CHROMATOGRAPHY DEVICES AND METHODS

Abstract

A device for automated or manual separations of complex chemical or biological mixtures and methods of use are described. The device is a chromatography pipette tip or column having a size exclusion media therein and held in place by a hydrophilic filter at the distal end of the pipette tip and an optional removable or pierceable barrier at the proximal end of the pipette tip. The size exclusion media is wetted by simply placing the tip into solvent and allowing capillary action to flow through the filter and into the SEC resin. The solvent is then allowed to gravity drain from the pipette tip, allowing the size exclusion media to reproducibly pack into a homogenous gel. Separations can then be performed with the gelled size exclusion media.

Claims

1. A method of preparing a chromatography column, comprising: a) inserting a chromatography column into a container of polar fluid, wherein said chromatography column contains a hydrophilic filter and a dry chromatography resin above said hydrophilic filter, wherein said hydrophilic filter is positioned at a distal end of said chromatography column so as to have <50 l dead volume below said hydrophilic filter; b) wetting said hydrophilic filter by capillary action pulling said polar fluid up through said hydrophilic filter and into said dry chromatography resin to form swelled resin; c) equilibrating said swelled resin in said polar fluid for a time sufficient to form a homogenous wetted resin (gel); d) optionally adding a small volume of said polar fluid to a top end of said chromatography column; and e) draining any excess polar fluid out of said distal end of said chromatography column.

2. The method of claim 1, wherein said chromatography column is a pipette tip.

3. The method of claim 1, wherein step d) is performed on a robotic liquid handling platform by a robotic liquid handler.

4. The method of claim 1, wherein said polar fluid is an aqueous buffer having a pH of 3-12 or said polar fluid is phosphate buffered saline.

5. The method of claim 1, wherein said hydrophilic filter is a porous plastic frit that has been modified with a surfactant, polysorbate, polyethylene glycol or polyvinyl alcohol or combinations thereof.

6. The method of claim 1, wherein said hydrophilic filter is a mesh screen, porous polymeric material, porous glass, porous membrane, glass microfibers, glass capillaries, or porous ceramic.

7. The method of claim 1, wherein said dry chromatography resin comprises agarose, sepharose, polyacrylamide, dextran, dextran crosslinked with epichlorohydrin, polystyrene, polyacrylate, cellulose, or a combination thereof.

8. The method of claim 1, wherein said draining step comprises gravity flow or by applying positive pressure to said top end of said chromatography column.

9. A method of chromatographic analyte separation, comprising: a) inserting a chromatography pipette tip into a container of polar fluid, wherein said chromatography pipette tip is a pipette tip containing a hydrophilic filter and a dry chromatography resin above said hydrophilic filter, wherein said hydrophilic filter is positioned at a distal end of said pipette tip so as to have <10 l dead volume below said hydrophilic filter; b) wetting said hydrophilic filter by capillary action pulling said polar fluid up through said hydrophilic filter and into said dry chromatography resin to form a swelled resin; c) equilibrating said swelled resin in said polar fluid for a time sufficient to form a homogenous wet resin (gel); d) optionally adding a small volume of said polar fluid to a top end of said chromatography pipette tip; e) draining any excess polar fluid out of said distal end of said chromatography pipette tip; f) adding a sample solution in said polar fluid to the top end of said chromatography pipette tip, said sample solution containing two or more analytes; g) allowing said sample solution to load into said gel by gravity flow or by applying positive pressure; h) optionally adding a wash solution (void volume) to the top end of said chromatography pipette tip, and allowing said wash solution to flow via gravity or positive pressure; i) adding an elution solution (collection fraction) to the top end of said chromatography pipette tip; and j) collecting one or more elution fractions having separated analytes via gravity flow or positive pressure in one or more containers.

10. The method of claim 9, further comprising the step of analyzing said separated analytes using enzymatic assay, immunoassay, affinity assay, refractive index detector, UV-Vis detector, viscometer, multi-angle light scattering detector, gel electrophoresis, mass spectrometer and combination(s) thereof.

11. The method of claim 9, wherein a pipetting aid is used in adding steps d), f), h) and i) and wherein said pipetting aid is a hand-held pipettor, a syringe, or a robotic liquid handler that directly attaches to a top end of said chromatography pipette tip, or indirectly attaches thereto through the use of an adaptor or a top-pipette-tip.

12. The method of claim 11, wherein said pipetting aid is a robotic liquid handler and steps d) through j) are performed on a robotic liquid handling platform.

13. The method of claim 9, wherein said sample solution, said wash solution and said elution solution are buffered to a pH of 3-12.

