Patent Abstract:
A bubbler is positioned within a solvent reservoir of a chromatographic system with its opening near the bottom of the system to measure the pressure of solvent. The bubbler may use air or may use helium or some other gas so that the solvent can be purged of excess air while its level is being monitored by the bubbler. The bubbler provides a depth signal to a microcontroller that records the drop in pressure and projects a low level of pressure at which point solvent should be replenished. The microprocessor may provide a signal to the operator or terminate operation or automatically replenish solvent depending upon the program.

Full Description:
BACKGROUND OF THE INVENTION  
       [0001]     This invention relates to liquid chromatographic methods and apparatuses and more particularly to methods and apparatuses for monitoring the solvent in a liquid chromatographic system.  
         [0002]     Liquid chromatographic apparatuses that automatically monitor the solvent are known. This feature has become increasingly significant with the increasing use of flash chromatography, arrays of columns that use solvent from the same reservoir and automated unattended operation of the chromatographic systems. Such systems are monitored to avoid having the system run out of solvent in the middle of a chromatographic run and nonetheless continue operation of some parts of the system without one or more of the solvents required. The increased rate at which solvent is used and the ability of some systems to automatically increase the amount of solvent needed during a chromatographic run has resulted in systems unexpectedly running out of solvent during chromatographic runs. For example, some systems can increase the length of a chromatographic run without operator intervention, such as when the programmed time has elapsed but a peak is being detected.  
         [0003]     One prior art system having the feature of monitoring the solvent during a chromatographic run tracks the amount of solvent used during a run in accordance with the program for the run and when the solvent is predicted to run out, terminates the chromatographic run. This system has several disadvantages, such as: (1) it requires that the operator correctly enter into the system the starting amount of solvent; (2) it requires operator intervention when the chromatographic system is terminated to replenish the solvent and reset the system; (3) it is more complicated than desired; and (4) it can fail to provide a warning ahead of time that the system needs to have solvent replenished when the run is automatically extended.  
       SUMMARY OF THE INVENTION  
       [0004]     Accordingly, it is an object of the invention to provide a novel chromatographic system and method.  
         [0005]     It is a still further object of the invention to provide a novel low-cost method for reliably providing substantial amounts of solvent to a chromatographic system.  
         [0006]     It is a still further object of the invention to provide a novel system for avoiding running out of solvent before a chromatographic run is completed.  
         [0007]     It is a still further object of the invention to provide a novel system for stopping a chromatographic run before the solvent is exhausted, which system is relatively simple and inexpensive.  
         [0008]     It is a still further object of the invention to provide a novel system for monitoring solvent that does not require entering the correct amount of solvent into the controller before starting operation.  
         [0009]     In accordance with the above and further objects of the invention, a chromatographic system includes as part of the system a solvent reservoir and a solvent level sensor. When the solvent is low, a solvent-level indicating signal is provided to the operator so that additional solvent can be added by the operator before the system runs out or additional solvent automatically is added. In the preferred embodiment, a bubbler is used to determine the depth of the solvent in a reservoir. The bubbler may also be used to purge the solvent of air and thus reduce the bubbles in the detector. For example, the gas used by the bubbler may be helium which will remove some air and avoid the introduction of air from an air-operated bubbler.  
         [0010]     One unexpected difficulty with this system occurs because the same system is intended to be used with different solvents and in some circumstances, different shaped solvent reservoirs. This circumstance, if not compensated for, increases operator&#39;s involvement to adjust readings in accordance with the density of the solvent and the shape of the containers. However, in one embodiment, the signal from the bubbler or other pressure sensing instrument is used to compensate for possible changes to solvents with different densities and/or changes in the reservoir shape. This is accomplished by determining the rate of change of pressure signal with respect to the rate of usage of solvent as known from the programmed run. These two parameters can be used to determine the time at which the solvent will reach a level that predicts a possible exhaustion of solvent. This information can be provided to the operator or it can be used to automatically replenish the solvent.  
         [0011]     It can be understood from the above description that the liquid chromatographic apparatus and technique of this invention has several advantages, such as: (1) it avoids having solvent run out during a chromatographic run, resulting in wasted solvent, a compromised column, lost sample and/or lost operator time; (2) it reduces the monitoring effort that must be supplied by persons operating the chromatograph; and (3) it is relatively inexpensive and can be implemented principally as software. 
