WO2008150766A1

WO2008150766A1 - Chromatography column

Info

Publication number: WO2008150766A1
Application number: PCT/US2008/064864
Authority: WO
Inventors: Edouard S.P. Bouvier; Johnathan Belanger
Original assignee: Waters Investments Limited
Priority date: 2007-05-29
Filing date: 2008-05-27
Publication date: 2008-12-11

Abstract

A device for chromatographically separating compounds held in a fluid sample. The device comprises a tube means having an inlet aperture for receiving a fluid sample, an outlet aperture and an internal wall. The internal wall defines a passage for receiving a monolithic separation medium. The passage has a first end and a second end, spaced from each other along a length, and a plurality of cross-sectional areas along the length. The inlet aperture is at the first end and the outlet aperture at the second end. A monolithic separation medium is positioned in said passage. Each of the cross-sectional areas of the passage decreases with each cross sectional area in a continuous or stepwise manner between the first end and the second end, such that a flow of a fluid sample through the passage from the inlet aperture to the outlet aperture pushes the monolithic separation medium against the internal wall.

Description

CHROMATOGRAPHY COLUMN

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit to U.S. Provisional Application No. 60/940,468, filed May 29, 2007. The contents of this application is expressly incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Embodiments of the present invention are directed to a device and methods of using and manufacturing such a device for the separation of chemical mixtures, for instance for use in chromatography systems.

This paper will use the term "monomer" to denote a molecule comprising one or more reactive functional groups capable of reacting with a similar or different reactive molecule to form a longer molecule comprising a plurality of repeating units.

The term "monolith" is intended to include a porous, three-dimensional material having a continuous interconnected pore structure in a single piece. A monolith is prepared, for example, by casting precursors into a mould of a desired shape. The term monolith is meant to be distinguished from a collection of individual particles packed into a bed formation, in which the end product still comprises individual particles in bed formation.

The term "hybrid" is intended to include structures which comprise both organic and inorganic functionalities which are integral to both the internal or "skeletal" structure as well as the material surface. The inorganic portion of the hybrid material may be, for example, alumina, silica, titanium or zirconium oxides, or ceramic material. Chromatography is an efficient tool for separation of samples and its use is widespread throughout the analytical community. Very generally a chromatographic separation is carried on with an instrument composed of solvent reservoirs, pumps, a mixing unit, an injection device, a chromatographic column, a detector and a data collecting device.

The sample is injected in a flow of an appropriate solvent going through the chromatographic column. The different components are separated in the column due to adsorption, absorption, size exclusion, ion exchange or other interactions with the packing material. The separated components are then detected in the detector. Some detectors which are commonly used include ultraviolet absorption, fluorescence, refractive index, conductivity, electrochemical and derivatization detectors, as well as mass spectrometers. The data obtained is processed with an appropriate data module.

Chromatography also has important uses in synthesis, where it can be used on a larger scale to separate target compounds from mixtures after synthetic reactions.

Columns used for chromatography can be divided for the purposes of this discussion into two groups: Packed columns and monolithic columns. Packed columns are cylindrical vessels with a frit or screen on one or both ends defining a chamber. Particles, beads, or some other packing material is placed soundly under pressure between the frits. A monolith column is a cylinder in which a permeable, substantially non-particulate polymer mass fills the cylinder.

Monolithic columns for chromatography exhibit several benefits over particle packed chromatography columns. Monolithic columns may typically exhibit four times the permeability of a particle packed column of similar dimension and chromatographic efficiency. This provides performance benefits, particularly when high throughput analyses are required.

An important limitation to the use of monolithic columns for chromatography lies in the synthesis of the monoliths. It is desirable to synthesise the monoliths inside the column housing, for ease of manufacturability and handling. The monoliths are typically synthesised in condensation reactions to form a permeable mass. The resulting solid may shrink upon curing owing to the loss of water in the condensation reactions, the shorter intra-molecular bonds compared to inter- molecular bonds, and a reduction in network swelling owing to solvent removal.

Shrinking in the monolith may cause cracks to appear, or cause a void to form between the monolith and portions of the housing wall. These voids and cracks have a significant detrimental effect upon chromatographic performance. The increased porosity of these cracks and voids allows a quantity of the analyte to bypass the separation medium.

There exists a need for a device and a method of manufacturing such a device having a monolithic separation medium which is resistive to the formation of voids.

SUMMARY OF THE INVENTION

Preferably, the passage has a first cross sectional area Ai and a second cross-sectional area A₂. The second cross sectional area A₂ is 1 unit of length closer to the inlet aperture than the first cross-sectional area Ai. Preferably, second cross-sectional area A₂ is at least 0.005 corresponding units of area greater than the first cross-sectional area Ai.

