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Numéro de publicationUS20060083663 A1
Type de publicationDemande
Numéro de demandeUS 11/184,170
Date de publication20 avr. 2006
Date de dépôt19 juil. 2005
Date de priorité19 oct. 2004
Autre référence de publicationEP1649911A2, EP1649911A3, US7563410, US20060083659
Numéro de publication11184170, 184170, US 2006/0083663 A1, US 2006/083663 A1, US 20060083663 A1, US 20060083663A1, US 2006083663 A1, US 2006083663A1, US-A1-20060083663, US-A1-2006083663, US2006/0083663A1, US2006/083663A1, US20060083663 A1, US20060083663A1, US2006083663 A1, US2006083663A1
InventeursRobert Ricker
Cessionnaire d'origineRicker Robert D
Exporter la citationBiBTeX, EndNote, RefMan
Liens externes: USPTO, Cession USPTO, Espacenet
Fluid processing devices with multiple sealing mechanisms and automated methods of use thereof
US 20060083663 A1
Résumé
Methods of and an apparatus formed by a combination of components for automated fluid processing through use of structures integrated within plates or cartridges receivable by autosamplers, that include at least one inlet, at least one outlet, and stationary phase material disposed therebetween. An enclosed fluid processing pathway is formed by automatically connecting an autosampler fluid transport connector, such as an autosampler needle, to each of the inlet(s) and outlet(s) and simultaneously injecting a fluid to be processed into the inlet(s) and extracting processed fluid from the outlet(s).
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Revendications(22)
1. An automated method of processing a fluid, comprising the steps of:
providing a structure having at least one inlet in fluid communication with at least one outlet, and at least one stationary phase disposed downstream from the at least one inlet and upstream from the at least one outlet such that fluid injected through the at least one inlet traverses the at least one stationary phase prior to transport to the at least one outlet;
automatically connecting one of a plurality of autosampler fluid transport connectors to each of the at least one inlet and the at least one outlet in a relatively pressure-tight, fluid communicable connection so as to form an enclosed fluid pathway through the structure; and
injecting a fluid through the at least one inlet and simultaneously extracting fluid from the at least one outlet.
2. The method of claim 1, wherein the structure is integrated within a plate or cartridge receivable by an autosampler.
3. The method of claim 1, wherein:
the structure is integrated within a plate; and
the enclosed fluid pathway between the at least one inlet and the at least one outlet traverses at least one volume disposed below a top surface of the plate.
4. The method of claim 1, further comprising the step of accessing via one of the autosampler fluid transport connectors at least one reservoir or liquid soluble sample position.
5. The method of claim 2, wherein each of the at least one inlet and the at least one outlet includes a sealing surface having an opening therein through which fluid may flow, said sealing surface at least partially conforming to the shape of the autosampler fluid transport connectors.
6. The method of claim 1, further comprising the steps of:
providing an additional structure having at least one inlet in fluid communication with at least one outlet, and at least one stationary phase disposed downstream from the at least one inlet and upstream from the at least one outlet such that fluid injected through the at least one inlet traverses the at least one stationary phase prior to transport to the at least one outlet;
automatically connecting one of the plurality of autosampler fluid transport connectors to each of the at least one inlet and the at least one outlet of the additional structure in a relatively pressure-tight, fluid communicable connection so as to form an enclosed fluid pathway through the additional structure; and
injecting the fluid extracted from the outlet of the first structure through the at least one inlet of the additional structure and simultaneously extracting fluid from the at least one outlet of the additional structure.
7. The method of claim 1, wherein:
the structure is integrated within a cartridge; and
the respective openings of the at least one inlet and the at least one outlet are each disposed on the same side of the cartridge.
8. An apparatus for use in automated fluid processing, comprising the combination of:
a plurality of fluid transport connectors of an autosampler;
a plate receivable by an autosampler, the plate having a structure integrated therein including at least one inlet in fluid communication with at least one outlet, each of the at least one inlet and the at least one outlet connectable in a relatively pressure-tight fluid communicable connection with one of the plurality of autosampler fluid transport connectors so as to form an enclosed fluid pathway through the structure; and
at least one stationary phase disposed downstream from the first inlet and upstream from the first outlet such that a fluid injected through the first inlet traverses the at least one stationary phase prior to transport to the first outlet.
9. The apparatus of claim 8, wherein at least a portion of the structure protrudes upwardly from a top surface of the plate.
10. The apparatus of claim 8, wherein the plate further includes at least one reservoir or liquid-soluble sample position accessible by one of the autosampler fluid transport connectors.
11. The apparatus of claim 8, wherein the at least one stationary phase comprises a plurality of distinct types of stationary phases disposed such that the injected fluid traverses the distinct types of stationary phases in a sequence.
