U.S. patent application number 16/049464 was filed with the patent office on 2020-01-30 for anion exchange stationary phases based on a polyalkylpolyamine polymer layer.
The applicant listed for this patent is DIONEX CORPORATION. Invention is credited to Jinhua CHEN, Christopher A. POHL.
Application Number | 20200031857 16/049464 |
Document ID | / |
Family ID | 67438233 |
Filed Date | 2020-01-30 |
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United States Patent
Application |
20200031857 |
Kind Code |
A1 |
POHL; Christopher A. ; et
al. |
January 30, 2020 |
ANION EXCHANGE STATIONARY PHASES BASED ON A POLYALKYLPOLYAMINE
POLYMER LAYER
Abstract
An anion exchange for separating a plurality of carbohydrates
includes a negatively charged substrate particle. A base polymer
layer includes a first plurality of quaternary amines. The
polyalkylpolyamine polymer layer is covalently attached to the base
condensation polymer layer. The polyalkylpolyamine polymer layer
includes a polymeric branch structure that includes a second
plurality of quaternary amines. A density of the second plurality
of quaternary amines increases in a direction away from the base
condensation polymer layer. The anion exchange stationary phase
does not have a hydroxy group spaced apart from any one of the
first or the second plurality of quaternary amines by an ethyl
group.
Inventors: |
POHL; Christopher A.; (Union
City, CA) ; CHEN; Jinhua; (San Jose, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DIONEX CORPORATION |
Sunnyvale |
CA |
US |
|
|
Family ID: |
67438233 |
Appl. No.: |
16/049464 |
Filed: |
July 30, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07H 1/06 20130101; B01J
41/05 20170101; B01D 15/363 20130101; B01J 41/07 20170101; B01J
41/13 20170101; B01J 41/20 20130101 |
International
Class: |
C07H 1/06 20060101
C07H001/06; B01J 41/13 20060101 B01J041/13; B01D 15/36 20060101
B01D015/36 |
Claims
1. An anion exchange stationary phase for separating a plurality of
carbohydrates comprises: a) a negatively charged substrate
particle; b) a base condensation polymer layer attached to the
negatively charged substrate particle, the base condensation
polymer layer comprises a first plurality of quaternary amines, in
which the first plurality of quaternary amines are spaced apart by
either a first spacer or a second spacer, in which the base
condensation polymer layer does not have a hydroxy group spaced
apart from one of the first plurality of quaternary amines by an
ethyl group; c) a polyalkylpolyamine condensation polymer layer
covalently attached to the base condensation polymer layer, the
polyalkylpolyamine condensation polymer layer comprises a polymeric
branch structure, the polymeric branch structure includes a second
plurality of quaternary amines, in which the second plurality of
quaternary amines are spaced apart by the first spacer or the
second spacer, in which a density of the second plurality of
quaternary amines increases in a direction away from the base
condensation polymer layer, in which the polyalkylpolyamine
condensation polymer layer does not have a hydroxy group spaced
apart from one of the second plurality of quaternary amines by an
ethyl group.
2. The anion exchange stationary phase of claim 1, in which the
negatively charged substrate particle comprises a crosslinked
divinylbenzene and ethylvinyl benzene copolymer, in which at least
a surface of the negatively charged substrate particle includes
sulfonate groups.
3. The anion exchange stationary phase of claim 1, in which the
negatively charged substrate particle comprises a crosslinked
divinylbenzene and ethylvinyl benzene copolymer, in which at least
a surface of the negatively charged substrate particle includes
carboxylate groups.
4. The anion exchange stationary phase of claim 1, in which the
base condensation polymer layer is positively charged and ionically
attached to the negatively charged substrate particle.
5. The anion exchange stationary phase of claim 1, in which the
first spacer comprises a chemical formula of (--CH.sub.2--).sub.x
and the second spacer comprises a chemical formula of
(--CH.sub.2--).sub.y, where x and y each independently range from 3
to 6.
6. The anion exchange stationary phase of claim 1, in which the
first spacer comprises a first alkyl and the second spacer
comprises a second alkyl, in which the first alkyl and the second
alkyl are both a linear and unsubstituted alkyl.
7. The anion exchange stationary phase of claim 1, in which the
first spacer comprises a linear and unsubstituted alkyl and the
second spacer comprises an arylalkyl.
8. The anion exchange stationary phase of claim 1, in which the
first spacer is selected from group consisting of an alkyl, a
dialkylether, a cycloalkyl, an arylalkyl, and a combination
thereof.
9. The anion exchange stationary phase of claim 1, in which the
first spacer comprises an alkyl and the second spacer is selected
from group consisting of an alkyl, an arylalkyl, and a combination
thereof.
10. An anion exchange stationary phase for separating a plurality
of carbohydrates, the anion exchange stationary phase formed by a
method comprising: reacting a polyhalohydrocarbon with a
polyalkylpolyamine to form a base condensation polymer layer on a
negatively charged substrate particle; and reacting the base
condensation polymer layer with a number of reaction cycles to form
a polyalkylpolyamine condensation polymer layer, in which the
number of reaction cycles ranges from about three to about ten and
each reaction cycle includes a polyhalohydrocarbon treatment and a
polyalkylpolyamine treatment.
11. The anion exchange stationary phase of claim 10 further
comprising: reacting the polyalkylpolyamine condensation polymer
layer with a monohaloalkane treatment.
12. The anion exchange stationary phase of claim 10, in which the
negatively charged substrate particles are contained as a packed
bed in a column, in which the reacting of the polyhalohydrocarbon
with the polyalkylpolyamine comprises: flowing a solution of the
polyhalohydrocarbon and the polyalkylpolyamine through the column
to form the base condensation polymer layer on the negatively
charged substrate particles.
13. The anion exchange stationary phase of claim 12, in which the
polyhalohydrocarbon treatment comprises: flowing a solution of the
polyhalohydrocarbon through the column; the polyalkylpolyamine
treatment comprises: flowing a solution of the polyalkylpolyamine
through the column; and the monohaloalkane treatment comprises:
flowing a solution of the monohaloalkane through the column.
14. The anion exchange stationary phase of claim 10, in which the
number of reaction cycles ranges from about 3 to about 4.
15. The anion exchange stationary phase of claim 10, in which the
negatively charged substrate particle comprises a crosslinked
divinylbenzene and ethylvinyl benzene copolymer, in which at least
a surface of the negatively charged substrate particle includes
sulfonate groups.
16. The anion exchange stationary phase of claim 10, in which the
negatively charged substrate particle comprises a crosslinked
divinylbenzene and ethylvinyl benzene copolymer, in which at least
a surface of the negatively charged substrate particle includes
carboxylate groups.
17. The anion exchange stationary phase of claim 10, in which the
polyhalohydrocarbon comprises a material selected from group
consisting of a dihaloalkane, a dihalodialkylether, a
dihalocycloalkane, a trihaloarylalkane, and a combination
thereof.
18. The anion exchange stationary phase of claim 10, in which the
polyalkylpolyamine comprises a material selected from group
consisting of a polyalkyltriamine, a polyalkyldiamine, and a
combination thereof.
19. The anion exchange stationary phase of claim 10, in which all
amines of the polyalkylpolyamine are tertiary amines.
20. The anion exchange stationary phase of claim 10, in which the
polyhalohydrocarbon is selected from the group consisting of a
dibromobutane, a dibromopentane, a dibromohexane, a
tribromomethylbenzene, and a combination thereof.
21. The anion exchange stationary phase of claim 10, in which the
polyalkylpolyamine is selected from the group consisting of a
pentamethyldipropyltriamine, a pentamethyldihexylltriamine, a
permethylated spermine, a permethylated spermidine, and a
combination thereof.
22. The anion exchange stationary phase of claim 10, in which the
polyhalohydrocarbon is dibromobutane and the polyalkylpolyamine is
pentamethyldihexylltriamine.
23. The anion exchange stationary phase of claim 10, in which the
polyhalohydrocarbon is a trihaloalkylaryl and the
polyalkylpolyamine is an alkyldiamine.
24. The anion exchange stationary phase of claim 10, in which the
polyhalohydrocarbon is tribromomethylbenzene and the
polyalkylpolyamine is tetramethylhexanediamine.
25. An anion exchange stationary phase for separating a plurality
of carbohydrates, the anion exchange stationary phase comprises: A)
a negatively charged substrate particle; B) a base condensation
polymer layer attached to the negatively charged substrate
particle, the base condensation polymer layer comprises a reaction
product of i) a first polyhalohydrocarbon, and ii) a first
polyalkylpolyamine; C) a first alkyl condensation reaction product
covalently attached to the base condensation polymer layer, the
first alkyl condensation reaction product comprises a reaction
product of i) an amine group of the base condensation polymer
layer, and ii) a second polyhalohydrocarbon, in which the amine
group of the base condensation polymer layer includes a positive
charge so that the base condensation polymer layer is ionically
coupled to the negatively charged substrate particle; D) a first
polyalkylpolyamine condensation reaction product covalently
attached to the first alkyl condensation reaction product, the
first polyalkylpolyamine condensation reaction product comprises a
reaction product of i) a halide group of the second
polyhalohydrocarbon, and ii) a second polyalkylpolyamine; and E) a
second alkyl condensation reaction product covalently attached to
the first polyalkylpolyamine condensation reaction product, the
second alkyl condensation reaction product comprises a reaction
product of i) an amine group of the first polyalkylpolyamine
condensation reaction product, and ii) a third polyhalohydrocarbon;
F) a second polyalkylpolyamine CRP covalently attached to the
second alkyl CRP, the second polyalkylpolyamine CRP comprises a
reaction product of i) a halide group of the third
polyhalohydrocarbon and ii) a third polyalkylpolyamine; G) a third
alkyl CRP covalently attached to the second polyalkylpolyamine CRP,
the third alkyl CRP includes a reaction product of i) an amine
group of the second polyalkylpolyamine CRP, and ii) a fourth
polyhalohydrocarbon; and H) a third polyalkylpolyamine CRP is
covalently attached to the third alkyl CRP, the third
polyalkylpolyamine CRP includes a reaction product of i) a halide
group of the fourth polyhalohydrocarbon, and ii) a fourth
polyalkylpolyamine.
26. The anion exchange stationary phase of claim 25, in which the
first polyhalohydrocarbon, second polyhalohydrocarbon, third
polyhalohydrocarbon, and fourth polyhalohydrocarbon comprise a
dihaloalkane; and the first polyalkylpolyamine, second
polyalkylpolyamine, third polyalkylpolyamine, and fourth
polyalkylpolyamine comprise a polyalkyltriamine.
