Abstract:
In a preferred embodiment, an eluter instrument for an SPE system, including: a housing; an upper seal mounted in the housing for up and down motion with respect thereto; a lower seal mounted in the housing for up and down motion with respect thereto, and the upper seal and the lower seal being moveable between a first position in which the upper seal and the lower seal are spaced apart and not in proximity and a second position in which the upper seal and the lower seal are in proximity and spaced apart sufficiently only to clamp therebetween a first area of a plurality of areas of an SPE medium, the first area containing therein one or more compounds of interest, such as to permit elution of the one or more compounds of interest to another instrument.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention. 
     The present invention relates to solid phase extraction generally and, more particularly, but not by way of limitation, to a novel eluter for a solid phase extraction system method that simultaneously aspirates multiple samples to be processed by solid phase extraction through a unique defined area for each sample in the defined area to contain various solid phase extraction elements, without an intervening liquid transfer step, and eluting the samples to an analyzer. 
     2. Background Art. 
     High performance liquid chromatography (HPLC) and mass spectrography (MS) are commonly used for the analysis of various chemical products. HPLC and MS have the unique ability to identify specific chemical entities within a mixture of components. A very common use is in drug research and development in the pharmaceutical industry. 
     In many cases, it is mandatory that the sample be preprocessed or “cleaned up” using solid phase extraction (SPE), prior to HPLC or MS. This is normally done by passing the sample through a silica bed. There are different silicas depending on the desired end product. C18 is a common SPE grade of silica and there are others. The component of interest, normally a chemical compound (e.g., a drug) is present mixed with other components within the sample. To analyze the compound of interest, it must first have some form of separation from the rest of the sample. Otherwise, the signal-to-noise ratio would be such that the component of interest could not be detected with sufficient precision. 
     SPE is the typical technique that is employed. The unique aspect of this technique is that the retention of the compound is a function of the solvent flowing through the silica. Thus, an aqueous solution flowing through the silica will cause certain compounds to be retained. If an organic solvent is then passed through the silica, the retained compound can be released or eluted into the solvent. This is SPE—the use of silica to separate compounds of interest from other materials. 
     In the late 1980′s, cartridges of silica came on to the market. A common format used was the barrel of a 10 mL disposable syringe. In the middle to late 1990′s, the need for higher throughput moved solid phase extraction to the 96-well format of microplates. The 10 mL syringe barrels were replaced by the columns in a deepwell microplate. This facilitated processing the samples through the silica columns with a 96-well pipettor. 
     Recently, 3M Company introduced its Empore® product in the 96-well format. This consists of silica supported in a Teflon® matrix. One advantage of this construction is that it provides for very small volume retention This is of critical importance as the total volume of the sample becomes smaller. Empore® elements are located in the bottoms of the SPE columns. Other techniques use more silica within the column. The end result serves the same purpose. 
     The incoming samples normally arrive in a 96-well format, although other formats can be employed as well Using individual or multi-well pipettors, the 96 samples are transferred to the 96 SPE columns Vacuum is used to move the samples through the silica. The compounds of interest are then retained on the silica, with the balance of the streams going to waste. 
     The next step is to place a capture plate under the SPE columns. Then, solvent of choice is added to the columns using the pipettor Vacuum is again used to create flow through the SPE columns This time, however, the eluent is captured in individual wells under each respective SPE column These 96 extracted samples are then transported to a sampling device that injects each sample individually into an analyzer. This normally is an HPLC instrument or an MS instrument or a combination of HPLC and MS instruments. 
     It will be appreciated that the above conventional techniques require a three-step process that involves three different types of equipment as well as involving multiple handling of the sample materials 
     Accordingly, it is a principal object of the present invention to provide an eluter for a simplified SPE system that reduces the different types of equipment required. 
     It is a further object of the invention to provide such eluter that reduces the handling of sample materials. 
     Another object of the invention is to provide such eluter that is simple and economical. 
     Other objects of the present invention, as well as particular features, elements, and advantages thereof, will elucidated in, or be apparent from, the following description and the accompanying drawing figures. 