12. The apparatus of claim 8, wherein each of the at least one inlet and the at least one outlet includes a sealing surface having an opening therein through which fluid may flow, said sealing surface at least partially conforming to the shape of the autosampler fluid transport connectors.
13. The apparatus of claim 8, wherein the structure further comprises:
a first chamber, defined in part by the at least one inlet, a second chamber, defined in part by the at least one outlet; and
at least one channel providing fluidic communication between the at least one chamber and the second chamber.
14. The apparatus of claim 8, further comprising at least one frit disposed within the structure at a position selected from the group consisting of between the at least one stationary phase and the at least one inlet, between the at least one stationary phase and the at least one outlet, and between two distinct types of stationary phase.
15. The apparatus of claim 8, further comprising at least one element simultaneously connectable in a relatively pressure-tight fluid communicable connection with another of the plurality of autosampler fluid transport connectors and selected from the group consisting of (a) an additional inlet in fluid communication with the at least one outlet and (b) an additional outlet in fluid communication with the at least one inlet.
16. The apparatus of claim 8, wherein the at least one inlet further comprises a plurality of inlets or the at least one outlet further comprises a plurality of outlets.
17. The apparatus of claim 8, wherein the inlet and the outlet are disposed at indexable positions of the plate.
18. An apparatus for use in automated fluid processing, comprising a combination of:
a plurality of fluid transport connectors of an autosampler;
a cartridge receivable by an autosampler, the cartridge having a structure integrated therein including at least one inlet in fluid communication with at least one outlet, each of the at least one inlet and the at least one outlet connectable in a relatively pressure-tight fluid communicable connection with one of the plurality of autosampler fluid transport connectors so as to form an enclosed fluid pathway through the structure; and
at least one stationary phase disposed downstream from the first inlet and upstream from the first outlet such that a fluid injected through the first inlet traverses the at least one stationary phase prior to transport to the first outlet.
19. The apparatus of claim 18, wherein the at least one stationary phase comprises multiple volumes of distinct types of stationary phases arranged such that the injected fluid traverses the multiple volumes in a sequence.
20. The apparatus of claim 18, further comprising at least one frit disposed within the structure at a position selected from the group consisting of between the at least one stationary phase and the inlet, between the at least one stationary phase and the outlet, and between two volumes of distinct types of stationary phase.
21. The apparatus of claim 18, wherein the respective openings of the inlet and the outlet are each disposed on the same side of the cartridge.
22. The apparatus of claim 18, wherein the cartridge further comprises:
an inlet chamber defined in part by the inlet;
an outlet chamber defined in part by the outlet; and
at least one channel providing fluidic communication between the inlet chamber and the outlet chamber.
Description
    CROSS-REFERENCE TO RELATED APPLICATIONS
  • [0001]
    This application is a continuation-in-part of, and claims the benefit of priority to, U.S. patent application Ser. No. 10/968,296, entitled “Fluid Processing Devices With Multiple Sealing Mechanisms And Automated Methods Of Use Thereof”, filed on 19 Oct. 2004, the contents of which are incorporated herein by reference in their entirety.
  • BACKGROUND
  • [0002]
    Many samples (e.g., of chemical, biological or environmental sample) cannot be injected into chromatographic, nuclear magnetic resonance, or other analytical equipment without prior offline fluid sample processing, including reaction, separation and/or fractionation processing. For example, pre-cleaning steps to remove interferences such as particulate matter and soluble contaminants from some samples is necessary prior to injection to avoid temporary or permanent system contamination. Thus labs dealing with such samples often spend 50-70% of their overall analysis time preparing samples for injection into these delicate instruments, including time for enriching and/or concentrating liquid-soluble samples.
  • [0003]
    After liquid-soluble samples have undergone reaction, separation (SPE) and/or fractionation processing, they may be injected into the chromatographic instruments manually or by means of autosamplers, any device that can automatically provide and/or retrieve samples to multiple containers in sequence or in parallel. Some autosamplers are additionally adapted to receive and/or grasp and manipulate sample containers, such as well plates, trays or individual vials containing the samples to be injected. Through alignment and movement (typically in multiple dimensions) of one or more injector syringes or probes with respect to indexed positions of the sample containers, metered aliquots may be withdrawn and injected into chromatographic instruments. The movement of one or more of the autosampler syringes is typically guided by a robotic controller executing user programming. Autosamplers that operate on stationary, indexed, multi-well trays or racks of sample vials, such as Series 1100 HPLC Autosamplers manufactured by Agilent Technologies of Palo Alto, Calif., and the Agilent 220 Micro Plate Sampler, are in wide use. Alternatively functioning autosamplers are also well known in the art, including those configured for use with rotatable trays.
