Abstract:
An apparatus and method for transferring one or more liquid samples in one or more sample containers to one or more measurement devices. The invention provides a sample transfer device that is small enough to be mounted over a microtiter plate in an array for simultaneous sampling. In a preferred embodiment, each sample transfer device in an array can communicate with a well of the microtiter plate simultaneously, such that in one or more cycles of operation, all of the samples in the wells in the microtiter place can be chemically analyzed. In the invention, the functions of valve actuator, sampling valve, syringe pump, and transfer device in a liquid chromatograph are all integrated into a single injector mounted at the end of the chromatographic column.

Description:
[0001]     This application claims priority to U.S. Patent Application No. 60/491,008 filed Jul. 29, 2003, the entire contents and substance of which are hereby incorporated in total by reference. 
     
    
     BACKGROUND OF THE INVENTION  
       [0002]     1. Field of the Invention  
         [0003]     This invention relates to separation-based chemical analyses of liquid samples, e.g., via liquid chromatography or electrophoresis.  
         [0004]     2. Description of Related Art  
         [0005]     Liquid chromatography is a vitally important technique used by scientists to identify unknown components in a sample. Chromatography in general is a separations method that relies on differences in partitioning behavior between a flowing mobile phase and a stationary phase to separate the components in a mixture. Typically, a chromatographic column holds the stationary phase and the mobile phase carries the sample through it. Sample components that partition strongly into the stationary phase spend a greater amount of time in the column and are separated from components that stay predominantly in the mobile phase and pass through the column faster. As the components elute from the column, they can be quantified by a detector and/or collected for further analysis. A chromatographic instrument is generally combined with a detection means for real-time analysis.  
         [0006]     Liquid chromatography, more particularly, is an analytical chromatographic technique that is useful for separating ions or molecules that are dissolved in a solvent. If the sample solution is in contact with a second solid or liquid phase, the different solutes will interact with the other phase to differing degrees due to differences in adsorption, ion-exchange, partitioning, mobility or size. These differences allow the mixture components to be separated from each other by using these differences to determine the transit time of the solutes through a column. Simple liquid chromatography consists simply of a column with a fritted bottom that holds a stationary phase in equilibrium with a solvent. Typical stationary phases (and their interactions with the solutes) include solids (adsorption), ionic groups on a resin (ion-exchange), liquids on an inert solid support (partitioning), and porous inert particles (size-exclusion). In this simple column chromatograph, the mixture to be separated is loaded onto the top of the column followed by more solvent. The different components in the sample mixture pass through the column at different rates due to differences in their partitioning behavior between the mobile liquid phase and the stationary phase. The compounds are separated by collecting aliquots of the column effluent as a function of time.  
         [0007]     Analytical separations of solutions for detection or quantification typically use a more sophisticated technique known as High-Performance Liquid Chromatography (“HPLC”). HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector. In a simple HPLC instrument, solid compounds may be separated by injecting a plug of the sample mixture onto the column. The different components in the mixture pass through the column at different rates due to differences in their partitioning behavior between the mobile liquid phase and the stationary phase. The pumps provide a steady high pressure with no pulsating, and can be programmed to vary the composition of the liquid phase (typically a solvent) during the course of the separation. Liquid samples are conventionally introduced into a sample loop of an injector with a syringe. When the loop is filled, the injector can be injected the sample into the stream by placing the sample loop in line with the mobile phase tubing. The presence of analytes in the column effluent is recorded by detecting a change in refractive index, UV-VIS absorption at a set wavelength, fluorescence after excitation with a suitable wavelength, or electrochemical response. Mass spectrometers can also be interfaced with liquid chromatography to provide structural information and help identify the separated analytes.  
         [0008]     In addition, the analysis of multiple chemical samples stored in arrays, as in microtiter plates, has become common. This arrangement is commonly used, for example, for analyses of large numbers of chemical samples in the pharmaceutical industry. Chromatographic and electrophoretic instruments have been widely adopted for use with such arrays of liquid samples.  
         [0009]      FIG. 1  depicts an example of a prior art HPLC system used with samples in a conventional microtiter plate. In such a system, the samples to be analyzed are contained in a microtiter plate or “microplate”  20  having a rectangular array of recesses or wells  21 . A syringe  30  is used to draw predetermined units of a liquid sample and deliver them to injection valves  6  at a first valve position  26  and a second valve position  27 . The syringe  30  comprises a syringe needle  31 , a syringe moving means  34  by which the liquid-tight plunger  32  can be moved within the syringe barrel  33 , causing liquid to be sucked into the syringe  30  or to be expelled. In this conventional HPLC system, microplate  20  has a series of wells  21  for the purpose of containing liquid samples  22 . An automated mechanism, not shown, moves the syringe to a microplate position  23  so that the syringe needle  31  comes in contact with a liquid sample  22 . The syringe system then aspirates a certain volume of the liquid sample  22   
         [0010]     The system shown in  FIG. 1  further includes multiple valves  3  suited to injecting liquid samples into HPLC analysis systems. Each valve  3  comprises a sampling loop  4  a valve exit tube  11  and a port  6  suitable for receiving a syringe needle. The syringe  30 , which contains a certain amount of sample, is moved to a first valve position  26 , where the syringe needle  31  is engaged in a port  6  so that a liquid sample can be injected into a sampling loop  4  with any excess, passing through the loop  4  and out a vent exit tube  12 . The plunger moving means  34  causes the plunger  32  to expel the liquid into the loop  4 .  
         [0011]     For each valve  3  there is a pump  1  and a delivery tube  2  that delivers a liquid flow to the valve  3 . From each valve  3 , a transfer tube  5 , a chromatographic column  7 , a column exit tube  8 , a detector  9 , and a detector exit tube  12  carries a liquid flow out of the valve  3 . When a valve  3  is caused to change its state (typically via an electrical solenoid), the loop  4  is inserted into the flow path between delivery tube  2  and transfer tube  5  so that the contents of the loop  4  including liquid sample injected by the syringe  30  are swept through the transfer tube  5  to the chromatographic column  7 .  
