U.S. patent application number 09/878495 was filed with the patent office on 2002-02-14 for surface modification of a porous polymer monolith and products therefrom.
Invention is credited to Huang, Xian.
Application Number | 20020017487 09/878495 |
Document ID | / |
Family ID | 22784713 |
Filed Date | 2002-02-14 |
United States Patent
Application |
20020017487 |
Kind Code |
A1 |
Huang, Xian |
February 14, 2002 |
Surface modification of a porous polymer monolith and products
therefrom
Abstract
A process is provided for modifying a porous polystyrene
monolith to render its internal pore surfaces grafted with alkyl
groups, preferably of at least four carbon atoms, which includes
the step of alkylating the monolith with a uniform liquid solution
containing a Friedel-Crafts catalyst and an alkyl halide. A
surface-modified monolith produced thereby is an efficient
separation medium in reversed-phase liquid chromatography for both
small and large biomolecules.
Inventors: |
Huang, Xian; (Ithaca,
NY) |
Correspondence
Address: |
Michael L. Goldman
NIXON PEABODY LLP
Clinton Square
P.O. Box 31051
Rochester
NY
14603
US
|
Family ID: |
22784713 |
Appl. No.: |
09/878495 |
Filed: |
June 11, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60210890 |
Jun 9, 2000 |
|
|
|
Current U.S.
Class: |
210/635 ;
210/639; 210/656; 436/161 |
Current CPC
Class: |
B01J 20/26 20130101;
B01J 2220/82 20130101; B01J 20/3219 20130101; B01J 20/327 20130101;
B01J 2220/54 20130101; B01J 20/287 20130101; B01J 20/3204 20130101;
B01J 20/3278 20130101; B01J 20/261 20130101; B01J 20/3282 20130101;
Y10T 428/249988 20150401; B01J 20/32 20130101; B01J 20/3276
20130101; B01J 20/3246 20130101; Y10T 428/249987 20150401; B01J
20/285 20130101; B01J 20/3217 20130101; B01D 15/325 20130101; B01J
20/28042 20130101; B01J 20/3208 20130101; B01J 20/3206 20130101;
B01J 20/28097 20130101 |
Class at
Publication: |
210/635 ;
210/639; 210/656; 436/161 |
International
Class: |
B01D 015/08 |
Claims
What is claimed:
1. A process for the surface modification of a porous polystyrenic
monolith, comprising the steps of: (a) wetting the monolith
internal through-pore surfaces with an organic solvent used in step
(b); (b) treating the internal through-pore surfaces of said
monolith by contacting said monolith pores with a uniform liquid
solution comprising a Friedel-Crafts catalyst, an alkyl halide, and
an organic solvent so as to alkylate the internal through-pore
surfaces; and (c) removing the post-reaction solution from the
alkylated through-pore surfaces by rinsing the organic solvent used
in step (b) through said monolith.
2. The process of claim 1, further comprising washing said
alkylated through-pore surfaces by sequentially rinsing a series of
solvents through said monolith.
3. The process of claim 1, wherein said treating of the internal
through-pore surfaces is performed with heating.
4. The process of claim 1, wherein said wetting of the monolith
internal through-pore surfaces comprises rinsing said organic
solvent through said monolith.
5. The process of claim 1, wherein said contacting of said monolith
through-pores with a uniform liquid solution comprises filling said
monolith with said uniform liquid solution and then rinsing said
uniform liquid solution through said monolith.
6. The process of claim 1, wherein said porous monolith comprises a
cross-linked polystyrene-based copolymer.
7. The process of claim 1, wherein said porous monolith comprises
poly(styrene-co-divinylbenzene).
8. The process of claim 1, further comprising preparing said porous
polystyrenic monolith in situ in a tube prior to said
alkylation.
9. The process of claim 1, wherein said monolith is contained in a
tube.
10. The process of claim 9, wherein said tube comprises a metal
tube, plastic tube, or capillary tube.
11. The process of claim 1, wherein said tube comprises a microchip
made from silicon, plastic, or glass.
