CN107709984A

CN107709984A - Extraction column

Info

Publication number: CN107709984A
Application number: CN201680034142.7A
Authority: CN
Inventors: 琼尼·兰尼斯托; 苏维·拉韦拉; 凯利·J·弗卢克; 约翰·芬耐尔; 莱纳·瓦尔穆; 鲍里斯拉夫·马里诺; 维尔·萨里嫩; 克里斯托夫·A·波尔
Original assignee: Thermo Fisher Scientific Oy
Current assignee: Thermo Fisher Scientific Oy
Priority date: 2015-06-10
Filing date: 2016-06-10
Publication date: 2018-02-16
Also published as: WO2016198748A1; CN112067731A; EP3308156A1

Abstract

The invention discloses the devices, systems, and methods for being related to extraction column.The embodiment of extraction column includes：Post body, the post body have storage part, Extraction medium part and collar portion, and the storage unit point includes entrance and storage room.The Extraction medium part includes elongate sleeve, and the elongate sleeve has cavity, and Extraction medium is set in the cavity.Extraction medium part has arrival end and the port of export.Collar portion is axially extending together with elongate sleeve.Collar portion has end, and the outlet end of the end and elongate sleeve is opened, and at least axially extends to the plane limited by the port of export of elongate sleeve.The embodiment of shell is also disclosed, shell is included by one layer of medium separated with the inner surface of shell, and the part that this layer is configured to be substantially prevented from sample bypasses the medium.

Description

Extraction column

Technical field

The present invention relates to a kind of device and system being used for from sample extraction component, and carried more particularly, to one kind Take post and the disposable end including extraction column.

Background technology

Liquid chromatography(LC) mass-spectrometry (liquid chromatography-mass spectrography) generally, based on protein group is referred to as from bottom to top Peptide is set as analyte in the processing of protein group.In managing in this place, make protein digestibility into contracting ammonia using protease Acid.The duration of the digestion can be very long and be frequently necessary to incubated overnight., it is necessary to short sample in clinical settings Processing time.Top-down protein group, which introduces, can wherein utilize LC-MS analysis former state protein etc. Option.This proteinoid group does not require very long digestion, it is particularly suited in Clinical practice and automatic system.However, Still have to extract protein particulate from another non-protein component in sample.Extraction column can be utilized from its in sample Protein is extracted in its component.Preferably, the extraction column will allow automatic sample to prepare and be directly integrated into liquid chromatogram.

Generally, pipette tip and similar running stores are sold, transported and are inserted into known for entering to each end In autoanalyzer in the pallet of row position and orientation or other devices.However, easier and cheap is to wrap end Fill in a reservoir and transported with container, wherein end is in high volume packaged into such as sack or box with random sequence.It is logical Often, pipette tip is the conical by its shape with tip and openend, and this causes when loosely being stored with random sequence and orientation, These ends stack and accumulated.This make it that automatic system is difficult one pipette tip of pickup in some time, and the end stacked can Automatic system can be caused to block.Another problem of this usually said " random encapsulation " is the suction pipe for including Extraction medium The very thin end of end may be damaged due to contacting other ends.

Further, since the problem of interface between Extraction medium and the wall of conduit wall it sometimes appear that sample flow around extraction Medium and cause post there is no effect.

The content of the invention

There is illustrated a kind of extraction column and system, the extraction column and system solve one or more problems or upper The needs that face is recognized.In one embodiment, extraction column includes post body, Extraction medium part and set with storage part Loop section.Storage unit point includes entrance and storage room.Extraction medium part includes elongate sleeve, and the elongate sleeve, which has, limits sky The inner surface of chamber.Extraction medium part has the arrival end being in fluid communication with storage room and the outlet for being provided remote from arrival end End.Extraction medium is arranged in the cavity of elongate sleeve.Collar portion is axial in a common direction together with elongate sleeve Extension.Collar portion has end, and the outlet end of the end and elongate sleeve is opened and at least axially extended to by elongate sheath The port of export of cylinder limits plane.In an embodiment of the present invention, the at the end of the axial of collar portion is extended beyond by elongate sleeve The plane that the port of export limits, and in some alternative embodiments, the end of collar portion is to be enough the outlet for preventing elongate sleeve End is extended axially beyond the plane limited by the port of export of elongate sleeve by the distance that any part of the second extraction column contacts. In embodiments of the invention, store part entrance have be not more than and be likely less than the interior of the external diameter of the end of collar portion Footpath.The embodiment of extraction column alternatively includes neighboring entry from the outstanding shoulder in storage part.The implementation of Extraction medium Example can include solid phase extraction medium, such as condensate monolith or multiple non-porous pearls, porous bead or its mixture.Porous bead Or non-porous pearl can be by secondary deformation and/or by using adhesive bonding together, so as to form overall support, the bonding Agent can be for example including latex formulations.

The other side of the present invention is related to a kind of system, and the system includes the implementation for the extraction column being combined with sample port Example, the sample port have the first chamber for the elongate sleeve for being configured to accommodate extraction column.The system alternatively includes liquid Conveying device, such as ceramic probe.The system can also alternatively include analytical equipment, for example, mass spectrograph, liquid-chromatography apparatus, And its combine.

In addition, illustrate the embodiment of shell, the shell includes the bed for being provided with medium, wherein medium by layer with The inner surface separation of shell, the layer are configured to be substantially prevented from sample flow to bypass medium.

Detailed description with reference to following exemplary embodiment is recognized to different other purposes, excellent of the present invention together with accompanying drawing Point and feature.

Brief description of the drawings

Those, which are merged into this specification and form the accompanying drawing of this part for specification, shows embodiments of the invention, And it is used for illustrating the present invention together with the content of the invention of the invention given above and detailed description given below.

Fig. 1 is the exploded perspective view of extraction column according to embodiments of the present invention；

Fig. 2 is the stereogram of the longitudinal cross-section of Fig. 1 extraction column being under assembled state；

Fig. 3 is according to embodiments of the present invention to include liquid transporting apparatus, extraction column, sample port and analytical equipment The longitudinal cross-section side view of a part for the embodiment of the system of a part；

Fig. 4 is the longitudinal cross-section side view of extraction column and a part of optional liquid transporting apparatus according to embodiments of the present invention Figure；

Fig. 5 is according to embodiments of the present invention including liquid transporting apparatus, the alternative embodiment of extraction column, sample and one The longitudinal cross-section side view of a part for the embodiment of the system of partial analysis device；

Fig. 6 A are the side views of Fig. 4 sample port；

Fig. 6 B be along the longitudinal cross-section side views of Fig. 4 A sample port that intercept of 6B-6B；

Fig. 7 is the chart of the back pressure in extraction column during liquid flows through porous Extraction medium；

Fig. 8 A are the chromatograms obtained from sample, and the sample includes 5 eluted from extraction column according to embodiments of the present invention The mixture of kind protein；

Fig. 8 B are the mass spectrums obtained from sample, and the sample includes 5 eluted from extraction column according to embodiments of the present invention The mixture of kind protein；

Fig. 9 is the chromatogram of insulin, and the insulin obtains from the sample that extraction column according to embodiments of the present invention elutes；

Figure 10 is the chromatogram of ubiquitin, and the ubiquitin obtains from the sample that extraction column according to embodiments of the present invention elutes；

Figure 11 is the chromatogram of myoglobins, the myoglobins from the sample that extraction column according to embodiments of the present invention elutes and Obtain；

Figure 12 is the chromatogram of carbonic anhydrase, the carbonic anhydrase from the sample that extraction column according to embodiments of the present invention elutes and Obtain；

Figure 13 is the scanning electron of the layer in the centre position between Extraction medium and conduit wall according to embodiments of the present invention MIcrosope image；And

Figure 14 is the comparison of chromatogram according to embodiments of the present invention, shows from using second medium and is protected against conduit wall Hold the difference of result that Extraction medium obtained relative to the result for not using second medium to be obtained.