         [0012]     The constituents of the liquid sample travel through the column at different rates. The effluent from the column  7  flows through column exit tube  8  to a detector  9 . The detector  9  is configured to measure the concentration of chemical samples in the liquid a continuous record of the varying level of concentration. Such record indicates three things: a variation in the measurement indicates the presence of various chemicals in the sample; the times of the variations indicate the identities of said chemicals; and the intensities of the variations indicate the concentration of the chemicals.  
         [0013]     It is further known to combine several chromatographic systems of the type shown in  FIG. 1  into a “multi-HPLC” analyzer. In such an analyzer, the syringe  30  delivers samples consecutively from the microplate to several chromatographic systems. For instance, the syringe  30  after delivering a sample from first microplate position  23  to first valve position  26  can deliver another sample from second microplate position  24  to a second valve position  27 . In a multi-HPLC analyzer, there is a multiplicity of pumps  1  delivery tubes  2  valves  3  transfer tubes  5  columns  7  and detectors  9 . In addition, in conventional multi-HPLC analyzers, multiple syringes, rather than a single syringe, may be used and operated simultaneously. In this case, the multiple syringes can take samples from several or all of the microplate position.  
         [0014]     It is further known for the syringe or syringes in an HPLC system to undergo a cleaning step between injections, consisting of rinsing the syringe with one or more solvents. It is further known that devices performing separation-based chemical analyses may additionally or even principally perform other chemical functions, such as reaction, filtration, purification, fractionation, or measurement of other properties, in addition to chemical analysis. It is further known that each sample in an HPLC system may also undergo subsequent further chemical analysis in a separate instrument, such as a mass spectrometer.  
         [0015]     Several disadvantages exist with conventional multi-BPLC systems, however. For example, the quality of the chromatographic measurement in traditional HPLC equipment may be negatively impacted by the transfer of the sample to the sampling valve (e.g., valve  3  in  FIG. 1 ). Either a moving syringe or a relatively long sample delivery tube is required to deliver the aliquot sample from the sample well to the sampling valve inlet (port  6  in  FIG. 1 ). In this syringe or delivery tube, however, the flow of sample can spread out, resulting in a lower-quality chromatographic separation. Conventional multi-HPLC systems further tend to be overly large, complex, and unwieldy, especially when there are more than 4 columns, since there is little or no sharing of common hardware. Some systems fail to allow the taking of liquid samples from all of the wells of a microtiter plate. Others fail to permit simultaneous multiple injection of samples into the measurement columns, or fail to perform simultaneous detection on the flow from multiple columns.  
         [0016]     Recently, integrated multi-column chromatographic instruments based on microfluidic technology have become available. These instruments, such as those from Nanostream and Eksigent, provide “nanoLC”, or liquid chromatography where the flow rates and volumes of sample injected are very much lower than with more conventional liquid chromatography. See, e.g., C. M. Harris, “Shrinking the LC Landscape”, Analytical Chemistry, vol. 75, pages 65A-69A, 2003. Such nanoLC systems typically include multiple columns that are combined together into an inseparable unit. All the columns must be replaced when only one needs to be replaced. Moreover, the column assemblies are only available from the instrument manufacturer, so that a unique column type developed by another manufacturer cannot be used in these instruments. In addition, the very small sample size, low column flow and non-standard columns of these instruments mean that an analyst with a validated and approved method, as by a government agency such as the U.S. Food and Drug Administration, must repeat the extensive work needed to achieve validation and approval of the method in order to use the nanoLC instrument.  
         [0017]     Many multi-channel separation instruments have been adapted to samples in microtiter plates, where the instruments are based on electrophoresis rather than chromatography. The instruments are typically fully integrated with the microtiter plates, and have simultaneous injection and detection. Existing electrophoretic instruments, however, typically do not use an integrated sample injection system, wherein the sample volume is determined by a fixed-volume sample reservoir. Furthermore, these electrophoretic instruments are limited to very small samples and restricted to only certain kinds of chemical samples.  
         [0018]     It would therefore be desirable to provide a multi-HPLC system that is simple and compact and that does not require a moveable sampling syringe or long sample delivery tube. It is further desirable to provide a multi-HPLC system that is capable of processing all of the wells in a microtiter plate. It is further desirable to provide a multi-HPLC system that simultaneously samples, separates and detects multiple samples. It is further desirable to provide a multi-HPLC system having individually replaceable measurement columns that may be inexpensively replaced and that comply with industry standards for HPLC measuring columns.  
       SUMMARY OF THE INVENTION  
       [0019]     The present invention solves these and other problems with conventional HPLC systems by providing a series of sampling devices that are small enough to be mounted in an array over a microtiter plate of standard size. In accordance with the invention, each sampling device can communicate with a well of the microtiter plate simultaneously, and in one or more cycles of operation, all of the samples in the wells in the microtiter place can be chemically analyzed. In accordance with the invention, the functions of valve actuator, sampling valve, syringe pump, and transfer device are all integrated into a single injector mounted at the end of the chromatographic column. The combination of the injector, chromatographic column, detector and auxiliary tubing and conduits needed to support the operation, all fit into sufficiently small space so that multiple combinations can be mounted side by side, and simultaneously service multiple chemical samples in a row of the microtiter plate.  
         [0020]     More particularly, the invention provides a method and apparatus for transferring one or more liquid samples in one or more sample containers to one or more measurement devices, comprising the steps of, and means for, (a) opening a first valve communicating a first sample container with a first sample reservoir; (b) drawing a first liquid sample from the first sample container through the first valve into the first sample reservoir; (c) closing the first valve; (d) pumping the first liquid sample from the first sample reservoir into the first measurement device.  