     SUMMARY OF THE INVENTION 
     The present invention achieves the above objects, among others by providing, in a preferred embodiment, an eluter instrument for an SPE system, comprising a housing, an upper seal mounted in said housing for up and down motion with respect thereto, a lower seal mounted in said housing for up and down motion with respect thereto, and said upper seal and said lower seal being moveable between a first position in which said upper seal and said lower seal are spaced apart and not in proximity and a second position in which said upper seal and said lower seal are in proximity and spaced apart sufficiently only to clamp therebetween a first area of a plurality of areas of an SPE medium, said first area containing therein one or more compounds of interest, such as to permit elution of said one or more compounds of interest to another instrument. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     Understanding of the present invention and the various aspects thereof will be facilitated by reference to the accompanying drawing figures, submitted for purposes of illustration only and not intended to define the scope of the invention, on which: 
     FIG. 1 is a top plan view of a solid phase extraction (SPE) plate. 
     FIG. 2 is an isometric view of a sample transfer instrument. 
     FIG. 3 is a front view of an elution instrument according to the present invention. 
     FIG. 4 is an isometric view of a stacker device used in the elution instrument of FIG.  3 . 
     FIG. 5 is a cut-away isometric view of the elution instrument, with the top and right side removed and with the front operator&#39;s panel folded down. 
     FIG. 6 is a fragmentary isometric view of an X-Y shuttle assembly, less the X-motion components and the Y-motion drive belt used in the elution instrument. 
     FIG. 7 is a front elevational view of the elution instrument, with the front panel thereof removed. 
     FIG. 8 is a front elevational view, in cross-section, of the air upper and lower cylinders that provide the closing action of the “O” rings clamping the SPE plate for elution, with an SPE material clamped in position. 
     FIG. 9 is a fragmentary, side elevational view, in cross-section, of a stacker assembly, less the cassette for handling SPE plates 
     FIG. 10 is a schematic flow diagram showing the elution pathway with a single well elution design. 
     FIG. 11 is a schematic flow diagram showing the elution pathway, with a design that can elute multiple wells in a row by moving the shuttle holding the SPE plate. 
     FIG. 12 is a side elevational view, in cross-section, of an eight-place elution head. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Reference should now be made to the drawing, on which similar or Identical elements are given consistent identifying numerals throughout the various figures thereof, and on which parenthetical references to figure numbers direct the reader to the view(s) on which the element(s) being described is (are) best seen, although the element(s) may be seen also on other views 
     This invention contributes toward streamlining the conventional SPE process. It minimizes the sample handling, allowing a higher number of samples to be efficiently processed. Instead of placing the Empore® material in the bottom of the wells in a 96 well plate, it is supplied in a sheet form. Additional manufacturing steps are required to provide 96 areas of Empore® material to isolate one from the other. This is done by forming a heat-sealed dam around each of the 96 areas, thereby preventing cross flow between wells. 
     This sheet of Empore® material is then placed in a modified conventional instrument, as further described below Using vacuum, the 96 original samples are passed from the sample plate directly through the Empore® material. The novel use of the conventional instrument has replaced the use of the pipettor for this transfer function. The modified conventional instrument is not only faster, but it is a less complex instrument than a multi-well pipettor and thus has a lower cost. The result is improved efficiency and a lower capital equipment cost 
     The next step in the process is to elute the sample of interest from the Empore® material into the analysis instrument This invention has created an eluter instrument to accomplish this function. The eluter instrument simply places the required Empore® element directly in the injection pathway to the analyzer (HPLC or MS.). This eliminates the prior art method of eluting the sample into a capture plate and then moving the capture plate to the point of injection 
     FIG. 1 illustrates a solid phase extraction (SPE) plate, generally indicated by the reference numeral  20 . Plate  20  comprises a sheet of SPE material  30  mounted in an injection molded rectangular frame  32  SPE material  30  may be the Empore® material furnished by 3M company, as described above. SPE material  30  is about 0.030-inch thick and has a plurality of circular areas, as at  34 , defined by a similar plurality of circular dams, as at  36 , formed by the heat-sealing of the SPE material. Areas  34  are arranged in the 96-well format of an 8×12 matrix on 9 mm centers, with each of the well locations being defined and separated from the adjacent wells by heat-sealed dams  36 , although other isolated well arrangements may also be employed. 