Embodiment

With reference to Fig. 1 and 2, embodiments of the invention are related to extraction column 10, and the extraction column 10 is used for from sample extraction such as egg The component of white matter or protein fragments, the sample include such as protein, peptide, carbohydrate, lipoid, nucleic acid, salt, With the mixture of the component of small molecule.Extraction column 10 includes post body 12, and post body 12 has storage part 14, Extraction medium portion Divide 16 and the collar 18.

Storage part 14 is usually located at the near-end 20 of post body 12, and the cavity including entrance 24 and as storage room 26. Storage room 26 by store part 14 inner surface 28 limit and with the distal end for being located substantially at post body 12 of Extraction medium part 16 Cavity 32 at 30 is in fluid communication.Storage room 26 can keep big quantity of fluid.In embodiment, the volume of storage room 26 is enough to protect Hold need be used for load Extraction medium 42 liquid volume, by the volume for the sample being extracted, the volume of washing lotion and elution it is molten The volume of liquid.For example, in some extracting methods, it is necessary to pass through forcing sample in the cavity 32 of Extraction medium part 16 Extraction medium 42 before utilize suitable solvent dilute sample.Generally, the step of dilute sample performs in a separate container. In the present example embodiment, the volume of storage room 26 is enough to be applied to storage room 26 in normal pressure is carried so that sample is sent to Take the medium direct dilute sample in storage room 26 before.In an embodiment of the present invention, storage room 26 has from about 50 μ l To the volume of about 1500 μ l scope.

In Fig. 1-3 exemplary embodiment illustrated, the inner surface 28 of storage room 26 includes storing up close to the offer of entrance 24 Deposit the Part I 34 of most of volume of room 26 and the Part II 38 close to Extraction medium part 16.Illustrated in Fig. 1-3 In exemplary embodiment, the inner surface 28 of the Part I 34 of storage room 26 generally has frusto-conical shape and first There is the meeting less than about 10 degree between internal diameter ID2 at the proximal end internal diameter ID1 of part 34 and the distal end 36 of Part I 34 Poly- angle, or in an alternative embodiment, the convergent angle is less than 4 degree.As used herein, the convergent angle is the surface and this being related to Angle between the central axis of structure, in this case, the central axis are the central axis 62 of post body 12.

As shown in figure 4, storage room 26 can have the shape for being configured to be sealed relative to liquid operating device 41, This can produce positive air pressure to force the liquid in storage room 26 to pass through Extraction medium 42 in storage room 26.For example, the The outer surface 43 of adjoining near-end 20 of a part 34 may be configured to and the phase of O-ring 44 on the mandrel of liquid operating device 41 45 Engage and form a seal with O-ring 44.In alternative embodiment, the inner surface 28 of the Part I 34 of storage room 26 can by with It is set to that either outer surface engages and forms a seal with O-ring or outer surface with the O-ring of the mandrel of liquid operating device.

As shown in Figures 2 and 3, the Part II 38 of storage room 26 can be with funnel shaped and with towards Extraction medium portion Divide 16 annular walls being inwardly tapered.In an alternative embodiment, Part II 38 can have frusto-conical shape.The infundibulate Shape or frusto-conical shape reduce the internal diameter of Part II 38 and help to guide the end of liquid transporting apparatus 46 to arrive Appropriate location is to form the tight seal between liquid transporting apparatus 46 and extraction column 10.The Part II 38 of storage room 26 is There is convergent angle between internal diameter ID3 at the near-end 48 of two parts 38 and the internal diameter ID4 at the distal end 50 of Part II 38.Showing In example property embodiment, the convergent angle of the Part I 34 of storage room is less than the convergent angle of the Part II 38 of storage room 26.

In Fig. 1 to 3 in embodiment illustrated, the Part II 38 of storage room 26 includes further reducing storage room 26 The support 52 of diameter, because the fluid that support 52 is transitioned between storage room 26 and the cavity 32 of Extraction medium part 16 leads to In road 54.Support 52 is used as sealing surfaces to be formed and liquid transporting apparatus 46 (such as pipette tip or similar hollow ceramic The hollow probe of probe) sealing.In certain embodiments, the inner surface of the far-end of storage room part can also aid in and liquid The sealing that body conveying device 46 is formed.It is enough shape when liquid transporting apparatus 46 as shown in Figure 3 and positioning utilizes against support 52 When being pushed into the active force of the sealing between post body 12 and liquid transporting apparatus 46 (and in certain embodiments, against When inner surface 40 at the distal end 50 of the Part II 38 of the storage room 26 of adjacent support 52 is pushed into) form sealing.It is applied to The active force of liquid transporting apparatus 46 can make the distal end of the Part II 38 of the storage room 26 of support 46 and/or adjacent support 52 The plastic construction of inner surface 40 at 50 is deformed into the shape of liquid transporting apparatus 46 to establish liquid transporting apparatus 46 and extraction Sealing between post 10.Support 52 in Fig. 1-3 exemplary embodiment is illustrated out with generally frustoconical shape, The generally frustoconical shape has the obtuse angle assembled with about 45 degree to about 90 degree of scope.

In the alternative embodiment illustrated in Figure 5,38 no support of Part II simultaneously transits directly to extraction as replacement The cavity 32 of media fraction 16.In the exemplary alternative embodiment, liquid transporting apparatus 46 is formed relative to Extraction medium 42 Near-end 53 sealing.Inner surface 40 at the distal end 50 of Part II 38 can engage a part for liquid transporting apparatus 46 simultaneously Promote the sealing between liquid 46 and Extraction medium 42.In the alternative embodiment, support 57 is positioned at the extraction of post body 12 At the port of export 60 of the cavity 32 of the elongate sleeve 55 of media fraction 16.Support 57 reduces going out for the cavity 32 of elongate sleeve 55 The internal diameter of the through hole 59 at mouth end 60.

In embodiment, the sealing formed between liquid transporting apparatus 46 and extraction column 10 allows from liquid transporting apparatus 46 are sent to the seepage of the volume no more than about 10% of the sample of extraction column 10.In another embodiment, about 70 Bar under about 200 bars of injection pressure, the sealing formed between liquid transporting apparatus 46 and extraction column 10 allows from liquid Conveying device 46 is sent to the seepage of the volume no more than about 10% of the sample of extraction column 10.

The embodiment illustrated in Fig. 2-4 includes the fluid passage between storage room 26 and the cavity 32 of Extraction medium part 16 54.In this exemplary embodiment, fluid passage 54 has frusto-conical shape, and convergent angle is generally less than about 10 Degree, and it is less than about 1 degree in an alternative embodiment.Fluid passage 54 has providing enough stream to prevent from passing through stream by limitation Back pressure caused by the fluid of body path 54 makes the length and internal diameter of the volume minimization of fluid passage 54 while growth.Fluid The dead volume that the volume of path 54 is minimized so that in extraction column minimizes.In embodiment, the volume of fluid passage 54 No more than about 1000nl and can be in the range of about 1nl to about 50nl.