14. The method of claim 9, wherein adding said elution solution step i) or adding said wash solution step h) or both are repeated 2 to 5 times.

15. The method of claim 9, wherein said hydrophilic filter is selected from a screen, porous frit, porous polymeric material, porous membrane, porous glass, sintered glass, glass microfibers, glass capillaries, or porous ceramic.

16. The method of claim 9, wherein said dry chromatography resin comprises agarose, sepharose, polyacrylamide, dextran, dextran crosslinked with epichlorohydrin, polystyrene, polyacrylate, cellulose, or a combination thereof.

17. A chromatography pipette tip, comprising: a pipette tip comprising a hollow tube with an open top end over a conical section and a bottom end that is smaller than said top end; said top end sized to fit over a separate pipette head; an exterior surface of said pipette tip above said conical section having an annular ridge or three or more vertical fins; a hydrophilic filter and a dry chromatography resin or media above said hydrophilic filter; said hydrophilic filter positioned at said bottom end of said pipette tip so as to have less than 10 l dead volume inside said pipette tip and below said hydrophilic filter; and a barrier at said top end to retain said dry chromatography resin.

18. The chromatography pipette tip of claim 17, wherein said hydrophilic filter is a porous plastic frit, a mesh screen, porous polymeric material, porous membrane, porous glass, glass microfibers, glass capillaries, or porous ceramic.

19. The chromatography pipette tip of claim 17, wherein said dry chromatography resin is a dextran crosslinked with epichlorohydrin based size exclusion resin.

20. A kit comprising a box with an array of holes therein, the chromatography pipette tip of claim 17 reversibly inserted into each hole of said array of holes, plus instructions for use of said chromatography pipette tip.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0094] FIG. 1A-B display a chromatography pipette tip having 180 mg of Sephadex G15 resin before swelling (1A) and after swelling (1B).

[0095] FIG. 2A-F display an embodiment of a chromatography pipette tip going through the method of swelling to form the gel, loading a sample solution, and separating compounds by size using e.g., size exclusion chromatography or SEC.

[0096] FIG. 3 illustrates the method of preparing and using a chromatography pipette tip for separation of target compounds in a sample.

[0097] FIG. 4 is an image of a chromatography pipette tip at various points in the method illustrated in FIG. 3.

[0098] FIG. 5A-B show the chromatograms for fentanyl and carboxy-THC from the loading solvent in Example 2.

[0099] FIG. 6 Gel electrophoresis separation result for Example 3.

DETAILED DESCRIPTION

[0100] FIG. 1A displays a chromatography pipette tip 100 with dry resin (or media) 102 loosely contained therein, and FIG. 1B displays the same chromatography pipette tip 100 after the resin 103 has been prepared. A hydrophilic filter 104 located at the distal narrow end prevents loss of the resin 102 and facilitates the flow of fluid into the tip via capillary action.

[0101] In FIG. 2 we discuss particular examples of resins and filters, but these are exemplary only and the devices and methods are not so limited. FIG. 2A, the chromatography pipette tip contains a porous glass microfiber filter at the wide bore narrow end of the tip, and 70 mg of Sephadex G15 resin. FIG. 2B shows this tip placed inside of a well containing PBS buffer, and the resin was completely swelled in about 3 min via capillary action. FIG. 2C shows the tip with 50 L of PBS buffer added to the top of the bed of gel. After allowing gravity drainage for this additional buffer, 75 L of a green dye solution was added to the top of the chromatography pipette tip in FIG. 2D. After about 3 min of gravity flow, the sample was absorbed into the resin in FIG. 2E. Elution buffer was added to the top of the chromatography pipette tip, and after collecting about 100 L of buffer, separation of blue dye (FD&C blue 1) from yellow dye (FD&C yellow 5) shown inside of the chromatography pipette tip in FIG. 2F. The 2 dyes were thus readily separated and collected into separate wells for further analysis.

[0102] The basic method of preparing the resin in the chromatography pipette tip for chromatography includes the steps of: [0103] 1) Placing the chromatography pipette tip into a container containing a polar fluid that is compatible with both the resin and the analytes to be separated. The level of fluid is lower than the height of the tip, so fluid may only enter via the delivery end; [0104] 2) Allowing the solvent to flow upwards into the chromatography pipette tip and fully wet and swell the resin; [0105] 3) Optionally adding a small volume of said polar fluid to the top of the chromatography pipette tip and allowing any excess to drain, thus producing a homogeneous gel-like column.

