WO2004002603A1

WO2004002603A1 - Separation system, components of a separation system and methods of making and using them

Info

Publication number: WO2004002603A1
Application number: PCT/US2003/020415
Authority: WO
Inventors: Robert W. Allington; Shaofeng Xie; Mingcheng Xu; Tao Jiang
Original assignee: Isco, Inc.
Priority date: 2002-06-26
Filing date: 2003-06-26
Publication date: 2004-01-08
Also published as: EP2664366A3; JP2005531011A; US7922909B2; US7922908B2; JP4721702B2; EP1515784A1; US20040020855A1; US20060207939A1; US20080179773A1; US7074331B2; EP2664366A2; US20080185327A1; US20040000522A1; US6749749B2; EP1515784A4; US8795529B2

Abstract

Permeable polymeric monolithic materials are prepared in a column casing. In one embodiment, the permeable polymeric monolithic materials are polymerized while pressure is applied through a piston having a smooth piston head in contact with the polymerization mixture. The pressure eliminates wall effect and changes the structure in the column. Similarly, some columns that have a tendency to swell in the presence of aqueous solutions and pressurized while the solution is applied to prevent swelling and wall effect. This procedure also changes the structure in the column. The size of the separation effective openings can be controlled by the amount of the pressure and pores eliminated. Uniformity in the direction flow is improved by controlling polymerization with radiation rather than with conducted heat.

Description

SEPARATION SYSTEM, COMPONENTS OF A SEPARATION SYSTEM AND METHODS OF MAKING AND USING THEM

RELATED CASES

This application is a continuation in part of United States patent

application 10/180,350 filed June 26, 2002, in the names of Shaofeng Xie and

Robert W. Allington for SEPARATION SYSTEM, COMPONENTS OF A

SEPARATION SYSTEM AND METHODS OF MAKING AND USING THEM.

BACKGROUND OF THE INVENTION

This invention relates to separation systems and their components and

more particularly to separation systems and components involving monolithic

permeable polymeric materials.

Monolithic macroporous materials such as for example organic

monolithic macroporous polymeric materials and monolithic silica packings are

known as components for separation systems such as chromatographic or

extraction systems. One class of such materials is formed as a monolithic

macroporous polymer plug or solid support produced by polymerizing one or

more monomers in a polymerization mixture that includes at least a porogen.

It is known for some polymerization mixtures, to include other materials such

as cross-linking agents, catalysts and small soluble polymers which can be

dissolved after polymerization to control the porosity and pore size distribution.

Moreover, the plug may be modified after being formed to add functional

groups. The plug or solid support is normally contained in a housing such as for

example a chromatographic column or a pressure vessel. The portion of the

housing where the plug resides acts as a reactor. In one prior art process for

making monolithic columns, the polymerization mixture may be added to the

column casing and polymerization initiated therein to form a macroporous

polymeric plug or solid support within the walls of the column.

There are wide applications of these plugs or solid supports including

gas, liquid and supercritical fluid chromatography, membrane chromatography

and filtration, solid phase extraction, catalytic reactors, solid phase synthesis

and others. The efficiency of the column or other container for the plug or solid

support, the time required for a separation, and the reproducibility of the

columns or other container for the plug or solid support are important

commercial factors. The efficiency of separation systems such as

chromatographic columns with porous polymer in them is related to both the

selectivity of the column or other component containing the macroporous

polymeric material and to zone spreading. Some of hese factors are affected

by molecular diffusion and velocity of the mobile phase in the plug or solid

support during a separation process.

The manner in which molecular diffusion and velocity of the mobile

phase affects column efficiency can be in part explained by showing the effect

of these factors on Height Equivalent to Theoretical Plates (HETP), the

conventional designation of column efficiency. The van Deemter Equation

shows the relationship between zone spreading, flow velocity and diffusion in terms of H (HETP) as follows:

H = A + B/u + Cu with low H corresponding to high efficiency

U= Flow velocity of the mobile phase

A=RadiaI Eddy Diffusion coefficient

B=Longitudinal Molecular Diffusion coefficient

C= The mass transfer coefficient

Molecular diffusion depends on the diffusion of the molecules but not on the

packing ofthe bed. Eddy diffusion depends on the homogeneity ofthe packing

of the particles.

Zone spreading from mass transfer can be minimized by using non-

porous particles and porous particle with sizes smaller than 1.5 microns.

However, packing with non-porous particles has extremely low surface area

which is detrimental to the purification process (as opposed to the analytical

process) because the purification process requires high sample loading. The

use of very small packed particles requires either high pressure which is difficult

in most ofthe separation process using current instrumentation or low velocity,

which can increase the time for a given separation (sometimes expressed in H

per minute).

The prior art separation systems that include as a component

macroporous polymeric monolithic plugs or solid supports use plugs or solid

supports formed from particles in the polymers that are larger than desired, less

homogenous and include micropores. The large size ofthe particles and their

lack of homogeneity result in a lack of homogeneity in the pore size distribution. The non-homogeneity ofthe pore sizes and large amount of micropores in the

prior art porous polymers contributes greatly to the zone spreading as shown

by the van Deemter Equation. The large number of micropores contributes to

zone spreading by capturing sample and retaining it for a time. This may be

stated conventionally as the non-equilibrium mass transfer in and out of the

pores and between the stationary phase and the mobile phase.

The prior art plugs or solid supports formed of porous polymers have

lower homogeneity of pore size, less desirable surface features and voids in

their outer wall creating by wall effect and thus higher zone spreading and

lower efficiency than desired in separation systems.

The prior art also fails to provide an adequate solution to a problem

related to shrinkage that occurs during polymerization and shrinkage that

occurs after polymerization in some prior art porous polymers. The problem of

shrinkage during polymerization occurs because monomers are randomly

dispersed in the polymerization solution and the polymers consist of orderly

structured monomers. Therefore, the volume of the polymers in most of the

polymerization is smaller than the volume of the mixed monomers. The

shrinkage happens during the polymerization in all of the above preparation

processes. One of the problems with shrinkage after polymerization occurs

because of the incompatibility of a highly hydrophilic polymer support with a

highly hydrophilic aqueous mobile phase or other highly polar mobile phase

such as for example, a solution having less than 5-8 percent organic solvent

content. Shrinkage ofthe porous polymeric materials used in separation systems

and their components during polymerization results in irregular voids on the

surface of the porous polymers and irregularity of the pore size inside the

polymer, which are detrimental to the column efficiency and the reproducibility

ofthe production process. One reason the column efficiency is reduced by wall

effect is that wall effect permits the sample to flow through the wall channels

and bypass the separation media. One reason the reproducibility of the

production process are reduced by wall effect is the degree of wall effect and

location of the wall effect are unpredictable from column to column.

The columns with large channels in the prior art patents cited above

have low surface area and capacity. The low capacities of the columns are

detrimental for purification process which requires high sample loadings. In

spite of much effort, time and expense in trying to solve the problems of

shrinkage, the prior art fails to show a solution to reduced capacity.

Because of the above phenomena and/or other deficiencies, the

columns prepared by the above methods have several disadvantages, such as

for example: (1) they provide columns with little more or less resolution than

commercially available columns packed with beads; (2) the separations

obtained by these methods have little more or no better resolution and speed

than the conventional columns packed with either silica beads or polymer

beads, particularly with respect to separation of large molecules; (3) the wide

pore size distribution that results from stacking of the irregular particles with

various shapes and sizes lowers the column efficiency; (4) the non- homogeneity of the pore sizes resulting from the non-homogeneity of the

particle sizes and shapes in the above materials contribute heavily to the zone

spreading; (5) the large amount of micropores in the above materials also

contributes greatly to the zone spreading; and (6) shrinkage of the material

used in the columns reduces the efficiency of the columns. These problems

limit their use in high resolution chromatography.

U.S. Patent 5,453, 185 proposed a method of reducing the shrinkage by

reducing the amount of monomers in the polymerization mixture using insoluble

polymer to replace part of the monomers. This reduces the shrinkage but is

detrimental to the capacity and retention capacity factor of the columns which

require high amount of functional monomers. There is nothing mentioned in

these patents regarding the detrimental effect of shrinkage on resolution and

the resulting irregular voids on the surface of the porous polymer and

irregularity of the pore size inside the polymer, which are detrimental to the

column efficiency and the reproducibility of the production process.

Prior art European patent 1 ,188,736 describes a method of making

porous poly(ethylene glycol methacrylate-co-ethylene glycol dimethacrylate) by

in situ copolymerization of a monomer, a crosslinking agent, a porogenic

solvent and an initiator inside a polytetrafuoroethylene tube sealed at one end

and open at the other end. The resulting column was used for gas-liquid

chromatography. This prior art approach has the disadvantage of not resulting

in materials having the characteristics desirable for the practical uses at least

partly because it uses polymerization in a plastic tube with an open end. U.S. Patent 2,889,632, 4,923,610 and 4,952,349 disclose a method of

making thin macroporous membranes within a sealed device containing two

plates and a separator. In this method the desired membrane support was

punched out of a thin layer of porous polymer sheet and modified to have

desired functional groups. The layers of porous sheets are held in a support

device for "membrane separation". These patents extended the method

described in European patent 1 ,188,736 to prepare a porous membrane and

improve the technique for practical applications in membrane separation. The

resulting material is a macroporous membrane including pores from micropores

of size less than 2 nanometers to large pores. The size of the particles of the

polymer is less than 0.5 micrometers. The separation mechanism of

membrane separation is different from that of conventional liquid

chromatography.

This porous material has several disadvantages, such as for example:

(1) the thinness of the membrane limits its retention factor; and (2) the pores

formed by these particles are small and can not be used at high flow rate with

liquid chromatography columns that have much longer bed lengths than the

individual membrane thicknesses. The micropores and other trapping pores

trap molecules that are to be separated and contribute to zone spreading. The

term "trapping pore" in this specification means pores that contribute to zone

spreading such as pores ranging in size from slightly larger than the molecule

being separated to 7 times the diameter of the pore being separated.

U.S. Patents 5,334,310; 5,453,185 and 5,728,457 each disclose a method of making macroporous poly(glycidyl methacrylate-co-ethylene glycol

dimethacrylate) polystyrene in situ within sealed columns. This method extends

the methods described in both the European patent 1 ,188,736 and US patents

2,889,632, 4,923,610 and 4,952,349 for preparing liquid chromatography

columns forthe separation of proteins. US patents 5,334,310, 5,453,185 and

5,728,457 profess the intention of improving the column efficiency by removing

the interstitial volume of conventional packed columns having beads. The

plugs formed according to these patents have a pore size distribution that is

controlled by the type and amount of porogens, monomers and polymerization

temperature. The macroporous polymers consist of interconnected aggregates

of particles of various sizes which form large pore channels between the

aggregates for the transport of the mobile phase. Among the aggregates or

clusters there exist small pores for separations. The small particles are formed

from tightly packed extremely small particles ca 100-300 nanometers.

The materials made in accordance with these patents have a

disadvantage in that the micropores within or between these particles physically

trap the sample molecules and degrade the separation. Although these patents

claim that there are no interstitial spaces in the monolithic media as in the

packed bed with beads, the large channels between the aggregates and

interconnected particles actually cause the same problem as the interstitial

spaces between the beads in conventional packed columns with beads. The

large channels formed from various size of aggregates or clusters are

in homogeneous and provide random interstitial spaces, even with narrow particle size distribution. Because ofthe random interstitial spaces the column

efficiency is poor.

U.S. Patent 5,334,310, 5,453,185 and 5,728,457 disclose the

preparation of the separation media inside a column with cross section area

from square micrometers to square meters. The processes disclosed in these

patents have some disadvantages. Some of the disadvantages were

disclosed by the inventors named in those patents in 1997 in Chemistry of

Materials, 1997, 9, 1898.

One significant disadvantage is that larger diameter (26 mm I.D.)

columns prepared from the above patented process have a pore size

distribution is too irregular to be effective in chromatography separation. The

irregular pore size distribution is caused by the detrimental effect of

polymerization exotherm, the heat isolating effect of the polymer, the inability

of heat transfer, autoaccelerated decomposition ofthe initiator and concomitant

rapid release of nitrogen by using azobisisobutyronitrile as initiator in a mold

with 26mm diameter. It has been found that the temperature increase and

differential across the column created by the polymerization exotherm and heat

transfer difficulties results in accelerated polymerization in large diameter

molds such as for example molds having a diameter of more than 15 mm and

in a temperature gradient between the center of the column and the exterior

wall of the column which results in inhomogeneous pore structure. It was

suggested in this article that the problem might be reduced by slow addition of

polymerization mixture. This helps to solve the problem partly but does not solve the problem completely. There is still a temperature gradient for the larger

diameter columns, which result in in-homogeneity of the pore size distribution.

This problem was also verified by theoretical calculations in the

publication of Analytical Chemistry, 2000, 72, 5693. This author proposes a

modular approach by stacking thin cylinders to construct large diameter

columns for radial flow chromatography. However, sealing between the discs

to form a continuous plug is difficult and time consuming.

US patents 5,334,310, 5,453,185 and 5,728,457 disclose the material

of weak anion exchange and reversed phase columns. The weak anion

exchanger prepared had low resolution, low capacity, low rigidity, slow

separation and very poor reproducibility. The reversed phase media has very

little capacity, non-ideal resolution, and very poor reproducibility. They can not

be used in mobile phase with high water content such as less than 8%

acetonitrile in water due to wall channeling effect resulting from shrinkage ofthe

very hydrophobic media in this very polar mobile phase. This media is also

compressed during separation and result in excess void volume in the head of

the column. The above patents provide little guidance on how to prepare a

weak cation exchanger, strong cation exchanger, strong anion exchanger,

normal phase media and hydrophobic interaction media. These media based

on membrane, beads or gels are known. However, the preparation are done by

off-line and can not be used for in situ preparation of monolithic columns. The

monolithic membrane prepared according to US patents 2,889,632, 4,923,610

and 4,952,349 has low capacity and resolution. SUMMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide an improved

separating system having synergistic relationships with a polymer having

separation-effective openings.

It is a further object of the invention to provide an improved

chromatographic column.

It is a still further object of the invention to provide an improved

apparatus for making a chromatographic column.

It is a further object of the invention to provide an improved method for

forming chromatographic columns.

It is a still further object of the invention to provide an improved

permeable monolithic medium.

It is a still further object of the invention to provide a permeable

monolithic column with improved resolution.

It is a still further object of the invention to provide a permeable

monolithic column with improved capacity.

It is a still further object of the invention to provide a column with

improved flow rate.

It is a still further object ofthe invention to provide a column with reduced

tendency to swell when used with aqueous solvent.

It is a still further object of the invention to provide a column with

improved reproducibility.

It is a still further object of the invention to provide an improved technique for the formation of permeable monolithic columns with controlled

pore size selected for the purpose of improving capacity or resolution or flow

rate.

It is a still further object ofthe invention to provide a method of preparing

large diameter columns for preparative separations.

It is a still further object ofthe invention to provide a method of preparing

polymeric materials including permeable polymer support using irradiation

methods.

It is a still further object of the invention to provide an improved weak

anion exchange column.

It is a still further object of the invention to provide an improved reverse

phase column.

It is a still further object of the invention to provide a high performance

strong anion exchange column.

It is a still further object of the invention to provide a high performance

weak cation exchange column.

It is a still further object of the invention to provide a high performance

strong cation exchange column.

It is a still further object of the invention to provide a high performance

normal phase column.

It is a still further object ofthe invention to provide a method of avoiding

a reduction in the quality of a separating medium caused by shrinkage during

polymerization or swelling ofthe medium during washing or during separation. It is a still further object of the invention to provide a chromatographic

system in which an array of columns, in which the columns are very close in

characteristics, are operated together.

It is a still further object of the invention to provide a novel process for

making monolithic permeable solid support for applications in chromatography

including liquid, gas and supercritical fluid chromatography,

electrochromatography, catalytic reactor, filtration or others requiring permeable

polymer supports or solid permeable polymer holders adjacent or positioned at

least partly horizontally from a sample .

It is a further object of the invention to provide a novel high resolution

media and novel method of obtaining it.

It is a further object of the invention to provide an improved permeable

solid support with homogeneous separation-effective opening size distribution

resulting from more homogeneous size and shape of the interconnected

aggregated particles.

It is a further object of the invention to provide an improved permeable

solid support with less or no micropore.

It is a further object of the invention to provide an improved permeable

solid support with no voids on the wall of the polymeric support.

It is a further object of the invention to provide an method for improving

the capacity of monolithic chromatography media.

It is a still further object of the invention to provide a permeable

monolithic column with improved resolution. It is a still further object of the invention to provide an improved

technique for the formation of permeable monolithic columns with controlled

size separation-effective openings selected for the purpose of improving

capacity or resolution or flow rate.

It is a still further object of the invention to provide a method of preparing

large diameter columns for preparative separations.

It is a still further object of the invention to provide a high performance

catalytic reactor.

It is a still further object of the invention to provide a high performance

solid phase extraction bed.

It is a still further object of the invention to provide an improved

permeable monolithic medium with covalently bonded particles having a controlled minute throughly convoluted surface configuration but with few or no

micropores.

It is still a further object of the invention to provide a monolithic

chromatographic bed with negligible nonuniformities due to thermal effects

during polymerization reaction.

It is a still further object of the invention to provide a novel method for

controlling polymerization in a chromatographic column with reliance on

conduction of heat into the column.

It is still a further object of the invention to control the rate of

polymerization, and resulting thermal ^' gradients, by means of controlled

radiation impinging on the bed. It is still a further object of the invention to control polymerization with a

relatively safe radiation source such as those providing medium energy x-ray

(e.g. below 200 kEV), UV or visible radiation.

It is a still further object of the invention to provide a permeable, high

capacity, column with few or no micropores.

It is a still further object ofthe invention to provide a method of preparing

columns with small diameters from nanometers to millimeters.

It is a still further object ofthe invention to provide a method of preparing

chromatographically uniform columns with medium diameters up to 100 mm

and large diameter columns up to 1000 mm.

In accordance with the above and further objects of the invention, a

polymerization mixture is polymerized in place with a porogen or solvent, to form

a polymer plug that has

separation effective openings. In this specification, "separation-effective

openings" means pores or channels or other openings that play a role in

separation processes such as for example chromatography. Pores generally

means openings in the particles that are substantially round and may be

through pores passing through particles (through pores) or openings into the

particles or in some cases, openings into or through aggregates of particles.

By being substantially round in cross-section, it is meant that the pores are not

perfect circles and for example may be bounded by sectors of imperfect

spheres with the pores being the open spaces between the adjacent spherical

surfaces. Some other terms are defined below as they are used in this

specification. Separation factors includes those factors that effect retention and

capacity or other factors that play a role in separation processes. The term

"macroporous" in this specification is given its usual meaning in referring to

monolithic materials in separation systems. Its usual meaning refers to pores

or other voids between globules of particles, which pores or other voids have

a diameter of over 50 nm. regardless of the length of an opening, rather than

its literal connotation that would limit the openings to pores with a substantially

circular cross section and no cross sectional dimension substantially longer

than the other. The term "permeable" in this specification shall be interpreted

in the same manner as "macroporous" with reference to monolithic materials

in the separation arts but is used in preference to the term "macroporous" to

distinguish materials having channels and other openings from those containing

pores to avoid confusion with the literal meaning of the term "macroporous".

In this specification the term "permeable non-porous" describes media having

openings such as channels or the like but not containing pores as defined

above.

In one embodiment of this invention, shrinkage during polymerization is

compensated for and In another embodiment of this invention, swelling after

polymerization, which might otherwise later result in shrinkage is avoided.

Shrinkage results in enlarged voids on the polymer surface and may result in

a lack of homogeneity of pore size distribution inside the polymer. The voids

are believed to be created by decreased volume of orderly structured polymer compared to the volume of monomers prior to polymerization when created

during polymerization. The voids are mostly located in between the column

wall and polymer due to the difference in surface free energy. The voids are

probably occupied by the nitrogen gas generated by azobisisobutyronitrile

(AIBN), which is a common initiator for the polymerization.

In a first embodiment, the compensation for shrinkage is accomplished

by applying sufficient pressure during polymerization to create uniformity in the

distribution of separation-effective openings and to avoid wall effect voids. This

pressure has been found to also control particle size and the nature and shape

of the openings in the plug to some extent. Maintaining the column at

atmospheric pressure during polymerization to accommodate shrinkage does

reliably prevent the formation of voids. Generally 250 psi pressure is used for

convenience but higher and lower pressures have been used successfully. The

voids are removed when the plug stops shrinking when put under even modest

amounts of pressure. In a second embodiment, shrinkage that otherwise would

occur after polymerization is avoided. For example, some plugs tend to expand

when exposed to some solutions such as organic solvent and then shrink later

such as during a separating run in aqueous mobile phase, causing voids. In

these embodiments, shrinkage is prevented by holding the column from

shrinkage when exposed to the solutions. The application of pressure is one

method of preventing shrinkage during exposure to the aqueous solutions.

Other methods for compensated for shrinking and/or swelling , for reducing

shrinking or for avoiding shrinking are also used as described in greater detail below. It is believed that the externally applied pressure overcomes uneven

forces internal to the reacting polymerization mixture and between the

polymerization mixture and internal wall of the column to maintain

homogeneous separation effective factors, separation-effective opening size

and distribution and uniform continuous contact of the polymer to the internal

wall of the column.

Surprisingly, some types of polymer plugs contain no pores if they are

subject to pressure during polymerization to compensate for shrinking or in the

case of some reversed phase columns to compensate for shrinkage when

exposed to hydrophillic solutions such as for example in the aqueous mobile

phase Instead, they contain solid particles ca 2 micrometers in diameter,

covalently bonded together with relatively large flow channels between them

(separation-effective openings) . The surprising thing is that, although these

particles have no pores, the chromatographic capacity ofthe plug is high. This

is believed to happen because of the unexpected formation of ca 50-200 nm

deep and wide grooves or corrugations and other odd surface features. A

typical particle resembles a telescopic view of a very small asteroid.

The pressure applied during polymerization is selected in accordance

with the desired result and may be, for example, a linearly increasing

pressure, a constant pressure or a step pressure gradient. In one

embodiment, separation-effective opening size is controlled by selecting the

type and proportion of porogen that generates the pores during polymerization

and the porogen that must be washed out ofthe plug after polymerization. This proportion is selected by trial runs to obtain the desired characteristic. The total

amount of porogen is also selected.

In another embodiment, some plugs tends to expand when exposed to

some solutions such as organic washing solutions and then shrinks later such

as during a separating run in the aqueous mobile phase, creating voids

between column wall and polymersupport and variations in separation-effective

opening size distribution. For example, some reverse phase plugs with

separation-effective openings may shrink when polymerized others may not,

and after polymerization, some of the plugs that did not shrink during

polymerization and some that did may shrink if exposed to water or some other

polar solutions. In this case, the compensation for this shrinkage is the

compression with a piston during polymerization and/or compression after

polymerization during conditions that would normally cause shrinking equal or

more than the shrinkage that could happen during the separation run to force

reordering or repositioning or to compensate for the shrinking. In either case

where shrinkage is compensated for with pressure or where shrinkage is

prevented to avoid causing voids, non-fluidic pressure such as with a piston is

preferred rather than pressure with fluid. The word "pressure" in this

specification excludes and differentiates from the term "compression" if the

word "compression" is used to indicate the application of salt solutions to gel

monoliths to open the pores of such gel monoliths. Another way of solving this

problem is to introduce hydrophilicity to the reversed phase media to result in

swelling and prevent the shrikage of the polymer in highly hydrophilic environment during the separation run.

More specifically, a polymerization mixture is applied to a column in the

preferred embodiment or to some other suitable mold and polymerization is

initiated within the column or mold. The column of mold is sufficiently sealed:

(1) to avoid unplanned loss by evaporation if polymerization is in an oven; or (2)

to avoid contamination or dilution if polymerization is in a water bath. During

polymerization, pressure is applied to the polymerization solution. Preferably

the pressure is maintained at a level above atmospheric pressure to prevent

the formation of voids by shrinkage until polymerization has resulted in a solid

plug of separating medium or polymerization is completed. The inner surface

ofthe column or mold with which the polymerization solution is in contact during

polymerization may be non-reactive or may be treated to increase adhesion to

the surface of the plug.

The polymerization mixture in some embodiments includes: (1) selected

monomers; (2) for some types of columns, an additive; (3) an initiator or

catalyst; and (4) a porogen or porogens to form separation-effective openings.

In some embodiments function groups can be added before or after

polymerization. The porogen, initiator, functional group to be added, additives,

and reaction conditions and the monomer and/or polymer are selected for a

specific type of column such as reverse phase, weak cation, strong cation,

weak anion, strong anion columns, affinity support, normal phase, solid phase

extraction and catalytic bed. The selection of components ofthe polymerization

mixture is made to provide the desired quality of column. A chromatographic column in accordance with this invention preferably

includes a casing having internal walls to receive a permeable monolithic

polymeric plug in which the separation-effective openings or surface features

are of a controlled size formed in the polymer by a porogen in the

polymerization mixture and are controlled in size by pressure during

polymerization. This plug serves as a support for a sample in chromatographic

columns. The permeable monolithic polymeric plug has smooth walls with no

visible discontinuity in the plug wall and substantially no discontinuity or

opening within the plug. Discontinuity in this specification means a raised

portion or opening or depression or other change from the normal smoothness

or pattern sufficient in size to be visible with the unaided eye. In this

specification, the term "size-compensated polymers" or "size-compensated

polymeric" means monolithic polymeric permeable material having separation-

effective openings in which discontinuities lack of homogeneity in the

separation-effective openings have been prevented by the methods referred to

in this specification such as for example applying pressure during

polymerization or after polymerization during exposure to polar solutions in the

case of some types of columns or by using a column that is prevented from

further shrinkage in the presence of an aqueous solution by the application of

pressure in the presence of the aqueous solution either during washing with an

aqueous solution or during use in a separation operation using an aqueous

solution.

One embodiment of column is made using a temperature controlled reaction chamber adapted to contain a polymerization mixture during

polymerization and means for applying pressure to said polymerization mixture

in said temperature controlled reaction chamber. The polymerization mixture

comprises at least a polymer forming material and a porogen. In one

embodiment, the pressure is applied by a movable member having a smooth

surface in contact with the polymerization mixture under external fluid or

mechanical pressure, although pressure can be applied directly to the

polymerization mixture with gas such as nitrogen gas or with a liquid under

pressure.

An embodiment of reversed phase media have been formed with

different hydrophobicity, and hydrophilicity from the prior art. The reversed

phase media include polystyrenes, polymethacrylates and their combinations.

