DE202016103304U1 - Extraction column assembly - Google Patents

Abstract

Sample cleaning device comprising:
a plurality of extraction columns disposed therebetween at a predetermined interval, and
a connection component extending between the plurality of extraction columns, the connection component holding the plurality of extraction columns with the predetermined distance therebetween;
wherein the plurality of extraction columns each comprise a column body having a reservoir portion and an extraction media portion,
wherein the reservoir portion includes an inlet and a reservoir, and
wherein the extraction media portion includes an extraction medium disposed within an elongate shell having an interior surface defining a cavity having an inlet end in fluid communication with the reservoir and an outlet end remote from the inlet end.

Description

BACKGROUND

1. Technical area

The invention relates to an apparatus and a system for extracting a component from a sample, and more particularly to a disposable tip having an extraction column therein and a plurality of extraction columns arranged in an array.

2. Relevant technology

A typical liquid chromatography-mass spectrometry (LC-MS) -based proteomics is performed with peptides as analytes in a process called bottom-up proteomics. In this process, proteins are digested to peptides using proteases. The disruption can take a long time and often requires an overnight incubation. In a clinical setting, a short sample processing time is desirable. Top-down proteomics offers the option of allowing intact proteins to be analyzed as such using LC-MS. This type of proteomics does not require a long term digestion, so it is beneficial for clinical use and also in automated systems. However, the protein fraction must continue to be extracted from the other non-protein components in the sample. Proteins can be extracted from other components in a sample by means of an extraction column. Preferably, such extraction columns permit automated sample preparation and integration directly into the LC.

Usually, pipette tips and similar consumables are sold, transported and plugged into trays or other devices that provide a known position and orientation for each tip and inserted into an automated analyzer. However, it is easier and less expensive to package and transport tips in a container in which the tips are packed in a large number and with any arrangement, such as in a bag or box. Usually, pipette tips are cone-shaped and have a pointed end and an open end so that the tips can interlock when loosely stored in a random arrangement and orientation. This makes it difficult for an automated system to pick up a single pipette, and the interlocking tips can also cause jams in an automated system. Another problem with such "random packaging" is that the sensitive end of a pipette tip may become damaged by the contact with other tips for receiving an extraction medium.

In addition, eluting or sifting eluent from multiple chromatography columns onto or into a multi-well sample plate can be costly and time consuming.

Accordingly, there are a number of limitations and / or disadvantages of the prior art, the removal of which would be advantageous.

SHORT SUMMARY

Extraction columns and systems are described herein which overcome one or more of the problems described above. In one embodiment, a plurality of extraction columns are arranged in an array (eg, in a linear array or in a two-dimensional array). The assembly may provide predetermined clearances suitable for use in a general application. The assembly may include two or more extraction columns each having a column body comprising a reservoir portion and an extraction media portion. The reservoir part includes an inlet and a reservoir. The extraction media portion comprises an elongate shell having an interior surface defining a cavity. The extraction media portion includes an inlet end in fluid communication with the reservoir and an outlet end remote from the inlet end. An extraction medium may be disposed in the cavity of the elongated shell. The extraction medium may be a solid phase extraction medium such as a polymer monolith, a plurality of non-porous beads or porous beads or a mixture of non-porous beads and porous beads. A connection component may extend between the respective extraction columns (or reservoir portions thereof (eg, adjacent to the respective inlets).) A shoulder may optionally project outwardly from the periphery of the assembly and / or a connection component thereof.

At least one embodiment includes a disposable, disposable solid phase extraction (SPE) seating assembly (eg, a linear array (arranged in a stripe format) or a two-dimensional array (in a plate format) for use in cleaning intact proteins positive blood culture samples, in particular after lysis of human cells and centrifugation of a microbial residue and before MS analysis of microbial proteins. In one embodiment, the SPE tip assembly may comprise two or more individual SPE extraction tips (or pillars) coupled together in a linear array with a selected spacing between the individual peaks. In one embodiment, a linear array of interconnected SPE tips may be spaced and arranged to fit the SPE tips with a standard pipette having a plurality of channels therein. In another embodiment, the SPE tip assembly may include individual, coupled SPE tips arranged in a grid (e.g., two-by-two, twelve-by-four, twelve-by-eight, etc.), the individual tips being spaced apart to correspond to the space dimensions of a multi-well format plate (e.g., a 96-well plate).

In one embodiment, proteins from the microbial extract are bound to the SPE peak, washed and eluted (directly) into the MS analysis (eg, MALDI). Accordingly, the predetermined spacing of the plurality of extraction columns in the assembly may be suitable for the application of eluted proteins directly to a MALDI-TOF analysis plate so that intermediate pipette steps can be avoided. The sample application workflow may be performed using a centrifugation or manifold (at a negative pressure or a positive pressure).

At least one embodiment includes an extraction column that includes a column body having a reservoir portion, an extraction media portion, and an optional sleeve portion. The reservoir portion may include an inlet and a reservoir, and / or the extraction media portion may include an extraction medium, which is optionally disposed in an elongate shell. The elongated shell may have an interior surface defining a cavity having an inlet end in fluid communication with the reservoir and an outlet end remote from the inlet end. The extraction medium may extend substantially to the outlet end of the elongate shell. The reservoir may include a sealing surface in fluid communication with the cavity of the extraction media portion.

The reservoir may include a sealing surface that is funnel-shaped and includes an annular wall that tapers inwardly toward the extraction media portion. The extraction column may provide fluid passage between the reservoir and the cavity of the extraction media portion. The fluid passage may be generally frusto-conical (i.e., have a convergence angle of less than about 10 degrees). The fluid passage may have a volume not greater than 1000 nl.

The reservoir may be defined by an inner surface comprising a first part near the inlet and a second part near the extraction medium part. The first part may have a first near end corresponding to the inlet and a first far end adjacent to the second part. A first convergence angle may be calculated as the difference between the inside diameters of the first reservoir part at the first near end and the first far end. The second part may have a second near end adjacent the first far end and / or a second far end near the extraction media part. A second convergence angle may be calculated as the difference between the inner diameters of the second reservoir part at the second near end and the second far end. The convergence angle of the first part may be smaller than the convergence angle of the second end. The second part may be funnel-shaped and includes an annular wall that tapers inwardly toward the extraction media part.

The reservoir inlet may have an inner diameter, the terminal end of the optional sleeve may have an outer diameter, and / or the inner diameter of the inlet may be smaller or (substantially) equal to the outer diameter of the terminal end of the sleeve. The inner diameter of the inlet may be smaller than the outer diameter of the terminal end of the optional sleeve part. The elongate shell cavity may be generally frusto-conical with a convergence angle of less than about 1 degree. The extraction media portion may have an outer surface configured to form a seal with an injection port of a sample analysis system. The extraction media portion may have an outer surface with a generally frustoconical shape.

The extraction medium may be or include a solid phase extraction medium. The solid phase extraction medium is a porous, solid polymer that defines a polymer monolith. The extraction medium may be contained in a generally cylindrical sleeve which is open at both ends and has an outer surface with a diameter corresponding to the diameter of the inner surface of the cavity of the elongate shell. The sleeve may be or include a portion of a tube. The sleeve may consist of polyethylene ether ketone, fused silica, fluoropolymers and cyclic olefin copolymers. The extraction medium may be covalently bound to the inner surface of the cavity of the polymer sleeve. The extraction medium may contain a plurality of non-porous beads or porous beads or a mixture of these. The extraction column may further include a first frit near the inlet end of the elongate shell and a second frit near the outlet end of the elongate shell.

The optional cuff portion may extend axially in the same direction as the elongated sleeve and / or may include a terminal end spaced from the outlet end of the elongated sleeve. The termination end may extend axially to at least one plane defined by the outlet end of the elongate shell. The terminal end of the collar portion may also extend axially beyond the plane defined by the outlet end of the elongate shell (eg, a distance sufficient to avoid contact of the outlet end of the elongate shell with a portion of a second extraction column). The extraction column may further include a shoulder projecting outwardly from the reservoir portion adjacent the inlet.

Certain aspects of the disclosure relate to systems that include embodiments of the extraction column in combination with a sample port having a first chamber configured to receive the elongated shell of the extraction column. The systems may optionally include a fluid delivery device such as a ceramic probe. Optionally, the systems may also include an analyzer such as a mass spectrometer, a liquid chromatographic apparatus, or combinations thereof.

A system for extracting a desired fraction from a liquid may include an extraction column, a sample port having a first chamber configured to receive the elongated shell of the extraction column, a liquid supply device, and / or an analyzer. The liquid supply device may be or comprise a ceramic probe. The analyzer may be selected from the group consisting of a mass spectrometer, a liquid chromatography apparatus, and combinations thereof. The sample port may further include a second chamber configured to receive a tube from the analyzer, and a fluid passageway between the first chamber and the second chamber. The sample port may have an outer surface that extends generally parallel to the axis of the first chamber. The outer surface may have a diameter that is smaller than the inner diameter of the cuff portion of the extraction column. The fluid passage may have a volume no greater than about 0.5 μl.

These and other objects, features, and advantages of the invention will become apparent from the following description, drawings, and claims, or may be learned during practice or implementation of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to describe how the above and other objects, features and advantages of the invention can be achieved, a more particular description of the invention will be given below by way of specific embodiments with reference to the accompanying drawings. The accompanying drawings, which form an integral part of the specification, illustrate embodiments of the invention and, together with the following detailed description, serve to clarify the invention. The figures are not necessarily drawn to scale. The drawings merely show typical embodiments of the invention and in no way limit the scope of the invention. The invention will now be described more concretely and in greater detail with reference to the accompanying drawings.