[0106] Steps 2 and 3 may be performed in a predetermined amount of time, which depends on the type and amount of resin and also on the fluid used. For amounts less than 100 mg, the swelling is almost immediate after capillary action fills the bed, which takes less than about 5 min. It is recommended to add a small volume of buffer to the top of the bed to ensure the resin is completely wetted and swollen, but it is not essential and depends on the application. This additional amount is recommended to be about 50 L or more, depending on the resin mass and composition, and only adds a couple of minutes to the overall equilibration time and gravity flow to make the chromatography pipette tip. It is also possible to control this flow using an attached pipettor or other positive pressure device.

[0107] The tip is then ready for sample loading once the excess fluid is drained.

[0108] Sample loading can be performed by adding the sample onto the top of the chromatography pipette tip. This step can be performed using either manual or robotic pipetting techniques.

[0109] The volume of sample loading is dependent on the dead volume of the resin column inside the pipette tip. As shown in FIG. 1B, the column of homogenous gel formed in the chromatography pipette tip is about 4.5 cm; however, it can be larger or smaller depending on the initial amount of dry chromatography resin and the size of the pipette tip being used. In some embodiments, the volume of sample loading will range between about 10 L to about 500 L, preferably in the range of 50 L to 300 L, or the range of 50 L to 150 L, or the range of 200 L to 300 L, or the range of 200 L to 250 L.

[0110] If too dilute the sample may first be concentrated, or the sample may be added to the tip in multiple doses to obtain complete loading.

[0111] Once the sample is fully loaded into the resin, the sample can be washed as needed, or otherwise treated, and then the analytes eluted. Wash and elution steps can be repeated 2 to 5 times as needed. As each target compound or analyte is eluted from the bottom of the chromatography pipette tip, it can be collected in one or more containers, such as wells, for further analysis.

[0112] The collected analytes can be analyzed by any method, including at least one of refractive index detector, UV-Vis detector, viscometer, multi-angle light scattering detector, gel electrophoresis and/or mass spectrometer, enzymatic assay, immunoassay, affinity assay, and the like. In some embodiments, a detector is used in tandem to analyze a sample.

EXAMPLE 1

[0113] The separation capabilities of the presently described device was evaluated using size exclusion media with a low molecular weight cutoff of 1500 Da, using a hand-held pipettor as the pipetting aid. The sample solution contained a mixture of FD&C red 40 and red 3 dyes (small molecules with molecular weights of 496 and 880 g/mol, respectively), and a blue stained protein standard (molecular weights 10-250 kDa).

[0114] The process for preparing the chromatography pipette tip and performing the separation is illustrated in FIG. 3 and real images of the chromatography pipette tip at different points in this process are shown in FIG. 4. The sample wells and pipetting aids are omitted for simplicity.

[0115] The chromatography pipette tip 310 utilized Sephadex G15resin 311 having a molecular weight cutoff of 1500 Da. About 180 mg of the Sephadex G15 resin was placed in a pipette tip having a porous hydrophilic frit (20 m and made of polyethylene modified with surfactants) 312 at the distal, narrow end of the pipette tip, as shown in step 301. Annular ridge 307 at the exterior of the pipette tip and three vertical ridges 308 are also shown.

[0116] The frit is friction fitted into the narrow end of the tip, with the frit pressed to the bottom so that the frit is flush or near flush with the end of the pipette tip. We are reliably able to obtain a tip with less than 20 l of dead volume using this method and this is low enough to readily allow wicking, but if a filter was placed over the tip (rather than inside) in a reliable leak proof manner, this could be reduced to zero. Use of a spherical filter or frit may also greatly reduce the dead space volume, and may be of simple manufacture.

[0117] The chromatography pipette tip was positioned into a well containing phosphate buffered saline (PBS) solution. In a matter of a few seconds, the buffer migrated up through the frit and was absorbed into the resin. The buffer continued to be absorbed into the resin via capillary action, and in a few minutes was completely absorbed and the resin swollen. A small volume of PBS was added to the top and allowed to gravity flow out, and this resulted in a homogenous gel-like resin 313 within the chromatography pipette tip, as shown in Step 302. It is also possible to use positive pressure from the pipettor to push the PBS solution through the wetted material and out of the pipette tip to form the final resin, however it is not necessary.