12. The process of claim 1, wherein said monolith is a molded in a
fused silica capillary.
13. The process of claim 1, wherein said monolith is contained
within a rigid micro-opening of a silicon or polymer chip
comprising a microfluidic separation device.
14. The process of claim 1, wherein said Friedel-Crafts catalyst is
aluminum chloride (AlCl.sub.3), aluminum bromide (AlBr.sub.3), or
antimony pentachloride (SbCl.sub.5).
15. The process of claim 1, wherein said alkyl halide is a primary,
secondary, or tertiary chloride or bromide having at least four
carbon atoms.
16. The process of claim 1, wherein said alkyl halide has 4, 8, or
18 carbon atoms.
17. The process of claim 1, wherein said alkyl halide is linear,
branched, cyclic, or combination thereof.
18. The process of claim 1, wherein said alkyl halide is octadecyl
chloride.
19. The process of claim 1, wherein said solvent is nitrobenzene
(C.sub.6H.sub.5NO.sub.2) or nitromethane (CH.sub.3NO.sub.2), or
mixture thereof.
20. The process of claim 1, wherein said solvent is capable of
dissolving said Friedel-Crafts catalyst and is miscible in said
alkyl halide.
21. The process of claim 2, wherein said series of solvents are
N,N-dimethylformamide, 1 M HCl aqueous solution, water, and
acetonitrile, respectively.
22. The process of claim 1, wherein said wetting and rinsing are
performed under pressure.
23. The product produced by the process of claim 1.
24. A porous polystyrenic monolith comprising alkylated internal
through-pore surfaces.
25. The product of claim 24, wherein said porous monolith comprises
a chromatography media.
26. The product of claim 24, wherein said porous monolith is a
cross-linked polystyrene-based copolymer.
27. The product of claim 24, wherein said porous monolith comprises
poly(styrene-co-divinylbenzene).
28. The product of claim 24, wherein said porous monolith is in
situ prepared in a tube.
29. The product of claim 24, wherein said monolith is contained in
a tube.
30. The product of claim 29, wherein said tube comprises a metal
tube, plastic tube, or capillary tube.
31. The product of claim 29, wherein said tube comprises a
microchip made from silicon, plastic, or glass.
32. The product of claim 24, wherein said monolith is a molded in a
fused silica capillary.
33. The product of claim 24, wherein said monolith is contained
within a rigid micro-opening of a silicon or polymer chip
comprising a microfluidic separation device.
34. The product of claim 24, wherein said alkylated pore surfaces
comprise alkyl groups having at least four carbon atoms.
35. The product of claim 34, wherein said alkyl group has 4, 8, or
18 carbon atoms.
36. The product of claim 34, wherein said alkyl group is linear,
branched, cyclic, or combination thereof.
37. The product of claim 34, wherein said alkyl group is octadecyl.
Description
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 60/210,890, filed Jun. 9, 2000, which
is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to a process for the surface
modification of a porous polymer monolith and products produced
therefrom. In particular, the process relates to the surface
modification of a polystyrenic monolith by alkylating its
through-pore surfaces and to products so modified.
BACKGROUND OF THE INVENTION
[0003] Liquid chromatography, as a technique for the separation of
soluble, non-volatile compounds, has become over the past 30 years
an indispensable tool for chemical and biochemical analyses in
numerous disciplines of chemistry and life sciences. Generally,
separation in liquid chromatography is achieved in a column by
selective distribution of the sample molecules between a stationary
phase and a mobile phase. In reversed-phase liquid chromatography,
the stationary phase is usually highly hydrophobic or non-polar.
Conventional reversed-phase liquid chromatography uses 5-10 .mu.m
spherical silica beads that have been modified by covalent
attachment of hydrocarbon chains including 4, 8, or 18 carbon atoms
to provide a non-polar surface.
[0004] In addition to silica beads, PS-DVB particles are also
widely used as a stationary phase support. Actually, the PS-DVB
surface can be directly used as the reversed-phase chromatography
stationary phase since it is highly hydrophobic. Maa and Horvth, J.