  • [0004]
    In light of the cost of manual labor, it would, therefore, be desirable to automate reaction, separation and/or fractionation liquid-soluble sample processing in a relatively inexpensive manner. A system that accurately, robustly and reproducibly moves such fluid processing into an online, standard analytical workflow, leveraging conventional autosampling equipment, would be of great benefit. A further benefit would accrue to any instrument that enables HPLC (LC/MS) analysis of biological samples, which typically would require prior removal of both particulate matter and soluble contaminants.
  • SUMMARY
  • [0005]
    The present invention provides integrated structures that are preferably dimensioned or otherwise adapted for receipt and movement by liquid chromatographic (LC) and mass spectrophotometric (MS) autosampling equipment such as, for example, the Agilent instruments mentioned above. The structures may be integrated within individual cartridges, for example, or within devices such as modified well plates.
  • [0006]
    The integrated structures each have at least one inlet and at least one outlet connected by an enclosed fluid pathway. Each inlet and outlet is mateable with a respective fluid transport connector to form a pressure-tight fluid communicable connection. By this, it is meant that the seal formed around the connection is able to withstand the fluid pressures typically encountered during autosampler injections and extractions while preventing air bubbles to penetrate the seal into the fluid pathway created, or the fluid being transported to escape the enclosed fluid pathway formed. A stationary phase is disposed downstream from the inlet and upstream from the outlet such that a fluid injected through the inlet traverses the stationary phase prior to transport to the outlet.
  • [0007]
    The connections formed between the autosampler fluid transport connectors and the inlet and outlet enable a fluid to be processed through the stationary phase in a single step of simultaneous injection and extraction, a process that can be very accurately controlled (e.g., rates and volumes) through use of metered pumping mechanisms of the autosampler. The enclosed fluid pathway formed also prevents fluids from flowing in directions or at times not intended due to, for example, gravity. Suitable stationary phases for use in the fluid processing include reversed phase, normal phase, affinity, chiral, gel filtration, ion exchange, size exclusion, HILIC, digestion, absorbent, non-polar, polar, cation exchange, anion exchange, antibody, enzymatic and reactive media and the like.
  • [0008]
    Modified well plates incorporating one or more of the integrated structures may be used with well plate autosamplers having the ability to simultaneously engage multiple fluid transport connectors (such as fixed or movable syringes, probes or other types of injection and/or extraction components) with indexed positions (e.g., inlets, outlets and/or reservoirs) on the well plate. For simplifying understanding, some of the descriptions provided herein may refer only to “syringes”, but use of the term is meant to encompass the broader class of fluid transport connectors. Engagement may be achieved by moving the syringes and/or the well plate via one or more robotic arms into engaged positions. The well plate and syringes may engage at positions along the top surface of the well plate, or alternatively on multiple surfaces (e.g., top and bottom) of the well plate.
  • [0009]
    The well plate may be configured with numerous such integrated structures in an indexed array or network that may additionally include sample positions, waste reservoirs, wash reservoirs, fractionation reservoirs, fraction-pooling reservoirs, reaction reservoirs, and solvent reservoirs. This allows a wide range of fluid processing operations, including solid phase extraction (SPE) and other operations, to be performed in an automated manner through use of existing autosampler capabilities.
  • [0010]
    In another aspect, the inventive structure may take the form of a stand-alone cartridge. Such a cartridge may similarly be used with well plate autosamplers (e.g., where one or more cartridges are seated in a rack that is transported by the autosampler), but are preferably designed for use with standard autosamplers, wherein robotic fingers operate to grasp the cartridge and transport it into a position of alignment with the autosampler's fluid transport connectors for simultaneous engagement of the inlet(s) and outlet(s). As in the well plate embodiment, multiple fluid transport connectors of the autosampler simultaneously engage the inlet and outlet to form the enclosed fluid pathway for processing the fluid through the stationary phase within the cartridge.
  • [0011]
    In another aspect, the present invention provides an automated fluid processing system including a standard or well plate autosampler equipped with multiple fluid transport connectors that may be sealably engaged with the inlet and outlet of fluid processing devices such as described above. A wide variety of automated fluid processing methods that employ injections and extractions of fluids (e.g., samples, solvents and waste) to and from the inventive structure are possible utilizing these devices. In SPE processing, for example, a separation material may be conditioned, then a sample loaded onto/through the separation material, after which matrix and analyte fractions may be sequentially eluted from the separation material (and optionally reconstituted in a more aqueous solvent composition.)
  • [0012]
    Thus, liquid-soluble sample preparation processes that have been performed manually such as, for example, SPE pre-cleaning of complex chemical and biological samples, can be advantageously integrated into a standard analytical workflow with reproducible sample preparation conditions (i.e., precisely controlled flow rates, solvent volumes, and timing between sample preparation and chromatographic analysis.) Driving the liquid-soluble sample flow through the integrated structures described herein with the metering piston of an autosampler, rather than by using a vacuum or gravity eliminates backpressure variations encountered in preexisting fluid processing cartridges or columns. The precise timing also eliminates the possibility that the stationary phase will dry out and lead to irreversible absorption of analytes on the stationary phase.