Preferably, the passage comprises a longitudinal axis. At least a portion of the internal wall is inclined to the longitudinal axis at an angle θ. Preferably, angle θ falls within the range 0.2° to 12°. In some preferred embodiments, angle θ is about 4°.

Preferably, the outlet aperture has an internal diameter, the internal diameter measuring between 0.5mm and 50mm. More preferably, the internal diameter measures less than 0.5mm.

In some preferred embodiments, the tube means is a capillary tube. In other preferred embodiments, the tube means is a pipe, tube, conduit or microfluidic chip.

Preferably, the tube means is constructed from fused silica, stainless steel or polyetheretherketone.

Preferably, the monolithic separation medium comprises a porous organic monolith, a porous inorganic monolith, a porous inorganic silica monolith, a porous inorganic-organic hybrid monolith, a porous organic hybrid monolith.

Preferably, the monolithic separation medium is shaped in a complementary manner to said passage.

Preferably, the device further comprises a detector in fluid communication with the outlet aperture. Preferably, detector is selected from the group comprising a mass spectrometer, ultra violet/visual spectroscope, evaporative light scattering detector, fluorescence detector, electrochemical detector, chemiluminescence detector, refractive index detector, or conductivity detector. In another aspect, the invention relates to a method of manufacturing a device for chromatographicaily separating compounds held in a fluid sample. The method comprises the steps of:

Providing a tube means having an inlet aperture for receiving a fluid sample, an outlet aperture and at least one internal wall. The internal wall defines a passage for receiving a monolithic separation medium. The passage has a first end and a second end, spaced from each other along a length, and a plurality of cross-sectional areas along the length. The inlet aperture is at the first end and the outlet aperture at the second end. Each of the cross-sectional areas of the passage decreases with each cross sectional area in a continuous or stepwise manner between the first end and the second end.

Providing a monolithic separation medium in said passage.

Preferably, the step of providing the monolithic separation medium comprises the steps of:

Providing a mould having a cavity for receiving a monomer mixture, the cavity being a scale replica of said passage, filling the cavity with a monomer mixture, and initiating a polymerisation reaction to produce a monolithic separation medium.

Preferably, the cavity is of similar dimensions to said passage. Alternatively, the cavity is of larger dimensions than said passage.

In an alternative embodiment, the step of providing the monolithic separation medium preferably comprises the steps of:

Providing at least one stop to seal at least the inlet aperture or the outlet aperture, filling the passage with a monomer mixture, and initiating a polymerisation reaction to produce said monolithic separation medium.

Preferably, the method further comprises the step of applying a force to the monolithic separation medium through said inlet aperture. The force may act such that at least a first portion of the monolithic separation medium is pushed against the internal wall and at least a second portion of the monolithic separation medium extends through the outlet aperture.

Preferably, the method further comprises the step of removing the second portion of the monolithic separation medium.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1A shows an aspect of the device of the present invention.

Figure 1 B shows a further aspect of the device of the present invention.

Figure 1C shows a further aspect of the device of the present invention.

Figure 1 D shows a further aspect of the device of the present invention.

Figure 2 shows an aspect of the device of present invention where the device comprises an injector and a detector.

Figure 3 shows steps in a method of the present invention.

Figure 4 shows steps in a further method of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a device for chromatographically separating compounds held in a fluid sample, as demonstrated in the embodiments shown in Figures 1 and 2. The device 110 as shown in Figure 1A comprises a tube means 112 having an inlet aperture 114, for receiving a fluid sample, at a first end 116, and an outlet aperture 118, for discharging a fluid sample, at a second end 120. Internal wall 122 defines a passage 124 between inlet aperture 114 and outlet aperture 118. Passage 124 has a plurality of cross sectional areas, each area decreasing with respect to the last, with distance from inlet aperture 114, in a continuous or step-wise manner. In a preferred embodiment, this decrease in cross sectional area makes passage 124 tapered in shape.

The same device 110 is shown in Figure 1B. Passage 124 comprises a longitudinal axis 126. At least a portion of the internal wall 122 is inclined to the longitudinal axis 126 at an angle θ. In preferred embodiments, angle θ is between 0.2° and 12°. In other preferred embodiments, angle θ is 4°.

The same device 110 is shown in Fig C. Passage 124 has a first cross- sectional area A₁. Passage 124 has a second cross-sectional area A₂ 1 unit of length closer to inlet aperture 114 than first cross-sectional area A-i. In preferred embodiments, second cross sectional area A₂ is at least 0.005 units of area greater than first cross-sectional area Ai, where the units of area correspond accordingly with the units of length.

