U.S. patent application number 09/924084 was filed with the patent office on 2002-02-14 for method for determination of chromatographic media parameters in packed beds of hydrophobic polymer particles.
Invention is credited to Maikner, John Joseph.
Application Number | 20020017149 09/924084 |
Document ID | / |
Family ID | 26918661 |
Filed Date | 2002-02-14 |
United States Patent
Application |
20020017149 |
Kind Code |
A1 |
Maikner, John Joseph |
February 14, 2002 |
Method for determination of chromatographic media parameters in
packed beds of hydrophobic polymer particles
Abstract
An improved method for measuring the chromatographic properties,
such as excluded volume, of hydrophobic polymer substrates is
disclosed. Use of 0.05-1 micron sized crosslinked ionically-charged
polymer particles, especially emulsion-form or ground polymer
particles, as a large-molecule probe, allows environmentally
friendly aqueous-based solvent systems to be used as mobile phases
to characterize hydrophobic polymer supports for use in analytical
or preparative scale chromatographic applications. This method
eliminates the use of non-polar organic solvents that is required
when conventional non-polar large-molecule probes (such as
polystyrene) are used to characterize chromatographic media.
Inventors: |
Maikner, John Joseph;
(Quakertown, PA) |
Correspondence
Address: |
ROHM AND HAAS COMPANY
PATENT DEPARTMENT
100 INDEPENDENCE MALL WEST
PHILADELPHIA
PA
19106-2399
US
|
Family ID: |
26918661 |
Appl. No.: |
09/924084 |
Filed: |
August 7, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60245288 |
Nov 2, 2000 |
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60224373 |
Aug 11, 2000 |
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Current U.S.
Class: |
73/866 |
Current CPC
Class: |
B01D 15/325 20130101;
B01J 2220/54 20130101; B01D 15/20 20130101; B01D 15/34 20130101;
B01J 20/26 20130101; G01N 30/02 20130101; G01N 30/89 20130101; G01N
30/02 20130101; B01J 20/285 20130101; G01N 30/482 20130101; B01D
15/327 20130101; B01D 15/166 20130101; B01D 15/34 20130101 |
Class at
Publication: |
73/866 |
International
Class: |
G01N 033/00 |
Claims
We claim:
1. A method for measuring chromatographic media parameters in a
packed bed of hydrophobic polymer particles comprising: (a) packing
a column with hydrophobic polymer particles and a first solvent to
provide a packed bed; (b) introducing a large-molecule probe
mixture, comprising a crosslinked ionically-charged polymer
particle probe and a second solvent, onto the packed bed; (c)
eluting the crosslinked ionically-charged polymer particle probe
from the packed bed by passing a third solvent through the packed
bed, wherein the third solvent is selected from one or more of
polar organic solvent and water; and (d) determining an elution
volume for the crosslinked ionically-charged polymer particle
probe; wherein the chromatographic media parameter is excluded
volume, corresponding to the elution volume for the crosslinked
ionically-charged polymer particle probe.
2. The method of claim 1 further comprising: (i) introducing a
small-molecule probe mixture, comprising a small-molecule probe and
the second solvent, onto the packed bed; (ii) eluting the
small-molecule probe from the packed bed by passing the third
solvent through the packed bed; (iii) determining an elution volume
for the small-molecule probe; and (iv) determining a pore volume
for the hydrophobic polymer particles by subtracting the elution
volume for the crosslinked ionically-charged polymer particle probe
from the elution volume for the small-molecule probe; wherein the
chromatographic media parameter is pore volume.
3. The method of claim 1 wherein the hydrophobic polymer particles
are selected from one or more of organic copolymer and
hydrophobically-modified inorganic polymer.
4. The method of claim 1 wherein the first solvent is selected from
one or more of non-polar organic solvent, polar organic solvent and
water.
5. The method of claim 1 wherein the second solvent is selected
from one or more of non-polar organic solvent, polar organic
solvent and water.
6. The method of claim 1 wherein the third solvent is a mixed
aqueous-organic solvent comprising from 2 to 99% polar organic
solvent, based on total weight of the mixed aqueous-organic
solvent.
7. The method of claim 1 wherein the crosslinked ionically-charged
polymer particle probe is selected from one or more of
anionically-charged emulsion-form polymer particles,
cationically-charged emulsion-form polymer particles,
anionically-charged ground polymer particles and
cationically-charged ground polymer particles, having a particle
size from 0.05 to 1 micron.
8. The method of claim 1 wherein the crosslinked ionically-charged
polymer particle probe is crosslinked polystyrenesulfonate.
9. The method of claim 2 wherein the small-molecule probe is
selected from one or more of inorganic salt and low-molecular
weight organic compound.
10. The method of claim 9 wherein the inorganic salt is selected
from one or more of sodium chloride, sodium nitrate and calcium
chloride.