1 FIG. 15 is a perspective view of a disassembled state of an extraction column according to embodiments of the invention. FIG.

2 is a perspective view of a longitudinal section of the extraction column of 1 in a mounted state.

3 FIG. 3 is a longitudinal cross-sectional side view of a portion of one embodiment of a system that includes a fluid delivery device, an extraction column, a sample port, and a portion of an analyzer, in accordance with embodiments of the invention. FIG.

4 is a longitudinal sectional side view of an extraction column and part of an alternative liquid supply device according to embodiments of the invention.

5 Fig. 3 is a longitudinal sectional side view of a portion of one embodiment of a system including a fluid delivery device, an alternative embodiment of an extraction column, a sample and a portion of an analyzer, in accordance with embodiments of the invention.

6 is a perspective view of a sample port according to embodiments of the invention.

6A is a side view of the sample port of 6 ,

6B is a longitudinal sectional side view of the sample port of 6A along the line 6B-6B.

7 Figure 11 is a graph of the back pressures in the extraction column during the passage of liquid through the porous extraction medium.

8A FIG. 12 is a chromatogram obtained from a sample having a mixture of proteins eluted from an extraction column contained therein according to embodiments of the invention. FIG.

8B is a mass spectrum obtained from a sample containing a mixture of five proteins eluted from an extraction column according to embodiments of the invention.

9 is a chromatogram of insulin obtained from a sample eluted from an extraction column according to embodiments of the invention.

10 is a chromatogram of ubiquitin obtained from a sample eluted from an extraction column according to embodiments of the invention.

11 Figure 12 is a chromatogram of myoglobin obtained from a sample eluted from an extraction column according to embodiments of the invention.

12 is a chromatogram of carbonic anhydrase obtained from a sample eluted from an extraction column according to embodiments of the invention.

13A is a perspective view of an extraction column assembly according to embodiments of the invention.

13B is a top plan view of the extraction column assembly of 13A ,

13C is a plan view from below of the extraction column assembly of 13A ,

13D is a first side view of the extraction column assembly of 13A ,

13E is a second side view of the extraction column assembly of 13A ,

DETAILED DESCRIPTION

Before describing in detail below various embodiments of the invention, it should be understood that the invention is not limited to the parameters of the particular systems, methods, devices, products, processes, and / or sets described herein by way of example, which of course may vary. Numerous methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, with only specific materials and methods being described herein. Particular embodiments of the invention will be described in detail with reference to specific configurations, parameters, features (e.g. Components, members, elements, parts and / or sections), etc., which descriptions are intended to be illustrative and not in any way limiting the scope of the invention. In addition, the terminology used herein is illustrative of the embodiments and does not necessarily limit the scope of the invention.

Unless defined otherwise, the technical and scientific terms used herein have the meanings familiar to those skilled in the art.

Various aspects of the invention including the devices, systems, and methods are described with reference to one or more embodiments or implementations, which are merely exemplary. By an "embodiment" or "implementation" is meant herein an example, instance, or illustration that is not necessarily to be construed as preferred or advantageous over other aspects described herein. An implementation of the invention may include one or more embodiments, and vice versa, each being illustrative examples that in no way limit the scope of the invention as defined by the appended claims and not by the description.

By "systems" are meant devices, assemblies, arrangements, sentences and vice versa. Accordingly, a "process" here means processes, procedures, steps and vice versa. Furthermore, "devices" here products, devices, compilations, arrangements, sentences and vice versa to understand.

The word "may" is to be understood in a permissive sense (i.e., optional) and not in a compelling sense (i.e., mandatory). Further, the words "including," "comprising," "including," "characterized by," and similar words are to be understood as inclusive herein, and thus have the same meaning as the word "include," so as additional, here unspecified elements or process steps are not excluded.

When singularizations are used in this specification and in the claims, it is to be understood that each may be multiple units, unless otherwise specified by context. Thus, the mention of a "seam" can be one, two or more seams. Accordingly, the naming of multiple units should be interpreted as being a single unit and / or a plurality of units, unless otherwise specified by the context. The term "stitching" is therefore not necessarily a multiplicity of seams. Regardless of the grammatical form, it may therefore be one or more seams.

Directions such as "top", "bottom", "left", "right", "top", "bottom", "inside", "outside", "inside", "outside", "near", "far", etc . can here be arbitrarily used to indicate relative directions, positions and / or orientations and in no way limit the scope of the invention.

Various aspects of the invention may be illustrated by the description of components that are "bound," "coupled," "attached," "connected," or "in communication" with each other. If the words "bound", "coupled", "attached", "connected" or "in a connection" are used, it can either be a direct association between two components or an indirect association between the two components intervene between other components. On the other hand, if a component is referred to as being "directly bound," "directly coupled," "directly attached," "directly connected," or "directly connected" with another component, there are no other components in between. Furthermore, a bond, coupling, attachment, connection or communicating connection may be a mechanical and / or fluid association.

To facilitate understanding, where possible, like reference numerals (eg, like numbering of components and / or elements) are used to indicate corresponding elements throughout the several figures. In particular, in the exemplary embodiments shown in the figures, similar structures or structures with similar functions are indicated, where possible, by similar reference numerals. Specific terminology is used herein to describe the exemplary embodiments. Nevertheless, it should be noted that this does not limit the scope of the invention in any way. The terminology used to describe the exemplary embodiments is to be construed as illustrative and does not limit the scope of the invention (unless it is specifically stated that the terminology used herein is to be understood in its narrow sense).

Furthermore, in alternative configurations, a particular element may be indicated by a reference number to which a letter is attached. Accordingly, an appended letter may be used to indicate an alternative of design, construction, function, implementation, and / or designation to the corresponding element or feature without the appended letter. Correspondingly, a plurality of instances of an element and / or subelements of a superordinate element can each be distinguished by different letters appended to the reference character. In any case, the reference numeral may be used without an appended letter to generally refer to different instances of the element or any of alternative elements. Reference numerals with an appended letter may be used to refer to a specific instance of the element or to distinguish between different uses of the element. However, elements indicated by a reference number with an appended letter are not limited to the specific and / or particular embodiments in which they are shown. In other words, a reference to a specific feature with respect to an implementation and / or embodiment is not to be interpreted as being limited in its application to this embodiment.

The headings used in the description have only the functions of a clear structure and limit the scope of the invention in any way.

The invention relates to an extraction column assembly for extracting one or more components from a plurality of samples. The extraction column assembly may comprise a disposable tip assembly comprising two or more extraction columns arranged and / or fixed in an array with predetermined intervals therebetween. The distances may be configured, adjusted and / or provided such that the column assembly is suitable for use in a general application. The assembly may comprise an appropriate number of (separate and / or individual) extraction columns and a shoulder extending between the respective extraction columns.

In certain embodiments, the assemblies are configured to be discarded after a single use. Therefore, some embodiments of the invention reduce the workload otherwise required for cleaning and sterilizing conventional columns or other systems. This will also ensure that sterility can always be maintained during repeated processing of multiple batches. In view of this, and because certain embodiments of the invention are readily scalable, relatively inexpensive, and easy to use, some embodiments of the invention may be employed in a variety of industrial and research facilities where such processing has hitherto been outsourced. Here, when some components are referred to as "disposable" components, it means that the components are designed and / or commercially compatible with disposable disposable applications. Thus, while strictly speaking, all materials are technically disposable, it should be apparent to those skilled in the art that the term "disposable" or "disposable" has the particular meaning that such components are designed for economical disposal after a single use.

extraction columns

As in 1 and 2 As shown, one embodiment of the invention relates to an extraction column 10 for extracting a component such as a protein or a protein fragment from a sample containing a mixture of components such as proteins, peptides, carbohydrates, lipids, nucleic acids, salts and small molecules. The extraction column 10 includes a column body 12 with a reservoir part 14 , an extraction media part 16 and an optional cuff 18 ,

The reservoir part 14 is generally at the near end 20 of the column body 12 arranged and includes an inlet 24 and a cavity acting as a reservoir 26 works. The reservoir 26 gets through the inner surface 28 of the reservoir part 14 defines and is in fluid communication with the cavity 32 of the extraction media part 16 , the general at the far end 30 of the column body 12 is arranged. The reservoir 26 can hold a volume of a liquid. In one embodiment, the volume of the reservoir is sufficient 26 to prepare it for the extraction medium 42 required volume of liquid to keep the volume of the liquid to be extracted, the volume of the washing solutions and the volume of the elution solution. For example, in some extraction procedures it is necessary to dilute a sample with a suitable solvent before passing the sample through the extraction medium 42 in the cavity 32 of the extraction media part 16 is forced. Usually, the step of diluting the sample is carried out in a separate vessel. In the exemplary embodiment, the volume of the reservoir is sufficient 26 out to the sample directly into the reservoir 26 to dilute before a positive pressure on the reservoir 26 is applied to transfer the sample to the extraction medium. In one embodiment of the invention, the reservoir 26 a volume of about 50 μl to about 1500 μl.