         [0021]     The invention further provides an assembly suitable for use in a separation-based measurement device, comprising a housing having an interior chamber connected to a sample inlet, a sample outlet, and a reservoir input/output port, a valve seal located between said sample inlet and said chamber; a hollow needle slideably mounted through said valve seal into said chamber; and a valve ball within said chamber, connected to the end of said hollow needle; whereby a mechanical pressure on the hollow needle tends to remove said valve ball from said valve seal, thereby creating a liquid flow passage through the hollow needle, past the valve ball and seal, to the interior chamber.  
         [0022]     The invention still further provides a multiple-column separation-based analyzer, comprising: a support and two or more measurement assemblies mounted on said support. Each measurement assembly respectively comprises an interior chamber, connected to a sample inlet, a sample outlet, and a reservoir input/output port, a valve seal located between said sample inlet and said chamber; a hollow needle slideably mounted through said valve seal into said chamber; a valve ball within said chamber, connected to the end of said hollow needle, whereby a mechanical pressure on the hollow needle tends to remove said valve ball from said valve seal, thereby creating a liquid flow passage through the hollow needle, past the valve ball and seal, to the interior chamber; a sample reservoir connected to said reservoir input/output port; and a chromatographic or electrophoretic column connected to said sample outlet.  
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0023]      FIG. 1 , discussed above, shows a schematic diagram of a conventional HPLC system.  
         [0024]      FIG. 2  is a block diagram depicting an exemplary HPLC system in accordance with the present invention, including an injector, a column, and a hollow needle.  
         [0025]      FIG. 3  is a cross-sectional view of an injector suitable for use in the present invention.  
         [0026]      FIG. 4  is an isometric view of an exemplary single-row multi-HPLC system in accordance with the present invention.  
         [0027]      FIG. 5  is an isometric view of an exemplary multiple-row multi-HPLC system in accordance with the present invention.  
         [0028]      FIG. 6  is a cross-sectional view of an alternative embodiment of a hollow needle suitable for drawing a liquid sample from a particular location in a well of a microtiter plate in accordance with the present invention.  
         [0029]      FIG. 7  is an isometric view of the multiple-row multi-HPLC system of  FIG. 5 , further including a microtiter plate for device washing and rinsing.  
         [0030]      FIG. 8  is an isometric view of an exemplary two-injector device including discrete tubing lengths and Tee connectors in accordance with the present invention.  
         [0031]      FIG. 9A  is an isometric view of an alternative embodiment of a multi-HPLC system in accordance with the invention, including a sample tube block having shared flow paths in accordance with the invention.  
         [0032]      FIG. 9B  is a cross-sectional view of the multi-HPLC system depicted in  FIG. 9A .  
         [0033]      FIG. 10  is a cross-sectional view of an alternative embodiment of a multi-HPLC system in accordance with the invention, including a sample tube block having individual source paths and shared aspirating paths.  
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0034]      FIG. 2  shows a liquid chromatograph that incorporates the injector according to this invention. A reservoir  40  contains a liquid  41 , used as the mobile phase for the liquid chromatographic process. A pump  42  withdraws said liquid  41  through a liquid supply tube  43 , which is connected both to the reservoir  40  and to the pump  42 . The pump  42  also delivers the liquid  41  to the injector  45  though a tube  44 , which is connected both to the pump  42  and to the injector  45 . The liquid  41  flows through the injector  45  and then to a chromatographic column  46  though an adaptor fitting  47 .  
         [0035]     From the chromatographic column  46  the liquid  41  flows into a detector  48  through a detector adaptor fitting  49 . By processes well known to those skilled in the art, continuous chemical measurements are made by the detector on any chemicals contained in the detector  48  by the flow of liquid  41 . An electronic readout of these measurements is carried to a computer  50  though a cable  51 . Liquid flows out of the detector  48  though vent tube  58 .  
         [0036]     In a preferred embodiment, the injector  45  is attached to an actuator  53  can move the injector  45  to a container  54  for the purpose of taking a sample of a liquid chemical  55  contained therein. In accordance with the invention, injector  45  also contains a hollow needle  52  and a mechanically activated valve. The actuator moves the injector  45  over the container  54  and lowers the injector  45  so that the needle  52  enters the container  54  and presses against the container bottom  56 . This causes the needle  52  to move upward within the injector  45 , opening the valve and creating a passage between the needle  52  and the tube  44 .  
         [0037]     In operation, a sample of the liquid chemical  55  is introduced onto the column  46  in several steps.  
         [0038]     (a) The liquid pumping system  42  interrupts the flow of liquid to the injector, and waits for a time sufficient for the liquid pressure within the injector  45  to drop below a low value convenient for introducing a sample, e.g., preferably less than about 600 p.s.i., and more preferably less than about 500 p.s.i.  
         [0039]     (b) The actuator  53  moves the injector  45  to a position over the container  54  and lowers the injector  45  so that the needle  52  presses against the container bottom  56 . This movement opens a passage from the chemical  55  through the needle  52  through the injector  45  to the tube  44 .  
         [0040]     (c) The pump  42  aspirates a certain amount of liquid out of tube  44  into pump  42 . This in turn sucks liquid out of the sample container  54  throgh the needle  52  through the injector  45  and into said tube  44 . During this process, a negligible amount of liquid is also sucked out of column  46  and column adaptor  47 , but the amount of this liquid flow is much less than the flow from the container  54  since the resistance to flow within the column  46  is much higher than the resistance to flow within the needle  52 . Pump  42  preferably provides an aspiration pressure greater than the residual pressure remaining after step (a) above.  
         [0041]     (d) Once the desired amount of liquid sample from the chemical  55  has entered the tube  44  the actuator  53  raises the injector  45  from the container  54  so that the needle  52  no longer holds open a passage from the needle  52  to the tube  44 .  
         [0042]     (e) The liquid pumping system  42  resumes delivery of liquid through tube  44  successively into the injector  45  column adaptor fitting  47  column  46  detector  48  and vent tube  58 . This flow of liquid carries the sample deposited in tube  44  into the column, for the purpose of chromatographic separation and detection of the constituents of the liquid chemical  55  as is well known to those skilled in the art. If a high-pressure liquid chromatograph measurement is required, pump  42  preferably should be capable of producing a pressure that is preferably between 1000-8000 p.s.i., more preferably between 3000-5500 p.s.i., and most preferably about 5000 p.s.i.  