  • BRIEF DESCRIPTION OF THE FIGURES
  • [0013]
    The various features and aspects of the present invention may be more readily understood with reference to the following figures, wherein:
  • [0014]
    FIGS. 1A,1B are illustrations of a modified well plate in accordance with embodiments of the present invention;
  • [0015]
    FIGS. 2A,2B are illustrations of alternative embodiments of structures including and inlet in fluid communication with an outlet in accordance with the present invention;
  • [0016]
    FIGS. 3A-3C are illustrations of alternative embodiments of inlet sealing mechanisms in accordance with the present invention;
  • [0017]
    FIG. 4A-4E are illustrations of layouts of well plates arranged in accordance with embodiments of the present invention;
  • [0018]
    FIG. 5 is a cross-sectional view of a structure integrated within a well plate;
  • [0019]
    FIGS. 6A-6E are sequential illustrations of a well plate in accordance with the present invention that exemplify an automated fluid process capable of being performed with devices in accordance with the present invention; and
  • [0020]
    FIGS. 7A-7F are illustrations of cartridge-type embodiments of devices for use in automated fluid processing in accordance with the invention.
  • DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
  • [0021]
    Referring now to the drawings, wherein like numbers designate like or corresponding parts throughout each of the several views, there is shown in FIGS. 1A and 1B well-plate embodiments of device 2 for use in automated processing of a volume of aqueous fluid containing suspended solids and/or solubles. The device 2 is shaped and dimensioned to be received by an autosampler 3. Alternative embodiments of the device 2, including cartridge-type embodiments, are described below. The terms “well plate” and “cartridge”, as used herein, are intended to have meanings more broad than their conventional meanings, wherein a conventional well plates might be understood to mean structures including arrays of independent wells, or wherein the meaning of the term cartridge might be limited to the conventional pipette-shaped bodies.
  • [0022]
    Each of the embodiments of device 2 described below are intended for use with chromatographic and spectrophotometric autosamplers, only the arms 4 of which are shown in FIGS. 1A and 1B. Modified well plates in accordance with the present invention may also be manipulated by well plate feeders. Cartridge embodiments of device 2 are preferably adapted for use with autosamplers equipped with robotic mechanisms (e.g., fingers) for grasping and transporting fluid containers such as, for example, sample vials, to positions whereby autosampler fluid connectors (e.g., needles 6′, 6″ and needle seats) may engage the device 2 so as to form an enclosed fluid pathway through the device 2.
  • [0023]
    As noted above, autosamplers that operate on stationary, indexed, multi-well trays or racks of sample vials, such as Series 1100 HPLC Autosamplers manufactured by Agilent Technologies of Palo Alto, Calif., are in wide use. Such autosamplers include a plurality of fluid transport connectors (i.e., needles 6′, 6″) for individually injecting fluid samples into an inlet 8 of a structure 10 integrated within the device 2. In the descriptions that follow, references to “device 2” may be referred to as “plate 2” or “cartridge 2” depending upon the embodiment of device 2 being described, as many of the properties are similar. Each time a different embodiment of device 2 is introduced, however, an attempt will be made to designate it with a distinct alphanumeric reference number of a format “2x”. The needles 6′, 6″ that deliver and extract the liquid-soluble sample are transported on one or more robotic autosampler arms 4, each of which may be equipped with multiple needles for performing simultaneously multiple series of injections and extractions. The autosampler preferably includes a processor for controlling the selection of sample(s) to be processed and the order of processing that is to be accomplished. The precision and reproducibility of such known automatic injection mechanisms is clearly superior to manual injection since variability of injection technique between operators is eliminated.
  • [0024]
    FIG. 2A illustrates a preferred embodiment of the structure 10 integrated within plate 2, which includes inlet 8 in fluid communication with an outlet 12 through a fluid pathway 14. The inlet 8, outlet 12, and fluid pathway 14 are integrated in a unitary structure that may be formed as a unitary element or as multiple components securely (and preferably unalterably) connectable together. At the top of the inlet 8 and outlet 12 are openings 16 through which autosampler needles 6A, 6B may respectively connect, in a relatively fluid-tight connection, to the inlet and outlet so as to form an enclosed fluid pathway through a corresponding inlet chamber 18 and outlet chamber 20 each defined in part by the inlet 8 and outlet 12. Note that the autosampler needles 6A,6B are illustrated as having different distances from plate 2, which is not a requirement of the invention (in fact, it may be preferable to arrange the needles such that they are equidistant from the plate.