     Frame  32  includes an angled corner  40  for proper orientation in a sample transfer instrument, described below, and includes a plurality of triangular indents  42  defined around the perimeter thereof for precise positioning on a shuttle assembly, also described below Also provided on frame  32  is a bar code  60  that contains information as to the processing protocol for plate  20  Plate  20 , as shown, meets the standard microplate footprint dimensions, established by the Society for BioMolecular Screening (SBS), and has a length of 5.030 inches and a width of 3.365 inches. 
     FIG. 2 illustrates a sample transfer instrument, generally indicated by the reference numeral  100 . The construction and operation of instrument  100  are similar to that of the instrument described in U.S. Pat. No. 5,648,266, issued Jul. 15, 1997, to Thomas W. Astle, and titled CELL HARVESTER SYSTEM, the disclosure of which is incorporated by reference hereinto The instrument described in the foregoing patent is modified for its use in the present invention by machining upper head assembly  110  and lower head assembly  112  to mate with SPE plate  20  (FIG.  1 ). Mating “O” rings  114  align with and mate to the 96-well configuration of SPE plate  20 . 
     The operation of sample transfer instrument  100  is as described in the foregoing patent. A set of incoming samples in the 96-well format of a microplate is placed on input platform  120  which is raised to come into contact with aspirating head assembly  120 . The incoming samples to be tested, contained in the sample plate, are drawn directly through the solid phase extraction media in SPE plate  20  (not shown) disposed between upper head assembly  110  and lower head assembly  114 , with one or more compounds of interest being retained in the solid phase extraction media. The use of a pipettor as is currently used to accomplish this task, is eliminated This adds further to the utility of the process by eliminating equipment and a process step. SPE plate  20  has a defined orientation in transfer instrument  100   
     In those applications in which a solvent or other prewet of SPE media  30  (FIG. 1) is required, a reservoir of the solvent may be provided and aspirated through the SPE media with vacuum. The solvent reservoir is then replaced with the sample microplate and the samples aspirated to SPE media  30  If desired, another wash solution may follow the sample. SPE plate  20  with its 96 contained samples is then moved to an elution instrument, described below 
     Other details of the construction and operation of sample transfer instrument  100  may be had by reference to the foregoing patent. 
     FIG. 3 illustrates an elution instrument, generally indicated by the reference numeral  150  for use following the above step. Elution instrument  150  includes a cabinet base  160  on which are mounted first and second stacker assemblies  162  and  164 , the latter having removably mounted thereon, respectively, first and second cassettes  166  and  168  one or both containing a plurality of stacked SPE plates  20  from the above step. Also shown extending from the top of cabinet base  160  is an upper air cylinder  170 , the function of which is described in detail below, and at the top of the upper air cylinder is an adjustable stop  172  and a cooperating adjustable stop nut  174  which limit the downward movement of a piston disposed in the upper air cylinder. A 0.010-inch diameter tube  180  extends from the upper end of upper air cylinder  170  and is connected (not shown) to an HPLC or MS instrument. 
     Mounted on the face of cabinet base  160  is an operator&#39;s panel  190  that includes a single line display  192  and a plurality of push button controls, as at  194 . Extending from the side of cabinet base  160  are two high-pressure chromatography fittings  200  and  202  for connection (not shown) to a bypass loop. 
     FIG. 4 illustrates the major elements of stacker assembly  162 . Stacker assembly  162  includes first and second side vertical support/mounting brackets  250  and  252  on which is mounted a horizontal intermediate plate  254 . Extending vertically from horizontal upper plate  254  are an air cylinder shaft  260  and an anti-rotation guide rod  262 , both with their distal ends attached to and terminating at a horizontal nest plate  266 . 