Description
BACKGROUND
[0001] This invention relates to an improved method for measuring
chromatographic media parameters, such as excluded volume and pore
volume, of hydrophobic polymer particles in packed beds. Excluded
volumes are useful in determining the performance characteristics
of hydrophobic polymer substrates (organic or inorganic polymers)
used as stationary phases in medium and high pressure reversed
phase liquid chromatography (RPC). Chromatographic media
properties, such as porosity, compressibility and permeability
(flow resistance), are important in selecting polymers for use in
high performance chromatographic preparative modes, such as is
required in the separation and purification of biomolecules.
[0002] Current methods for measuring excluded volumes of
chromatographic media in packed beds involves the use of
conventional large-molecule "probe" or "marker" materials, such as
linear polystyrene or polyethylene glycol. These probe compounds
require the use of non-polar mobile phases (such as tetrahydrofuran
and toluene) in chromatographic operations; however, the use of
non-polar solvents often presents operational problems, such as
toxicity, flammability and incompatibility (with aqueous systems).
A discussion of performance evaluation (including void-volume
determinations) of hydrophobic polymer supports in size-exclusion
chromatography is given by E. Meehan, Size Exclusion Chromatography
Columns from Polymer Laboratories in Column Handbook for Size
Exclusion Chromatography, pp 349-366, Academic Press (1999).
[0003] The problem addressed by the present invention is to provide
an improved method for determining chromatographic media parameters
that does not require the use of non-polar solvents and that can be
used in aqueous-based solvent systems, for example, water-alcohol
mixtures, such as 20% aqueous ethanol.
SUMMARY OF INVENTION
[0004] The present invention provides a method for measuring
chromatographic media parameters in a packed bed of hydrophobic
polymer particles comprising (a) packing a column with hydrophobic
polymer particles and a first solvent to provide a packed bed; (b)
introducing a large-molecule probe mixture, comprising a
crosslinked ionically-charged polymer particle probe and a second
solvent, onto the packed bed; (c) eluting the crosslinked
ionically-charged polymer particle probe from the packed bed by
passing a third solvent through the packed bed, wherein the third
solvent is selected from one or more of polar organic solvent and
water; and (d) determining an elution volume for the crosslinked
ionically-charged polymer particle probe; wherein the
chromatographic media parameter is excluded volume, corresponding
to the elution volume for the crosslinked ionically-charged polymer
particle probe.
[0005] In a preferred embodiment, the crosslinked ionically-charged
polymer particle probe of the aforementioned method is selected
from one or more of anionically-charged emulsion-form polymer
particles, cationically-charged emulsion-form polymer particles,
anionically-charged ground polymer particles and
cationically-charged ground polymer particles, having a particle
size from 0.05 to 1 micron.
[0006] In another embodiment, the aforementioned method further
comprises (i) introducing a small-molecule probe mixture,
comprising a small-molecule probe and the second solvent, onto the
packed bed; (ii) eluting the small-molecule probe from the packed
bed by passing the third solvent through the packed bed; (iii)
determining an elution volume for the small-molecule probe; and
(iv) determining a pore volume for the hydrophobic polymer
particles by subtracting the elution volume for the crosslinked
ionically-charged polymer particle probe from the elution volume
for the small-molecule probe; wherein the chromatographic media
parameter is pore volume.
DETAILED DESCRIPTION
[0007] In the determination of the interparticle void volume
("excluded volume") and other chromatographic media performance
parameters, we have discovered that the use of a large-molecule
probe (or marker) based on crosslinked ionically-charged polymer
particles allows for quick and efficient measurements using polar
organic solvents and aqueous-based solvent systems that are
environmentally friendly. Use of the aforementioned large-molecule
probe particles in place of conventional probe materials provides a
probe material that (1) is totally excluded from entering the
polymer matrix being evaluated, (2) allows the use of polar
solvents during the chromatographic process; and (3) does not
contribute to hydrophobic interactions between the probe material
and the polymer matrix of the packed chromatography column.
[0008] In particular, we have discovered that emulsion-form or
ground crosslinked polystyrene particles containing ionizable
functional groups (such carboxylate, sulfonate or quaternary
ammonium chloride) are particularly useful as large-molecule probe
materials in the evaluation of various hydrophobic solid media,
particularly macroporous polyvinylaromatic polymers, for
chromatographic applications. Carboxylate and sulfonate functional
groups are representative of "anionically-charged" probe particles
and quaternary ammonium functional groups are representative of
"cationically-charged" probe particles.
[0009] As used throughout the specification, the following terms
shall have the designated meanings, unless the context clearly
indicates otherwise.
[0010] All percentages referred to will be expressed in weight
percent (%), based on total weight of polymer or composition
involved, unless specified otherwise. The term "(meth)acrylate" or
"(meth)acrylic" refers to either the corresponding acrylate or
methacrylate derivatives: such as the corresponding acids, esters,
amides, substituted esters or substituted amides. The term
"copolymer" refers to polymer compositions containing units of two
or more different monomers, including positional isomers. The
following abbreviations are used herein: g=gram; ppm=parts per
million by weight/volume, cm=centimeter, cc=cubic centimeter,
mm=millimeter, ml=milliliter, .mu.m=microns, min=minute. Unless
otherwise specified, ranges listed are to be read as inclusive and
combinable and temperatures are in degrees centigrade (.degree.
C.).
[0011] As used herein, chromatographic media parameters include
those properties that are typically used to characterize
hydrophobic polymer substrates for suitability in specific end use
applications, such as the separation and purification of
biomolecules using size exclusion chromatography, gel filtration
chromatography, gel permeation chromatography, hydrophobic
interaction chromatography or reversed phase chromatography.
Typical chromatographic media performance parameters of interest to
the chromatography practitioner include, for example,
compressibility, permeability (flow resistance), % polymer pore
volume (% porosity of polymer bed), pore volume, interparticle void
volume (% interstitial volume or excluded volume), % polymer solids
(volume), polymer porosity (volume pores/volume polymer).
[0012] In general, to determine various chromatographic media
parameters of polymer substrates, mobile phases (solvents used to
transport molecules through the polymer matrix) must be selected
for compatibility with the probe molecules such that interactions
of the probe molecule with the hydrophobic surface of the polymer
particles is minimized and preferably eliminated, otherwise
chromatographic performance parameter measurements may be
inaccurate and imprecise. Conventional large-molecule probe
materials, such as linear polystyrene and polyethylene glycol, may
be used for interparticle void volume determinations; however these
types of probe materials require the use of nonpolar mobile phases
(such as tetrahydrofuran, dichloromethane and toluene) to minimize
hydrophobic interactions between the probe material and the polymer
surfaces. The probe materials used in the method of the present
invention, however, do not require the use of non-polar solvents
and can be used in a wide range of polar organic solvents and
aqueous-based solvent systems, for example, water or 20% aqueous
ethanol, thus eliminating hydrophobic interactions between the
probe molecule and the stationary phase (polymer matrix).
[0013] In practicing the method of the present invention,
small-molecule and large-molecule probe materials may be introduced
onto the packed chromatography column containing hydrophobic
polymer particles in any manner convenient to obtain the required
information on elution times that is needed for determination of
chromatographic media parameters. For example, small-molecule and
large-molecule probe mixtures may be (a) added onto a packed column
concurrently (that is simultaneously), (b) in a staggered fashion
(addition of each probe mixture overlapping with the other, with
each addition starting and finishing at different times), (c)
addition of the large-molecule probe mixture first, followed by
addition of the small-molecule probe mixture, or (d) addition of
the small-molecule probe mixture first, followed by addition of the
large-molecule probe mixture. If characterization of the
hydrophobic polymer matrix is desired for excluded volume only,
then only the large-molecule mixture need be loaded onto the column
and eluted. It will be understood by one skilled in the art of
chromatographic separations that selection of addition sequence and
flow rates may be varied over a range of conditions in order to
obtain the required data.
[0014] "Large-molecule" probe materials useful in the method of the
present invention include crosslinked ionically-charged polymer
particle probes selected from one or more of anionically-charged
emulsion-form polymer particles, cationically-charged emulsion-form
polymer particles, anionically-charged ground polymer particles and
cationically-charged ground polymer particles, having a particle
size from 0.05 to 1 .mu.m. Preferably the particles have a particle
size ranging from 0.1 to 0.9, more preferably from 0.1 to 0.5 and
most preferably from 0.1 to 0.2 .mu.m. Suitable crosslinked
ionically-charged polymer particles include, for example,
crosslinked polystyrene with ionizable functional groups, such as
weak-acid functional group (carboxylate group), strong acid
functional group (sulfonate) or quaternary ammonium halide
functional group. The polystyrene is typically crosslinked with
polyvinylaromatic or aliphatic crosslinking monomers. Suitable
polyvinylaromatic crosslinkers include, for example,
divinylbenzene, trivinylbenzene, divinyltoluene,
divinylnaphthalene, divinylanthracene and divinylxylene; preferably
the crosslinking monomer is divinylbenzene. Suitable aliphatic
crosslinking monomers include, for example, ethyleneglycol
diacrylate, ethyleneglycol dimethacrylate, trimethylolpropane
triacrylate, trimethylolpropane trimethacrylate. The crosslinking
monomers are typically used at levels of 1 to 80%, preferably from
1 to 50% and more preferably from 2 to 25%, based on total weight
of the emulsion-form or ground crosslinked ionically-charged
polymer.
[0015] The crosslinked ionically-charged polymers may be used in
their various salt forms: ammonium, alkali (such as sodium and
potassium) or alkaline earth (such as calcium, magnesium) metal
salts; preferably the crosslinked ionically-charged polymers are at
least 50% neutralized (salt form), and more preferably at least 75%
neutralized. Optionally, the crosslinked ionically-charged polymer,
if based on a strong acid such as a sulfonic acid, may be used in
its free acid form.
[0016] Typical crosslinked anionically-charged polymer probe
materials would include, for example, crosslinked polystyrene
containing sodium carboxylate, sodium sulfonate or ammonium
sulfonate groups; preferably the anionically-charged polymer probe
material is crosslinked polystyrenesulfonate. Additional
crosslinked anionically-charged polymer probe materials include,
for example, crosslinked poly(meth)acrylic acid in its various salt
forms (sodium, potassium or ammonium salts). Additional crosslinked
anionically-charged polymer probe materials include, for example,
copolymers of styrene or (meth)acrylic acid, where one or more of
the following comonomers have been incorporated into the polymer:
2-acrylamido-2-methyl-1-propanesulfonic acid,
2-methacrylamido-2-methyl-1-propanesulfonic acid,
3-methacrylamido-2-hydr- oxypropanesulfonic acid, allylsulfonic
acid, methallylsulfonic acid, allyloxybenzenesulfonic acid,
methallyloxybenzenesulfonic acid,
2-hydroxy-3-(2-propenyloxy)propanesulfonic acid,
2-methyl-2-propene-1-sul- fonic acid, styrene sulfonic acid,
vinylsulfonic acid, 3-sulfopropyl acrylate, 3-sulfopropyl
methacrylate, sulfomethyl acrylamide and sulfomethyl
methacrylamide.
[0017] Typical crosslinked cationically-charged polymer probe
materials include, for example, ammonium salts of crosslinked
polyvinylbenzyl chloride; ammonium salts of crosslinked
poly(dialkylaminoalkyl(meth)acryl- amide) such as
poly(3-acrylamidopropyl-trimethylammonium chloride),
poly(3-methacrylamidopropyl-trimethylammonium chloride); and
ammonium salts of (meth)acrylate esters, such as
poly(2-(N,N,N-trimethylammonium chloride)-ethylmethacrylate). By
"ammonium" salts, we mean that both quaternary (such as
trialkylammonium) and acid (such as dialkylammonium hydrochloride)
salts of the amine derivatives may be used.
[0018] Suitable "small-molecule" probe materials useful in the
method of the present invention include water-soluble inorganic
salts (such as, for example, halide, nitrate, sulfate, borate,
phosphate salts of ammonium, alkali and alkaline earth metals) and
low-molecular weight organic molecules (such as C.sub.1-C.sub.2
organic acids and salts thereof, C.sub.1-C.sub.3 alcohols, acetone,
toluene and benzene); suitable low-molecular weight organic
molecules typically include those having molecular weights below
about 200. Typically, the small-molecule probe material is an
inorganic salt selected from one or more of sodium chloride, sodium
nitrate and calcium chloride.
[0019] Solvent systems used in the process of the present invention
may vary depending on the purpose or use of the solvent. For
example, when packing the column with hydrophobic particles,
solvents (designated as first solvent) may include one or more of
non-polar organic solvent, polar organic solvent and water,
including mixtures of polar organic solvents and water (designated
as mixed aqueous-organic solvents). Typical non-polar organic
solvents include, for example, tetrahydrofuran, dichloromethane,
chloroform, benzene and toluene. Suitable polar organic solvents
include those that are water-soluble or water-miscible, such as,
for example, C.sub.1-C.sub.3 alcohols (methanol, ethanol,
n-propanol and isopropanol), C.sub.1-C.sub.2 organic acids (formic
acid, acetic acid), C.sub.3-C.sub.4 ketones (acetone, methylethyl
ketone) and acetonitrile. Preferably, the polar organic solvents
are selected from one or more of methanol, ethanol, n-propanol and
isopropanol. Typical mixed aqueous-organic solvents include for
example, 2 to 99% polar organic solvent in water, preferably 5 to
50%, and more preferably 10 to 30% polar organic solvent, such as
C.sub.1-C.sub.3 alcohol. Preferably, the first solvent is an
aqueous-based solvent, that is, water or a mixed aqueous-organic
solvent.
[0020] When introducing large-molecule or small-molecule probe
mixtures onto the packed bed, solvents (designated as second
solvent) used to make up the probe mixtures may include any of the
solvent systems described above as first solvents. Preferably, the
second solvent is an aqueous-based solvent, that is, water or a
mixed aqueous-organic solvent.
[0021] When eluting the large-molecule or small-molecule probe from
the packed bed, solvents used for elution (designated as third
solvent) comprise one or more of polar organic solvent and water,
including mixed aqueous-organic solvents, as described above.
Preferably, the third solvent is an aqueous-based solvent, that is,
water or a mixed aqueous-organic solvent.
[0022] Typically, the first and second solvents used in the process
of the present invention are the same as the third solvent as a
matter of convenience; however, each solvent system may be selected
independently of the other.
[0023] As described above, suitable aqueous-based solvents include
water solutions and mixed aqueous-organic solvent systems.
Preferably, the aqueous-based solvent is a mixed aqueous-organic
solvent system since the presence of some polar organic solvent
provides improved wettability of the hydrophobic polymer particles
during packing and operation of the chromatography column.
[0024] Concentrations of polar organic solvent in the mixed
aqueous-organic solvent mixtures may vary, depending on the type of
small-molecule probe used in characterization of the
chromatographic media. For example, when low-molecular weight
organic molecules are used as small-molecule probes, from 75-100%
polar organic solvent may be used to elute the small-molecule probe
from the packed column. When inorganic salts are used as
small-molecule probes, typically from zero (that is, 100% water) up
to 75%, preferably from 5 to 50% and more preferably from 10 to 30%
polar organic solvent, based on total weight of the mixed
aqueous-organic solvent, may be used to elute the small-molecule
probe.
[0025] When the aqueous-based solvent system is water, that is,
contains little or no organic solvent, the solvent system may
optionally contain water-soluble salts, such as buffer agents, for
example, phosphate, carbonate, bicarbonate, borate and acetate
salts. When used, these salts are typically present at levels from
50 ppm to 5%, and preferably from 1000 ppm to 1%, in the
aqueous-based solvent system.
[0026] Suitable detection methods useful for detecting the presence
of the probe materials in the effluents of the chromatography
columns include, for example, UV spectrophotometry, infrared
spectrophotometry, conductivity and refractive index techniques.
The particular detection method selected will depend on the type of
probe particle being detected and may be any detection method
sufficient to sense the small-molecule or large-molecule probe. For
detection of the inorganic salt probes, conductivity and refractive
index methods are preferred. For detection of the crosslinked
ionically-charged polymer particle probes, UV spectrophotometry,
refractive index or conductivity methods are preferred.
[0027] A chromatography column packed with polymeric particles may
be visualized as having four different types of "volumes."
[0028] The liquid volume between the particles of the stationary
phase (polymer beads or polymer matrix) is known as the
interstitial volume, interparticle void volume, void volume or
excluded volume (V.sub.o).
[0029] The liquid volume within the pores of the stationary phase
is the pore volume (V.sub.i).
[0030] The volume of the solid portion of the stationary phase is
the polymer skeletal volume (V.sub.g).
[0031] The total volume of the packed bed (V.sub.t) corresponds to
the sum of the above volumes: V.sub.t=V.sub.o+V.sub.i+V.sub.g.
[0032] All molecules, regardless of size, have access to V.sub.o
and none of the molecules have access to V.sub.g. Access to V.sub.i
depends on the size of the molecules and the size of the pores in
the polymer matrix. Large molecules (for example, molecular weights
larger than about 20,000 daltons) generally cannot penetrate the
pores of the polymer matrix due to pore size limitations (such as
where average pore sizes are typically less than about 100 Angstrom
units) and the accessible volume for large molecules is typically
equal to V.sub.o. Medium-size molecules (for example, molecular
weights of 2,000 to 10,000 daltons) may partially penetrate the
pore structure and the "apparent" volume (elution volume) will be
equal to V.sub.o plus the part of V.sub.i that is accessible to
medium-size molecules. Small molecules (such as those having
molelcular weights less than about 1,000 daltons) typically have
complete access to the polymer matrix and the corresponding elution
volume will be equal to Vo.sub.o+V.sub.i. Molecules passing through
the column will exit in order of their accessible volumes (or
"apparent" volumes), and the latter are measured as the
corresponding elution volumes The largest molecules (for example,
the large-molecule probe) will elute first with a volume equal to
V.sub.o and the smallest molecule (for example, inorganic salt or
small-molecule probe) will elute last with a volume equal to
V.sub.o+V.sub.i.
[0033] The choice of an appropriate hydrophobic polymer matrix for
chromatographic purposes depends on the molecular size and the
chemical properties of the substances to be separated. Most polymer
matrices will fractionate (separate) target molecules within a
particular molecular weight range, depending on the pore size
distribution in the hydrophobic polymer particles. The function of
the hydrophobic polymer matrix is to provide a continuous decrease
in accessibility for the targeted molecules (for example,
biomolecules) of increasing size where the largest molecules are
eluted from a chromatographic column first and the smallest are
eluted last.
[0034] Among the hydrophobic solid media useful as substrates for
chromatographic applications are, for example:
[0035] (a) hydrophobically-modified silica-based polymers, such as
those based on silica particles that have been surface treated with
trimethylsilyl groups, (C.sub.8-C.sub.20)alkyl groups or other
hydrophobic groups;
[0036] (b) polyvinylaromatic polymers, such as crosslinked
polystyrene and polydivinylbenzene copolymers;
[0037] (c) poly(meth)acrylate or acrylic-based copolymers, such as
alkyl(meth)acrylate copolymers and trimethylolpropane
tri(meth)acrylate copolymers.
[0038] By "hydrophobic" we mean polymers that are substantially
non-polar in nature, that is, they contain little or no polar,
ionizable or hydrogen-bonding functionality (the latter are
typically characteristic of hydrophilic characteristics). For the
purposes of the present invention, hydrophobic polymers also
include hydrophilic polymer substrates that have had their surfaces
hydrophobically-modified, such as silica or other hydrophilic
substrates whose surfaces have been chemically modified or coated
with hydrophobic (water-insoluble) functionality (such as
fluorocarbon or alkyl groups).
[0039] Hydrophobic polymer particles selected as stationary phases
for chromatography applications are typically based on polymers
selected from one or more of organic copolymer and
hydrophobically-modified inorganic polymer. Preferably, the organic
copolymer is selected from one or more of polyvinylaromatic
copolymer and poly(meth)acrylate copolymer. When
hydrophobically-modified inorganic polymers are used, the
hydrophobic polymers are typically selected from one or more of
silica-based, alumina-based and zeolite-based polymer.
[0040] Hydrophobic polymers useful for the separation and
purification of biomolecules via high performance reverse phase
liquid chromatography (such as in columns from 2 to 100 cm in
diameter) typically have average particle size diameters from 2 to
150 .mu.m. Hydrophobic polymers useful for the separation and
isolation of biomolecules via large scale adsorption processes
(such as in columns up to several meters in diameter or in
fermentation broths) typically have average particle size diameters
from 150 up to 600 .mu.m. Preferably, the hydrophobic polymers are
spherical copolymer beads having particle diameters from 2 to 150
.mu.m, more preferably from 5 to 100 .mu.m and most preferably from
10 to 75 .mu.m. Particularly preferred are macroporous polymers
that are produced by suspension polymerization and possess surface
areas from 200 to 1500 square meters per gram (m.sup.2/g) and
preferably from 300 to 1200 m.sup.2/g. The macroporous polymers are
preferably those of the type described in U.S. Pat. No. 4,382,124,
for example, in which porosity is introduced into the copolymer
beads by suspension-polymerization in the presence of a porogen
(also known as "phase extender" or "precipitant"), that is, a
solvent for the monomer but a non-solvent for the polymer.
Conventional macroporous polymers, such as those prepared according
to U.S. Pat. No. 4,382,124, typically encompass the use of a wide
range of porogen types, porogen concentrations relative to the
monomer phase, monomer types, crosslinking monomer types,
crosslinker levels, polymerization initiators and initiator
concentrations.
[0041] Polyvinylaromatic monomers that may be used in the
preparation of macroporous polymers suitable for use in
chromatographic separations include, for example, divinylbenzene,
trivinylbenzene, divinyltoluene, divinylnaphthalene,
divinylanthracene and divinylxylene; preferably the
polyvinylaromatic monomer is divinylbenzene. Suitable macroporous
polymers typically comprise 50 to 100% and preferably 75 to 90%
polyvinylaromatic monomer units.
[0042] Monounsaturated vinylaromatic monomers that may be used in
the preparation of macroporous copolymers suitable for use in
chromatographic separations include, for example, styrene,
.alpha.-methylstyrene, (C.sub.1-C.sub.4)alkyl-substituted styrenes,
halo-substituted styrenes (such as dibromo- and tribromostyrene),
vinylnaphthalene and vinylanthracene; preferably the
monounsaturated vinylaromatic monomer is selected from one or more
of styrene and (C.sub.1-C.sub.4)alkyl-substitut- ed styrenes.
Included among the suitable (C.sub.1-C.sub.4)alkyl-substitute- d
styrenes are, for example, ethylvinylbenzenes, vinyltoluenes,
diethylstyrenes, ethylmethylstyrenes and dimethylstyrenes;
preferably the monounsaturated vinylaromatic monomer is
ethylvinylbenzene. Typically, suitable macroporous polymers
comprise zero to 50% and preferably 10 to 25% monounsaturated
vinylaromatic monomer units.
[0043] Preferred macroporous polymers useful for chromatographic
separations include divinylbenzene copolymer,
styrene-divinylbenzene copolymer, divinylbenzene-ethylvinylbenzene
copolymer and styrene-ethylvinylbenzenedivinylbenzene copolymer.
Most preferable are macroporous divinylbenzeneethylvinylbenzene and
styrene-ethylvinylbenzene- -divinylbenzene copolymers. These
macroporous polymers are especially useful in packed chromatography
column applications where porosity and mechanical strength of the
polymer allows for high performance separation and purification of
biomolecules at high throughput rates without pressure buildup on
prolonged use.
[0044] Acrylic-based monomers that may be used in the preparation
of suitable hydrophobic polymer substrates include, for example,
(C.sub.1-C.sub.20)alkyl (meth)acrylates. In addition, the alkyl
(meth)acrylate monomers may be copolymerized with functionalized
(meth)acrylate derivatives, such as hydroxyalkyl (meth)acrylates,
amides of ethylenically unsaturated (C.sub.3-C.sub.6)carboxylic
acids that are substituted at the nitrogen by one or two
(C.sub.1-C.sub.4)alkyl groups, dimethylaminopropyl(meth)acrylamide
and 2-(dimethylamino)ethyl(meth)acryl- ate. When used as
comonomers, the functionalized (meth)acrylate derivatives typically
comprise up to 50% and preferably less than 20%, based on total
weight of the polymer.
[0045] Among the chromatographic media parameters of interest to
the chromatography practitioner are "permeability" or "flow
resistance" properties. The permeability (K) is related to the
backpressure generated in a column through Darcy's Law (Equation
1):
.DELTA.P=.mu.V/[K(d.sub.p).sup.2] Equation 1
[0046] where:
[0047] .mu.=viscosity (milliPascal.multidot.second or
centipoise)
[0048] V=linear velocity (cm/hr)
[0049] .DELTA.P=pressure drop (bars)
[0050] d.sub.p=mean particle size of the polymer (microns)
[0051] The units of the above variables are expressed in their
common form; it is understood that unit conversion is required to
render Equation 1 dimensionless. Under laminar flow conditions,
which are typical for chromatographic separation and purification
applications, the backpressure in a column can also be expressed by
the Carman-Kozeny Equation (Equation 2):
.DELTA.P=150.multidot.[(1-.epsilon.).sup.2/.epsilon..sup.3].multidot..mu.V-
/(d.sub.p).sup.2 Equation 2
[0052] where:
[0053] .epsilon.=interparticle void volume (cc/cc)
[0054] References, such as Fundamentals of Preparative and
Nonlinear Chromatography, G. Guiochon, S. Goshan Shirazi and A.
Katti; Academic Press (1994) and Unit Operations in Chemical
Engineering, W. L. McCabe, J. C. Smith and P. Harriott; McGraw Hill
(1985), may be consulted for further general and specific details
on Darcy's Law and the Carman-Kozeny Equation (Equations 1 and
2).
[0055] By combining Equations 1 and 2, it can be seen that
permeability (or flow resistance) in the chromatography column is
related to the interparticle void volume of the polymer resin bed
(that is, the volume between polymer particles); .epsilon. is
expressed as volume of voids per unit volume of polymer bed in this
case. This relationship is expressed by Equation 3:
1/K=150.multidot.[(1-.epsilon.).sup.2/.epsilon..sup.3] Equation
3
[0056] The characteristic "flow resistance" value of a polymer
(inverse of the permeability) is an indication of how well the
polymer will perform under medium to high pressure conditions: low
flow resistance values represent low compressibility and high flow
resistance values represent poor compressibility.
[0057] Some embodiments of the invention are described in detail in
the following Example. All ratios, parts and percentages are
expressed by weight unless otherwise specified, and all reagents
used are of good commercial quality unless otherwise specified.
Abbreviations used in the Examples and Tables are listed below with
the corresponding descriptions:
[0058] v/v=volume/volume
[0059] w/v=weight/volume
[0060] nm=nanometer
EXAMPLE 1
[0061] This example describes how polyvinylaromatic macroporous
polymers were evaluated for their permeability characteristics,
that is, resistance to compression. In order for the polymers to be
characterized by their "flow resistance" or 1/K values (see
Equation 3), accurate determinations of void volumes were needed.
In this case, the polymer tested was a polymer of 80%
divinylbenzene and 20% ethylvinylbenzene, having a porosity of 1.9
cc/g, pore volume of 0.67 cc/cc, surface area of 947 m.sup.2/g and
an average particle size of about 30 .mu.m.
[0062] In industrial high pressure liquid chromatography, columns
are equipped with a piston that exerts a force (pressure) directly
onto the resin. It is preferred to keep the piston actively
compressing the bed at a pressure that is equal to or greater than
the maximum anticipated flow pressure throughout the
chromatographic cycle. In order to test the permeability
characteristics of the polymers, polymer resin was packed into a
ProChrom.TM. Dynamic Axial Compression column (Model LC.50) and
compressed with the piston set first at 10 and then at 60 bar
compression pressure. A detailed description follows:
[0063] Approximately 100 g of dry polymer resin (corresponding to
approximately 500 ml wet resin) was added to 700 ml of a solution
of 20% ethanol/water (v/v) and allowed to stand at ambient
temperature for at least 2 hours. This polymer sample was agitated
into slurry form and poured into a 5 cm (internal
diameter).times.54 cm (length) Prochrom.TM. Dynamic Axial
Compression L.C.50 316 L stainless steel column (manufactured by
Prochrom S.A., France). A piston assembly (driven by external air
pressure converted into hydraulic oil pressure) was activated to
apply a variable pressure to the polymer resin bed. The piston was
first set to deliver approximately 10 bar of hydraulic pressure and
the resin bed was considered packed when the piston no longer
moved. The height of the bed was then measured at 21.5 cm,
corresponding to a total packed bed volume of 421.9 cc wet polymer
(=V.sub.t=V.sub.o+V.sub.i+V.sub.g). A flow of 10 ml/min of a
solution of 20% ethanol/water (v/v) was passed through the resin
bed for 30 minutes to equilibrate the bed.