  • [0025]
    A stationary phase 22 contained in the inlet chamber 18 serves to process the liquid-soluble sample injected via needle 6′ into inlet chamber 18 as the sample traverses the stationary phase 22 as it flows along the fluid pathway 14 through the outlet chamber 20 to the outlet 12. As used herein, the phrase “stationary phase” includes any material that facilitates separations and/or reactions including, but not limited to, reversed phase, normal phase, affinity-based (for biological sample processing), chiral, size exclusion, HILIC, digestion media, reactive, ion-exchange, etc. Among these are solid phase extraction (SPE) media that cause suspended solids and/or solubles to separate from the solution in which they are suspended. This includes chromatographic sorbents such as porous silica derivatized with octadecyl (C18) or octyl (C8) functional groups, or porous particles based on organic polymer. The stationary phase may consist of a plurality of the materials listed above, arranged so that the liquid-soluble sample traverses the distinct media in a predetermined sequence (such as shown in FIG. 2B, wherein a plurality of distinct stationary phase materials 22A, 22B are separated by a frit 42.)
  • [0026]
    Fluid pathway 14 may include a conduit 24, or a plurality of such channels, of any geometry but having sufficient cross-sectional area to permit fluid flow commensurate with the injection rate, connecting respective openings 26, 28 at or near the bottoms of inlet chamber 18 and outlet chamber 20. Note that the fluid pathway is not necessarily limited to conduit connections at the bottoms of the respective chambers. For example, certain embodiments of structure 10 (as shown in FIG. 2B) do not utilize conduits or distinct inlet and outlet chambers. The injected fluid, however, is required to traverse the stationary phase 22 along the fluid pathway 14 from the inlet 8 to the outlet 12.
  • [0027]
    FIGS. 3A-3C, illustrate various non-limiting embodiments of inlet 8 of structure 10, which includes a sealing surface 30 at least partially conforming to the shape of the autosampler needle 6A. The sealing surface 30 and needle 6A axially engage to form a pressure tight seal and an enclosed fluid pathway along which injected fluids will flow. As used herein, the phrase “pressure tight” means leak free up to about 10 bar (150 psi), and preferably pressures much higher (e.g., 100 to 200 bar.) A metering pump of the autosampler provides tight control over the volume and flow rates of fluids injected through needle 6A. Typical autosampler injection volumes are on the order of 0.2 to 100 μL, but may be lower or higher (e.g., up to about 3 mL.) In certain preferred embodiments, needles 6A, 6B have sufficiently wide inner diameters to transport volumes of conditioners, solvents and potentially viscous, complex chemical and biological (e.g., whole blood, urine, plasma, tissue, etc.) matrices involved in stationary phase (e.g., SPE) fluid processing. Simultaneously injecting and extracting fluids through the structure 10 provides highly precise and repeatable automated fluid processing.
  • [0028]
    In certain embodiments, such as shown in FIG. 3A, inlet 8 comprises a rigid cap integrally-formed with the remainder of structure 10 or subsequently insertable, whose sealing surface 30 includes a tapered bore 32 substantially conforming to the shape of the tip 34 of needle 6A. The cap may be a press fit component placed in the inlet chamber 18 before or after filling the chamber with the stationary phase material 22. The bore 32 of the cap may be tapered to mate with the taper of needle tip 34, preferably in a conical shape having an inner diameter greater than the diameter 36 of the needle at the top of the bore but narrower than the needle diameter at the bottom of the bore. A wide variety of alternative configurations of the inlet 8 are also possible, such as, for example: as shown in FIG. 3B, wherein the bore 33 has no taper, or as shown in FIG. 3C, wherein the inlet 8 has a circular groove 37 into which the needle tip 34 may be seated. The angle of the taper of the bore 32 that contacts the tip of needle 6A is preferably chosen so that self-locking will not occur and the needle will be retractable without damaging the structure 10, but which allows the fluid-tight seal to be formed with axial compression only. Alternative sealing surface configurations (e.g., luer compatible) may be utilized to mate with non-tapered needles or other autosampler connectors, such as needle seats, which are present on many autosamplers and typically are configured to receive autosampler needles transporting a volume of a liquid-soluble sample, but which, in preferred embodiments of a complete inventive system, are adapted to receive the outlet 12 of cartridge-type embodiments of the invention.
  • [0029]
    Similar configurations may be utilized to form a pressure tight seal between outlet sealing surface 13 and needle 6B (as shown in FIG. 2A.) Outlet chamber 20 is partially defined by sealing surface 13 which preferably has a taper or conical shape arranged such that fluids (e.g., eluted fractions) can be more efficiently withdrawn by constraining the volume of processed fluid to a region where it is more easily extractable. Alternative non-linear geometries that work on a similar principle(s) of picking up eluting fractions (and other fluids) with needles 6B shaped to reversibly mate with sealing surface 13. Outlet chambers 20 having no taper (and no frit 42) may also be used, as the enclosed fluid pathway 14 from injector needle 6A to extractor needle 6B through structure 10 reduces the need for extraction-enhancing or backflow-preventing features.