In preferred embodiments, the internal diameter of outlet aperture 118 is between 0.5mm and 50mm. In other preferred embodiments, the internal diameter of outlet aperture is less than 0.5mm.

The same device 110 is shown in Figure 1 D. In passage 124 is monolithic separation medium 128. Monolithic separation medium 128 is shaped in a complementary manner to passage 124, such that voids between monolithic separation medium 128 and internal wall 122 are minimised.

Tube means 112 is preferably a capillary tube. Such a capillary tube is preferably made from stainless steel. In other preferred embodiments, such a capillary tube is constructed from fused silica, glass or polyetheretherketone. In further preferred embodiments, tube means 112 is a pipe, tube, conduit or microfluidic chip. The cross-section of such a tube means may take any shape.

Monolithic separation medium 128 preferably comprises a porous organic monolith, a porous inorganic monolith, a porous inorganic silica monolith, a porous inorganic-organic hybrid monolith, a porous organic hybrid monolith, or a porous inorganic silica monolith.

In the preferred embodiment shown in Figure 1 D, device 110 further comprises an outlet frit 138, fixed to the second end 120 of tube means 112 by endfitting 136. Inlet frit 130, positioned in passage 124 at the first end 116 of tube means 112, prevents any particulate matter entering monolithic separation medium 128 in use. Flow conduit 132 packs any remaining space in passage 124. Closure fitting 134 seals the device 110 and provides a means for ensuring even flow distribution and connection to the remainder of a chromatographic system.

Another embodiment of the invention is shown in Figure 2. Tube means 112 having an inlet aperture 114, for receiving a fluid sample, at a first end 116, and an outlet aperture 118, for discharging a fluid sample, at a second end 120. Internal wall 122 defines a passage 124 between inlet aperture 114 and outlet aperture 118. Passage 124 has a plurality of cross sectional areas, each area decreasing with respect to the last, with distance from inlet aperture 114, in a continuous or step-wise manner. In a preferred embodiment, this decrease in cross sectional area makes passage 124 tapered in shape. In passage 124 is monolithic separation medium 128. Monolithic separation medium 128 is shaped in a complementary manner to passage 124, such that voids between monolithic separation medium 128 and internal wall 122 are minimised. Injector 140, for injecting a fluid sample, is in fluid communication with inlet aperture 114. Pump 141 , for pumping solvent through tube means 112, is in fluid communication with inlet aperture 114, via injector 140. Detector 142, for detecting compounds after separation, is in fluid communication with outlet aperture 118.

In use, injector 140 injects a fluid sample containing at least one compound through inlet aperture 114. Pressure exerted by the injection of fluid sample pushes monolithic separation medium 128 against internal wall 122. Pump 141 may pump a suitable chromatographic solvent through inlet aperture 114 to affect a chromatographic separation of the compounds in the fluid sample. Compounds may be in a state of separation when discharged through outlet aperture 118. Fluid flow maintained by pump 141 transports compounds to detector 142 for detection. In preferred embodiments, detector 142 is a mass spectrometer. In other preferred embodiments, detector 142 is an ultra violet/visual spectroscope, fourier transform ultra violet/visual spectroscope, infra red spectroscope, fourier transform infra red spectroscope, nuclear magnetic resonance spectroscope, fourier transform nuclear magnetic resonance spectroscope, raman spectroscope, evaporative light scattering detector, fluorescence detector, electrochemical detector, chemiluminescence detector, refractive index detector, or conductivity detector.

In another embodiment, the invention is a method of manufacturing a device 110 for chromatographically separating compounds held in a fluid sample. Steps for the manufacture of this method are shown in Figure 3. A tube means 112 having an inlet aperture 114, for receiving a fluid sample, at a first end 116, and an outlet aperture 118, for discharging a fluid sample, at a second end 120. Internal wall 122 defines a passage 124 between inlet aperture 114 and outlet aperture 118. Passage 124 has a plurality of cross sectional areas, each area decreasing with respect to the last, with distance from inlet aperture 114, in a continuous or step-wise manner. In a preferred embodiment, this decrease in cross sectional area makes passage 124 tapered in shape.

Stop 154 is provided to seal outlet aperture 118 or inlet aperture 114.

Monomer mixture 156 is then introduced to passage 124. Monomer mixture 156 preferably comprises at least one monomer compound and a porogen compound. A polymerisation reaction is then initiated. The polymerisation reaction may be initiated by means including, but not limited to, heat or ultra-violet radiation. The result of the polymerisation is monolithic separation medium 128.