[0118] A volume of 0.2 mL of sample solution 314 was dispensed slowly onto the top of the size exclusion resin, and loaded into the gel using gravity flow, per step 303. The smaller red dyes 315 were retained by the size exclusion resin and remain at the top while the larger proteins 316 began to move through the column and separate from the red dyes, as shown in step 304. After loading the sample, the waste solvent reservoir was replaced with a sample vial. Then, 0.3 mL (300 L) of the PBS solution 317 was added to the top of the resin, and passed through the resin using positive pressure from the handheld pipettor (not shown). The smaller red dyes were retained by the size exclusion resin and remained at the top of the column bed while the larger, blue stained proteins were able to exit the chromatography pipette tip and be collected in the sample vial as the target compounds eluted from the gel.

[0119] FIG. 4 displays photographs of the chromatography pipette tip at various points in this separation process, including step 306 collection of target compounds. The targeted compounds, blue stained proteins, are clearly visible in the sample vial 401, while the red dyes remain at the top 402.

EXAMPLE 2

[0120] This example describes methods for using the chromatography pipette tip to remove proteins and collecting small drugs from a sample for chemical analysis by liquid chromatography with tandem mass spectrometry (LC/MS/MS). To 25 L of urine (containing a drug mixture at 10 ng/mL), 25 L of a commercial enzyme and buffer solution for hydrolyzing glucuronide conjugated drugs (B-glucuronidase) was added, along with 25 L of water (to help minimize inhibitors). The solution was held at room temperature for 10 minutes to complete the hydrolysis step.

[0121] While hydrolysis was ongoing, a chromatography pipette tip containing 50 mg G-15 resin with a hydrophilic frit, was inserted into a well of PBS buffer and allowed to swell. After a couple of minutes, the PBS buffer flowed into the tip and the sorbent was swollen. Then 25 L of PBS was added to the top and allowed to gravity drain. The wetted resin appeared completely homogenous and took less than 5 minutes to make.

[0122] The sample was added to the top of the chromatography pipette tip and allowed to gravity flow thereinto. Then 75 L of PBS buffer (wash solution) was added and allowed to gravity flow through the tip for sample loading. Afterwards, 250 L of 20% methanol in water (elution solution) was added to the top of the chromatography pipette tip and collected at the bottom via gravity flow, and this was analyzed as fraction 1. A second aliquot of 250 L of 20% methanol was added to the top and collected via gravity flow, and this was analyzed as fraction 2.

[0123] The loading buffer (wash solution) and the elution fractions were injected into a LC/MS/MS system for analysis. Negligible amounts of drugs were detected in the 75 L of PBS loading buffer, but fraction 1 contained the majority of drugs (over 70% recovery of over 45 drugs of abuse were detected). The major metabolite of tetrahydrocannabinol (THC), carboxy-THC, was not detected in the fraction 1. Fraction 2, however, contained a large amount of carboxy-THC. FIG. 5A and 5B show the chromatograms for fentanyl and carboxy-THC from the loading solvent, fraction 1 and fraction 2, respectively.

EXAMPLE 3

[0124] The method of Example 2 was used herein, except the resin was replaced with 50mg Sephadex G-10. Also, instead of elution with 20% methanol, elution was done using PBS buffer. The same protein and small molecule drug samples were used in this example.

[0125] A sample volume of 75 L was added to the bed, and the well collected any excess PBS buffer as the sample loaded. A loading volume of 75 L of PBS buffer was added to the wetted resin, and the gravity flow of fluid was collected into a second well. This represents the dead volume of compounds that are greater than the molecular weight cutoff (meaning these compounds would pass freely through the resin without partitioning into the pores). Then 3 subsequent aliquots of 75 L of PBS buffer was added to the chromatography pipette tip and collected as fractions in wells 3, 4 and 5.

[0126] The 5 eluates were loaded onto a protein gel and separated by gel electrophoresis. The first lane, which should contain only PBS buffer as long as the dead volume of the column bed was at least 75 L, did not contain any protein as shown in FIG. 6. The second lane was expected to contain protein since its molecular weight is much greater than 700 Da, the molecular weight cutoff of the G-10 resin. A dark blue band appeared in the protein gel for well 2, which shows the majority of protein passed through in this loading buffer.

[0127] The third well appeared to contain a very small amount of protein, which is barely noticeable in the gel. The fourth and fifth wells show no protein is present. The small drugs were found in wells 3 and subsequent fractions, and therefore the use of this chromatography pipette tip is suited for removing protein from samples being prepared for analysis of drugs and other small molecules.

[0128] While multiple embodiments and examples are disclosed herein, still other embodiments will become apparent to those skilled in the art from the above detailed description and drawings. As will be apparent, certain embodiments, as disclosed herein, are capable of modifications in various aspects, without departing from the spirit and scope of the claims as presented herein. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not restrictive.

[0129] The following references are incorporated by reference in their entirety for all purposes.

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