  • [0030]
    Structure 10 may also include a blocking element preventing gravitational flow of fluids between the inlet chamber 18 and outlet chamber 20 that potentially could corrupt the fluid stationary phase processing. FIG. 7A shows a flap mechanism 40 disposed at the base of outlet chamber 20 that allows fluids to flow into the chamber 20 but blocks gravitational flow back out of the chamber. Flap mechanism 40 could also be disposed at some other position between the inlet chamber and outlet chamber. Reverse or gravitational flow may also be prevented by one or more frits 42 disposed in either or both of the inlet chamber 18 and/or outlet chamber 20 on either side of the stationary phase material 22. The frits 42 may exhibit hydrophobic properties and/or block fluid flows not driven by sufficient fluid pressure. Hydrophobic properties may be inherent in the materials selected for forming the device, or may result from chemical treatment (e.g.. with silicone or Teflon™.) The stationary phase material is preferably retained by two porous discs of frits, and another frit is disposed in the outlet chamber to prevent gravitational backflow, but in simultaneous injection/extraction operation fluid flow is precisely controlled, thus reducing the need for backflow prevention.
  • [0031]
    A top view of a well plate 2 in accordance with the present invention is illustrated in FIG. 4A. Well plate 2 is preferably formed of standard polymeric materials, such as polyethylene or polypropylene that are relatively rigid, resistive to wear, and having a low coefficient of friction. Plate 2 is shown configured to process 24 samples, however plate design choices could lead to a greater or fewer number of pairs 44A,44B of inlets 8 and outlets 12. Plate 2 is additionally configured with a number of reservoirs 46, including reservoirs for waste 46-1, conditioner 46-2, wash/rinse fluid 46-3, solvent 46-4, and/or any other fluid 46-5 desired, such as, for example, for reaction processing steps. Obviously, a plate 2 could be configured with none, some or all of these reservoirs 46 as desired or required by the particular fluid processing being performed. Well plates such as plate 2 are easily adopted into a standard, analytical workflow including analytical equipment with only minor modifications, thereby increase reproducibility of sample preparation, as all samples can be processed under precisely reproducible conditions (i.e., flow rates, defined solvent volumes, timing between sample preparation and chromatographic analysis, etc.) Obviously, draw volumes and draw rates should be matched to the sample to be processed and/or chamber sizes being utilized. Although sample positions are not integrated in the embodiment of plate 2 shown in FIG. 4A, samples can be drawn from a feeder plate or other sample source within the capability of the autosampler fluid transport mechanism.
  • [0032]
    A wide variety of alternative configurations of, and uses for, well plate 2 are within the scope the invention, certain preferred embodiments of which will be described below. If the particular autosampler involved in the fluid processing has the capability, for example, an injection and extraction of a liquid-soluble sample through the inlet/outlet pair 44A can be coupled with injection and extraction of the processed liquid-soluble sample immediately through one or more additional inlet/outlet pairs 44B.
  • [0033]
    In an example by no means meant to limit the scope of the invention:
      • the conditioner 46-2, wash 46-3 and elute 46-4 reservoirs each may have 16 ml volumes;up to 0.6 ml may be injected into each inlet 8 from each reservoir (injection volumes often are selected to be roughly three times (3×) the volume of stationary phase media utilized);the stationary phase (e.g., separation material) comprises 100-200 mg of C18, C8, SiOH, or similar media);the waste reservoir may hold a volume equal to the combined volumes of the three other reservoirs (˜50 ml); and the autosampler needle wash can be accomplished in a conventional autosampler wash port.
  • [0035]
    FIG. 5 illustrates a partial cross-sectional view of plate 2, which integrates inlet chamber 18/outlet chamber 20 and fluid pathway 14. The fluid pathway 14, in this preferred embodiment, extends downward from a top surface 48 of plate 2 through the volume of the inlet chamber 18 containing the stationary phase 22. Fluid pathway 14 may include a transfer channel 50 imprinted, ablated, or otherwise formed in a polypropylene base 52 of the plate 2. Both the frit 42 and inlet 8 may also be composed of polypropylene. Through controlled, simultaneous injections into inlet 8 and extractions from outlet 12 by autosampler needles 6A,6B, automated fluid processing, such as, for example, SPE processing comprised of sequential absorption/desorption of analytes and matrix compounds on the separation medium (stationary phase 22) can be performed.