         [0043]     (f) When the chromatographic separation and detection of the sample is completed, the injector and needle are rinsed. The liquid pumping system  42  interrupts the flow of liquid to the injector  45  and the pressure drops. The actuator  53  moves the injector  45  to a rinsing container  60  where the needle  52  is pressed against the rinsing container bottom  61  thereby opening a passage between the needle  52  and the tube  44 . Now the liquid pumping system sends liquid through the tube  44  successively through the injector  45  and needle  52 . The amount of this flow is set to be sufficient to remove residues of the sample taken during the aspiration of sample from the liquid chemical  55  so that subsequent samples will be substantially free of contamination from previous samples.  
         [0044]      FIG. 3  shows a further embodiment of the injector  45 , which is mounted on a mechanical transporter  53 . There is an upper liquid flow path, composed of the tube  44  connected to an injector body  45  with a tube fitting  69 . The tube  44  is capable of sustaining the high pressures typical of HPLC, e.g., from 1000-8000 p.s.i., and has sufficient internal volume to contain the volume of sample liquid used for chromatography, such as  10  microliters.  
         [0045]     The flow path includes a flow passage  70  within the injector body  45 . The flow passage  70  is limited to a very small volume, with all parts well-swept by the liquid flow, so that when liquid samples are transferred to the column through the flow passage  70 , there is no broadening of the chromatographic output due to dispersion of the sample while in the flow passage  70 . The flow passage  70  communicates with an adaptor fitting  47 . During operation, the tube  44  conducts a solvent liquid into the injector body  45 . The solvent liquid flows through the flow passage  70  and then through the adaptor fitting  47  and into the column.  
         [0046]     A ball  71  makes an intermittent seal against a valve seat  72 . The ball  71  is rigidly attached to a hollow needle  52 . The ball  71  is sufficiently round, smooth and concentric with respect to the hollow needle  52  to serve as a high-pressure seal, in combination with the valve seat  72 .  
         [0047]     The valve seat  72  is designed to form a high-pressure seal with ball  71 . So it is either hard and very smooth or somewhat smooth and resilient, as is necessary in forming a high-pressure seal. The seal between the ball  71  and value seat  72  is suitable for intermittent operations, with many thousands of cycles before replacement, and can seal against the high pressures typical in HPLC, e.g., 1000-8000 p.s.i.  
         [0048]     The hollow needle  52  has a bottom opening  80  communicating with a top opening  74 . The hollow needle  52  is sufficiently narrow near the bottom opening, so that the hollow needle can easily be positioned within a well in a microtiter plate. In operation, a mechanical transporter  53  moves the injector  45  against a microtiter plate, so that the hollow needle  52  is positioned within a well of the microtiter place, and is pressed against the bottom of said well to open the seal of the ball  71 . The hollow needle  52  is sufficiently rigid that it will not buckle when pressed against the bottom of the well.  
         [0049]     The hollow needle  52  is positioned so that it penetrates a central hole in the valve seat  72 , and it is spring loaded by spring  77  to pull the ball  71  against the valve seat  72 , so that said ball  71  and said valve seat  72  form a liquid-tight seal.  
         [0050]     In operation, the valve seat  72  is mounted in the injector body  45  and pressed against the injector body  45 , forming a seal. The two seals, between the ball  71  and the valve seat  72 , and between the valve seat  72  and the injector body  45 , prevent flow from the flow passage  70  from entering the hollow needle  52 .  
         [0051]     The combination of the ball  71 , hollow needle  52 , valve seat  72 , and spring mounted into the injector nut  75 . The valve seat  72  is first mounted in or next to a back ferrule  73 , which is in turn pressed against the injector nut  75 . The injector nut  75  is a threaded part, designed to be attached to the injector body  49  for the purpose of supporting the ball  71 , valve seat  72 , and hollow needle  52 . The back ferrule  73  prevents torque being applied to the valve seat  72  when it is being sealed against the injector body  49 .  
         [0052]     The ball  71  is positioned against the valve seat  72 , so that the hollow needle  52  penetrates the valve seat  72 , the back ferrule  73 , and the injector nut  75 , and protrudes beyond the far edge of the injector nut  75 . The spring  77  is mounted around the hollow needle  52  from the far side and is compressed between the injector nut  75  and a collar  78  and a crimp  79 . The combination of collar  78  and crimp  79  are attached to the hollow needle  52  in such a way that the compressed spring  77  applies a force to the hollow needle  52  and thence to the ball  71 , which induces the ball  71  to make a seal against the valve seat  72 . For instance, with a 2 mm diameter ball, a force of 2 pounds is sufficient to make a seal. The crimp  79  may be attached to the hollow needle  52  by brazing, crimping, gluing, or by fitting into a groove in the hollow needle, as with a retaining ring, snap ring, or locking ring.  
         [0053]     An O-ring  76  is mounted around the hollow needle  52  and between the injector nut  75  and the back ferrule  74 . The O-ring  76  seals to the back ferrule  73 . The outside of the hollow needle  52  is smooth so that it can f a sliding seal against the O-ring  76 . Advantageously, O-ring  76  need only withstand lower pressures, such as about 500 p.s.i., since in normal operation, the high pressures used in HPLC are turned off before the ball  71  opens and exposes the O-ring  76  to the higher pressures that the ball  71  is designed to seal against.  
         [0054]     The top opening  74  in the hollow needle  52  is located below the seal between the valve seat  72  and the ball  71  but above the seal between the hollow needle  52  and the back ferrule  73 . The bottom opening  80  allows sample to enter from a well, but the edge of the hollow needle  52  around the bottom opening must be robust enough to support pressure between the well bottom and the hollow needle  52 . The space around the hollow needle  52  below the ball  71  and above O-ring  76  needs to have minimal volume and be easily cleaned by rinsing, so that residual liquid from one sample may be prevented from substantially contaminating a subsequent sample.  