Chromatography, 445 (1988), 71-86, disclose that PS-DVB particles
are very effective for the rapid analysis of proteins in the
reversed-phase mode. However, for separation and identification of
smaller biomolecules like peptides, which have become more crucial
to the new emerging field of proteomics, the unfunctionalized
PS-DVB particles may present unacceptable poor resolution. It has
been shown by Huber et al., Chromatographia, 44 (7/8) (1997),
438-448, that alkylation of PS-DVB particles to graft octadecyl
chains on their surface is necessary to achieve good resolution for
reversed-phase liquid chromatography of peptides.
[0005] The increasing demand for more efficient and rapid
separations in many areas especially for the pharmaceutical
industry has initiated research towards column consolidation and
miniaturization. In recent years, column consolidation has been
achieved as a result of the introduction or invention of porous
polymer continuous beds or monoliths. Hjertn, J. Chromatography,
473 (1989), 273-275 introduces a polymer gel continuous bed
prepared by in situ polymerization of an aqueous solution of
acrylamide derivatives. Svec and Frchet disclosed in 1994 and 1995
(U.S. Pat. Nos. 5,334,310 and 5,453,185) a continuous liquid
chromatographic column containing a separation medium in the form
of a macroporous polymer plug. The column miniaturization has also
been achieved by a porous polymer monolith prepared by free radical
polymerization in situ in a fused silica capillary. The development
of fritless columns with a polymer-based porous monolith rather
than conventional spherical beads has become more and more
important since it meets the requirement of today's micro-scale
liquid chromatography and capillary electrochromatography as
described by Liao and Hjertn (1997 U.S. Pat. No. 5,647,979); Peters
et al., Analytical Chemistry, 70 (1998), 2288-2295; Gusev et al.,
J. Chromatography A, 855 (1999), 273-290; and Zhang et al., J.
Chromatography A, 887 (2000), 465-477.
[0006] Practicable methods for preparing a PS-DVB monolith have
been published. The PS-DVB monolith has become an acceptable
consolidated column packing material. However, the art lacks a
method to effectively impart alkyl chains onto the through-pore
surfaces of a PS-DVB monolith so as to provide an effective column
packing capable of enhancing the resolution of peptides in
reversed-phase liquid chromatography.
[0007] Several methods have been proposed to introduce alkyl
functional groups to the polymer monolith. In one method, the alkyl
groups are directly imparted from a co-monomer, e.g., alkylene
dimethacrylate, rather than divinylbenzene used for the initial
polymerization as claimed in U.S. Pat. No. 5,453,185 (Svec and
Frchet). However, the introduction of a new monomer to the initial
polymerization mixture may require redesign of the formulation and
conditions including the selection of a new porogen. Moreover, only
those alkyl chains in the polymer surfaces are needed for the
separation, while the alkyl chains involved inside the bulk solid
support are not necessary. It has been found that alkyl chains
imparted by this process do not appreciably improve the resolution
of peptides. Thus, this method for alkylating the monolith surfaces
forms an ineffective stationary phase for separation of relatively
smaller biomolecules like peptides.
[0008] The reaction of benzene with "amyl chloride" in the presence
of aluminum chloride to produce "amylbenzene" was carried out by
Charles Friedel and James Mason Crafts in 1877. For over a century,
Friedel-Crafts alkylation chemistry has been one of the most
interesting aspects of modern organic theory (R. M. Roberts and A.
A. Khalaf, "Friedel-Crafts Alkylation Chemistry", Mercel Dekker,
Inc., New York, 1984). Major processes for the production of
high-octane gasoline, synthetic rubber, plastics, and synthetic
detergents are applications of Friedel-Crafts chemistry.
[0009] The Friedel-Crafts reaction has also been adopted for the
surface modification of PS-DVB particles or beads to form
reversed-phase column packing materials as disclosed by Huber et
al., Analytical Biochemistry, 212 (1993), 351-358. According to the
Huber et al. process, the solid aluminum chloride is directly added
to the suspension of PS-DVB particles in an alkyl chloride
(1-chlorooctadecane). No other solvent was added during the
multiphase reaction. Since the reactantion is controlled by
diffusion, the size of the particles which can be modified is
limited.