27. A method of using an anion exchange stationary phase for
separating a plurality of carbohydrates in a sample, the anion
exchange stationary phase comprising: a) a negatively charged
substrate particle; b) a base condensation polymer layer attached
to the negatively charged substrate particle, the base condensation
polymer layer comprises a first plurality of quaternary amines, in
which the first plurality of quaternary amines are spaced apart by
either a first spacer or a second spacer, in which the base
condensation polymer layer does not have a hydroxy group spaced
apart from one of the first plurality of quaternary amines by an
ethyl group; c) a polyalkylpolyamine condensation polymer layer
covalently attached to the base condensation polymer layer, the
polyalkylpolyamine condensation polymer layer comprises a polymeric
branch structure, the polymeric branch structure includes a second
plurality of quaternary amines, in which the second plurality of
quaternary amines are spaced apart by the first spacer or the
second spacer, in which a density of the second plurality of
quaternary amines increases in a direction away from the base
condensation polymer layer, in which the polyalkylpolyamine
condensation polymer layer does not have a hydroxy group spaced
apart from one of the second plurality of quaternary amines by an
ethyl group, the method comprising: flowing an eluent through a
chromatography column, the chromatography column containing the
anion exchange stationary phase, in which the eluent includes a
hydroxide; injecting the sample comprising a plurality of
carbohydrates into the chromatography column; separating at least
one carbohydrate from the sample injected into the chromatography
column; and detecting the at least one carbohydrate at a
detector.
28. The method of claim 27, in which the at least one carbohydrate
is a branched glycan.
Description
FIELD OF THE INVENTION
[0001] The invention generally relates to anion exchange stationary
phases based on a polyalkylpolyamine layer for applications such as
chromatographically separating samples that include anions, and in
particular a combination of carbohydrates, and more particularly a
combination of branched glycans.
BACKGROUND
[0002] Chromatography is a widely used analytical technique for the
chemical analysis and separation of molecules. Chromatography
involves the separation of one or more analyte species from other
matrix components present in a sample. A stationary phase of a
chromatography column is typically selected so that there is an
interaction with the analyte. Such interactions can be ionic,
hydrophilic, hydrophobic, or combinations thereof. For example, the
stationary phase can be derivatized with ionic moieties that
ideally will bind to ionic analytes and matrix components with
varying levels of affinity. A mobile phase is percolated through
the stationary phase and competes with the analyte and matrix
components for binding to the ionic moieties. The mobile phase or
eluent are terms used to describe a liquid solvent or buffer
solution that is pumped through a chromatography column. During
this competition, the analyte and matrix components will elute off
of the stationary phase as a function of time and then be
subsequently detected at a detector. Examples of some typical
detectors are a conductivity detector, a UV-VIS spectrophotometer,
and a mass spectrometer. Over the years, chromatography has
developed into a powerful analytical tool that is useful for
creating a healthier, cleaner, and safer environment where complex
sample mixtures can be separated and analyzed for various
industries such as water quality, environmental monitoring, food
analysis, pharmaceutical, and biotechnology.
[0003] U.S. Pat. No. 7,291,395 "Coated ion exchanged substrate and
method of forming" described a hyperbranched anion exchange
material based on a crosslinked layer that includes quaternary
amine groups and hydroxy groups. In an embodiment, the anion
exchange material was formed using a reaction between a diepoxide
reagent and an amine group. The ring opening reaction results in a
hydroxy group that is spaced apart from a quaternary amine group by
a two carbon spacer. The hydroxy group produced in this type of
reaction can be referred to as a beta hydroxy group with respect to
a quaternary amine, which makes the hydroxy groups more acidic and
in turn influences the anion binding characteristics of the anion
exchange resin. During an anion exchange chromatographic
separation, a hydroxide eluent is typically used. When the pH of
the hydroxide eluent is sufficiently high, the beta hydroxy group
can be deprotonated causing the formation of a zwitterionic ion
pair with the quaternary amine, which decreases the anion binding
to the quaternary amine ion exchange site. The zwitterionic ion
pair has a positively charged quaternary amine that is stabilized
by the negatively charged and deprotonated beta hydroxy group. The
proximity of the positively charged quaternary amine and the
deprotonated hydroxy group form a relatively stable zwitterionic
pair that reduces the anion binding strength of the quaternary
amine. Thus, the anion binding capability of anion exchange resins
can be tuned based on the concentration (or pH) of the hydroxide
eluent making it useful for various anion exchange chromatography
tests.
[0004] However, under certain circumstances, Applicant has found
that carbohydrates have little if any retention with hyperbranched
anion exchange materials containing beta hydroxy groups, and thus,
was not useful for this application. As such, Applicant believes
that there is a need for hyperbranched anion exchange materials
that do not contain beta hydroxy groups so that chromatographic
analysis can be performed for carbohydrates such as branched
glycans.
SUMMARY
[0005] A first embodiment of an anion exchange stationary phase for
separating a plurality of carbohydrates includes a) a negatively
charged substrate particle, b) a base condensation polymer layer,
and c) a polyalkylpolyamine condensation polymer layer. The base
condensation polymer layer is attached to the negatively charged
substrate particle. The base condensation polymer layer includes a
first plurality of quaternary amines, in which the first plurality
of quaternary amines are spaced apart by either a first spacer or a
second spacer. The base condensation polymer layer does not have a
hydroxy group spaced apart from one of the first plurality of
quaternary amines by an ethyl group. The polyalkylpolyamine
condensation polymer layer is covalently attached to the base
condensation polymer layer. The polyalkylpolyamine condensation
polymer layer includes a polymeric branch structure that includes a
second plurality of quaternary amines, in which the second
plurality of quaternary amines are spaced apart by the first spacer
or the second spacer. A density of the second plurality of
quaternary amines increases in a direction away from the base
condensation polymer layer. The polyalkylpolyamine condensation
polymer layer does not have a hydroxy group spaced apart from one
of the second plurality of quaternary amines by an ethyl group.
[0006] In regards to any of the first embodiments, the first spacer
can be an alkyl, a dialkylether, a cycloalkyl, an arylalkyl, and
combinations thereof.
[0007] In regards to any of the first embodiments, the second
spacer can be an alkyl, a dialkylether, a cycloalkyl, an arylalkyl,
and combinations thereof.
[0008] In regards to the first embodiment, which the first spacer
includes a first alkyl and the second spacer includes a second
alkyl, in which the first alkyl and the second alkyl are both a
linear and unsubstituted alkyl.
[0009] In regards to the first embodiment, the first spacer
includes a linear and unsubstituted alkyl and the second spacer
includes an arylalkyl.
[0010] In regards to the first embodiment, the first spacer
includes a chemical formula of (--CH.sub.2--).sub.x and the second
spacer comprises a chemical formula of (--CH.sub.2--).sub.y, where
x and y each independently range from 3 to 10, and preferably range
from 3 to 6.
[0011] A second embodiment of an anion exchange stationary phase
for separating a plurality of carbohydrates is formed by a method
including reacting a polyhalohydrocarbon with a polyalkylpolyamine
to form a base condensation polymer layer on a negatively charged
substrate particle. The base condensation polymer layer is reacted
with a number of reaction cycles to form a polyalkylpolyamine
condensation polymer layer. The number of reaction cycles ranges
from about 3 to about 10 and each reaction cycle includes a
polyhalohydrocarbon treatment and a polyalkylpolyamine treatment.
After the reaction cycles, the polyalkylpolyamine condensation
polymer layer is reacted with a monohaloalkane treatment. An
example of a monohaloalkane is methyliodide.
[0012] In regards to the second embodiment, the negatively charged
substrate particles are contained as a packed bed in a
chromatography column. The reacting of the polyhalohydrocarbon with
the polyalkylpolyamine includes flowing a solution of the
polyhalohydrocarbon and the polyalkylpolyamine through the column
to form the base condensation polymer layer on the negatively
charged substrate particles.
[0013] In regards to the second embodiment, the polyhalohydrocarbon
treatment includes flowing a solution of the polyhalohydrocarbon
through the column. The polyalkylpolyamine treatment includes
flowing a solution of the polyalkylpolyamine through the column.
The monohaloalkane treatment includes flowing a solution of the
monohaloalkane through the column.
[0014] In regards to any of the second embodiments, the number of
reaction cycles may range from about 3 to about 4.
[0015] In regards to any of the second embodiments, the
polyhalohydrocarbon can be a dihaloalkane, a dihalodialkylether, a
dihalocycloalkane, a trihaloarylalkane, and a combination
thereof.
[0016] In regards to any of the second embodiments, the
polyalkylpolyamine can include an ether group, a cycloalkane group,
an arylalkane, and a combination thereof.
[0017] In regards to any of the second embodiments, the
polyalkylpolyamine can be a polyalkyltriamine, a polyalkyldiamine,
and a combination thereof.
[0018] In regards to the second embodiment, all amines of the
polyalkylpolyamine can be tertiary amines (e.g.,
polyalkylpolytertiaryamine).
[0019] In regards to the second embodiment, the polyhalohydrocarbon
can be a dibromobutane, a dibromopentane, a dibromohexane, a
tribromomethylbenzene, and a combination thereof.
[0020] In regards to the second embodiment, the polyalkylpolyamine
can be a pentamethyldipropyltriamine, a
pentamethyldihexylltriamine, a permethylated spermine, a
permethylated spermidine, and a combination thereof.
[0021] In regards to the second embodiment, the polyhalohydrocarbon
is dibromobutane and the polyalkylpolyamine is
pentamethyldihexylltriamine.
[0022] In regards to the second embodiment, the polyhalohydrocarbon
is a trihaloalkylaryl and the polyalkylpolyamine is an
alkyldiamine.
[0023] In regards to the second embodiment, the polyhalohydrocarbon
is tribromomethylbenzene and the polyalkylpolyamine is
tetramethylhexanediamine.