  • [0036]
    FIG. 4B illustrates an alternative version of well plate 2. This variant integrates indexed sample wells 46-6 on the plate that may be filled manually prior to initiating fluid processing, or which may be filled automatically by the autosampler, as many autosamplers have the capability to dispense fluids from cartridges or containers (not shown) separate from the well plates upon which they operate. Another feature of note is bar code 33, which serves as a unique identifier of well plate 2 and each processed sample. Reading the bar code 33 with a bar code reader (not shown) would assist an operator in integrating the fluid processing into a standard analytical workflow by creating an electronic record of the processing conditions (e.g., the number of channels, etc.) utilized. Alternative identification means could be employed. Certain autosamplers, for example, have the ability to uniquely identify well plates by detecting the relative positions of mechanical tabs present on the surface of well plates. The unique identifier could also comprise some other form of identification (e.g., radiofrequency tag or magnetic label.) The identifier facilitates compliance with governmental record-keeping requirements, such as Good Laboratory Practices (GLP's) and electronic recordkeeping requirements (e.g., “Part 11” FDA regulations.)
  • [0037]
    Although the well plates illustrated in certain of the figures are rectangularly shaped, as noted above, the present invention is by no means limited to such geometries. For example, rotary autosamplers are configured to receive circular trays, and could easily be adapted to receive circular versions of well plate 2. The only requirement is that each position on the plate 2 be individually addressable by the needle controller of the autosampler. In addition, FIGS. 4C-4E illustrate that the one-to-one paired relationship described thus far between inlets 8 and outlets 12 is not a requirement of the present invention. At least one inlet 8 and one outlet 20 connected by a fluid pathway 14 are essential elements, but in certain embodiments multiple outlets 12-1, 12-2,12-3 (FIG. 4C) and/or multiple inlets 8-1, 8-2, 8-3 (FIG. 4D.) The layout of well plate 2 illustrated in FIG. 4E is intended to demonstrate a few of the high number of possible inlet/outlet and/or reservoir arrangements, and that the geometries and volumes of the inlets, outlets, and/or reservoirs are not limited, except by the ability of the autosampler needle(s) to access positions on the plate. There may even be configurations, such as the “8X” and “11X”, wherein outlet chambers are concentrically disposed around an inlet chamber with which it is in fluid communication (i.e., exemplary fluid pathways are indicated by the dotted lines.)
  • [0038]
    Referring now to FIGS. 6A-6F, an automated SPE processing method which utilizes the described apparatus in accordance with the invention will now be explained. The process utilizes the well plate 2 and any conventional means for supplying a liquid-soluble sample in an autosampler environment such as, for example, a commercially-available sample plate 50 consisting of an indexed network of sample wells accessible by the autosampler needle(s). Note that samples could be dispensed automatically from a sample container other than sample plate 50, provided the autosampler has such a capability. Each of the operations that follow is performed automatically by the autosampler controller that controls the movements and fluid flows in the autosampler needle. Solvent and sample transfer between the different positions on plate 2A is ideally performed by an autosampler system suited for larger volume injections (e.g., the 1100 Series Autosampler with 900 μl upgrade from Agilent Technologies.) Clearly, a vast number of alternative steps could be envisioned by those of skill in the art depending on the sample size and required amounts of separation material and solvent needed, including, but not limited to: (a) repetitions of particular steps; (b) cleaning of the needle (or needles) used; and/or (c) extraction of waste from outlet 12 to waste reservoir 46-1 after (or contemporaneously in multi-needle autosamplers) each injection.
  • [0039]
    In an optional but preferable conditioning step shown in FIG. 6A, a solvent (e.g., water or an organic solvent such as methanol or acetonitrile), usually but not necessarily containing buffers (salts) for pH definition, is withdrawn/extracted from the conditioner reservoir 46-2 and injected by the autosampler needle (not shown) into the inlet 8 to “wet” the SPE separation material and rinse any contaminants from inlet chamber and prepare the separation material to preferentially retain the target components by defining the loading properties of polar functions (e.g., silanol groups) of the separation material. It is advantageous for the SPE device 2A to have a high capacity for retaining target compounds of a wide range of chromatographic polarities and to be capable of maintaining target compound retention as sample interferences are washed to waste.
  • [0040]
    In a loading step shown in FIG. 6B, a sample (including analytes and matrix) will be loaded onto the separation material, through pickup at sample position 50-1 and injection into inlet 8. During the loading step, either sample molecules or matrix molecules or sample molecules and matrix molecules will be absorbed by the separation material in the inlet chamber.
  • [0041]
    In subsequent steps shown in FIGS. 6C-6E typically referred to as “washing” or “elution” steps, the matrix and the analyte molecules absorbed by the separation material will be sequentially eluted therefrom and retrieved from outlet 12 for possible immediate injection onto, for example, an HPLC column, or for reconstitution. During these steps, the robotic SPE system (including the well plate 2 and the autosampler) will deliver wash (from reservoirs 46-3 and/or 46-5) and elute (from elute reservoir 46-4) solutions to the inlet 8, and retrieve waste and eluting fractions from outlet 12. The eluted fraction, containing analytes, can be reconstituted in a weaker or stronger solvent, using the standard autosampler functions. In many cases, a higher aqueous content of solvent will improve HPLC performance with large volume injections. Sample focusing can be done either on the analytical column or on the fluid-handling devices described above.