When polymerisation is complete, a force is applied to the first end 144 of monolithic separation medium 128 through inlet aperture 114. Monolithic separation medium 128 is thus pushed against internal wall 122. At least a first portion 146 of monolithic separation medium 128 remains inside tube means 112, pushed against internal wall 122. At least a second portion 148 of monolithic separation medium 128 extends through outlet aperture 118 to protrude clear of second end 120 of tube means 112. This second portion 148 of monolithic separation medium 128 is removed. This removal step may be performed by means including, but not limited to shaving, cutting filing or breaking.

The device may then be finished for use. This involves the addition of outlet frit 138, held in place by endfitting 136. Endfitting 136 may be attached to second end 120 of tube means 112 by such means as a screw fitting, adhesive or a clip. Endfitting 136 allows easy connection into a chromatographic system. First end 144 of monolithic separation medium 128 may require some finishing to ensure that it is of regular shape. Any space remaining within passage 124 that is not filled by monolithic separation medium 128 is filled with flow conduit 132. Inlet frit 130 is fixed to the first end 116 of tube means 112 by closure fitting 134. Closure fitting 134 also seals the device 110 and provides a means for ensuring even flow distribution and connection to the remainder of a chromatographic system.

In an alternative embodiment, the invention is a method of manufacturing a device 110 for chromatographically separating compounds held in a fluid sample. Steps for the manufacture of this method are shown in Figure 4. A tube means 112 having an inlet aperture 114, for receiving a fluid sample, at a first end 116, and an outlet aperture 118, for discharging a fluid sample, at a second end 120. Internal wall 122 defines a passage 124 between inlet aperture 114 and outlet aperture 118. Passage 124 has a plurality of cross sectional areas, each area decreasing with respect to the last, with distance from inlet aperture 114, in a continuous or step-wise manner. In a preferred embodiment, this decrease in cross sectional area makes passage 124 tapered in shape. Further provided is a mould 150 having a cavity 152. Cavity 152 is a scale replica of passage 124 defined by internal wall 122 of tube means 112. This is such that a monolith fabricated in mould 150 is shaped in a complementary manner to passage 124. The dimensions of cavity 152 may be substantially the same as the dimensions of passage 124. Alternatively, the dimensions of cavity 152 may be larger than the dimensions of passage 124, thus minimising the effect of shrinkage of the resulting monolith.

Monomer mixture 156 is then introduced to cavity 152. Monomer mixture 156 preferably comprises at least one monomer compound and a porogen compound. A polymerisation reaction is then initiated. The polymerisation reaction may be initiated by means including, but not limited to, heat or ultra-violet radiation. The result of the polymerisation is monolithic separation medium 128. When polymerisation is complete, monolithic separation medium 128 is removed from cavity 152 of mould 150 and introduced to passage 124 defined by internal wall 122 of tube means 112. A force is applied to the first end 144 of monolithic separation medium 128 through inlet aperture 114. Monolithic separation medium 128 is thus pushed against internal wall 122. At least a first portion 146 of monolithic separation medium 128 remains inside tube means 112, pushed against internal wall 122. At least a second portion 148 of monolithic separation medium 128 extends through outlet aperture 118 to protrude clear of second end 120 of tube means 112. This second portion 148 of monolithic separation medium 128 is removed. This removal step may be performed by means including, but not limited to shaving, cutting filing or breaking.

The device may then be finished for use. This involves the addition of outlet frit 138, held in place by endfitting 136. Endfitting 136 may be attached to second end 120 of tube means 112 by such means as a screw fitting, adhesive or a clip. Endfitting 136 allows easy connection into a chromatographic system. First end 144 of monolithic separation medium 128 may require some finishing to ensure that it is of regular shape. Any space remaining within passage 124 that is not filled by monolithic separation medium 128 is filled with flow conduit 132. Inlet frit 130 is fixed to the first end 116 of tube means 112 by closure fitting 134.

Closure fitting 134 also seals the device 110 and provides a means for ensuring even flow distribution and connection to the remainder of a chromatographic system.

In one preferred embodiment, monolithic separation medium was prepared by mixing 1.6 mL of tetramethoxysilane (TMOS), 0.464 g polyethylene oxide (molecular weight 10,000) and 3.95 ml of 0.01 M acetic acid together at 0 ⁰C for 30 minutes. The mixture was introduced to the passage of a tube means, constructed of stainless steel, of length 100mm, outlet aperture 2mm and angle θ of 0.5°. The tube means was then heated to 40 ⁰C for 20 hours. The resulting solid monolithic separation medium was then flushed with 1.5 M urea using an HPLC pump. The monolithic separation medium was then heated at 110 ⁰C in a sealed container for 24 hours. Subsequently, the device 110 was washed with water, followed by methanol.

Claims

1. A device for chromatographically separating compounds held in a fluid sample comprising: a tube means having an inlet aperture for receiving a fluid sample, an outlet aperture and an internal wall, said internal wall defining a passage for receiving a monolithic separation medium, said passage having a first end and a second end spaced from each other along a length and having a plurality of cross sectional areas along said length, said inlet aperture at said first end and said outlet aperture at said second end; and, a monolithic separation medium positioned in said passage, wherein each of said cross-sectional areas decreases for each cross sectional area in a continuous or stepwise manner between said first end and said second end, such that a flow of a fluid sample through said passage from said inlet aperture to said outlet aperture pushes said monolithic separation medium against said internal wall.

2. The device of claim 1 where said passage comprises a longitudinal axis and at least a portion of said internal wall is inclined to said longitudinal axis at an angle θ, wherein said angle θ falls within the range 0.2° to 12°.

3. The device of claim 2 where said angle θ is about 4°.

4. The device of claim 1 a first of said cross sectional areas Ai is 1 unit of length further from said inlet aperture than a second of said cross-sectional areas A₂, wherein said second of said cross-sectional areas A₂ is at least 0.005 corresponding units of area greater than said first of said cross-sectional areas Ai.

5. The device of claim 1 where said outlet aperture has an internal diameter, said internal diameter measuring between 0.5mm and 50mm.

6. The device of claim 1 where said outlet aperture has an internal diameter, said internal diameter measuring less than 0.5mm.

7, The device of claim 1 wherein said tube means is a capillary tube.

8. The device of claim 1 wherein said tube means is a microfluidic chip.

9. The device of claim 1 wherein said tube means is constructed from fused silica.

10. The device of claim 1 wherein said tube means is constructed from stainless steel.

11. The device of claim 1 wherein said tube means is constructed from polyetheretherketone.

12. The device of claim 1 where said monolithic separation medium comprises a porous organic monolith.

13. The device of claim 1 where said monolithic separation medium comprises a porous inorganic monolith.

14. The device of claim 1 where said monolithic separation medium comprises a porous inorganic silica monolith.

15. The device of claim 1 where said monolithic separation medium comprises a porous inorganic-organic hybrid monolith.

16. The device of claim 1 where said monolithic separation medium comprises a porous organic hybrid monolith.

17. The device of claim 1 where said monolithic separation medium comprises a porous inorganic silica monolith.

18. The device of claim 1 where said monolithic separation medium is shaped in a complementary manner to said passage.

19. The device of claim 1 further comprising a detector in fluid communication with said outlet aperture.

20. The device of claim 19 where said detector is selected from the group comprising a mass spectrometer, ultra violet/visual spectroscope, evaporative light scattering detector, fluorescence detector, electrochemical detector, chemiluminescence detector, refractive index detector, or conductivity detector.

21. A method of manufacturing a device for chromatographicaliy separating compounds held in a fluid sample comprising the steps of: providing a tube means having an inlet aperture for receiving a fluid sample, an outlet aperture and an internal wall, said internal wall defining a passage for receiving a monolithic separation medium, said passage having a first end and a second end spaced from each other along a length and having a plurality of cross sectional areas along said length, said inlet aperture at said first end and said outlet aperture at said second end, wherein each of said cross-sectional areas decreases for each cross sectional area in a continuous or stepwise manner between said first end and said second end; providing a monolithic separation medium in said passage.

22. The method of claim 21 where the step of providing said monolithic separation medium comprises the steps of: providing a mould having a cavity for receiving a monomer mixture, said cavity being a scale replica of said passage; filling said cavity with a monomer mixture, initiating a polymerisation reaction to produce said monolithic separation medium.

23. The method of claim 22 where said cavity is of similar dimensions to said passage.

24. The method of claim 22 where said cavity is of larger dimensions than said passage.

25. The method of claim 21 where the step of providing said monolithic separation medium comprises the steps of: providing at least one stop to seal at least said inlet aperture or said outlet aperture, filling said passage with a monomer mixture, initiating a polymerisation reaction to produce said monolithic separation medium.

26. The method of claim 21 further comprising the step of applying a force to said monolithic separation medium through said inlet aperture such that at least a first portion of said monolithic separation medium is pushed against said internal wall and at least a second portion of said monolithic separation medium extends through said outlet aperture,

27. The method of claim 26 further comprising the step of removing said second portion of said monolithic separation medium.