[0024] A third embodiment of an anion exchange stationary phase for
separating a plurality of carbohydrates includes A) a negatively
charged substrate particle, B) a base condensation polymer layer
300, C) a first alkyl condensation reaction product (CRP) 400, D) a
first polyalkylpolyamine CRP 500, E) a second alkyl CRP 600, F) a
second alkyl CRP 700, G) a third alkyl CRP, and H) a third alkyl
CRP. The base condensation polymer layer 300 is attached to the
negatively charged substrate particle. The base condensation
polymer layer includes a reaction product of i) a first
polyhalohydrocarbon, and ii) a first polyalkylpolyamine. The first
alkyl CRP 400 is covalently attached to the base condensation
polymer layer 300. The first alkyl CRP 400 includes a reaction
product of i) an amine group of the base condensation polymer layer
300, and ii) a second polyhalohydrocarbon. The amine group of the
base condensation polymer layer 300 includes a positive charge so
that the base condensation polymer layer is ionically coupled to
the negatively charged substrate particle. The first
polyalkylpolyamine CRP 500 is covalently attached to the first
alkyl CRP 400. The first polyalkylpolyamine CRP 500 includes a
reaction product of i) a halide group of the second
polyhalohydrocarbon, and ii) a second polyalkylpolyamine. The
second alkyl CRP 600 is covalently attached to the first
polyalkylpolyamine CRP 500. The second alkyl CRP 600 includes a
reaction product of i) an amine group of the first
polyalkylpolyamine CRP 500 and ii) a third polyhalohydrocarbon. The
second polyalkylpolyamine CRP 700 is covalently attached to the
second alkyl CRP 600. The second polyalkylpolyamine CRP 700
includes a reaction product of i) a halide group of the third
polyhalohydrocarbon, and ii) a third polyalkylpolyamine. The third
alkyl CRP is covalently attached to the second polyalkylpolyamine
CRP 700. The third alkyl CRP includes a reaction product of i) an
amine group of the second polyalkylpolyamine CRP and ii) a fourth
polyhalohydrocarbon. The third polyalkylpolyamine CRP is covalently
attached to the third alkyl CRP. The third polyalkylpolyamine CRP
includes a reaction product of i) a halide group of the fourth
polyhalohydrocarbon, and ii) a fourth polyalkylpolyamine.
[0025] In regards to the third embodiment, the first
polyhalohydrocarbon, second polyhalohydrocarbon, third
polyhalohydrocarbon, and fourth polyhalohydrocarbon can include a
dihaloalkane. The first polyalkylpolyamine, second
polyalkylpolyamine, third polyalkylpolyamine, and fourth
polyalkylpolyamine can include a polyalkyltriamine.
[0026] A fourth embodiment of an anion exchange stationary phase
for separating a plurality of carbohydrates includes A) a
negatively charged substrate particle, B) a base condensation
polymer layer 1500, C) a first polyalkylpolyamine CRP 1600, D) a
first polyalkylaryl CRP 1700, E) a second polyalkylpolyamine CRP
1800, F) a second polyalkylaryl CRP 1900, G) a third
polyalkylpolyamine CRP, and H) a third polyalkylaryl CRP. The base
condensation polymer layer 1500 is attached to the negatively
charged substrate particle. The base condensation polymer layer
includes a reaction product of i) a first polyhalohydrocarbon, and
ii) a first polyalkylpolyamine. The amine group of the base
condensation polymer layer 1500 includes a positive charge so that
the base condensation polymer layer is ionically coupled to the
negatively charged substrate particle. The first polyalkylpolyamine
condensation reaction product 1600 is covalently attached to the
base condensation polymer layer 1500. The first polyalkylpolyamine
condensation reaction product 1600 includes a reaction product of
i) a halide group of the base condensation polymer layer 1500, and
ii) a second polyalkylpolyamine. The first polyalkylaryl
condensation reaction product 1700 is covalently attached to the
first polyalkylpolyamine condensation reaction product 1600. The
first polyalkylaryl condensation reaction product 1700 includes a
reaction product of i) an amine group of the first
polyalkylpolyamine condensation reaction product 1600, and ii) a
second polyhalohydrocarbon. The second polyalkylpolyamine
condensation reaction product 1800 is covalently attached to the
first polyalkylaryl condensation reaction product 1700. The second
polyalkylpolyamine condensation reaction product 1800 includes a
reaction product of i) a halide group of the first polyalkylaryl
condensation reaction product 1700, and ii) a third
polyalkylpolyamine. The second polyalkylaryl CRP 1900 is covalently
attached to the second polyalkylpolyamine CRP 1800. The second
polyalkylaryl CRP 1900 includes a reaction product of i) an amine
group of the second polyalkylpolyamine CRP 1800, and ii) a third
polyhalohydrocarbon. The third polyalkylpolyamine CRP is covalently
attached to the second polyalkylaryl CRP 1900. The third
polyalkylpolyamine CRP includes a reaction product of i) a halide
group of the second polyalkylaryl CRP 1900, and ii) a fourth
polyalkylpolyamine. The third polyalkylaryl CRP is covalently
attached to the third polyalkylpolyamine CRP. The third
polyalkylaryl CRP includes a reaction product of i) an amine group
of the third polyalkylpolyamine, and ii) a fourth
polyhalohydrocarbon.
[0027] In regards to the fourth embodiment, it may further includes
a reaction product of i) a halide group of the third polylalkylaryl
CRP, and ii) a tertiary amine.
[0028] In regards to the fourth embodiment, the first
polyhalohydrocarbon, second polyhalohydrocarbon, third
polyhalohydrocarbon, and fourth polyhalohydrocarbon can include a
trihaloalkylaryl, and the first polyalkylpolyamine, second
polyalkylpolyamine, third polyalkylpolyamine, and fourth
polyalkylpolyamine can include a polyalkyldiamine.
[0029] A fifth embodiment is a method of using an anion exchange
stationary phase for separating a plurality of carbohydrates in a
sample with the anion exchange stationary phase of any of the
first, second, and third embodiments. The method includes flowing
an eluent through a chromatography column containing the anion
exchange stationary phase. The eluent includes a hydroxide. A
sample is injected that includes a plurality of carbohydrates into
the chromatography column. At least one carbohydrate is separated
from the sample injected into the chromatography column. The at
least one carbohydrate is detected at a detector.
[0030] In regards to the fifth embodiment, the at least one
carbohydrate is a branched glycan.
[0031] In regards to the above embodiments, the negatively charged
substrate particle can include a crosslinked divinylbenzene and
ethylvinyl benzene copolymer, in which at least a surface of the
negatively charged substrate particle includes sulfonate
groups.
[0032] In regards to the above embodiments, the negatively charged
substrate particle comprises a crosslinked divinylbenzene and
ethylvinyl benzene copolymer, in which at least a surface of the
negatively charged substrate particle includes carboxylate
groups.
[0033] In regards to the above embodiments, in which the base
condensation polymer layer is positively charged and ionically
attached to the negatively charged substrate particle.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] The accompanying drawings, which are incorporated herein and
constitute part of this specification, illustrate presently
preferred embodiments of the invention, and, together with the
general description given above and the detailed description given
below, serve to explain features of the invention (wherein like
numerals represent like elements).
[0035] FIG. 1 illustrates various chemical structures of
polyalkylpolyamine reagents that can be used in forming polymers
and reaction products for anion exchange resins.
[0036] FIG. 2 illustrates various chemical structures of
polyhalohydrocarbon reagents that can be used in forming polymers
and reaction products for anion exchange resins.
[0037] FIG. 3 illustrates a schematic of a base polymer layer
formed from a first polyakyltriamine and a first dihaloalkane where
the base polymer layer is attached to a negatively charged
substrate particle.
[0038] FIG. 4 illustrates a schematic of a first alkyl reaction
product that has pendant halide groups and is covalently attached
to the base polymer. The first alkyl reaction product is a reaction
product of an amine group of the base polymer and a second
dihaloalkane.
[0039] FIG. 5 illustrates a schematic of a first polyalkylpolyamine
reaction product that is covalently attached to the first alkyl
reaction product. The first polyalkylpolyamine reaction product is
a reaction product of the pendant halide group of the second
dihaloalkane and an amine group of a second polyalkyltriamine.
[0040] FIG. 6 illustrates a schematic of a second alkyl reaction
product that has a pendant halide group and is covalently attached
to the first polyalkylpolyamine reaction product. The second alkyl
reaction product is a reaction product of an amine group of the
first polyalkylpolyamine reaction product and a third
dihaloalkane.
[0041] FIG. 7 illustrates a schematic of a second
polyalkylpolyamine that is covalently attached to the second alkyl
reaction product. The second polyalkylpolyamine reaction product is
a reaction product of the pendant halide group of the third
dihaloalkane and an amine group of a third polyalkyltriamine.
[0042] FIG. 8 illustrates six chromatograms where each one uses a
different NaOH eluent concentration and a standard solution
containing five different carbohydrates. The chromatograms were
performed using an anion exchange resin that includes
pentamethyldihexyltriamine PMDHTA and 1-4-dibromobutane DBB.
[0043] FIG. 9 illustrates two chromatograms using the anion
exchange resin that includes PMDHTA and DBB. A standard solution
was injected into a chromatography column that contained either
five carbohydrates (upper chromatogram) or two carbohydrates (lower
chromatogram).
[0044] FIG. 10 illustrates three chromatograms where each one uses
a different NaOH eluent concentration and a standard solution
containing five different carbohydrates. The chromatograms were
performed using an anion exchange resin that includes
N,N,N',N'-tetramethyl-1,6-hexanediamine TMHDA and
1,3,5-tri(bromomethyl)benzene TBMB.
[0045] FIG. 11 illustrates two chromatograms using an anion
exchange resin that includes TMHDA and TBMB. A standard solution
was injected into a chromatography column that contained either
five carbohydrates (upper chromatogram) or two carbohydrates (lower
chromatogram).
[0046] FIG. 12 illustrates a chromatogram using an anion exchange
resin that includes PMDHTA and DBB that shows 38 peaks. A sample
solution was injected into a chromatography column that contained
fetuin alditols.
[0047] FIG. 13 illustrates a chromatogram using an anion exchange
resins that include TMHDA and TBMB that shows 28 peaks. A sample
solution was injected into a chromatography column that contained
fetuin alditols.
[0048] FIG. 14 shows an ion chromatography system suitable for
analyzing samples with an ion exchange chromatography column and an
electrochemical detector using quadruple voltage waveforms.
[0049] FIG. 15 illustrates a schematic representation of a base
polymer layer formed from N,N,N',N'-tetramethyl-1,6-hexanediamine
TMHDA and 1,3,5-tri(bromomethyl)benzene TBMB where the base polymer
layer is attached to a negatively charged substrate particle.
[0050] FIG. 16 illustrates a schematic of a first
polyalkylpolyamine reaction product that has pendant tertiary amine
groups and is covalently attached to the base polymer. The first
polyalkylpolyamine reaction product is a reaction product of a
halide group of the base polymer and a second TMHDA.
[0051] FIG. 17 illustrates a schematic of a first polyalkylaryl
reaction product that is covalently attached to the first
polyalkylpolyamine reaction product. The first polyalkylaryl
reaction product is a reaction product of the pendant tertiary
amine of the second TMHDA and a halide group of a second TBMB.