     
    
     SUMMARY OF THE DRAWINGS  
       [0012]     The above noted and other features of the invention will be better understood from the following detailed description when considered with reference to the accompanying drawings in which:  
         [0013]      FIG. 1  is a block diagram of a liquid chromatographic system in accordance with an embodiment of the invention;  
         [0014]      FIG. 2  is a flow diagram of a process of controlling a run in accordance with the invention;  
         [0015]      FIG. 3  is a flow diagram of the operation of the solvent monitoring technique useful in the embodiment of  FIG. 1 ; and  
         [0016]      FIG. 4  is a flow diagram of a program for determining the solvent volume indicating a signal in a manner independent of the density of solvent and the shape of the reservoir used in the embodiment of  FIG. 1 . 
     
    
     DETAILED DESCRIPTION  
       [0017]     In  FIG. 1 , there is shown a block diagram of a preparatory liquid chromatographic system  10  having a pumping system  12 , a column and detector array  14 , a collector system  16 , a controller  18 , a purge system  20 A and  20 B and a liquid level sensor  22 A and  22 B. The pumping system  12  supplies solvent to the column and bands are sensed by a detector array  14  under the control of the controller  18 . The purge system  20 A and  20 B communicates with a pump array  34  to purge the pumps and the lines between the pumps and the columns between chromatographic runs. The pump array  34  supplies solvent to the column and detector array  14  from which effluent flows into the collector system  16  under the control of the controller  18 . The controller  18  receives signals from detectors in the column and detector array  14  indicating bands of solute and activates the fraction collector system  16  accordingly in a manner known in the art. One suitable fraction collection system is the FOXY®  200  fraction collector available from Isco, Inc., 4700 Superior Street, Lincoln, Nebr. 68504. A chromatographic system which may use the novel solvent monitor is described in greater detail in U.S. Pat. No. 6,427,526, to Davison, et al., the disclosure of which is incorporated by reference.  
         [0018]     To detect if solvent in either of solvent reservoirs within solvent reservoir and manifold  30  or  32  is running low, the liquid level sensors  22 A and  22 B are in communication with the solvent reservoir and manifold  30  and the solvent reservoir and manifold  32  respectively to receive signals indicating the pressure near the bottom of the reservoirs  30  and  32  and to supply that information to the controller  18  to which each of them is electrically connected. In the preferred embodiment, the liquid level sensors are bubblers which have Teflon tubing or other tubing that is compatible with the solvent extending to the bottom of the reservoirs to measure the pressure at the bottom of the reservoirs. The use of bubblers is advantageous since they are inexpensive and only the tubing, which can be selected for compatibility with the solvent extends into the reservoir. They may operate from a gas supply which, in some embodiments, may operate the purge systems  20 A and  20 B as well. A suitable bubbler is disclosed in U.S. Pat. No. 5,280,721 to Douglas T. Carson, the disclosure of which is incorporated by reference although many bubblers are available on the market and are suitable for use in this invention.  
         [0019]     Generally, the bubblers will operate from an air supply that may also be used in the purge system. However, an additional benefit can be obtained by using helium or other suitable gas in the bubbler. This will permit the gas escaping from the end of the tubing in the bubbler to also remove air or moisture or other undesirable substances within the reservoir. For example, helium is commonly used to remove air from solvent.  
         [0020]     To supply solvent to the pump array  34 , the pumping system  12  includes a plurality of solvent reservoirs and manifolds, a first and second of which are indicated at  30  and  32  respectively, a pump array  34  and a motor  36  which is driven under the control of the controller  18  to operate the array of pumps  34 . The controller  18  also controls the valves in the pump array  34  to control the flow of solvent and the formation of gradients as the motor  36  actuates pistons of the reciprocating pumps in the pump array  34  simultaneously to pump solvent from a plurality of pumps in the pump array  34  and to draw solvent from the solvent reservoirs and manifolds such as  30  and  32 . Valves in the pump array  34  control the amount of liquid, if any, and the proportions of liquids from different reservoirs in the case of gradient operation that are drawn into the pump and pumped from it. The manifolds communicate with the reservoirs so that a plurality of each of the solvents such as the first and second solvents in the solvent reservoir manifold  30  and  32  respectively can be drawn into the array of pumps  34  to permit simultaneous operation of a number of pumps. In some embodiments, the controller  18  may provide a signal on conductor  97  to cause solvent to flow from a large source of solvent into individual reservoirs that are low on solvent. In some embodiments, the controller  18  stops the run when a low level signal is received or causes the read-out display  125  to indicate a low solvent level.  