Extraction medium part 16 includes the elongate sleeve 55 with inner surface 56, and inner surface 56 limits cavity 32, and extraction is situated between Matter 42 is arranged in cavity 32.Cavity 32 includes the arrival end 58 and the port of export 60 being in fluid communication with storage room 26, the port of export 60 have the through hole 59 set away from arrival end 58.In embodiment, cavity 32 has frusto-conical shape, the frustum of a cone Shape shape has the convergent angle less than about 5 degree, and in one alternate embodiment, convergent angle can have about 0.2 degree To 1 degree of scope.In another alternative embodiment, the convergent angle of the cavity 32 of frusto-conical shape can be about 0.4 Degree.In the embodiment with frusto-conical shape cavity 32, the larger diameter end portion of cavity 32 is towards Extraction medium 42 Insertion point opening.Internal diameter for the cavity 32 of alternative embodiment also has about 0.5 millimeter to about 2.0 millimeters of scope, And preferably there is about 0.75 millimeter to about 0.85 millimeter of scope.Post body 12 has central axis 62, central shaft Line 62 extends through the cavity 32 of Extraction medium part 16.Cavity 32 has along the central shaft between arrival end 58 and the port of export 60 The length L1 of line 62.In embodiment, the length L1 of cavity 32 has about 1 millimeter to 10 millimeters of scope.In another reality Apply in example, the length L1 of cavity 32 has about 3 millimeters to about 5 millimeters of scope, and preferably has about 3.5 millimeters To about 4.5 millimeters of scope.In embodiment, the length L1 of cavity is corresponding with the length of elongate sleeve 55.

Extraction medium part 16 also has outer surface 66, and the base section of the Extraction medium part 16 is configured to and sample The sample port 68 of analysis system (Fig. 3) forms sealing.In embodiment, outer surface 66 has the port of export in elongate sleeve 55 The generally frustoconical shape to narrow at 60.Elongate sleeve 55 can have about 1 millimeter to about 2 millis at the port of export 60 The external diameter of the scope of rice.In another embodiment, elongate sleeve 55 has about 1.2 millimeters to about at the port of export 60 The external diameter of 1.5 millimeters of scope.In another embodiment, elongate sleeve 55 has about 1.4 millimeters at the port of export 60 External diameter.The outer surface 66 of elongate sleeve 55 can have the convergent angle of about 1 degree to about 20 degree of scope, and preferably, Convergent angle with about 5 degree to about 15 degree of scope.In the embodiment of elongate sleeve 55, the wall of elongate sleeve 55 is formed Thickness increase from the port of export 60 of cavity 32 to the arrival end 58 of cavity 32.

The collar 18 of post body 12 is axially extending on the common direction of elongate sleeve 55.In the implementation that Fig. 1-4 is illustrated In example, the collar 18 has the outer surface that neighbouring transition between Part I 34 and Part II 38 is connected to storage room part 14 78 blind end 72.In the illustrated embodiment, the outer surface 74 of the collar 18 and the outer surface 78 of storage room part 14 are mutually continuous. Storage room part 14 and the outer surface of the collar 18 78,74 can be tapered in the case of the convergent angle less than 15 degree, and optional In embodiment, the convergent angle has about 0.1 degree to about 5 degree of scope.

The collar 18 has the open end 70 being spaced apart with the port of export 60 of elongate sleeve 55.The port of export of elongate sleeve 55 60 limit plane P1.The restriction of end 70 of the collar 18 at least extends to the plane P1's limited by the port of export 60 of elongate sleeve 55 Plane P2.In embodiment, the plane P2 of the end 70 of the collar 18 extends beyond the plane P1 of the port of export 60 of elongate sleeve 55. In the embodiment illustrated in Fig. 1-4, the plane P2 of the end 70 of the collar 18 extends beyond the flat of the port of export 60 of elongate sleeve 55 Face P1 distance D1, distance D1 are enough to prevent the port of export 60 of elongate sleeve 55 from contacting any part of the second extraction column.For example, Distance D1 can have about 0.1 millimeter to about 2 millimeters of scope, this internal diameter ID5 depended at the end 70 of the collar 18, With the external diameter OD2 at the near-end 20 of smaller external diameter OD1 or post body 12 at the end 70 of the collar 18, and distance D1 is enough Prevent the port of export 60 of elongate sleeve 55 from being contacted by any part of the second extraction column.When extraction column 10 is loosely stored in sack Or when in box, the structure prevents the infringement to the port of export 60 of elongate sleeve 55.Plane P2 extends beyond plane P1 volume Outer advantage is the pollution that the port of export 60 of elongate sleeve 55 is prevented when extraction column 10 is being handled by automatic sample analysis system. Make extraction column 10 by conduit or flexible pipe from system for example, the method for transporting the extraction column 10 in automatic analysis system has A position drop to another position.If the port of export 60 of elongate sleeve 55 is exposed and (that is, not protected by the collar 18), If the port of export 60 contacts the surface of transporting tube or flexible pipe, the notable risk transferred the pollution with the port of export 60.This hair The bright collar 18 prevents the contact on surface of the port of export 60 of elongate sleeve with transporting flexible pipe, and thereby reduces in identical flexible pipe The risk transferred the pollution between the different extraction columns of middle transport.

The end 70 of the collar 18 has external diameter OD1, and the entrance 24 of storage room 26 has internal diameter ID1 so that the collar 18 End 70 can not be inserted completely into the entrance 24 of storage room 26.In embodiment, the internal diameter of the entrance 24 of storage room External diameter OD1s of the ID1 no more than the end 70 of the collar 18.In another embodiment, the internal diameter ID1 of the entrance 24 of storage room is less than The external diameter OD1 of the end 70 of the collar 18.Similarly, the near-end 20 of post body 12 has an external diameter OD2, and the open end of the collar 18 70 have internal diameter ID5 so that the near-end 20 of post body 12 can not be inserted completely into the open end 70 of the collar 18. In the embodiment that Fig. 1-4 is illustrated, the external diameter OD2 at the near-end 20 of post body 12 includes optional shoulder 76.In embodiment, set External diameter OD2s of the internal diameter ID5 of the open end 70 of ring 18 no more than the near-end 20 of post body 12.In another embodiment, cover The internal diameter ID5 of the open end 70 of ring 18 is less than the external diameter OD2 of the near-end 20 of post body 12.When multiple extraction columns 10 are stored in When being encapsulated in together and at random in such as sack or box, in the event of an end of extraction column second can be coordinated to carry The situation in the opening in the end of post is taken, then extraction column 10 will not the inside that be stacked on another extraction column.Extraction column 10 this respect causes automatic system is obvious more easily once to pick up an extraction column 10, and allows the extraction column 10 to be automatically Use unite without classifying and being placed into bracket.Therefore, extraction column 10 can loosely be packed, sell, stores in batch Be inserted into automatic analysis system without they are packaged in into bracket or pallet according to regulation position and regulation orientation In, which thereby enhance efficiency, save money, space and labour.

In the embodiment shown in Fig. 1-4, the near-end 20 of post body 12 includes neighboring entry 24 from the outer of storage part 14 The outstanding optional shoulder 76 in surface 74.Shoulder 76 adds the external diameter of the near-end 20 of post body 12 and offer can be by Automatics using with processing procedure either if necessary to extraction column 10 is suspended in pallet or rack system when hang Hang the surface of extraction column 10.

