BACKGROUND OF THE INVENTION

Over the past 40 years, the practice of chromatography has witnessed a continuing growth in almost every respect. These changes have included the number of chro atographers; the amount of published work in the field of chromatography; increases in the variety and complexity of the samples which have been separated; and increases in separation speed, convenience, and accuracy. The term liquid chromatography refers to any chromatographic procedure in which the moving phase is a liquid in contrast to a moving gas as in gas chromatography. There are a number of liquid chromatographic techniques including absorption chromatography, partition chromatography, ion exchange chromatography, thin layer chromatography, paper chromatography and modern liquid chromatography.

Typically, a chromatographic system includes a sample introduction system and an LC column. An important factor in obtaining good column performance is the proper introduction of a sample into an LC column. Ideally, the sample injector should

(a) introduce any sample into the column as a narrow plug, and

(b) operate at high back pressures.

Frequently, samples require or benefit from additional treatment prior to their injection into the column. The initial sample should preferably be a homogenous solution contained in a solvent that is no stronger than the initial mobile phase for the separation. For adequate sensitivity, trace assays may require a reconcentration of the sample. Very complex samples, containing many components, may require pre-separation into simpler fractions to avoid overlapping bands in the final LC separation. Sample pretreatment has, historically, been carried out manually. More recently, the procedure has become automated. The full automation of sample pretreatment has provided many advantages apart from merely reducing the time and effort required for sample preparation. Through automation, the precision of sample assays can be dramatically improved in many cases, inasmuch as manual pretreatment commonly introduces a large source of imprecision into an overall LC assay.

The simplest form of automation in LC is automatic sampling. Automatic samplers permit several samples to be loaded onto the system, following which each sample is automatically injected and run on the LC system. Various automatic devices are on the market which simplify the repetitive steps of extraction, dilution, and reaction which are involved in typical pretreatment procedures. These include automated or semi-automated diluters and dispensers. Complete systems that combine automated sample- pretreatment plus HPLC are commercially available such as the FAST-LC ^t from Technicon.

U.S. Patent Nos. 4,740,025; 4,727,494; 4,689,755 and 4,507,044, issued to Zymark, Inc. of Hopkinton, MA, are directed to robotic-based controlled sample systems which may be used for HPLC applications. These systems are characterized by their use of mechanical manipulators to mix and control sampling of an HPLC mixture. These systems, while providing some measure of precision in the sampling process, are both complex and include a number of moving parts.

One of the problems which has been associated with automatic sampling systems has been the difficulty in accounting for

wastage and excess during the measurement procedure. This wastage may often be found in feed conduits which carry reagent to the metering system. For example, while many systems utilize precision metering pumps to measure the quantity of desired reagent in an HPLC sample, none of the prior art systems account for traces of excess reagent in the feed conduits. None of the systems appearing in the prior art address the problem of eliminating excess reagent from the system which may impact on proper measurement procedures or provide a purging system to eliminate excess reagent in the system. None of the prior art systems previously disclosed teach the use of a purging system comprising a peristolic pump to eliminate excess reagent in the system. Such a system would provide greatly enhanced precision in the reagent metering process in an HPLC system. The prior art is further characterized by an inability to easily utilize reagent vials having varying sizes and diameters. It would further be desirable to provide an HPLC sampling system having modular adapters which could be utilized to facilitate storage vials having different shapes and diameters. Prior art automated systems are further characterized by their difficulty in loading reagent samples onto the system. It would be desirable to produce an HPLC sampling system having a circular-shaped sample table comprising a plurality of wedge- shaped members which facilitate ease of loading and unloading. The present invention is directed to addressing these and other problems . The advantages of the present invention will become apparent from the following summary and detailed description which follow.

SUMMARY OF THE INVENTION In accordance with the present invention, a system for purging the excess fluid in an HPLC sample is disclosed. The system comprises : a vial for holding an HPLC reagent sample to be metered; means for drawing in a metered amount of reagent sample; a conduit between the vial and the drawing means for transporting the HPLC reagent sample between the vial and the drawing means; and means for purging the conduit of excess reagent such that when said drawing means draws in a metered

amount of HPLC reagent, the excess reagent is removed from the conduit .

In a more preferred embodiment, the present invention is directed to a system for metering an HPLC sample comprising: a rotatable table holding vials a plurality of HPLC samples; means for selectively rotating said table; a syringe pump for drawing a metered amount of HPLC sample from said vials; a plurality of conduits, each of which is associated with a suction probe for transporting HPLC reagent samples between said vials and said syringe pump; at least one controllable valve for switching the flow of reagents between said syringe pump and said plurality of vials; and means for purging the plurality of conduits of excess reagent such that said syringe pump only pumps a metered amount of fluid through the system and excess HPLC reagent is removed from the conduits.