         [0055]     Preferably, the clearance between the hollow needle  52  and injector nut  76  is sufficient to allow freely sliding relative motion but narrow enough to prevent the tilt of the hollow needle sufficient to interfere with the hollow needle passing into a well in a microtiter plate.  
         [0056]     When multiple injectors  45  are installed together, the flow characteristics, especially in the hollow needles  52  and the tubes  44  preferably are substantially the same, so that when sample is aspirated, and the samples enter the tubes, the flows will be substantially the same in each injector  54 ,and substantially the same amounts of samples will enter each tube.  
         [0057]     The ball  71  is preferentially kept small, such as 2 mm in diameter, so that lower forces are required from the spring  77  to make a seal, and to minimize the volume of the flow passage  70 , which is typically less than 10 microliters. The tube  44  should be narrow to minimize broadening of the sample distributions during chromatography, such as 0.1 to 0.5 mm diameter. The volume of the tube  44 , however, needs to be sufficiently large to contain the largest sample injection volume, such as 20 microliters. The above numbers have been found suitable for a certain size of chromatography column, 2 mm diameter and 20 mm length, with a flow rate of 1 to 2 mL per minute. It will be appreciated by those skilled in the art that the above-mentioned dimensions should be scaled, when other chromatographic conditions are used, such as a chromatographic column of different diameter. For instance, with a 4.6 mm diameter chromatographic column, the volumetric dimensions should be about 10 times larger; with a 1 mm column, they should be about 10 times smaller.  
         [0058]      FIG. 4  shows the invention used for multi-channel chromatographic analysis of liquid chemical samples contained in a microtiter plate  103  which has a pattern of 96 wells that are arranged in a rectangular array of 8×12 wells, based on a unit cell of 9 by 9 mm square. Other versions of microtiter plates have 384 wells in a 16×24 array on 4.5 mm centers, or 1536 wells, in 32×48 array, on 2.25 mm centers.  
         [0059]     Several columns  86  such as the four columns shown in  FIG. 4  are mounted on a series of injectors  83 . The injectors  83  may be fashioned out of separate parts, and mounted together as shown in  FIG. 4 , or machined in a common block. The injectors  83  are rigidly mounted onto a mechanical transporter  82 . The spacing between the hollow needles  84  in said injectors is preferably 9 mm, in order to match the standard spacing between the wells of the microtiter plate  103 .  
         [0060]     The injectors  83  can each supply liquid to, and receive liquid from, tubes  92 . These tubes  92  all connect to a union fitting  93 , which then is connected to a supply tube  94 .  
         [0000]     Pump Selection Valve.  
         [0061]     During chromatography, the liquid chromatography pump  96  through pump tube  99  supplies flow at high pressure to the injectors  83  and columns  86 . The syringe pump  100  and syringe control valve  112  are generally not designed to withstand the pressures used in liquid chromatography, which can exceed 5000 p.s.i.  
         [0062]     Therefore a pump selection valve  95  is interposed between the flow from the pump tube  99  and the supply tube  94 . During chromatography, the pump selection valve  95  is in the “pump” position  97  permitting flow from the liquid chromatography pump  96 . When the syringe pump  100  is in use, the pump selection valve  95  connects to said syringe pump  100  in place of the liquid chromatography pump  96  by switching to the “aspirate” position  98 .  
         [0000]     Syringe Control Valve.  
         [0063]     The flow from the pump selection valve  95  through the aspiration tube  111  the syringe tube  109  to the syringe pump  100  goes through the syringe control valve  112  when said syringe control valve is in the “fill” valve position  113 . Said valve  112  in its “vent” valve position  114  can divert this flow through the vent tube  116  to vent to waste  117  by. Said valve can also, it its “empty” valve position  115  can allow the syringe pump to empty to vent to waste  117 .  
         [0000]     Syringe Pump.  
         [0064]     The syringe pump  100  has a syringe plunger  101  connected to a syringe driver  102 . The syringe driver  102  can move the syringe plunger  101  with respect to the syringe pump  100  so that the syringe pump  100  can either aspirate liquid when the syringe plunger  101  is pulled out, or can deliver liquid, when the syringe plunger  101  is pushed in.  
         [0065]     Some types of syringe pumps are designed for high pressures. In this case, the pump selection valve  95  can be replaced by a “Tee” connection between the two pump tubes  99  and the supply tube  94 . Alternatively, one or more syringe pumps  100  can be adapted to serve as both the liquid chromatography pump and the pump used to aspirate and deliver samples.  
         [0000]     Aspiration  
         [0066]     In preparing for chromatographic injection, the mechanical transporter  82  moves the injectors  83  so that the hollow needles  84 , which are incorporated into said injectors, simultaneously press against the bottom of a series of wells  104  in the microtiter plate  103 . Then, with the pump control valve  95  in the “aspirate ” valve position  98  and the syringe control valve is in the “fill” valve position  113 , the syringe pump  100 , the syringe plunger  101  and syringe driver  102  cause liquid to be aspirated from the sample wells  104  through the injectors  83  into tubes  92 .  
         [0000]     Injection and Chromatographic Separation  
         [0067]     When the mechanical transporter  82  moves the injectors  83  so that the hollow needles  84  no longer press against the bottom of a series of wells  104  the injectors  83  are no longer connected to the wells  104 . When the pump control valve  95  is returned to the “pump” position  97  the flow is resumed from the liquid chromatography pump  96 , through the pump selection valve  95  the supply tube  94  union fitting  93  tubes  92  injectors  83  and columns  86 . Said flow carries the liquid samples left in tubes  92  and moves it into the columns  86  thereby injecting the sample into the column. As the flow continues, chromatographic separations take place in the columns  86 . The flow through the columns then traverses the second adaptor fittings  87  to the detectors  88  where the passage of various chemical components within the flow is detected. The detectors  88  are connected to a detector controller  90  through detector cables  91 .  
         [0000]     Other operations  
         [0068]     Several auxiliary operations are necessary in liquid chromatography, and that these can easily be performed with the device of  FIG. 4 . For example, the tubes  92  injectors  83  and the needles  84  can be rinsed with a rinsing liquid. This is often but not always the same liquid as is used for chromatography. The mechanical transporter  83  moves said injectors to a series of wells, used as rinse wells  105 . The needles are pressed against the bottoms of the wells  105  in the same way as during aspiration. However, instead of aspirating sample, either the liquid chromatography pump or the syringe pump causes rinsing liquid to flow through tubes  92  injectors  83  and hollow needles  84  into the rinse wells  105 . In addition, other manipulations of valves and pumps can be used to rinse aspiration tube  111  and syringe tube  109 .  
         [0000]     Series of Injections.  
         [0069]     The device of  FIG. 4 , can inject from more than one series of samples wells  104 . For instance, if the mechanical transporter  82  moves the injectors  83  to a second set of wells  106  then the contents of said second set of wells can be chromatographed. In this way, the contents of all or substantially all of the wells in the microtiter plate  103  can be chromatographed.  
         [0070]     When a microtiter plate with a cell spacing of a half or one-quarter of the standard 9-mm cell spacing is used, the series of injectors, 9 mm apart, may still be used to chromatograph all the wells in the microtiter plate. For instance, with a 16-cell row in a 384 well plate, with 4.5 mm spacing, the device of  FIG. 4  can inject and chromatographically separate samples from wells #  1 ,  3 ,  5  and  7 , in the first row. In a second operation, with the mechanical transporter moved by 4.5 mm, the first-row wells #  2 ,  4 ,  6 , and  8  can be processed. Then the mechanical transporter moves so that the device is aligned with wells  9 ,  11 ,  13 , and  15 . After a third movement, the wells at #  10 ,  12 ,  14 , and  16  are processed. With a movement in the other axis, all of the wells in the other 23 columns can be processed.  
         [0071]     While  FIG. 4  shows four injectors  83 , multiple analyses may readily be performed by another number of injectors, such as two, three, six, eight or twelve, where the number of injectors is an even divisor of the number of rows or of columns in the microtiter plate.  
         [0000]     Multiple Rows of Injectors.  
         [0072]     While  FIG. 4  shows the injectors  83  arranged in a line, the injectors may be arranged in more than one line. For example,  FIG. 5  shows a valuable realization of the invention that has an arrangement of two rows of eight injectors, with the spacing between the rows determined by the spacing of the wells. There are eight injectors in a first row  110  with the eight tubes connected into common union fitting  111 . The spacing between the injectors is 9 mm. A second set of injectors is arranged into a second row  112 . The second row  112  is spaced 27 mm from the first row which is triple the spacing between wells in the microtiter plate  103 . The sixteen injectors are all mounted to a mechanical transporter  113 . The mechanical transporter  113  can move the injectors down to press against the wells of the microtiter plate  103  in order to aspirate a liquid sample into each injector.  
         [0073]     Two flow systems  114  are used, one for the first row  110  and the other for the second row  112 . Alternatively, a single flow system may be used, or a larger number of flow systems. For instance, four flow systems could be used, each of which is connected to four injectors.  
         [0000]     Alternative Injector Valve Embodiment  
         [0074]      FIG. 6  shows an alternative embodiment of hollow needle  84  in injector  83 . There may be cases where it is not desirable to press a hollow needle  84  against the bottom of a well  126 . For instance, some samples may contain contamination by solid particles  125  which it would be disadvantageous to aspirate along with the liquid  124 . In this case, it would be desirable to position the tip of the hollow needle  84  above the bottom of the well  126  during aspiration.  
         [0075]     In  FIG. 6 , a flat boss  121  is attached to the hollow needle  84 . The flat boss is attached by means of a threaded collar  120 , which allows the position of the flat boss along the hollow needle  84  to be adjusted. When the needle is inserted into the well  126  the flat boss  121  contacts the upper edge of the well  126  before said hollow needle contacts the bottom of said well. The pressure of this contact opens the ball valve. One advantage of this variation is to provide for use with samples known to have immiscible liquid phases  123  and  124  where the aspirating position of the hollow needle  84  can be adjusted to sample from a particular liquid phase  124 .  
         [0000]     Washing Station  
         [0076]     In  FIG. 4 , some of the wells  105  in a microtiter plate  103  were dedicated to being used as a rinsing station for the hollow needles  84 .  FIG. 7  shows an alternative design, which provides a rinsing station, separate from the wells in the microtiter plate. Two rows of samplers  110  and  112  are rigidly mounted on a common transporter  113 . The transporter  113  not only can move the injectors  110  and  112  to different positions over the microtiter plate  103  but can also move the injectors to a separate washing station  131 , which has receiving wells disposed to permit the injectors to press down on the washing station  131  and to dispense waste rinsing liquid into the receiving wells. In addition, this arrangement may be used to aspirate a washing liquid from the washing station  131 .  
         [0000]     Control of Injection Volume.  
         [0077]     With further reference to the embodiment shown in  FIG. 4 , it is highly preferable for sample to be aspirated only into the tubes  92 , not into the union fitting  93  or supply tube  94 . Otherwise, when flow is resumed from the liquid chromatography pump  96 , a residual mixture from all the aspirated samples will be introduced into each column  86 . However, if samples are aspirated only part way through tube  92 , it may be difficult to control the volume of sample.  
         [0078]     In  FIG. 8 , the difficulty of controlling the sample volume is addressed.  FIG. 8  shows a two-channel system, with columns  140  attached to an injector block  141 , which contains several injectors, two of which are connected. Sample tubes  142  are of definite volume, and will determine the volume of the injection. Flow from the LC Pump  155  goes through the solvent delivery tube  147 , the solvent tee  146  and the two sample-solvent tubes  144  to the sample tees  143 . The sample tees  143  also connect to the syringe pump  157  through the aspiration tubes  145 , two-position valve  154 , valve tubes  149 , syringe tee  150 , syringe tube  151 , syringe valve  156  and tube  158 .  