Extraction medium 42 is arranged in the cavity 32 of Extraction medium part 16.Extraction medium 42 allows from biased sample Extract the component needed.Extraction medium 42 can pass through chromatographer protein isolate from other components in Mixed biological sample Matter.The embodiment of Extraction medium 42 can reversibly combine the molecular weight such as with about 1kDa to about 200kDa scopes The small molecule or macromolecular of peptide and protein.When sample is with the flowing in the range of about 50 μ l/min to 200 μ l/min When speed is by Extraction medium 42, Extraction medium 42 can reversibly combine the sample in the range of about 10 μ l to about 100ml At least 1 μ g protein in product volume.Extraction medium 42 is also based on the suitable component of suitable feature elution, such as base In the affinity of the molecular weight of component, hydrophobicity, electric charge or component for Extraction medium 42 in terms of certain.In embodiment, When model of the eluting solvent using about 1 μ l to about 100 μ l scope inner volumes with about 0.1 μ l/min to about 20 μ l/min When flow in enclosing is eluted, the protein that Extraction medium 42 can elute at least 20% from biased sample combines.

In embodiment, Extraction medium 42 is solid phase extraction medium, and more specifically, is with the enough samples of permission Flow through the porous monolith medium 80 of high internal void of the porous monolith medium 80 without generating bad high back pressure.Implementing In example, back pressure is no more than about 5 bars under 200 μ l/min flow.Porous monolith medium 80 is preferably able to bear at least 200 Bar.The pore structure of porous monolith medium 80 has an advantage that it has zigzag channel for sample, and this allows under fast flow Rapid convective mass transmission.In addition, porous monolith medium 80 does not need melt that its position is maintained in post body 12.

Porous monolith medium 80 can be prepared to the continuous bed of enclosure, such as the one of pipe 82 section.This causes inciting somebody to action When pipe 82 cuts into multistage, porous monolith medium 80 is present in the whole length of the micro-column as Extraction medium 42.

In the embodiment that Fig. 1-4 is illustrated, Extraction medium 42 is from the outlet of the elongate sleeve 55 of Extraction medium part 16 Porous monolith medium 80 in the cavity 32 for the Extraction medium part 16 that end 60 is inserted into post body 12.In the implementation shown in Fig. 5 In example, Extraction medium 42 is due to that support 57 is there are at the port of export 60 of elongate sleeve 55 and is inserted from the arrival end 58 of cavity 32 Enter to the porous monolith medium 80 in the cavity 32 of the Extraction medium part 16 of post body 12.Porous monolith is prepared inside pipe 82 Medium 80 and pipe 82 is cut to micro-column it has an advantage that this method is allowed to directly such as there is porous monolith medium Connect when being aggregated in inside pipette tip, avoid the boundary between air and polymeric porous monolith medium formed it is semi-permeable or Non-porous layer.Semi-permeable or non-porous layer can adversely affect to the flow behavior of caused porous monolith medium.Wherein Porous monolith medium is formed directly into the embodiment inside the cavity 32 of Extraction medium part 16, semi-permeable or non-porous layer Side effect can increase the flow of the sample by Extraction medium by being formed through the path at the center of porous monolith medium And it is reduced.However, a high proportion of component to be collected can be retained in path and bypass porous monolith medium, it is therefore desirable to sample Product are by multiple passages of medium, for maximum extracted.By contrast, preparing porous monolith medium 80, (pipe 82 is in porous monolith Media interior is cut into part) porous circular cylinder is generated, wherein the length across diameter and along porous monolith medium 80 Pore structure and porosity are all uniform.The preparation process causes sample flow to cross porous monolith medium 80 without by half Infiltration or the obstruction of pore-free surface, and substantially uniform combination is produced across porous monolith medium 80, so as to by porous The maximum extracted of suitable component is provided in the single passage of monolithic medium 80.

Pipe 82 on the outside of porous monolith medium 80 provides protective layer, and the protective layer helps to handle porous monolith medium 80. For example, during manchining feature post 10, post body 12 can be formed independently of porous monolith medium 80.Then, it is porous Monolithic medium 80 is inserted into the cavity 32 of the Extraction medium part 16 of post body 12.Porous monolith medium 80 is formed wherein In embodiment for the part of pipe 82, pipe 82 allows porous monolith medium 80 to be more readily disposed, and is situated between for being inserted into extraction In the cavity 32 of matter part 16.In addition, when in the cavity 32 that porous monolith medium 80 is inserted into storage part 14, the wall of pipe 82 It may be used as supporting porous monolith medium 80 and may be used as the sealing surfaces of the inner surface against cavity 32, it is porous so as to prevent Backflow around monolithic medium 80.

When in the cavity 32 that the porous monolith medium 80 for the part for being formed as pipe 82 is inserted into storage part 14, pipe 82 Compress around porous monolith medium 80 to prevent porous monolith medium 80 to be extruded from pipe 82.

Porous monolith medium 80 can optionally be connected to the inner surface of pipe 82 to improve to porous monolith medium 80 The resistance of extraction.For example, the inner surface of pipe 82 can be processed to establish the covalent bond for polymeric porous monolith medium 80 Tie point.In order to activate the inner surface of such as pipe of polyethylene ether ketone (PEEK) pipe, the inside of pipe can be with including such as acetonitrile Either the solvent of propionitrile (alkyl carbonitrile derivatives) and such as Vazo-64 or Vazo-55 (WakoV-70) azo group classification are drawn The reaction solution for sending out agent is filled with the concentration in the range of about 1% to about 10% (volume).The pipe of filling can then by The 80% of 10 hours half life temperatures of minimum solvent is heated to, such as Vazo-64 had for 10 small time-divisions under 64 Celsius temperatures Solve half-life period.Reaction can be allowed to carry out at least 30 minutes.Reaction solution can be replaced and permitted by new reaction solution Perhaps the additional continuous periods of at least 30 minutes are carried out.In embodiment, the end of pipe is produced at least 5psi inside back of the body by sealing Pressure.In another embodiment, reaction solution is continuously re-filled with ambient pressure.Then, reaction solution is removed from pipe, and And allow to utilize a branch of nitrogen drying tube.After drying, polyblend is injected into the inner chamber for the pipe being activated.Porous In the polymerization process of monolithic medium 80, the activation tie point on the inner surface of pipe is merged into Extraction medium.

In certain embodiments, the inner surface of pipe 82 can have medium, and extraction Jie is improved with further using the medium Combination of the matter to pipe 82.The inner surface of pipe 82 can scribble material, and the material is by substantially removing monolith medium 80 and pipe 82 Wall surface between gap or duct and improve performance, wherein the elimination in gap or duct substantially prevent sample flow around Cross monolith medium 80.In certain embodiments, coating includes being formed the barrier between the inner surface of monolith medium 80 and pipe 82 The non-porous layer hindered, the coating can utilize above-mentioned " activation " be processed at least two double combination polymerising ethylene system monomer and by Apply.For example, the surface of pipe 82 can include managing via the polyethylene ether ketone (PEEK) of initiator activation before polymerization. As set forth above, it is possible to use acetonitrile either propionitrile solvent and such as Vazo-64 or Vazo-55 (Wako V-70) azo group Classification initiator.Activation is appeared between the surface of pipe 82 and at least one vinyl groups, and this causes monomer to be anchored into surface. Then start the polymerization of the grappling crosslinking of vinyl monomer, and produce and precipitate cambial diffusion condensate on the surface.It is poly- Close layer to be formed because of condition so that condensate precipitation is assembled to form monolith knot on the surface rather than in dissolved state Structure.In this example, the thickness of layer depends, at least partially, on activating the concentration of the vinyl crosslinking in solution.

Those skilled in the art will be recognized that polymerizing condition can be affected according to result.In different application In, the scope that vinyl monomer can include about 1% to about 50% concentration (volume) in the solvent with initiator is dense Degree.If using too many cellular construction, as a result can include across the gel of pipe diameter or the formation of mesh, rather than The formation of layer.In this example, the result might also depend on channel internal diameter, and wherein smaller channels need the ethene of low concentration It is monomer, for successful polymerization into suitable layer.For example, the passage with about 1 millimeter of internal diameter can allow from about The monomer concentration of 30% to about 40%, and about 0.50 millimeter of internal diameter can allow the list from about 10% to about 20% Bulk concentration.In addition, other conditions of such as polymerization time and temperature can influence result.

In the illustrated embodiment, pipe 82 is activated using polymer layer as described above, is then mixed thing filling with shape Into continuous integral medium 80.At least partially due to steric hindrance, the double combination of the vinyl monomer of polymer layer is only used for A part merge in the course of the polymerization process, and the remainder of double combination in the forming process of monolith medium 80 via Copolymerisation is merged into monolith medium 80.

When PEEK pipes are used as exemplary tube, can also use includes cycle olefin copolymer (" COC ") and such as ethene Tetrafluoroethene (ETFE), PEP (FEP) and the polymeric fluorescent lamp of other fluorescent lamps are polymeric other types of Polymer pipe.In addition it is possible to use fused quartz tube, fused quartz tube is included by covalent bond after being hydrolyzed in vitreous silica The acrylates or methacrylate of silylating reagent and be activated.

Pipe 82 can the internal diameter with about 0.1 millimeter to about 1 millimeter of scope.In embodiment, the internal diameter of pipe 82 With about 0.4 millimeter to about 0.6 millimeter of scope.In another embodiment, the internal diameter of pipe is about 0.5 millimeter.Tool There is the pipe 82 for being internally formed porous media to be cut into certain length, when when the internal diameter of pipe is combined, the pipe of the length Suitable volume for porous Extraction medium is provided.In embodiment, there is the length quilt of the pipe 82 of porous monolith medium 80 The part with about 6 millimeters to about 2 millimeters of scope is cut into, and in an alternative embodiment, length has about 3 millimeters To about 5 millimeters of scope, or about 4 millimeters.After porous Extraction medium polymerize, such as IDEX A- can be utilized 350 tubing cutters cut into pipe with suitable length.

In certain embodiments, porous bead or non-porous pearl can be combined together by secondary modificalion and/or to pass through Integral medium 80 is formed using adhesive, adhesive can be prepared for example including latex, wherein example is in the U.S. the 7th, 303,671 Illustrated in the patent of number entitled " the ion exchange combination particle for flowing through porous monolith ", for all purposes, its Full content is herein incorporated by being referenced here.

In identical or alternative embodiment, porous monolith medium 80 can be by molten with initiator and stomatal limiting value Polymerization includes being adapted to the mixture of monomer and preparing in the case of agent (pore former).Caused porous monolith medium 80 has small Hole, aperture have about 50nm to about 20,000nm diameter range.Porous monolith medium 80 can have about 50nm- The aperture of 200nm or about 750-10000nm scope.Porous monolith medium 80 can be condensate bead, the condensate Bead has from about 20nm to the diameter of about 10,000nm scope.Porous monolith medium 80 should be able to bear at least big About 200 bars of pressure.

Monomer can be selected from the vinyl comprising monomer, the acrylates comprising monomer, the methacrylate comprising monomer, third Acrylamide, polyolefin, polyester, polyurethanes, polyamide, fluorinated ethylene and combinations thereof.Vinyl comprising monomer can be with Vi-ny l aromatic monomers including a such as vinyl for aromatic monomer and divinyl for aromatic monomer and combinations thereof.It is exemplary Vi-ny l aromatic monomers include divinylbenzene, styrene, the alkyl of such as ethyl styrene for styrene, Alpha-Methyl benzene second Alkene, alkyl for α-methylstyrene, such as 1-chloro-4-methyl-benzene halogen for α-methylstyrene, and combinations thereof.Alkyl substitutes Thing can include up to 18 carbon atom.Acrylates comprising monomer include it is single-, double-and three-acrylates.Methacrylate Monomer includes such as GMA, dimethyl ethyl, trimethylolpropane, methacrylate, first The list of base hydroxy-ethyl acrylate-, double-and three-methacrylate.In embodiment, the mixture of monomer or at least two monomers Substantially existed with about 10%vol. to about 60%vol. amount in polyblend, and in an alternative embodiment, with big About 20%vol. to about 70%vol. amount is present.

Pore former can be selected from a variety of different types of materials.For example, suitable liquid pore former includes aliphatic hydrocarbon, virtue Hydrocarbon, ester, alcohol, ketone, aether, the solution and its mixture of soluble polymer.Exemplary pore former includes 2,2,4- trimethyls Pentane, alcohol, toluene, butyl acetate, the Isosorbide-5-Nitrae with 1 to 12 carbon atom, butanediol, water, acetone, hexane, hexamethylene, ring Hexanol, tetrahydrofuran (THF), and combinations thereof.In embodiment, amount of the pore former with about 20%vol. to about 90%vol. It is present in polyblend, and in an alternative embodiment, exist with about 60%vol. to about 80%vol. amount.

Initiator can include thermal polymerization, conventional free radical triggers polymerization initiator, light trigger and oxidation are gone back Former initiator.The example of suitable initiator includes such as OO-t shapes-amyl group-O- (2 ethylhexyl) one and aoxidizes two carbonic esters (monoperoxycarbonate), the carbonic acid dipropyl of peroxide two and benzoyl peroxide, such as azodiisobutyronitrile (Du Pont Vazo-64 azo-compound, 2,2 ' azodiisobutyronitriles (2- amidine propanes), dihydrochloride and 2), the isobutyl of 2 ' azo two Nitrile (isobutyramide) dihydrate and ammonium persulfate and tetramethylethylenediamine (TMEDA).In embodiment, initiator is with about The amount of the monomer weight of 0.2% monomer weight to about 5% is present in polyblend, and in an alternative embodiment, with The amount of the monomer weight of about %1 monomer weight to about 2% is present.

The component of polyblend mixes according to routine techniques and is injected into the inside of pipe to be allowed to polymerize.For example, In embodiment, pipe is filled by polyblend and is applied in about 100psi pressure.Due to initiator in the course of the polymerization process Nitrogen can be formed during decomposition, so pressurization helps prevent the bubble formation in polyblend.In other embodiments, Guan Youju Close mixture filling, and when allowing to be polymerize seal pipe both ends.The both ends of seal pipe cause to polymerize when carrying out in pipe Pressure increase in portion, this prevents the formation of nitrogen bubble.In yet another embodiment, pipe is filled by polyblend, and is managed An end sealed and another end of pipe open and be put into finger pipe.The pipe of filling is heated during polymerization procedure. The openend that positioning refers to the pipe in pipe allows liquid to expand when mixture is heated in the course of the polymerization process, and this prevents from being drawn by heating The pressure increase risen, the pressure increase can have a negative impact to the porosity of porous monolith medium.Using light trigger In embodiment, the pipe of filling can undergo ultraviolet irradiation.The example of porous monolith medium and its manufacture method the 7,922nd, Illustrated in No. 908 United States Patent (USP)s, the patent is incorporated by herein by quoting.

Porous monolith medium can also be functionalized.For example, porous monolith medium 80 can be produced wherein epoxidation ring Either halide function can react oxygen derivative with amine or sulfide, so as to produce such as anionic exchange medium, or Person reacts to produce cation exchange medium with such as carboxylic acid, phosphoric acid, sulfonic acid.Then, these groups can further be changed To allow protein, peptide or the connection of immunoglobulin, so as to produce affinity isolation and Extraction medium.Epoxide group Directly either it can be reacted after aldehyde is converted into protein, peptide or immunoglobulin.Possible other affinitys Medium includes immobilized metal ion affinity chromatography (IMAC) state and borate state.