These media are prepared by direct polymerization of monomers containing

desired functionalities including phenyl, C4, C8, C12, C18 and hydroxyl groups

or other combination of hydrophobic and hydrophilic groups to have different

selectivity and wetability in aqueous mobile phase. The polymerization

conditions and porogens are investigated and selected to give the high

resolution separation of large molecules, in particular, the proteins, peptides,

oligonucleotides and synthetic homopolymers. In one embodiment a reversed

phase media is based on poly(styrene-co-divinylbenzene). In another

embodiment of this patent, a reversed phase media is based on poly(stearyl

methacrylate-co-divinylbenzene). In another embodiment, a reversed phase

media is based on poly(butyl methacryalate-co-ethylene glycol dimethacrylate). A reverse phase plug with exceptional characteristics is principally

formed of copolymers of crosslinkers including divinylbenzene (DVB), and

ethylene glycol dimethacrylate and monomers including styrene (ST) or

methacrylates (MA) containing different carbon chain length. Generally, the

best results are when the crosslinkers are greater than 40 percent by weight

Preferably the ratio of divinylbenzene and styrene is a value of divinylbenzene

in a range between 7 to 1 and 9 to 1 and preferably 4 to 1 by weight, but may

instead be 64 DVB or 40 percent styrene and 72 percent by weight DVB or 1

g divinylbenzene, 1 g styrene. The column may also be in the range of ratios

between 17 to 3 and 19 to 1 and preferably 9 parts divinylbenzene to 1 part

styrene. Monomers with hydrophilic functional groups can be added to reduce

shrinkage ofthe polymeric medium in aqueous mobile phase to prevent the wall

effect during separations. The content of DVB in total monomers is preferably

from 40% to 100%. In one preferred embodiment, the content of DVB is 80%

(which is the highest commercially available) to improve the loading capacity

ofthe column. The plug may also include methacrylates with hydrophobic

surface groups or instead of being a vinyl compound including urea

formaldehyde or silica.

Ion exchange plugs are formed principally of methacrylate polymers. A

weak anion exchange plug is principally formed of polymers of glycidyl

methacrylate (GMA) and of ethylene glycol dimethacrylate (EDMA). A strong

anion exchanger plug is principally polymers of glycidyl methacrylate, 2-

(acryloyloxyethyl) trimethylammonium methyl sulfate (ATMS), ethylene glycol dimethacrylate. The polymerization mixture may also include 1 , 4-butanediol,

propanol and AIBN. A weak cation exchanger plug is formed principally of

glycidyl methacrylate, acrylic acid (AA) and ethylene glycol dimethacrylate. A

strong cation exchanger plug is formed principally of glycidyl methacrylate, 2-

Acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and ethylene glycol

dimethacrylate. In all these ion exchangers, functional groups can be added

before or after the plug is formed. The content of EDMA in total monomers is

preferably from 40% to 80%.

An increase ofthe content of crosslinker, such as EDMA, increases the

rigidity ofthe column by reducing the swelling ofthe media in aqueous phase.

Each of the polymerization mixtures is modified under the pressurized

polymerization to obtain high flow rate and high resolution at both high and low

flow velocity. The coupling of copolymerization of the monomers containing

desired functional groups for interaction and the controlled modification of other

functional monomers to contain the desired interactive functional groups

increases the capacity of the column while improving the rigidity of the

separation media. This controlled modification may also improve the

hydrophilicity of the columns in general by covering the potential hydrophobic

surface area with hydrophilic functional groups. The modification conditions are

chosen to not only provide the higher capacity and higher hydrophilicity of the

media but also to prevent the swelling of polymer matrix in aqueous solution,

which happens in other highly hydrophilic polymer matrices including both

beads and monolith. The polymer plugs may be formed in a column of any size or shape

including conventional liquid chromatographic columns that may be circular

cylinders, or coiled, bent or straight capillary tubes, or microchips or having any

dimension or geometry. The sample or mixture to be separated into its

components is injected into the column and the liquid phase is moved through

the column to separate the sample into its components. The components may

be detected and/or collected in a fraction collector and/or inserted into another

device such as a gas chromatograph or mass spectrometer. In one

embodiment, a plurality of columns is connected in parallel in a

chromatographic system that includes a pumping system, solvent system and

detecting system. The columns are permeable polymeric columns with high

reproducibility so as to enable them to work together for related separations.

In one embodiment, chromatographic discs or plugs having diameters much

greater than 25 mm are produced.

In one embodiment, the reaction is controlled by independent means

such as for example electromagnetic radiation such as for example UV-vis, X-

ray, or γ-ray instead of or in addition to reliance only on time, temperature of

a water bath and the reactants in the polymerization mixture. In one form of

this embodiment, heat may be added from a heat source or removed by

cooling means in contact with a significantly large portion of coolant of the

thermal mass and in the reactor under the control of feedback to maintain the

temperature of the reaction mass in the desired temperature range or to vary

it during the reaction if desired. In another form, variable intensity or variable wavelength X-rays may be used to control the polymerization rates of the

mixing reactions at a rate such that the exotherm is under control. X-ray

radiation penetrates the column to impart energy throughout the column or at

a selected location to increase of decrease polymerization rates. This may be

done by irradiating the monomer sufficiently to disassociate its double bonds

to make monomers free radicals and thus increase their reactivity. Another way

is to use an initiator sensitive to the radiation that is activated by the radiation

in the temperature region to be used for the reaction mass. The initiator is

chosen to have an activation time and temperature considerably less than that

of the monomers alone. Because the initiator forms free radicals only upon

radiation of sufficient intensity, the radiation may be used to control the

polymerization reaction independently of the other factors. Another way is to

use the radiation sensitizers or scintillators in combine with photo-initiators to

initiate the polymerizations. The radiation sensitizers such as x-ray scintillators

transfer the energy of radiations to photo-initiators by luminescence of the

photos at the desired wavelength after absorbing the radiation energy

transferred through the solvents. The wavelength of the luminescence should

be the same as the absorption wavelength of the photo-initiator.

Polymerization using irradiation such as x-ray is used for preparing

monolithic materials with cross sections from micrometers to meters. X-rays can

penetrate the materials in depth. Both organic and inorganic polymers can be

prepared using x-ray or γ-ray. High energy x-ray and γ-ray can travel the

materials in high depth. Low energy to medium x-ray penetrates the materials in less depth resulting in a longer polymerization time but is safer to use. In

one embodiment, a lower energy x-ray is used to initiate the polymerizations

using the combination of x-ray scintillator and photo initiators. We have

discovered that non-thermal (photoinitiation) control of polymerization times

from less than 12 hours to more than one week provides satisfactory

chromatographic columns. Thermal polymerization of columns usually suffers

from runaway exothermic reaction and extreme temperature gradients with

columns over 20 mm in diameter. This causes un uniformities which degrade

chromatographic properties. The slower, controlled, polymerization rate

available with x- or γ-rays, or even UV causes a slower polymerization with

tolerable rates of exotherm while still maintaining reasonable rates of

polymerization. Thermal gradients to exotherm maybe made small enough to

not degrade the properties of columns over 1 meter in diameter.

Excessive rates of exotherm and resulting process (polymerization)

temperature and temperature gradient may be prevented with choice of a

stabilizing additive. This stabilizing additive should have properties such that

the reaction can proceed freely up to rate at which the desired polymer is

formed, but not at a higher rate producing too high a temperature. For

example, with peroxide initiators Disterylhiodipropionate (DSTDP) quenches

the hydroxyl radical which results from a side reaction, which later would go on

to produce the further heat per event compared to the main reaction. Another

approach is to use a stabilizer for the main reaction. This stabilizer is selected

for limited solubility in the primary solvents or activity at the reaction temperature and more solubility above the reaction temperature. Under

analogous conditions a stabilizer, preferentially soluble in the porogen and

having a temperature dependent of solubility or activity may be used.

It is desirable to scale up the size of the column to have higher volume

of media is highly desired in preparative chromatography and catalytic reactors.

In one embodiment ofthe invention, the large diameter column is prepared by

two staged polymerization inside the column. First, multiple thin cylindrical

columns with the diameter smaller than that of the targeted column are

prepared in a mold under pressure or without pressure. The thin columns are

placed inside a large column filled with the same polymerization solution as

used in formation of the thin columns. The thickness in one side of the thin

column should not exceed the 8mm which is the known maximum to prevent

the formation of temperature gradient due to the difficulty in heat dissipation

during exothermic polymerization. The temperature gradient results in vary

inhomogeneous pore size distribution which is detrimental to chromatography

use.

In making size-compensated polymers for use in separation systems, the

characteristics for a given type of separation can be tailored with a given

polymer to the application, by altering the amount of pressure applied during

polymerization or and in the case of some polymers such as used in forming

reverse phase separation media applying pressure when used or when

otherwise brought into contact with a polar solvent such as an aqueous solvent

or washing fluid. After the nature of the polymer itself has been selected for a class of applications, columns can be made and tested. Based on the tests,

the characteristics can be altered in some columns by applying pressure. It is

believed that the application of pressure in some columns increases the

uniformity of particle size and either because of the change in particle size of

for other reasons, the size distribution and uniformity of separation effective

openings throughout the polymer is increased. The increase in homogeniety

ofthe particle size and pore size improves resolution. An increase in pressure

generally improves capacity and resolution and the pressure-time gradient. It

is believed that in some columns micropores are greatly reduced or eliminated

thus reducing zone spreading by the application of pressure during

polymerization and/orduring use orwashing ofthe polymerwith polarsolutions.

From the above description it can be understood that the novel

monolithic solid support of this invention has several advantages, such as for

example: (1) it provides chromatograms in a manner superior to the prior art;

(2) it can be made simply and inexpensively; (3) it provides higher flow rates for

some separations than the prior art separations, thus reducing the time of some

separations; (4) it provides high resolution separations for some separation

processes at lower pressures than some prior art processes; (5) it provides high

resolution with disposable columns by reducing the cost of the columns; (6) it

permits column of many different shapes to be easily prepared, such as for

example annular columns for annular chromatography and prepared in any

dimensions especially small dimensions such as for microchips and capillaries and for mass spectroscopy injectors using monolithic permeable polymeric tips;

(7) it separates both small and large molecules rapidly; (8) it can provide a

superior separating medium for many processes including among others

extraction, chromatography, electrophoresis, supercritical fluid chromatography

and solid support for catalysis, TLC and integrated CEC separations or

chemical reaction; (9) it can provide better characteristics to certain known

permeable monolithic separating media; (10) it provides a novel approach for

the preparation of large diameter columns with homogeneous separation-

effective opening size distribution; (11) it provides a separation media with no

wall effect in highly aqueous mobile phase and with improved column

efficiency: (11) it improves separation effective factors; and (12) it reduces the

problems of swelling and shrinking in reverse phase columns.

BRIEF DESCRIPTION OF THE DRAWINGS

The above noted and other features of the invention will be better

understood from the following detailed description, when considered with

reference to the accompanying drawings in which:

FIG. 1 is a schematic diagram of one embodiment of a process for

making a chromatographic column in accordance with an embodiment of the

invention;

FIG.2 is an assembly of a fixture for applying pressure to a glass column

during polymerization;

FIG. 3 is an assembly of another fixture for applying pressure to a stainless steel column during polymerization;

FIG. 4 is an assembly of still another fixture for applying pressure to a

glass column during polymerization;

FIG. 5 is a Scanning Electron Microscopy (SEM) picture of the strong

cation exchanger polymerized inside a cylinder column under 120 psi hydraulic

pressure.

FIG.6 is a chromatogram showing peaks from a protein sample with a

column in which distortions have been avoided by pressure during

polymerization; FIG. 7 is a photograph showing three columns with the one on the left

made with pressure during polymerization and the two on the right polymerized

without pressure;

FIG. 8 is a block diagram of a chromatographic system with an array of

columns and having reproducible characteristics which are similarto each other

in the array.

Fig. 9 is a chromatogram showing the chromatography separation

achieved with medium energy (110 kEV)x-ray irradiated polymerization.

FIG. 10 is a top view of a UV or visible light polymerization apparatus for

chromatographic columns; FIG. 11 is a sectional side elevational view of the apparatus of FIG. 10.

FIG. 12 is a schematic elevational view of an x-ray polymerization

apparatus for chromatographic columns;

FIG. 13 is a top view of a portion of the apparatus of FIG. 12; FIG. 14 is an elevational sectional view of a portion of the apparatus of

FIG. 12 taken through lines 14-14; and

FIG. 15 is an elevational sectional view taken through lines 15-15 of

FIG. 13.

DETAILED DESCRIPTION

Broadly, a polymerizable mixture is placed in a container with a porogen

or solvent and polymerized to form a plug having separation-effective openings

for use in a separation system such as for example a chromatographic column.

Advantageously, the polymerization is done in a container in which the plug is

to be used such as a chromatographic column or extraction chamber or the like.

In one embodiment of the invention, the mixture is polymerized while

compensating for the effect of shrinkage during polymerization to form a size

compensated polymeric plug. In other embodiments shrinkage is avoided by

applying pressure to materials that tend to swell in the presence of water to

form another embodiment of size compensated polymeric plug. In still other

embodiments separation-effective opening size and distribution is controlled

by pressure and/or by selection ofthe ingredients ofthe polymerization mixture

and/or by processes using external influences such as electromagnetic

radiation. Shrinking is compensated for or avoided because it may causes

enlarged voids adjacent to the wall of the container and have a deleterious

effect on pore size distribution within the column.

In a preferred embodiment, the compensation is accomplished by applying pressure during polymerization to at least maintain the integrity ofthe

material having separation-effective openings as it shrinks during

polymerization. Maintaining the column at atmospheric pressure to

accommodate shrinkage may not prevent the formation of voids in every case

and may provide poor reproducibility. The polymers, monomers, initiators and

porogens have been selected to improve the characteristics ofthe column and

may be used with the embodiments of polymerization under pressure during

polymerization or with other processes. In another embodiment, pressure is

applied to the plug after it has been formed and after or during swelling caused

by other reactions such as washing with an aqueous solution after

polymerization in the case of some reverse phase plugs. The surface of the

column or mold with which the polymerization solution is in contact during

polymerization may be non-reactive or may be treated to increase adhesion.

In one embodiment, the polymerization mixture includes at least one

vinyl compound and a porogen. An initiator is included in the polymerization

mixture or an initiation process is applied to a vinyl monomer and a porogen to

form a monolithic plug for a chromatographic column or other device using a

polymeric plug for separation. Generally the polymerization mixture includes,

in addition to the vinyl monomer, a vinyl polymer, or mixture of monomers and

polymers, an initiator and a porogen. However, other approaches to

polymerization without incorporating an initiator in the polymerization mixture

are known in the art and can be used, such as radiation to form polymers.

Moreover, some of the aspects of this invention may be applied to monomers and polymers in a polymerization reaction other than vinyl groups such as for

example urea formaldehyde or silica to form urea formaldehyde or silica plugs.

A chromatographic column formed by these processes includes a

support having internal walls to receive a permeable monolithic polymeric plug

having separation-effective openings of a preselected size distribution formed

during polymerization and controlled in size partly by pressure during

polymerization and partly by selection ofthe components and proportions ofthe

components ofthe polymerization mixture. For example, separation-effective

opening size is controlled by the amount and type of porogen in the

polymerization mixture in proportion to the amount of some of the other

ingredients. The pressures may be selected in a range of slightly above

atmospheric pressure to a value within the strength ofthe walls ofthe column.

The permeable monolithic polymeric plug has smooth walls and substantially

no micropores within the plug. The plugs may have surface functional groups.

For example, hydrophobic surface groups such as phenolic groups may be

added to decrease swelling with aqueous based solvents in reverse phase

plugs but capacity may also be decreased in this case. Similarly, hydrophillic

surface groups may be added to increase capacity in reverse phase plugs.

One embodiment of permeable, monolithic, polymeric plug that is free

of micropores or channeling openings in the walls over is formed principally of

vinyl polymers although many other polymers may be used in practicing the

invention. A weak ion exchange permeable monolithic polymeric plug is

principally formed of polymers of methacrylate such as glycidyl methacrylate and ethylene dimethacrylate in the ratios by weight of a value in the range

between 2.5 and 3.5 to a value between 1.8 and 2.2 and preferably 3 to 2. A

reverse phase permeable monolithic polymeric plug with exceptional

characteristics is principally formed of polymers of divinylbenzene, and of

styrene. Preferably the ratio of divinylbenzene and styrene is approximately in

a range of a value between 3.5 and 4.5 to a value between 0.8 and 1.2 and

preferably 4 to 1 by weight, but may instead be 64 DVB (divinylbenzene) or 40

percent styrene and 72 percent by weight DVB or in the ratio of divinylbenzene

to styrene in the range of a ratio of 2 to 3 and a ratio of 3 to 2 or preferably 1 to

1. The column may also be in the ratio of divinylbenzene to styrene in a range

of the ratio of 8 to 1 and 10 to 1 and preferably 9 to 1. One hundred percent

DVB is also preferred.

In FIG. 1 there is shown a block diagram of one embodiment 10 of a

method for making chromatographic columns comprising the step 12 of

preparing a polymerization mixture, the step 14 of polymerizing the mixture, the

step 16 of preparing the column for chromatographic run and the step 18 of

performing a chromatographic run. The polymerization mixture used in step 14

of polymerizing a mixture includes a monomer and or polymer capable of

polymerization, an initiator or initiation process such as radiation, and a

porogen, many of which are known in the art. The step 12 of preparing the.

chromatographic mixture, the step 14 of polymerizing, the step 16 of preparing

for a chromatographic run and the step 18 of performing the chromatographic

run all may take different forms. Some of these variations will be described hereinafter.

In the step 14 includes the substeps 20 of reacting the polymerization

compound while compressing it to reduce voids, the substep 22 of washing the

polymer, and in some embodiments, the step 24 of reacting the polymer to add

certain functional groups. While a workable column may be obtained without

compressing the polymerization mixture while polymerizing, significant

improvements have been obtained by applying this compression. These

improvements have been significant enough so as to make the difference

between a competitive commercial column and one which would not be

competitive in some types of columns and for some applications The voids and

inhomogeneous separation-effective openings that are prevented from forming

by this compression may result from the inhomogeneous distribution of the

empty space created by shrinkage of the polymer during polymerization in a

sealed container. The inhomogeneous distribution ofthe empty space may be

due to the differences in surface tension between the column wall surface,

polymer surface, nitrogen and porogens. The compression must be sufficient

to take up this shrinkage and thus reduce the total volume of the column during

polymerization. This process may also affect the separation-effective opening

size in the column and can be used in a step by step process to create

variations in separation-effective opening size if desired.

The washing step 22 is a conventional step intended to remove

porogens and unreacted monomers or other ingredients that may be used for

a specific column but are not intended to remain in the column. This step may be followed by reacting in a manner to add functional groups such as the

groups described above that are important if the column is intended to separate

proteins. In some embodiments, the washing step causes swelling ofthe plug

followed by later shrinkage. Because the shrinkage may cause channeling

voids, pressure is applied to the swollen plug in one embodiment to prevent

the formation of voids during shrinking.

In FIG. 2, there is shown a block diagram of a polymerizing apparatus

28 having a pressure source 23, a pressure transfer mechanism 25 for applying

pressure to a polymerization mixture 32 compression piston 27and a

confinement vessel 21. In the preferred embodiment, the source of pressure

23 is a regulated source of constant hydraulic pressure but other sources such

as a spring or source of air or of an inert gas may be used. Similarly, in the

preferred embodiment, the mechanism 25 is a piston with a smooth surface to

provide a smooth surface to the polymerized plug that the piston surface has

pressed against during polymerization but other sources such as a gas applied

directly to the permeable monolithic polymeric plug may be used. In the

preferred embodiment , the compression piston 27 moves inwardly into the

column department 21 to exert pressure on the polymerization mixture within

the compartment 32 during the polymerization reaction. When the

polymerization reaction is complete, the porogen can be removed by a solvent

pumped through the column. The polymerization occurs in a temperature

controlled environment 29, which is the preferred embodiment is a water bath

but can be any such temperature control mechanism such as a heated chamber. The materials for this device can be any conventional materials know

in the art.

In FIG. 3 there is shown a sectional view of one embodiment of the

polymerizing apparatus 28 having a metal column casing 1022, a confinement

vessel 88, a transfer mechanism 25, a compression piston 112 and a pressure

cap 80. The metal column 1022 is tightly held against the confinement vessel

88 with a seal 1021 between them. Compression of the seal 1021 is provided

by a shoulder 1052 in the barrel 122 and wrench flats 1023 of the apparatus,

which is attached to the column 1022 with threads 1053, thus providing a leak

free connection between the column 1022 and the confinement vessel 88. A

transfer mechanism 25 consisting of a compression piston 112, an o-ring 110,

a rod 106, a retaining collar 104, another o-ring 100, and a hydraulic piston

head 602, all of which are arranged and fitted into the barrel 122 such that the

compression piston 112 and o-ring 110 form a tight seal inside the confinement

vessel 88. The pressure cap 80 contains a fluid inlet port 33 fitted to the barrel

122, with a gasket 94 between them.

The pressure cap 80 and the barrel 122 are tightly connected,

preventing the leakage of pressurized fluid applied through the fluid inlet 33.

The transfer mechanism 25 is then positioned as shown, creating a volume in

the confinement vessel 88. The square of the ratios of the inside diameter of

the barrel 122 at o-ring 100 to inside diameter of confinement vessel 88

provides a pressure multiplication factor.. The opposite end ofthe column 1022

is filled with the polymerization reactants in the column compartment 32, and a containment plug 1024 is fitted in the opening. A containment cap 604 is

threaded onto the column 1022, forcing the containment plug to seal the

opening. Although the preferred embodiment here shows a tight fitting plug

1024 to provide the sealing, an alternate sealing arrangement, such as an o-

ring, could as easily be used to provide either a face seal or a radial seal. The

fluid inlet 33 is connected to a controlled pressure source, such as a

controllable fluid pump or regulated bottle of compressed gas.

This description of the preferred embodiment employs a fluid source;

either compressed gas or compressed liquid applied through the fluid inlet 33,

however the compressive force could as easily be supplied by alternate means;

such as, but not limited to a spring pressing on the transfer mechanism 25,

weights stacked on the transfer mechanism 25 utilizing gravity to provide the

compression , or centripetal force arranged to cause the transfer mechanism 25

to compress the monolithic polymeric column material inside the column

compartment 32.

Once assembled, the apparatus 28 is placed in a temperature-controlled

environment 27, which is a thermally controlled water bath in the preferred

embodiment. Fluid pressure is then applied through the fluid inlet port 33, which

is contained by the pressure cap 80, the gasket 94 the hydraulic piston head

602 and the o-ring 100. This applied force causes the hydraulic piston head

602 to move away from the pressure cap 80, and exerts force on the end ofthe

rod 106. This rod 106 communicates the force to the compression piston,

applying compressive pressure to the monolithic polymeric column material, preferably at the smooth surface 1101. This smooth surface causes a

continuous, uniform surface to be created on the monolithic polymeric material

exposed to the analytical fluids in the ultimate application and reduces the

adhesion ofthe monolithic polymeric column material to the compression piston

112.

The containment plug 1024, column 1022, seal 1021 confinement vessel

88, and the compression piston 112 confine the monolithic polymeric material.

As the chemical reaction proceeds, the volume of the monolithic polymeric

material decreases, and the transfer mechanism 25 moves further into the

confinement vessel 88. Air trapped between the o-ring 100 and the o-ring 110

is allowed to escape through an air escape opening 603 in the barrel 122. The

compression ofthe reactant materials in this manner prevents the formation of

undesirable voids in the monolithic polymeric material and eliminates wall

effects between the monolithic polymeric material and the column 1022, which

would reduce the performance of the column in use.

As the reaction proceeds and the monolithic polymeric material volume

reduces, the compression piston moves closer to the column 1022. Near the

end ofthe polymerization, the retaining collar 104 contacts the shoulder 608 in

the barrel 122, halting the forward motion of the transfer mechanism 25.

Crushing ofthe newly formed monolithic polymeric material is prevented by this

action. At this position, the smooth surface 1101 ofthe compression piston 112

is approximately even with the end ofthe column, and the monolithic polymeric

material fills the column 1022 without undesired voids in the material or wall effects between the material and the column 1022. Using the wrench flats 1023

and 604, the polymer apparatus 28 is separated from the column 1022 as an

assembly. Chromatographic fittings are then installed on both ends.

In FIG. 4 there is shown a sectional view of another embodiment of

polymerizing apparatus 28A similar to the embodiment of polymerizing

apparatus 28 having a glass column casing 922, a piston head assembly 401 ,

a displacement piston 40 and a containment plug 923. . In some analytical

chemistry applications, the wetted surfaces must not contain metal

components. Although the present discussion of this preferred embodiment

refers to a glass column 922 and plastic pieces, any non-metallic material; such

as, but not limited to glass, ceramic, or plastic which provides acceptable

mechanical properties can be used. Further, the discussion here refers to the

pressure applied being provided by a suitable fluid pressure source, alternative

means of providing compression; including, but not limited to springs, weights,

or mechanical means could as easily be used.

The piston head assembly 401 comprises a piston 76, an o-ring 38 and

an intermediate portion 50, assembled and fitted into the column 922. A plunger

assembly 20 consisting of the displacement piston 40 and an o-ring 64 are

assembled and fitted into the hydraulic cylinder portion 21 such that the recess

92 is away from the fluid inlet port 33 in the hydraulic cylinder portion 21. This

plunger assembly 30 is pushed fully into the displacement chamber 60.

The hydraulic cylinder portion 21 and plunger assembly 30 are then

threaded onto the column 922. Using a suitable tool, the piston assembly 401 is pushed into the hydraulic cylinder portion 21 until the annular shoulder 42

contacts the displacement piston 40, with the reduced diameter neck 48 fitting

into the recess 42.

The column 922 is filled with the reactant, and the containment plug 923 is

inserted into the open end of the column 922. A containment cap 924 is then

threaded onto the end ofthe column 922, tightly holding the containment plug

923 to the column 922. Although this embodiment utilizes a tight fit between the

containment plug 923 and the column 922, alternate methods; including, but

not limited to an o-ring creating a face or radial seal can as easily be used.

A fluid pressure source is then applied through the fluid inlet port 33. The

fluid is contained within the displacement chamber 60 by the hydraulic cylinder

portion 21 , the displacement piston 40 and the o-ring 64. The assembled

components are then placed in a temperature-controlled environment 27. For

this embodiment, a thermally controlled water bath was used, but any suitable

method of controlling the reaction temperature can be employed.

The application of such fluid pressure causes the displacement piston

40 to move away from the fluid inlet port 33. This movement of the

displacement piston 40 applies force to the annular shoulder 42 of the

intermediate portion 50, which then applies pressure to the monolithic polymeric

material in the column 922 through the piston 76.

As the reactant chemicals polymerize, the volume decreases. With the

controlled application of pressure to the monolithic polymeric material prevents

the formation of undesirable voids within the monolithic polymeric material and the formation of wall effects between the monolithic polymeric material and the

wall ofthe column 922. As the reaction progresses, the piston 76 moves further

into the column 922 to displace this reduction in volume. A smooth surface 74

on the piston creates a uniform surface of the monolithic polymeric material to

provide a consistent interface to the analytic fluids in its final use, and to

prevent the monolithic polymeric material from adhering to the surface of the

piston 76.

Near the end of the reaction, the annular shoulder 42 comes in contact

with the end of the column 922, preventing any further movement of the

intermediate portion 50 into the column 922. This prevents the crushing of the

monolithic polymeric material after the voids and wall effects have been

eliminated. This annular shoulder 42 also limits the distance that the piston can

travel, allowing control the porosity and size of the resultant monolithic

polymeric material in the column 922.