         [0079]     In this realization, the flow from the LC Pump  155  always flows during sample aspiration. This requires that the rate of aspiration by the syringe pump  157  is higher than the flow rate from said LC pump. Unlike the realization of  FIG. 4 , sample is aspirated until the flow of sample over fills the sample tubes  142  and flows into the aspiration tubes  145  or even further. After sufficient sample has been aspirated, the injector block  141  is raised, so that the valves within the injector block close. This ends the aspiration of sample.  
         [0080]     The flow from the LC pump  155  continues, and is directed through the aspiration tubes  145  ultimately to the syringe valve  156 , where it is either aspirated into the syringe pump  157  or directed to the vent tube  152  to vent to waste  153 . This process rinses any remaining sample in the sample tees  146 , aspiration tubes  145  and other parts of the flow system.  
         [0081]     Next, the two-position valve  148  switches, so that the syringe pump  157  and syringe valve  156  are isolated from the aspiration tubes  145 . Now the flow from the LC Pump  155  has nowhere to go except through the columns  140 . So the flow sweeps the sample that resides in the sample tubes into the columns. Note that the volume of each sample is precisely set by the volume of each sample tube, along with a small, defined volume in the injector block. In this way, the two problems of a defined sample volume, and cross-sample contamination are eliminated.  
         [0082]     The following components are suitable for use with the invention:  
                                                                         Identifier/Name   Description                                140   column   Thermo Hypersil-Keystone Javelin column,               2.1 mm id, 20 mm long, 5-micron packing,               coated with Beta Basic C18       141   injector block   —       142   sample tube   8.3 cm long, 0.25 mm id. PEEK       143   sample tee,   three way, for 1/16″ tubing, 10-32               fittings, PEEK       146   solvent tee   three way, for 1/16″ tubing, 10-32               fittings, PEEK       150   syringe tee   three way, for 1/16″ tubing, 10-32               fittings, PEEK            Other Tubes   0.5 mm id, PEEK, length as follows:            144   solvent-sample tube   30 cm long       145   aspiration tube   30 cm long       147   solvent delivery   25 cm long           tube       149   valve tube   20 cm long       151   syringe tube   30 cm long       158   tube   30 cm long       148   two-position valve   Valco, Cheminert, rotary valve, 6-port       152   vent tube   17 cm long, 1/16″ o.d. PTFE       153   vent to waste   —       154   plug   —       155   LC Pump   Pharmacia “Bromma” 2249 Gradient               Pump       156   syringe valve and   both part of Cavro XP3000 modular digital       157   syringe pump:   pump                  
 
 Integrated Sample Tubes 
 
         [0083]      FIGS. 9A and 9B  show another realization of the invention. Here the sample tubes have been incorporated into a sample tube block. In  FIG. 9A  the sample tube block  168  is rigidly fastened to the injector block  167  by a fastener  169 . The sample tube block has three sets of interconnected channels lying in a plane. Multiple cross channels  176  contain the sample tubes  175 . The sample tubes meet with or penetrate into the injector block  167 . Sample tube seals  170  seal the sample tubes to the injector block  176  and to the sample tube block  168 . They also seal between the injector block  167  and the sample tube block  168 .  
         [0084]     Each cross channel  176  is intercepted by a solvent channel  177 , which in turn communicates with the solvent-sample tube  163 , a solvent flow circuit  162  and an LC Pump  161 . Note that the solvent channel  177  intercepts the cross channel  176  between the sample tube seal  170  and the mixing point  179  where the flow from the LC Pump, through the solvent channel  177  meets with the flow to or from the syringe pump  164  through the aspiration channel  178 .  
         [0085]     Each cross channel  176  is intercepted by the aspiration channel  178  which also connects to the aspiration tube  166  which connects to a syringe flow circuit  165  and thence to the syringe pump  164 . Note that the aspiration channel  178  intercepts the cross channel  176  beyond the end of the sample tube  175  and the mixing point  179 .  
         [0086]     With this realization, sample can be aspirated through each sample tube  175  so that some sample flows past the mixing point  179  into the aspiration channel  178  and aspiration tube  166 . The portion of this flow path past the mixing point  179  can be rinsed by flow from the LC pump  161  solvent-sample tube  163  solvent channel  177  and thence, along the outside of the sample tube  175  to the mixing point  179 .  
         [0087]     This realization does not depend on tees for the mixing point  179  as were used in  FIG. 8  and permit the sample tube  175  and mixing point  179  to be made compactly and precisely, with little dead volume. It also has the advantage that, the volume of the sample tube  175  can be adjusted simply by changing the length or inside diameter of said sample tube with no change to other dimensions.  
         [0000]     Unequal Column Flow  
         [0088]     In  FIG. 9A , where a source of flow is split between columns, a possible difficulty is that the flows may not be the same in each column. This difference in flows may cause solvent flows from one cross channel  176  and sample tube  177  where the column flow is lower, to flow over to another cross channel, where the column flow is higher.  
         [0089]     One way this difficulty can be eliminated or greatly reduced is by arranging, during chromatography, to have some bleed flow out of the sample tube block  168  into the aspiration tube  166 . For instance, a tube with restricted flow can be mounted in the syringe flow circuit  165  so that said tube can be connected from the aspirator tube to vent to waste, thereby permitting a low bleed flow to sweep the aspirator channel  178  and aspirator tube  166 . Alternatively, the bleed flow can flow in the alternative direction. With this arrangement, a difference in flow in a column is accommodated by adjustments in the bleed flow.  
         [0000]     Independent Flow Sources  
         [0090]     Another realization uses independent flow sources for each column, but still uses a common syringe pump for aspiration. This permits independent control over the chromatographic flow in each channel.  