[0052] FIG. 18 illustrates a schematic of a second
polyalkylpolyamine reaction product that has a pendant tertiary
amine groups and is covalently attached to the first polyalkylaryl
reaction product. The second polyalkylpolyamine reaction product is
a reaction product of the halide groups of the first polyalkylaryl
reaction product and a third TMHDA.
[0053] FIG. 19 illustrates a schematic of a second polyalkylaryl
that is covalently attached to the second polyalkylpolyamine
reaction product. The second polyalkylaryl reaction product is a
reaction product of the pendant tertiary amine groups of the third
TMHDA and a halide group of a third TBMB.
DETAILED DESCRIPTION OF EMBODIMENTS
[0054] The following detailed description should be read with
reference to the drawings, in which like elements in different
drawings are identically numbered. The drawings, which are not
necessarily to scale, depict selected embodiments and are not
intended to limit the scope of the invention. The detailed
description illustrates by way of example, not by way of
limitation, the principles of the invention. This description will
clearly enable one skilled in the art to make and use the
invention, and describes several embodiments, adaptations,
variations, alternatives and uses of the invention, including what
is presently believed to be the best mode of carrying out the
invention. As used herein, the terms "about" or "approximately" for
any numerical values or ranges indicate a suitable dimensional
tolerance that allows the part or collection of components to
function for its intended purpose as described herein.
[0055] Herein the term "alkyl," by itself or as part of another
substituent, means, unless otherwise stated, a straight or branched
chain, or cyclic hydrocarbon radical, or combination thereof, which
may be fully saturated, mono- or polyunsaturated and can include
di- and multivalent radicals, having the number of carbon atoms
designated (i.e., C.sub.1-C.sub.10 means one to ten carbons).
Examples of saturated hydrocarbon radicals include, but are not
limited to, groups such as methyl, ethyl, n-propyl (e.g.,
--CH.sub.2--CH.sub.2--CH.sub.3, --CH.sub.2--CH.sub.2--CH.sub.2--),
isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,
(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for
example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An
unsaturated alkyl group is one having one or more double bonds or
triple bonds. Examples of unsaturated alkyl groups include, but are
not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,
2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-
and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The
term "alkyl," unless otherwise noted, is also meant to include
those derivatives of alkyl defined in more detail below, such as
"heteroalkyl". Alkyl groups that are limited to hydrocarbon groups
are termed "homoalkyl". The term "alkyl" can also mean "alkylene"
or "alkyldiyl" as well as alkylidene in those cases where the alkyl
group is a divalent radical.
[0056] Herein the term "alkylene" or "alkyldiyl" by itself or as
part of another substituent means a divalent radical derived from
an alkyl group, as exemplified, but not limited, by
--CH.sub.2CH.sub.2CH.sub.2-- (propylene or propane-1,3-diyl), and
further includes those groups described below as "heteroalkylene".
Typically, an alkyl (or alkylene) group will have from 1 to about
30 carbon atoms, preferably from 1 to about 25 carbon atoms, more
preferably from 1 to about 20 carbon atoms, even more preferably
from 1 to about 15 carbon atoms and most preferably from 1 to about
10 carbon atoms. A "lower alkyl", "lower alkylene" or "lower
alkyldiyl" is a shorter chain alkyl, alkylene or alkyldiyl group,
generally having about 10 or fewer carbon atoms, about 8 or fewer
carbon atoms, about 6 or fewer carbon atoms or about 4 or fewer
carbon atoms.
[0057] Herein the term "alkylidene" by itself or as part of another
substituent means a divalent radical derived from an alkyl group,
as exemplified, but not limited, by CH.sub.3CH.sub.2CH.sub.2.dbd.
(propylidene). Typically, an alkylidene group will have from 1 to
about 30 carbon atoms, preferably from 1 to about 25 carbon atoms,
more preferably from 1 to about 20 carbon atoms, even more
preferably from 1 to about 15 carbon atoms and most preferably from
1 to about 10 carbon atoms. A "lower alkyl" or "lower alkylidene"
is a shorter chain alkyl or alkylidene group, generally having
about 10 or fewer carbon atoms, about 8 or fewer carbon atoms,
about 6 or fewer carbon atoms or about 4 or fewer carbon atoms.
[0058] Herein the terms "alkoxy," "alkylamino" and "alkylthio" (or
thioalkoxy) are used in their conventional sense, and refer to
those alkyl groups attached to the remainder of the molecule via an
oxygen atom, an amino group, or a sulfur atom, respectively.
[0059] Herein the term "heteroalkyl," by itself or in combination
with another term, means, unless otherwise stated, a stable
straight or branched chain, or cyclic hydrocarbon radical, or
combinations thereof, consisting of the stated number of carbon
atoms and at least one heteroatom selected from the group
consisting of O, N, Si, S and B, and wherein the nitrogen and
sulfur atoms may optionally be oxidized and the nitrogen heteroatom
may optionally be quaternized. The heteroatom(s) O, N, B, S and Si
may be placed at any interior position of the heteroalkyl group or
at the position at which the alkyl group is attached to the
remainder of the molecule. Examples include, but are not limited
to, --CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NHCH.sub.3, --CH.sub.2--
CH.sub.2--N(CH.sub.3)--CH.sub.3, --CH.sub.2--S--CH.sub.2--CH.sub.3,
--CH.sub.2--CH.sub.2, --S(O)--CH.sub.3,
--CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3,
--CH.dbd.CH--O--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3, and
--CH.dbd.CH--N(CH.sub.3)--CH.sub.3. Up to two heteroatoms may be
consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3 and
--CH.sub.2-- O--Si(CH.sub.3).sub.3. Similarly, the term
"heteroalkylene" by itself or as part of another substituent means
a divalent radical derived from heteroalkyl, as exemplified, but
not limited by, --CH.sub.2-- CH.sub.2--S--CH.sub.2--CH.sub.2-- and
--CH.sub.2--S--CH.sub.2--CH.sub.2--NH--CH.sub.2--. For
heteroalkylene groups, heteroatoms can also occupy either or both
of the chain termini (e.g., alkyleneoxy, alkylenedioxy,
alkyleneamino, alkylenediamino, and the like). Optionally, for
alkylene and heteroalkylene linking groups, no orientation of the
linking group is implied by the direction in which the formula of
the linking group is written. For example, the formula
--CO.sub.2R'-- optionally represents both --C(O)OR' and
--OC(O)R'.
[0060] Herein the terms "cycloalkyl" and "heterocycloalkyl", by
themselves or in combination with other terms, represent, unless
otherwise stated, cyclic versions of "alkyl" and "heteroalkyl",
respectively. Additionally, for heterocycloalkyl, a heteroatom can
occupy the position at which the heterocycle is attached to the
remainder of the molecule. Examples of cycloalkyl include, but are
not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl,
3-cyclohexenyl, cycloheptyl, and the like. Examples of
heterocycloalkyl include, but are not limited to,
1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,
3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,
tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,
1-piperazinyl, 2-piperazinyl, and the like.
[0061] Herein the terms "halo" or "halogen," by themselves or as
part of another substituent, mean, unless otherwise stated, a
fluorine, chlorine, bromine, or iodine atom. Additionally, terms
such as "haloalkyl," are meant to include monohaloalkyl and
polyhaloalkyl. For example, the term "halo(C.sub.1-C.sub.4)alkyl"
is mean to include, but not be limited to, trifluoromethyl,
2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the
like.
[0062] Herein the term "aryl" means, unless otherwise stated, a
polyunsaturated, aromatic, substituent that can be a single ring or
multiple rings (preferably from 1 to 3 rings), which are fused
together or linked covalently. The term "heteroaryl" refers to aryl
groups (or rings) that contain from one to four heteroatoms
selected from N, O, S, Si and B, wherein the nitrogen and sulfur
atoms are optionally oxidized, and the nitrogen atom(s) are
optionally quaternized. A heteroaryl group can be attached to the
remainder of the molecule through a heteroatom. Non-limiting
examples of aryl and heteroaryl groups include phenyl, 1-naphthyl,
2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,
3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,
4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl,
4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,
2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl,
4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl,
2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,
2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.
Substituents for each of the above noted aryl and heteroaryl ring
systems are selected from the group of acceptable substituents
described below.
[0063] For brevity, herein the term "aryl" when used in combination
with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes
both aryl and heteroaryl rings as defined above. Thus, the term
"arylalkyl" is meant to include those radicals in which an aryl
group is attached to an alkyl group (e.g., benzyl, phenethyl,
pyridylmethyl and the like) including those alkyl groups in which a
carbon atom (e.g., a methylene group) has been replaced by, for
example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl,
3-(1-naphthyloxy)propyl, and the like).
[0064] Each of the above terms (e.g., "alkyl," "heteroalkyl,"
"aryl" and "heteroaryl") are meant to include both substituted and
unsubstituted forms of the indicated radical. Preferred
substituents for each type of radical are provided below.
[0065] Substituents for the alkyl and heteroalkyl radicals
(including those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are
generically referred to as "alkyl group substituents," and they can
be one or more of a variety of groups selected from, but not
limited to: substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heterocycloalkyl, --OR', .dbd.O, .dbd.NR', .dbd.N--OR', --NR'R'',
--SR', -halogen, --SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R',
--CONR'R'', --OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''',
--NR''C(O).sub.2R', --NR-- C(NR'R''R''').dbd.NR'''',
--NR--C(NR'R'').dbd.NR''', --S(O)R', --S(O).sub.2R',
--OS(O).sub.2R', --S(O).sub.2NR'R'', --NRSO2R', --CN and --NO.sub.2
in a number ranging from zero to (2m'+1), where m' is the total
number of carbon atoms in such radical. R', R'', R''' and R''''
each preferably independently refer to hydrogen, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g.,
aryl substituted with 1-3 halogens, substituted or unsubstituted
alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a
compound of the invention includes more than one R group, for
example, each of the R groups is independently selected as are each
R', R'', R''' and R'''' groups when more than one of these groups
is present. When R' and R'' are attached to the same nitrogen atom,
they can be combined with the nitrogen atom to form a 5-, 6-, or
7-membered ring. For example, --NR'R'' is meant to include, but not
be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above
discussion of substituents, one of skill in the art will understand
that the term "alkyl" is meant to include groups including carbon
atoms bound to groups other than hydrogen groups, such as haloalkyl
(e.g., --CF.sub.3 and --CH.sub.2CF.sub.3) and acyl (e.g.,
--C(O)CH.sub.3, --C(O)CF.sub.3, --C(O)CH.sub.2OCH.sub.3, and the
like).