In the exemplary embodiment of 1 - 3 contains the inner surface 28 of the reservoir 26 a first part 34 near the inlet 24 that accounts for much of the volume of the reservoir 26 provides, and a second part 38 near the extraction media part 16 , In the exemplary embodiment of 1 - 3 is the inner surface 28 of the first part 34 of the reservoir 26 is generally frusto-conical and has a convergent angle between the inner diameter ID1 at the proximal end of the first part 34 and the inner diameter ID2 at the far end 36 of the first part 34 which is less than about 10 degrees or, in an alternative embodiment, less than 4 degrees. The angle of convergence here is to be understood as meaning the angle between said surface and the center axis of the structure, which in this case is the center axis 62 of the column body 12 is.

As in 4 shown, the reservoir can 26 have a shape that is configured to oppose a liquid handling device 41 to be sealed, which has a positive air pressure in the reservoir 26 can generate fluid in the reservoir 26 through the extraction medium 42 to force. For example, the outer surface 43 in the neighborhood of the near end 20 of the first part 34 configured to be in an O-ring 44 at the spindle 45 the liquid handling device 41 to intervene and form a seal with this. Alternatively, the inner surface 28 of the first part 34 of the reservoir 26 configured to engage and seal with an O-ring or an outer surface of the spindle of a liquid handling device.

The second part 38 of the reservoir 26 can be like in 2 and 3 shown funnel-shaped and having an annular wall which extends inwardly to the extraction media part 16 rejuvenated. In an alternative embodiment, the second part 38 be frustoconical. The funnel shape or truncated cone shape reduces the inside diameter of the second part 38 and helps keep the end of the liquid supply device 46 in a position to a tight seal between the liquid supply 46 and the extraction column 10 to build. The second part 38 of the reservoir 26 has a converging angle between the inner diameter ID3 at the near end 48 of the second part 38 and the inner diameter ID4 at the far end 50 of the second part 38 on. In the exemplary embodiment, the convergence angle of the first part is 34 of the reservoir is smaller than the convergence angle of the second part 38 of the reservoir 26 ,

In the embodiment of 1 to 3 includes the second part 38 of the reservoir 26 a paragraph 52 that the diameter of the reservoir 26 further reduced, if this to the fluid passage 54 between the reservoir 26 and the cavity 32 of the extraction media part 16 passes. Paragraph 52 functions as a sealing surface for forming a seal with the liquid supply device 46 such as a pipette tip or a hollow probe such as a hollow ceramic probe. In some embodiments, the inner surface at the distal end of the reservoir portion may also be that with the liquid supply device 46 Contribute formed seal. The seal is formed when one as in 3 shown positioned liquid supply 46 against the paragraph 52 and in some embodiments, the inner surface 40 at the far end 50 of the second part 38 of the reservoir 26 in the neighborhood of the paragraph 52 is pushed with a force sufficient to make a tight seal between the column body 12 and the liquid supply device 46 to build. The on the liquid supply 46 exerted force can cause the plastic structures of the paragraph 52 and / or the inner surface 40 at the far end 50 of the second part 38 of the reservoir 26 in the neighborhood of the paragraph 52 to the shape of the liquid supply device 46 deform to a seal between the liquid feed 46 and the extraction column 10 to build. Paragraph 52 is in the exemplary embodiment of 1 - 3 generally frustoconical having a blunt convergence angle in a range of about 45 degrees to about 90 degrees.

In an in 5 shown alternative embodiment, the second part 38 no paragraph on and instead goes directly to the cavity 32 of the extraction media part 16 above. In this exemplary alternative embodiment, the liquid supply means 46 a seal against the near end 53 of the extraction medium 42 , The inner surface 40 at the far end 50 of the second part 38 can be in a part of the liquid supply 46 engage and contribute to the seal between the liquid supply 46 and the extraction medium 42 at. In this alternative embodiment is a paragraph 57 at the outlet end 60 of the cavity 32 the elongated shell 55 of the extraction media part 16 of the column body 12 positioned. Paragraph 57 reduces the inside diameter of the opening 59 the outlet end 60 of the cavity 32 the elongated shell 55 ,

In one embodiment, this allows between the liquid supply device 46 and the extraction column 10 Sealing does not leak more than about 10% of the volume of the sample coming from the liquid supply device 46 to the extraction column 10 is transmitted. In another embodiment, the between the Flüssigkeitszuführeinrichtung allows 46 and the extraction column 10 The seal formed will not leak more than about 10% of the volume of the fluid delivery device 46 to the extraction column 10 transferred sample under injection pressures of about 70 bar to about 200 bar.

In the 2 - 4 embodiment shown comprises a fluid passage 54 between the reservoir 26 and the cavity 32 of the extraction media part 16 , In this exemplary embodiment, the fluid passage is 54 truncated cone shape and the angle of convergence is usually less than about 10 degrees and in an alternative embodiment less than about 1 degree. The fluid passage 54 has a length and an inner diameter that is the volume of the fluid passage 54 reduce and at the same time provide sufficient flow to build one up by obstructing a flow through the fluid passageway 54 caused back pressure to prevent. The volume of the fluid passage 54 is minimized to minimize the dead volume in the extraction column. In one embodiment, the volume of the fluid passage is 54 not greater than about 1000 nl and may range from about 1 nl to about 50 nl.

The extraction media part 16 includes an elongated shell 55 with an inner surface 56 that have a cavity 32 with an extraction medium provided therein 42 Are defined. The cavity 32 includes an inlet end 58 in fluid communication with the reservoir 26 and an outlet end 60 with an opening 59 away from the inlet end 58 is arranged. In one embodiment, the cavity is 32 truncated cone with a convergence angle of less than about 5 degrees, in another embodiment, the convergence angle may be from about 0.2 degrees to 1 degree. In another alternative embodiment, the angle of convergence of the frusto-conical cavity 32 about 0.4 degrees. In embodiments having a frusto-conical cavity 32 opens the larger-diameter end of the cavity 32 to the point of introduction for the extraction medium 42 , The inner diameter of the cavity 32 for the alternative embodiment is also from about 0.5 mm to about 2.0 mm, and preferably from about 0.75 mm to about 0.85 mm. The column body 12 has a center axis 62 on, extending through the cavity 32 of the extraction media part 16 extends. The cavity 32 has a length L1 along the center axis 62 between the inlet end 58 and the outlet end 60 on. In one embodiment, the length L1 of the cavity is located 32 in the range of about 1 mm to about 10 mm. In another embodiment, the length L1 of the hol space 32 in the range of about 3 mm to about 5 mm, and preferably in the range of about 3.5 mm to about 4.5 mm. In one embodiment, the length L1 of the cavity corresponds to the length of the elongated shell 55 ,

The extraction media part 16 also has an outer surface 66 whose bottom part is configured to seal with a sample port 68 a sample analysis system ( 3 ) to build. In one embodiment, the outer surface 66 a generally frustoconical shape narrower than the outlet end 60 the elongated shell 55 is. The elongated shell 55 may have an outside diameter at the outlet end 60 from about 1 mm to about 2 mm. In another embodiment, the elongated sheath 55 an outer diameter at the outlet end 60 from about 1.2 mm to about 1.5 mm. In another embodiment, the elongated sheath 55 an outer diameter at the outlet end 60 of about 1.4 mm. The outer surface 66 the elongated shell 55 may have a convergence angle of about 1 degree to about 20 degrees, with the convergence angle preferably from about 5 degrees to about 15 degrees. In embodiments of the elongated shell 55 takes the thickness of the elongated shell 55 forming wall from the outlet end 60 of the cavity 32 to the inlet end 58 of the cavity 32 towards.

The cuff 18 of the column body 12 extends axially in the same direction as the elongated shell 55 , In the embodiment of 1 - 4 shows the cuff 18 a closed end 72 on, with the outside surface 78 of the reservoir part 14 in the neighborhood of the transition between the first and second parts 34 . 38 is coupled. In the embodiment shown, the outer surface closes 74 the cuff 18 to the outer surface 78 of the reservoir part 14 at. The outer surfaces 78 . 74 of the reservoir part 14 and the cuff 18 may taper with a convergence angle of less than 15 degrees and, in an alternative embodiment, in a range of about 0.1 degrees to about 5 degrees.

The cuff 18 has an open completion date 70 on, that from the outlet end 60 the elongated shell 55 is spaced. The outlet end 60 the elongated shell 55 defines a plane P1. The terminal end 70 the cuff 18 defines a plane P2 at least that of the outlet end 60 the elongated shell 55 defined plane P1 extends. In one embodiment, the plane P2 of the terminal end extends 70 the cuff 18 over the plane P1 of the outlet end 60 the elongated shell 55 out. In the in 1 - 4 In the embodiment shown, the plane P2 of the terminal end extends 70 the cuff 18 over the plane P1 of the outlet end 60 the elongated shell 55 with a distance D1 sufficient to contact the outlet end 60 the elongated shell 55 to prevent with any part of a second extraction column. For example, the distance D1 may be from about 0.1 mm to about 2 mm, depending on the inner diameter ID5 at the terminal end 70 the cuff 18 and the smaller of the outer diameter OD1 at the terminal end 70 the cuff 18 or the outer diameter OD2 at the near end 20 of the column body 12 can be sufficient to make a contact of the outlet end 60 the elongated shell 55 to prevent with any part of a second extraction column. This structure prevents damage to the outlet end 60 the elongated shell 55 if the extraction columns 10 loosely stored in a bag or box. A further advantage of the plane P2 extending beyond the plane P1 is that contamination of the outlet end 60 the elongated shell 55 is prevented when the extraction column 10 is handled by an automated sample analysis system. For example, there is a method of transporting extraction columns 10 in automated analysis systems in it, the extraction column 10 dropping from one position in the system to another through a guide tube or tube. When the outlet end 60 the elongated shell 55 exposed, ie not by a cuff 18 is protected, there is the risk of carryover contamination of the outlet end 60 big when the outlet end 60 touches the surfaces of the transport tube or hose. The cuff 18 The invention protects the outlet end 60 the elongate sheath against contact with the surfaces of the transport tube, thereby reducing the risk of carryover contamination between different extraction columns transported in the same tube.