         [0091]      FIG. 10  shows a sample tube block  182  mounted on an injection block  183  with sample tubes  184  located in cross channels  185 . Instead of a single solvent-sample tube feeding connecting to multiple cross channels, as in  FIG. 9 , in  FIG. 10  there are multiple solvent-sample tubes  187  each of which is joined to a cross channel  185 . There are multiple aspiration tubes  186  each of which is joined to a cross channel  185 . Seals  181  are used to seal the various components. Each solvent-sample tube  187  is connected to a solvent inlet port  188  where a source of solvent flow can be connected.  
         [0092]     Each aspiration tube  186  connects to a separate port  194  on a valve  190 . The valve  190  in one position closes off the aspiration tubes  186 . In another position, said valve connects each aspiration tube  186  to another valve port  195  which connects to a syringe valve  191  and syringe pump  192 . For example, the valve  190  may be a rotary valve, with a rotor with a cross-shaped groove  196  which can simultaneously connect the separate ports  194  to another valve port  195 .  
         [0093]     This realization permits totally independent control of the chromatographic flow in each column, since independent sources of chromatographic flow are connected to each column, via solvent inlet ports  188 . During aspiration of samples, only a single syringe pump  191  is needed. Even if the flows in the sample tubes  184  are not identical during aspiration, this realization permits substantially known and equal injection volumes, since the sample tubes  184  can be overfilled, and the excess sample can be washed out of the aspiration tubes  186  by flow of solvent, through the aspiration tubes  186  and the valve  190 .  
         [0000]     Flow Normalization  
         [0094]     Beneficially, the flow and parameters of the injectors and columns in the present invention are the same or very similar. It is an advantage of this invention that it takes advantage of this similarity to control flows to each column simply by spitting the flow into several nearly equal flows, relying on the similarity of the column flow resistances to give nearly equal flows. Since the flows may not be identical, however, software adjustments may used to compensate for the small differences.  
         [0095]     As is commonly done in liquid chromatographic or electrophoretic instruments, in this invention the output of the each detector connected to a column produces a series of data-point pairs, T and S, which record the time of the measurement, T, and the signal level, S. A separate series of (T,S) pairs is produced for each column.  
         [0096]     In the data processing step, these series of data-point pairs are analyzed for the presence of chemical compounds, as indicated by an increase in signal level, S, over several measurements. The response of the chemical compound is recorded as the Retention Time, which is the time of the maximum response, and the Area, which is the sum of the signal levels showing a response, with the baseline response subtracted out.  
         [0097]     In order to correct for differences in the flow between columns, as well as for other differences that affect the times, T, a correction is made to each data-point series, prior to the determination of Retention Times and Areas of the chemical compounds.  
         [0098]     A chemical sample containing at least two known compounds is analyzed by all of the columns. The first calibration compound is virtually unretained by the columns, and transits each column at substantially the same rate as a segment of the solvent flow. For each column, this time, T 1 , is measured. The retention time of the first compound under standard conditions, T 1 std, is known. The second compound is retained by the column for a time that is substantially longer than T 1 . The retention time of the second compound, T 2 , is measured. The retention time of the second compound under standard conditions, T 2 std, is known.  
         [0099]     A flow-normalization is then made to the chromatograms, that is, sereies of data-pairs (T, S) of samples containing mixtures of unknown compounds. In each data-pair, (T, S), each time, T, is replaced by a corrected value, T′, where T′=−(T 1 −T 1 std)+T*(T 2 std−T 1 std)/(T 2 −T 1 ). In this way, the flow normalization correction terms, (T 1 −T 1 std) and (T 2 std −T 1 std)/(T 2 −T 1 ), may be customized for each column.  
         [0100]     Once the chromatograms have been flow-normalized, their Retention Times and Area are determined in the normal way. It is noted that normal chromatographic signal processing also calibrates and corrects for variations in signal level, S, based on a calibration sample. This normal procedure can easily be merged with the flow-normalization process.  
         [0101]     It has been found that the Retention Times, T 1  and T 2 , for a calibration sample normally vary slowly over many hours, so that it is feasible and practical to run a flow normalization measurement a few times a day, while processing many sets of unknown samples mixtures in between. The combination of flow normalization and signal-level calibration may also be used to also compensate for other environmental or operational changes that may occur over the course of a day, week or other period of time.  
         [0000]     Advantages of the Invention  
         [0102]     The present invention described above has numerous benefits in comparison with conventional HPLC systems. First, whereas many conventional HPLC systems require successive sample injection steps to process multiple samples, the HPLC system of the present invention allows multiple samples to be injected into a chromatography measurement device (e.g., chromatographic column) simultaneously, in a single step. This simultaneous processing reduces the duration and cost of analyses on multiple samples.  
         [0103]     Second, whereas many conventional HPLC systems require a separate sample loading and transporting step, typically involving the use of a syringe to aspirate a liquid sample and transport it to the input port of an injector valve, the present invention allows the liquid samples to be aspirated and immediately analyzed, without a transport step. This improvement results in a lowered process time and in a higher measurement quality.  
         [0104]     Third, in conventional HPLC systems, variations in flow, viscosity, and pressure make it difficult to inject a known volume. Conventional injectors therefore fill a known volume with the sample liquid at low pressure, and then insert the volume into a high-pressure flow path. In the multiplexed operation according to the invention, the physical conditions of flow, viscosity, and pressure are much more controlled. Therefore it is possible to use a known volume (created by a device like a syringe pump) to set the total volume, and use the uniformity of the columns to allocate this volume among the injection volumes of each injector.  
         [0105]     Fourth, in a conventional injection valve, an electrical solenoid is commonly used to switch the state of the injection valve. Because the multiplexed chromatography in accordance with the present invention uses a known physical environment (taking samples from a microtiter plate), the need for an electrical solenoid can be avoided through the use of the pressure-sensitive injection needle described above.  
         [0106]     While the invention has been described with reference to the preferred embodiment thereof, it will be appreciated by those of ordinary skill in the art that modifications can be made to the structure and elements of the invention without departing form the spirit and scope of the invention as a whole.