In yet a further embodiment, the present invention is directed to a system for metering an HPLC sample comprising: a rotatable table for placing a plurality of HPLC reagent samples held in vials; means under the control of a computer program for rotating said table; a plurality of suction probes for selectively removing HPLC sample from said vials; a plurality of conduits, each of which is associated with a suction probe for transporting HPLC reagent samples between said vials and a plurality of syringe pumps under the control of said computer program, each of said syringe pumps being associated with a conduit so as to draw a metered sample of HPLC reagent through a conduit; at least one valve under the control of said computer program for switching the flow of reagents between said syringe pump and said vials; and means for purging the conduits of excess reagent such that said plurality of syringe pumps only pump a metered amount of reagent through said system.

In still a further embodiment, the present invention is directed to a system for metering an HPLC sample comprising: a rotatable table comprising a plurality of modular removable wedge-shaped members for placing a plurality of HPLC reagent samples held in vials; means under the control of a computer program for rotating said table; a plurality of suction probes

affixed above said table for selectively removing the HPLC samples from said vials; a plurality of syringe pumps under the control of said computer program, each of said syringe pumps being associated with a flow conduit so as to draw a metered sample of HPLC reagent through a conduit; a plurality of flow conduits, each of which is associated with a suction probe for transporting HPLC reagent samples between said vials and said syringe pumps; at least one valve under the control of said computer program for switching the flow of reagents between said syringe pumps and said vials; and means for purging the conduits of excess reagent such that said plurality of syringe pumps only pump a metered amount of reagent through said system.

In a further embodiment, the present invention is directed to a system for precisely metering an HPLC sample comprising: a rotatable table comprising a plurality of modular removable wedge-shaped members for placing a plurality of HPLC reagent samples held in vials; means under the control of a computer program for rotating said table; a plurality of suction probes affixed above said table for selectively removing the HPLC samples from said vials; a plurality of syringe pumps under the control of said computer program, each of said syringe pumps being associated with a flow conduit so as to draw a metered sample of HPLC reagent through a conduit; a plurality of flow conduits, each of which is associated with a suction probe for transporting HPLC reagent samples between said vials and said syringe pumps; at least one valve under the control of said computer program for switching the flow of reagents between said syringe pumps and said vials; and means for purging the conduits of excess reagent such that said plurality of syringe pumps only pump a metered amount of reagent through said system.

In a further embodiment, the present invention is directed to a system for precisely metering an HPLC sample comprising: a rotatable table comprising a plurality of removable modular wedge-shaped members for placing a plurality of HPLC reagent samples held in vials; drive motor means under the control of a computer program for rotating said table; a plurality of suction probes affixed above said rotational table for selectively

removing the HPLC samples from said vials; a plurality of conduits, each of which is associated with a suction probe for transporting HPLC reagent samples between said vials and a plurality of syringe pumps, said plurality of syringe pumps being under the control of said computer program, each of said pumps being associated with a conduit so as to draw a metered sample of HPLC reagent through a conduit; at least one valve under the control of said computer program for switching the flow of reagents between said vials and said plurality of syringe pumps; and a plurality of peristolic pump means for purging the conduits of excess reagent such that said plurality of syringe pumps only pump a metered amount of reagent through said system.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a perspective view of a typical liquid HPLC system.

Figure 2 is a perspective view of the HPLC sampling system of the present invention.

Figure 3 is a block diagram of the HPLC sampling preparation system of the present invention. Figure 4 is a block diagram of an alternative HPLC sampling preparation system of the present invention.

Figure 5 is a block diagram of a control system for the valve and pump systems of the present invention.

Figure 6 is a front perspective view of the syringe pump in accordance with the present invention.

Figure 6A is a side perspective view of the syringe pump in accordance with the present invention.

Figure 6B is a top perspective view of the syringe pump in accordance with the present invention. Figure 7 is a front perspective view of a controllable valve in accordance with the present invention.

Figure 7A is a side perspective view of a controllable valve in accordance with the present invention.

Figure 8A is a plan view of a modular radial sample table in accordance with the present invention.

Figure 8B is a section view along line 8B-8B of Figure 8A. Figure 9A is a plan view of a wedge-shaped modular table

segment in accordance with the present invention.

Figure 9B is a section view of the wedge-shaped member of Figure 9A.

Figure 10 is a perspective view of a wedge-shaped modular table segment in accordance with the present invention.

Figure 11 is a perspective view of a gripper assembly to be used with the wedge-shaped modular table segment in accordance with the present invention.

Figures 12A-12C is an algorithmic flow chart of a computer control system in accordance with the present invention.