     Disposed generally within a volume  270  defined by first and second side support/mounting brackets  250  and  252  and intermediate plate  254  is a printed circuit board  272  on which is mounted a solenoid air valve  273  having connections  274  to supply control air to a square air cylinder  276  in which air cylinder shaft  260  is disposed for up and down movement (tubing between the connections and the air cylinder not shown) Also mounted on printed circuit board  272  are a electronic connector  280  and first and second position light switches  282  and  284 . The latter two elements cooperate with a flag  290  operatively connected to air cylinder shaft  260  to indicate upper and lower positions of the shaft. 
     FIG. 5 illustrates the major internal elements of cabinet base  160  (FIG. 3) which include stacker assemblies  162  and  164 , the major elements of which have been described with reference to FIG.  4 . Disposed generally centrally of cabinet base  160 , as shown on FIG. 5, is a horizontal shuttle  300  having defined therethrough a shuttle opening  302 . Shuttle  300  is fixedly disposed between two horizontal Y-motion bearing blocks  310  that are journaled on two horizontal Y-motion guide rods  312  fixedly disposed between two horizontal X-motion bearing blocks  314 . X-motion bearing blocks  314  are journaled on two X-motion guide rods  320  disposed between opposite sides of cabinet base  160 . A software-controlled X-motion stepper motor  330 , an X-motion drive belt  332 , a software-controlled Y-motion stepper motor  334 , and a Y-motion drive belt  336  provide the necessary X-Y motion for shuttle  300 . Cabinet base  160  also houses four cassette escapement control valves  340 , a rotary valve  342  connected to external connections  200  and  202 , a rotary valve drive motor  344 , rotary valve drive motor power supply and controller  346 , an electronics power supply  348 , a cooling fan  350 , and a connection panel  352   
     In operation, one of stacker assemblies  162  and  164 , say stacker assembly  162 , serves as an infeed to the system, while the other stacker, say stacker assembly  164 , may serves as an outfeed to the system. Shuttle  300  is moved over nest plate  266  on stacker assembly  162 . Nest plate  266  is moved through shuttle opening  302  and an air cylinder (not shown on FIG. 5) opens an escapement releasing a SPE plate  20  (FIG. 1) from cassette  166  (FIG. 3) to the nest plate. The escapement closes, to retain the next plate in cassette  166 . As nest plate  266  lowers, it passes through shuttle opening  302 , depositing SPE plate  20  on shuttle  300 . Triangular points  42  (FIG. 1) formed on SPE plate  20  precisely position the SPE plate on shuttle  300 . Shuttle  300  is then moved to the position shown on FIG. 5 for elution, as is described below. After all areas  34  have been sampled, finished SPE plate  20  is positioned by shuttle  300  under outfeed stacker,  164 . The process then repeats for the next SPE plate  20   
     FIG. 6 illustrates in more detail the construction of shuttle  300 . Here it can be seen that one set of ends of Y-motion guide rods  312  terminates at a horizontal member  360 . A tab  370  extending outwardly from horizontal member  360  is pinned at a single point to front X-motion bearing block  314  (FIG. 5, not shown on FIG.  6 ). This single point of attachment accommodates misalignment of X-motion bearing blocks  314 . A second tab  380  extends outwardly from front bearing block  314  and activates an optical switch to indicate when shuttle  300  is fully forward. Four locating triangles  390  engage notches  42  (FIG. 1) on SPE plate  20  to align the SPE plate on shuttle  300 . 
     FIG. 7 illustrates further the arrangement of some of the elements shown on FIGS. 3-6 and further illustrates the location of a lower air cylinder  140 . 