The final 70 the cuff 18 has an outer diameter OD1, and the inlet 24 of the reservoir 26 has an inner diameter ID1 so that the terminal end 70 the cuff 18 not completely in the inlet 24 of the reservoir 26 can be inserted. In one embodiment, the inner diameter ID1 of the inlet 24 of the reservoir not larger than the outer diameter OD1 of the terminal end 70 the cuff 18 , In another embodiment, the inner diameter ID1 of the inlet 24 of the reservoir smaller than the outer diameter OD1 of the terminal end 70 the cuff 18 , Accordingly, the near end 20 of the column body 12 an outer diameter OD2 and has the open terminal end 70 the cuff 18 an ID5 inner diameter so that the near end 20 of the column body 12 not completely in the open completion 70 the cuff 18 can be inserted. In the embodiment of 1 - 4 the outer diameter OD2 contains at the near end 20 of the column body 12 an optional shoulder 76 , In one embodiment, the inner diameter is ID5 of the open termination end 70 the cuff 18 not larger than the outer diameter OD2 of the near end 20 of the column body 12 , In another embodiment, the inner diameter ID5 of the open termination end 70 the cuff 18 smaller than the outer diameter OD2 of the near end 20 of the column body 12 , If several extraction columns 10 When packed randomly, for example, in a bag or a box, the extraction columns grip 10 not in one another, as would be the case if one end of the extraction column would fit into an opening at one end of a second extraction column. This aspect of the extraction columns 10 makes it much easier for an automated system, one extraction column each 10 pick up and allow the extraction columns 10 sorted by automated systems and placed in racks. Therefore, the extraction columns can be packed, sold, stored and fed into an automated analysis system in loose batches and do not need to be packed in specified positions and with a specified orientation in a rack or on a tray, thus improving the efficiency and the monetary, Space and labor costs is reduced.

In the embodiment of 1 - 4 includes the near end 20 of the column body 12 an optional shoulder 76 that from the outside surface 74 of the reservoir part 14 in the neighborhood of the inlet 24 protrudes. The shoulder 76 increases the outside diameter of the near end 20 of the column body 12 and provides an area that can be used by automated devices around the extraction column 10 hang during handling or the extraction column 10 to hang in a tray or rack system.

The extraction medium 42 is in the cavity 32 of the extraction media part 16 arranged. The extraction medium 42 allows the extraction of a desired component from a mixed sample. The extraction medium 42 can chromatographically proteins from other components in a mixed separate biological sample. Embodiments of the extraction medium 42 may reversibly bind small molecules or macromolecules such as peptides and proteins having a molecular weight in the range of about 1 kDa to about 200 kDa. The extraction medium 42 can reversibly bind at least 1 μg of protein in a sample volume of from about 10 μl to about 100 ml when the sample is passed through the extraction medium 42 at a flow rate in the range of about 50 μl / min to 200 μl / min. The extraction medium 42 Also, the desired component may be based on a desired property such as the molecular weight of the component, the hydrophobicity, the charge, or the affinity of the component for an aspect of the extraction medium 42 elute. In one embodiment, the extraction medium 42 elute at least 20% of the bound proteins from the mixed sample when eluting at a flow rate of about 0.1 μl / min to about 20 μl / min with a volume of elution solution of about 1 μl to about 100 μl.

In one embodiment, the extraction medium is 42 a solid phase extraction medium, and more particularly, a porous monolith medium 80 with a high internal porosity, which allows a sufficient flow of a sample through the porous monolith medium 80 allowed without generation of undesirably high back pressures. In one embodiment, the backpressure is no greater than about 5 bar at a flow rate of 200 μl / min. The porous monolith medium 80 may preferably withstand at least 200 bar. An advantage of pore construction of the porous monolith medium 80 This is because it provides a tortuous path for the sample, allowing fast convective mass transfer at fast flow rates. Furthermore, requires a porous monolith medium 80 no frit to his position in the column body 12 maintain.

The porous monolith medium 80 can be considered as a continuous bed in a sleeve such as in a section of a tube 82 to get prepared. That is why when cutting the pipe 82 in sections the porous monolith medium 80 present in the full length of a microcolumn and can be used as the extraction medium 42 serve.

In the embodiment of 1 - 4 is the extraction medium 42 a porous monolith medium 80 that in the cavity 32 of the extraction media part 16 of the column body 12 from the outlet end 60 the elongated shell 55 of the extraction media part 16 is inserted. In the embodiment of 5 is the extraction medium 42 a porous monolith medium 80 that in the cavity 32 of the extraction media part 16 of the column body 12 from the inlet end 58 of the cavity 32 is plugged in because of paragraph 57 at the outlet end 60 the elongated shell 55 is provided. An advantage of preparing the porous monolith media 80 in the tube 82 and cutting the pipe 82 to a microcolumn is that this method avoids the formation of a semipermeable or non-porous layer at the interface between the air and the polymerizing porous monolith medium that occurs when the porous monolith medium polymerizes directly inside a pipette tip. The semipermeable or non-porous layer may affect the flow characteristics of the resulting porous monolith medium. In embodiments in which the porous monolith medium is directly in the cavity 32 of the extraction media part 16 is formed, the negative effects of the semipermeable or non-porous layer can be reduced by forming a passage through the center of the porous monolith medium to increase the flow rate of the sample through the extraction medium. However, much of the component to be collected can remain in the passageway and bypass the porous monolith medium, requiring multiple passes of the sample through the medium for maximum extraction. In contrast, preparing the porous monolith media creates 80 in the tube 82 , which is cut into segments, a porous cylinder in which the pore structure and the porosity along the diameter and along the length of the porous monolith medium 80 are even. This preparation process allows flow of a sample through the porous monolith medium 80 which is not hindered by semipermeable or non-porous surfaces, thus allowing substantially uniform bonding in the porous monolith medium 80 to maximize extraction of the desired component in a single pass through the porous monolith medium 80 provided.

The pipe 82 outside the porous monolith medium 80 provides a protective layer that helps to provide the porous monolithic medium 80 to handle. For example, during the preparation of the extraction column 10 the column body 12 independent of the porous monolith medium 80 be formed. The porous monolith medium 80 is then in the cavity 32 of the extraction media part 16 of the column body 12 plugged in. In embodiments in which the porous monolith medium 80 in a section of a pipe 82 is formed, allows the tube 82 a simpler handling of the porous monolith medium 80 when plugging into the cavity 32 of the extraction media part 16 , And if the porous monolithic medium 80 in the cavity 32 of the reservoir part 14 can be plugged in, the wall of the pipe 82 as a holder for the porous monolith medium 80 serve and can act as a sealing surface against the inner surface 56 of the cavity 32 act to return to the porous monolith medium 80 to prevent around.

If that is in a section of the pipe 82 formed porous monolith medium 80 in the cavity 32 of the reservoir part 14 is inserted, the pipe becomes 82 around the porous monolith medium 80 compressed around to prevent the porous monolith medium 80 from the pipe 82 is extruded.

The porous monolith medium 80 Can be optional with the inner surface of the tube 82 be coupled to the resistance to extrusion of the porous monolith medium 80 to improve. For example, the inner surface of the pipe 82 be treated to covalent bonding adhesion points for the polymerizing porous monolith medium 80 to create. In order to activate the inner surface of a pipe such as a polyethylene ether ketone (PEEK) pipe, the inside of the pipe may be filled with a reaction solution containing a solvent such as acetonitrile or propionitrile (alkylnitrile derivatives) and an azo class initiator such as Vazo-64 or Vazo-55 at a concentration of about 1% to about 10% (volume). The filled tube may then be heated to at least 80% of the 10 hour half life temperature of the solvent, with Vazo-64, for example, having a 10 hour degradation half life at 64 degrees Celsius. The reaction can proceed for at least 30 minutes. The reaction solution can be replaced with a fresh reaction solution and allowed to proceed for at least another 30 minutes. In one embodiment, the ends of the tube are sealed to produce an internal back pressure of at least 5 psi. In another embodiment, the reaction solution is continuously filled with ambient pressure. The reaction solution is then removed from the tube and the tube is dried with a stream of nitrogen. After drying, the polymerization mixture is injected into the lumen of the activated tube. During the polymerization of the porous monolith medium 80 The activated adhesion points on the inner surface of the tube are integrated into the extraction medium.

As an example, a PEEK tube is used but other types of polymeric tubes may be used, including, for example, cyclic olefin copolymers ("COC") and fluoropolymers such as ethylene tetrafluoroethylene (ETFE), fluorinated ethylene propylene (FEP), and other fluoropolymers , In addition, a fused silica tube activated by the covalent bonding of an acrylate or methacrylate-containing silanizing reagent after hydrolysis of the fused silica may be used.