[0010] That process is not suitable for alkylating the through-pore
surfaces of a porous PS-DVB monolith. First, the solid state
catalyst is difficult to introduce into the internal pores of a
monolith since the solid catalyst is not very soluble in the
alkylating solution. Second, the internal pores of the monolith may
become clogged with precipitated solid that remains inside the
monolith during the Friedel-Crafts reaction. Although there are a
few liquid-state Friedel-Crafts catalysts, these are not suitable
for this purpose. For example, tin(IV) chloride is a liquid and is
easily filled into the monolith porous structure, but it may
precipitate insoluble substances during the Friedel-Crafts reaction
and it is a very weak catalyst as well. Unlike a free benzene ring
which can be easily alkylated in a few minutes through the
Friedel-Crafts reaction at below room temperature, a polymer-based
benzene ring is much more difficult to alkylate since the
Friedel-Crafts reaction is controlled by diffusion. Heating is
preferred for speeding the Friedel-Crafts reaction on a PS-DVB
surface.
[0011] It would be desirable, therefore, to develop a process that
can be used for alkylating through-pore surfaces of a porous PS-DVB
monolith as the one-piece packing material of a liquid
chromatographic column. A strong catalyst solution is needed.
SUMMARY OF THE INVENTION
[0012] One aspect of the present invention relates to a process for
the surface modification of a porous polystyrenic monolith. The
process includes wetting the monolith internal pore (through-pore)
surfaces with an organic solvent used in the uniform liquid
solution. The pore surfaces of the monolith are treated by
contacting the monolith pores with a uniform liquid solution
containing a Friedel-Crafts catalyst, an alkyl halide, and an
organic solvent so as to alkylate the internal pore surfaces. The
post-reaction solution is removed from the alkylated pore surfaces
by rinsing the organic solvent through the monolith. Preferably,
the alkylated pore surfaces are further washed by sequentially
rinsing a series of solvents through the monolith,
respectively.
[0013] Another aspect of the present invention relates to a porous
polystyrenic monolith having alkylated internal pore surfaces.
[0014] The present invention results in a number of advantages over
the prior art. It is an advantage of the present invention to
provide a simple and reliable process for alkylating internal pore
surfaces of a porous polystyrenic monolith. The monolith to be
alkylated may be already molded in a fused-silica capillary, a
plastic tubing, a micro-channel or hole of a silicon/polymer chip,
or a similar opening in a separation device. The monolith is
preferably a cross-linked polystyrene copolymer having internal
through-pores that a liquid can pass through.
[0015] It is a further advantage of the present invention to
provide surface-alkylated porous PS-DVB monoliths as consolidated
and miniaturized columns for liquid chromatographic separation,
especially in the reversed-phase mode. Advantages of this invention
include the improved separation and identification of peptides by
using such alkylated monolithic columns in reversed-phase liquid
chromatography hyphenated with electrospray ionization mass
spectrometry.
[0016] Other advantages of the present invention will be apparent
to those skilled in the art from the following description and the
appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is the total ion current (TIC) profile from the
reversed-phase LC-ESI/MS of a tryptic digest cytochrome c described
in Example 2. The peaks representing different peptide fragments
(T1-12) are labeled. The capillary column having an alkylated
porous PS-DVB monolith was produced in Example 1.
[0018] FIG. 2 is the TIC profile of the tryptic digest cytochrome c
from FIG. 1 and the extracted ion current profiles for the selected
peptide fragments.
[0019] FIG. 3 is the reversed-phase LC-ESI/MS of the tryptic digest
cytochrome c using the same conditions as for FIG. 1 with the
unmodified monolithic PS-DVB column described in Example 2.
[0020] FIG. 4 is the reconstructed full-scan LC-ESI/MS total ion
current (TIC) profile and extracted ion current profiles from the
analysis of the synthetic peptide mixture described in Example 4.
The capillary column having the alkylated porous PS-DVB monolith
was produced in Example 3.
[0021] FIG. 5 is the mass spectra related to the chromatograms of
FIG. 4 and described in Example 4.
[0022] FIG. 6 is a cross-section view of the monolith showing
liquid forced through the monolith molded in a chip with a liquid
delivery probe in Example 5.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention provides a process for alkylating
outer and internal (through-pore) surfaces of a porous polystyrene
monolith using the Friedel-Crafts alkylation reaction. The process
is preferably effective on a highly cross-linked porous polystyrene
monolith. The process includes the step of treating internal pore
surfaces of the monolith, preferably, by filling them and then
rinsing a uniform liquid solution containing a Friedel-Crafts
catalyst, an alkyl halide, and an organic solvent through the
monolith. Such a process was made possible by the discovery of the
formulation of a suitable uniform liquid alkylating reagent
solution. Preferably, a specific organic solvent is used. In this
solvent, both the selected highly strong Friedel-Crafts catalyst
and an alkyl halide (e.g., linear and primary octadecyl chloride)
can be dissolved and a uniform liquid solution can be formed. By
using the present process the alkylation reaction can be completed
inside the monolith through-pores which eliminates clogging
problems while the pore surfaces can be grafted with the desired
alkyl groups. The process also includes a subsequent washing step
in which the post-alkylation mixture is removed and the
functionalized polystyrenic through-pore surfaces are cleaned
without precipitation or clogging.
[0024] In a preferred embodiment of the present invention, a
uniform liquid alkylating reagent solution is formulated for
treating a porous polymer monolith. The monolith is a
polystyrene-based copolymer, preferably,
poly(styrene-co-divinylbenzene) (PS-DVB) with a molecular ratio of
about 10% to about 50% divinylbenzene. The internal pore size
distribution and the porosity can be varied with the processes by
which the monolith is prepared. Examples of such polystyrenic
monoliths include those covered by U.S. Pat. No. 5,334,310 and No.
5,334,310 (Frchet and Svec) and introduced by J. Chromatography A,
855 (1999), 273290 (Gusev et al.) and J. Chromatography A, 887
(2000), 465-477 (Zhang et al.), which are each herein incorporated
by reference in their entirety. The processes for preparing such
monoliths have been modified at Advion BioSciences, Inc. (formerly
Advanced BioAnalytical Services, Inc., Ithaca, N.Y.) based on the
company's licensed US Patents and other published literature, which
monoliths are commercially available from this company.
[0025] The porous polystyrenic monolith can be prepared in situ in
a tube or open ended structure. Suitable tubes include metal tubes,
plastic tubes, capillary tubes, and the like. Also suitable are
microchips made from silicon, plastic, glass, and the like.
Preferably, the diameter of the tube is about several millimeters
or less. For example, a PS-DVB monolith covalently bonded in a
fused silica capillary can be prepared by first silanizing the
internal wall of the capillary with the method introduced by Huang
and Horvth, Journal of Chromatography A, 788 (1997) 155-164. In
accordance with the present invention, the PS-DVB monolith is
prepared in situ inside a pretreated and silanized fused silica
capillary having an inner diameter, preferably the commercially
available sizes of about 50 .mu.m or about 75 .mu.m. In the initial
polymerizing solution, the monomer ratio is 40% (v/v) and the
porogen is the co-solvents 1-propanol and formamide. The initiator
used is 2'2-azobisisobutyronitril- e (AIBN).
[0026] With the same polymerization solution, the PS-DVB monolith
is also prepared in situ in commercially available PEEK tubing or
stainless steel tubing. In this case, the monolith is not
covalently bonded onto the inner wall of the PEEK or stainless
steel capillary. It is remarkable that a 10 cm long PEEK or
stainless steel column (125 .mu.m I.D., {fraction (1/16)}" O.D.)
containing such polymer monolith is mechanically stable under a
back pressure as high as 200 bar delivered by acetonitrile
flow.
[0027] Prior to alkylation, the through-pore surfaces of the porous
polystyrenic monolith are wetted or swelled by the solvent that is
used to make the uniform liquid catalyst solution. Preferably the
monolith is washed sequentially with several organic solvents, such
as, methylene chloride, N,N-dimethylformamide, and nitrobenzene or
nitromethane. The final pre-wash not only improves surface wetting
characteristics but is also preferably compatible with the
subsequent alkylating solution. Washing with the initial solvents
enables the relatively quick loading of the desired solvent prior
to loading the alkylating solution. Preferably, the solvent for the
final pre-wash, nitrobenzene or nitromethane, is also the solvent
for preparing alkylating solution. In rinsing with the solvent, the
flow is driven through the monolith, usually by pressure. This step
is preferably completed at room temperature.
[0028] A mixture of a Friedel-Crafts catalyst and an alkyl halide
is formulated for use with the alkylating process. Suitable
Friedel-Crafts catalysts include those that can be dissolved in the
solvent and are strong enough to achieve suitable alkylation. As a
rule, the mixture is preferably prepared as a uniform liquid from
the Friedel-Crafts catalyst and alkyl halide selected. To guarantee
a strong alkylating reaction under mild conditions, the
Friedel-Crafts catalysts are preferred to be as strong as possible.
Preferred are those commercially available strongest Friedel-Crafts
catalysts such as aluminum chloride (AlCl.sub.3), aluminum bromide
(AlBr.sub.3) and antimony pentachloride (SbCl.sub.5).
[0029] Suitable solvents include those solvents that can dissolve
the Friedel-Crafts catalyst and the alkyl halide and form a uniform
liquid reactant. Nitromethane and nitrobenzene can dissolve most
alkyl halides, the commercially available strongest Friedel-Crafts
catalysts, and are most preferred. For example, nitrobenzene can
dissolve an 18-carbon halide such as octadecyl chloride. With
nitromethane or nitrobenzene as the solvent, a uniform liquid
solution containing the catalyst and the alkyl halide can be formed
from a wide variety of catalyst and alkyl halide combinations.
Suitable high concentrations of the catalyst and alkyl halide can
be preferably chosen to avoid precipitation during the
treatment.
[0030] Preferred alkyl halides include primary, secondary, or
tertiary chloride or bromide. The alkyl groups include linear,
branched, cyclic chains, or combinations thereof. For
reversed-phase chromatography, hydrophobic groups preferably having
4, 8, or 18 carbon atoms are chosen for the stationary phase.
Additionally preferred alkyl halides for preparing the uniform
liquid alkylating solution include linear primary chloride or
bromide having 4, 8, or 18 carbon atoms.
[0031] The internal pore surfaces of the porous polystyrenic
monolith are filled with the uniform liquid alkylating solution
described above and then the solution is rinsed through the
monolith. Although theoretically the benzene ring can be alkylated
under room temperature, gentle heating is preferred to speed and
enhance the diffusion-dominated reaction with the polymer surfaces.
The preferred reaction temperature for the alkylating solution
contacting the pore surfaces, is from about 45.degree. C. to about
70.degree. C. Typically, the heating step can take several hours or
longer.
[0032] The reaction solution is then removed from the monolith. A
post-alkylation wash is preferred for making highly cleaned
alkylated monoliths. The risk for pore-clogging is high if the
wrong solvent is used, since the residual alkylating mixture can
produce solid precipitates or a highly viscous liquid. A preferred
solvent for the post reaction wash is the same solvent as that used
for preparing the alkylation solution, e.g., nitrobenzene or
nitromethane, which removes the post-alkylation solution from the
monolith and rinses the alkylated pores by being passed through the
monolith. Typically, this wash may not completely remove the
residual mixture in the pores. An additional wash is preferably
sequentially applied, for example, with N,N-dimethylformamide, 1 M
HCl aqueous solution, water, and acetonitrile, respectively.
[0033] The following examples illustrate several embodiments of the
present invention. However, the invention should not be limited to
the embodiments illustrated.
EXAMPLE 1
[0034] This example illustrates a process for octadecylating a
porous PS-DVB monolith formed in situ in a PEEK capillary.
[0035] A PEEK capillary (internal diameter 0.005-in or 125 .mu.m,
outer diameter {fraction (1/16)}-in, and length 10 cm) containing a
PS-DVB monolith was used for the alkylation. Specifically, the
monolith was prepared from heating the solution sealed inside the
capillary containing 20% v/v styrene, 20% v/v DVB (80%, mixture of
isomers), 40% v/v 1-propanol, 20% v/v formamide, and 0.3% w/v
2'2-azobisisobutyronitrile (AlBN), for 24 hours at 70.degree. C.
After the residual mixture was removed, the porous monolith was
thoroughly washed with methylene chloride and
N,N-dimethylformamide.
[0036] A screw top glass vial (1.5- to 3-ml) having an open-top cap
with a Teflon-faced plastic septum was used for delivering liquid
into the monolithic column. Typically, a liquid contained in the
vial was pressurized by a nitrogen source of 60-100 psi introduced
with a fused silica capillary inserted through the septum.
[0037] Each end of the monolithic column was extended with a fused
silica capillary by using a connection union. With one end (the
fused silica capillary) inserted into the capped vial containing
1.0 ml nitrobenzene, the column was rinsed with nitrobenzene for 1
hour before the alkylation.
[0038] 25 mg of aluminum chloride powder was put into another screw
top glass vial. 0.5 ml of nitrobenzene was added. After stirring
for a few minutes, 0.5 ml of 1-chlorooctadecane (liquid) was added.
A uniform solution was formed after further stirring. The vial was
then closed using the open-top cap with the septum. In accordance
with the procedure described above, the column was rinsed with the
prepared solution for about 1 hour with an inlet pressure from 60
to 100 psi. The column was then filled with the same solution and
sealed at both ends to prevent leakage. Subsequently, the column
was placed in an oven and heated at 60.degree. C. for 12 hours.
[0039] The post-alkylation mixture was removed by pressurized
nitrobenzene liquid. The alkylated column was finally washed
sequentially with nitrobenzene, N,N-dimethylformamide, 1 M HCl
aqueous solution, water, and acetonitrile.
EXAMPLE 2
[0040] This example illustrates a typical application of the
monolithic capillary column prepared from Example 1. Peptides from
a tryptic digest of cytochrome c were separated and analyzed by
LC-ESI/MS using the octadecylated monolithic column.
[0041] The octadecylated PS-DVB monolithic column (125 .mu.m in
inner diameter and 10 cm in length) produced from Example 1 was
used as a reversed-phase liquid chromatographic column. The column
was attached to a Micromass LCT mass spectrometer with a tapered
fused silica capillary (tip end inner diameter, 10 .mu.m,
flame-pulled from a fused silica capillary having 150 .mu.m outer
diameter and 50 .mu.m inner diameter) as the electrospray
interface. A micro gradient pump, Eldex MicroPro 1000 syringe
pumping system, was used to deliver the mobile phase to the column.
In the mobile phase flow line, a Valco micro-electric two position
valve actuator with 1 .mu.L injection volume was connected after
the pump. A split valve was connected after the sample injector and
right before the column inlet, which split {fraction (1/100)} of
the main flow into the column while the remainder went into the
waste. All connection capillaries were nonconductive fused silica
capillaries. The mobile phase flow rate before the split was
typically 30 .mu.L/min. The flow rate for the column was maintained
at 300 nL/min. The applied high voltage for the electrospray
ionization was 3.5 kV. Peptide mass spectra were recorded in the
range of 380 to 1700 m/z.
[0042] A Sigma standard protein, cytochrome c, was digested in the
presence of trypsin under denaturating conditions with 7 M urea.
The sample mixture of the tryptic digest for the LC-ESI/MS test
contained 70 pmol/.mu.l each of the fragments (peptides) (injected
before the 1:100 split valve).
[0043] The separation results are presented as the chromatogram and
the mass spectra shown in FIGS. 1 and 2. The mobile phases for the
gradient elution were: A=0.1% v/v acetic acid and 0.01% v/v
heptafluorobutyric acid in water; and B=0.1% v/v acetic acid and
0.01% v/v heptafluorobutyric acid in acetonitrile. The gradient
elution was programmed as: 0.fwdarw.10.fwdarw.15 min; 5%.fwdarw.40%
.fwdarw.70% B (i.e., acetonitrile concentration
5%.fwdarw.40%.fwdarw.70% v/v).
[0044] For comparison, prior to the octadecylation (described in
Example 1) the monolithic PS-DVB column was tested with the same
sample and experimental conditions (see the description for FIG.
1). The separation results are shown in FIG. 3. By the comparison
of FIG. 3 with FIG. 1 it is shown that the PS-DVB stationary phase
without octadecyl groups provided very poor resolution for the
reversed-phase chromatographic separation of peptides from the
cytochrome c digest.
EXAMPLE 3
[0045] This example illustrates a process for octadecylating a
porous PS-DVB monolith covalently bonded in a fused silica
capillary.
[0046] A fused silica capillary (inner diameter 75 .mu.m, outer
diameter 375 .mu.m, and length 20 cm) containing covalently bonded
PS-DVB monolith was prepared as described above and used for the
octadecylation.
[0047] The procedure for octadecylating a porous PS-DVB monolith in
a fused silica capillary was the same as that described in Example
1. The removal of post-alkylation mixture and the wash of the
column were operated under relatively higher pressure (100 psi) due
to the smaller column inner diameter.
EXAMPLE 4
[0048] This example illustrates the use of the surface-alkylated
porous PS-DVB monolith in a fused silica capillary prepared from
Example 3. By using the column, standard peptides were separated
and characterized in liquid chromatography hyphenated with
electrospray ionization mass spectrometry (LC-ESI/MS).
[0049] The monolithic capillary column with octadecyl groups as the
stationary phase produced from Example 3 was cut to 10 cm in
length. The testing system and procedure were the same as that of
Example 2.
[0050] A synthetic mixture containing 7 Sigma standard peptides (14
pmol each in water, injected before the 1:100 split valve) was
separated in reversed-phase LC mode under gradient elution
conditions. The injected sample mixture contained: 1) methionine
enkephalin, 2) leucine enkephalin, 3) oxytocin, 4) bradykinin, 5)
LH-RH, 6) angiotensin II, and 7) angiotensin I. The separation
results are presented as the chromatogram and the mass spectra
shown in FIGS. 4 and 5. The mobile phases for the gradient elution
were: A=0.1% v/v trifluroacetic acid in water; and B=0.1% v/v
trifluroacetic acid in acetonitrile. The gradient elution was
completed in 10 minutes while the mobile phase was changed from 1
0%B to 50%B (i.e., acetonitrile concentration from 10 to 50%
v/v).
EXAMPLE 5
[0051] In accordance with the same procedure as described in
Example 1, 25 mg of aluminum chloride powder was put into a screw
top glass vial. 0.5 ml of nitromethane was added. After stirring
for a few minutes, 0.5 ml of 1-chlorobutane was added. A uniform
solution was formed after further stirring. The vial was then
closed using the open-top cap with the septum. The other steps for
treating the capillary monolith were the same as that in Example 1.
The PS-DVB surfaces were modified with butyl chains.
EXAMPLE 6
[0052] This example illustrates a process for alkylating a porous
PS-DVB monolith molded in a micro-opening of a silicon or polymer
chip.
[0053] A porous PS-DVB monolith was formed in situ in a vertical
cylindrical through-opening with a diameter of 100 .mu.m in a
silicon chip or wafer. The process for preparing the chip-molded
monoliths was modified from that for capillary monoliths.
[0054] A liquid was forced into the monolith by using a liquid
delivery probe as shown in FIG. 6. The liquid in the probe was
driven by a nitrogen source with a pressure of up to 30 psi. The
chip-molded monolith was wetted, treated, and washed with the
liquid delivery probe as described in Example 1.
* * * * *