After the polymerization is completed, the hydraulic cylinder portion 21

is removed from the column 922, together with the displacement piston 40 and

its o-ring 64. The confinement cap 924 and confinement plug 923 are then

removed, and finally the piston head assembly 401 is removed.

Chromatographic fittings are then installed on both ends.

It is also possible to provide compression on the reactant chemicals by

the direct application of compressed gas directly to the reactant chemical's

surface. Such a method is considered inferiorto the above techniques because

the surface of the resultant monolithic polymeric material will not be smooth or even, and may be more porous than the body of the monolithic polymeric

material, when particular column formats are chosen. In other column formats

the direct application of gas may be more applicable. It may be necessary to

cut off the end of the polymer rod to achieve high resolution separation. I n

FIG. 5 there is shown a Scanning Electron Microscopy (SEM) picture of a

strong cation exchanger polymerized inside a column casing under 120 psi

hydraulic pressure magnified 9,000 times to show globules of particles with no

pores but with channels between them having high surface area because ofthe

irregular surface area and to a lesser extent the more stacked-plate like

configurations of the globules of particles. The rough surface area of the

particles with projections covering their surface area shows signs that may

indicate growth by accretion.

In FIG. 6, there is shown a chromatogram having peaks from a protein

sample separated in a column in which the problems of swelling and shrinking

avoided by the application of pressurel. The peaks are distinctive and relatively

high with good resolution. This particular chromatogram is for gradient elusion

at a flow rate of 3 ml/min on a protein sample of conalbumin, ovalbumin and

tripsin inhibitor using a 0.01 MTris buffer of pH 7.6 as one solvent and a 1 M

sodium chloride as the other solvent with a gradient of 0 to 50 percent the

second solvent in 5 minutes time. The back pressure is 250 pounds per square

inch in this column whereas a column without such compensation would be

expected to have a higher back pressure for the same gradient.

In FIG. 7, there is shown three plugs with the one on the left made with pressure during polymerization and the two on the right polymerized without

pressure. FIG. 7 illustrates the discontinuities formed on the surface of

columns caused by shrinkage during formation ofthe column. There are similar

discontinuities inside the column in the form of relatively large openings

unpredictably spaced. These figures also illustrate that the discontinuities can

be removed, resulting in better reproducibility between columns of the same

composition and the same size and improved resolution during

chromatographic runs.

In FIG. 8, there is shown a block diagram of a preparatory liquid

chromatographic system 101 having a pumping system 121 , a column and

detector array 141 , a collector system 117, a controller 119, and a purge

system 123. The column and detector array 141 includes a plurality of columns

with permeable plugs in them. Preferably the plugs are size-compensated

polymeric plugs. The pumping system 121 supplies solvent to the column and

detector array 141 under the control of the controller 119. The purge system

123 communicates with a pump array 135 to purge the pumps and the lines

between the pumps and the columns between chromatographic runs. The

pump array 135 supplies solvent to the column and detector array 141 from

which effluent flows into the collector system 117 under the control of the

controller 119. The controller 119 receives signals from detectors in the column

and detector array 141 indicating bands of solute and activates the fraction

collector system 117 accordingly in a manner known in the art. One suitable

fraction collector system is the FOXY7 200 fraction collector available from Isco, Inc., 4700 Superior Street, Lincoln, NE 68504.

To supply solvent to the pump array 135, the pumping system 121

includes a plurality of solvent reservoirs and manifolds, a first and second of

which are indicated at 131 and 133 respectively, a pump array 135 and a motor

137 which is driven under the control of the controller 119 to operate the array

of pumps 135. The controller 119 also controls the valves in the pump array

135 to control the flow of solvent and the formation of gradients as the motor

actuates the pistons of the reciprocating pumps in the pump array 135

simultaneously to pump solvent from a plurality of pumps in the array and to

draw solvent from the solvent reservoirs and manifolds such as 131 and 133.

While in the preferred embodiment, an array of reciprocating piston

pumps are used, any type of pump is suitable whether reciprocating or not and

whether piston type or not. A large number of different pumps and pumping

principles are known in the art and to persons of ordinary skill in the art and any

such known pump or pumping principle may be adaptable to the invention

disclosed herein with routine engineering in most cases. While two solvents

are disclosed in the embodiment of FIG. 1 , only one solvent may be used or

more than two solvents.

To process the effluent, the collector system 117 includes a fraction

collector 141 to collect solute, a manifold 143 and a waste depository 145 to

handle waste from the manifold 143. One or more fraction collectors

communicate with a column and detector array 143 to receive the solute from

the columns, either with a manifold or not. A manifold may be used to combine solute from more than one column and deposit them together in a single

receptacle or each column may deposit solute in its own receptacle or some of

the columns each may deposit solute in its own corresponding receptacle and

others may combine solute in the same receptacles. The manifold 143

communicates with the column and detector array 141 to channel effluent from

each column and deposit it in the waste depository 145.

Because the system of FIG. 8 includes an array of columns each

involved in a similar task, reproducibility of the column is particularly important

since it is desirable for each column performing a single task to have

characteristics as similar to all of the other columns performing that task as

possible. Consequently, there is a substantial advantage in any group of

columns that are intended to cooperate in the performing of a separation of

samples closely related to each other, the permeable polymeric columns of this

invention have particular application.

To make a column or other device having a polymeric plug as a

separating medium without openings in the side walls or large openings in the

body of the plug, one embodiment of polymerization equipment includes a

temperature controlled reaction chamber adapted to contain a polymerization

mixture during polymerization and means for applying pressure to said

polymerization mixture in said temperature controlled reaction chamber. The

polymerization mixture comprises a monomer , polymer and a porogen. In a

preferred embodiment the means for applying pressure is a means for applying

pressure with a movable member. The polymerization mixture comprises includes a cross-linking reagent and a cross-linking monomer. The rigidity,

capacity and separation-effective opening distribution are controlled by the

amount of cross-linking reagent, monomer, and pressure.

The polymerization takes place in a closed container to avoid loss of

solvent in the case or an oven or to avoid dilution or contamination of the

mixture with water in the case of a water bath reaction chamber. Pressure is

applied during polymerization of some mixtures such as mixtures for ion

exchange columns to balance vacuum formed by shrinkage. The polymer plug

is washed after polymerization to remove the porogen. In the case of some

polymer, the plug may have a tendency to swell during washing or during a

chromatographic run if aqueous solutions are applied such as if the plug is a

reverse phase plug.

To separate a mixture into its components, a permeable polymeric plug

is formed as described above. It may be formed in a column of any size or

shape including conventional liquid chromatographic columns that are right

regular tubular cylinders, or capillary tubes, or microchips or having any

dimension or geometry. A sample is located in juxtaposition with the plug and

the components of the sample are separated one from the other as they are

moved through the plug.

For example, to separate proteins^' from a mixture of proteins in a sample

by liquid chromatography, a column is formed as a plug and polymerized in

place with a porogen. Shrinkage is compensated for before use. The sample

is injected into the column and a solvent caused to flow through the column, whereby the sample is separated into its components as it is carried through

the plug. In one embodiment, a plurality of samples are separated

simultaneously in separate columns with high reproducibility.

One embodiment of chromatographic columns used in separation

processes have a chromatographic casing with internal casing walls and have

a permeable monolithic polymeric plug in the casing walls. The plug is a

polymer having separation-effective openings which may be of a controlled size

formed in the polymer by a porogen in the polymerization mixture before

polymerization and controlled in size at least partly by pressure during

polymerization. The permeable monolithic polymeric plug has smooth walls

and substantially no pores within the permeable monolithic polymeric plug. In

the preferred embodiment, the plug is formed of vinyl polymers but may be

formed of others such as urea formaldehyde or silica. The may include surface

groups such as hydrophobic groups to reduce swelling with aqueous solvents

or hydrophillic groups to increase capacity.

Some examples of the proportions of the ingredients in polymerization

mixtures and the ingredients in different plugs are illustrative. For example, a

weak ion exchanger permeable monolithic polymeric plug that is free of

channeling openings is formed principally of methacrylate polymer.

Advantageously, his permeable monolithic polymeric plug is principally formed

of polymers of glycidyl methacrylate and of ethylene dimethacrylate in the ratio

by weight in a range of ratios between 1 to 1 and 7 to 3 and preferably 3 to 2.

A strong anion exchanger includes as its principal ingredients glycidyl methacrylate (GMA) and ethylene divinylmethacrylate (EDMA) in the ratio of a

value in the range of .8 to 1.2 to a value in the range of 2.8 to 3.2 and

preferably a ratio of 1 to 3. The polymerization solution in the preferred

embodiment includes 0.4 grams GMA, 0.5 grams of 2-(acryloyloxyethyl)

trimethylammonium methyl sulfate, 1.2 grams EDMA, 1.5 grams of 1 , 4-

butanediol, 1.35 grams propanol, 0.15 grams water and 0.02 grams AIBN.

A weak cation exchanger included a polymerization solution of methyl

methacrylate (MMA), GMA and EDMA in the ratio of a value of MMA in the

range of 4.5 to 5.5 to a value of GMA in the range of .8 to 1.2 to a value of

EDMA in a range of 11 to 13, and preferably a ratio of 5 to 1 to 12. The

polymerization solution includes 0.2 grams AA, 0.5 grams methyl methacrylate

(MMA), 0.1 grams GMA, 1.2 grams EDMA, 2.55 grams dodecanol, 0.45 grams

cyclohexanol and 0.02 grams AIBN. After polymerization, this column is

hydrolyzed in a 0.25 M sodium chloroacetate in 5 M (molar) sodium hydroxide

NaOH at 60°C for six hours.

Other examples are: (1) glycidyl methacrylate and of ethylene

dimethacrylate in a ratio by weight in the range of from 1 to 1 and 2 to 1 ; or (2)

divinylbenzene and styrene in a ratio in the range of 3 to 1 and 9 to 1 ; or (3)

divinylbenzene and styrene in a ratio in the range of 4 to 1 or with the amount

divinylbenzene being in the range of 35 percent and 80 percent by weight

divinylbenzene and preferably 64 percent by weight divinylbenzene; or (4)

divinylbenzene, styrene and porogen are in the ratio of 8 to 2 to 15 respectively;

or (5) divinylbenzene, styrene and dodecanol in a proportion within the range of 7 to 9 units of divinylbenzene to 1.5 to 2.5 units of styrene to 13-17 units of

dodecanol combined with an initiator; or (6) divinylbenzene, styrene and

dodecanol in the range of 8 to 2 to 15 respectively combined with an initiator;

or (7) divinylbenzene, styrene dodecanol and toluene in the proportions of 7-9

to 1.5-2.5 to 9-13 to 2.5-3.5 respectively, combined with an initiator; or (8)

divinylbenzene, styrene dodecanol and toluene combined in the proportions of

of 8 to 2 to 11 to 3 respectively, combined with an initiator; or (8) glycidyl

methacrylate, ethylene dimethacrylate, cyclohexanol and dodecanol in the

proportions of 0.5-0.7 to 0.3-0.5 to 1-2 to 0.1-2.5, combined with an initiator; or

(9) glycidyl methacrylate, ethylene dimethacrylate, cyclohexanol and

dodecanol in the proportions of 0.6 to 0.4to 1.325 to 0.175 respectively.

In general, a process of preparing a monolithic polymer support having

separation-effective openings for a targeted application may include the

following steps: (1 ) preparing a polymerization mixture with a selected formula;

(2) placing the mixture in a container, sometimes referred to as a column in the

embodiments of this inventions, with desired shape and size; (3) sealing the

column with pressurizing fittings or non-pressure sealing; (4) polymerizing the

polymerization mixture in a heating bath or oven with controlled temperature

under selected pressure or without pressure; (5) taking the columns from the

heating bath oroven and applying selected or specially designed fittings for the

desired function; (6) washing the porogens and soluble materials out of the

columns with selected solvent preferably by programmed flow; (7) in some

embodiments, pumping a formulated modification solutions to obtain the desired functionality for interaction; (8) performing special modification in a

heating bath or oven under controlled conditions; (9) washing the modification

solutions out of the columns preferably with a programmed flow; (10)

stabilizing, assembling and conditioning the column for its use at desired

conditions with high resolution; (11) characterizing the columns with sample

separation in the target application; (12) replacing the liquid in the column with

selected storage solution. Steps 7 to 9 ares optional or repetitive depending on

the functionality of the media to be used. Steps 1 to 5 are modified and

repeated in the two or multiple staged polymerization process.

In some of the embodiments of this invention, a polymerization mixture

includes single or plurality of: (1 ) monomers; (2) porogens; (3) initiators or

catalysts; and/or (4) additives or fillers (optional). The polymerization mixture

may be degassed with helium for more than 15 minutes, or by vacuum, or by

combination of both prior to be filled or injected to the column. The goal of this

degassing is to get rid ofthe oxygen inside the mixture. The oxygen can act as

an inhibitor or initiator at different situation resulting in some unpredictable

behavior of the polymerization, which is detrimental to the resolution and

reproducibility of the columns.

The suitable monomers for the above process comprise mono, di and

multiple functional monomers known in the art, preferably monomers containing

the vinyl or hydroxyl silica functional groups, which might be generated in situ

as an intermediate. The typical monovinyl monomers include styrene and its

derivatives containing hydroxyl, halogen, amino, sulfonic acid, carboxylic acid, nitro groups and different alkyl chain such as c4, c8, c12 and c18, or their

protected format which could be used to generate those functionalities before

or after polymerization; and include acrylates, methacrylates, acrylamides,

methacrylamides, vinylpyrolidones, vinylacetates, acrylic acids, methacrylic

acids, vinyl sulfonic acids, and the derivatives or these groups which could be

used to generate these compounds in situ. The mixture of these monomers can

be used. Siloxanes with hydroxyl group, vinyl groups, alkyl groups or their

derivative and mixture thereof are preferred. The amount ofthe monofunctional

monomers are varied from 2% to 60% of the total monomers in the

embodiments of this invention. They vary dramatically depend on the type of

media.

The typical di or multifunctional monomers are preferably the di or

multiple vinyl- containing monomers with a bridging moiety such as benzene,

naphthalene, pyridine, alkyl ethylene glycol or its oligoes. Examples of these

polyvinyl compounds are divinylbenzene, divinylnaphthalene, alkylene

diacrylates, dimethacrylates, diacrylamides and dimethacrylamide,

divinylpiridine, ethylene glycol dimethacrylates and diacrylates, polyethylene

glycol dimethacrylates and acrylates, pentaerythritol di-, tri-, or

tetramethacrylate and acrylate, trimethylopropane trimethacrylate and acrylate,

and the mixture of these compounds. Siloxanes with di, tri and tetrahydroxyl

groups, which are often generated in situ are also preferred in this invention.

The typical amount ofthe multifunctional monomers are from 40% to 80% in the

embodiments of this invention. The initiators comprise all the initiators known in the art such as azo

compounds, peroxides. Example of the typical intiators are

azobisisobutylonitrile, benzoyl peroxide, 2,2'-azobis(isobutyramide)dehydrate,

2,2'-azobis(2-amidinopropane)dihydrochloride. The typical amount of the

initiator is from 0.5% to 2% of the total monomers in the embodiments of this

invention. When siloxane is used, a catalyst such as an acid is used instead of

an initiator. The amount of catalyst is from milimoles to moles per liter of

polymerization mixture. Other approaches to polymerization without

incorporating an initiator in the polymerization mixture are known in the art and

can be used, such as radiation to form polymers.

The porogen is any material or compound which can be removed after

polymerization to generate separation-effective opening structures. The typical

porogens may be used are organic solvents, water, oligomers, polymers,

decomposable or soluble polymers. Some example ofthe organic solvents are

alcohols, esters, ethers, aliphatic and aromatic hydrocarbons, ketones, di, tri,

tetraethylene glycols, butane diols, glycerols or the combination of these

solvents. The choice of porogens depends on the separation-effective opening

size and separation-effective opening distribution needed.

In some embodiments, a single or a combination of porogenic solvents

which are mixable with the monomers and initiators to form a homogeneous

solution but have poor solvating power to the polymers formed is chosen. The

polymerization usually starts from the initiator. The formation of oligomers is

followed by crosslinking forming crosslinked polymer or nuclei, and the continuous growth of the polymer or nuclei. These polymer chains and nuclei

precipitate out of the solution at the size allowed by the solvating power of the

porogenic solvents. These polymer chains and nucleis are suspended in the

solution first and form small particle through collision and crosslinking. The

small particles are swelled by the porogens and monomers, and continue to

grow by both polymerization and aggregation with other nucleis or particles.

The larger particles aggregate together by collision and held in place by

crosslinking. The time and speed of the precipitation of the polymer and nuclei

dramatically affect the size of particles, aggregates or clusters and the

separation-effective opening size formed among these particles and

aggregates as well as the separation-effective opening size distribution.

It has been discovered that the combination of a very poor solvent and

a fairly good solvent are usually better to tune the solubility or swellability of the

polymer in the solution, which result in desired porosity and separation-effective

opening size distribution. The choice of poor solvent is more important since

generation of the large separation-effective opening is the most important.

After the generation of large separation-effective opening, it is always easier to

find a good orfairiy good solvent to tune the separation-effective opening size

down. It has been discovered that the alcohols or the neutral compounds

containing a hydroxyl group or multiple hydroxyl groups are the better choice

of the poor solvents for the media made of polystyrenes, polymethacrylates,

polyacrylates, polyacrylamides and polymethacrylamides. The solubility or

solvation power can be easily tuned to using alcohols of different chain length and number of hydroxyl groups. A good solvent for the polymers can be chosen

from many conventional good solvents such as toluene, tetrahydrofuran,

acetonitrile, formamide, acetamide, DMSO. They typical amount of the

porogens vary from 20% to 80%, more preferably 40% to 60% in the

embodiments of this invention.

The additives or fillers used in this invention are those materials which

can add a specifically desired feature to the media. One important characteristic

of polymers having separation-effective openings is the rigidity ofthe polymer.

Insoluble rigid polymer particles, silica particles, or other inorganic particles can

be added into the polymerization mixture to strengthen the polymer having

separation-effective openings after the polymerization. Polymers with a very

large number or amount of separation-effective openings usually do not have

good strength or toughness. They are fragile most of the time. The rigid

particles can act as framework for the polymers. In another embodiment, the

resolution ofthe large columns and reduce the problem of heat transfer during

the preparation of large diameter columns are reduced by adding very mono-

dispersed nonporous particles to the polymerization mixture for. Quite often, a

large diameter column is required for high flow preparative chromatography or

catalytic bed to allow high flow rate with only low back pressure.

In an embodiment of polymer having separation-effective openings,

mono-dispersed large non-porous particles or beads are packed tightly with the

pattern of close to dense packing. The polymerization mixture is filled into the

interstitial space ofthe large beads and polymerized in these spaces. The flow pattern and column efficiency are improved by the densely packed

monodispersed beads. Materials with a very large number of or amount of

separation-effective openings can be prepared in this large diameter columns

without fear of collapse of the media with low rigidity since the large

monodispersed beads are the supporting materials for the large columns. High

flow rate can be achieved owing to the large number or amount of separation-

effective openings but robust structure of the polymer.

In another embodiment, the heat dissipation problem is avoided in

preparation of the large columns with two or multiple staged polymerization

incorporating polymers having separation-effective openings as fillers. In one

embodiment ofthe invention, multiple thin columns having separation-effective

openings prepared from the same polymerization mixture are used as a filler

to reduce the heat dissipation problem during in situ preparation of the large

columns. In another emobodiment of the invention, a polymer rod is used as

the filler for the same purpose. In one embodiment of this invention, the filler

material is large non-porous silica beads.

The polymers, monomers, initiators, porogens, additives and

polymerization temperature are selected to improve the characteristics of the

column and may be used with an embodiment of polymerization using pressure

during polymerization or with other processes. Some of the aspects of this

process may be applied to monomers and polymers formed in a polymerization

reaction other than free radical reactions such as the polycondensation

reactions and sol gel process which form silica monolith. The column hardware in one embodiment ofthe invention includes rigid

tubes to be used as chromatographic columns, with various shapes including

cylindrical, conical, rectangular, and polygonal or an assembly of these tubes.

The tube may be made from any conventional materials know in the art

including metal, glass, silica, plastic or other polymers, more preferably the

stainless steel or glass. The inner dimension of this tube can be from

micrometers to meters in diameter, thickness, width, or depth. The permeable

solid material may span the entire cross-section area of the tube where the

separation of the samples take place by passing through the tube axially or

radially (Lee, W-C, et al, "Radial Flow Affinity Chromatography for Trypsin

Purification", Protein Purification (book), ACS Symposium Series 427, Chapter

8, American Chemical Society, Washington, D.C., 1990.) depending on the

mode of separation, more specifically the axial or direct flow chromatography

or the radial flow chromatography. The inner surface of the column or mold

with which the polymerization solution is in contact during polymerization may

be non-reactive or may be treated to increase adhesion to the surface of the

plug. The tube can incorporate any usable fittings know in the art to connect

it with other instruments, more specifically chromatography instruments.

In an embodiment of this invention, the monolithic permeable solid

polymer is formed in a capillary tube, which can be for example a capillary tube

with an internal diameter if 150 micons. In another, very significant,

embodiment, the monolithic permeable rigid material is formed and sealed,

often under pressure, in a removable 80. mm i.d. Teflon® sealing ring. This ring and column may be sold as a low-cost, reliable, high capacity, high resolution,

very fast, easily replaceable, replacement chromatographic colmn. In another

embodiment, the diameter of the tube is 10 mm. In another embodiment, the

tube diameter is 4.6 mm and the material is stainless steel. In another

embodiment of this invention, a plastic syringe barrel is used as a column. In

another embodiment, the monolithic matrix is formed in a mold containing a

metal container, a sealing plate and an insert with multiple cylindrical holes. The

thickness of the insert varies from 1 to 10 mm. A mold can be a micro device

with plurality of channels or grooves on a plate made of silica or rigid polymers.

The monolithic materials can be formed in any sizes and shapes make it

suitable for a specially designed micro-sized device, for example a micro-titer

plates with multiple wells containing the subject media and optionally having a

small elution port in the bottom. There is no limit for the designed shape and

size or the applications with these devices.

The polymerization mixture is filled or injected into a column with desired

shape and size depending on the final use of the product to be polymerized to

form a plug having separation-effective opening for use as a solid support.

Advantageously, the polymerization is done in a column in which the plug is to

be used such as a chromatographic column, catalytic bed, extraction chamber

or the like. In one embodiment ofthe invention, the positive pressure is exerted

to the polymerization mixture during polymerization to control the particle size

of the aggregates and to compensate for volume shrinkage during

polymerization. The particle size of the aggregate has been found to be more homogeneous and larger than that from non-pressurized polymerization. The

volume shrinkage during polymerization is compensated by a positive air

pressure or a moving piston with positive pressure.

More specifically, a polymerization mixture is applied to a column in the

preferred embodiment or to some other suitable mold. Polymerization is

initiated within the column or mold. The column or mold is sufficiently sealed

to avoid unplanned loss by evaporation of porogens or monomers if the

polymerization is in an oven, or to avoid contamination or dilution if

polymerization is in a water bath. During polymerization, pressure is applied

to the polymerization solution. Preferably the pressure is maintained at a level

above atmospheric pressure to control the size of the aggregates and its

distribution in the polymer, and to prevent the formation of voids on the polymer

wall surface and inside the media by shrinkage, and to prevent the media from

separating from the wall of the column which forms alternative fluid path

through the gap or wall channels, until polymerization has been completed.

Maintaining the column at atmospheric pressure to accommodate shrinkage did

not prevent the formation of voids in every case and provided poor

reproducibility. The pressure source can be a gas pressure, a pressure from

non-compatible liquid, a piston driven by air pressure, sprint force or hydraulic

pressure.

In one embodiment of the invention any number of pressurized molds

(tubes) can be kept at constant or controlled temperature in a single water bath,

and identically pressurized from a single (e.g. nitrogen, water, etc.) manifold. This increases both uniformity and speed of production.

In another embodiment of the invention, a selected pressure is exerted

on the polymerization mixture by high pressure nitrogen. In still another

embodiment of the invention, a selected pressure is exerted on the

polymerization mixture during polymerization by a pressurization device shown

in FIG. 2,3 or 4. The column is sealed in one end and the polymerization

mixture is filled into this column. The other end is sealed by the device shown

in FIG. 2,3 or 4. The whole assembly of the polymerization fixture including the

column is shown in FIG. 2,3 or 4. The pressure is applied to the polymerization

by a piston with a smooth Teflon plug driven by a hydraulic pressure from a

syringe pump. The polymerization mixture was sealed in the column by the

Teflon plug and an O-ring. When a constant positive pressure is applied to the

polymerization mixture, the actual pressure is the difference between the

hydraulic pressure and the friction. During the polymerization, the piston moves

into the column upon the conversion of the monomers to polymers to

compensate the voids generated due to the shrinkage of the polymers. This

prevents any negative pressure and void space generated inside the sealed

column due to this shrinkage thus improves the column efficiency.

It is believed that the shrinkage is in every direction. The resulting voids

are probably occupied by the nitrogen gas generated by AIBN or by solvent

vapor with negative pressure inside the column. The voids can be a large

irregular dents on the polymer wall or small irregular dents spreading the entire

polymer surface. The voids can also be distributed inside the polymer resulting in inhomogeneity of the separation-effective opening size distribution. These

irregular voids and gaps result in the wall effect or zone spreading of the

column. They are detrimental to the column efficiency and lower the resolution

of the column. These voids and gaps also result in low reproducibility of the

column performance from one to the other in the same batch of production or

from batch to batch of the productions.

In one embodiment of the invention, a selected pressure is exerted to

the polymerization mixture to control the size of the aggregates and the

separation-effective opening size distribution. The particle size changes with

the change ofthe pressure on the polymerization mixture during polymerization.

The particle size is larger at higher pressure. Under the positive pressure, the

shrinkage of the polymer during polymerization happens only at the direction

of the pressure force. This prevents the formation of voids inside the polymer

and the voids/gaps on the wall surface adjacent to the column wall.

During the polymerization process, the monomer concentration

continues to decrease with the increasing conversion of the monomers to

polymers. The crosslinked polymers continue to precipitate out of the solution

and aggregate with each other to form larger particles or clusters. These

particles precipitate and linked to each other by crosslinking agents such as an

active polymer chain with a vinyl group. These interconnected particles

sediment to the bottom of the column, which result in the lower monomer

concentration at the top part of the column.

The separation-effective opening size is highly affected by the total monomer concentration and their ratios. An in-homogeneous separation-

effective opening size gradient is formed along the direction of gravity, which

results in zone spreading. Since the particle size is partly controlled by the

pressure of polymerization, the gradient of separation-effective opening size

can be corrected by adjusting the pressure during the polymerization. In one

embodiment, the linearly increased pressure is exerted to the polymerization

mixture during polymerization. In another preferred embodiment, the step

pressure gradient is exerted to the polymerization mixture during

polymerization. The speed and pattern of increasing/decreasing the pressure

is chosen to control the particle size of the aggregates and its distribution

during the entire polymerization process. When a linear gradient of separation-

effective opening size distribution is desired, it can also be achieved by

changing the pressure during the polymerization with different speed and

different maximum pressure.