         [0021]     While in the preferred embodiment, arrays of pumps, columns and detectors are used, any type of pump, column or detector is suitable. A large number of different liquid chromatographic systems are known in the art and to persons of ordinary skill in the art and any such known system may be adaptable to the invention disclosed herein with routine engineering. . While two solvents are disclosed in the embodiment of  FIG. 1 , only one solvent may be used or more than two solvents.  
         [0022]     To process the effluent, the collector system  16  includes a fraction collector  40  to collect solute, a manifold  42  and a waste depository  44  to handle waste from the manifold  42 . One or more fraction collectors  40  communicate with the column and detector array  14  to receive the solute from the columns, either with a manifold or not. A manifold  42  may be used to combine solute from more than one column and deposit them together in a single receptacle or each column may deposit solute in its own receptacle or some of the columns each may deposit solute in its own corresponding receptacle and others may combine solute in the same receptacles. The manifold  42  communicates with the column and detector array  14  to channel effluent from each column and deposit it in the waste depository  44 . The fraction collector  40  may be any suitable fraction collector such as that disclosed in U.S. Pat. No. 3,418,084 or the above-identified FOXY fraction collector.  
         [0023]     In  FIG. 2 , there is shown a block diagram of a purge system  20 A, a liquid level sensor  22 A,  22 B, and the solvent reservoir and manifold  30 . In  FIG. 1 , two such identical systems are used but for clarity only one will be described herein. As explained in conjunction with  FIG.1 , the purge system  20 A supplies gas to any of a plurality of liquid level sensors, liquid level sensors  22 A and  22 B being shown in  FIG. 2  for illustration. Each of the liquid level sensors  22 A and  22 B communicates with a different reservoir and with the controller  18  ( FIG. 1 ) to monitor the solvent level in a corresponding reservoir. Several reservoirs may be associated with a single manifold so that different solvents may be utilized in chromatographic runs by operation of a valve with purge operation occurring between runs. Thus, different reservoirs with different solvents in them may be selected easily. In a simplified system, only one reservoir would be utilized with a single solvent. In any of the systems, the reservoirs may communicate with a large source of solvent and a signal indicating a low solvent level, instead of only informing the operator, could cause a replenishment of solvent into the reservoir from a larger supply or a main supply  95  illustrated with respect to reservoir  50 A in  FIG. 2 .  
         [0024]     In one embodiment, the purge system  20 A includes a gas source  90 , a plurality of three-way valves, two of which are shown at  94 A and  94 B, a tank pressure monitor  92  and a gas pump  99 . The gas source  90  may be a reservoir which is supplied by the gas pump  99  and may contain air or in some embodiments, helium. In a simplified version, the gas pump  99  alone pumps air that may or may not be utilized to supply air for purging purposes. A simple diaphragm pump without pressure regulation or a reservoir may be adequate for some applications. For a more reliable operation, the tank pressure monitor  92  is connected to the gas source  90  and controls the pressure of the gas in the gas source  90  so that the purge operation may be done reliably at a selected gas pressure and more significantly, the gas pressure used in the bubblers may be reliably controlled. The embodiment shown in  FIG. 2  has the gas pump  99  communicating through the tank pressure monitor  92  with the gas source  90  which is a gas reservoir to maintain a reliable preset gas pressure in the gas source. The gas source is then connected to the liquid level sensors, two of which are shown at  22 A and  22 B. In the preferred embodiment, the liquid level sensors are bubblers and the gas source supplies gas to the bubblers for the purpose of sensing the amount of solvent in the reservoirs. A plurality of three-way valves, valves  94 A and  94 B being shown by way of example in  FIG. 2 , may communicate with the gas source  90  to receive gas and, in one position of the three-way valve, supply it to purge systems through corresponding ones of check valves  101 A and  101 B.  
         [0025]     The liquid level sensor  22 A includes an adjustable or fixed orifice  115  connected to the gas source  90  to supply a continuous flow of air through tubing  103  to the bottom of reservoir  50 A to cause bubbles to flow from the outlet at  105  of the tubing  103  against the pressure of liquid  54  at the bottom of the reservoir  50 A. A transducer in the bubbler console  107  provides a signal indicating the pressure needed to maintain the air flow from the head of solvent in the reservoir. This signal may be used by the controller  18  ( FIG. 1 ) to indicate when solvent is low and the system should be stopped or the solvent should be automatically replenished by main supply  95  and/or a message provided to the operator in a display on the controller  18  ( FIG. 1 ). The signal representative of the pressure as measured by the bubbler console  107  is supplied to the controller  18  ( FIG. 1 ) through a conductor  117 A to indicate the level of the solvent in reservoir  50 A. The second liquid level sensor  22 B functions in the same manner as the first liquid level sensor  22 A and is shown only generally in  FIG. 2 .  