[0064] To determine the total volume of voids in the packed bed
(sum of intraparticle (V.sub.i) and interparticle (V.sub.o) voids),
2 ml of 1% sodium chloride (w/v in 20% aqueous ethanol),
corresponding to the small-molecule probe mixture, was injected
into the system (flowing at 10 ml/min). The sodium chloride
(small-molecule probe) was detected by a conductivity detector;
elution time for the sodium chloride was 31.1 minutes, which
corresponded to an elution volume of 311 ml [=V.sub.sm].
[0065] To determine the interparticle (interstitial or excluded)
void volume only (V.sub.o), a solution of 20% ethanol (aqueous)
containing 1% (w/v) of a 0.1-0.9 .mu.m crosslinked sulfonated
polystyrene (emulsion-form or ground) particles, corresponding to
the large-molecule probe mixture, was injected into the stream of
20% ethanol (aqueous) flowing through the bed at 10 ml/min. The
large-molecule probe were detected by UV spectrophotometer, set at
280 nm. The elution time for the large-molecule probe was 13.7
minutes, which corresponded to an elution volume of 137 ml
[=V.sub.lm].
[0066] The calculated volume [V.sub.g] of polymer solids was:
[V.sub.t-V.sub.sm]=421.9-311=110.9 cc.
[0067] The calculated volume [V.sub.i] of polymer pores was:
[V.sub.sm-V.sub.lm]=174 cc.
[0068] The calculated volume [V.sub.o] of interstitial voids
(excluded volume) was:
[V.sub.t-V.sub.i-V.sub.g]=V.sub.lm=137 cc.
[0069] Calculated % polymer solids
volume=V.sub.g/V.sub.t=110.9/421.9=26.3- %
[0070] Calculated % polymer pore volume (%
porosity)=V.sub.i/V.sub.t=174/4- 21.9=41.2%
[0071] Calculated % interstitial volume (% void
volume)=V.sub.o/V.sub.t=17- 4/421.9=32.5%
[0072] Calculated porosity of
polymer=V.sub.i/(V.sub.i+V.sub.g)=174/(174+1- 10.9)=0.61 cc/cc
[0073] The above process was repeated again at 60 bar pressure,
with the following results:
[0074] The height of the bed (60 bar) was measured at 17.0 cm,
corresponding to a total packed bed volume of 333.6 cc wet polymer
[=V.sub.t].
[0075] The elution time for the sodium chloride was 22.3 minutes,
which corresponded to an elution volume of 223 ml [=V.sub.sm].
[0076] The elution time for the large-molecule probe was 7.4
minutes, which corresponded to an elution volume of 74 ml
[=V.sub.lm].
[0077] The calculated volume [V.sub.g] of polymer solids was:
[V.sub.tV.sub.sm]-333.6+223=110.6 cc.
[0078] The calculated volume [V.sub.i] of polymer pores was:
[V.sub.sm-V.sub.lm]=149 cc.
[0079] The calculated volume [V.sub.o] of interstitial voids
(excluded volume) was:
[V.sub.t-V.sub.i-V.sub.g]=V.sub.lm=74 cc.
[0080] Calculated % polymer solids
volume=V.sub.g/V.sub.t=110.6/333.6=33.2- %
[0081] Calculated % polymer pore volume (%
porosity)=V.sub.i/V.sub.t=149/3- 33.6=44.7%
[0082] Calculated % interstitial volume (% void
volume)=V.sub.o/V.sub.t=74- /333.6=22.2%
[0083] Calculated porosity of
polymer=V.sub.i/(V.sub.i+V.sub.g)=149/(149+1- 10.6)=0.57 cc/cc
[0084] The various chromatographic polymer properties measured
above may be used to determine the value of ".epsilon." that is
needed for calculations based on Equations 2 and 3. It is important
to generate accurate and precise values for V.sub.o and V.sub.i in
order to provide reliable permeability and compressibility
characteristics of polymer supports used in chromatographic
separations. As would be recognized by the skilled chromatography
practitioner, the volumes referred to (V.sub.t, V.sub.o, V.sub.i
and V.sub.g) are independent of and exclusive of other components
of the "total chromatographic column system" volume, that is:
"dead" volumes, transfer line volumes and column head volumes--the
latter are taken into account during measurement of the
chromatographic media parameters.
* * * * *