The polymerization temperature depends on the choice of initiator. When

AIBN and Benzoyl Peroxide are used, the typical temperature range is from 50

to 90 degree C. The heating source can be any known in the art. The preferred

ways are temperature controlled heating bath or oven. The reaction time can

be from 0.5 to 48 hours depending on the choice of initiator and reaction

temperature. In one embodiment of this invention, the polymerization is carried

out in a temperature controlled water bath at 60 °C for 20 hours.

Irradiation, such as IR, UV-vis or X-ray, is used as the source for

polymerization when light sensitive initiator is used. In one embodiment the reaction starts by thermal activation of the initiator. In another it starts by the

application of energetic radiation such as x-rays, either with or without a

chemical initiator. If x-rays are used the initiator should selected to thermally

activate at temperatures well above, the polymerization temperature. On the

other hand the initiator activates when under x-ray irradiation at temperatures

in the given region desirable for the reaction mass to receive activation. The

initiator is also selected so that activation time and temperature for dissociation

is considerably less than for the monomers alone. The production of active

initiator (free radical) is controlled only by the X-rays intensity. Since the X-ray

intensity is controllable, the reaction rate is controllable and won't "run away"

or overheat. Moreover, the initiator may be chosen to activate when under X-

ray irradiation at temperatures in the given region desirable for the reaction

mass to receive activation.

In one embodiment, x-rays are used as the energy source for

polymerization. Energy of the x-ray photons are varied with the preparation of

the polymers with difference thickness or cross-sections. Lower energy x-ray

is used for preparation of smaller diameter polymer rods and higher energy x-

ray or exposure to lower energy x-ray for a longer period of time may be used

for preparation of large diameter polymer rods. In the preferred embodiment,

the polymerization temperature is controlled by switching the x-ray on/off. When

x-ray is switched off, the polymerization is quickly shut off. within several

seconds since the lifetime of the free radical is typically around one second.

In one embodiment, photo initiator is used to initiate the polymerization using x-ray as the energy source. The photo initiators are the typical photo

initiators used in photo polymerizations- in the polymer field including γ-ray, x-

ray, UV, Visible and IR sensitive photoinitiators. The photo initiators include azo

compounds such as azobisisobutylonitrile, peroxides such as Diphenyl (2,4,6, -

Trimethyl Benzoyl) Phosphine Oxide, ketones such as phenanthrenequinone,

2-chlorothioxanthen-9-one, 4,4'-bis(dimethylamino)benzophenone, 4,4'-

bis(diethylamino)benzophenone, 4,4'-bis(dimethylamino)bezebophenone, 2-

chlorothioxanthen-9-one, Benzil Dimethyl ketal, Organic metallic complexes.

These photo initiators include the ones used in both cationic and free radical

polymerizations. In one embodiment, AIBN is used as the photo initiator. In

another embodiment, phenanthrenequinone is used as the photo initiator.

In one embodiment, x-ray sensitizer or scintillator is used in combination

with the photo initiator. The scintillators which can be used include all the

luminescence materials in the prior art. The scintillators include the compounds

containing benzene rings such as terphenyls, quarter phenyls, naphthalenes,

anthracenes, compounds containing heterocycles, compounds with a carbonyl

group, compoumnds with two or more lurorophors and organometallic

compounds, and inorganic compounds such as ZnTe, ZnSe, ZnS, Csl, Gd₂0₂S

and CaWO₄. In some embodiments, 2,5-diphenyloxazole (PPO), 2-phenyl-5-(4-

biphenylyl ) 1 ,3,4-oxadiazole (PBD), 2-(1-Naphthyl)-5-phenyloxazole (a-NPO

are used as scintillators. In one embodiment, terphenyl is used as the

scintillator. In another embodiment, ZnSe is used as the scintillator. The

mechanism of the initiation using the combination of scintillators and photoinitiators is believed to be a multiple step initiation process. First, the x-

rays activate the solvent molecules to form electronicvally excited solvent

molecules. The excited solvent molecules rapidly transfer their excitation

energy to. the scintillator forming electronically excited scintillator. Then, the

excited state of the scintillator relax to ground state by emission of photons.

The emitted photons were aborbed by the photo initiators and forming active

free radicals. The free initiator free radicals contact the vinyl function groups of

the monomers and start the polymerization process. The process can be

depicted as followings:

X-ray hγ - excited solvent molecule ^* - excited scintillator molecules^* - UV or

Visible hγ' - excited initiator molecules ^* - initiator free radicals - polymer

radicals

x-ray irradiated polymerization can be used for preparing homopolymers

such as polystyrene, polytetrahydrofuran, resins such epoxy or other

crosslinked materials, and porous polymer support such as separation media

in the shape of columns, membranes, or materials in any other housings. The

method can be used for preparation of molded parts in any shape. In one

embodiment, polystyrene is prepared by using x-ray irradiation as energy

source. Polystyrene is prepared in a mold of a glass vial with narrow opening

and in a shape of a cylinder. Both solution polymerization and bulk

polymerization are used to prepare homopolystyrene materials. In another

embodiment, polyglycidylmethacrylate resin is prepared by using the same method. In another embodiment, porous poly(styrene-co-divinylbenzene) is

prepared. In another embodiment, porous poly(glycidyl methacrylate-co-

ethylene glycol dimethacrylate) polymer support is prepared.

X-rays can be used for preparing porous monolithic polymer supports

with cross sections from micrometers to meters, and the particle-shaped

polymer supports as well. X-ray irradiated polymerizations are used to prepared

monolithic liquid chromatography columns including capillary and microbore

analytical columns, conventional analytical columns, and preparative and

process columns, In one embodiment, porous monolithic support in a glass

column with inner diameter of 1 cm is prepared. The column is characterized

by SEM, porosimetry and chromatography. The poly(styrene-co-

divinylbenzene) monolithic column in this glass housing shows excellent

separation of peptides. X-rays can penetrate the materials in depth. Both

organic and inorganic polymers can be prepared using x-ray or γ-ray. High

energy x-ray and γ-ray can travel the materials in high depth. If a greater such

depth is required, the unpolymerized mixture can be placed in the x-ray beam,

and rotated and translated in a way chosen that the overall, time-averaged

irradiation is sufficiently uniform throughout the body being polymerized. Note

that radiation aligned in the intended direction of chromatographic flow does not

cause significant chromatographic nonuniformity in the bed because of

absorbance of the radiation beam. Axial nonuniformities do not necessarily

degrade performance as radial nonuniformities do. Low energy x-ray

penetrates the materials in less depth but is safer to use. In one embodiment, low energy x-ray was used to prepare a porous monolithic support in a glass

column with the diameter of 3.5 cm. In another embodiment, a monolithic

support in a polymeric columns housing with 88 mm diameter is prepared. The

temperature of polymerization is controlled by controlling the intensities and

energies of the x-ray photons and by a water jacket so at least one side of the

reaction mixture. Preferably such water cooling cools the column in its axial

direction but not the radial direction to avoid thermal gradient and resulting

inhomogenuity. All flow paths experience the same inhomogenuity in the

direction of chromatographic flow, and thus have no effect upon the

separation.The polymerization temperature in the center of this large diameter

column is about the same as the polymerization temperature on the edge ofthe

column during the whole polymerization process. The conversions of the

monomers are completed after 4 days of polymerization.

X-ray irradiated polymerization can be combined with thermal

polymerization. Polymerization rate and porosities can be controlled by the rate

of polymerization, which can be controlled by the energies and intensities of x-

ray, and the temperature of polymerization. In consideration of the economy

and safety of irradiated polymerization process, lower energy and intensity x-

ray is preferably used in preparation of large diameter monolithic polymer

support. The conversion of monomers is more than 70% complete after 48

hours of polymerization in the 3.5 cm diameter column. In order to speed up the

polymerization rate and finish the conversion in a short time, thermal

polymerization at desired temperature corresponding to the initiators is used. In one embodiment, porous poly(styrene-co-divinylbenzene) is prepared by

using x-ray irradiation followed by thermal polymerization at 70 °C after the x-

ray irradiated polymerization. The porosity ofthe polymer can be controlled by

monomer compositions, x-ray intensity and energy, choice of initiator and

scintillator combination, and the polymerization temperature in both x-ray

irradiated polymerization and thermal polymerization. The porogenic solvents

used in this polymerization process is the same as those used in pure thermal

polymerization described earlier. Excellent polymer support is obtained for

liquid chromatography use.

Ultraviolet and visible light radiation has been used in the past for

preparing polymers including homopolymers, slab-shaped polymer resins and

porous polymer surface coatings but not the polymer materials in this

invention. Both UV and visible light can penetrate thin, solid, clear materials.

Ultraviolet and visible lights have been used to prepare thin, clear materials

such as membranes. The homogeneity of a porous polymer becomes more of

a problem when the thickness of materials increases. This is due to the

scattering and attenuation of lights through the porous solid materials. The

depth that the light can travel through the solid materials decreases quickly

depending on the functional groups and the refractive indexes ofthe materials.

Consider an acrylic, dry porous column, made of two phases: acrylic resin

refractive index about 1.5 and air with refractive index of 1.00. This scatters

light so completely that this material looks like chalk, white and opaque.

Heretofore then these apparent differences have prevented the use of UV or visible light for polymerization. This patent application discloses a method of

using ultraviolet and visible lights to prepare polymer materials including

materials listed above with high homogeneity. This patent application also

discloses a method of preparing polymer materials many inches thick.

In this invention, a solvent of very similar or the same refractive index to

the targeted polymer resins at the selected wavelength of light is chosen for the

polymerizations. The lights at the selected wavelength can travel though the

polymers during the polymerization due to the little used Christiansen Effect,

wherein the targeted porous resin becomes quite translucent or transparent.

Light in a particular wavelength range show transparency because the pores

are full of solvent. . Preferably, monochromatic light, of which the indices ofthe

refraction in both the polymer solid and solvents are very close, is chosen for

the polymerization. An initiator activated by light rather than heat is used with

a photoinitiator. If white light is used, the transmitted light corresponds to the

particular wavelength at which the refractive indexes ofthe light in both polymer

solid and the solvent or solvent combinations are very close. A photo initiator

is chosen to absorb and be activated by light in the near transparent polymer

and solvent wavelength range. The initiator absorbs this wavelength range.

Preferably, when a photoinitiator is activated it breaks up onto fragments

lacking in the original chromophores. Such fragments have no absorption in the

range of selected wavelength of the light. When a light ray is absorbed by the

chromophore in a photo initiating molecule the result is local polymerization at

the surface. If the chromophores survive this reaction they will be there to block the next ray of light from activating the next photo initiator molecule, hence

limiting polymerization to the original surface. Therefore, the light can travel

through the polymer mixture in more depth and the transmission of the light

increases. This effect is known as Bleaching Effect. It has been found that a

good photo initiator in this respect is 2,4,6-trimethylbenzoyldiphenylphosphine

oxide. The bleaching effect and the Christiansen effect are synergistic in the

production of very thick layer, or deep, porous polymers by photoinitiation ore-

ray initiation. In the latter case x-rays excite the solvent which then transmits

secondary energy to a so-called "sensitizer" which then fluoresces to activate

the initiator. It can also be seen that if the solvent when excited by x-ray also

in itself fluoresces, an additional degree of synergy exists. However, often the

use of p-terphenyl as a sensitizer for x-ray polymerizations. Fluorescing

solvents that are used include many aryl compounds such as toluene, o-, m-,

or p-xylene, or the 1 ,2,3 (or other isomers of) mestylene. In one embodiment,

homo-poly(glycidyl methacrylate) is prepared by ordinary solution

polymerization using o-xylene as the porogenic solvent. The refractive indexes

of 0-xylene, the monomer and especially the polymer are close. The resulted

polymer in solution is close to transparent but a little translucent due to minor

scattering of the white light.

The refractive indexes of target polymers are measured. Both the good

and poor solvents are selected to have the refractive indexes close to the

refractive indexes of the target polymers. By tuning the ratio ofthe solvents, the

refractive indexes of the solvent mixture will be almost the same as the polymer. Therefore, the transmission of the light through the polymer swelled

by the solvents can reach the maximum. In one embodiment, porous

polymethacrylate based monolith is prepared by using the combination of

solvents.

The resulting permeable polymer may have more than one suitable

configuration. One has a desired separation-effective opening size distribution

for target applications. In general, it includes small separation openings less

than 300 nm in at least one direction which provide high surface area for

separation, and large separation openings such as larger than 500 nm for the

majority mobile phase to go through. Preferably the sizes of large separation-

effective opening are between 2 to 5 microns for medium and low pressure

separation media, and 0.6 to 2 microns for high and medium pressure

separation. The separation-effective opening size distribution and the irregular

feature size distribution can be controlled by the types and amount of porogens,

monomers, initiators and polymerization temperature, time and pressure. In one

embodiment, the monomers are selected not only to have desired functionality,

but also to help improve the column efficiency by changing the kinetics of

polymerization and polymer structure, which leads to more ideal separation-

effective opening size distribution. The ratio of monomers is selected for the

same purpose. The type and amount of porogens are selected after careful

investigation forthe generation ofthe desired separation-effective opening size

distribution. The use and selection of pressure during polymerization is

particularly important for the generation of the desired separation-effective opening size distribution and the homogeneity of the separation-effective

opening size distribution through out the whole column. It also prevents the

formation of irregular voids and wall channels which happened in conventional

sealed or open polymerization.

It has been found that the formation of micropores can be drastically

reduced or prevented by using pressure during polymerization and the careful

tuning of the other polymerization conditions and reagents. From Scanning

Electron Microscopy (SEM) studies of this polymer having separation-effective

openings, it is found that the polymer particle morphology is similar to some

form of coral. The coral-like polymer is formed of interconnected corrugated

particles which apparently have grown by accretion. The interior of these

particles are non-porous. The surface is highly corrugated and contains huge

number of small open shallow grooves with various sizes of openings. In SEM

study of one of the polymers the particles are core-shell structured with short

depth ofthe openings. These unusual corrugated polymer structures and core-

shell particle structures without through pores in the individual particles greatly

improve the capacity of the monolith compared to regular non-porous

separation media while avoiding the mass transfer problem in conventional

macroporous media containing a large number of micropores and mesopores

inside the particle or beads. These structures are also dramatically different

from so called "Perfusion Beads" or monoliths with though pores disclosed in

the literatures. The aggregates of particles in some columns conditioned by the

application of pressure or by holding the volume against expansion when internal forces tend to swell or expand the polymer results in stacked generally

flat or nested configuation rather than the aggregated substantially round

aggregates of particles.

The monolith in this invention combines the advantages of high

resolution in non-porous particle packing and the high capacity of macroporous

packing while avoiding their problems. These corrugated particles may grow by

accretion from polymer nucleus which are swelled and surrounded by

monomers and active oligomers, and merging with other polymer nucleus.

These particles aggregate with each other and are reinforced by crosslinking.

This structure improves the column efficiency greatly by prevention of the

trapping of sample molecules in the micropores, and in the pores inside the

particles, which are one ofthe major reasons for zone spreading according to

the theoretical model. The size of the particles, aggregates or clusters can be

finely tuned to be more homogeneous, and the separation-effective opening

size distribution can be improved to give high resolution separation by careful

control of pressure in combining with the selections of other factors discussed

earlier.

Satisfactory separation of more hydrophilic compounds in liquid

chromatography often requires the starting mobile phase to be 100% water with

no organic solvent. This type of separation can not be achieved to a

satisfactory extent with reversed phase media based on pure

styrene/divinylbenzene, polymethacrylates and their derivatives containing C4,

C8, C12 and C18, which are very hydrophobic. The polymers shrink in aqueous mobile phase with low organic solvent contents. The shrinkage results in void

space between the column wall and the polymer media, which leads to so

called "wall channeling" in chromatography. As the consequence, the sample

and mobile phase bypass the media and go through wall channels instead of

the media. The sample is not retained or partly retained which results in

multiple peaks for each pure sample.

In one embodiment, wall effect is avoided by first shrinking the media

down to the maximum extent by passing pure water or salt solution through the

media, and then followed by compression ofthe media with piston fittings until

the void space between the wall and polymer media is sealed. The piston is

held in place by a nut fitting. The fittings are shown in FIGS. 3 and 4. The

shrinkage and sealing ofthe wall void can be monitored by first decreasing then

increasing the back pressure of the column. This process prevents the

formation of the "wall channeling" during the separation process. In one

embodiment, the polymer is shrunk in pure water and compressed with PEEK

piston fittings. In another embodiment of this invention, the polymer is shrunk

in 1 mol/l NaCl solution and compressed with PEEK piston fittings.

In highly polar environments, the linear polymer chain, and the chains

of C4, C8, C12, C18 on the polymer surface collapse to the surface resulting

in poor interaction between the sample molecules and the surface ofthe media.

Also, the bed is very poorly wetted by the mobile phase due to the extreme

difference in polarity of the media and mobile phase. The mass transport and

interactions between the sample and stationary phase is very poor, which result in low column efficiency and resolution. These problems and the problem of

"wall channelling" are reduced by increasing the hydrophilicity of the polymer

matrix while maintaining the desired hydrophobicity for sample retention and

separation.

The polymer matrix contains hydrophilic functional groups such as

hydroxyl, amide, carbamide or hydrophilic moieties in the polymer repeating

units. The polymer can be wetted and swelled by water due to the hydrophilicity

of the polymer matrix. The surface of the polymer media still contain highly

hydrophobic polystyrene chain, or C4, C8, C12 and C18 chains for the

hydrophobic interation in reversed phase chromatography. The shrinkage ofthe

polymer in water is reduced or completely prevented, which also solve the

problem of wall channeling. The hydrophilicity can be improved by direct

copolymerization of monomers containing hydrophilicity moieties, or by

modification of polymers to incorporate the hydrophilic moieties.

In one embodiment of this invention, hydrophilic hydroxylethyl

methacrylate is copolymerized with styrene and divinylbenzene for the

prevention of "wall channeling" and collapse of the hydrophobic interaction

chains in water. In one embodiment of this invention, stearyl acrylamide is

copolymerized with stearyl methacrylate and ethylene glycol dimethacrylate.

The addition of hydrophilic monomers also may decrease protein denaturation

in reversed phase columns. In one preferred embodiment, acrylonitrile is

copolymerized with styrene and divinylbenzene. The polymers swell more and

more with the increase of the hydrophilicity of the hydrophilic monomers. The reverse phase separation media constructed with the polymer containing

hydrophilic moieties will swell in both aqueous and non-aqueous solutions. This

enlarges the applicability of reversed phase separation media in aqueous

mobile phase.

In Situ polymerization can be used to prepare the columns with any sizes

and shapes. In one embodiment, a capillary column with cylindrical shape of

inner diameter of 75 μm was prepared. In another embodiment, a column with

4.6 mm ID was prepared. In another embodiment, a column with 88 mm ID was

prepared. In another embodiment, a square capillary column with cross-section

of 100 μ and on up to 700 μm was prepared. In another embodiment, a

polymer disk with 3 mm thickness and 1 cm inner diameter was prepared. In

another embodiment, a donut shape monolith with 1 cm outer diameter and 4.6

mm inner diameter was prepared. In another embodiment, a microchip column

with 100 μm inner diameter grooves was prepared.

Hydrophobic interaction chromatography requires very hydrophilic

separation media with mild hydrophobicity. Upon further increase of the

hydrophilicity of the matrix with less hydrophobic carbon chain or polymer

chain, the reversed phase media can be turned into hydrophobic interaction

media.

Normal phase chromatography requires hydrophilic media, whose

surface is fully covered by hydrophilic functional groups. Hydrolysis of the

epoxy group in poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate)

was used in prior art to obtain normal phase separation media. The Normal Phase separation media is prepared by in situ direct polymerization of

hydrophilic monomers containing hydrophilicfunctional groups such as hydroxyl

and amide. In one embodiment hydroxylethyl methacrylate is copolymerized

with a crosslinker, such as EDMA, to obtain normal phase media. In one

embodiment of this invention, the column hardware is a polypropylene barrel

reinforced with glass fibers.

The reversed phase monolithic media prepared according to prior art has

extremely low capacity and is compressed during separation. The loading

capacity of the liquid chromatography media and the rigidity of the media is

increased by increasing the crosslinking density ofthe media. The crosslinking

density is increased by using higher amount of crosslinker, such as

divinylbenzene, in poly(styrene-co-divinylbenzene) monolith. In one

embodiment of this invention, 100% of divinylbenzene (80% purity. The rest of

them are mostly ethylstyrene. It is the highest purity grade available

commercially.) in total monomer is used. The capacity is six times higher than

the monolith prepared in the prior art. The high capacity monolith prepared

under pressurized polymerization has high resolution as well as high capacity.

In one embodiment of this invention, 90% of the divinylbenzene (80% purity)

in total monomer is used. In another embodiment, 80% of the divinylbenzene

(80% purity) in total monomer is used.

Another method of improving the rigidity and resolution of the media is

by increasing the total polymer density in the column. The total polymer content

is increased by increasing the total monomer content in the mixture. By increasing the total monomer content in the polymerization mixture, the

resolution of column is improved as well. The separation-effective opening size

and its distribution are highly affected by the total monomer concentration in the

polymerization mixture. In one embodiment of this invention, 46% weight

percent of total monomers is used to improve the rigidity and resolution of the

media.

The monolithic media prepared in the prior art has poor resolution, low

speed of separation, low rigidity and extremely low capacity. The prio r- a rt

monolithic polymethacrylate based weak anion exchanger was prepared by

modification of poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate)

with neat diethylamine. It swells extensively in water and can not be used at

high flow velocity. This medium has very low rigidity and is not stable at flow

velocities more than 6 cm/min. The back pressure of the column keeps

increasing during the runs. Two methods of improving the rigidity of the

hydrophilic medium are provided.

First, the rigidity of the medium can be improved by increasing the

crosslinking density ofthe polymer matrix. At low crosslinking density, there are

a lot of non-cross linked linear polymer chains which are solvated by water and

extend out to the solvents. The polymer matrix expands due to this extensive

solvation. The expansion of polymer matrix in a polymer narrows the size of

the separation-effective openings and interstitial spaces between the

interconnected particles. The porosity is also decreased. These result in high

back pressure. The highly solvated porous polymer has characteristics of soft gel. Under a pressure, the soft polymer can be compressed easily and leads

to higher pressure. The increase of pressure will further compress the medium

and lead to even higher pressure. The cycle of pressure increase and

compression make the prior art monolith not useful for the application in high

speed separation.

Upon the increase of crosslinking density, the swellable linear polymer

chains are shortened and the swelling is reduced. The polymerwith separation-

effective openings becomes more rigid. The structure with separation effective

openings is maintained in aqueous solvents. High porosity and larger

separation-effective opening size can be obtained with highly crosslinked

polymer matrix, which allows higherflow rateto be used without the detrimental

cycles of the compression and pressure increase. High speed separation can

be achieved by using high flow rate.

In one embodiment of this invention, the cross linking density of the ion

exchanger is greatly improved by using 70% crosslinker, ethylene glycol

dimethacryalte (EDMA). In a preferred embodiment, the amount of EDMA is

50%. In another preferred embodiment, the amount of EDMA is 60%. EDMA

is more hydrophobic than its copolymers containing ion exchange groups. By

increasing the amount of EDMA, the hydrophilicity of the polymer matrix is

reduced. This results in decrease of swelling and improves the rigidity. This is

the additional advantage from using more hydrophobic cross linker instead of

hydrophilic crosslinker when high rigidity of the polymer in aqueous phase is

highly desired. Second, the rigidity and column efficiency of the polymer separation

media are both improved by controlled modification in this invention. The

glycidyl methacrylate (GMA) is hydrophobic before it is modified to contain ion

exchange functional groups. The GMA in prior art monolithic weak anion

exchanger (WAX) is modified by reaction with neat diethylamine at 60 °C for 3

hours. Neat diethylamine can swell the polymer and diffuse into the polymer

particles to access the GMA epoxide groups. This modification reaction

modifies not only the GMA moieties on the separation-effective opening

surface ofthe polymer but also those inside the polymer matrix. The hydrophilic

moieties containing the ion exchange functional groups intermingle with

hydrophobic backbone both inside and outside the separation-effective

openings after modification. This non-selective modification makes the whole

polymer matrix swell extensively in water while some hydrophobic backbones

are exposed on the surface of the separation effective openings. The

hydrophobic patches on the surface of the separation openings can result in

secondary hydrophobic interaction during ion exchange chromatography

separations, which leads to zone-broadening.

A controlled modification of the GMA on the surface of the polymer

particles can keep the internal part of the particle more hydrophobic and less

swellable in water. After modification of the surface GMA, the surfaces of the

particles become much more hydrophilic and attract the water molecules.

Those more hydrophobic backbones retreat to inner part of the polymer

particles to stay with more hydrophobic cores of the particles, and get away from the very polar buffer environment during chromatography separation. This

increases the coverage of surface with hydrophilic ion exchange groups and

prevents the zone-broadening from secondary hydrophobic interaction. The

controlled surface modification is accomplished by catalyzed modification

reaction in aqueous solution at lower temperature. The catalyst is preferably an

acid or reagent which can generate protons in situ.

In the preferred embodiments of this invention, a dialkyl amine hydrogen

chloride salt is used as a catalyst. The salt solution is very polar and has less

tendency to swell the hydrophobic polymer. The ionic catalyst tends to stay in

solution instead of diffusing into the very hydrodrophobic internal matrix. The

lower reaction temperature reduces the swellability of the polymer. Diethyl

amine might diffuse into the polymer matrix but the reaction of diethyl amine

with GMA at low temperature is very slow and insignificant. In one embodiment

of this invention, the dialkyl amine hydrogen chloride catalyst is diethyl amine

hydrogen chloride. In another embodiment of this invention, the catalyst is

trimethyl amine hydrogen chloride. In one embodiment of this invention, the

reaction temperature is 25 °C. In another embodiment of the invention, the

modification temperature is 30 °C.

The prior art processes for the synthesis of monolithic weak cation

exchangers are based on membrane, beads and gels and none of them can be

used directly in the in situ preparation process of monolithic columns. Seven

methods for preparing a weak cation exchanger a described below. . All the

modification reactions in these methods are carried out by pumping the modification solution through the column continuously at a selected

temperature, or the reagents are sealed inside the column and heated in a

heating bath or oven after the reagents are pumped into the column.

The first method is the two-step modification of poly(glycidyl

methacrylate-co-ethylene glycol dimethacrylate) (PGMAEDMA) with

chloroacetate salt. Sodium chloroacetate has been used to modify

hydroxylethyl methacrylate based material to obtain carboxylic acid groups in

the literature. In order to take advantage of the above reaction for the

modification of monolith , the epoxide ring in GMA is first opened to obtain

hydroxyl group by hydrolysis using 1 M H₂SO₄ aqueous solution. In the second

step, chloroacetate couples with the hydroxyl group in the polymerto attach the

carboxylic group to the polymer. This reaction is catalyzed by strong base such

as sodium hydroxide. The reaction temperature is from 40 to 80 °C, preferably

from 50 to 70 °C. The reaction time is varied from 1 to 24 hours, preferably less

than six hours. In one embodiment of this invention, the modification reaction

takes place by pumping 5 M sodium hydroxide aqueous solution through the

monolithic column at 60 °C for 2 hours. The capacity of this media is not ideal

although the column efficiency is good. The capacity can be increased by

longer reaction and higher reaction temperature. However, the separation

medium becomes soft due to the side hydrolysis reaction of the esters in the

polymer. The crosslinking density is lowered since the crosslinker EDMA is

hydrolyzed as well.