     FIG. 8 illustrates a fixed body  410  of upper air cylinder  170  (FIG. 7) disposed vertically over a fixed body  412  of lower air cylinder  400  (FIG.  7 ). Upper air cylinder body  410  has disposed therein for up and down motion with respect thereto an upper piston  420 , while lower air cylinder body  412  has disposed therein for up and down motion with respect thereto a lower piston  430 , the upper and lower pistons being vertically aligned. Extending downwardly coaxially from upper piston  420  is an upper connector  440  and extending upwardly coaxially from lower piston  430  is a lower connector  442 . Upper and lower O-rings  450  and  452  disposed, respectively, around the distal ends of upper and lower connectors  440  and  442  are shown clamping therebetween a heat-sealed dam  36  (FIG. 1) and, therefore, an area  34 . Indexing of sheet  30  will permit other areas  34  to be clamped between upper and lower O-rings  450  and  452 . It will be understood that area  34  has been clamped between upper and lower O-rings  450  and  452  by means of upper piston  440  moving downwardly and lower piston  442  moving upwardly. O-rings  450  and  452  prevent flow between areas  34 , commonly referred to as “cross talk”. 
     Upper and lower horizontal circular stainless steel frits  460  and  462  are provided, respectively, in the distal ends of upper and lower connectors  440  and  442  to spread out liquid flowing upwards from tube  470  connected to rotary valve  342  (FIG.  5 ), across area  34 , and into tube  180  Tubesl  80  and  47   o  are disposed, respectively, in upper and lower chromatography connectors  480  and  482  axially centrally disposed in upper and lower connectors  440  and  442  Annular upper and lower piston guides and bearing and seals  490  and  492  are disposed, respectively, between upper and lower pistons  420  and  422  and upper and lower air cylinder bodies  410  and  412 . 
     A software program activates upper air cylinder  170  (FIG. 7) first. It has a larger bore than lower air cylinder  400 , thus upper air cylinder  170  can generate more force with an equivalent air pressure Piston  420  moves downwardly to a depth set by adjustable stop  172 . This brings upper O-ring  450  into contact with sheet  30 . Lower air cylinder  400  is then energized, forcing lower O-ring  452  to mate with upper O-ring  450 , thereby sealing a specific area  34  in sheet  30  to be eluted. Motor operated rotary valve  342  (FIG.  5 ), as commonly used in high performance liquid chromatography, switches the solvent flow path to the analysis instrument (not shown), so as to put the desired clamped area  34  in the flow path The solvent elutes the sample from area  34 , transporting it to the analysis instrument, usually HPLC or MS. After the operator-set elution time expires, the next sequence is enabled. 
     If eluter  150  is set up to clamp a row of areas  34  at a time, then another selector valve, common to HPLC use, switches the flow path to the next clamped area. The elution step repeats for this area  34  and the next, until all areas in that clamped row have been eluted. 
     After all clamped areas  34  have been eluted (single area or multiple areas), the solvent flow path to the analysis instrument is switched to bypass eluter  150 . Upper and lower air cylinders  170  and  400  retract to clear plate  20  on shuttle  300 . Stepper motors  330  and/or  334  move shuttle  300  to the next desired position, and the above stated sequence repeats for the next clamped area or areas  34   
     Following elution of all areas  34  in the program for that particular plate  20 , shuttle  300  moves the plate to align with whichever of stacker assemblies  162  and  164  is the outfeed. An operating air cylinder associated with that stacker lifts plate  20  from shuttle  300  by passing through shuttle opening  302 . Plate  20  is carried up into cassette  166  or  168 . An escapement in that stacker opens to allow incoming plate  20  to pass through and then closes to retain the plate. The entire sequence is then repeated for the next plate  20   
     FIG. 9 illustrates upper air cylinder  170  mounted on upper deck  500  of cabinet base  160  (FIG. 3) and also a bar code reader  510  that scans bar code  60  (FIG. 20) on incoming SPE plates  20  while on shuttle  300  (FIG. 5, not shown on FIG.  9 ). Also shown on FIG. 9 are stacker  164 , with first and second escapement assembly air cylinders  520  and  522 , respectively, and a fitting  530  for air to upper air cylinder  170 . 