The Exemplary porous material of porous monolith medium 80 and the manufacture method of this material are suitable as in United States Patent (USP) Illustrated in 5th, 334,310 and 5,633, No. 290, each patent is incorporated by herein by quoting.

In an alternative embodiment, Extraction medium 42 can include multiple porous beads and/or non-porous pearl, such as included in extraction Glass, silica or polymeric beads in the cavity 32 of media fraction 16.In this embodiment, the arrival end of cavity can include First frit, and the port of export of cavity can include the second frit.Frit has the function of preventing that pearl from spilling from cavity.Enough Integument is encapsulated into cavity to allow enough flow rates, while will not produce bad back pressure.Exemplary pearl is special in the U.S. Illustrated in profit the 6th, 783,672, the patent is incorporated by herein by quoting.

In use, before sample is by extraction column 10,42 wetted wet with solvent of Extraction medium.Carry wherein Medium 42 is taken to include in the embodiment of porous monolith medium 80, porous monolith medium 80 is soaked by the wetting solvents of enough volumes, The wetting solvents can include aqueous group of the formic acid (FA) of such as organic solvent of acetonitrile (acrylonitrile) and such as 0.2% volume Point.Generally, porous monolith medium 80 is soaked using about 10 μ l to about 100 μ l wetting solvents.Then, porous monolith medium 80 are balanced by balanced solvent, and the balanced solvent can include water and the FA of about 0.2% volume.Generally, using about 10 μ L to about 100 μ l balanced solvent balance porous monolith medium 80.Then, volume including in the range of 10 μ l to 100 μ l The sample of the interesting mixture of such as protein is forced past the porous monolith medium 80 in extraction column 10.Come from The stream of sample can optionally be collected for supplement analysis.Then, using including water and about 0.1% volume is to about The FA of 0.2% volume wash liquid porous monolith medium 80.Generally, the μ l of about 10 μ l about 100 washing lotion is used to wash Wash the sample being collected in porous monolith medium 80.Then using elute solution elute past porous monolith medium 80 collection Mixture.The content and volume and elution time of elution solution can be changed based on suitable type of elution.Wherein quickly wash It is suitable to take off, and elution solution can include water, the organic mixture in about 20%-60% volume ranges and about 0.2% The FA of volume；About 1 μ l to about 100 μ l elution solution is used to elute the seizure mixture from porous monolith medium 80. Liquid solution including sample can be quickly propelled by Extraction medium 42.The mixture combined in Extraction medium 42 quickly goes out It is existing, and elution time depends on suitable application.Isocratic elution can be with the table being exceedingly fast using the elution of fast gradient Existing, this can be used for wherein being adapted to the automatic system in the case of high throughput., can when using the combination of high de-agglomeration mass-spectrometry The mixture of such as protein is identified using fast elution.This method is useful, and wherein rapid analysis method needs such as micro- Bio-identification.When needing more detailed analysis, elution can be slowly performed, it is allowed to according to such as molecular weight, electric charge, hydrophobic Property interaction or with the suitable features of other affinity types of the component phase reaction of extraction column 10 from the order of extraction column 10 Elute mixture.Short (for example, about 5 minutes) or long (such as about 30 minutes) gradient can be utilized to complete to wash slowly It is de-.For example, in embodiment, the percentage of the organic component in eluting solvent can increase continuously or gradually throughout whole ladder Degree, to allow to elute lasting reasonable time.The longer elution duration can analyze single target mixture, such as micro- life Antibiotic-resistance marker in thing sample or the cancer biomarkers from biopsy.Effective mixture is isolated and applied The possibility of gradient allows user to avoid using costly and time-consuming analytical column.Analytical column needs to use the purge step of multiple time Suddenly to avoid being transferred to another sample from a sample.This extraction column 10 is accessible and allows users to avoid cleaning Step and it is transferred to next sample from a sample.

The mixture of elution can be collected for subsequent analysis, or preferably, (will be under via sample port 68 Fang Jinhang is described in more detail) it is passed directly in analysis system.Elution samples are passed directly to have the advantage that in analysis system： Ability of the extraction column 10 from the rapid extraction mixture of sample；And extraction column 10 is mutual based on such as molecular weight, electric charge, hydrophobicity Effect or the ability with the suitable features of other affinity types of the component phase reaction of post chromatographically elution mixture. Exemplary analysis system is liquid chromatogram (LC) system.When extraction column 10 is used to mixture being directly eluted in LC systems, Extraction column 10 allows for bearing the high pressure used in LC systems.In addition, the fluid flow in LC systems can be very low, therefore Space and dead volume need to minimize.The embodiment of this extraction column 10 by Extraction medium 42 by being positioned at the close of storage room 26 Seal between surface and the port of export 60 of Extraction medium part 16 and make the fluid passage between sealing surfaces and Extraction medium 42 Volume minimization and realize space and dead volume and minimize.Using this structure, sample is substantially from Extraction medium with most Small liquid volume is passed directly in the sample port of liquid chromatographic system.

Reference 3 and 5 to 6B, extraction column 10 can be connected by use with sample port 68, and one as sample analysis system Part.Sample port 68, which has, to be configured to the proximal part 94 for sealingly engaging extraction column 10 and is configured to joined sample point The distal portions 96 of the part of analysis system.Proximal part 94 includes being configured to the Extraction medium part 16 of receiving extraction column 10 The first chamber 98 of elongate sleeve 55.Distal portions 96 have the second chamber of the part for being configured to accommodate sample analysis system 92 Room 100.First chamber 98 is in fluid communication via low volume fluid delivery path 104 and second chamber 100.In embodiment, the first chamber Fluid passage 104 between room 98 and second chamber 100 has about 0.5 microlitre or less of volume.

First chamber 98 has an inner surface 106, and inner surface 106 is outer with the sleeve of the Extraction medium part 16 of extraction column 10 The size and shape on surface 66 is corresponding.In embodiment, inner surface 106 has generally frustoconical shape.In addition, first Chamber 98 has sealing surfaces 108, and fluid passage 104 is formed in the distal end of sealing surfaces 108.From the near-end of sample port 68 110 measure to sealing surfaces 108, and first chamber 98 has depth D2.The depth D2 of first chamber 98 is less than Extraction medium part The length L1 of 16 elongate sleeve 55 so that when active force (such as is applied to the work of extraction column 10 by liquid transporting apparatus 46 When firmly) being applied to extraction column 10, the outer surface 66 of the port of export 60 and elongate sleeve 55 engages and with the first of sample port 68 The inner surface 106 and/or sealing surfaces 108 of chamber 98 form sealing.In embodiment, between extraction column 10 and sample port 68 The sealing of formation allows to be transferred into the sample of sample port 68 with the sample volume no more than about 10% from extraction column 10 Seepage.In another embodiment, the sealing formed between liquid transporting apparatus 46 and extraction column 10 allows at about 70 bars The sample for being transferred into sample port 68 from extraction column 10 under pressure in the range of to about 200 bars has no more than about 10% Sample volume seepage.

The proximal part 94 of sample port 68 has the internal diameter of the open end 70 of the collar 18 than extraction column 10 smaller External diameter.Therefore when extraction column 10 is positioned in sample port 68, the collar 18 will surround the proximal part 94 of sample port 68 At least a portion of outer surface 112.Proximal part 94 is prevented to elongate sheath from the projection of distal portions 96, the distance at a certain distance Formed between the port of export 60 of cylinder 55 and the inner surface 106 and/or sealing surfaces 108 of the first chamber 98 of sample port 68 close Envelope is disturbed.

The distal portions 96 of sample port 68 have the external diameter of the typically larger than external diameter of proximal part 94.Distal portions 96 Second chamber 100 is configured to accommodate the part of sample analysis system, i.e., can be by the sample delivery of elution to analytical equipment.The Two chambers 100 are configured to be sealingly engaged the part of sample analysis system.In embodiment, second chamber 100 has screw thread knot Structure.In embodiment, the part that is contained in second chamber is can be by the pipe 114 of the sample delivery of elution to analysis system. Exemplary analysis system includes liquid chromatographic system.

Example 1

Prepared and carried using the Extraction medium micro-column formed by forming the porous monolith medium in the PEEK pipes of part Take post.Porous monolith medium is prepared by polyblend, and the polyblend includes 37.5 percentage by weights of mixture Monomer (vinyl xylene, ethyl vinyl benzene of the divinylbenzene of 55 percentage by weights and 45 percentage by weights), 1.75 weights per monomer weight Measure the pore former of the initiator (azodiisobutyronitrile (Du Pont Vazo-64)) of percentage and 62.5 percentage by weights of mixture. Polyblend is injected into the inner chamber of the activating part of pipe, and allows to polymerize 18 hours under 76 Celsius temperatures.

The inner surface of PEEK pipelines is activated before polyblend injection so that porous Extraction medium will polymerize The inner surface of pipe is covalently bound in journey.In order to activate the inner surface of pipe, PEEK pipes are filled by reaction solution, the reaction solution Comprising solvent, acetonitrile either propionitrile (alkyl carbonitrile derivatives) and with 1-10% concentration such as Vazo-64 or Vazo-55 (Wako V-70) azo group classification initiator.Then, the PEEK pipes of filling are heated to the minimum of 10 hours half life temperatures 80%, such as Vazo-55 has the half life of decomposition of 10 hours under 55 Celsius temperatures.The reaction allows to carry out 30 minutes.Instead Answer solution to be replaced by new reaction solution and allow to carry out extra 30 minutes.Reaction solution is removed from pipe and allows to utilize A branch of nitrogen drying tube.After drying, polyblend is injected into the inner chamber of the pipe of activation.

After porous Extraction medium is by polymerization, pipe is cut into suitable length using IDEX A-350 tubing cutters.

Managed using the PEEK of the internal diameter with 0.75 millimeter, 1 millimeter, 0.5 millimeter and 0.4 millimeter.Porous monolith medium shape In Cheng Guan inner chamber, and using IDEX A-350 tubing cutters pipe cut into 2 millimeters, 4 millimeters, 5 millimeters or The part of 6 millimeters of length is to form Extraction medium micro-column.Extraction medium micro-column is inserted into the Extraction medium of post body In partial cavity, to form the extraction column with the size shown in table 1.With the Extraction medium micro-column 2 in table 1,3, Tested with the extraction column of 4 size using bacteria samples, as described below.

Test specimen is prepared by extracting Escherichia coli.The extraction is by formic acid (FA) and 25% volume comprising 50% volume Acetonitrile (acrylonitrile) solvent in lysine Escherichia coli prepare.FA and ACN concentration is adjusted to the third of 37.5% volume The formic acid of alkene nitrile and 25% volume.Protein concentration in the test specimen is based on BCA analyses and determined in 2mg/ml and 3mg/ml Between.Test specimen is diluted, and 0.5 μ g or 1 μ g protein are applied to extraction column.

Before test specimen is applied into extraction column, porous list is soaked with the 50 μ l of the FA with 0.2% volume ACN Block medium.Then, with the 50 μ l of the FA comprising water and 0.2% volume solution equilibria porous monolith medium.It is 0.5 with total amount The sample (volume is in the range of 50 μ l to 100 μ l) of μ g or 1 μ g protein is pushed through the porous list in extraction column Block medium.Stream from sample is collected for analyzing.Solution washing using the 50 μ l FA comprising water and 0.1% volume is more Hole monolithic medium.Using 10 μ l the FA comprising water, the acrylonitrile of 60% volume and 0.2% volume solution from porous monolith Medium elutes protein example.The protein of elution is collected for analyzing.

The sample of circulation (FT) and the sample of elution are analyzed to protein concentration.In some results, multiple samples The eluate of (3-5) combined before analyzed and by local desiccation to about its initial volume 1/3, then according to combination As a result the albumen quality per extraction column is calculated.Protein is determined using ultramicron biology BCA detection methods (the silent winged generation that science and technology of match) Concentration.Bovine serum albumin standard curve is prepared using with identical FA and the ACN concentration used in FT and elution samples. Result is provided in table 2.

Then Polyacrylamide Gel Electrophoresis circulation and elution samples are passed through.Combine before analysis and drying comes from The circulation and elution of 5 kinds of samples.Sample flows on the gel from 4% to 20% gradient.Then silver is carried out to gel Colour and analyzed.As a result (not shown) is consistent with protein assay result.

Example 2

Include protein mixture (including insulin, ubiquitin, myoglobins, carbonic anhydrase and bovine serum albumin) Sample is produced in the solution of the acrylonitrile with 37.5% volume and the formic acid of 25% volume.Gross protein in sample is 0.5μg.The test sample on extraction column, the extraction column are produced as described in Example 1, have the chi of No. 5 extraction column in table 1 It is very little.The porous Extraction medium of extraction column is wetted and is balanced, and applies sample, and the porous Extraction medium in extraction column is strictly according to the facts It is washed described in example 1.In just wheel experiment, pressure while manual elution samples in monitoring extraction terminus inner.Can be with It was observed that 50 μ l sample can be pushed through extraction column being shorter than in the time of 15 seconds, as shown in fig. 7, caused by end Back pressure, which can be used for monitoring whole liquid, passes through moment during porous Extraction medium.

Then liquid is made to pass through extraction column using automatic sample device.The Force measurement applied by liquid transmission probe is to know Do not need to be formed sealing between liquid transporting apparatus and extraction column and extraction column and sample port to liquid chromatographic system it Between sealing active force amount.It is close more particularly to being formed between these components under the high operation back pressure of liquid chromatographic system Envelope, the back pressure is generally in the range of about 70 bars to about 200 bars.It is observed that extraction is applied to by liquid transmission probe The sealing that the 30N of post active force is formed between part enough under 210 bars of back pressure.

In other experiment, during elution step, extraction column is inserted into the sample port of liquid chromatographic system In, and eluted using the aqueous solution of the acrylonitrile comprising 50% volume and the formic acid of 0.2 volume with 1.5 μ l/min speed.Figure 8A is the chromatogram obtained from the sample of the elution from protein mixture.Fig. 8 B are obtained from the protein mixture of elution Mass spectrum.Fig. 9 is the chromatogram obtained from the insulin sample of elution.Figure 10 is the chromatogram obtained from the ubiquitin sample of elution.Figure 11 It is the chromatogram obtained from the myoglobins sample of elution.Figure 12 is the chromatogram obtained from the carbonic anhydrase sample of elution.

When illustrating of the invention by one or more embodiments of specification, and explaining very much reality When applying, these do not mean limitation or limit the protection domains of appended claims in any way into this details.To this For the technical staff in field, other advantages and modification will be apparent.Therefore, wider aspect of the invention is not limited to In detail, the apparatus and method represented and the illustrative example of display and explanation.Therefore, can be without departing substantially from conventional hair These details are changed in the case of the protection domain or spirit of bright design.

Example 3

Figure 13 provides the scanning electron microscope diagram for the monolith medium 80 that display is separated by layer 1305 with tube wall 1310 The illustrative examples of picture.Figure 13 also show the interface intersected in wherein material and be merged into the element of monolith medium 80 Non-porous layer 1305.In Figure 13 example, layer 1305 be configured to make liquid flow turn to return in most of media 80 without It is to be flowed along wall 1310.

Figure 14 is provided by being obtained in the application of chromatogram reverse phase HPLC with coating and without the coating The illustrative examples to compare of the result obtained.Top call wire is shown in the case of inapplicable polymer layer by preparing The result of the monolith medium 80 obtained in PEEK pipes.The peak asymmetry of reduction shows and eluted before analyte blocking A part for analyte fluid.This is allowed through smaller resistance path (such as by wall stream) in a part for analyte fluid Produced during around monolith medium 80.Bottom call wire, which is shown, have been lacked front end to indicate that whole analyte fluids pass through most of whole The peak value of body material medium 80.

Separation condition：

Mobile phase A：0.1% trifluoroacetylacetone (TFA) in 95/5 water/acetonitrile (v/v)

Mobile phase B：0.08% trifluoroacetylacetone (TFA) in 95/5 acetonitrile/water (v/v)

Gradient：1% to 99%B in 9 minutes, 99%B in 3 minutes

Flow rate：0.20mL/min temperature：30℃

Detection：Ultraviolet 214nm

Injection volume：1.0μL

Claims

1. a kind of extraction column, including：

Post body, the post body have storage part, Extraction medium part and collar portion；

Wherein described storage unit point includes entrance and storage room, and the Extraction medium part includes being arranged on carrying in elongate sleeve Medium is taken, the elongate sleeve has inner surface, and the inner surface limits the cavity with arrival end and the port of export, the entrance End is in fluid communication with the storage room, and the port of export is provided remote from the arrival end, and the Extraction medium is substantially Extend to the port of export of the elongate sleeve；And

Wherein described collar portion is axially extending along common direction together with the elongate sleeve and has end, the end Opened with the outlet end of the elongate sleeve and at least extend axially to the port of export limit by the elongate sleeve Fixed plane.

2. extraction column according to claim 1, wherein, the storage room includes the sky with the Extraction medium part The sealing surfaces that chamber is in fluid communication.

3. extraction column according to claim 1 or 2, the described of the storage room and the Extraction medium part is additionally included in Fluid passage between cavity.

4. extraction column according to claim 3, wherein, the fluid passage substantially has frusto-conical shape, this section Head coniform shape has the convergent angle less than about 10 degree.

5. the extraction column according to claim 3 or 4, wherein, the fluid passage has the appearance no more than about 1000nl Product.

6. extraction column according to any one of claim 1 to 5, wherein, the at the end of the axial extension of the collar portion is super Cross the plane limited by the port of export of the elongate sleeve.

7. extraction column according to any one of claim 1 to 6, wherein, the end of the collar portion is to be enough to prevent The port of export of the elongate sleeve is extended axially beyond by the elongate sheath by the distance that any part of the second extraction column contacts The plane that the port of export of cylinder limits.

8. extraction column according to any one of claim 1 to 7, wherein, the entrance of the storage room part has Internal diameter, the end of the collar portion has external diameter, and the internal diameter of the entrance is not more than the end of the collar portion The external diameter at end.

9. extraction column according to claim 8, wherein, the internal diameter of the entrance is less than the end of the collar portion External diameter.

10. extraction column according to any one of claim 1 to 9, in addition to shoulder, the shoulder enters described in Mouth is outstanding from the storage room part.

11. extraction column according to any one of claim 1 to 10, wherein, the storage room includes sealing surfaces, described Sealing surfaces have funnel shaped and are inwardly tapered including annular wall, the annular wall towards the Extraction medium part.

12. the extraction column according to any one of claim 1 to 11, wherein, the storage room by including close to it is described enter Mouthful Part I and close to the Extraction medium part Part II inner surface limit,

The Part I have first near-end corresponding with the entrance, the neighbouring Part II the first distal end, with And first convergent angle, first convergent angle be calculated as the first storage part in the first proximal end and in the first distal end Difference between the internal diameter at place,

The Part II have neighbouring first distal end the second near-end, close to the Extraction medium part the second distal end, with And second convergent angle, second convergent angle be calculated as the second storage part in the second proximal end and in the second far-end Difference between internal diameter, and

The convergent angle of Part I is less than the convergent angle at the second end.

13. extraction column according to claim 12, wherein, the Part II has infundibulate shape and including towards institute State the annular wall that Extraction medium part is inwardly tapered.

14. the extraction column according to any one of claim 1 to 13, wherein, the cavity of the elongate sleeve has Generally frustoconical shape, the frusto-conical shape have the convergent angle less than about 1 degree.

15. the extraction column according to any one of claim 1 to 14, wherein, the Extraction medium part has outer surface, The outer surface is configured to be formed with the injection port of sample analysis system and sealed.

16. the extraction column according to any one of claim 1 to 15, wherein, the Extraction medium part has outer surface, The outer surface has generally frustoconical shape.

17. the extraction column according to any one of claim 1 to 16, wherein, Extraction medium is solid phase extraction medium.

18. extraction column according to claim 17, wherein, the solid phase extraction medium is the more of restriction condensate monolith Hole solid polymer.

19. extraction column according to claim 18, wherein, the Extraction medium is accommodated in substantial cylindrical shell, The substantial cylindrical shell is in both ends open and has an outer surface, the diameter of the outer surface and the sky of the elongate sleeve The diameter of the inner surface of chamber is corresponding.

20. extraction column according to claim 19, wherein the shell is a part for pipe.

21. the extraction column according to any one of claim 19 or 20, wherein, the shell is by polyethylene ether ketone, consolidation Silica, fluoropolymer and cyclic olefin copolymer composition.

22. the extraction column according to any one of claim 19 to 21, wherein, the Extraction medium is covalently coupled to institute State the inner surface of the cavity of polymer shell.

23. the extraction column according to any one of claim 19 to 22, wherein the medium is substantially prevented by being configured to Only sample flow separates around the layer of the medium with the inner surface of the shell.

24. the extraction column according to any one of claim 1 to 23, wherein, the Extraction medium include multiple non-porous pearls, Porous bead or its mixture.

25. extraction column according to claim 24, in addition to the first frit of the arrival end of the neighbouring elongate sleeve and Second frit of the port of export of the neighbouring elongate sleeve.

26. a kind of system being used for from liquid extraction desired component, the system are included such as any one of claim 1 to 25 institute The extraction column and sample port stated, the sample port have the first chamber of the elongate sleeve for being configured to accommodate the extraction column Room.

27. system according to claim 26, in addition to liquid transporting apparatus.

28. system according to claim 27, wherein, the liquid transporting apparatus is ceramic probe.

29. the system according to any one of claim 26 to 28, in addition to analytical equipment.

30. system according to claim 29, wherein, the analytical equipment be selected from by mass spectrograph, liquid-chromatography apparatus and Its group being bonded.

31. the system according to any one of claim 26 to 30, wherein, the sample port also include second chamber and Fluid passage between first chamber and second chamber, the second chamber are configured to accommodate the pipe from analytical equipment.

32. system according to claim 31, wherein, the fluid passage has the volume no more than about 0.5 μ l.

33. the system according to any one of claim 26 to 32, wherein, the sample port includes being roughly parallel to institute The outer surface of the axis extension of first chamber is stated, wherein the outer surface has the internal diameter of the collar portion less than the extraction column Diameter.