The pipe 82 may have an inner diameter between about 0.1 mm and about 1 mm. In one embodiment, the inner diameter of the tube is sufficient 82 from about 0.4 mm to about 0.6 mm. In another embodiment, the inner diameter of the tube is about 0.5 mm. The pipe 82 with the porous media formed therein can be cut to a length which, in combination with the inner diameter of the tube, provides the desired volume for the porous extraction media. In one embodiment, the length of the tube becomes 82 with a porous monolith medium 80 cut into sections of about 6 mm to about 2 mm, the length in an alternative embodiment ranging from about 3 mm to about 5 mm or about 4 mm. After the polymerization of the porous extraction medium, the pipe can be cut to the desired length using a pipe cutter such as an IDEX A-350 pipe cutter.

The porous monolith medium 80 can be prepared by polymerizing a mixture containing suitable monomers as well as an initiator and a pore-forming solvent (porogen). The resulting porous monolith medium 80 has pores whose diameter ranges from about 50 nm to about 20,000 nm. The porous monolith medium 80 may have pores in the range of about 50 nm-200 nm or about 750-10000 nm. The porous monolith medium 80 may consist of polymer beads ranging in diameter from about 20 nm to about 10,000 nm. The porous monolith medium 80 should withstand a pressure of at least about 200 bar.

The monomers may be selected from vinyl-containing monomers, acrylate-containing monomers, methacrylate-containing monomers, acrylamide, polyolefin, polyester, polyurethane, polyamide, fluoro-substituted ethylene, and combinations thereof. The vinyl-containing monomers may include vinyl aromatic monomers such as monovinyl-substituted aromatic monomers and divinyl-substituted aromatic monomers, and combinations of these. Exemplary vinyl aromatic monomers are divinylbenzene, styrene, alkyl-substituted styrene such as ethylvinylbenzene, alpha-methylstyrene, alkyl-substituted alpha-methylstyrene, halo-substituted alpha-methylstyrene such as chloromethylstyrene, and combinations thereof. The alkyl substitutions may include up to 18 carbon atoms. The acrylate-containing monomers are mono-, di- and triacrylates. The methacrylate monomers are mono-, di- and trimethacrylates such as glyidyl methacrylate, ethylene dimethacrylate, trimethylolpropane, trimethyl acrylate, hydroxyethyl methacrylate. In one embodiment, the monomers or mixtures of at least two monomers are generally present in the polymerization mixture at a level of from about 10 volume percent to about 60 volume percent, and in an alternative embodiment at a level of from about 20 volume percent to about 70 volume percent.

The porogen can be selected from various types of materials. Suitable liquid porogens are, for example, aliphatic hydrocarbons, aromatic hydrocarbons, esters, alcohols, ketones, ethers, solutions of soluble polymers and mixtures of these. Exemplary porogens are 4,4,4-trimethylpentane, alcohols having 1 to 12 carbon atoms, toluene, butyl acetate, 1,4-butanediol, water, acetone, hexane, cyclohexane, cyclohexanol, tetrahydrofuran (THF), and combinations thereof. In one embodiment, the porogen is generally present in the polymerization mixture at a level of from about 20 volume percent to about 90 volume percent and in an alternative embodiment from about 60 volume percent to about 80 volume percent.

The initiators may be thermal polymerization initiators, conventional radical polymerization initiators, photoinitiators and redox initiators. Examples of suitable initiators are peroxides such as OO-t-amyl-O- (2-ethylhexyl) monoperoxycarbonate, dipropyl peroxydicarbonate and benzoyl peroxide, azo compounds such as azobisisobutyronitrile (Dupont Vazo-64), 2,2'-azobis (2-amidinopropane) dihydrochloride and 2,2'-azobis (isobutyramide) dihydrate and ammonium persulfate and tetramethylethylenediamine (TMEDA). In one embodiment, the initiator is generally present in the polymerization mixture at a level of from about 0.2% to about 5% by weight of the monomers, and in an alternative embodiment from about 1% to about 2% by weight of the monomers.

The components of the polymerization mixture are mixed according to routine techniques and injected into the interior of the tube where they can then polymerize. For example, in one embodiment, the tube is filled with a polymerization mixture and a pressure of about 100 psi is applied. The application of pressure helps to prevent the formation of bubbles in the polymerization mixture when nitrogen is formed during the degradation of the initiator during the polymerization. In other embodiments, the tube is filled with the polymerization mixture and sealed off both ends of the tube while the polymerization can proceed. The sealing of both ends of the tube results in an increased pressure inside the tube during the progress of the polymerization, thereby preventing the formation of nitrogen bubbles. In another embodiment, the tube is filled with the polymerization mixture and one end of the tube is sealed while the other end of the tube is left open but placed in a vial. The filled tube is heated during the polymerization step. Because the open end of the tube is placed in a vial, the liquid may expand while the mixture is heated during polymerization. This prevents pressure increases during heating that could affect the porosity of the porous monolith media. In embodiments with a photoinitiator, the filled tube may be subjected to UV irradiation. Examples of porous monolith media and methods of making the same are disclosed in U.S. Pat U.S. Patent No. 7,922,908 which is hereby incorporated by reference in its entirety.

The porous monolith medium 80 can also be functionalized. For example, the porous monolith medium 80 wherein the epoxide or halide functionality can react with amines or sulfides to produce, for example, anion exchange media or with, for example, carboxylic acid, phosphoric acid or sulfonic acid to produce cation exchange media. These groups can then be further modified to allow binding of proteins, peptides or immunoglobulins, thereby producing affinity separation and extraction media. Epoxide groups can react either directly with proteins, peptides or immunoglobulins or after conversion to aldehyde. Other possible affinity media are immobilized metal ion affinity chromatography (IMAC) phases and boronate phases.

Exemplary porous materials suitable for use as the porous monolith medium 80 are suitable. Methods for producing such materials are described in U.S. Patent Nos. 4,135,774 U.S. Patent Nos. 5,334,310 and 5,633,290 described in detail herein.

In an alternative embodiment, the extraction medium 42 a plurality of porous and / or non-porous beads of, for example, glass, silica or a polymer material contained in the cavity 32 of the extraction media part 16 are included. In this embodiment, the inlet end of the cavity may include a first frit, and the outlet end of the cavity may be a second frit contain. The frits prevent the beads from escaping from the cavity. The beads are sufficiently packed in the cavity to allow for a sufficient flow rate and to produce no undesirable back pressure. Exemplary beads are used in the U.S. Patent No. 6,783,672 which is hereby incorporated by reference in its entirety.

During use, before a sample passes through the extraction column 10 is guided, the extraction medium 42 wetted with a wetting agent. In embodiments in which the extraction medium 42 a porous monolith medium 80 contains, becomes the porous monolith medium 80 with a sufficient volume of a wetting agent, which may contain an organic solvent such as acetonitrile (ACN) and an aqueous component such as 0.2 volume percent formic acid (AS). Usually, about 10 μl to about 100 μl of the wetting agent is used to form the porous monolith medium 80 to wet. The porous monolith medium 80 is then equilibrated with an equilibrium solution which may contain water and about 0.2% by volume of AS. Usually, about 10 μl to about 100 μl of the equilibrium solution is used to construct the porous monolith medium 80 compensate. Samples containing interesting compounds such as proteins having a volume of 10 μl to 100 μl are then passed through the porous monolith medium 80 in the extraction column 10 forced. The flow through the samples can optionally be collected for additional analysis. The porous monolith medium 80 is then washed with a wash solution containing water and about 0.1 volume percent to about 0.2 volume percent AS. Usually, about 10 μl to about 100 μl of the wash solution is used to replace that in the porous monolith medium 80 wash collected sample. The through the porous monolith medium 80 Trapped compounds are then eluted with an elution solution. The proportion of elution solution and the volumes and elution times may vary depending on the type of elution desired. If rapid elution is desired, the elution solution may contain water, an organic component in a range of about 20-60 volume percent, and about 0.2 volume percent AS, using about 1 μL to about 100 μL of the elution solution to collect the trapped compounds from the porous monolith medium 80 to elute. The liquid solutions including the sample can be rapidly passed through the extraction medium 42 be pressed. Binding of the compounds in the extraction medium 42 occurs rapidly, with the elution time depending on the desired application. Isocratic elution or elution using fast gradients allows extremely fast performance, which can be used in automated systems where high throughput is desired. When used in conjunction with high resolution mass spectrometry, compounds such as proteins can be identified using rapid elution. Such methods may be useful when rapid analysis such as microbial identification is required. If a more detailed analysis is desired, the elution can be carried out slowly so that the compounds are sequential from the extraction column 10 according to a desired property such as molecular weight, charge, hydrophobic interaction or other affinity-type interaction with components of the extraction column 10 can elute. The slow elution can be performed with a short (eg, about 5 minutes) or long (eg, about 30 minutes) elution gradient. For example, in one embodiment, the percentage of organic component in the eluent may be increased continuously or stepwise across the gradient to provide elution for a desired duration. The longer elution times allow analysis of a single desired compound such as an antibiotic resistance marker in a microbial sample or a biopsy cancer biomarker. Because a gradient can be applied for effective connection separation, the user does not have to use a costly and time-consuming analytical column. Analysis columns are used several times, with washes required to avoid transfer from one sample to another. The extraction column 10 can be disposable so that the user does not have to do washes and a transfer from one sample to another is avoided.

The eluted compounds may be collected for later analysis, or preferably directly via a sample port 68 into an analysis system, which will be described in greater detail below. When the eluted sample is fed directly into the analytical system, the beneficial properties of the extraction column become 10 for rapid extraction of compounds from a sample and also for the chromatographic elution of compounds based on a desired property such as molecular weight, charge, hydrophobic interaction or other affinity type reactions with components of the column. An exemplary analytical system is a liquid chromatography (LC) system. If the extraction column 10 used to elute a compound directly into an LC system, the extraction column must 10 can withstand the high pressure used in the LC system. In addition, the liquid flow in an LC system can be extremely low, so that voids and dead volumes must be minimized. Embodiments of the Extraction Column 10 do this by they are the extraction medium 42 between the sealing surfaces of the reservoir 26 and the outlet end 60 of the extraction media part 16 position and the volume of the fluid passage between the sealing surfaces and the extraction medium 42 minimize. In this configuration, the sample is essentially directly from the extraction medium 42 into the sample port of the LC system with a minimum fluid volume.

As in 3 and 5 to 6B shown, the extraction unit 10 in connection with a sample connection 68 be used as part of a sample analysis system. The sample connection 68 includes a near part 94 which is configured to seal into the extraction column 10 to intervene, and a distant part 96 , which is configured to intervene in a component of the sample analysis system. The near part 954 contains a first chamber 98 that is configured to the elongated shell 55 of the extraction media part 16 the extraction column 10 take. The distant part 96 contains a second chamber 100 , which is configured to be a component of a sample analysis system 92 take. The first chamber 98 is in fluid communication with the second chamber 100 via a fluid passage 104 with a small volume. In one embodiment, the fluid passageway 104 between the first chamber 98 and the second chamber 100 a volume of about 0.5 μl or less.

The first chamber 98 has an inner surface 106 On, the general in their size and shape of the outer surface 66 the shell of the extraction media part 16 the extraction column 10 equivalent. In one embodiment, the inner surface is 106 generally frusto-conical. The first chamber 98 also has a sealing surface 108 at its far end with the fluid passageway formed therein 104 on. The first chamber 98 has a depth D2 measured from the near end 110 the sample connection 68 to the sealing surface 108 on. The depth D2 of the first chamber 98 is smaller than the length L1 of the elongated shell 55 of the extraction media part 16 So if there is a force on the extraction column 10 is exercised, such as by a liquid supply 46 on the extraction column 10 applied force, the outlet end 60 and the outer surface 66 the elongated shell 55 with the inner surface 106 and / or the sealing surface 108 the first chamber 98 the sample connection 68 engage and form a seal with these. In one embodiment, this allows between the extraction column 10 and the sample port 68 The seal formed licks no more than about 10% of the volume of the extraction column 10 to the sample port 68 transferred sample. In another embodiment, the between the Flüssigkeitszuführeinrichtung allowed 46 and the extraction column 10 The seal formed licks no more than about 10% of the volume of the extraction column 10 to the sample port 68 transferred sample under injection pressures of between about 70 bar up to about 200 bar.

The near part 94 the sample connection 68 has an outer diameter smaller than the inner diameter of the open terminal end 70 the cuff 18 the extraction column 10 is. So if the extraction column 10 at the sample port 68 is positioned, surrounds the cuff 18 at least part of the outer surface 112 of the near part 94 the sample connection 68 , The near part 94 is from the far part 96 at a distance that hinders the formation of the seal between the outlet end 60 the elongated shell 55 and the inner surface 106 and / or the sealing surface 108 the first chamber 98 the sample connection 68 prevented.

The distant part 96 the sample connection 68 has an outside diameter that is generally larger than the outside diameter of the near part 94 is. The second chamber 100 the distant part 96 is configured to receive a component of the sample analysis system that can transmit an eluted sample to the analyzer. The second chamber 100 is configured to sealingly interfere with the component of the sample analysis system. In one embodiment, the second chamber 100 a thread on. In one embodiment, the component received in the second chamber is a tube 114 which can transfer an eluted sample to an analytical system. An example of an analytical system is a liquid chromatography system.

example 1

Extraction columns were prepared using extraction medium microcolumns from a porous monolith medium formed in a section of a PEEK tube. The porous monolith medium was prepared from a polymerization mixture with monomers ( 55 Percent by weight divinylbenzene and 45 weight percent ethylvinylbenzene) at a level of 37.5 weight percent of the mixture, an initiator (asobisisobutyronitrile (Dupont Vazo-64)) at a level of 1.75 weight percent by weight of the monomers and a porogen at a level of 62; 5 weight percent of the mixture vorbreitet. The Polymerization mixture was injected into the lumen of a portion of an activated tube and polymerized for 18 hours at 76 degrees Celsius.

The inner surface of the PEEK tube was activated prior to injection of the polymerization mixture so that the porous extraction medium becomes covalently bound to the inner surface of the tube during polymerization. In order to activate the inner surface of the tube, the PEEK tube was filled with a reaction solution containing a solvent, acetonitrile or propionitrile (alkylnitrile derivatives) and an azo-class initiator such as Vazo-64 or Vazo-55 (Wakko V). 70) at a concentration of 1-10%. The filled PEEK tube was then heated to at least 80% of the 10 hour half life temperature, for example, with Vazo-55 having a 10 hour degradation hold time at 55 degrees Celsius. The reaction was allowed to proceed for 30 minutes. Then, the reaction solution was replaced with a fresh reaction solution, and the reaction was allowed to proceed for another 30 minutes. Then, the reaction solution was removed from the tube, and the tube was dried with a nitrogen stream. After drying, the polymerization mixture was injected into the lumen of the activated tube.

After the porous extraction medium was polymerized, the tube was cut to the desired length using an IDEX A-350 tube cutter.

A PEEK tube with internal diameters of 0.75 mm, 1 mm, 0.5 mm and 0.4 mm was used. The porous monolith medium was formed in the lumen of the tube and the tube was cut with an IDEX A-350 tube cutter into lengths 2mm, 4mm, 5mm or 6mm long to form the extraction medium microcolumns. The extraction medium microcolumns were inserted into the cavity of the extraction media portion of a column body to form extraction columns having the dimensions given in Table 1. The extraction columns with the dimensions of extraction medium microcolumns 2, 3 and 4 from Table 1 were tested with bacterial samples as described below. Extraction Medium Micro Column No. Inner tube diameter (mm) Pipe length (mm) Tube volume (μl) 1 0.75 2 0.9 2 1 2 1.6 3 1 4 3.1 4 0.4 6 0.8 5 0.5 4 0.8

Test samples were prepared from extracts of Eschericia coli. The extract was prepared by lysis of E. coli in a solvent containing 50% by volume of formic acid (AS) and 25% by volume of acetonitrile (ACN). The AS and ACN concentrations were adjusted to 37.5% by volume ACN and 25% by volume AS. The protein concentration in the test samples was 2 mg / ml and 3 mg / ml as determined by BCA analysis. Test samples were diluted and either 0.5 μg or 1 μg protein was applied to the extraction columns.

Before the test samples were applied to the extraction columns, the porous monolith medium was wetted with 50 μl of 0.2% ASA ACN. The porous monolith medium was then equilibrated with 50 μl of a solution containing water and 0.2% by volume of AS. Samples containing a total of 0.5 μg or 1 μg of protein in volumes of 50 μl to 100 μl were forced through the porous monolith medium in the extraction column. The flow from the samples was collected for analysis. The porous monolith medium was washed with 50 μl of a solution containing water and 0.1% by volume of AS. Protein samples were eluted from the porous monolith medium with 10 μl of a solution containing water, 60% by volume ACN and 0.2% by volume AS. The eluted proteins were collected for analysis.

The flow (DF) samples and the eluted samples were analyzed for protein concentration. In some of the results, the eluates of several samples (3-5) were combined and partially dried to approximately 1/3 of their original volume before being analyzed, with the amount of protein per extraction column later calculated from the combined results. Protein concentration was determined using a nanodroplet BCA assay (Thermo Fisher Scientific). The bovine albumin standard curves were prepared using the same AS and ACN concentrations as for the DF and eluate samples. The results are shown in Table 2.

The DF and eluate samples were then analyzed by SDS-PAGE. The flow and eluate from 5 samples were combined and dried prior to analysis. The samples were provided on a gel with a gradient of 4% to 20%. The gel was then silver stained and analyzed. The results (not shown) were consistent with protein determination results. Microcolumn No. Pipe length (mm) Tube volume (μl) protein samples Sample volume (μl) Eluted (μg) Yield% (eluted μg / applied μg) Eluted (μg) / peak volume (μl) 2 2 1.6 0.55 50 0.35 65% 0.22 3 4 3.1 0.55 50 0.34 62% 0.11 2 2 1.6 1.67 100 0.8 48% 0.51 3 4 3.1 1.67 100 0.56 34% 0.18 4 6 0.8 1 50 0.27 27% 0.36

Example 2

Samples containing a mixture of proteins including insulin, ubiquitin, myoglobin, carbonic anhydrase and bovine albumin were prepared in a solution containing 37.5% by volume of ACN and 25% by volume of AS. The total protein amount in the samples was 0.5 μg. The samples were tested on an extraction column prepared as described above for Example 1 and having the dimensions of Extraction Column # 5 in Table 1. The porous extraction media from the extraction columns were wetted and equilibrated, the samples were applied and the porous extraction media in the extraction columns were washed as described above for Example 1. In an initial round of tests, samples were eluted manually, with the pressure observed inside the extraction tip. It was found that 50 μl samples could be pushed through the extraction column in less than 15 seconds, using as in 7 shown, the back pressure generated in the tip can be used to determine the time at which all the liquid has passed through the porous extraction medium.

Then, a sampler was used to pass liquids through the extraction column. The force exerted by the liquid delivery means was measured to identify the force needed to form a seal between the liquid delivery means and the extraction column and between the extraction column and the sample port of a liquid chromatography system. It is of great importance that a seal be provided between these components under the high operating pressures of liquid chromatography systems, which usually range from about 70 bar to about 200 bar. It was found that a force of 30 N applied to the extraction column by the liquid supply means was sufficient to form a seal between the components at a back pressure of 210 bar.

In another test, during the elution step, the extraction column was plugged into a sample port of a liquid chromatography system and eluted with an aqueous solution containing 50% by volume ACN and 0.2% by volume AS at a rate of 1.5 μl / min. 8A is a chromatograph obtained from the eluted sample of the protein mixture. 8B is a mass spectrograph obtained from the eluted protein mixture. 9 is a chromatograph obtained from the eluted insulin sample. 10 is a chromatograph obtained from the eluted Ubiquitin sample. 11 is a chromatograph obtained from the eluted myoglobin sample. 12 is a chromatograph obtained from the eluted carbonic anhydrase sample.

Multiple column arrangements

As in 13A - 13E As shown, certain embodiments of the invention relate to a sample cleaning apparatus having a multi-tip plate 200 , The plate 200 includes a variety of extraction columns 10 , The extraction columns 10 may be configured substantially similar to one or more of the embodiments described herein. For example, every extraction column 10 a pillar body 12 comprising: a reservoir portion 14 who has an inlet 24 and a reservoir (as in 13A - 13B shown); and an extraction media part 16 , which is an elongated shell with an outlet end with an opening 49 (as in 13C shown) and an inlet end in fluid communication with the reservoir, wherein an extraction medium in an inner cavity of the elongated shell is arranged. The opening 59 can with the inlet 24 be aligned. In at least one embodiment, the reservoir part 14 truncated cone in an extraction media section 16 go over or rejuvenate to this (eg with a suitable convergence angle).

In at least one embodiment, the plate comprises 200 a connection component 202 that is between the multitude of extraction columns 10 or corresponding reservoir parts 14 extend (eg, adjacent to corresponding inlets 24 the same). In at least one embodiment, the connection component 202 an upper plate surface part 204 and optionally a shoulder 76a that depends on the size of the plate 200 , the connection component 202 or the upper plate part 204 protrudes outward.

The plate 200 may also include at least one cuff, that of the cuff 18 is similar (see eg 1 - 3 and 5 ). The cuffs may be different from the reservoir part 14 one or more of the extraction columns 10 extend. Alternatively, the sleeves may be off the circumference of the plate 200 , the connection component 202 or the upper plate part 204 extend. As described herein, the cuff may extend in the same direction as the elongated sheath and / or may include a terminal end extending from the outlet end of the elongate sheath (or outlet port) 59 ) is spaced. The termination end of the collar portion may extend axially toward or beyond the outlet end of the elongated shell (or outlet port) 59 ) beyond a distance sufficient to allow contact of the outlet end of the elongate sheath (or the outlet opening) 59 ) with any part of a second (adjacent) extraction column 10 to prevent.

As shown for this embodiment, the plurality of extraction columns may be in one or more (linear) rows 206 be arranged. For example, the center of each extraction column 10 or the corresponding inlet 24 and / or the opening 59 essentially with the other extraction columns 10 or the corresponding inlets 24 and / or openings 59 in the same row 206 be aligned (stand in a linear relationship with these). As shown, each row includes 206 twelve (12) extraction columns 10 , In alternative embodiments, each row 206 also two, three, four, five, six, seven, eight, nine, ten, eleven or more extraction columns 10 include. Certain embodiments may include at least four, at least eight, or at least twelve extraction columns 10 per row 206 include.

In some embodiments, the centers of each adjacent extraction column 10 (in every row 206 ) or the corresponding inlets 24 and / or openings 59 the same be spaced by a predetermined (uniform) distance. For example, as in 13D shown the centers of the neighboring extraction columns 10a and 10b (in line 206 ) be spaced apart by a distance T1. Accordingly, adjacent extraction columns 10b and 10c be spaced apart by the distance T1. Accordingly, further extraction columns 10 similarly from adjacent extraction columns 10 be spaced. Accordingly, in at least one embodiment, the centers of all adjacent extraction columns 10 in every row 206 or the corresponding inlets 24 and / or openings 59 be spaced from each other by a predetermined, uniform distance T1.

In some embodiments, the (uniform) distance T1 may be between about 2 mm and about 13 mm, between about 2.25 and about 12.73 mm, or between about 4.5 mm and about 9 mm. In certain embodiments, the (uniform) spacing may be about 4.5 mm or about 9 mm. It should be noted that the center-to-recess center distance in a standard 96-well plate (or similar sized and / or configured product such as a MALDI plate) may be about 9 mm. Similarly, the center-to-recess center distance in a standard 384-well plate (or similar sized and / or configured product, such as a MALDI plate) may be about 4.5 mm. Accordingly, at least one embodiment may comprise a plurality of extraction columns 10 with corresponding outlet openings 59 contain, with the centers of adjacent extraction columns 10 or corresponding outlet openings 59 they are regularly spaced so that they correspond to the regularly spaced sample receiving areas (eg, pits or dots) on a sample analysis plate or other product or device (eg, sample strips or tube strips).

As further shown for this embodiment, the plurality of extraction columns 10 also be arranged in an array. The arrangement may be at least two rows 206 of extraction columns 10 include. For example, the arrangement may be a first row 206a and an adjacent (aligned and / or parallel) second row 206b include. The arrangement can also be a third row 206c in Neighborhood to a second row 206b and a fourth row 206d in the neighborhood of the third row 206c include. Accordingly, as shown, the arrangement comprises four (aligned and / or parallel) rows 206 , However, in other embodiments, the assembly may also include (at least) three, (at least) four, (at least) five, (at least) six, (at least) seven, (at least) eight or more rows 206 of extraction columns 10 include.

In an alternative embodiment, the assembly may comprise a single row of extraction columns 10 include. For example, as in 13C shown the row 206a an arrangement 208 form the twelve extraction columns aligned in a linear relationship 10 includes. In alternative embodiments, the arrangement 208 a single row with (at least) two, (at least) three, (at least) four, (at least) five, (at least) six, (at least) seven, (at least) eight, (at least) nine, (at least) ten, ( at least) eleven or more extraction columns 10 include. It should also be clear that the shoulder 76a can extend around the (entire) arrangement (around the circumference of the same) around.

For example, in other embodiments, the array may include two rows of (at least) two, (at least) three, (at least) four, (at least) five, (at least) six, (at least) seven, (at least) eight, (at least) nine , (at least) ten, (at least) eleven or more extraction columns 10 as described above. Other embodiments may include (at least) three, (at least) four, (at least) five, (at least) six, (at least) seven, (at least) eight, (at least) nine, (at least) ten, (at least) eleven, (at least twelve) or more rows of (at least) two, (at least) three, (at least) four, (at least) five, (at least) six, (at least) seven, (at least) eight, (at least) nine, (at least) ten , (at least) eleven or more extraction columns 10 include.

Further embodiments may be at least one row with (at least) one extraction column 10 and a series with (at least) two extraction columns 10 include. Thus, certain embodiments may have an unequal number of extraction columns 10 in adjacent (or other) rows. At least one embodiment may include a first row with an extraction column 10 , a second adjacent row with two extraction columns 10 , a third adjacent row with three extraction columns 10 etc. include. Other embodiments may include a first row with two extraction columns 10 , a second, adjacent row with four extraction columns 10 , a third, adjacent row with eight extraction columns 10 etc. include.

In some embodiments, the centers of the extraction columns 10 in adjacent rows 206 or corresponding inlets 24 and / or openings 59 by a predetermined (uniform) distance from each other. For example, as in 13E shown the centers of the extraction columns 10a (the series 206a ) and 10d (the series 206b ) are spaced apart by a distance T2. Accordingly, adjacent extraction columns 10d (the series 206b ) and 10e (the series 206c ) spaced apart by a distance T2. Further extraction columns 10 can be similar to neighboring extraction columns 10 be spaced. Accordingly, in at least one embodiment, the centers of all adjacent extraction columns 10 in every adjacent row 206 or corresponding inlets 24 and / or openings 59 be spaced from each other by a predetermined, uniform distance T2.

In some embodiments, the distance T2 may be (substantially) equal to the distance T1. Alternatively, the distances T1 and T2 may be different. As further shown, the rows 206 be arranged parallel to each other and / or aligned. In addition, corresponding (eg, first, second, etc.) extraction columns may be used 10 in adjacent rows 206 also be arranged parallel to each other and / or aligned. Accordingly, the extraction columns 10 The array may be configured in a regularly spaced grid (eg, at least some of the evenly spaced sample receiving areas (eg, pits or spots) on a sample analysis plate (96-well, 384-well, etc.) or other product other device (eg sample strips (tube strips))). The assembly may provide predetermined clearances suitable for use in a general application.

At least one embodiment comprises a disposable, disposable solid phase extraction (SPE) tip assembly (or multi-tip plate) for use in purifying intact proteins from positive blood culture samples, especially after lysing human cells and centrifuging a microbial residue and before MS. Analysis of microbial proteins. Proteins from the microbial extract are bound to the SPE peak, washed and eluted (directly) into an MS analysis (eg MALDI). Accordingly, the predetermined distance of the plurality of Extraction columns in the assembly may be suitable for the application of eluted proteins directly to a MALI-TOF analysis plate, thereby avoiding intermediate pipette steps. By avoiding intervening pipette steps, the sample (extracted or purified) can maintain its purity and / or appropriate sterility levels for MS analysis (eg, MALDI).

The sample application workflow may be performed using a centrifugation or manifold (with a vacuum or positive pressure) or other device. In some embodiments, a sample purification device having a plurality of extraction columns disposed at a predetermined distance and a connection component extending between the plurality of extraction columns and holding the plurality of extraction columns therebetween at the predetermined distance may be provided with one (a plurality of channels ) Pipette (or one end thereof) or placed in a row (in fluid communication) with a sample source. The connection end of the pipette may be plugged into or otherwise connected to the extraction columns, and in particular at the inlet thereof. A sample pump or vacuum may force a sample through the extraction columns (eg, push or pull).

Certain aspects of the invention relate to systems that include embodiments of the extraction column in combination with a sample port having a first chamber configured to receive an elongated shell of the sample cleaning device or extraction columns thereof. The systems may optionally include a fluid delivery device, such as a ceramic probe. Optionally, the systems may also include an analysis device such as a mass spectrometer, a liquid chromatography device, or combinations thereof.

In one exemplary method, one or more (liquid) biological samples (as fluids) may be introduced (i.e., coated, placed, poured, dropped, dropped, etc.) into one or more extraction columns of a sample cleaning device. For example, the biological samples may be introduced into (corresponding) reservoir portions or (corresponding) reservoirs or first portions thereof (e.g., at the proximal end of the column body) of one or more extraction columns (eg, via an inlet thereof). The samples may pass (as fluids) through the second (funnel or frustoconical) portion of the reservoir and / or into the cavity of the extraction media portion (eg, at the far end of the column body). For example, a pipette (multiple channels), a sample pump, or a vacuum may be connected to the sample contaminant or one of its multiple extraction columns, and / or a sample may be forced through the extraction column (s).

The sample may pass (as a fluid) through the extraction medium disposed in the cavity (eg, by means of a pipette, a pump, a vacuum, etc.) and / or be extracted or purified thereby. The (extracted or purified) sample may exit (as fluid) from the cavity (or extraction medium located therein) and / or the extraction columns via the (lower) orifice. In at least one embodiment, the samples may be taken directly from the void (or extraction medium) and / or extraction columns into or onto sample receiving areas (e.g., pits or spots) on a sample analysis plate (96-well, 384-well, etc.) or another product or device (eg, sample strips) and the samples can then be analyzed.

The invention has been described with reference to one or more embodiments, without thereby limiting the scope of the invention. The person skilled in the art can create further advantages and modifications. Thus, the invention is not limited to the specific details of the apparatus and methods described herein and the examples described herein. Accordingly, the embodiments described herein may be modified without departing from the scope of the invention.

Those skilled in the art can make various changes and / or modifications to the inventive features described herein and realize additional applications of the principles described herein without, however, departing from the scope of the invention as defined by the claims. Thus, while various aspects and embodiments have been described herein, other aspects and embodiments are possible. Numerous methods and components similar or equivalent to those described herein may be used to form further embodiments of the invention.

It is further to be noted that the systems, processes and / or products according to certain embodiments of the invention also feature described for other embodiments (eg. Components, links, elements, parts and / or sections). The various features of certain embodiments can therefore be combined with each other and / or integrated into other embodiments of the invention. The mention of certain features in relation to a specific embodiment of the invention does not therefore mean that the features are limited to this specific embodiment. Rather, these features may be included in other embodiments without, therefore, the scope of the invention is abandoned. Unless specifically stated that one particular feature must be combined with another, any of the features mentioned may be combined with any other features of the same or different embodiment. Furthermore, well-known aspects of example systems, processes, products, etc., have not been described in detail herein, not to distract from the aspects of the exemplary embodiments, but such aspects are nevertheless contemplated herein.

The invention can also be realized by other embodiments without, therefore, departing from the scope of the invention. The described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the invention is defined by the following claims rather than by the foregoing description. Each of the claims and the elements recited therein may be combined with any other claims and / or the elements recited therein in any combination or dependency, without regard to the stated dependency of the claims. Certain embodiments and details are described and shown herein, it being understood by those skilled in the art that various changes may be made to the methods and apparatus described and illustrated without departing from the scope of the invention as defined by the claims, which may include many others Changes within the meaning and equivalency of the claims.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 7922908 [0080]
• US 5334310 [0082]
• US 5633290 [0082]
• US Pat. No. 6783672 [0083]

Claims (15)

Sample cleaning device comprising: a plurality of extraction columns disposed therebetween at a predetermined interval, and a connection component extending between the plurality of extraction columns, the connection component holding the plurality of extraction columns with the predetermined distance therebetween; wherein the plurality of extraction columns each comprise a column body having a reservoir portion and an extraction media portion, wherein the reservoir portion includes an inlet and a reservoir, and wherein the extraction media portion includes an extraction medium disposed within an elongate shell having an interior surface defining a cavity having an inlet end in fluid communication with the reservoir and an outlet end remote from the inlet end. The sample cleaning apparatus of claim 1, wherein the plurality of extraction columns are arranged in a linear row. A sample cleaning device according to claim 2, wherein the linear array contains at least four extraction columns, preferably at least eight extraction columns, and more preferably at least twelve extraction columns. A sample cleaning apparatus according to claim 1 or 2, wherein the centers of the respective outlet ends of adjacent extraction columns are spaced apart by a uniform distance. The sample cleaning apparatus of claim 4, wherein the uniform spacing is between about 2 mm and about 13 mm, preferably between about 2.25 mm and about 12.73 mm, more preferably between about 4.5 mm and about 9 mm, and even better about 4.5 mm or about 9 mm. The sample cleaning apparatus of any of claims 1-5, wherein the plurality of extraction columns are arranged in an array. The sample cleaning apparatus of claim 6, wherein the assembly comprises at least two rows of extraction columns, preferably at least four rows of extraction columns and more preferably at least eight rows of extraction columns. A sample cleaning device according to claim 7, wherein the rows are arranged parallel to each other. A sample cleaning apparatus according to claim 7, wherein each row comprises at least two extraction columns, preferably at least four extraction columns, more preferably at least eight extraction columns and more preferably at least twelve extraction columns. A sample cleaning apparatus according to any one of claims 6-9, wherein the centers of the respective outlet ends of adjacent extraction columns are spaced apart by a uniform distance. The sample cleaning apparatus of claim 10, wherein the uniform spacing is between about 2 mm and about 13 mm, preferably between about 2.25 mm and about 12.73 mm, more preferably between about 4.5 mm and about 9 mm, and even better about 4.5 mm or about 9 mm. A sample cleaning device according to any one of claims 6-11, wherein the array comprises at least four extraction columns arranged in a 2 x 2 array, preferably at least six extraction columns arranged in a 3 x 2 array, more preferably at least eight extraction columns located in More preferably, at least nine extraction columns arranged in a 3 × 3 array, more preferably at least ten extraction columns arranged in a 5 × 2 array, more preferably at least 12 extraction columns, are arranged More preferably, at least 16 extraction columns arranged in a 4 × 4 array or in an 8 × 2 array, more preferably at least 18 extraction columns, are arranged in a 4 × 3 array or in a 6 × 2 array in a 6x3 array or in a 9x2 array, more preferably at least 20 extraction columns arranged in a 5x4 array or in a 10x2 array More preferably, at least 24 extraction columns arranged in a 6x4 array or in an 8x3 array or in a 12x2 array, more preferably at least 48 extraction columns arranged in a 12x4 array and more preferably at least 96 extraction columns arranged in a 12x8 array. A sample cleaning device according to any one of claims 1-12, wherein the extraction medium is selected from the group consisting of: a solid phase extraction medium, which is preferably a porous solid polymer defining a polymer monolith, preferably the extraction medium is contained in a generally cylindrical shell which is open at both ends and has an outer surface with a diameter corresponding to the diameter of the inner surface of the cavity of the elongate shell, and wherein the shell preferably consists of a polyethylene ether ketone, fused silica, fluoropolymers and cyclic olefin copolymers, and a plurality of non-porous Globules, porous beads or a mixture of these. The sample cleaning apparatus of any one of claims 1-13, wherein the extraction medium extends substantially to the outlet end of the elongate shell. A system for extracting a desired fraction from a liquid, the system comprising: an extraction column assembly according to any one of claims 1-14, and a sample analysis plate having a plurality of sample receiving regions, wherein the sample receiving areas are spaced such that the position of each of the sample receiving areas corresponds to a position of a corresponding outlet end of the plurality of extraction columns.

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