limited by the
container volume and can range from about 0.001 g to about 50 g,
but is preferably between about 0.025 g and about 1 g. The amount
of polymer suitable for a given extraction depends upon the amount
of solute to be adsorbed, the available surface area of the polymer
and the strength of the interaction between the solute and the
polymer. This can be readily determined by one of ordinary skill in
the art.
[0034] The present invention includes a solid phase extraction
cartridge as described above, wherein the polymer is a
water-wettable polymer formed by copolymerizing at least one
hydrophobic monomer and at least one hydrophilic monomer. The
polymer can be, for example, a
poly(divinylbenzene-co-N-vinylpyrrolidone) copolymer comprising
about 12 mole percent or more N-vinylpyrrolidone. In a preferred
embodiment, the copolymer comprises from about 15 mole percent to
about 30 mole percent N-vinylpyrrolidone. The cartridge can be a
single use cartridge, which is used for the treatment of a single
sample and then discarded, or it can be used to treat multiple
samples.
[0035] A preferred embodiment of the solid phase extraction
cartridge of the present invention is illustrated in cross section
in the FIGURE. Container 1 is a syringe barrel which can be formed
of molded polypropylene and can have a volume ranging from about 1
cm.sup.3 to about 50 cm.sup.3. Water wettable polymer 2 is prepared
by the copolymerization of N-vinylpyrrolidone and divinylbenzene
and comprises from about 12 mole percent to about 30 mole percent
N-vinylpyrrolidone. Polymer 2 is packed within the container as
porous beads of diameter between about 20 .mu.m and about 200
.mu.m. The mass of polymer 2 packed within the container can range
from about 0.025 g to about 10 g, depending upon the volume of the
container. Frits 3 and 4 are formed of porous high density
polyethylene.
[0036] The solution to be treated is added to the top of the solid
phase extraction cartridge and allowed to flow through the
cartridge, bringing the solute to be adsorbed into contact with the
polymer. The solution can flow through the cartridge under the
force of gravity. Increased flow rates can be achieved by
establishing a pressure difference between the ends of the
cartridge. Such a pressure difference can be established by
attaching a vacuum source to the lower end of the cartridge or by
applying positive pressure to the upper end of the cartridge, for
example, by applying a pressurized gas, such as air or nitrogen, to
the top of the cartridge, or by compressing the air within the
cartridge above the polymer with a piston or plunger. The flow rate
of the solution through the cartridge can be adjusted by regulating
the pressure difference across the cartridge. Suitable solution
flow rates, given in terms of the linear velocity of the solution,
range up to about 14 mm/second, but are preferably in the range
from about 0.7 to about 3.5 mm/second.
[0037] Another aspect of the present invention is a method for
forming a solution of a solute which is suitable for quantitative
analysis. In one embodiment, the solute is of a polarity suitable
for adsorption onto the polymer. The method comprises contacting a
first solution which includes the solute with a polymer formed by
copolymerizing at least one hydrophobic monomer and at least one
hydrophilic monomer, whereby the solute is adsorbed onto the
polymer. This is followed by washing the polymer with a suitable,
stronger solvent or mixture of solvents, thereby desorbing or
eluting the solute from the polymer and forming a second solution
which contains the solute. This second solution is suitable for the
quantitative analysis of the solute.
[0038] The solution contacted with the polymer can comprise the
solute of interest in dilute form, for example, at a concentration
too low for accurate quantitation. By adsorbing the solute onto the
polymer and then desorbing the solute with a substantially smaller
volume of a less polar solvent, a solution which includes the
solute of interest can be prepared having a substantially higher
concentration of the solute of interest than that of the original
solution. The method also results in solvent exchange, that is, the
solute is removed from a first solvent and re-dissolved in a second
solvent
[0039] The polymer need not be pretreated or wetted prior to
contacting the solution with the polymer. In one embodiment, the
polymer is treated with a water-miscible organic solvent, followed
by water or aqueous buffer, prior to contacting the solution with
the polymer. In another embodiment, the solution is contacted with
dry polymer, that is, the polymer is not wetted prior to treatment
of the solution.
[0040] The solution contacted with the polymer can comprise a polar
solvent and is preferably predominately, i.e. greater than 50% by
volume, an acidic, basic or neutral aqueous solution or aqueous
buffer. The solution can also comprise a water-miscible polar
organic solvent such as methanol, ethanol, acetonitrile,
N,N-dimethylformamide, or dimethylsulfoxide, or a mixture of such a
solvent and water.
[0041] The solution comprising the solute of interest can further
comprise one or more additional solutes. In one embodiment, the
additional solute or solutes are more polar than the solute of
interest, and, thus, adsorb more weakly to the polymer than the
solute of interest. Such an additional solute can be desorbed from
the polymer by washing the polymer with a solvent which does not
desorb the compound of interest, thereby forming a solution of the
additional solute or solutes which is substantially free of the
solute of interest. A suitable solvent for the desorption of the
additional solute will typically be sufficiently polar that it does
not desorb the compound of interest.
[0042] After desorption of the additional solute or solutes, the
compound of interest can be desorbed by washing the polymer with a
suitable, i.e., less polar, solvent. This forms a solution of the
organic solute which is substantially free from more polar solutes
and is suitable for the quantitative analysis of the organic
solute.
[0043] In one embodiment, the solute of interest adsorbs onto the
polymer, but one or more additional solutes do not. Such an
additional solute can be, for example, of sufficiently high
polarity that it does not adsorb onto the polymer. The additional
solute can also comprise large molecules, for example,
macromolecules such as proteins, which are unable to pass through
the pores within the polymer, and, thus, have access to only a
small fraction of the overall polymer surface area. Such molecules
are typically retained poorly, if at all, by the polymer.
[0044] In a further embodiment, the additional solute or solutes
are less polar than the solute of interest and, thus, adsorb to the
polymer more strongly than the compound of interest. The compound
of interest can be weakly to moderately adsorbed or not adsorbed.
If adsorbed, the solute of interest is desorbed from the polymer by
washing the polymer with a solvent of sufficient polarity that it
does not desorb the additional solute or solutes. Thus, the
compound of interest can be desorbed from the polymer without
desorbing the other solutes.
[0045] In one embodiment, the additional solute or solutes are also
analytes of interest. Thus a series of solutes initially present in
a solution can be separated, and solutions of each suitable for
quantitative analysis can be formed using the method of the present
invention. In this case, the solution is contacted with the polymer
so that the solutes adsorb to the polymer. The solutes are then
desorbed from the polymer in order of decreasing polarity (i.e.,
most polar solute first, followed by solutes of successively
decreasing polarity) by washing the polymer with a sequence of
solvents of decreasing polarity.
[0046] Polymers, solutions and solutes which are suitable for this
method include those described above. Solvents which are suitable
for desorbing the solute from the polymer will typically be polar
water-miscible organic solvents, such as alcohols, for example,
methanol, ethanol, and isopropanol, acetonitrile, acetone, and
tetrahydrofuran, or mixtures of water and these solvents. The
desorbing solvent can also be a nonpolar or moderately polar
water-immiscible solvent such as dichloromethane, diethylether,
chloroform, or ethylacetate. Mixtures of these solvents are also
suitable. Preferred solvents or solvent mixtures must be determined
for each individual case. A suitable solvent can be determined by
one of ordinary skill in the art without undue experimentation, as
is routinely done in chromatographic methods development (McDonald
and Bouvier, supra, (1995); Snyder and Kirkland, Introduction to
Modern Liquid Chromatography, New York: J. Wiley and Sons
(1974)).
[0047] The methods of the present invention can be used to prepare
solutions of a solute which are suitable for quantitative analysis
via a variety of techniques, including high performance liquid
chromatography, gas chromatography, gas chromatography/mass
spectrometry, and immunoassay.
[0048] The sorbent polymers used in the methods of the present
invention can be prepared via standard synthetic methods. For
example, a poly(divinylbenzene-co-N-vinylpyrrolidone) copolymer can
be synthesized by copolymerization of divinylbenzene and
N-vinylpyrrolidone using standard methods of free radical
polymerization which are well known in the art. One method for
forming copolymers of this type is disclosed in U.S. Pat. No.
4,382,124, issued to Meitzner et al., the contents of which are
incorporated herein by reference. The composition of the resulting
copolymer depends upon the starting stoichiometry of the two
monomers and can be readily varied. The composition of the product
copolymer in some cases will not be substantially the same as the
proportion of the starting materials, due to differences in
reactivity ratios among the monomers.
[0049] The invention will now be further and specifically described
by the following example.
EXEMPLIFICATION
Materials
[0050] The model solutes procainamide, acetaminophen, m-toluidine,
m-toluamide, propranolol, caffeine, and 2,7-dihydroxynaphthalene
were obtained from Aldrich Chemical Company (Milwaukee, Wis.),
while doxepin, ranitidine, and betamethasone-17-valerate were
purchased from Sigma Chemical Company (St. Louis, Mo.). The
tC.sub.18 bonded silica solid phase extraction cartridge was
obtained from Waters Corporation (Milford, Mass., catalogue no.
WAT054960). A poly(divinylbenzene-co-N-vinylpyrrolid- one)
copolymer comprising about 9 mole percent N-vinylpyrrolidone was
obtained from Waters Corporation (Porapak.RTM.R).
Poly(divinylbenzene) was also obtained from Waters
(Styragel.RTM.).
[0051] Preparation of Poly(Divinylbenzene-Co-N-Vinylpyrrolidone)
Copolymers
[0052] To a 3000 mL flask was added a solution of 5.0 g
hydroxypropylmethylcellulose (Methocel E15, Dow Chemical Co.,
Midland, Mich.) in 1000 mL water. To this was added a solution of
175 g divinylbenzene (DVB HP-80, Dow), 102 g N-vinyl-2-pyrrolidone
(International Specialty Products), and 1.85 g
azobisisobutyronitrile (Vazo 64, Dupont Chemical Co, Wilmington,
Del.) in 242 g toluene. The resulting biphasic mixture was stirred
for 30 minutes at room temperature using sufficient agitation to
form oil droplets of the desired micron size. The resulting
suspension was then heated under moderate agitation to 70.degree.
C. and maintained at this temperature for 20 hours. The suspension
was then cooled to room temperature, filtered and washed with
methanol. The filter cake was then dried in vacuo for 16 hours at
80.degree. C. The composition of the product polymer was determined
by elemental analysis. Elemental analysis: N: 2.24%; mole percent
N-vinylpyrrolidone: 20%.
[0053] A series of poly(divinylbenzene-co-N-vinylpyrrolidone)
copolymers comprising about 13, 14, 16, and 22 mole percent
N-vinylpyrrolidone was also prepared by this method by varying the
starting ratio of the divinylbenzene and N-vinylpyrrolidone
monomers.
[0054] A 50 mg amount of each polymer was packed into a 1 cc
Sep-Pak Vac.RTM. cartridge container (Waters Corporation) having a
polyethylene frit at both the inlet and the outlet of the polymer
bed to form a solid phase extraction cartridge.
METHOD
[0055] Each model compound was dissolved in 20 mM phosphate buffer,
pH 7, to form a solution having a concentration of 10 .mu.g/mL.
[0056] Solid Phase Extraction of Model Solutes
[0057] The solutions of the model solutes were subjected to solid
phase extraction on solid phase extraction cartridges conditioned
under two sets of conditions. In both cases the cartridge was
attached to a vacuum manifold and treated with 1 mL methanol. The
vacuum was set to about 4" Hg, to give a methanol flow rate of 1 mL
/minute. Under the first set of conditions ("wet conditions"), the
vacuum was released when the methanol level reached the top of the
sorbent. Under the second set of conditions ("dry conditions") the
sorbent was allowed to dry out under vacuum following conditioning
with methanol. As in the first method, the cartridge was treated
with 1 mL methanol, at a flow rate, under reduced pressure (4" Hg),
of about 1 mL/minute. When the methanol level reached the top of
the sorbent, the vacuum was set to 10" Hg and maintained for 10
minutes to dry the polymer bed.
[0058] In both wet and dry cases, 1 mL of the model compound
solution was applied to the cartridge at a flow rate of 1
mL/minute. A 1 mL portion of 20 mM phosphate buffer, pH 7 was then
added at a flow rate of 1 mL/minute. A 1 mL portion of methanol was
then added at a flow rate of 1 mL/minute to desorb and eluate the
model compound. To the eluate was added an internal standard, and
the model compound within the eluent was quantitated by high
performance liquid chromatography.
RESULTS
[0059] The results are summarized in the table below, which lists
polar compounds (procainamide, acetaminophen and ranitidine),
moderately polar compounds (caffeine, m-toluamide, m-toluidine,
2,7-dihydroxynaphthalene, and propranolol) and nonpolar compounds
(dipropylphthalate, doxepin and betamethasone-13-valerate). When
the sorbent was poly(divinylbenzene) all compounds except doxepin
showed greater than 89% recovery when the sorbent was conditioned
under wet conditions. Recovery of oxepin, as shown, was
significantly lower because this compound required greater than 1
mL methanol for quantitative elution. When the sorbent was treated
under dry conditions, only dipropylphthalate and betamethasone
valerate were recovered in greater than 80% yield, and recovery of
procainamide, acetaminophen and ranitidine fell below 10%.
[0060] When the sorbent was tC.sub.18-bonded silica, each compound
tested was recovered in high yield (>85%) under wet conditions.
Allowing the sorbent to dry out had negligible effect on the
recovery of dipropylphthalate, doxepin and betamethasone valerate,
but reduced the yield of caffeine, m-toluidine, m-toluamide,
2,7-dihydroxynaphthalene and propranolol to about 13% or less.
[0061] When the sorbent was a
poly(divinylbenzene-co-N-vinylpyrrolidone) copolymer, recovery of
each compound was in the range of about 80-100% when the sorbent
was kept wetted. When the copolymer composition was 9 mole percent
N-vinylpyrrolidone, high recovery of the nonpolar compounds was
noted under both wet and dry conditions. The more polar compounds
were recovered in high yield under wet conditions but in sharply
reduced yield under dry conditions. Recovery of these compounds
under dry conditions dramatically increased when the
N-vinylpyrrolidone component of the copolymer was increased to
about 13 mole percent or greater. The recovery of these compounds
under wet conditions was essentially invariant as the copolymer
composition was changed.
1TABLE Comparison of SPE recoveries for various model compounds
Analyses performed in triplicate. DVB = divinylbenzene, NVP =
N-vinylpyrrolidone-% NVP given as mole percent NVP. Percent
Recovery (Average) Poly(DVB- Poly(DVB- co-NVP) co-NVP) Poly(DVB) 9%
NVP 13% NVP Compound wet dry wet dry wet dry Procainamide 95.4 2.5*
90.8 4.9* 84.8* Acetaminophen 98.0 2.0* 93.8* 10.4* 92.3 Ranitidine
95.0 5.4* 89.6 13.4* 99.7 Caffeine 95.6* 30.8* 101.0 25.6* 96.0
95.7 Toluamide 98.5 54.8* 101.1 74.4* 96.5 96.3 Toluidine 95.6
80.7* 102.8 96.8 97.6 98.0 2,7-Dihydroxynaphthalene 99.5 46.5*
103.6 89.6* 96.8 96.3 Propranolol 92.3 55.8* 102.1 94.5 94.2 92.4
Dipropylphthalate 91.5 99.2 102.1 101.6 93.1 100.0 Doxepin 47.5+
55.4+ 85.3 86.8 77.5+ 78.0 Betamethasone-13-valerate 89.1 83.6 93.3
97.1 85.9+ 89.8 Percent Recovery (Average) Poly(DVB- Poly(DVB-
Poly(DVB- co-NVP) co-NVP) co-NVP) 14% NVP 16% NVP 20% NVP Compound
wet dry wet dry wet dry Procainamide 94.2 84* 99.9 98.8 89.1 88.3
Acetaminophen 97.9 89.4* 104.5 104.4 96.2 94.8 Ranitidine 93.5 88.4
98.3 97.6 86.0 85.2 Caffeine 98.7 96.8 99.7 97.3 Toluamide 100.8
96.4 100.0 97.0 Toluidine 96.5 93.4 94.0 93.2
2,7-Dihydroxynaphthalene 95.9 94.2 96.7 95.4 Propranolol 94.1 95.4
Dipropylphthalate 89.5 89.3 Doxepin 84.1 81.8
Betamethasone-13-valerate 92.7 87.0 Percent Recovery (Average)
Poly(DVB- Poly(DVB- co-NVP) co-NVP) 20% NVP 22% N tC.sub.18
Compound wet dry wet dry wet dry Procainamide 92.3 85.7 93.3
Acetaminophen 93.4 96.7 102.2 Ranitidine 92.1 76.9 86.6 Caffeine
97.7 98.2 99.2 95.4 103.9 1.5* Toluamide 97.0 97.3 100.5 96.3 103.7
2.8* Toluidine 95.1 96.7 91.9 90.3 101.7 13.4*
2,7-Dihydroxynaphthalene 95.7 94.5 94.4 90.0 102.8 0* Propranolol
88.5 85.5 88.0 88.3 98.2 9.3* Dipropylphthalate 88.9 86.0 89.1 98.5
92.2 98.4 Doxepin 84.7 77.5 78.6 79.9 95.1 104.0
Betamethasone-13-valerate 84.0 85.0 85.9 85.9 86.6 88.3
*Breakthrough in load/wash +Requires greater than 1 mL methanol for
complete elution
EQUIVALENTS
[0062] Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed in the
scope of the following claims.
* * * * *