         [0026]     In one embodiment, the solvent reservoir and manifold  30  utilizing more than one reservoir with the more than one solvent as indicated at reservoirs  50 A and  50 B includes a valve  119  communicating with all of the reservoirs, two of which are shown at  50 A and  50 B as well as with manifold  52  and the liquid level sensor  22 B so that the valve  119  receives fluid from the plurality of reservoirs, two of which are shown at  50 A and  50 B containing different solvents and selects one ofthem forapplicationtothe manifold  52 . Eachofthe reservoirs such as  50 A and  50 B is connected to a corresponding liquid level sensor such as  22 A or  22 B, which in turn provides signals to the controller  18  through their electrical outlets  117 A and  177 B respectively.  
         [0027]     The first solvent reservoir and manifold  30  includes a first manifold  52  having one inlet and ten outlets  58 A- 58 J, a conduit  56  and a first solvent reservoir  50 A, which solvent reservoir  50 A holds a first solvent  54 . The conduit  56  communicates with the solvent  54  in the solvent reservoir  50 A through the valve  119  on one end and communicates with the interior of the manifold  52  at its other end. Each of the outlets  58 A- 58 J of the manifold  52  communicate with the interior of a different one of ten cylinders of the pumps (not shown in  FIG. 2 ) through appropriate valves. Similarly, the second manifold ( FIG. 1 ) communicates with a second solvent in a second solvent reservoir through a another conduit. The second manifold also includes a plurality of outlet conduits that communicate with the interiors of a corresponding number of pump cylinders through appropriate valves as described in more detail in the aforesaid U.S. Pat. No. 6,427,526, to Davison, et al., so that the solvent from the reservoir  50 A and the solvent from the second reservoir may be mixed together in a proportion that is set in accordance with the timing of the valves.  
         [0028]     The check valves  101 A and  101 B communicate with purge manifolds (not shown in  FIG. 2 ) to provide communication with the gas source  90  through conduits  91 A, and  91 B and the pressure monitor  92  and the three-way valves  94 A and  94 B to maintain an appropriate pressure for purging the lines. These purge manifolds each have ten outlets, each communicating with a different one of the ten conduits connecting a corresponding one of the corresponding pumps to a corresponding one of ten corresponding columns to transmit gas back through the piston pumps to purge the cylinders of the piston pumps and the conduits connecting the pumps to the columns. Each of the conduits connected to the purge connector arrangement lead to a corresponding pump in the pump array  34  ( FIG. 1 ) which in turn communicates with the corresponding one of the columns in the column and detector array  14  ( FIG. 1 ). Between chromatographic runs, the pressurized gas source  90 , which is commonly a source of air, nitrogen or helium gas, communicates through the pressure regulator  92  and the three-way valves  94 A, and  94 B with the manifold to provide purging fluid to each of the corresponding outlets for each of the pump and column combinations.  
         [0029]     While in the embodiment shown in  FIG. 2  as an example, the manifolds each have ten outlet conduits which communicate with ten pump cylinders through appropriate valves as will be described hereinafter, each could have more or less than ten outlets and a manifold is not required for many chromatographic systems in which this novel solvent monitoring system has utility. Each of the reservoirs in the embodiment of  FIG. 2  is similar to the reservoir  30  and operates in a similar manner to provide the same solvent from the same reservoir to a plurality of pump cylinders for simultaneous pumping of the solvent into a plurality of columns and therefore only one is shown in detail in  FIG. 2  for simplicity.  
         [0030]     In  FIG. 3 , there is shown a flow diagram  120  of the operation of the solvent monitoring technique having the step of selecting a solvent  122 , the step of programming a chromatographic run  124 , the step  126  of measuring the volume of solvent in the reservoir with a solvent compatible sensor, the step  128  of generating a solvent level indicator signal when the solvent is low and the step  130  of stopping the chromatographic run at a preset solvent level. With this process, in some embodiments, a particular solvent may be selected as shown at step  122  from selection valves which connect different solvent reservoirs through a multi-position valve to supply fluid from any of the reservoirs into the system. Each of the reservoirs has associated with it a solvent monitoring system, which in the preferred embodiment, includes a bubbler to sense the depth of the solvent.  