The second method is a one-step modification of the GMA in the polymer to obtain the carboxylic functional groups using glycolic acid as

reagent. Glycolic acid is reacted with PGMEDMA at temperature between 40

to 90 °C for 1 to 24 hours. This reaction is a self catalyzed reaction since

glycolic acid is a catalyst itself. The reaction can be catalyzed by other stronger

acid such trifluroacetic acid (TFA). The reaction is simple but the capacity ofthe

weak cation exchanger is low due to a parrarel side reaction. The epoxide ring

can be opened by hydrolysis reactions as the side reaction. In order to prevent

this reaction, the non aqueous solvent is used. Preferably, solvent containing

protons is used. In one embodiment of this invention, glycolic acid solution in

formic acid containing TFA catalyst is pumped through the column for 3 hours

at 80 °C.

The third method is a double modification of the PGMEDMA with both

glycolic acid and choroacetate. There are several advantages of the double

modification reactions. First, they can all be performed in aqueous solution;

Second, both reaction steps lead to desired product; Third, the side reaction of

the first step leads to the desired functional group for the second step

modification; Fourth, the conditions of double modification reaction can be

milder than the single reaction to obtain the same or higher capacity while

avoiding the hydrolysis of the backbone which maintains the rigidity of the

matrix. In one embodiment of this invention, the first reaction is performed in

glycolic acid aqueous solution containing TFA as catalyst, and the second

reaction is the substitution reaction of chloroacetate by NaOH aqueous

solution. The fourth method is a one-pot reaction of glycolic acid and

chloroacetate. Instead ofthe double sequential reactions, both reagents are put

into the solution together during reaction. The reaction with glycolic acid is

base-catalyzed instead of acid-catalyzed. This method has the the advantage

of the third method but with lower capacity due to less reactivity of the base-

catalyzed ring-opening reactions by glycolic acid in water.

The fifth method is a hydrolysis reaction of acrylates or methacrylates.

The hydrolysis of the ester groups leads to the carboxylic functional groups.

The direct hydrolysis of PGMEDMA membrane or beads is known in the prior

art. However, the resulting media did not have either good capacity or

separation. It is discovered in our work that both the resolution and capacity

can be dramatically improved by hybriding the hydrophilic and hydrophobic

acrylates or methacrylates. The hydrolysis reaction is much more efficient since

the water molecule can diffuse into the surface of the particles and wet the

surface much better due to the hydrophilic moieties of the acrylates. The

reaction can be catalyzed by both acid and base, such as TFA or NaOH

aqueous solution. In one embodiment of this invention, poly(methyl

methacrylate-co-hydroxylethyl methacrylate-co-ethylene glycol diemthacrylate)

is prepared and hydrolyzed to obtain weak cation exchanger. In another

embodiment of this invention, poly(hydroxylethyl methacrylate-co-ethylene

glycol dimethacrylate) is hydrolyzed. In another embodiment of this invention,

PGMEDMA is hydrolyzed by acid first and base in the second step. The weak

cation exchanger obtained in this method is softer due to the hydrolysis of the backbone crosslinker.

The sixth method is a direct copolymerization of acrylic or methacrylic

acid. The direct copolymerization of the acid leads to the weak cation

exchanger in one step. This method greatly simplifies the preparation method.

The capacity of the weak cation exchanger is relatively higher than the

modification method but still not ideal. The ratio of the acidic monomer to

crosslinking monomer is between 2% to 30%, preferably 5% to 15%. With the

higher content of the acid monomer, the capacity is higher but the media is

softer. The direct polymerization method is applicable to the preparation of

monolithic membranes, columns, chips, tubes or any format known in the art.

The seventh method is the combination of direct copolymerization and

the controlled modification. It was discovered in this invention that this

combination leads to high capacity while maintains the rigidity of the media. The

resulting media can be used for high-throughput separation using high flow

velocity. The improved capacity by direct polymerization of acrylic or

methacrylic acid reaches a limit due to the softness of the media containing

high amount of the acid. The acidic monomers are randomly polymerized and

dispersed throughout these matrixes. These acids are converted to salts in

buffer and resulted in extensive swelling ofthe media in aqueous mobile phase.

Also, the hydrophobic backbone consists of carbon chains and esters are

exposed on the surface resulting in secondary hydrophobic interaction during

ion exchange chromatography separations. This leads to zone-broadening and

tailing. The hydrophobic surface can be further modified to become hydrophilic while improving the capacity. The controlled modification improves the capacity

and hydrophilicity ofthe media while preventing the softness ofthe media. Over

modified media will leads to the modification inside the particles besides the

modification on the surface. As discussed in the preparation of weak anion

exchanger, the modification inside the particle results in extensive swelling

which blocks the separation-effective opening or changing of the morphology

which leads to detrimental cycle of pressure increase and compression. In one

embodiment of this invention, weak cation exchanger is prepared by

copolymerization of acrylic acid, methyl methacrylate (MMA) and EDMA in the

first step, and hydrolysis of the methyl methacrylate in the second step. The

hydrolysis of MMA is base-catalyzed and accelerated by the presence of very

hydrophilic acrylate salt, which is the conversion product ofthe acrylic acid after

reaction with NaOH.

This methodology of combining direct polymerization and modification

is applicable to the preparation of all hydrophilic polymer supports which require

high number of functional groups and rigidity of the matrix at the same time. It

is applicable in preparation of monolithic tubes or columns, monolithic

membranes, monolithic capillary and chips or any other monolith in different

format, as well as polymeric particles, gels, membranes or any other type of

polymeric separation media. In particular, the resolution and capacity of

monolithic membrane can be improved with this method. The improvement can

be achieved by in situ process of by off-line process. The monoliths or beads

obtained by direct polymerization can be modified by pumping the reaction solution through the packed columns or membranes, or immerge them into the

modification solution. The beads can be suspended in the modification solution.

By using this methodology, we developed high capacity strong anion exchanger

(SAX) and strong cation exchanger (SCX).

The prior art processes for making monolithic strong anion exchangers

are based on membrane, beads and gels and are not applicable to the

manufacture of monolithic strong anion exchange column. Three methods of

preparing monolithic strong anion exchange column in situ are provided below.

Method one is the combination of highly rigid polymer and controlled

modification of the surface. Modification of PGMEDMA with trimethylamine

hydrogen chloride has been used to obtained membrane and bead based

strong anion exchanger. When the reaction is used for preparation of monolithic

strong anion exchangers, the resulting media is soft and can not be used for

high speed separation. The rigidity is improved in two ways as in the

preparation of monolithic weak anion exchanger in this invention: High

crosslinking density and Controlled Modification reaction.

The basic polymer for SAX is formulated to contain high crosslinking

density by using higher ratio of crosslinking monomer to functional monomer.

The amount of crosslinker is increased to more than 50% of the total monomers

in the polymer. In one embodiment of this invention, 60% EDMA in total

monomers is used. The porogens and their ratios are selected to offer optimal

resolution at relatively low pressure.

The controlled modification is accomplished by catalyzed amination of the PGMEDMA. The catalyst can be any base known in the art. In one

embodiment of this invention, trimethyl amine is used as catalyst. The amount

of the catalyst is from 1% to 50% volume of the solution, preferably between

10% and 30%. The reaction temperature is between 10 to 60 °C, preferably

between 20 to 50 °C. The reaction time is between 10 minutes to 24 hours,

preferably between 1 to 4 hours. The selected catalytic reaction modifies the

surface ofthe particles more than the internal part ofthe particles, which results

in the media to be used at high flow rate. In one embodiment of this invention,

the reaction is carried out at 40 °C for 3 hours.

Method two is the direct copolymerization of monomers containing the

quarternary amine, or their intermediate which can generate the quaternary

amine in situ. In one embodiment of this invention, the functional monomer

containing quaternary amine is 2-(acryloyloxyethyl] trimethylammonium methyl

sulfate (ATMS). The polymer has high crosslinking density. The ratio of the

crosslinking monomer in the total monomers is between 50% to 70%. In one

embodiment of this invention, 60% EDMA is used. The amount of ATMS is

between 2% to 20%, preferably between 5% to 15%. The third monomer which

makes up the rest of monomer is preferred to be hydrophilic monomers such

as HEMA although hydrophobic monomer can be used as well.

Method three is the combination of direct polymerization and controlled

surface modification shown in method two and one. The strong anion

exchanger obtained by method one is rigid and have high resolution. However,

it suffers from non-ideal capacity. Method two improves the capacity but not sufficient and suffers from lower resolution. The combination of direct

polymerization and controlled surface modification doubles the capacity and

improves the resolution and recovery. The recovery of proteins is improved

since the surface is fully covered by hydrophilic protein benign groups.

Secondary hydrophobic interaction, which is the main reason for lower protein

recovery, is minimized. The porogens are researched and selected to offer the

desired flow rate. In one preferred embodiment of this invention, the

combination of butanediol, propanol and water is used as porogens. The

polymerization mixture and conditions are formulated to offer the optimal

resolution at the desired flow rate.

Prior art processes for preparing monolithic strong cation exchangers are

based on membrane, beads and gels and are not transferable to the in situ

preparation of monolithic strong cation exchange columns. Three methods for

preparing monolithic strong cation exchange columns in situ are provided

below.

Method one is the modification of PGMEDMA with butane sultone or

propane sultone catalyzed by strong base soluble in organic solvent.

Modification of PGMEDMA with propane sultone using NaOH solution as the

catalyst has been used to prepared membrane or bead-based strong cation

exchanger. The reaction was a two-phase reaction since propane sultone is not

soluble in NaOH aqueous solution. The two-phase reaction mixture can not be

pumped through the column to carry out the modification. Several approaches

have been taken to carry out the modification reaction. Approach one is a two- step modification reactions consist of activation of the media with strong base

such as potassium t-butoxide in the first step followed by nucleophilic ring-

opening reaction with butane sultone. Butane sultone is preferred since it is a

liquid but propane sultone is a solid at room temperature. Potassium t-butoxide

is preferred since it has higher solubility than it sodium counterpart. The solvent

is a good solvent of the reagent such as dimethylsulfone (DMSO). The

modification solution has to be homogeneous in order to be pumped through

the column for in situ modification. Approach two is a one-pot reaction consist

of both activation and modification steps. Both strong base and butane sultone

are dissolved in a strong solvent. The solution is pumped through the column

continuously or sealed at a selected temperature for several hours. The

reaction temperature is preferably between 80 and 120 °C. In one embodiment

of this invention, 90 °C is used. In another embodiment of this invention, 120

°C is used.

Method two is a direct polymerization of monomers containing strong

cation exchange group. In the preferred embodiment of this invention, 2-

Acrylamido-2-methyl-1-propanesulfonic acid (AMPS) is used as the functional

monomer containing sulfonic group. The amount of AMPS is between 2% to

20%, preferably 5% to 15%. The capacity of this polymer is greatly improved

comparing to the first method. The polymerization mixture is formulated to offer

the optimal resolution at desired flow rate.

Method three is the combination of direct polymerization and controlled

modification. In one embodiment of this invention, AMPS is copolymerized with GMA and EDMA. The polymer is further modified by controlled modification as

described in method 1. Both the capacity and resolution is greatly improved

comparing to the method 1 and 2. The amount of EDMA is preferably to be

between 50% to 70%. The amount of AMPS is preferably between 2% to 15%.

The rest ofthe monomer is GMA. In another embodiment of this invention, the

AMPS is copolymerized with HEMA and EDMA. The porogens are researched

and selected to offer the desired flow rate. The polymerization mixture and

conditions are improved to offer high resolution and high speed

chromatography.

Preparation of large diameter monolithic columns for effective

chromatography separation by in situ polymerization method has been a very

difficult task due to the heat isolating effect of polymer formed by exothermic

polymerization. It was found that the heat transfer is fast enough to prevent the

inhomogeneity of the polymerization temperature as long as the shortest

distance (defined as radius in this writing) between the center of the monolith

to the surface ofthe monolith is less than 8 mm depending on the materials of

column hardware.

To prepare large diameter monolithic columns, with reduced heat of

polymerization problems, multiple staged polymerizations are used. The

resulting polymer monoliths in each stage of polymerization have radius up to

8 mm if the mold for polymerization is made of good heat transfering material.

This is accomplished by first preparing columns with radius less than 8 mm and

using them as fillers for the second stage polymerization, in which the radius of the polymerization solutions between the fillers and the column wall is also

less than 8mm, and the distance between the fillers is less than 2 mm. It is

found in this work that the thickness ofthe polymerization solution between the

fillers less than 2 mm has insignificant effect on the variations of the

polymerization temperature. Multiple thin polymer columns are filled into a large

diameter column and filled with the second stage polymerization mixture. The

column is sealed with regular fittings or fittings to allow pressurization during

polymerization. The large diameter column is then placed into a temperature

controlled heating bath or oven to carry out the second stage polymerization.

The thin columns are prepared by the process disclosed above with

pressurized or non-pressurized polymerization. The thin monolithic columns

prepared in the first stage polymerization are preferably preserved without

washing and further modification. A polymerization mixture for the second stage

polymerization is the same or different from the polymerization mixture of the

first stage polymerization depending on the types of media. The thin columns

with radius less than 8 mm can be solid rods, discs, hollow tubes with thickness

of the cylinder wall less than 8 mm, or a membrane. The shapes of the above

thin columns can be any shape known in the art, such as round, rectangular,

triangle, etc. It is perceivable that the fillers can be other particles described in

the section of filler materials in this specification.

In one embodiment of this invention, multiple thin columns with radius

of 5 mm are used. In one preferred embodiment of this invention, monolithic

polymer rods with size of 50 mm x 10 mm I.D. are used as fillers. In another preferred embodiment of this invention, monolithic polymer rods with size of 10

mm x 34 mm I.D. are used as fillers. In another preferred embodiment of this

invention, the monolithic polymer cylinder with various inner and outer

diameters are used as fillers.

The performance of capillary columns can be improved by our inventions

described earlier. One method is to choose right combination of porogenic

solvents to generate separation media with and without pressure. The choice

of solvents with right polarity and solventing power of the polymers can result

in porous polymer support with no micropores or small pores which can affect

separation efficiency ofthe column. Exertion of pressure during polymerization

will further improve the uniformity of the media and avoid the formation of

micropores. In one embodyment, the capillary columns of internal diameter 320

μm is prepared with the combination of solvents including chlorocyclohexane

and 1-decanol to generate the monolithic porous polymer support containing

no micropores or small pores which result in poor mass transfer of the sample

molecules with and without the pressure of 120 psi. In another embodyment,

the combination of solvents including 1-ethylhexanoic acid and mineral oil has

been used to prepare monolithic porous materials containing no micropores

with and without pressure.

The employment of x-ray, UV-vis can improve the performance of

capillary greatly. X-ray can penetrate materials low energy and intensity loss.

X-ray can penetrate a capillary with almost no intensity loss. Sensitizers or

scintillators can absorb x-ray energy transferred by the solvents effectively and emit fluorescence or phosphorescence light homogeneously in the solutions.

The homogeneous fluorescence and phosphorescence light can be absorbed

by initiators which initiate the polymerization homogeneously in the

polymerization solutions. This leads to homogeneity of the porous structure of

the porous polymer support including monolithic separation media and

particles. This improves the column efficiency of the monolithic capillary greatly.

The low temperature polymerization using x-ray as energy source results in

slow polymerization rate due to the lower polymerization temperature. Fine

tuning the intensity and energy of x-ray results in the desired polymerization

rate which can lead to the formation of homogeneous separation media. The

empolyment of x-ray, scintillators/sensitizers, solvents with right solventing

powers and the pressure during polymerization can leads to the formation of

separation media with no micropores or small pores with similar size to the

sample molecules which leads to greatly improved performance. In one

embodyment, capillary columns have been prepared using x-ray as energy

source. In another embodiment, microchip columns have been prepared with

x-ray as energy source.

The right choice of solvents with reflective index very close to the

polymers allows the light to travel through the capillary with little loss of intensity

of the light, which is known as Christiansen Effect and described earlier. The

use of initiators having Bleaching Effect allows the UV-vis light to travel through

the capillary columns with negligible loss of intensity. The lights are

homogeneous in the polymerization solutions. The absorptions of the homogeneous light result in homogeneous initiations of the polymerization

which is unlike the initiation promoted by thermal heating or the UV-vis initiated

polymerization without the consideration Christiansen and bleaching effect. This

leads to the formation of homogeneous polymerization in the solution. As the

result, more homogeneous porous structure can be formed. This leads to the

much improved performance of the capillary columns. The combination of the

above method with solvents of good solvating power, pressurized

polymerization can lead to formation ofthe homogeneous media containing no

micropores or small pores with having no impact on column performance. In

one embodyment, capillary columns have been prepared using UV-visible light.

In another embodyment, microchip columns have been prepared using UV-vis

light.

EXAMPLES

While many other values ofthe variables in the following examples can

be selected from this description with predictable results, the following non-

limiting examples illustrate the inventions:

GENERAL

In general, the preparation of each type of media in the following

examples include three major steps, which are:(1 ) preparation of polymer matrix; (2) modification of the polymer matrix to contain desired functional

groups; and (3) characterization of the media.

Firstly, the preparation major step, includes several substeps, which are:

(1 a) formulation of the polymerization mixture by varying the types and amount

of monomers, porogens and initiators; (1b) degassing the polymerization

mixture by vacuum and helium purge. (1 c) assembly of the empty column with

different diameter and material of tubing as a mold, frequently with one end of

the column sealed with a cap or stopper; (1d) filling the column with the

polymerization mixture; (1 e) sealing the other end of the mold with a cap or a

specially designed fitting to add pressure during the polymerization; (1f)

preheating the solution in the mold with one open end if a glass column is used

and applying a selected pressure using various pressure sources including

hydraulic pressure, air pressure or mechanic pressure; (1g) placing the mold

in a temperature-controlled heating bath or oven at a selected temperature;

(1 h) polymerizing for various amount of time; (1 i) taking the column out of the

heating bath after polymerization and replacing the sealing cap or

pressurization device with column fittings for pumping the washing liquid

through; (1j) washing the column with organic solvents and/or water.

Secondly, the modification process includes: (2a) formulating the

modification reaction mixture with various types and amount of reactants and

catalysts; (2b) pumping more than 5 bed volume modification solution through

the column and sealing it, or pumping more solution continuously. (2c) carrying

out the modification reaction at various temperature and time in a temperature- controlled heating bath; (2d) washing the column with organic and water.

The columns are characterized using varieties of methods including

liquid chromatography separation, porosimetry, BET surface area

measurement, Scanning Electron Microscopy, UV spectroscopy and visual

observation. Liquid chromatography characterization includes various modes

of separation at different speeds. The commonly used devices, processes and

methods are described in the following preceding the specific examples.

DEGASSING OF THE POLYMERIZATION SOLUTION

The polymerization mixture is degassed by vacuum generated by water

aspirator for 5 minutes using an ultrasonic degasser. It is followed by purging

the solution for minimum of 20 minutes.

STABILIZING AND CONDITIONING METHODS

The ion exchange columns were subject to a stabilizing and conditioning

procedure following the washing step after modification reaction. The stabilizing

and conditioning procedure for a glass column (100 x 10 mm I.D.) of strong

anion exchanger was as following: The flow rate of 0.01 mol/l Tris. HCI buffer

at pH 7.6 was increased linearly from 0 ml/min to 20 ml/min in 1 minute, and

kept for 0.5 minutes. The compression liquid was changed to 1 mol/l NaCl in

the same buffer by gradient in 2 minutes, kept for 0.5 minutes. The stabilizing

and conditioning procedure for a PEEK-lined stainless steel column is the same

as above except the maximum flow rate was 5 ml/min instead of 20 ml/min. The procedures for other ion exchange columns depend on the maximum flow rate

allowed.

CHARACTERIZATION PROCEDURES

1. Characterizations with liquid chromatography (LC) separations

1a. LC Characterization Method 1 : Liquid chromatography separation of

proteins and peptides

Mobile phases:

Mobile phase A (or Buffer A):

Anion Exchange Chromatography: 0.01 M Tris.HCI (pH 7.6)

Cation Exchange Chromatography: 0.01 M sodium phosphate (pH 7.0)

Reversed Phase Chromatography: 0.15% Trifluoroacetic acid (TFA) in water

Mobile phase B (or Buffer B):

Ion Exchange Chromatography: 1 M NaCl in Buffer A

Reversed Phase Chromatography: 0.15% TFA in acetonitrile (ACN) chromatography

Samples:

Anion Exchange Chromatography:

0.6 mg/ml myoglobin, 1 mg/ml conalbumin, 1 mg/ml ovalbumin and 1 mg/ml

trypsin inhibitor.

Cation Exchange Chromatography:

1 mg/ml connalbumin, 1 mg/ml ovalbumin and 1 mg/ml trypsin inhibitor. Reversed Phase Chromatography for proteins:

1.5 mg/ml Ribonuclease A, 0.5 mg/ml Cytochrome C, 1.5 mg/ml BSA, 0.9

mg/ml Carbonic Anhydrase, 1.5 mg/ml Ovalbumin.

Reversed phase chromatography for peptides:

33 ?g/ml Met-Enkephaiin, Let-Enkephalin, Angiotensin II, Physalaemin,

Substance P

Sample preparation:

Filled 8 ml of buffer A in a 15 ml graduated plastic sample tube; Weighed

appropriate amount of protein samples and placed them into this sample

tube; Sealed the tube with cap and tumbled the tube gently until all the

proteins dissolved; Added in more buffer solution until the 10 ml mark on the

sample tube was reached.

A column was characterized by protein separation according to the

following procedure: The column was attached to an Isco 2350 Two Pump

System. Pump A contained 0.01 mol/l Tris.HCI buffer (Buffer A) and Pump

B contained 1 mol/l NaCl in Buffer A (Buffer B). The mobile phases were

degassed with helium purging for more than 20 minutes before use. The UV

detector was set at 0.05 sensitivity and 280 nm wavelength for protein

separation (214 nm for peptide separation). The volume of the sample

injection was 20 micro liters. The column was first cleaned by 20 bed

volume of Buffer B and conditioned by 15 bed volume of Buffer A at the 3 ml/min for 4.6 mm I.D. column (10 ml/min for 10 mm I.D. column). The

separation was achieved by a gradient from 0 to 50% Buffer B for 20 bed

volume at the flow rate of 3 ml/min for 4.6 mm I.D. column and 10 ml/min for

10 mm I.D. column.

1 b. LC Characterization Method 2: Binding Capacity Measurement of Ion

Exchangers

The binding capacity of an ion exchange column was measured by

frontal analysis. The column was cleaned with 20 bed volume of Buffer B

and conditioned with 15 bed volume of Buffer A. This columns was

saturated with the sample protein by pumping 5 mg/ml BSA or lysozyme

solution (BSA for anion exchanger and reversed phase, and lysozyme for

cation exchanger) in Buffer A through the column until no further increase

of the absorbance of the eluent, followed by cleaning the non-adsorbed

proteins with 100% Buffer A. The protein bound to the columns was eluted

by a gradient from 0 to 50% Buffer B for 20 bed volume. The eluted protein

was collected in a sample vial and the protein concentration was determined

by UV spectrometer at 280 nm. The total binding capacity of the column

was calculated by multiplying the concentration of collected protein with the

volume of collection.

1 c. LC Characterization Method 3: Hydrophobic Interaction Chromatography

This column was characterized for hydrophobic interaction chromatography of proteins. A mixture of proteins containing Ribonuclease,

Cytochrome C, Lysozyme, Bovine Serum Albumin and Carbonic Anhydrase

(1 , 0.3, 0.2, 1 and 0.5 mg/ml in 0.01 M Tris.HCI buffer solution at pH 7.0.)

was separated by a 15 minute gradient of 0.5 mol/l NaCl in 0.01 mol/l

Tris.HCI buffer (pH 7.6) to the same buffer at the flow rate of 1 ml/min.

1d. LC Characterization Method 4: Polymer molecular weight determination by

precipitation-redissolution chromatography

Characterization method 6 was used for polymer molecular weight

determination using Precipitation/Redissolution Chromatography. Seven

polymer standards (Mp: 12,900, 20,650, 34,500, 50,400, 96,000, 214,500,

982,000) were separated by a 6 minute gradient from 15% to 80% THF in

methanol at the flow rate of 2.6 ml/min. The polymer standards were dissolved

in 50% THF in methanol with the total concentration of 56 mg/ml. The injection

volume was 20 FI.

1e. LC separation of nucleotides

A sample of Pd(A)_12-18 (2.4 mg/ml in water) was separated by a gradient

from 30% to 60% Buffer B in Buffer A (A=20% acetonitrile and 80% 20

mmol/l sodium phosphate; B=1 mol/l NaCl in A) at the flow rate of 1 ml/min.

1f. LC separation of nucleotides with anion exchanger

A sample of AMP, ADP and ATP (0.4, 0.8, 0.8 mg/ml in water respectively) was separated in a gradient of 0 to 50% Buffer B in Buffer A

as specified in LC Characterization Method 1.

NUMBERED EXAMPLES

Example 1

A polymerization solution was prepared as following: Weighed 1.2 g of

glycidyl methacrylate (GMA), 0.80 g of ethylene dimethacrylate (EDMA)

(polymerization mixture 1) and 0.02 g of 2,2=-azobisisobutyronitrile (AIBN) into

a 20 ml sample vial and shook the mixture gently until it became a

homogeneous solution; Weighed 2.55 g of cyclohexanol (CHOH) and 0.45 g

of dodecanol (DODOH) into this solution and shook it until it is homogeneous.

The polymerization mixture was degassed as in Degassing Procedure.

An empty stainless steel column (4.6 mm inner i.d. and 50 mm length),

one end of which was sealed by a pressuring device shown in FIG.4, was filled

with this solution until the column is full. A PEEK-plug contained in the stainless

steel screw cap, which was the original cap for the column shown in the same

figure, was used to seal the other end ofthe column. The device of FIG. 3 was

connected to a syringe pump. Water was used as the medium to generate the

pressure of 120 psi. No air was inside the column. This column was placed into

a water bath upright at 60?C and kept for 20 hours. After polymerization, the

column was taken out of the water bath and cooled to room temperature.

The device of FIG. 3 was detached from the syringe pump after the

pressure was released. The device of FIG. 3 was opened and carefully removed from the column. White polymer extended outside the column. The

length ofthe polymer was found to be about 2mm shorter than the height ofthe

polymerization solution inside the column and the device of FIG. 3. This

extended part of polymer was removed by razor blade. The column was then

fitted with the original HPLC column fittings. The column was connected to a

HPLC pump and washed with acetonitrile at 0.5 ml/min for 20 minutes at 45?C.

The fittings from one end of the column were detached and the media

was pressed out of the column by pumping 10 ml/min acetonitrile into the

column through the other end. The wall surface of the polymer media was

found to be smooth. The top of polymer was flat.

Comparative Versions of Example 1

A polymerization solution was prepared as following: Weighed 1.2 g of

glycidyl methacrylate (GMA), 0.80 g of ethylene dimethacrylate (EDMA)

(polymerization mixture 1 ) and 0.02 g of 2,2=-azobisisobutyronitrile (AIBN) into

a 20 ml sample vial and shook the mixture gently until it became a

homogeneous solution; Weighed 2.55 g of cyclohexanol (CHOH) and 0.45 g

of dodecanol (DODOH) into this solution and shook it until it is homogeneous.

The polymerization solution was degassed with the above Degassing

Procedure.

An empty stainless steel column (4.6 mm inner i.d. and 50 mm length),

one end of which was sealed by a PEEK-plug contained in the stainless steel

screw cap which was the original cap for the column. This column was filled with the above solution until it was full. A PEEK-plug was carefully placed on

the top of column and sealed with another screw cap. No air should be kept

inside the column. This column was placed into a water bath upright at 60?C

and kept for 20 hours. After polymerization, the column was taken out of the

water bath and cooled to room temperature.

The PEEK-plugs were detached and white polymer was observed in the

column. The column was then fitted with the original HPLC column fittings. The

column was connected to a HPLC pump and washed with tetrahydrofuran

(THF) at 0.5 ml/min for 20 minutes.

The fitting from one end ofthe column was detached and the media was

pressed out of the column by pumping 10 ml/min acetonitrile (ACN) into the

column through the other end. The wall surface of the media was found to

contain many small irregular dents.

Other columns were prepared using different monomers. Irregularvoids

were found on the wall surface of the polymer rods. Pictures of two of these

rods and one rod made under pressure were taken and shown in the Figure 7.

Alternative Versions of Example 1

The procedure of Example 1 was followed except that different

polymerization mixtures were used having different proportions and

combinations of the functional monomers and crosslinkers. The functional

monomers used includes glycidyl methacrylate (GMA), 2-hydroethyl

methacrylate (HEMA), methyl methacrylate (MMA), 2- (acryloyloxyethyl)trimethylammonium methyl sulfate (ATMS), acrylic acid

(AA), 2-Acrylamido-2-methyl-1-propanesulfonic acid (AMPS), stearyl

methacrylate (SMA), lauryl methacrylate (LMA), butyl methacrylate (BMA),

styrene (ST) and 4-ethylstyrene (EST). The crosslinking monomers

(crosslinkers) used include ethyleneglycol dimethacrylate (EDMA), divinyle

benzene (DVB). Different proportions of functional monomers, crosslinking

monomers and porogens were used. The porogens includes different

alcohols such as cyclohexanol, dodecanol, decanol, 1-hexadecanol, butanol,

propanol, iso-propanol, ethanol, methanol, 1 , 4-butanediol and others such as

toluene, N,N-Dimethyl acetamide, acetonitrile, 1 ,2-dimethoxyethane, 1 ,2-

dichloroethane, dimethyl phthalate, 2,2,4-trimethylpentane, 1 ,4-dixane, 2-

methyloxyethanol, 1 , 4-butanediol, m-xylene, diisobutyl phthalate,

tetra(ethylene glycol) dimethyl ether, tetra(ethylene glycol), poly(propylene

glycol) (F.W. 1000), polypropylene glycol) monobutyl ether (F.W. 340, 1000,

2500). The initiators used included 2, 2-prime-azobisobutyronitrile (AIBN)

and benzoyl peroxide.

For each ofthe combinations of monomers and porogens, columns were

prepared, examined and characterized.

Several columns were made with each polymerization mixtures under

several pressure conditions. The pressure conditions include: (1) the column

opened to the atmosphere during polymerization; (2) the column sealed during

polymerization; (3) pressure being applied to the column with gas applied

directly to the polymerization mixture using nitrogen as the gas; (4) each of rubber, plastic and metal pistons being in contact with the polymerization

mixture and applying pressure from either a spring, hydraulic pressure, gas

pressure or by threading the piston downwardly using the device described

above or the modified device when the mechanical force such as spring was

used.

The results were: (1 ) for cases when atmospheric pressure was

present there were discontinuities on the surface of a high percentage of

columns. Column efficiencies and resolutions were not reproducible; (2) for

pressure conditions in which the column was sealed, a higher percentage of

columns had discontinuities on the surface, column efficiencies and

resolutions were not reproducible; (3) for columns in which gas pressure was

applied, the top end surfaces of the columns were soft and irregular, and the

wall surfaces were smooth. The separation chromatograms had better

reproducibility. The elution peaks are sharper than when pressure was not

applied; (4) reproducibility was very high when pistons were used and the

resolution was better than all the above methods used.

Example 2

A polymerization solution was prepared as Example 1 with the

following polymerization solution: 2.0g GMA, 2.5g 2-

(acryloyloxyethyl)trimethylammonium methyl sulfate (ATMS, 80%), 6.0g

EDMA, 7.5g 1 , 4-butanediol, 6.75g propanol, 0.75g water and 0.1g AIBN.

An empty glass column (10 mm inner i.d. and 100 mm length), one end of which was sealed by a pressuring device shown in FIG.4, was filled with this

solution until the column is full. A TEFLON-plug contained in the PEEK screw

cap shown in the same figure, was used to seal the other end of the column.

The device of FIG. 4 was connected to a syringe pump. No air was inside the

column. This column was placed into a water bath upright at 60?C and kept for

15 minutes. Then the column was pressurized to 120 psi by syringe pump using

water as the medium, and kept for 20 hours. After polymerization, the column

was taken out of the water bath and cooled to room temperature.

The device of FIG. 4 was detached from the syringe pump after the

pressure was released. The device of FIG. 4 was opened and carefully

removed from the column. It was found that the height ofthe polymer rod was

4mm shorter than the height of the polymerization solution inside the column.

The column was then fitted with the original HPLC column fittings. The column

was connected to a HPLC pump and washed with acetonitrile at 2 ml/min for

20 minutes at 45?C.

The fittings from one end of the column were detached and the media

was pressed out of the column by pumping 10 ml/min acetonitrile into the

column through the other end. The wall surface of the polymer media was

found to be smooth. The top of polymer was flat.

Alternative Versions of Example 2

The method of Example 2 was followed with the change of pressures

and methods of applying the pressures. Different constant pressures were used during polymerization. The pressures used include 80 psi, 150 psi, 180

psi, 200 psi, 240 psi and 300 psi. The back pressures of these columns are

different. The Scanning Electron Microscopy examination of the polymer

structure revealed that the particle sizes of these polymers are also different.

A step gradient of pressure was applied to the polymerization mixture

during polymerization. The gradient is as following: 4 psi/min increase from

10 psi for 5 min, 2 psi/min increase for 10 min, 1 psi/min increase for 20 min,

0.8 psi/min increase for 30 min and then increase the pressure to 180 psi

within an hour. The final pressure of 180 psi was kept for 20 hours during

polymerization.

A linear gradient from 15 psi to 180 psi for 2 hours was used to

pressurize the reaction at early stage of polymerization. 180 psi was kept for

another 18 hours during the rest of polymerization.

All these columns were modified by pumping in 5 ml solution of

0.45g/ml trimethylamine hydrochloride in trimethylamine aqueous solution

(50% volume). The columns were sealed and heated in a water bath at 40 °C

for 3 hours. They were washed with 20 bed volume of water right after

modification. These columns were subjected to Bed Stabilization and

Conditioning as described above. They were characterized as above LC

Characterization Method for ion exchangers-. The backpressures, particle

sizes and separation resolutions of these columns all varied with the

pressurization methods.

Different polymerization time was also used. A column was prepared as in example 2 except that the polymerization time was 44 hours instead of

20 hours.

Ten columns were prepared in parallel using a ten channel manifold

with only one pressure source. The columns prepared have better

reproducibility than individual preparation process.

The polymer morphologies and the internal structures of the particles

were examined with Scanning Electron Microscopy (SEM). It was found that

the internal structures of these particles are non-porous instead of porous in

the other monolithic media known in the art.

Example 3

Two columns were prepared as in Example 1 and 2 with the following

solution: 17.5g DVB, 19.8g tetra(ethylene glycol), 10.2g tetra(ethylene glycol)

dimethyl ether, and 0.18g AIBN. After wash with acetonitrile, the column was

further washed with 20 bed volume of water at the flow rate of 16 ml/min. The

manually positioned pistons in both ends were compressed into the column.

This column was then washed with acetonitrile containing 0.15% trifluoroacetic

acid and characterized by LC characterization method 1a and 1 b. The

resolutions of these columns for both protein and peptide separation were

improved greatly over the columns made in the Comparative Examples. The

capacities were more than 5 times higher. The back pressures of these

columns were low. High resolutions were achieved with the mobile phase

gradient starting from 100% water containing 0.15% TFA. No wall effect was found in these columns. The proteins and peptides pre-eluted out ofthe column

in the columns from Comparative Examples due to wall effect in aqueous

phase.

Comparative Versions of Example 3

A polymerization solution was prepared as Comparative Example of Example 1

with the following reagents: 3ml styrene, 2 ml divinylbenzene, 7.5 ml

dodecanol and 0.5 g AIBN. The column was characterized with reversed-

phase protein and peptide separation described as LC Characterization 1a

and 1 b.

Alternative Versions of Example 3

Example 3 was followed with different combinations of porogens,

different monomers containing different carbon chain length, different

amount of total monomer contents, different initiators and different shrinkage

solvents.

The porogens used includes: alcohols containing C1 to C12, N,N-

Dimethyl acetamide, acetonitrile, 1 ,2-dimethoxyethane, 1 ,2-dichloroethane,

dimethyl phthalate, 2,2,4-trimethylpentane, 1 ,4-dixane, 2-methyloxyethanol,

1 , 4-butanediol, toluene, m-xylene, diisobutyl phthalate, tetra(ethylene glycol)

dimethyl ether, tetra(ethylene glycol), poly(propylene glycol) (F.W. 1000),

polypropylene glycol) monobutyl ether (F.W. 340, 1000, 2500). The

combination of some of these solvents led to high resolution columns as

well. The alcohols and their combinations can provide large channels for mobile phase to flow through with low back pressure while providing high

resolutions. The resolution can be finely tuned with other good solvents as

well.

The monomers used include butyl methacrylate and stearyl

methacrylate with the above combination of porogens. One column was

prepared with the following polymerization solution: 7g SMA, 10.5g DVB,

19.5g ethanol, 13.0g butanol and 0.18g AIBN. Another column was prepared

with the following polymerization solution: 7g lauryl methacrylate (LMA), 1g

HEMA, 12g EDMA, 30g dodecanol and 0.2g AIBN. Another column was

prepared with the following polymerization solution: 7g butyl methacrylate

(BMA), 1g HEMA, 12g EDMA, 3g water, 16.5g propanol, 10.5g 1 ,4-

butanediol and 0.2g AIBN. The combinations of these monomers containing

different carbon chain length provide different hydrophobicity and interaction,

which offer high resolution and recovery toward samples with different

hydrophobicity and characteristics. For example, the butyl methacrylate

based media was used for more hydrophobic protein separation and the

stearyl methacrylate based media can be used for more hydrophilic protein,

peptide or oligonuleotide separations.

Different ratios of monomer to crosslinker were also used to tune the

selectivity and resolution. One column was prepared with the following

polymerization solution: 1.05g SMA, 0.7g DVB, 3.25g ethanol and 0.018g

AIBN.

Different initiator was also used. A column was prepared with the following polymerization solution: 10 ml divinylbenzene (80% purity), 30 ml

dodecanol, 10 ml styrene and 0.20 g benzoyl peroxide.

Different polymerization time was also used. A column was prepared as in example 3 except that the polymerization time was 44 hours instead of 20 hours.

Different polar solvents were used to shrink the polymer before

compression. A column was prepared as Example 3 and washed with 20

bed volume of 1 M NaCl after water wash. Manually positioned pistons were

compressed into the column after the salt wash. The column show no wall

effect when 0.1 M NaH₂PO₄(pH 4.0) was used as the starting mobile phase.

These experiments were repeated with different washing solutions

and different catalysts with and without pressure in the presence of an

aqueous solution after the plug was polymerized as well as with different

pressures. In each case, when no pressure was applied, there were

discontinuities in the outer wall, and upon characterization, there was a lack

of repeatability and the peaks of the chromatograms were less pronounced

when no pressure was applied after swelling by washing with an aqueous

solution.

Tests have been run at a plurality of pressures both low pressures

and high pressures including 60 psi (pounds per square inch) and 120 psi

and 600 psi with good results. It is believed that the amount of pressure

needed will vary with the diameter of the column and the particular

polymerization mixture but satisfactory results can be obtained at a very low

pressure in all cases. The upper limit on pressure is the strength of the column walls and fittings.

The amount of pressure also affects the pore size so that the

pressure should be selected together with desired pore size, distribution and

reproducibility of the column.

Example 4

A column was prepared as Example 2 with the following solution: Nine g of

glycidyl methacrylate, 9 g of ethylene dimethacrylate, 0.18 g, 21.6 g of

cyclohexanol and 6.3 g of dodecanol. The length of the polymer was found to

be about 7 mm shorterthan the height ofthe polymerization solution inside the

column. The column was then fitted with the original column fittings. The

column was connected to a HPLC pump and washed with acetonitrile at 4

ml/min for 20 minutes at 45?C. The column was further modified as following:

A solution containing 570 mg trimethylamine hydrochloride, 24 ml

diethylamine and 6 ml water was pumped into these columns at the flow rate

of 2 ml/min for 18 minutes. These columns were then placed in a water bath at

30?C for 3 hour. Each column was washed with 100 ml water. The columns

were further washed with 0.01 mol/l Tris.HCI buffer at pH 7.6 at 4 ml/min for 30

minutes. The column was stabilized and conditioned by STABILIZING AND

CONDITIONING METHODS. The pistons from original column fittings were

compressed in completely after the wash. The column back pressures were

about 360 psi at 10 ml/min in this buffer. The column was characterized as LC

Characterization Method described above. Example 5

A polymerization solution was prepared by mixing 1 g styrene, 1 g

divinylbenzene (DVB) (80% divinyl benzene and 20% ethylstyrene (EST)), 3 g

dodecanol and 0.02 g AIBN.

This solution was degassed by N₂ purging for 20 minutes and filled into

a stainless steel column (50 x 4.6 mm i.d.), one end of which was sealed with

a PEEK plug inside the screw cap from the column fittings. The other end ofthe

column was sealed with another PEEK plug. It was polymerized at 70 degrees

C in a water bath for 24 hours. This column was fitted with the original column

fittings and washed with THF at the flow rate of 1 ml/min for 10 minutes before

it was used for separation of proteins. The back pressure of this column was

about 230 psi at the flow rate of 10 ml/min. The compression of the polymer at

10 ml/min of acetonitrile was about 2.9 mm. This column was used for

reversed phase protein and peptide separation as LC Characterization Method

1a and 1 b.

Alternative Versions of Example 5

Another column was prepared as in example 5 but with higher total

monomer contents. The polymerization solution contained 1.2 g styrene, 1.2 g

divinylbenzene, and 2.6 g dodecanol and 0.024g AIBN. The back pressure of

the column was 220 psi at 10 ml/min acetonitrile and the compression of the

polymer was only 0.9 mm. The highertotal monomer content makes the column

less compressible. Columns of different diameters including 22mm, 15mm, 10mm, 8mm,

2.1 mm, 1 mm, 542 ?m, and 320 ?m were prepared as in Example 4. Shorter

columns with the size of 10 mm x 2.1 mm i.d. were also prepared. These

columns were characterized with reversed phase protein separation by LC

Characterization Method but at the same flow velocity corresponding to the

diameters of the columns. The in situ polymerization method is applicable in

columns with different diameters. It is especially useful for smaller diameter

columns or microfluid channels since there is no other packing step involved.

Example 6

Another column was prepared according to Example 5.

This column was fitted with the original column fittings containing a

piston and washed with acetonitrile at the flow rate of 1 ml/min for 10 minutes.

The column was further washed with water for 10 minutes. The pistons in both

ends were compressed into the column. This column was characterized by LC

Characterization Method. The compression of the polymer in water by piston

and held by this piston after compression avoid the wall effect resulted from the

shrinkage of polymer in water.

Alternative Versions of Example 6

Another column was prepared as Example 5 with the following solution: 1.8 g

divinylbenzene, 0.2 g of styrene, 2.3375 g dodecanol, 0.6625 g toulene, 0.02

g of AIBN and 3.0 g of dodecanol. -All the reagents were degassed by vacuum using aspirator for five minutes, followed by purging with Helium for 20 minutes,

before weighing. The polymerization solution was filled into a stainless steel

column (50 x 4.6 mm i.d.), one end of which was sealed by PEEK-plug

contained in the screw cap. The other end of the column was sealed with

another PEEK plug. It was polymerized at 66 degrees C in a water bath for 24

hours. This column was fitted with the original column fittings containing a

The column was further washed with water for 10 minutes. The pistons in both

ends were compressed into the column. This column was characterized by the

LC Characterization Method. The higher content of crosslinker improved the

compacity. This version of a column has more than 3 times higher capacity than

the some other columns prepared in accordance with Example 6.

Another column was prepared with the following polymerization solution:

10 ml divinylbenzene (80% purity), 10 ml styrene and 0.20 g benzoyl peroxide.

This polymerization solution was purged by N₂ for 20 minutes. It was

filled into a stainless steel column (50 x 4.6 mm i.d.), one end of which was

sealed by PEEK-plug contained in the screw cap. The other end ofthe column

was sealed with another PEEK plug. It was polymerized at 70?C in a water

bath for 24 hours. This column was fitted with the original column fittings and

washed with tetrahydrofuran at the flow rate of 1 ml/min for 10 minutes. It was

characterized by reversed phase protein and peptide separation as described

in LC Characterization Method. Different initiators such as benzoyl is also

effective in making the monolithic media. A stainless column of smaller size (50 x 2.1 mm i.d.) and a PEEKcolumn

(50 x 4.6 mm i.d.) were prepared and characterized 8-as above example.

Example 7

A column was prepared as in Example 6 with the following solution:

divinylbenzene, 0.2 g of hydroxylethylmethacrylate and 0.02 g of AIBN. It was

polymerized at 70?C in a water bath for 24 hours. This column was fitted with

the original column fittings containing pistons. It was washed with acetonitrile

at the flow rate of 1 ml/min for 10 minutes, and further washed with water and

0.5 mol/l NaCl in 0.01 mol/l Tris.HCI buffer (pH 7.6). This column was

characterized by reversed-phase protein separation as in LC Characterization

Method.

Alternative Versions of Example 7

A column was prepared and characterized according to the above

procedure except the weight of hydroxylethylmethacrylate and divinylbenzene

were changed to 0.4 g and 1.6 g.

Another- column was prepared and characterized according to the above

procedure except the weight of hydroxylethylmethacrylate and divinylbenzene

were changed to 1 g and 1 g.

Another column was prepared as in Example 7 with the following

polymerization solution: -1.8 g divinylbenzene, 0.16 g styrene, 0.04 g of

hydroxylethylmethacrylate, and 0.02 g of AIBN and This column was fitted with the original column fittings containing pistons after

polymerization.T It was washed with acetonitrile at the flow rate of 1 ml/min for

10 minutes, and further washed with water and 0.5 mol/l NaCl in 0.01 mol/l

Tris.HCI buffer (pH 7.6). This column was compressed with pistons and

characterized by reversed phase separations of proteins and peptides as in LC

Characterization Method.

Example 8

An empty syringe barrel (70 x 12 mm i.d. Redisep barrel for Combiflash

chromatography from Isco, Inc., 4700 Superior Street, Lincoln, NE 68504) was

sealed at one end and filled with the following polymerization solution: 1.6 g

hydroxylethyl methacrylate, 6.4 g divinylbenzene, 89 mg AIBN, 12 g dodecanol

after degassing with N₂ for 20 minutes. The tip ofthe barrel was sealed with a

blocked needle. This barrel was heated in a water bath at 70?C for 24 hours.

It was connected to a HPLC pump and washed with THF at the flow rate of 1

ml/min for 30 minutes. It was then used for both normal phase and reversed

phase separation of phenolic compounds.

Example 9

A polymerization solution was prepared as following: Weighed 1 g of

hydroxylethyl methacrylate, 1 g of ethylene dimethacrylate and 0.02 g of AIBN

into a 20 ml sample vial and shook the mixture gently until it became a

homogeneous solution; Weighed 1 g of cyclohexanol and 2 g of dodecanol into this solution and shook it until it is homogeneous. All the reagents were

degassed by vacuum using aspirator for five minutes, followed by purging with

Helium for 20 minutes, before weighing.

An empty stainless steel column (4.6 mm inner i.d. and 50 mm length),

one end of which was sealed by a device of FIG. 4 was filled with this solution

until the column is full. A PEEK-plug contained in the stainless steel screw cap,

which was the original cap for the column, was used to seal the other end ofthe

column. The device of FIG. 4 was connected to a syringe pump. Water was

used as the medium to generate the pressure of 120 psi. No air should be kept

inside the column. This column was placed into a water bath upright at 60?C

and kept for 20 hours. After polymerization, the column was taken out of the

water bath and cooled to room temperature. The column was connected to a

HPLC pump and washed with dry THF (dried by molecular sieve) at 0.5 ml/min

for 20 minutes. The column was used for Normal Phase separation of drugs.

Alternative Versions of Example 9

A column was prepared as in Example 9 with the following

polymerization solution: 0.5 g GMA, 0.5g HEMA, 1 g EDMA, 1.8g cyclohexanol,

1.2g dodecanol, 0.02 g AIBN.

The columns were washed with water after THF wash at the flow rate of

0.5 ml/min for 20 minutes. 10 ml of 1.0 mol/l sulfuric acid in water was pumped

through the columns. The columns were sealed with column plugs and placed

into a water bath at 80?C for 3 hours. They were washed with 20 ml water after the modification reaction and further washed with dry THF before

characterization with Normal Phase separation.

Another column was prepared with the following polymerization solution:

1 g glycidyl methacrylate, 1 g ethylene dimethacrylate, 2.4 g cyclohexanol, 0.6

g dodecanol, 0.02 g AIBN.

The columns were washed with water after THF wash at the flow rate of

0.5 ml/min for 20 minutes. 10 ml of 1.0 mol/l sulfuric acid in water was pumped

through the columns. The columns were sealed with column plugs and placed

into a water bath at 80?C for 3 hours. They were washed with 20 ml water after

the modification reaction and further washed with dry THF before

characterization with Normal Phase separation.

Example 10

A stainless steel column (50 x 4.6 mm i.d.) was prepared as in

Example 3 with the following polymerization solution contained 0.7g lauryl

methacrylate (LMA), 0.1g HEMA, 1.2g EDMA, 3g dodecanol and 0.02g

AIBN.

Alternative Versions of Example 10

A column was prepared with the following polymerization solution

contained 0.8g lauryl methacrylate (LMA), 1.2g EDMA, 3g dodecanol and 0.02g

AIBN. Another column was prepared with the following polymerization solution

containing 0.175g stearyl methacrylate (SMA), 1.575g DVB (80% pure), 3.25g

1-hexadecanol and 0.018 mg AIBN to form a mixture with a ratio SMA/DVB=10/90. Another column was prepared with the following

polymerization solution containing 0.7g SMA, 1.05g DVB, 3.25g octanol and

0.018g AIBN to provide a ratio of 40/60 SMA/DVB.

Another column was prepared with the following polymerization solution

containing 1.05g SMA, 0.7g DVB, 3.25g ethanol and 0.018g AIBN to provide

a ratio of 60/40 SMA/DVB.

Another column was prepared with the following polymerization solution

containing 0.7 SMA, 1.05g DVB, 1.95g ethanol, 1.30g butanol and 0.018g AIBN

to provide a ratio of 40/60 SMA/DVB.

Other columns were prepared as above using tetradecanol, decanol,

octanol, hexanol, butanol, propanol, ethanol and methanol and their

combinations as porogenic solvents.

Another column was prepared as above with the following

polymerization solution: 0.7 SMA, 1.05g DVB, 2.925g ethanol, 0.33g

methanol, 0.33g isopropanol and 0.018g AIBN to provide ratio of 40/60

SMA/DVB.

Another column was prepared as above with the following polymerization

solution: 0.7 SMA, 1.05g DVB, 2.6g ethanol, 0.33g methanol, 0.33g propanol,

0.33g butanol and 0.018g AIBN to provide a ratio of 40/60 SMA/DVB.

Another column was prepared as above with the following polymerization

solution: 0.7 SMA, 1.05g DVB, 2.762g ethanol, 0.33g methanol, 0.33g

propanol, 0.33g butanol, 0.33g hexanol and 0.018g AIBN to provide a ratio of

40/60 SMA/DVB. Another column was prepared as above with the following polymerization

solution: 0.7 SMA, 1.05g DVB, 2.435g ethanol, 0.33g methanol, 0.33g

propanol, 0.33g butanol, 0.33g hexanol, 0.33g octanol and 0.018g AIBN to

provide a ratio of 40/60 SMA/DVB.

Another column was prepared as above with the following polymerization

solution:-! .05g DVB, 3.08g ethanol, 0.0.16g ethyl ester and 0.018g AIBN. This

column was used for peptide separation as in Example 10, to provide a ratio of

40/60 SMA/DVB.

Another column was prepared as above with the following polymerization

solution: 1.75g DVB, 3.25g dodecanol and 0.018g AIBN. This column was used

for peptide separation as in the first version ofExample 10. A series of columns

with alcohols containing C1 to C12 were prepared as above and characterized

with peptide separation.

A series of columns were prepared as above using the following

porogens instead of dodecanol: iso-propanol, N,N-Dimethyl acetamide,

acetonitrile, 1 ,2-dimethoxyethane, 1 ,2-dichloroethane, dimethyl phthalate,

2,2,4-trimethylpentane, 1 ,4-dixane, 2-methyloxyethanol, 1 , 4-butanediol,

toluene, m-xylene, diisobutyl phthalate, tetra(ethylene glycol) dimethyl ether,

tetra(ethylene glycol), poly(propylene glycol) (F.W. 1000), poly(propylene

glycol) monobutyl ether (F.W. 340, 1000, 2500).

Another column was prepared as above with the following polymerization

solution: 1.75g DVB, 2.925g isopropanol, 0.325g 1 , 4-butanediol and 0.018g

AIBN. Another column was prepared as above with the following polymerization

solution: 1.75g DVB, 2.275g isopropanol, 0.975g 2-methyloxyethanol and

0.018g AIBN.

Another column was prepared as above with the following polymerization

5 solution: 1.75g DVB, 2.60g isopropanol, 0.65 dimethyl phthalate and 0.018g

AIBN.

Another column was prepared as above with the following polymerization

solution: 1.75g DVB, 2.7g tetraethylene glycol, 0.3g diethylene glycol and

0.018g AIBN.

o Another column was prepared as above with the following polymerization

solution: 1.75g DVB, 2.7g tetra(ethylene glycol), 0.3g glycerol and 0.018g

AIBN.

Another column was prepared as above with the following polymerization

solution: 1.75g DVB, 1.98g tetra(ethylene glycol), 1.02g tetra(ethylene glycol)

5 dimethyl ether, and 0.018g AIBN.

Another column was prepared as above with the following polymerization

solution: 1.75g DVB, 1.98g tetra(ethylene glycol), 1.02g tetra(ethylene glycol)

dimethyl ether, and 0.018g AIBN.

Example 11

0 A-column (50 x 4.6 mm i.d. stainless steel, which is 50 mm length and

4.6 mm inner i.d.) was prepared as Example 1 with the following polymerization

solution containing 1g methyl methacrylate (MMA), 1g EDMA, 1.8g cyclohexanol, 1.2g dodecanol and 0.02g AIBN-lt was connected to a HPLC

pump and washed with THF and water at 0.5 ml/min for 20 minutes in

sequence.

This column was subjected to a hydrolysis reaction as following: 2 ml 6

mol/l NaOH was pumped through the column at the flow rate of 0.5 ml/min; The

column was sealed by two column plugs and placed in a water bath at 80?C for

1 hour. It was washed with 20 ml water at the flow rate of 0.5 ml/min and

characterized with protein separation and binding capacity measurement

described in LC Characterization Method.

Alternative Versions of Example 11

A column was prepared as Example 11 with the following polymerization

solution: 0.1 g acrylic acid (AA), 0.9g methyl methacrylate (MMA), 1g EDMA, 3g

dodecanol and 0.02g AIBN. The capacity of the column was measured before

and after hydrolysis. The capacity before hydrolysis was about 10 mg

lysozyme per ml column volume. It was about 30 mg after hydrolysis.

Another column was prepared as Example 11 with the following

polymerization solution: 0.2g AA, 0.8g MMA, 1g EDMA, 3g dodecanol and

0.02g AIBN. The capacity of the column was measured as in

characterization method 11. The capacity before hydrolysis was about 27

mg lysozyme per ml column volume. It was about 50 mg after hydrolysis.

Another column was prepared as Example 11 with the following polymerization solution: 0.3g AA, 0.7g MMA, 1g EDMA, 3g dodecanol and

0.02g AIBN. The capacity of the column was measured before and after

hydrolysis The capacity before hydrolysis was about 43 mg lysozyme per ml

column volume. The capacity was more than 60 mg after hydrolysis.

Another column was prepared as above with the following polymerization

solution: 0.4g AA, 0.6g MMA, 1g EDMA, 3g dodecanol and 0.02g AIBN.

Another column was prepared as above with the following polymerization

solution: 0.1g AA, 0.9g tert-butyl acrylate, 1g EDMA, 3g dodecanol and 0.02g

AIBN. Another column was prepared as above with the following

polymerization solution: 0.3g AA, 0.3g MMA, 1.4g EDMA, 3g dodecanol and

0.02g AIBN.

Another column was prepared as above with the following polymerization

solution: 0.2g AA, 0.7g MMA, 0.1g HEMA, 1g EDMA, 2.85g dodecanol, 0.15g

cyclohexanol and 0.02g AIBN. Another column was prepared as above with

the following polymerization solution: 0.4g AA, 1.6g DVB, 3gdodecanol and

0.02g AIBN.

Another column was prepared as above with the following polymerization

solution: 1g GMA, 1g EDMA, 2.4g cyclohexanol, 0.6g dodecanol, 0.02g AIBN.

This column was subjected to an acid-catalyzed ring opening reaction as in

aAlternative versions of Example 9 before base-catalyzed hydrolysis reaction

as in Example 11. Another column was prepared as above with the following polymerization

solution: 0.5 g GMA, 0.5g HEMA, 1g EDMA, 1.8g cyclohexanol, 1.2g

dodecanol, 0.02 g AIBN.

Another column was prepared as above with the following polymerization

solution: 0.2g AA, 0.6g MMA, 0.2g GMA, 1g EDMA, 3g dodecanol and 0.02g

AIBN. This column was subjected to an acid-catalyzed ring opening reaction

before base-catalyzed hydrolysis reaction as above. It was further hydrolyzed

with the following solution: 0.25M Sodium chloroacetate in 5M NaOH at 60°C

for 6 hours.

Another column was prepared as above with the following polymerization

solution: 0.2g AA, 0.5g MMA, 0.1 g GMA, 1.2g EDMA, 2.55g dodecanol, 0.45g

cyclohexanol and 0.02g AIBN. It was hydrolyzed by 0.25M Sodium

chloroacetate in 5M NaOH at 60?C for 6 hours.

All these columns were subjected to Bed Stabilization and Compression

Method and characterized with protein separation and binding capacity

measurement as in LC Characterization Method described above.

Example 12

A column (PEEK-lined stainless steel, 50 x 4.6 mm ID) was prepared as

Example 1 with the following polymerization solution: 3 g GMA, 3 g EDMA, 6.9

g cyclohexanol, 2.1 g dodecanol and 0.06 g AIBN.

This column was first modified with ring-opening reaction under acidic condition. Five bed volume of the solution of 0.5 M sulfuric acid in water was

pumped through the columns. The column was sealed and heated in a water

bath at 50 °C for 4 hours. It was washed with 20 bed volume of water after

modification.

This column was further modified with a etherification reaction. Five bed

volume of a solution containing 20 g sodium chloroacetate, 20 g NaOH and 64

ml water was pumped through the column. The column was sealed with column

plugs and heated in a water bath at 60 °C for 2.5 hours. It was washed with

water, stabilized and conditioned as Stabilization and Conditioning Methods.

This column was characterized as in LC Characterization Method.

Alternative Versions of Example 12

The Example 12 was followed with different modification methods.

A column prepared as in Example 12 was modified by a ring-opening

reaction with the following solution: 6 mol/l glycolic acid and 0.5 M TFI in water

for 3 hours.

Another column was modified with the above ring-opening reaction and

hydrolysis reaction in 5 M NaOH solution at 60 °C for 2.5 hours.

Another column was first modified by ring-opening reaction with the

following solution containing 40 g glycolic acid, 60 ml 0.5 M trifuoroacetic acid

(TFA) for 2 hours. It was further modified with a solution containing 20g

ClCH₂COONa and 60 ml 5M NaOH for 3 hours. Example 13

Thirty columns were prepared as Example 2 with the following solution:

12g AA, 30g MMA, 6g GMA, 72g EDMA, 27g cyclohexanol, 153g dodecanol

and 1.2g AIBN. These columns were prepared by parallel synthesis at the

same time using three manifolds connecting to one syringe pump to obtain 120

psi pressure during polymerization. After polymerization, polymers were pushed

out ofthe columns by a syringe piston (about 9 mm i.d.) for the following uses.

One polymer rod from above was trimmed to be smaller with the

diameter about 8mm. It was cut to 1 cm thick discs. These discs were used as

fillers for a second stage polymerization to prepare another column. 1.8 ml

solution was filled into a glass column (100 x 10 mm i.d.), one end of which was

sealed by pressurization device shown in Figure 3. Six polymer discs were filled

into the column one by one. All these discs should be covered by the solution.

A Teflon stopper was used to seal the other end of the column. This

pressurization device was connected to a syringe pump which was used to add

120 psi pressure to polymerization solution at constant pressure mode. The

column was heated in a water bath at 60 °C for 20 hours. After polymerization,

the column was taken out of the water bath. The pressurization device was

detached from the syringe pump after the pressure was released. The

pressurization device was opened and slowly removed from the column while

the column is still warm. This column was washed with 20 bed volume of

acetonitrile and water at the flow rate of 1 ml/min in sequence. It was stabilized and conditioned as in Stabilization and Conditioning Method. This column was

modified with 0.25 mol/l Sodium chloroacetate in 5M NaOH at 60°C for 6 hours.

It was characterized as in LC Characterization method.

Alternative Examples of Example 13

Another column (100 mm x 35 mm ID, glass) was prepared with the two

stage polymerization method with the polymer rods as the fillers. This column

was sealed with TEFLON plug in one end. The other end of the column was

connected to N₂ tank. The polymerization was under 120psi for 20 hours at 60

°C.

Example 14

Eight short polymer rods (10 mm x 34 mm ID) were prepared with the

following solution: 8 g acrylic acid, 20 g methyl methacrylate, 4 g glycidyl

methacrylate, 48 g ethylene glycol dimethacrylate, 102 g dodecanol, 18 g

cyclohexanol and 0.8 g AIBN. The polymer rods were prepared under 120 psi

N₂ pressure. The rods were used as fillers for the preparation of a large

diameter long column using the two stage polymerization method as in

Example 12. A glass column (100 mm x 35 mm ID) was filled with the short

columns and the same polymerization as above. One end of the column was

sealed with a TEFLON plug and the other end was connected with a N₂tank.

The polymerization was carried out under 120 psi pressure at 60 °C for 20 hours. The column was washed with 20 bed volume acetonitrile and water. It

was subjected to hydrolysis reaction as following: 0.25M Sodium chloroacetate in 5M NaOH at 60?C for 6 hours. The column was characterized as LC Characterization Method described above. Example 15

A column (PEEK-lined stainless steel, 50 mm x 4.6 mm ID) was

prepared as in Example 1 with the following solution: 4 g GMA, 4 g EDMA, 2.8

g dodecanol, 9.2 g cycohexnanol and 0.08 g AIBN.

This column was first hydrolyzed by 1 M H₂SO₄ solution at 40 °C for 3

hours. After hydrolysis, it was activated by pumping 5 bed volume of 5%

sodium t-butoxide solution in DMSO through the column and heated in a water

bath at 90 °C for 1 hour. Then it was modified with the solution containing 20%

of the activation solution and 80% of butane sultone at 80 °C for 20 hours.

Alternative Versions of Example 15

A column was prepared as in Example 15 except that propane sultone

was used instead of butane sultone.

Another column was prepared as in Example 15 except the modification

and activation temperature was 120 °C instead of 90 °C in an oil bath.

Another column was prepared as in Example 15 with the following

solution: 4 g HEMA, 4 g EDMA, 9.4 g dodecanol, 2.6 cyclohexanol and 0.08 g

AIBN. It was modified as in Example 15. Another column was prepared as in Example 1 with the following

solution: 0.55 g GMA, 1.2 g EDMA, 0.25 g 2-Acrylamido-2-methyl-1-

propanesulfonic acid (AMPS), 0.48 g NaOH, 0.5 g water, 1.86 g propanol, 0.64 g

butanediol and 0.02 g AIBN.

Another column was prepared by direct copolymerization of AMPS as

above but was further modified with the modification method described in

Example 15.

All these columns were characterized with the strong cation exchange

protein separation and binding capacity measurement described in LC

Characterization Method.

Example 16

A column was prepared as Example 1 with the following polymerization

solution: 45 ml tetramethoxysilane, 100 ml of 0.01 mol/l aqueous acetic acid,

9g urea and 11.5g poly(ethylene oxide) (MW 10000). This solution was

prepared by stirring this mixture in ice bath for 30 minutes. The polymerization

was carried out in the column under 600 psi pressure and at 40 °C for 24 hours.

The column was then washed with 20 ml water at the flow rate of 0.5 ml/min

and pumped in 5 ml of 0.01 mol/l aqueous ammonium hydroxide solution. The

column was sealed and kept at 120 °C for 3 hours followed by ethanol wash. Example 17

The inhibitors such as methyl ether hydroquinone or tert-butylcatecol

were removed from monomers by distillation or normal phase chromatography

before uses.

A polymerization solution was prepared as Example 1 , but with the

following polymerization solution: 240 mg p-terphenyl, 800 mg AIBN, 16 g

styrene and 16 g divinylbenzene (80%), 26.4 g mineral oil, and 21.6 g 2-

ethylhexanoic acid.

An empty glass column (10 mm inner i.d. and 100 mm length), one end

of which was sealed by a pressuring device shown in FIG. 4, was filled with this

solution until the column was full. A TEFLON-plug contained in the PEEK screw

cap shown in the same figure, was used to seal the other end of the column.

The polymerization was allowed to expose to x-ray with a dosage of 600 R/hour

for 72 hours at an x-ray tube voltage-of 111 k\lρ. The obtained column was

further heated to 70 °C for 2 hours. Then the column was washed with hexane

followed by hexane/acetone (50/50), acetone, acetonitrile, respectively with 20

bed volume of each solvent.

The device of FIG. 4 was detached from the syringe pump after the

pressure was released. The device of FIG. 4 was opened and carefully

removed from the column. It was found that the height of the polymer rod was

4mm shorter than the height of the polymerization solution inside the column.

The column was then fitted with the original HPLC column fittings. The column was connected to a HPLC pump and washed with acetonitrile at 2 ml/min for

20 minutes at 45 degrees C.

The prepared column was further washed with 20 bed volume of water

and compressed with the piston to get rid of the void volume. The column was

then characterized using LC characterization method described in 1a. The

chromatogram is attached in Fig. 9.

Alternative Versions of Example 17

Another column was prepared as in example 17 in a glass column

(35mm i.d. x 100 mm length) with the following mixture: 30 g styrene, 30 g

divinylbenzene, 38.05 g mineral oil and 22.18 g 2-ethylhexanoic acid, 0.518 g

p-terphenyl and 1.31 g AIBN. The columns was washed with 20 bed volume of

acetonitrile and water respectively. It is characterized with LC characterization

method 1a. The separation is shown in Fig. 9.

Another column was prepared as in example 17. The fittings from one

end ofthe column were detached and the media was pressed out ofthe column

by pumping 10 ml/min acetonitrile into the column through the other end. The

separation media was submitted for porosimetry studies using SEM and

mercury porosimeter after drying in vaccum at 50 °C for 24 hours.

Another column was prepared as in example 17 using larger diameter

glass column (35mm i.d. x 100 mm length). The column was sealed with two

TEFLON-plug contained in the TEFLON screw caps instead of the device in Fig. 4. The polymer was pushed out of the column and dried as above

example. The polymer was submitted for SEM and porosimetry studies.

Another column was prepared as above example using large diameter

column (35mm x 100 mm) but using the following polymerization solution: 240

mg p-terphenyl, 800 mg AIBN, 3.2139 g styrene, 28.8088 g divinylbenzene

(80% pure), 37.4060 g 1 -dodecanol, 13.25 g toluene.

Another column was prepared as above example using large diameter

column (35mm x 100 mm) but using the following polymerization solution: 240

mg p-terphenyl and 800 mg AIBN was dissolved in the monomer mixture of

32.0034 g divinylbenzene (80%). Into the monomer mixture, 31.6926 g

tetraethylene glycol, 16.3224 g tetraethylene glycol dimethyl ester was added.

Another column was prepared as above example within a polymeric

housing described in Figs. 12-15. The polymerization solution contains 73.2

g divinylbenzene, 73.4 g styrene, 85.2 g mineral oil, 60.8 g 2-ethylhexanoic

acid, 0.882 g p-terphenyl and 2.94 g AIBN. The temperature of polymerization

in the center of the column was about the same as the one on the edge of the

column. The conversions of monomers were almost complete after 4 days of

110 kV x-ray irradiated polymerization. Complete reaction is attained thermally

as the further exotherm has no significant bad effect.

Many other columns using different scintillators, photo initiators,

monomers and porogenic solvents have been prepared. The porogenic

solvents used include other alkane such as octane, alcohols such methanol, propanol and cyclohexanol, ethers such as tetrahydrofuran, dioxane, oligomers

such tetraethylene glycol, tetraethylene glycol dimethyl ether. Photo initiators

u s e d i n c l u d e 2 - c h I o r o t h i o x a n t h e n - 9 - o n e , 4 , 4 ' -

bis(dimethylamino)benzophenone, 4,4'-bis(diethylamino)benzophenone,

phenanthrenequinone, diphenyl(2,4,6 trimethylbenzoyl)phosphine oxide and

azo bisisobutylronitrile (AIBN). The scintillators used include 2,5-

diphenyloxazole (PPO), 2-phenyl-5-(4-biphenylyl ) 1 ,3,4-oxadiazole (PBD), 2-

(1-Naphthyl)-5-phenyloxazole (a-NPO) besides p-terphenyl and ZnSe. The

monomers used include acrylonitrile, butyl methacrylate besides glycidyl

methacrylate, ethylene glycol dimethacrylate, styrene, divinylbenzene, ethyl

styrene.

In FIG. 9, t here is shown a chromatogram of a separation in two

different diameter columns of the mixture of (1) Met-Enkephalin, (2) Leu-

Enkephalin, (3) Angiotensin, (4) Phyusalaemin and (5) Substances P on Poly

(DVB-co-St) monolithic columns prepared by X-ray irradiation initiated

polymerization in column with a diameter of 10 mm and 35 mm and a length of

65 mm; Mobile phase: (A) water with 0.15% (v/v) TFA ; Gradient: 10-40% B in

A in 7 bed volume at a flow rate of 5 ml/min I.D. column and 50 ml/min for 35

I.D. column: Detection: UV at 214 nm.

In FIG. 10, there is shown a top view of an ultraviolet or visible light

polymerization apparatus 150 having a stationary top surface 152, a rotating

top surface 156, a support member 157 connected to the stationary support surface 152 and pinned to the rotating surface 156 to permit rotation thereof

and four fluorescent lamp holders 154A-154D. Visible or ultraviolet fluorescent

lamps are inserted in these holders.

In FIG. 11 , there is shown a schematic representation of a side sectional

view of the polymerization apparatus 150 showing the driven member 156

rotated reciprocally by a motor 159 to rotate the polymerization apparatus 162.

Two of the four lamps 166D and 166B are shown mounted to the lamp holders

154D and 154B and corresponding holders at the bottom end ofthe elongated

lamps 166D and 166B. A piston 164 is used to pressurize the polymerization

mixture at 162 during polymerization. A fan 158 aids in cooling the

polymerization apparatus and reflective coatings on the cover, the sides and

the lamps reflect light back into the light conducting walls of the container 162.

With this arrangement, the lamps 166A-166D cause light to impinge on the

polymerization mixture to initiate and control the polymerization reaction. The

polymerization of relatively large diameter columns may be performed while

maintaining radial uniformity in the final plug at all locations along the column

in the direction of flow of the solvent and analyte . The light may be turned on

and off as desired to control the temperature gradients so that the

polymerization may take place under a combination of light and temperature

in a controlled manner for uniformity.

In FIG. 12, there is shown a simplified elevational view of an apparatus

170 for polymerization using principally x-ray radiation having a radiation proof cabinet 172, with a door 174, an upper window 176, a holder 178 and a

container 180 to contain a reaction mixture at 182. A piston 184 may be

utilized in some embodiments to pressurize the reaction mixture 182. In the

apparatus 170, x-rays or other suitable radiation such as gamma rays may be

used to control the reaction in the polymerization container in a safe convenient

manner.

In some embodiments, pressure may be applied through the piston 184

by applying air through the conduit 186 to move the piston inwardly against the

reaction mixture 82 in a manner described above in connection with other

embodiments. In the preferred embodiment, the apparatus 170 is a small user

friendly cabinet x-ray system resembling a microwave in that it has a door and

controls mounted on the cabinet. It uses low voltage levels and can be

operated by personnel safely from next to the cabinet because it has low

penetration which is sufficient however for large columns. It is suitable for the

polymerization of this invention because processes described hereinabove use

added substances to aid in polymerization such as photo initiators, fluorescing

solvents, or porogens, x-ray sensitizers and/or scintillators. This unit permits

x-ray control ofthe polymerization and other units such as those of FIG. 10 and

11 permit other radiation control of polymerization, thus permitting for example

control of polymerization with the aid of radiation up to a point and finishing the

polymerization using heat to decrease the time and yet avoid destructive head

build-up.. In FIG. 13, there is shown a top view of the reaction vessel 180 having

a reactant entry opening 200, a coolant fluid inlet 203, a coolant outlet 205, a

casing 204 and an overflow outlet 202. The coolant is preferably water. An

opening 206 for air to move the plug 182 (FIG. 14) against the polymerization

mixture AT 212 for pressure thereon is provided. A thermocoouple can be

provided through the opening 200 after the reactant mixture is place by turning

over the vessel 180 and a plug can be inserted there as well. With this

arrangement, the reaction mixture may be irradiated under pressure if desired

and subjected to x-rays axially for initiation and control of the polymerization.

Water flows through it as a coolant so that the combination of radiation and

pressure can control thermal gradients and promote uniformity in the final

chromatography plug or support.

In FIG. 14, there is shown a sectional view taken through section lines

14-14 of FIG. 13 showing the transparent x-ray radiation window 192, the port

202 for the coolant water, the opening 200 for reactant, a thermocouple 215

and its conductor 218 and a pin in that sequence, the air pressure opening 206

to move the plug 182 to pressurize the reaction mixture in 212, the reactant

housing at 212 for receiving reactant mixture, a first distribution plate at 213 to

distribute the solvent and analyte during chromatography when the vessel 180

is used as a column and the second distributor plate 215 to receive the solvent

and analyte after separation if the column at 212 after polymerization. With this

arrangement, radiation passes through the window 192 to control the polymerization of the reactant 182. This may be done under pressure applied

by the plug 182.

In FIG. 15, there is shown a sectional view ofthe polymerization vessel

180 taken through lines 15-15 of FIG. 13 showing a piston 182, an air space

210 applying pressure to the plug 182 to pressurize the reactant at 212 while

it is being irradiated by x-rays and cooled by flowing water in reservoir 190.

As can be understood from the above description, a polymerization

mixture which may include a porogen or solvent is polymerized into a porous

plug under the control of radiation. For this purpose, there is at least one

substance that is caused by the radiation to effect polymerization. Some ofthe

substances may emit radiation that effect other substances which in turn initiate

or promote polymerization. For this purpose, the radiation mixture should

include at least one monomer, at least a porogen or a solvent and a substance

that effects polymerization. X-rays may be used with only a monomer to

prepare a support. If a porogen or solvent is included, the support may be

porous. The x-rays are a particularly safe type of radiation and mayhave wide

application in forming polymeric supports. The radiation may cause

polymerization or irradiate a substance such as a solvent that emits further

energy that causes initiation or promotion of polymerization. Example 18

Homopolymers, polymer resins and porous polymer support have been

prepared using 1 10 kV x-ray irradiation. The polymers were prepared in the

glass vials and columns. A polymerization mixture containing 1 % AIBN and

0.1 % p-terphenyl in styrene was degassed as described in the degassing

method and filled into and 1 ml glass vial. The vial was sealed with a screw cap.

The vial was exposed in 600 R/hour x-ray using 1 1 1 kVp power for two days.

A rigid bulk polystyrene polymer was obtained in the shape of the vial after

breaking the glass of the vial.

Alternative Versions of Example 18

A homopolyglycidyl methacrylate was prepared according to the above

method.

A homopolystyrene was prepared using solution polymerization

according to the above method. 50% of styene in toluene containing 1 % of

AIBN and 0.1 % of p-terphenyl was polymerized under 600 R h x-ray for two

days. Polystyrene was obtained after polymerization.

A poly(styrene-co-divinylbenzene) resin was prepared according to the

above method using the following 1 :1 ratio of styrene and divinyl benzene. The

polymerization mixture contains 0.9 g styrene, 0.9g divinylbenze, 17 mg AIBN,

5.7mg p-terphenyl. The polymer resin was obtained after polymerization. Gelation happened after 5 hours of polymerization.

A poly(glycidyl-co-ethylene glycol dimethacrylate) resin was prepared

according to the above method using the following 1 :1 ratio of glycidyl

methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA). The

polymerization mixture contains 0.9 g GMA, 0.9g EDMA, 17 mg AIBN, 5.7mg

p-terphenyl. The polymer resin was obtained after polymerization. Gelation

happened after 5 hours of polymerization.

A poly(glycidyl-co-ethylene glycol dimethacrylate) porous support was

prepared according to the above method using the following 1 :1 ratio of glycidyl

methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA). The

polymerization mixture contains 0.45 g GMA, 0.46g EDMA, 0.91 g

cyclohexanol, 20 mg AIBN, 1.5 mg p-terphenyl. A porous polymer was obtained

after polymerization. Gelation happened after 6 hours of polymerization.

A poly(styrene-co-divinylbenzene) porous support was prepared

according to the above method using the following polymerization mixture

contains 0.45 g styene, 0.45g divinylbenzene (80% pure), 0.91 g cyclohexanol,

20 mg AIBN, 6.5mg p-terphenyl. A porous polymer was obtained after

polymerization. Gelation happened after 4.5 hour of polymerization.

Example 19

Silica capillary with different inner diameters including 75 m, 100/iim,

200 m, 250μm, 320 m, 530μm and 700 m were modified with 1 M sodium hydroxide solution at 90 oC for 2 hours in an oven. The capillary was then washed with 60 column volumes of deionized water and acetone. It was dried by nitrogen purging through the column for 20 minutes. The capillary tubing was filled with a silanizing solution containing 50% (v/v) 3-(trimethoxysilyl)propyl methacrylate and 0.02% (w/v) hydroquinone in N,N-dimethylformamide (DMF). After both ends ofthe capillary were sealed, it was heated in an oven at 100 °C for about 10 h, and then washed with DMF and acetone. The capillary was dried with a nitrogen purging after wash.

Polymerization solutions were prepared as described in Example 1 with the composition of components listed in Table 1. Each modified capillary (usually 15~20cm) was filled with above polymerization solution. Two ends of the capillary were sealed in two 1.8mL glass vials, respectively, which were also filled with polymerization solution. Teflon and parafilm were used to double seal the cap ofthe vial. About 500μL empty space was left in each vial and the ends of the capillary locate at the half height of the vial. Two to four columns can be prepared by using the same sealing vials. The capillaries were hung vertically in the water bath at certain temperature listed in Table 1 for 20 hours by clamping the top vial on a stand. After polymerization, the monolithic capillary columns were washed with about 20 column volume organic solvent, usually acetonitrile, sometimes hexane when mineral oil was used as porogen.

The columns were characterized with the LC characterization method 1 a at the flow rate of 3, 5 and 10 μl/min. Great resolutions have been achieved.

Table 1. Polymerization solution composition and polymerization conditions.

Reactants I il III IV V vol% g vol% g wt% g vol% g wt% g

Styrene 20 2.1 16 1.6 20 2.0 20 2.1 8 0.8

Divinylbenzene 20 2.1 24 2.5 20 2.0 20 2.1 32 3.2

Tetrahydrofuran 7.5 0.8 9.5 1.1 ne

THP 7.0 0.7

1-decanoI 52.5 5.0 50.5 4.8 53 5.1

Toluene 8 0.8

1 -dodecanol 52 5.2

1-ethylhexanoic acid 27 2.7

Mineral oil 33 3.3

AIBN 0.04 g

P Poollyymmeerriizzaattiioonn 7700 70 70 70 80 Temp, °C

Alternative Versions of Example 19

A monolithic capillary column was prepared as in the above example with the following polymerization solution: .40g BMA, 2.6g EDMA, 3.8g 1- propanol, 1.6g 1 , 4-butanediol, 0.6g water and 0.04g AIBN. The polymerization was carried out at 60 °C. This column was characterized with the LC characterization method 1a with a microanalytical liquid chromatography system. The flow rate was 5 μl/min. Excellent separations of proteins were achieved.

Many other poly(acrylate) based capillary monoliths were synthesized with the variation of BMA /EDMA ratios (1/3 to 3/2) and different porogen concentrations (1 -propanol with 34 to 39 wt% ofthe total reaction mixture while

1 , 4-butanediol with 20 to 15 wt%).

Another column was prepared as in Example 19 with the following polymerization mixture: 2.2g DVB, 1.3g styrene, 0.9g acrylonitrile, 4.7g mineral oil, 0.8g toluene and 0.044g AIBN. The polymerization temperature is 75 °C. Many other columns of this type were prepared with the variation of acrylonitrile content in the polymer matrix from 0 to 50%. The polymers showed increasing hydrophilicity but different retentativities of proteins. Another column was prepared as in Example 19 with the following polymerization mixture: 0.8 g GMA, 2.4g EDMA, 0.8g ATMS, 3.1g 1-propanol, 2.6g 1 , 4-butanediol, 0.3g water and 0.04g AIBN. The polymerization reaction was carried out at 60 °C for 20 hours. After polymerization, the porogenic solvents in columns were washed away with aceonitrile. Then they were filled with modification solution, which is a mixture of TMA and water with volume ratio of 1 to 2 and with 0.45g/mL TMA

HCI. The modification was carried out at 40°C for 4 hours.

Example 20

The polymerization solution is prepared as in Example 1 but with the following mixure: 3.2154g acrylic acid, 8.0026 g methyl methacrylate, 1.6064 g glycidyl methacrylate, 19.2055 g ethylene glycol dimethacrylate, 43.8952 g

1 -dodecanol, 9.0017 g cyclohexanol, 320 mg diphenyl (2,4,6 trimethyl(benzole) phosphine oxide. The polymerization mixture was sonicated for 5 minutes and poured into the column, one end of which was sealed by a TEFLON cap, then the column was sealed with another TEFLON cap. The polymerization was allowed to expose to ordinary ceiling light for 7 days. The column was washed with 20 bed volume of acetonitrile followed by 20 bed volume of water. The column was characterized with chromatography.

Alternative Versions of Example 20

A glass column of size 10 cm x 1 cm ID was prepared as the above example with the following mixture: 2.5074 g glycidyl methacrylate, 2.5003 g ethylene glycol dimethacrylate, 7.5003 g p-xylene, and 58.2 mg diphenyl (2,4,6 trimethyl(benzole) phosphine oxide. The polymerization mixture was exposed to ordinary ceiling light for 24 hours. The same polymerization was carried out in another 2 ml vial for 24 hours. The conversion of monomers was 92%. A homopolyglycidyl methacrylate was prepared in a vial with the following polymerization mixture: 10 g GMA and 0.1 g diphenyl (2,4,6 trimethyl(benzole) phosphine oxide. The solution was exposed to ordinary ceiling light for 24 hours. The polymer gelled up after 3 hours. Very clear polymer was obtained. A homopoly(glycidyl methacrylate) was prepared in a vial with following polymerization mixture: 10 g GMA, 10 g xylene and 0.1 g diphenyl (2,4,6 trimethyl(benzole) phosphine oxide. The solution was exposed to ordinary ceiling light for 24 hours. The polymer gelled up after 3 hours. Clear polymer was obtained. From the above description it can be understood that the novel

monolithic solid support of this invention has several advantages, such as for

example: (1) it provides chromatograms in a manner superior to the prior art;

(2) it can be made simply and inexpensively; (3) it provides higherflow rates for

some separations than the prior art separations, thus reducing the time of some

separations; (4) it provides high resolution separations for some separation

resolution with disposable columns by reducing the cost of the columns; (6) it

permits column of many different shapes to be easily prepared, such as for

example annular columns for annular chromatography and prepared in any

dimensions especially small dimensions such as for microchips and capillaries

and for mass spectroscopy injectors using monolithic permeable polymeric tips;

(7) it separates both small and large molecules rapidly; (8) it can provide a

superior separating medium for many processes including among others

extraction, chromatography, electrophoresis, supercritical fluid chromatography and solid support for catalysis, TLC and integrated CEC separations or

chemical reaction; (9) it can provide better characteristics to certain known

permeable monolithic separating media; (10) it provides a novel approach for

the preparation of large diameter columns with homogeneous separation-

effective opening size distribution; (11 ) it provides a separation media with no

wall effect in highly aqueous mobile phase and with improved column

efficiency: (11 ) it improves separation effective factors; and (12) it reduces the

problems of swelling and shrinking in reverse phase columns.

Although preferred embodiments of the inventions have been described

with some particularity, many variations in the invention are possible within the

light of the above teachings. Therefore, it is to be understood that, within

the scope of the appended claims, the invention may be practiced other than

as specifically described.

Claims

What is claimed is:

1. A chromatographic system comprising:

a pumping system;

a column and detector array;

a collector system; and

a controller;

said column and detector array including a plurality of columns;

each of said columns containing a corresponding one of a plurality of

size-compensated polymeric plugs;

each of said size-compensated polymeric plugs having the same

characteristics, whereby said columns separate sample in a similar manner;

a source of solvent;

said pumping system being arrange to supply solvent from the source

of solvent to the column and detector array under the control of the controller

wherein effluent flows into the collector system from the column and detector

array under the control of the controller;

said controller being connected to receive signals from detectors in the

column and detector array indicating bands of solute and to activate the

fraction collector accordingly.

2. A chromatographic system according to claim 1 in which;

said solvent is supplied to a pump array having a different one of a plurality of pumps in said pump array communicating with each a corresponding

one of said plurality of columns in said column and detector array; and

a motor driven under the control of said controller to drive the array of

pumps.

3. A chromatographic system comprising:

a sample injector;

at least one chromatographic column having a column casing and at

least one permeable monolithic polymeric plug inside the column casing

positioned to receive a sample from the sample injector;

a solvent system in communication with the chromatographic column for

supplying a solvent to the at least one column, whereby the sample is

separated into its components within the at least one column;

said column casing including a column casing wall with an internal

column casing surface;

said at least one permeable monolithic polymeric plug with smooth

walls without discontinuities in contact with an internal surface of the column

walls; and

a utility device for receiving fluid from said column.

4. A chromatographic system according to claim 3 in which said at least

one permeable monolithic polymeric plug is a size-compensated polymeric

plug.

5. A chromatographic system in accordance with claim 3 in which said

at least one chromatographic column includes a plurality of columns.

6. A chromatographic system in accordance with claim 3 in which the

utility device is a detector.

7. A chromatographic system in accordance with claim 3 in which the

utility device is a fraction collector.

8. A chromatographic system in accordance with claim 3 in which the

at least one column is a capillary column.

9. A chromatographic system in accordance with claim 3 in which the

at least one column is a portion of a microchip.

10. A chromatographic system in accordance with claim 3 in which the

permeable monolithic polymeric plug includes divinylbenzene.

11. A chromatographic system in accordance with claim 3 in which the

permeable monolithic polymeric plug includes glycidyl methacrylate with

hydrophobic surface groups.

12. A chromatographic system in accordance with claim 3 in which the permeable monolithic polymeric plug includes urea formaldehyde.

13. A chromatographic system in accordance with claim 3 in which the

permeable monolithic polymeric plug includes silica.

14. A chromatographic system in accordance with claim 9 in which the

column casing is glass.

15. A chromatographic system in accordance with claim 9 in which the

column casing is steel.

16. A separating system comprising:

a permeable size-compensated polymeric support;

a sample injector positioned to inject sample onto the support of

permeable size-compensated polymer;

a solvent system in communication with the support of permeable size-

compensated polymer, whereby the sample is separated into its components

within the separating system; and

a utility device positioned to receive fluid from said permeable size-

compensated polymeric support.

17. A separating system in accordance with claim 16 in which the utility

device is a detector.

18. A separating system in accordance with claim 16 in which the

permeable size-compensated polymeric support has the structure of FIG. 5.

19. A method of performing chromatography comprising the steps of:

applying a sample to a chromatographic column having column casing

and a size compensated polymer monolithic packing; and

supplying a solvent to the chromatographic column, whereby the sample

is separated into its components within the chromatographic column.

20. A method in accordance with claim 19 further including the step of

causing said solvent to flow into a utility device.

21. A method in accordance with claim 20 in which the step of causing

said solvent to flow into a utility device includes the step of causing said solvent

to flow into a detector.

22. A method in accordance with claim 20 in which the step of causing

to flow into a fraction collector.

23. A method in accordance with claim 20 in which the step of causing said solvent to flow into a utility device includes the step of causing said solvent

to flow into a detector.

24. A method in accordance with claim 20 in which the step of causing

to flow into another instrument.

25. A method in accordance with claim 19 in which the step of

applying a sample to a chromatographic column having column casing and a

size compensated polymer monolithic packing includes the step of applying a

sample to permeable monolithic polymeric plug that includes a vinyl group.

26. A method in accordance with claim 19 in which the step of

applying a sample to a chromatographic column having column casing and a

size compensated polymer monolithic packing includes the step of applying a

sample to permeable monolithic polymeric plug that includes urea

formaldehyde.

27. A method in accordance with claim 19 in which the step of

applying a sample to a chromatographic column having column casing and a

size compensated polymer monolithic packing includes the step of applying a

sample to permeable monolithic polymeric plug that includes a silica polymer.

28. A chromatographic system comprising:

a sample injector;

at least one chromatographic column having a column casing and at

least one permeable monolithic polymeric plug inside the column casing

positioned to receive a sample from the sample injector;

a solvent system in communication with the chromatographic column for

supplying a solvent to the at least one column, whereby the sample is

separated into its components within the at least one column;

said column casing including a column casing wall with an internal

column casing surface;

said at least one permeable monolithic polymeric plug having the

structure shown in FIG. 9 when magnified 4.5 thousand times;; and

a utility device for receiving fluid from said column.

29. A chromatographic system according to claim 3 in which said at

least one permeable monolithic polymeric plug is a size-compensated

polymeric plug.

30. A separating system comprising:

a permeable size-compensated polymeric support having the internal

structure shown in FIG. 5 when magnified 9 thousand times; a sample injector positioned to inject sample onto the support of

permeable size-compensated polymer;

a solvent system in communication with the support of permeable size-

compensated polymer, whereby the sample is separated into its components

within the separating system; and

a utility device positioned to receive fluid from said permeable size-

compensated polymeric support.

31. A chromatographic column comprising:

a chromatographic column support having internal column walls;

a monolithic permeable plug in said column walls;

said permeable monolithic polymeric plug being a polymer having

separation-effective openings of a controlled size formed in the polymer by a

porogen in the polymerization mixture before polymerization and controlled in

size at least partly by pressure during polymerization.

32. A chromatographic column in accordance with claim 31 in which the

permeable monolithic polymeric plug has smooth walls.

33. A chromatographic column in accordance with claim 31 wherein

there are substantially no pores within the permeable monolithic polymeric

plug.

34. A chromatographic column in accordance with claim 31 wherein the

permeable monolithic polymeric plug is free of channeling openings in the walls

of the plug.

35. A chromatographic column in accordance with claim 31 wherein the

permeable monolithic polymeric plug is formed principally of methacrylate.

36. A chromatographic column in accordance with claim 31 wherein the

permeable monolithic polymeric plug is formed principally of methacrylate with

hydrophobic surface groups.

37. A chromatographic column in accordance with claim 31 wherein the

permeable monolithic polymeric plug is formed principally of urea

formaldehyde.

38. A chromatographic column in accordance with claim 31 wherein the

permeable monolithic polymeric plug is formed principally of silica.

39. A chromatographic column in accordance with claim 31 wherein the

permeable monolithic polymeric plug is principally formed of polymers of

glycidyl methacrylate and of ethylene dimethacrylate in a ratio by weight in the

range of from 1 to 1 and 2 to 1.

40. A chromatographic column in accordance with claim 31 wherein the

permeable monolithic polymeric plug is principally formed of polymers of

glycidyl methacrylate and of ethylene dimethacrylate in a ratio by weight of 3

to 2.

41. A column in accordance with claim 31 in which the permeable

monolithic polymeric plug includes as its principal component a mixture of

divinylbenzene and styrene in a ratio in the range of 3 to 1 and 9 to 1.

42. A column in accordance with claim 31 in which the permeable

monolithic polymeric plug includes as its principal component a mixture of

divinylbenzene and styrene in a ratio in the range of 4 to 1.

43. A column in accordance with claim 31 in which the column is in the

range of 35 percent and 80 percent by weight divinylbenzene.

44. A column in accordance with claim 43 in which the column is 64

percent by weight divinylbenzene.

45. A weak ion exchange chromatographic column comprising:

a chromatographic column support having internal column walls;

a permeable monolithic polymeric plug in said column walls; said permeable monolithic polymeric plug being formed principally of

methacrylate polymer and having smooth walls.

46. A weak ion exchange chromatographic column comprising:

a chromatographic column support having internal column walls;

a permeable monolithic polymeric plug in said column walls;

said permeable monolithic polymeric plug being formed principally of

polymers of glycidyl methacrylate and of ethylene dimethacrylate in a ratio by

weight in the range of from 1 to 1 and 2 to 1.

47. A chromatographic column in accordance with claim 45 wherein the

permeable monolithic polymeric plug is principally formed of polymers of

glycidyl methacrylate and of ethylene dimethacrylate in a ratio by weight of 3

to 2.

48. A chromatographic column comprising

a chromatographic column support having internal column walls;

a permeable monolithic polymeric plug in said column walls;

said permeable monolithic polymeric plug being formed principally of

a mixture of divinylbenzene and styrene and having smooth walls.

49. A chromatographic column in accordance with claim 65 in which the

ratio of divinylbenzene and styrene is in the range of 3 to 1 and 9 to 1 by weight.

50. A column in accordance with claim 51 in which the column is in the

range of 30 percent and 80 percent by weight divinylbenzene.

51. A chromatographic column comprising:

a chromatographic column support having internal column walls;

a permeable monolithic polymeric plug in said column walls;

said permeable monolithic polymeric plug being formed principally of a

mixture of divinylbenzene and styrene in a ratio of divinylbenzene and styrene

in the range of 3 to 1 and 9 to 1 by weight.

52. A column in accordance with claim 51 in which the column is in the

range of 30 percent and 80 percent by weight divinylbenzene.

53. A column in accordance with claim 31 further including hydrophobic

surface groups.

54. A column in accordance with claim 51 further including hydrophobic

surface groups.

55. A strong anion exchange column comprising: a column support having internal column walls;

a permeable monolithic polymeric plug in said column walls;

said permeable monolithic polymeric plug being formed principally of a

mixture of glycyl methylacrylate, ethyl divinylmethylacrylate in a range of ratios

of a value of glycyl

methylacrylate to ethyl divinylmethylacrylate in the range of ratios between 2

to 3 and 5 to 7.

56. A column in accordance with claim 55 in which the ratio of glycyl

methylacrylate to ethyl divinylmethylacrylate is substantially 1 to 3.

57. A weak cation chromatographic column comprising a

chromatographic column support having internal column walls;

a permeable monolithic polymeric plug in said column walls;

said permeable monolithic polymeric plug being formed principally of a

mixture of methyl methacrylate, glycyl methylacrylate and ethyl

divinylmethylacrylate in the ratio of a value of methyl methacrylate in a range

between 9 to 11 and 2 to 3 and 11 to 13.

58. A column in accordance with claim 57 in which the ratio of methyl

methacrylate, glycyl methylacrylate and ethyl divinylmethylacrylate is

substantially in the range of 5 to 1 to 12.

59. Apparatus for making a chromatographic column comprising:

a temperature controlled reaction chamber adapted to contain a

polymerization mixture during polymerization; and

means for applying pressure to said polymerization mixture in said

temperature controlled reaction chamber.

60. Apparatus according to claim 59 in which said polymerization

mixture comprises a polymer, a cross-linking reagent and a cross-linking

monomer, whereby rigidity, capacity and separation-effective opening

distribution are controlled by the amount of cross-linking reagent, monomer,

and pressure are aided forming mer and a porogen.

61. Apparatus according to claim 59 wherein said means for applying

pressure is a mean for applying pressure with a movable member.

62. Apparatus according to claim 59 in which said movable member has

a smooth surface positioned to contact said polymerization mixture as pressure

is applied during polymerization.

63. Apparatus for making a chromatographic column comprising:

a temperature controlled reaction chamber adapted to contain a

polymerization mixture during polymerization to form a plug; aqueous processing means for applying an aqueous solution to the plug;

means for applying pressure to said plug to reduce voids in the plug.

64. A method of making a monolithic chromatographic column

comprising the steps of:

preparing a polymerization mixture;

performing polymerization under pressure, whereby sufficient pressure

is applied to prevent voids from being formed caused by vacuum due to

shrinkage during polymerization.

65. The method of claim 64 in which the step of performing

polymerization includes the step of inserting a polymerization mixture into the

walls of a chromatographic column and polymerizing in a temperature

controlled chamber during at least part of the time pressure is being applied to

the polymerization mixture.

66. The method of claim 65 in which the step of inserting a

polymerization mixture includes the step of inserting at least one vinyl

monomer, an initiator, and a porogen.

67. A method of making a monolithic chromatographic column

comprising the steps of:

adding a polymerization mixture containing a porogen to a closed

container; polymerizing the mixture under pressure to form a polymer plug,

whereby sufficient pressure is applied to prevent voids from being formed

caused by vacuum due to shrinkage during polymerization; and

washing the polymer plug to remove the porogen.

68. A method of making a monolithic chromatographic column

comprising the steps of:

preparing a polymerization mixture including a porogen;

performing polymerization to form a polymer plug;

washing the polymer plug to remove the porogen, wherein the plug

tends to swell; and

applying pressure to prevent swelling ofthe polymer plug and eliminate

voids.

69. A method of making a monolithic permeable device for separating

the components of a sample, comprising the steps of:

preparing a polymerization mixture including a monomer and a porogen

wherein the porogen includes a mixture of alcohols;

said porogen comprising first and second alcohols in a ratio of a first

value in the range of a value between 1.8 and 2 to a value between 1.25 and

1.35, wherein the first value wherein the first alcohol is ethanol and the second

alcohol is butanol.

70. A method of making a monolithic permeable device for separating

the components of a sample, comprising the steps of:

preparing a polymerization mixture including a monomer and a porogen

wherein the porogen includes a mixture of alcohols;

said porogen comprising first, second and third alcohols in a ratio of a

first value in the range of a value between 2.8 and 3 to a second value in the

range of 0.3 to 0.35 and a third value in the range of 0.3 to 0.35 wherein the

first alcohol is ethanol, the second alcohol is methanol and the third alcohol is

isopropanol.

71. A polymerization mixture comprising divinylbenzene, styrene, an

initiator and a porogen, wherein the divinybenzene and styrene are in the ratio

in the range of 3 to 1 and 9 to 1.

72. A polymerization mixture in accordance with claim 71 wherein the

divinybenzene and styrene are in the ratio of 4 to 1.

73. A polymerization mixture in accordance with claim 72 in which the

proportions of divinylbenzene, styrene and porogen are in the ratio of 8 to 2 to

15 respectively.

74. A polymerization mixture comprising divinylbenzene, styrene and

dodecanol in a proportion within the range of 7 to 9 units of divinylbenzene to

1.5 to 2.5 units of styrene to 13-17 units of dodecanol combined with an

initiator.

75. A polymerization mixture according to claim 74 in which the

proportions of divinylbenzene, styrene and dodecanol are 8 to 2 to 15

respectively combined with an initiator.

76. A polymerization mixture comprising divinylbenzene, styrene

dodecanol and toluene in the proportions of 7-9 to 1.5-2.5 to 9-13 to 2.5-3.5

respectively, combined with an initiator.

77. A polymerization mixture according to claim 76 in which the

divinylbenzene, styrene dodecanol and toluene are combined in the proportions

of of 8 to 2 to 11 to 3 respectively, combined with an initiator.

78. A polymerization mixture comprising glycidyl methacrylate,

ethylene glycol dimethacrylate, cyclohexanol and dodecanol in the proportions

of 0.5-0.7 to 0.3-0.5 to 1-2 to 0.1-2.5, combined with an initiator.

79. A polymerization mixture in accordance with claim 78 in which the

glycidyl methacrylate, ethylene dimethacrylate, cyclohexanol and dodecanol

are in the proportions of 0.6 to 0.4 to 1.325 to 0.175 respectively.

80. A method of making a column, comprising the steps of:

weighing divinylbenzene, styrene and an initiator into a sample vial in the

proportions of 3 to 5 parts of divinylbenzene to one part of styrene;

shaking the mixture of divinylbenzene, benzene, styrene and initiator

gently until the mixture becomes a homogeneous solution;

weighing 13 to 17 parts of porogen into the homogeneous solution and

shaking until the new mixture is homogeneous to form a polymerization

solution;

degassing;

filling a stainless steel column with the polymerization solution;

sealing the stainless steel column;

polymerized the polymerization solution in the stainless steel column to

form a plug;

washing the porogen from the plug using at least some water in at least

one phase of the washing; and

compressing the plug by applying pressure to it in the stainless steel

column.

81. The method of claim 80 in which the step of weighing

divinylbenzene, styrene and an initiator into a sample vial comprises the step

of weighing the divinylbenzene and styrene in the proportions of approximately

4 parts of divinylbenzene to one part of styrene, the step of weighing 13 to 17

parts of porogen comprisies the step of weighing 15 parts of dodecanol into the homogeneous solution; and the step pf polymerizing includes the step of

polymerizing the polymerization solution at between 40 and 75 °C in a water

bath for between 14 and 34 hours.

82. The method of claim 81 in which the step of weighing 13 to 17 parts

of porogen into the homogeneous solution and shaking until the new mixture

is homogeneous to form a polymerization solution comprises the step of mixing

between 10 and 14 parts dodecanol and 2 and 3 parts toluene together to form

a polymerization solution.

83. A method of making a column, comprising the steps of:

weighing glycidyl methacrylate, ethylene dimethacrylate and an initiator

into a sample vial in the proportions of 1.5 to 4 parts of glycidyl methacrylate,

to 1 to 2 parts of ethylene dimethacrylate;

shaking the mixture of glycidyl methacrylate, ethylene dimethacrylate

and initiator gently until the mixture becomes a homogeneous solution;

weighing a porogen into the homogeneous solution and shaking until the

new mixture is homogeneous to form a polymerization solution;

degassing;

filling a column with the polymerization solution, wherein the column

includes means for applying pressure to the polymerization solution;

sealing the column;

polymerized the polymerization solution in the column under pressure to form a plug;

washing the plug to remove the porogen.

84. A method according to claim 83 in which the step of weighing

glycidyl methacrylate, ethylene dimethacrylate and an initiator into a sample vial

comprises the step of weighing 0.6 g of glycidyl methacrylate, 0.4 g of ethylene

dimethacrylate and an initiator into a sample vial in the proportions of 3 parts

of divinylbenzene to one part of styrene.

85. A method according to claim 83 in which the step of weighing a

porogen into the homogeneous solution and shaking until the new mixture is

homogeneous to form a polymerization solution comprises the step of weighing

cyclohexanol and dodecanol into the homogeneous solution in proportions in

the range of 10 parts of cyclohexanol to 1 part of dodecanol and 17 parts of

cyclohexanol to 1 part dodecanol by weight.

86. A method according to claim 83 in which the step of weighing a

porogen into the homogeneous solution and shaking until the new mixture is

homogeneous to form a polymerization solution comprises the step of weighing

cyclohexanol and dodecanol into the homogeneous solution in proportions in

the range of 10 parts of cyclohexanol to 1 part of dodecanol and 17 parts of

cyclohexanol to 1 part dodecanol by weight.

87. A method according to claim 83 in which the step of weighing a

porogen into the homogeneous solution and shaking until the new mixture is

homogeneous to form a polymerization solution comprises the step of weighing

cyclohexanol and dodecanol into the homogeneous solution in proportions of

13 parts of cyclohexanol to 1.7 parts of dodecanol.

88. A method according to claim 84 in which the step of polymerizing

the polymerization solution in the column under pressure to form a plug

comprises the steps of polymerizing the solution under a pressure within the

column of between 0.1 psi and 600 psi upright at a temperature in the range of

200 °C to 100 degrees C for a time of between 15 hours and 48 hours.

89. A method according to claim 86 further including the step of adding

a catalyst to the polymerization solution diethylamine hydrogen chloride as

catalyst.

90. A method according to claim 89 wherein the catalyst is diethylamine

hydrogen chloride.

91. A method according to claim 89 wherein the catalyst is

trimethylamine hydrogen chloride.

92. A separating medium that has the structure shown in FIG. 5.

93. A method of making a monolithic permeable device for separating

the components of a sample, comprising the steps of:

preparing a polymerization mixture including a monomer and a porogen

wherein the porogen includes a mixture of alcohols;

said porogen comprising first, second, third and fourth alcohols in a ratio

of a value between 6 to 7 and 3 to 35 and 3 to 35 and 3 to 35 wherein the

alcohols are ethanol, methanol, propanol and butanol respectively.

94. A method of controlling polymerization of a porous medium

comprising the steps of:

establishing a polymerization mixture including a porogen;

controlling the rate of the polymerization with radiation and at least one

substance that is caused by the radiation to affect the polymerization.

95. A method in accordance with claim 94 wherein at least one of the

at least one substance that is affected by the radiation is energized to emit

further radiation.

96. A method in accordance with claim 95 in which the further radiation

initiates polymerization.

97. A method in accordance with claim 95 wherein at least one of the

at least one substance emits radiation that energizes a second substance to

emit radiation.

98. A method of controlling polymerization of a porous medium

comprising the steps of:

establishing a polymerization mixture including a solvent;

controlling the rate of the polymerization with radiation and at least one

substance that is caused by the radiation to affect the polymerization.

99. A polymerization mixture comprising at least one monomer, at least

one porogen and at least one substance that affects polymerization in response

to radiation.

100. A polymerization mixture comprising at least one monomer, at least

one solvent and at least one substance that affects polymerization in response

to radiation.

101. A method of making a monolithic permeable device for separating

the components of a sample, comprising the steps of:

preparing a polymerization mixture including a monomer, a porogen and

at least one radiation sensitive substance;

said radiation sensitive substance comprising one or more substances selected from a group consisting of photo initiators, solvents, x-ray sensitizers

and scintillators; and irradiating the polymerization mixture.

102. A method of polymerizing a mixture to form a solid support

containing at least one monomer and a porogen comprising the step of

irradiating the mixure with X rays.

103. A method of polymerizing a mixture to form a solid support

containing at least one monomer and a solvent comprising the step of

irradiating the mixure with X rays.

104. A method in accordance with claim 103 in which the x-rays are

applied axially to an elongated mixture.

105. An apparatus for polymerizing chromatographic columns

comprising:

a temperature controlled reaction chamber adapted to contain a

polymerization mixture during polymerization to form a plug;

radiation means for applying, radiation to the plug; and

control means for controlling the radiation.

106. An apparatus in accordance with claim 105 further including means

for applying pressure to the plug to reduce voids in the plug.