Figure 13 is a plan view of a plasticware adapter in accordance with the present invention for use in solid phase extraction.

DETAILED DESCRIPTION OF THE PRESENT INVENTION The present invention is described with reference to the Figures attached hereto wherein the same numbers are utilized where applicable. Figure 1 illustrates a typical HPLC system. The system includes the HPLC sampling system of the present invention 10. The system further comprises a filtration module 12, rheodyne valve 14 and an HPLC column 16.

Referring to Figure 2, a perspective view of the HPLC sampling system 10 of the present invention is shown in a preferred embodiment. In a preferred embodiment, the system 10 comprises a radial sample holding storage table 18 and housing 17 holding modular digital pumps 20 and suction probes 22 configured as a modular unit. As shown in Figures 8A and 10, in a preferred embodiment, the storage table 18 is rotatable and comprises a plurality of wedge-shaped members 18a. The rotatable table 18 is preferably controlled by a stepper motor (not shown) . In the preferred embodiment, the wedge-shaped members 18a comprise dual rows 18b of radially extending vial storage and holding positions 19. In the embodiment shown, each row 18b contains four vial holding positions 19. The rotating storage table 18 rotates the vials into position under the suction probes 22 which, as will be discussed below, facilitate the removal and transport of HPLC reagent components from the vials.

The system of the present invention is utilized in

conjunction with an operating computer program residing on a personal computer 24 which may operate under a graphical user interface (GUI) package such as Microsoft Windows Version 3.0. The system, under the control of the computer program, can then be configured to selectively draw, meter and move HPLC reagent samples between the vials 21. A commercially available software package such as the Autoprep 286 sample preparation system, manufactured by Cardinal Instruments, assignee of the present invention, may be used as the operating computer of the present invention.

Referring now to Figure 3, a block diagram of the HPLC sampling system of the present invention is shown. As shown, the system comprises the rotatable table 18 with four vial positions 19 and vials 21. Positions 1, 2 and 3 hold reagent components with position 4 holding the final HPLC reagent which will be run through the LC column 16. The vial positions 19 are positioned directly below a plurality of suction probes 22.

The present invention can further be utilized for solid phase extraction or membrane filtration. In this application, a variable depth plunger 22a applies positive pressure to the top of a column filter bed. The plunger 22a allows any depth of bed material to be used without a loss of functionality. The plunger 22a is combined with variable pump 30a-c that facilitates unique probe height adjustment and flow rate. The system 10 includes a plurality of flow conduits 26 which facilitate the flow of HPLC reagent through controllable valves 28, 28a and onto syringe pumps 30 which precisely meter HPLC reagent. Controllable valve 28 is a 4-port valve and control valves 28a are 6-port valves. The six port valves 28a include access ports for the four reagent vial positions 19 as well as waste and clean ports. The system further includes means for purging the conduits of reagent 32 associated with each syringe pump to be described below.

Referring to Figures 5-7B, the controllable valves 28, 28a and syringe pumps 30 of the present invention are shown in greater detail. In a preferred embodiment, the valves comprises valves such as the SV (Smart Valve) valve manufactured by Cavro

Scientific Instruments, Inc. This valve is a compact, stepper motor driven module for OEM liquid handling applications. The Smart Valve is available in three, four or six port valves. Figures 7 and 7A illustrate a four port valve 28. The valve utilizes the same communication characteristic as the Cavro XL 3000 modular digital pump to be discussed below. As shown in Figure 5, the valve 28, 28a operates with an RS-232 or RS-485 bus 34, and a choice of two communication protocols. The valve 28, 28a includes an internal ROM 36 and CPU 38. Up to 15 XL-type devices can be addressed on the same bus 34. The valves 28, 28a use a single 24 VDC power supply and contain a buffered output which can drive an additional relay or solenoid. The valves interface with computer 24 and system software.

Referring to Figures 5-6B, a preferred syringe pump 30 in accordance with the present invention is shown. The system of the present invention 10 may utilize up to four syringe pumps. In a preferred embodiment, the pumping system comprises a pump such as the XL 3000 modular digital pump by Cavro Scientific Instruments, Inc. This pump is a fully programmable, open-framed precision liquid handling instrument, it is a stepper motor driven syringe pump. The pump 30 operates from a single 24 volt power supply and is controlled by an external computer or microprocessor and interfaces with bus 34. The syringe pump 30 has 3000 steps with a high resolution mode of 12,000 steps. The pump can use a variety of syringes up to 25ml and has plunger speed range of between less than one second to 10 minutes in duration.

Referring to Figure 3 and 4, a key feature of the present invention is the utilization of means for purging the conduit lines 26 of excess reagent 32, 32a. Means 32 operates to remove the excess reagent such that the only reagent in the system is that which has been precisely metered by pumps 30. In a preferred embodiment, means 32 comprises a peristolic pump for purging the conduit lines 26 of excess reagent. In an alternative embodiment, the purging means 32 comprises a syringe pump 32a with valve 33 which eliminates excess reagent from conduits 26.

An operational example of the present invention is now described with reference to flow chart of Figures 12A-12C and Figures 2 and 3. In the present example it is assumed that a reagent sample having 2 milliliters of 20% glucose is in vial position 1; 5 milliliters of 15% plasma is in vial position 2; and 7 milliliters of 5% caffeine is in vial position 3. The three will be mixed at vial position 4.

The samples are set in positions 1-3 on the table 18 under the probes 22. The HPLC sample vial will be set on table position 4. Referring to Figure 12, the table 18 is rotated such that the vials are under probes 22. The first syringe pump 30a will then be activated to draw in 2 milliliters of 20% glucose solution. After this is accomplished, the valve 28a associated with pump 30a will be rotated to the waste position. Purging means 32 associated with pump 30a is then activated to eliminate the excess sample from the conduit 26. The control valve switches the conduit to line 4. The syringe pump then pumps the 2ml sample in to the reagent vial situated at position 4.

The computer then switches valve to position 2. The second syringe pump 30b will then be activated to draw in 5 milliliters of 15% plasma solution. After this is accomplished, the valve 28a associated with pump 30b will be rotated to the waste position. Purging means 32 associated with pump 32b is then activated to eliminate the excess sample from the conduit 26. The control valve switches the conduit to line 4. The syringe pump 32b then pumps the 5ml sample in to the reagent vial situated at position 4.

Under the control of the computer program the, system then switches valve 28 to position 3. The third syringe pump 30c will then be activated to draw in 7 milliliters of 15% caffeine solution. After this is accomplished, the valve 28a associated with pump 30c will be rotated to the waste position. Purging means 32 associated with this pump is then activated to eliminate the excess sample from the conduit 26. The control valve switches the conduit to line 4. The syringe pump then pumps the 7ml sample in to the reagent vial situated at position 4. Utilizing the purging system 32 of the present invention, only

precisely metered reagent is transported to the HPLC sample.

While the present invention is described in the context of an air purging means, it is to be appreciated that a number of purging systems can be used with the present invention. For example, the line could be filled with a gas such as nitrogen or a liquid. Such a system could minimize volumetric changes in the reagent and could further be utilized with gas chromatographic systems.

Referring now to Figures 8A-11, a further feature of the present invention is the design of the rotating table 18 of the present invention. As shown in Figures 8A and 9A, the rotating table 18 is radial in design and comprises a plurality of wedge- shaped members 18a which are attached to form a circular shaped unit. As shown, the wedge-shaped members 18a hold dual rows of vials 18b. The wedge-shaped members 18a are separately removable and thus facilitate ease of loading and unloading.

As shown in Figure 10, each wedge member includes a plurality of interchangeable adapters 18c which facilitate the use of vials 19 having different diameters and shapes. The adapters define a plurality of circular receptacles 18d which hold cylindrical grippers 18e, which hold the vials. The cylindrical grippers 18e are hollow and contain a slit diaphragm 18f which holds and firmly supports the vials 21 and facilitates vials 21 of different sizes and shapes. Referring to Figure 13, an adapter 40, sometimes referred to as a plasticware component, which is utilized in accordance with HPLC systems of the present invention for solid phase extraction or membrane filtration is interfaced to wedge-shaped member 18a having dual rows 18b of radially extending vial storage and holding positions. In this fashion, many adapters 40 can fit snugly in the storage holding positions 18b. .An over cap 44 seals the adapter 40 and contains an opening 46 through which a syringe body 42 can be securely placed.

In accordance with solid phase extraction or membrane filtration techniques, syringe body 42 is filled with a resin that is adapted to extract unwanted materials from any type of fluid which is filtered through syringe 42 and is utilized in

conjunction with plunger 22a of Figure 3. The fluid enters the syringe 42 at an opening shown generally at 48 and the resin extracts unwanted materials from the fluid. Prior techniques for solid phase extraction could only heretofore be accomplished manually. By interfacing adapter 40 with the plurality of holding positions 18b, solid phase extraction can be automated for high volume extraction of unwanted materials. Such results have not previously been achieved in the art, and greatly streamline solid phase extraction techniques. When utilized for solid phase extraction applications, the system can utilize multiple gas or fluid reagents, temperature controlled gases or inert drying agents or reagents.

While the present invention has been described in the context of the above-described preferred embodiments, it is to be appreciated that other embodiments fulfill the spirit and scope of the present invention and that the true nature and scope of the present invention is to be determined with reference to the claims appended hereto.