         [0031]     With this arrangement, the chromatographic run is programmed as shown at step  124 . For example, a gradient may be programmed into the controller  18  ( FIG. 1 ) to draw one selected fluid from one reservoir and supply it to a mixer together with another solvent from another reservoir, typically with the strongest solvent starting at zero and the weakest solvent starting at 100% and then gradually increasing the stronger solvent and decreasing the weaker solvent to maintain the total flow equal. The timing of the mixture is programmed in accordance with the separation to be performed.  
         [0032]     As shown in step  126 , as the volume of the solvent in each of the reservoirs is supplied to the chromatographic system, the volume left in the reservoir is measured. This measurement may be accomplished in several different ways. In a preferred embodiment, the change in pressure with respect to the volume of solvent supplied to the columns during the chromatographic run is determined by measuring the pressure at several points during the run. This rate of change of pressure with respect to volume of solvent can be used to predict when the level when the reservoir will run out of solvent, which corresponds to zero pressure, or when it will reach a value preset by the operator to stop the run or provide a message to the operator or automatically supply more solvent to the reservoir. The pressure measured at any point minus the product of the rate of decrease of the pressure per unit of solvent supplied to the columns and the amount of solvent that will be supplied to the columns as programmed in the chromatographic run provides an indication of how close to zero pressure which is an empty reservoir the system will be at any point in the run. Thus a signal indicating a low level of solvent can be given when needed without knowing the exact amount of solvent is in the reservoir at the start of the run.  
         [0033]     Of course the signal can be generated in other ways such as by filling the reservoir to a predetermined level and measuring the amount of solvent that is being supplied to the columns by measuring the change in pressure. The amount of the solvent is programmed and the sensor can determine the change in pressure in the reservoirs. From these determinations, based on a change from maximum pressure to a zero pressure, the curve indicating the drop with respect to solvent used indicates the amount of solvent left in a manner independent of the density of the solvent and the shape of the container. On the other hand, the system may be programmed to take into account the density of the liquid and thus indicate the height of the liquid, and the shape of the container can be programmed so as to easily calculate the amount of solvent that is left in the reservoir.  
         [0034]     As shown at step  128 , when the solvent reaches a generally low level, a low volume indicator signal may be generated to stop the system. Also, as the solvent is depleted, the controller can generate an indication of the amount of solvent left and indicate the amount of solvent to the operator. Thus, as shown at step  130 , the chromatograph may be automatically stopped so as to avoid ruining the column and the run until an operator can replenish the reservoir. In the alternative, the solvent level indicating signal can be used to automatically open a valve to a master source of solvent so that the solvent can replenish the reservoirs.  
         [0035]     In  FIG. 4 , there is shown a flow diagram of a program  140  for determining the solvent volume indicating signal in a manner independent of the density of the solvent and the shape of the reservoir. As shown in flow diagram  140 , a volume of solvent is supplied to the reservoir. A pressure measurement is taken before the chromatographic run so as to obtain a signal indicating the total amount of solvent in the reservoir as shown as step  144 . As shown at step  146 , a series of pressure measurements at known volumes in the reservoir during the chromatographic run are taken as the program proceeds and each of these steps is correlated with the programmed amount of solvent to be supplied from the reservoir. As shown in step  148 , at least an approximate rate of change of pressure is determined with respect to the volume of solvent. As shown in step  150 , the remaining volume of solvent in the reservoir is projected from the rate of change of said starting volume. This succession of steps and progress through the program indicates a rate of use of the solvent with respect to the stage of the chromatographic run so as to be able to predict when a low volume of solvent will be left. At that point in time, a low solvent signal may be provided to inform the operator that the solvent is low. The stage of the chromatographic run may be determined in either of terms of volume or in terms of time of the run. In the preferred embodiment, it is determined in terms ofvolume and the read-out with peaks is correlated with the volume of fluid that has flown through the column. The starting solvent minus the rate of change of pressure with respect to time or volume programmed multiplied by the starting volume in the run indicates the amount of solvent left.  
         [0036]     It can be understood from the above description that the liquid chromatographic apparatus and technique of this invention has several advantages, such as: (1) it avoids having solvent run out during a chromatographic run, resulting in wasted solvent, a compromised column, lost sample and/or lost operator time; (2) it reduces the monitoring effort that must be supplied by persons operating the chromatograph; and (3) it is relatively inexpensive and can be implemented principally as software.  
         [0037]     Although a preferred embodiment of the invention has been described with some particularity, it is to be understood that the invention may be practiced other than as specifically described. Accordingly, it is to be understood that, within the scope of the appended claims, the invention may be practiced other than as specifically described.

Technology Classification (CPC): 6