[0066] Similar to the substituents described for the alkyl radical,
substituents for the aryl and heteroaryl groups are generically
referred to as "aryl group substituents." The substituents are
selected from, for example: substituted or unsubstituted alkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heterocycloalkyl, --OR',
.dbd.O, .dbd.NR', .dbd.N--OR', --NR'R'', --SR', -halogen,
--SiR'R''R''', --OC(O)R', --C(O)R', --CO2R', --CONR'R'',
--OC(O)NR'R'', --NR''C(O)R', --NR'-- C(O)NR''R''',
--NR''C(O).sub.2R', --NR--C(NR'R''R''').dbd.NR'''',
--NR--C(NR'R'').dbd.NR''', --S(O)R', --S(O).sub.2R',
--S(O).sub.2NR'R'', --NRSO.sub.2R', --CN and --NO.sub.2, --R',
--N.sub.3, --CH(Ph).sub.2, fluoro(C.sub.1-C.sub.4)alkoxy, and
fluoro(C.sub.1-C.sub.4)alkyl, in a number ranging from zero to the
total number of open valences on the aromatic ring system; and
where R', R'', R''' and R'''' are preferably independently selected
from hydrogen, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl and
substituted or unsubstituted heteroaryl. When a compound of the
invention includes more than one R group, for example, each of the
R groups is independently selected as are each R', R'', R''' and
R'''' groups when more than one of these groups is present.
[0067] A combination of multi-halogen reagents and multi-amine
reagents are described that produce hyperbranched polymers
containing no beta hydroxy groups. A hyperbranched anion exchange
polymer has a structure that has an increasing number of branches
as the polymer layer extends outwardly from the substrate. The
increasing number of branches causes an increasing density of anion
exchange sites towards the outer portion of the hyperbranched
structure. A wide variety of different capacities and selectivities
for hyperbranched structure can be achieved using various
combinations of reagents, reagent concentrations, and the number of
reaction cycles. For instance, hyperbranched anion exchange
structures can be generated with a high ion exchange capacity and
high stationary phase pH. Hyperbranched anion exchange structure
have a structure similar to highly branched glycans derived from
glycoproteins, and thus, hyperbranched anion exchange structures
are suitable for separating highly branched glycans by providing
selectivity without unduly restricting mass transport. The anion
exchange stationary phase may be referred to as a resin.
[0068] Glycans include a large number of carbohydrates linked to a
glycoprotein. A glycan may include saccharide subunits such as
glucose, galactose, fructose, mannose, galactosamine, and
glucosamine. It is believed that the pKa of beta hydroxy groups
have a pKa of about 13.9, which is close to the pKa's of the
saccharide subunits that are attached to glycans. Thus, pH values
that cause portions of a glycan to be ionized (as an anion) can
also cause beta hydroxy groups to be ionized resulting in low
retention at the anion exchange resin.
[0069] In an embodiment, hyperbranched polymers can be formed from
dihaloalkyls and permethylated triamines. Exemplary reagents,
pentamethyldipropylenetriamine 102 and 1,4-dibromobutane 202 can be
used to produce a hyperbranched polymer, as illustrated in FIGS. 1
and 2, respectively. Initially, these the two reagents together in
a 1:1 mole ratio to form a ground layer on a surface sulfonated
super macroporous resin, and then alternate reactions with
1,4-dibromobutane followed by reaction with
pentamethyldipropylenetriamine to produce a hyperbranched anion
exchange polymer that is free from having any beta hydroxy groups.
It should be noted that dihaloalkane is water-insoluble, and thus,
an organic solvent was used to dissolve the dihaloalkanes for
reacting with a permethylated triamine. A reaction product of the
dihaloalkanes and the permethylated triamine is a halide salt,
which tends to precipitate in organic solvents and causes the
reaction to terminate. The halide salt is more soluble in polar
solvents. However, the use of polar solvents tend to excessively
reduce the reaction rate making it difficult to produce suitable
molecular weight polymers. Thus, to prepare the ground layer, it is
necessary to react the two reagents in a solvent (or solvent
mixture) that balances all of these requirements of being able to
dissolve the reactants, reaction products, and have a suitable
reaction rate to reach the desired polymer molecular weight. In an
embodiment, a solvent mixture having a minimum amount of methanol
necessary to keep the polymer in solution with acetonitrile (e.g.,
4 parts acetonitrile to 1 part methanol) allows for the preparation
of suitable polymers.
[0070] Applicant believes that the charge density of the quaternary
ion exchange sites in a hyperbranched structure can be too high,
which would result in excessive stress on the bonds between the two
ion exchange sites. In an embodiment, Applicant believes that a
spacer length between pairs of quaternary amine groups can be about
greater than or equal to a propyl group so that the ion exchange
site density is not too high. However, if the spacer is group is
too long, then the ion exchange site density can be too low for
effectively separating anions and providing a sufficiently high ion
exchange capacity.
[0071] In another embodiment, the permethylated amines such as
pentamethyldibutylenetriamine or pentamethyldihexamethylenetriamine
may be used in conjunction with 1,4-dibromobutane. This would allow
positioning of the quaternary amine ion exchange sites to be spaced
apart by either a butyl or hexyl group. The permethylated amine
reagent can be in the form of tritertiary amines where the
alkylation by 1,4-dibromobutane results in the liberation of a
bromide ion that can ionically associate with the formed quaternary
amine. These reagents will also allow the formation of
hyperbranched polymers free from any beta hydroxy groups.
[0072] In another embodiment, permethylated diamines may be used in
conjunction with 1,3,5-tribromomethylbenzene to create
hyperbranched structures. Examples of permethylated diamines are
tetramethyl-1,4-butanediamine or tetramethyl-1,6-hexanediamine 106.
These reagents will also allow the formation of hyperbranched
polymers free from any beta hydroxy groups.
[0073] In an embodiment, a series of condensation reaction products
or polymer layers can be formed on a substrate. A polymer formed in
a polymerization reaction with a polyalkylpolyamine and a
polyhalohydrocarbon may be referred to as a condensation polymer or
a condensation polymer reaction product or a condensation polymer
layer. Similarly, a condensation reaction product CRP can be a
product from a condensation reaction between a polymer and a
reagent such as an alkylhalide or amine based reagent chemical,
which results in a loss of a halide or a halide and a hydrogen. It
should be noted that the reaction of an alkylhalide and a tertiary
amine based reagent results in a loss of a halide and does not
generate the loss of hydrogen. The reaction of an alkylhalide and a
primary or secondary amine based reagent results in a loss of a
halide and a hydrogen, where the generated hydrogen may interfere
in polymerization processes.
[0074] A polyalkylpolyamine can be a polyalkyltriamine or a
polyalkyldiamine. FIG. 1 illustrates polyalkyltriamine in the form
of 2,6,10-trimethyl-2,6,10-triazaundecane
(pentamethyldipropyltriamine, PMDPTA 102) and
2,9,13-Trimethyl-2,9,13-triazaheptadecane
(pentamethyldihexyltriamine, PMDHTA 104). Referring to FIG. 1, the
alkyl spacer for polyalkyltriamine can have an n value that ranges
from about 3 to about 10, and preferably from about 3 to about 6.
FIG. 1 also illustrates a polyalkyldiamine in the form of
N,N,N',N'-tetramethyl-1,6-hexanediamine TMHDA 106.
[0075] A polyalkylpolyamine may be a reagent compound that include
two or more alkyl groups and two or more amine groups. In an
embodiment, the polyalkylpolyamine may have a portion or all of the
amine groups be alkylated. The alkylated amine groups of the
polyalkylpolyamine may have a portion or all of the amine groups be
tertiary amines. Although FIG. 1 only illustrates
polyalkylpolyamine reagents that have alkyls attached to the
amines, a portion or all of the alkyls can be an alkylether,
cycloalkyl, arylalkyl, and combinations thereof. In an embodiment,
the alkanes attached in between two amine groups (i.e., spacer) can
have a length of 3 to 10 atoms, and preferably have a length of 3
to 6 atoms.
[0076] A polyhalohydrocarbon can be a polyhaloalkane or a
polyhaloalkylaryl. The polyhaloalkane can be a dihaloalkane such as
1-4-dibromobutane DBB 202, as illustrated in FIG. 2. The
polyhaloalkylaryl can be a trihaloalkylaryl such as
1,3,5-tri(bromomethyl)benzene TBMB 204, as illustrated in FIG. 2.
In an embodiment, a dihaloalkane can be represented by a chemical
formula 206 as illustrated in FIG. 2. The value n as it relates to
chemical formula 206 can range from 3 to 10, and preferably range
from 3 to 6.
[0077] A polyhalohydrocarbon may be a reagent compound that include
two or more halide groups attached to a hydrocarbon. The halide
group can be a bromide or iodide group. The polyhalohydrocarbon may
have 2 to 10 halide groups, and preferably 2 to 3 halide groups. In
an embodiment, the spacer between two halide groups of a
polyhalohydrocarbon can have a length of 3 to 10 atoms, and
preferably have a length of 3 to 6 atoms. The hydrocarbon portion
(spacer) of the polyhalohydrocarbon in between a pair of halide
groups may include an alkyl, a cycloalkyl, an arylalkyl, a dialkyl
ether group, and combinations thereof.
[0078] FIG. 3 illustrates a schematic representation of a base
condensation polymer layer 300 formed from a reaction with a first
PMDPTA 102 and a first DBB 202 where the base polymer layer 300 is
attached to a negatively charged substrate particle. The negatively
charged substrate particle can be contained as a packed bed in a
chromatography column. A solution of the PMDPTA 102 and
dihaloalkane 202 can be flowed through the column to form the base
condensation polymer layer on the negatively charged substrate
particle. It is worthwhile to note that the base condensation
polymer layer 300 does not have beta hydroxy groups since no
epoxide reagents were employed. As illustrated in FIG. 3, the base
condensation polymer layer 300 includes quaternary amines and
tertiary amines. In an aspect, a mole ratio can be a 1:1 mole ratio
of PMDPTA 102 and DBB 304 to form the base polymer layer. Although
the base polymer layer 300 is depicted as linear (by reacting only
the terminal end amines of PMDPTA 102), as illustrated in FIG. 3,
it is possible for some of the middle amine groups (e.g., 302) to
be quaternized and form either a branched or crosslinked portion.
The base layer 300 can be formed in the presence of a negatively
charged polymeric particle where the base layer associates and/or
partially binds with the negatively charged polymeric particle, as
illustrated in FIG. 3. For simplicity, the bromide salt associated
with quaternary amine is not shown in the figures. The term x in
FIG. 3 may range from about 5 to about 150.
[0079] The negatively charged polymeric particle can be any inert
polymeric substrate particle that is chemically stable under the
intended conditions of use (e.g., pH 0 to 14). The polymeric
particle may be based on a divinylbenzene (DVB) crosslinking
monomer and a support resin monomer where the support resin monomer
may be an ethylvinylbenzene (EVB) monomer, a styrene monomer, and a
combination thereof. The mole percent of DVB can be 55% and EVB can
be 45%. The support resin particles may have a diameter ranging
from about 1 micron to about 20 microns, preferably from about 2
microns to about 10 microns, and more preferably from about 3
microns to about 7 microns. The support resin particles may have a
surface area ranging from about 20 m.sup.2/g to about 800
m.sup.2/g, preferably from about 20 m.sup.2/g to about 500
m.sup.2/g, more preferably from about 20 m.sup.2/g to about 100
m.sup.2/g, and yet more preferably be about 20 m.sup.2/g to about
30 m.sup.2/g. The support resin particles may have a pore size
ranging from about 1000 angstroms to about 2000 angstroms.
[0080] In some embodiments, the negatively charged substrate
particle may include one or more super macroporous particles (SMP).
SMP can be obtained from commercial sources, including Agilent
PLRP-s1000A and Waters Styragel HR4-HR6. The super macroporous
particle can have a diameter of 4-6 .mu.m, a surface area of 20-30
m.sup.2/g, pore sizes of 1000 .ANG.-2000 .ANG., and a crosslinking
mole ratio of 55% of the divinylbenzene and a mole ratio of 45% of
the ethylvinylbenzene.
[0081] Alternatively, the polymeric particles may be based on other
vinylaromatic monomers such as alpha-methylstyrene, chlorostyrene,
chloromethylstyrene, vinyltoluene, vinylnaphthalene, and a
combination thereof. The polymeric particles may also be based on
unsaturated monomers, and copolymers of the above vinylaromatic
monomers and unsaturated monomers. Preferably such monomers will be
copolymerized with a vinylaromatic crosslinking monomer such as
divinylbenzene but other vinylaromatic crosslinking monomers such
as trivinylbenzene, divinylnaphthalene, and a combination thereof
may also be used.
[0082] The polymeric particles can be sulfonated to create a
negative charge at least on the surface of the particle. For
example, particles made with 55% DVB and 45% EVB can be sulfonated
by treating the particles with glacial acetic acid and concentrated
sulfuric acid.
[0083] Base layer 300 can be reacted with a number of reactions
cycles of reagents to form a polyalkylpolyamine condensation
polymer layer. In an embodiment, the number of reaction cycles may
range from 1 to 10, preferably 3 to 10, and more preferably 3 to 6.
Each reaction cycle includes a) a dihaloalkane treatment and b) a
polyalkyltriamine treatment. After performing the number of
reaction cycles, the polyalkylpolyamine condensation polymer layer
may be reacted with a monohaloalkane treatment to alkylate any
residual tertiary amines.
[0084] For the first step a) of a first cycle, a second DBB 202 can
be reacted with a tertiary amine of base layer 300 to form a first
alkyl condensation reaction product (CRP) 400 having pendant halide
groups 402, as illustrated in FIG. 4. In addition, at least a
portion of the tertiary amines 302 of base layer 300 are alkylated
to form quaternary amines that have a positive charge. It is
worthwhile to note that positive charges from the quaternary amines
are believed to help base layer 300 to ionically bond to the
negatively charged particles. The quaternary amines of the base
condensation polymer layer 300 are spaced apart by either a first
spacer 406 or a second spacer 408. The first spacer 406 is derived
from PMDPTA 102 and the second spacer 408 is derived from DBB 202,
as shown in FIGS. 3 and 4.
[0085] For a second step b) of the first cycle, the pendant halide
groups 402 of the first alkyl CRP 400 can be reacted with a second
PMDPTA (a second polyalkyltriamine) to form a first
polyalkylpolyamine CRP 500, as illustrated in FIG. 5. The first
polyalkylpolyamine CRP 500 is covalently attached to the first
alkyl CRP 400. Each branch of the first polyalkylpolyamine CRP 500
includes a quaternary amine 502 and two tertiary amines 504 and
506, as illustrated in FIG. 5. Although FIG. 5 illustrates PMDPTA
as only reacting with only one terminal amine group 502, it is
possible for some of the PMDPTA compounds to crosslink by reacting
two or three of the amine groups with the halide groups of the
first alkyl CRP 400. In addition, it is possible for only the
middle amine group 504 of the PMDPTA to react with a halide group
of the first alkyl CRP 400.
[0086] Now that one reaction cycle of two steps has been performed,
a second cycle of two steps may be performed to create a
hyperbranched structure. For a first step a) of a second cycle, the
two tertiary amines 504 and 506 of the first polyalkylpolyamine CRP
500 can each be reacted with two dihaloalkanes (third DBB) to form
a second alkyl condensation reaction product 600, as illustrated in
FIG. 6. The second alkyl CRP 600 is covalently attached to the
first polyalkylpolyamine CRP 500.
[0087] Although FIG. 6 illustrates each dihaloalkane compound as
only reacting with only one halide group (of the two halide
groups), it is possible for a some of the dihaloalkane compounds to
crosslink by reacting both of two halide groups with the first
polyalkylpolyamine CRP 500, or one halide group of the dihaloalkane
with one portion of the first polyalkylpolyamine CRP 500 and the
other halide group of the same dihaloalkane with another portion of
the first polyalkylpolyamine CRP 500.
[0088] For a second step b) of the second cycle, the pendant halide
groups of the second alkyl CRP 600 can then be reacted with a third
PMDPTA to form a second polyalkypolyamine CRP 700 that has a
quaternary amine 702 and two tertiary amines 704 and 706, as
illustrated in FIG. 7. The second polyalkylpolyamine CRP 700 is
covalently attached to the second alkyl CRP 600.
[0089] A third reaction cycle of two steps can be performed with
the second polyalkypolyamine CRP 700. For a first step a) of a
third cycle, the two tertiary amines 704 and 706 of first
polyalkypolyamine CRP 700 can each be reacted with two
dihaloalkanes (fourth DBB) to form a third alkyl CRP (not shown).
It should be noted that the structure of the third alkyl CRP is
analogous to the structure of the second alkyl CRP 600.
[0090] For a second step b) of the third cycle, the pendant halide
groups of the third alkyl CRP can then be reacted with a fourth
PMDPTA to form a third polyalkypolyamine CRP that has two tertiary
amines (not shown). It should be noted that the structure of the
third polyalkypolyamine CRP is analogous to the structure of the
second polyalkypolyamine CRP 700.
[0091] In an embodiment, more than 3 cycles may be performed by
repeating steps a) and b). It should be noted that the reaction
using PMDPTA, as illustrated in FIGS. 3 to 7, can be modified by
substituting PMDPTA with PMDHTA. The anion exchange stationary
phases using PMDPTA has a higher density of anion exchange sites
than anion exchange stationary phases using PMDHTA because of the
increased length of the spacer chain from C3 to C6. It should also
be noted that FIGS. 3 to 7 are exemplary and that PMDPTA may be
substituted with other polyalkylpolyamines described herein and
that DBB may also be substituted with other polyhalogenhydrocarbons
described herein.
[0092] In another embodiment, a hyperbranched ion exchange
stationary phase will be described that uses a trihaloalkylaryl
compound and a polyalkyldiamine. FIG. 15 illustrates a schematic
representation of a base condensation polymer layer 1500 formed
from a reaction with a first TMHDA 106 and a first TBMB 204 where
the base polymer layer 1500 is attached to a negatively charged
substrate particle. As illustrated in FIG. 15, the base
condensation polymer layer 1500 includes quaternary amines. The
term x in FIG. 15 may range from about 5 to about 150.
[0093] Base layer 1500 can be reacted with a number of reactions
cycles of reagents to form a polyalkylpolyamine condensation
polymer layer. In an embodiment, the number of reaction cycles may
range from 1 to 10, preferably 3 to 10, and more preferably 3 to 6.
Each reaction cycle includes a) a polyalkyldiiamine treatment and
b) a trihaloalkylaryl treatment. After performing the number of
reaction cycles, the polyalkylpolyamine condensation polymer layer
may be reacted with a tertiary amine treatment to convert the
remaining bromomethyl groups to quaternary amines.
[0094] For the first step a) of a first cycle, a second TMHDA 106
can be reacted with pendant halide groups of base polymer layer
1500 to form a first polyalkylpolyamine CRP 1600, as illustrated in
FIG. 16. The quaternary amines of the base condensation polymer
layer 1500 are spaced apart by either a first spacer 1602 or a
second spacer 1604. The first spacer 1602 is derived from TBMB 204
and the second spacer 1604 is derived from TMHDA 106, as shown in
FIGS. 15 and 16.
[0095] For a second step b) of the first cycle, a pendant tertiary
amine 1606 of the first polyalkylpolyamine CRP 1600 can be reacted
with a second TBMB 204 (a second trihaloalkylaryl) to form a first
polyalkylaryl CRP 1700, as illustrated in FIG. 17. The first
polyalkylaryl CRP 1700 is covalently attached to the first
polyalkylpolyamine CRP 1600. The first polyalkylaryl CRP 1700
includes two halide groups per benzyl ring, as illustrated in FIG.
17.
[0096] Now that one reaction cycle of two steps has been performed,
a second cycle of two steps may be performed to create a
hyperbranched structure. For a first step a) of a second cycle, two
halide groups per benzyl ring of the polyalkylaryl CRP 1700 can be
reacted with a third TMHDA 106 to form a second polyalkylpolyamine
CRP 1800, as illustrated in FIG. 18. The second polyalkylpolyamine
CRP 1800 is covalently attached to the first polyalkylaryl CRP
1700.
[0097] For a second step b) of the second cycle, the pendant
tertiary amine groups of the second polyalkylpolyamine CRP 1800 can
then be reacted with a third TBMB 204 to form a second
polyalkylaryl CRP 1900, as illustrated in FIG. 19. The second
polyalkylaryl CRP 1900 is covalently attached to the second
polyalkylpolyamine CRP 1800.
[0098] A third reaction cycle of two steps can be performed with
the second polyalkylaryl CRP 1900. For a first step a) of a third
cycle, two halide groups per benzyl ring of the second
polyalkylaryl CRP 1900 can be reacted with a fourth TMHDA 106 to
form a third polyalkylpolyamine CRP (not shown). The third
polyalkylpolyamine CRP is covalently attached to the second
polyalkylaryl CRP 1900. It should be noted that the structure of
the third polyalkylpolyamine CRP is analogous to the structure of
the second polyalkylpolyamine CRP 1800.
[0099] For a second step b) of the third cycle, the pendant
tertiary amine groups of the third polyalkylpolyamine CRP can then
be reacted with a fourth TBMB 204 to form a third polyalkylaryl CRP
(not shown). The third polyalkylaryl CRP is covalently attached to
the third polyalkylpolyamine CRP (not shown). It should be noted
that the structure of the third polyalkylaryl CRP is analogous to
the structure of the second polyalkylaryl CRP 1900.
[0100] In an embodiment, more than 3 cycles may be performed by
repeating steps a) and b). When the number of reaction cycles are
completed, the polyalkylpolyamine condensation polymer layer may be
reacted with a tertiary amine treatment to convert the remaining
bromomethyl groups to quaternary amines. It should also be noted
that FIGS. 15 to 19 are exemplary and that TMHDA may be substituted
with other polyalkylpolyamines having at least two amine groups and
that TBMB 204 may also be substituted with other
polyhalogenhydrocarbons having three or more halide groups.
[0101] The following will describe chromatography systems suitable
for use with the waveforms that includes a four voltage. FIG. 14
illustrates an embodiment of a chromatography system, which is an
ion chromatography system 1400 that includes a pump 1402, an
electrolytic eluent generating device 1403, a degas assembly 1410,
an injection valve 1412, a chromatography separation device 1414, a
detector 1416, and a microprocessor 1418. A recycle line 1420 may
be used to transfer the liquid from an output of detector 1416 to a
regenerant channel of degas assembly 1410. The degas assembly may
also be in the form of a vacuum degasser.
[0102] Pump 1402 can be configured to pump a liquid from a liquid
source and be fluidically connected to electrolytic eluent
generating device 1403. Electrolytic eluent generating device 1403
is configured to generate an eluent such as for example NaOH or
methanesulfonic acid. Details regarding electrolytic eluent
generating devices (e.g., eluent generator) can be found in U.S.
Pat. Nos. 6,225,129 and 6,682,701. In an embodiment, a residual gas
such as carbon dioxide, hydrogen, and oxygen may be generated or be
present in the eluent. This gas can be swept out with degas
assembly 1410 using a recycled liquid via a recycle line 1420 that
is downstream of detector 1416. Injection valve 1412 can be used to
inject an aliquot of a sample into an eluent stream. Chromatography
separation device 1414 (e.g., ion exchange chromatography column)
can be used to separate various matrix components present in the
liquid sample from the analytes of interest. An output of
chromatography separation device 1414 can be fluidically connected
to detector 1416 to measure the presence of the separated chemical
constituents of the liquid sample. Chromatography separation device
1414 can separate one or more analytes of a sample that is
outputted from chromatography separation device 1414 at different
retention times.
[0103] Detector 1416 can be in the form of an electrochemical
detector that includes a flow channel configured to be in fluidic
contact with at least a working electrode, a reference electrode,
and optionally a counter electrode. Details regarding an
electrochemical detector flow cell and a disposable working
electrode can be found in U.S. Pat. Nos. 8,925,374; 8,342,007; and
6,783,645, which are hereby fully incorporated by reference herein.
The electrochemical detector also includes a potentiostat or an
analytic device to apply a voltage waveform across the working
electrode and reference electrode, and optionally passes a current
between the counter electrode and working electrode. Details
regarding an analytic device to apply a quadruple voltage waveform
can be found in U.S. Pat. No. 8,636,885, which is hereby fully
incorporated by reference herein.
[0104] An electronic circuit may include microprocessor 1418 and a
memory portion. Microprocessor 1418 can be used to control the
operation of chromatography system 1400. Microprocessor 1418 may
either be integrated into chromatography system 1400 or be part of
a personal computer that communicates with chromatography system
1400. Microprocessor 1418 may be configured to communicate with and
control one or more components of chromatography system such as
pump 1402, electrolytic eluent generating device 1403, injection
valve 1412, and detector 1416. Memory portion can contain
instructions on the magnitude, polarity, and timing for how to
apply one or more voltage waveforms. In terms of measurement,
memory portion can also contain instructions regarding which time
periods to sample current values to integrate the signal and/or
measuring a total charge for a particular time period.
Example 1--Synthesis of the Anion Exchange Resin Using
Pentamethyldipropyltriamine and 1,4-Diaminobutane
[0105] A 4.times.250 mm (diameter.times.length) chromatography
column was packed with 6.5 .mu.m diameter particles with surface
sulfonated (one hour at room temperature) 20.8 m.sup.2/g wide-pore
(DVB/EVB). The base condensation layer was applied to a packed
column by flowing a pentamethyldipropyltriamine (PMDPTA):
1,4-diaminobutane (DBB) solution mixture (1:1 mole ratio in 20%
methanol/80% acetonitrile, 50% (wt/wt %) with respect to PMDPTA:
38% (wt/wt %) with respect to DBB) at 0.5 mL/minute through the
column at 70.degree. C. for 90 minutes to form a base condensation
polymer 300 (see FIG. 3). Next, 4 cycles of reagent treatment were
flowed at 0.5 mL/minute through the column at 70.degree. C. A
single cycle of reagent treatment included a first step a) 38%
(wt/wt %) DBB solution in 20% methanol/80% acetonitrile was flowed
through the column for 60 minutes to form a first alkyl
condensation reaction product 400 (see FIG. 4), and a second step
b) 50% (wt/wt %) PMDPTA solution in 20% methanol/80% acetonitrile
was flowed through the column for 60 minutes to form a first
polyalkylpolyamine condensation reaction product 500 (see FIG. 5).
After completing the first cycle of reagent treatment (steps a) to
b)), three additional cycles of reagent treatment were
performed.
Example 2--Synthesis of Pentamethyldihexyltriamine (PMDHTA)
[0106] Formic acid (150 mL) was added dropwise to a mixture of
bis(hexamethylene)triamine (86 grams) and formaldehyde solution
(37%, 223 mL) at 65.degree. C. during a one hour time period. The
reaction was allowed to continue for an additional three hours.
After cooling the reaction to ambient temperature, sodium hydroxide
(solid, flake, purity: 97%) was added until the pH was 14. The
reaction mixture was extracted with diethyl ether, washed with
brine (saturated NaCl solution), and then dried with magnesium
sulfate. After the volatiles were removed with a rotary evaporator,
the residue was purified by Kugelrohr distillation at 1.2 mm Hg and
140.degree. C. to yield 63 grams of PMDHTA.
Example 3--Synthesis of the Anion Exchange Resin Using
Pentamethyldihexyltriamine and 1,4-Diaminobutane
[0107] 6.2 grams of super macroporous resin particles were used
that have a surface sulfonation (one hour at room temperature), 6.5
.mu.m diameter, and 20.8 m.sup.2/g wide-pore (DVB/EVB). The resin
was rinsed with isopropyl alcohol (IPA) to remove water. The rinsed
resin was transferred to a vial. Next, the following ingredients
were added to the vial, which were 6.0 grams of IPA, 2.9 grams
PMDHTA, and 2 grams DBB. The vial with added ingredients was heated
to 70.degree. C. and allowed to react for 165 minutes. The reacted
ingredients from the vial formed the base condensation layer
attached to the resin particles, which were filtered and then
washed with IPA.
[0108] Next, the filtered particles with the base condensation
layer were added to a vial where three cycles of reagent treatment
were performed at 70.degree. C. A single cycle of reagent treatment
included a first reaction step a) 6 grams of IPA and 2 grams DBB
were added to the vial containing the filtered particles and
allowed to react for 120 minutes at 70.degree. C. After the
reaction, the contents of the vial were filtered and washed with
IPA. The second reaction step b) 2 grams of IPA and 1.5 grams
PMDHTA were added to the vial containing the filtered particles of
step a) and allowed to react for 120 minutes at 70.degree. C. After
the reaction, the contents of the vial were filtered and washed
with IPA. After completing the first cycle of reagent treatment
(steps a) to b)), two additional cycles of reagent treatment were
performed that resulted in a polyalkylpolyamine layer being bound
to the base condensation layer.
[0109] Next, the filtered particles with the polyalkylpolyamine
layer were added to a vial so that any remaining tertiary amines
can be quaternized. 2 grams of IPA and 1.5 grams methyliodide was
added to the vial containing the filtered particles and allowed to
react for 120 minutes at 70.degree. C. After the reaction, the
contents of the vial were filtered and washed with IPA, water, and
1 M NaOH. The filtered and washed particles were packed into a
chromatography column.
Example 4--Synthesis of the Anion Exchange Resin Using
N,N,N',N'-Tetramethyl-1,6-hexanediamine and
1,3,5-Tri(bromomethyl)benzene
[0110] 6.4 grams of super macroporous resin particles were used
that have a surface sulfonation (one hour at room temperature), 6.5
.mu.m diameter, and 20.8 m.sup.2/g wide-pore (DVB/EVB). The resin
was rinsed with a solvent mixture (20% methanol/80% acetonitrile)
to remove water. The rinsed resin was transferred to a vial. Next,
6.0 grams of the solvent mixture and 1.35 grams of
1,3,5-tri(bromomethyl)benzene (TBMB) were added to the vial and
heated in a capped vial at 50.degree. C. in an oven for 10 minutes
to dissolve the TBMB. 0.65 grams of
N,N,N',N'-tetramethyl-1,6-hexanediamine (TMHDA) was dissolved in
another vial using 1.5 grams of the solvent mixture. The dissolved
TMHDA was added dropwise to the vial containing TBMB while
stirring. The vial was then capped and allowed to react at
50.degree. C. for 30 minutes. After the reaction, the contents of
the vial were filtered and washed with solvent mixture to yield
resin particles with a base condensation layer.
[0111] Next, the filtered particles with the base condensation
layer were added to a vial where three cycles of reagent treatment
were performed at 50.degree. C. A single cycle of reagent treatment
included a first reaction step a) 3 grams of the solvent mixture
(20% methanol/80% acetonitrile) and 0.9 grams TMHDA were added to
the vial containing the filtered particles and allowed to react for
30 minutes at 50.degree. C. After the reaction, the contents of the
vial were filtered and washed with the solvent mixture. The second
reaction step b) 3 grams of the solvent mixture and 0.9 grams TBMB
were added to the vial containing the filtered particles of step a)
and allowed to react for 30 minutes at 50.degree. C. After the
reaction, the contents of the vial were filtered and washed with
the solvent mixture. After completing the first cycle of reagent
treatment (steps a) to b)), two additional cycles of reagent
treatment were performed that resulted in a polyalkylpolyamine
layer bound to the base condensation layer.
[0112] Next, the filtered particles with the polyalkylpolyamine
layer were added to a vial so that any remaining bromomethyl groups
can be reacted. 2 grams of the solvent mixture and 1.5 grams of 33%
trimethylamine in ethanol was added to the vial containing the
filtered particles and allowed to react overnight at ambient
temperature. After the reaction, the contents of the vial were
filtered and washed with the solvent mixture, water, and 1 M NaOH.
The filtered and washed particles were packed into a chromatography
column.
Example 5--Chromatograms of a Standard Solution Containing Various
Carbohydrates Using a Range of Eluent Concentrations with Anion
Exchange Resin Based on PMDHTA and DBB
[0113] The chromatography column of Examples 3 was installed into a
Thermo Scientific Dionex ICS-5000.sup.+ ion chromatography system
(commercially available from Thermo Fisher Scientific, Sunnyvale,
Calif.) with a format similar to FIG. 14. A pump was used to pump
deionized water into a Thermo Scientific Dionex EGC 500 NaOH
cartridge (Thermo Fisher Scientific, Sunnyvale, Calif.) for
generating a NaOH at one of six different concentrations that were
10, 20, 30, 40, 50, and 60 mM NaOH. A temperature regulator was
used to maintain a column temperature of 30.degree. C. The flow
rate was 1 mL/min and the injection volume was 25 .mu.L. The
detector was a Thermo Scientific electrochemical detector fitted
with disposable PTFE gold electrodes. The detector was operated in
the integrated pulsed amperometric mode using a quadruple waveform.
Table 1 shows the applied potentials and time durations of the
quadruple voltage waveform.
TABLE-US-00001 TABLE 1 Time (s) Potential (V) Integration 0.00 0.10
0.20 0.10 Start 0.40 0.10 End 0.41 -2.00 0.42 -2.00 0.43 0.60 0.44
-0.10 0.50 -0.10
[0114] FIG. 8 illustrates six chromatograms where each one uses a
different NaOH eluent concentration. The chromatograms were
performed using an anion exchange resins that include PMDHTA and
DBB and a standard solution containing five different
carbohydrates, which were dulcitol (peak 1 at 10 ppm), mannitol
(peak 2 at 10 ppm), glucose (peak 3 at 10 ppm), fructose (peak 4 at
10 ppm), sucrose (peak 5 at 10 ppm), and lactose (peak 6 at 10
ppm). The dulcitol and mannitol (peaks 1 and 2, respectively) had
relatively constant retention times over the tested range of NaOH
concentration. In contrast, the glucose, fructose, and sucrose
peaks (peaks 3, 4, and 5, respectively) had increasing retention
time with decreasing NaOH eluent concentration. The increase of
retention time for peaks 3-5 each had a different dependence on
eluent concentration so that the peak elution order was different
for 60 mM NaOH and 10 mM NaOH. This change in order of the peaks
3-5 provides an advantage of more flexibility in manipulating the
retention time of the peaks that may be needed to avoid potentially
interfering anions that can be present. The fact that sucrose (pKa
of 12.62) elutes well before fructose (pKa of 12.03) with a 10 mM
NaOH eluent (pH 12) indicates that the stationary phase pH is less
than the pH of the latex-based Thermo Scientific Dionex CarboPac
PA1 stationary phase. When using CarboPac PA1 stationary phase,
Applicant found that fructose and sucrose co-elute under the same
conditions. Such differences in stationary phase pH are useful in
providing alternative separation selectivity in cases where
existing commercial columns cannot separate important mixtures of
carbohydrates at low eluent concentrations (e.g., 10 mM).
Example 6--Chromatograms of Standard Solutions Containing Various
Carbohydrates Using the Anion Exchange Resin Based on PMDHTA and
DBB
[0115] The chromatography column of Example 3 was installed into a
Thermo Scientific Dionex ICS-5000.sup.+ ion chromatography system
(commercially available from Thermo Fisher Scientific, Sunnyvale,
Calif.) with a format and conditions that were similar as Example
5. However, the eluent concentration was held constant at 60 mM
NaOH and an additional standard solution that contained sucrose and
lactose was used.
[0116] FIG. 9 illustrates two chromatograms where the upper
chromatogram used the standard solution of Example 5 and the lower
chromatogram used a standard solution containing sucrose and
lactose (peaks 5 at 10 ppm and peak 6 at 10 ppm, respectively). The
chromatograms were performed using a chromatography column
containing the same anion exchange resin of Example 5. The upper
and lower chromatograms together suggest that all six carbohydrates
can be resolved in one chromatography run. In the lower
chromatogram, it should be noted that sucrose and lactose (peaks 5
and 6, respectively) were fully resolved, which is an advantage in
that these carbohydrates are difficult to resolve under the
chromatographic conditions.
Example 7--Chromatograms of a Standard Solution Containing Various
Carbohydrates Using a Range of Eluent Concentrations with Anion
Exchange Resin Based on TBMB and TMHDA
[0117] The chromatography column of Examples 4 was installed into a
Thermo Scientific Dionex ICS-5000.sup.+ ion chromatography system
(commercially available from Thermo Fisher Scientific, Sunnyvale,
Calif.) with a format similar to Example 5. In contrast to Example
5, only three NaOH concentrations were used instead of six. The
three NaOH concentrations were 10, 30, and 60 mM NaOH.
[0118] FIG. 10 illustrates three chromatograms where each one uses
a different NaOH eluent concentration. The chromatograms were
performed using an anion exchange resins that include TBMB and
TMHDA and a standard solution containing five different
carbohydrates, which were dulcitol (peak 1 at 10 ppm), mannitol
(peak 2 at 10 ppm), glucose (peak 3 at 10 ppm), fructose (peak 4 at
10 ppm), sucrose (peak 5 at 10 ppm), and lactose (peak 6 at 10
ppm). Similar to Example 5, the dulcitol and mannitol (peaks 1 and
2, respectively) had relatively constant retention times over the
tested range of NaOH concentration. Similar to Example 5, the
glucose, fructose, and sucrose peaks (peaks 3, 4, and 5,
respectively) had increasing retention time with decreasing NaOH
eluent concentration. The increase of retention time for peaks 3-5
each had a different dependence on eluent concentration so that the
peak elution order was different for 60 mM NaOH and 10 mM NaOH.
This change in order of the peaks 3-5 provides an advantage of more
flexibility in manipulating the retention time of the peaks that
may be needed to avoid potentially interfering anions that can be
present.
Example 8--Chromatograms of Standard Solutions Containing Various
Carbohydrates Using the Anion Exchange Resin Based on TBMB and
TMHDA
[0119] The chromatography column of Example 4 was installed into a
Thermo Scientific Dionex ICS-5000.sup.+ ion chromatography system
(commercially available from Thermo Fisher Scientific, Sunnyvale,
Calif.) with a format and conditions that were similar as Example
6.
[0120] FIG. 11 illustrates two chromatograms where the upper
chromatogram used the standard solution of Example 5 and the lower
chromatogram used a standard solution containing sucrose and
lactose (peaks 5 at 10 ppm and peak 6 at 10 ppm, respectively). The
chromatograms were performed using a chromatography column
containing the same anion exchange resin of Example 4. The upper
and lower chromatograms together suggest that all six carbohydrates
can be resolved in one chromatography run. In the lower
chromatogram, it should be noted that sucrose and lactose (peaks 5
and 6, respectively) were fully resolved, which is an advantage in
that these carbohydrates are difficult to resolve under the
chromatographic conditions.
Example 9--Chromatograms of Sample Solutions Containing Fetuin
Alditols Using Anion Exchange Resin Based on PMDHTA and DBB
[0121] The chromatography column of Example 4 was installed into a
Thermo Scientific Dionex ICS-5000.sup.+ ion chromatography system
(commercially available from Thermo Fisher Scientific, Sunnyvale,
Calif.) with a format and conditions that were similar as Example
5. A pump was used to pump a mixture of three solutions (A.
deionized water, B. 0.1 M NaOH, and C. 0.1 M NaOH and 0.25 M sodium
acetate) with a gradient elution of 20 to 225 mM sodium acetate at
0.1 M NaOH in 80 minutes. A temperature regulator was used to
maintain a column temperature of 30.degree. C. The flow rate was 1
mL/min and the injection volume was 10 .mu.l of 50 .mu.M fetuin
alditols. The alditols from fetuin were released using the enzyme
PNGase F. The detector was a Thermo Scientific electrochemical
detector fitted with disposable PTFE gold electrodes. The detector
was operated in the integrated pulsed amperometric mode using a
quadruple waveform as described in Table 1 of Example 5.
[0122] FIG. 12 illustrates a chromatogram using the anion exchange
resin that includes PMDHTA and DBB. A sample solution was injected
into a chromatography column that contained fetuin alditols that
resulted in the measurement of 38 peaks. The chromatogram of FIG.
12 showed the utility of using the chromatography column of Example
4 to characterize glycans released from fetuin.
Example 10--Chromatograms of Sample Solutions Containing Fetuin
Alditols Using Anion Exchange Resin Based on TBMB and TMHDA
[0123] The chromatography column of Example 5 was installed into a
Thermo Scientific Dionex ICS-5000.sup.+ ion chromatography system
(commercially available from Thermo Fisher Scientific, Sunnyvale,
Calif.) with a format and conditions that were similar as Example
9. FIG. 13 illustrates a chromatogram using the anion exchange
resin that includes TBMB and TMHDA. A sample solution was injected
into a chromatography column that contained fetuin alditols that
resulted in the measurement of 28 peaks. The chromatogram of FIG.
13 showed the utility of using the chromatography column of Example
5 to characterize glycans released from fetuin.
[0124] While preferred embodiments of the present invention have
been shown and described herein, it will be apparent to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. While the invention has been described in terms of
particular variations and illustrative figures, those of ordinary
skill in the art will recognize that the invention is not limited
to the variations or figures described. In addition, where methods
and steps described above indicate certain events occurring in
certain order, those of ordinary skill in the art will recognize
that the ordering of certain steps may be modified and that such
modifications are in accordance with the variations of the
invention. Additionally, certain of the steps may be performed
concurrently in a parallel process when possible, as well as
performed sequentially as described above. Therefore, to the extent
there are variations of the invention, which are within the spirit
of the disclosure or equivalent to the inventions found in the
claims, it is the intent that this patent will cover those
variations as well.
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