U.S. patent application number 11/184170 was filed with the patent office on 2006-04-20 for fluid processing devices with multiple sealing mechanisms and automated methods of use thereof.
Invention is credited to Robert D. Ricker.
Application Number | 20060083663 11/184170 |
Document ID | / |
Family ID | 35695740 |
Filed Date | 2006-04-20 |
United States Patent
Application |
20060083663 |
Kind Code |
A1 |
Ricker; Robert D. |
April 20, 2006 |
Fluid processing devices with multiple sealing mechanisms and
automated methods of use thereof
Abstract
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).
Inventors: |
Ricker; Robert D.;
(Middletown, DE) |
Correspondence
Address: |
AGILENT TECHNOLOGIES, INC.;INTELLECTUAL PROPERTY ADMINISTRATION, LEGAL
DEPT.
P.O. BOX 7599
M/S DL429
LOVELAND
CO
80537-0599
US
|
Family ID: |
35695740 |
Appl. No.: |
11/184170 |
Filed: |
July 19, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10968296 |
Oct 19, 2004 |
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11184170 |
Jul 19, 2005 |
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Current U.S.
Class: |
422/400 |
Current CPC
Class: |
B01D 15/3804 20130101;
G01N 1/34 20130101; G01N 2030/009 20130101; Y10T 436/255 20150115;
B01D 15/362 20130101; B01D 15/363 20130101; B01D 15/3833 20130101;
B01D 15/325 20130101; G01N 35/028 20130101; B01D 15/12
20130101 |
Class at
Publication: |
422/100 |
International
Class: |
B01L 3/02 20060101
B01L003/02 |
Claims
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 (C.sub.18) or
octyl (C.sub.8) 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 .mu.L, 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.TM..) 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: [0034] 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.times.) the volume of
stationary phase media utilized);the stationary phase (e.g.,
separation material) comprises 100-200 mg of C.sub.18, C.sub.8,
SiOH, or similar media);the waste reservoir may hold a volume equal
to the combined volumes of the three other reservoirs (.about.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 .mu.l
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.
* * * * *