  • [0042]
    FIGS. 7A-7F illustrates other embodiments of structure 10, configured for use with an autosampler of a type well known in the art that operates by gripping and transporting a fluid container (e.g., a sample vial) into precise alignment with the autosampler needles 6A,6B′ through use of robotics (arms and fingers). No teaching of such robotic means is deemed necessary as such means are presently well known in the field. In these embodiments, structure 10, rather than being embedded in a well plate, is integrated into a free-standing module or cartridge 2B. Cartridge 2B preferably has an external geometry approximating that of a conventional autosampler vial for easier adoption by standard autosamplers. Any exterior surface 60 of the cartridge 2A may be grasped by the robotic fingers or arms of the autosampler, and the handling of the cartridge 2 may be further enhanced by providing a lip 62 or other feature on the exterior surface 60 of the cartridge that forms a defined juncture at which the cartridge 2B may be grasped.
  • [0043]
    As with the other embodiments of device 2, only minor modification of current autosamplers, i.e., providing one or more additional syringe needles to the moving arms, or providing other connectors such as, for example, needle seats adapted to receive a cartridge 2B in a manner that engages a sealing surface of the cartridge for injection and/or extraction. Adding a second metering system would provide the capability of accurately delivering and removing mobile phase and eluted samples.
  • [0044]
    The inlet chamber 18 and outlet chamber 20 (frit-less in this example) are integrally formed (formed together or securely connected) with, and protrude upwardly from the surface of a base of plate 62. Plate 62 can be sized to be not much larger than the base of the inlet and outlet chambers, or alternatively could be the size of a well plate and integrate numerous inlet/outlet structures (in which case robotic grasping fingers would be unnecessary, as automated fluid processing to operate much like the processing utilizing a modified well plate.)
  • [0045]
    FIG. 7B illustrates an embodiment of cartridge 2C configured to be, although not necessarily required to be, transportable by some form of tray 64. Lower portions of the inlet chamber 18 and outlet chamber 20 are dimensioned to allow stable seating into one or more wells 66A,66B in order that the tray 64 may be manipulated without spillage. The conduit 24 connecting inlet chamber 18 and outlet chamber 20 is illustrated as bridging a well divider 68, however well divider 68 could have a groove (not shown) to accommodate the conduit 24 (and provide further mechanical stability.)
  • [0046]
    As with the well plate embodiment of device 2, the cartridge embodiment also lends itself to many applications and/or configurations, certain of which are illustrated in FIGS. 7C-7F, which are meant only to convey general concepts of alternative designs. FIG. 7C is intended to illustrate, for example, the concept that a cartridge 2D may be formed having multiple inlets 8A, 8B in fluid communication through a fluid pathway 14′ with a single outlet 12 (or, not shown, multiple outlets in connected to a single inlet) Such configurations would require additional autosampler needles to for the enclosed fluid pathway necessary for effective simultaneous injection/extraction. FIG. 7D shows a slight variation upon this theme, wherein multiple stationary-phase-holding chambers 70A,70B are utilized to process the liquid-soluble sample through cartridge 2E. FIGS. 7E and 7F illustrate single-chamber cartridges 2F and 2G, respectively, which contain one or more volumes of stationary phase material 22A, 22B separated and constrained by one or more frits 46.
  • [0047]
    Multiple cartridges, such as cartridges 2G, for example, shown in FIG. 7F, as well as certain embodiments of the well plates made in accordance with the present invention can be “stacked” to create a customized flow path and separation/processing arrangement. Thus, for example, two cartridges 2G containing distinct types of stationary phase materials 22 can be stacked together. The permutations and possibilities are thus almost infinite. In such embodiments, however, there is a small void volume between each body.
  • [0048]
    Some embodiments constructed in accordance with the present invention may provide the ability to perform extremely accurate high or low volume separations, fractionations and/or reactions, and is amenable to analyses where the sample is limited and may include samples for genomic or proteomic assays. Although the invention has been described with respect to various SPE embodiments, it should be realized this invention is also capable of a wide variety of further and other embodiments within the spirit and scope of the appended claims.
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Classifications
Classification aux États-Unis422/400
Classification internationaleB01L99/00, B01L3/02
Classification coopérativeG01N1/34, B01D15/12, B01D15/363, B01D15/3833, Y10T436/255, B01D15/3804, G01N2030/009, B01D15/325, G01N35/028, B01D15/362
Classification européenneG01N1/34, G01N35/02P
Événements juridiques
DateCodeÉvénementDescription
29 août 2005ASAssignment
Owner name: AGILENT TECHNOLOGIES, INC., COLORADO
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:RICKER, ROBERT D.;REEL/FRAME:016464/0577
Effective date: 20050714