     FIG. 10 illustrates the flow pathway for a single elution. When rotary valve  600  is set so that the solvent pathway is along the solid lines in the valve, the solvent will flow through bypass loop  610  When, however, rotary valve is rotated 180 degrees, solvent flow will be through cluter pathway  620  and an area  34  (FIG.  8 ). 
     FIG. 11 illustrates the same rotary valve  600 , bypass loop  610  and eluter pathway  620 . In this case, however, a second rotary valve  630  is disposed in eluter pathway  620  to select one of eight areas  34  through which solvent will flow. In this case, it is assumed that all eight areas  34  shown are clamped in a suitable elution instrument and that the eight areas represent one eight-area row on an SPE plate  20  (FIG.  1 ). If a 12-area row on SPE plate  20  is clamped, any one of twelve areas  34  could accessed and second rotary valve  630  would have twelve positions. The advantage of either arrangement is time-savings Second rotary valve  630  can switch sample locations faster than the eluter head can open to permit shuttle  300  (FIG. 5) to reposition and then close to sample the next well location 
     FIG. 12 illustrates an eight-place elution head Here eight areas, as at  34 , are clamped Clamping is accomplished by means of an upper clamping member  700  being lowered by upper air cylinder  170  (FIG. 7) and a lower clamping member  702  being raised by lower air cylinder  400  (FIG.  7 ). The various elements of upper and lower clamping members  700  and  702  are similar to those described with reference to FIG.  8 . 
     The control system for eluter  150  (FIG. 7) is an embedded microprocessor and its associated circuitry, combined with a specific software program. This allows an operator to control the system as deemed necessary Individual sequences can be performed for trouble shooting or set up. Preset sequences can be executed for the convenience of the operator. The software provides various elution sequences that may be programmed and operated by the user. Sequences of areas by row or individually can be set Single line display  192  (FIG. 3) is used for communication between the operator and the controlling software. Preset programs can be created and run at the press of a START button. A stack of SPE plates  20  can be loaded and processed automatically without operator attention. 
     Data tracking is provided by means of bar code reader  510  (FIG. 9) that reads identifying bar code  60  (FIG. 1) on each frame  32 . Bar code identifier  60  provides an audit trail of a specific plate  20  and this data may be printed out through an RS232 port on the instrument to a receiving device Complex programs may be created to sample only certain defined wells on specific frames  32 . The ability of stackers  162  and  164  (FIG. 7) to serve as either infeed or outfeed permits plates  20  to move back and forth between cassettes  166  and  168 , while searching for a specific bar code  60 . Eluter  150  then performs the desired operation on that specific SPE plate  20 . 
     To summarize the differences between this system and the prior art, this system is used in a two-step direct pathway. The sample is collected on the SPE medium and eluted directly from that medium into the analyzer. This achieves both throughput and efficiency in the process. The multi-well pipettor used for sample transfer has been replaced with a faster, lower-cost instrument. The auto sampler, normally used to feed samples to the injector, has been replaced with the eluter, described by this patent application. The Empore® media in a plate type format combined with the eluter provides a very high throughput efficient system. The eluter stackers can completely automate an extensive run of samples 
     A side benefit of the efficiency of this new system is the conservation of disposables. The Empore® media plate consumes far less plastic than is presently used in SPE plates. The pipettor tips and the capture plates of conventional methods are also eliminated. 
     In the embodiments of the present invention described above, it will be recognized that individual elements and/or features thereof are not necessarily limited to a particular embodiment but, where applicable, are interchangeable and can be used in any selected embodiment even though such may not be specifically shown. 
     Terms such as “upper”, “lower”, “inner”, “outer”, “inwardly”, “outwardly”, “vertical”, “horizontal”, and the like, when used herein, refer to the positions of the respective elements shown on the accompanying drawing figures and the present invention is not necessarily limited to such positions. 
     It will thus be seen that the objects set forth above, among those elucidated in, or made apparent from, the preceding description, are efficiently attained and, since certain changes may be made in the above construction without departing from the scope of the invention, it is intended that all matter contained in the above description or shown on the accompanying drawing figures shall be interpreted as illustrative only and not in a limiting sense 
     It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween.