Abstract:
Described are a method and system for diluting a sample in a liquid chromatography system. A sample flowing at a first flow rate is combined for a predetermined time with a diluent flowing at a second flow rate to generate a volume of diluted sample. The diluted sample has a dilution ratio determined according to the two flow rates. A portion of the volume is loaded into a sample loop of an injection valve and subsequently injected into a mobile phase flowing to a chromatography column. The method eliminates the need to have a technician available to perform dilutions or to transfer the sample to a remote location for dilution. Advantageously, manufacturing or processing downtime to perform the dilution is not required and the amount of sample flowing to waste is reduced.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to and benefit of U.S. Provisional Application No. 61/156,040, filed Feb. 27, 2009, the contents of which are expressly incorporated herein by reference in their entirety. 
    
    
     FIELD OF THE INVENTION 
     The invention relates generally to liquid chromatography systems. More particularly, the invention relates to a method for conveniently and efficiently diluting a sample for injection into a mobile phase in a liquid chromatography system. 
     BACKGROUND OF THE INVENTION 
     High pressure liquid chromatography (HPLC) systems sometimes require that a sample be diluted before it is injected into the mobile phase flowing to a chromatography column. Various reasons exist for diluting the sample prior to injection. For example, a system may not be capable of injecting a sufficiently small quantity of sample (e.g., picoliters) to avoid a mass overload condition for the chromatography column. Alternatively, the native solvent containing the sample may be inappropriate for the stationary phase due to a physical property (e.g., pH level) of the solvent. In another example, the sample may be dissolved in a strong solvent that interacts with the stationary phase, leading to ambiguous results in the chromatogram. 
     Dilutions can be performed manually by a skilled technician; however, it is not always practical to keep a technician available to perform dilutions. In many instances, the technician and equipment for performing the dilution are located an inconvenient distance from the HPLC system. Significant delay can occur if the sample is transferred to a remote location for dilution, potentially resulting in manufacturing or processing downtime. Moreover, the additional inconvenience of tracking the transported sample is often necessary. 
     The present invention addresses the problems set forth above and provides additional advantages. 
     SUMMARY OF THE INVENTION 
     In one aspect, the invention features a method of diluting a sample in a liquid chromatography system. A sample flowing at a first flow rate is combined for a predetermined time with a diluent flowing at a second flow rate to generate a volume of diluted sample. The diluted sample has a dilution ratio responsive to the first and second flow rates. A portion of the volume is loaded into a sample loop of an injection valve and subsequently injected into a mobile phase flowing to a chromatography column. 
     In another aspect, the invention features a method of diluting a sample in a liquid chromatography system. A sample flowing at a first flow rate and a diluent flowing at a second flow rate are combined for a predetermined time to generate in a fluid channel a volume of diluted sample. The diluted sample has a dilution ratio responsive to the first and second flow rates. A portion of the volume is loaded into a sample loop of an injection valve. The injection valve is switched so that the sample loop is inserted into a channel for a mobile phase flowing to a chromatography column and the portion of the volume is thereby injected into the mobile phase. 
     In yet another aspect, the invention features a system for injecting a diluted sample in a liquid chromatography system. The system includes a sample source, diluent source, control module, fluid combiner and injection valve. The sample source and diluent source supply sample and diluent, respectively, during a dilution time. The control module is in communication with the sample source and the diluent source to control the first flow rate, the second flow rate and the dilution time. The fluid combiner has a first inlet port in communication with the sample source, a second inlet port in communication with the diluent source and an outlet port. The fluid combiner supplies a volume of a diluted sample through the output port during the dilution time. The injection valve has a sample loop and is in communication with the outlet port of the fluid combiner. The injection valve is configured to load a portion of the volume of the diluted sample into the sample loop during a loading time. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The above and further advantages of this invention may be better understood by referring to the following description in conjunction with the accompanying drawings, in which like numerals indicate like structural elements and features in the various figures. For clarity, not every element may be labeled in every figure. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. 
         FIG. 1  is a schematic illustration of a portion of a liquid chromatography system according to an embodiment of the invention. 
         FIG. 2  is a flowchart representation of an embodiment of a method of diluting a sample in a liquid chromatography system according to the invention. 
         FIG. 3  is an illustration of the portion of the liquid chromatography system shown in  FIG. 1  configured for dilution of a sample. 
         FIG. 4  shows an alternative embodiment to  FIG. 3  that enables sampling of a flowing stream in a process line. 
         FIG. 5  is an illustration of the portion of the liquid chromatography system shown in  FIG. 3  after configuring the injection valve in a load position. 
         FIG. 6  is an illustration of the portion of the liquid chromatography system shown in  FIG. 5  after configuring the injection valve in an inject position. 
         FIG. 7  illustrates a configuration of a portion of a liquid chromatography system during preparation for a wash stage. 
         FIG. 8  illustrates the system of  FIG. 7  during a wash stage. 
     
    
    
     DETAILED DESCRIPTION 
     In brief overview, the invention relates to a method of diluting a sample in a liquid chromatography system. A sample flowing at a sample rate and a diluent flowing at a diluent rate are combined for a certain time to generate a volume of diluted sample. A portion of the volume is loaded into a sample loop of an injection valve and subsequently injected into a mobile phase flowing to a chromatography column. The method reduces the amount of sample wasted in conventional dilution processes for liquid chromatography. 
       FIG. 1  is a schematic illustration of a portion of a liquid chromatography system according to an embodiment of the invention. The system includes a sample reservoir  14  and diluent reservoir  18 . The sample reservoir  14  can be a vial or other container that holds a quantity of sample to be injected into a chromatography column (not shown) after dilution. Similarly, the diluent reservoir  18  can be a container that holds a quantity of diluent used to dilute the sample. The system also includes a sample syringe  22  that is coupled through a sample valve  26  and tubing  30  (or other fluid channel or conduit) to the sample reservoir  14 . In the illustrated embodiment, the tubing  30  is coupled to a sample needle  34  that is positioned in the sample reservoir  14  when the sample is drawn into the tubing  30  although in other embodiments the sample needle  34  may be absent so that the tubing  30  is directly coupled to the sample reservoir  14 . A diluent syringe  38  is coupled through a diluent valve  42  and tubing  46  to the diluent reservoir  18 . The system also includes an injection port  50  configured to receive the sample needle  34  at certain times during a chromatography cycle as described in more detail below. 
     The diluent valve  42  and the injection port  50  are each coupled by tubing or other fluid channel  54  and  58 , respectively, to a respective inlet port of a “tee” or other mixing component  60 . An outlet port of the tee  60  supplies a diluted sample to an input port  72  of a six-port injection valve  68  through tubing or fluid channel  64 . In general, the dilution ratio of the diluted sample provided to the injection valve  68  is determined according to the flow rates of the sample and the diluent at the tee  60 . 
       FIG. 2  is a flowchart representation of an embodiment of a method  200  of diluting a sample in a liquid chromatography system according to the invention. Referring also to  FIG. 1 , the sample and diluent valves  26  and  42  are initially configured so that the sample is drawn into holding loop  30  and diluent is drawn (step  210 ) into the syringe  38 , respectively. Preferably, the sample syringe  22  starts at a minimum volume (i.e., “home”) position and a quantity of sample is drawn into and through the sample needle  34  during a portion of the intake stroke. The volume of drawn sample is not sufficient to reach the sample syringe  22 . 
     Subsequently, the needle valve  34  is removed from the sample reservoir  14  and positioned in the injection port  50  before starting dilution as shown in  FIG. 3 . The sample valve  26  and diluent valve  42  are configured so that the sample and diluent are pushed at a predetermined flow rate from their syringes  22  and  38 , and combined (step  220 ) at the tee  60 , yielding after a predetermined duration (i.e., a dilution time) a known volume of diluted sample in the tubing  64  connected to the tee outlet port. Since the sample syringe  22  was activated at its home position during the draw stage ( FIG. 1 ) and because the sample syringe  22  is limited to a maximum of a single stroke during the dilution stage, only sample is pushed through the sample needle  34 . The flow rates of the sample and the diluent are controlled to obtain the desired dilution ratio. For example, the syringes  22  and  38  can be operated by stepper motors under the management of a control module to generate the desired flow rates. 
     In many applications, the amount of sample and/or diluent available in the reservoirs  14  and  18  is limited. In addition, the sample, diluent or both sample and diluent may have special handling and disposal requirements. Thus it can be desirable to limit the amount of sample and diluent consumed during chromatography measurements. According to an embodiment of the invention, the time during which the syringes  22  and  38  push the sample and diluent is accurately controlled to ensure sufficient volume of diluted sample to fill the tubing  64  between the tee  60  and injection valve  68 , and to pass through inlet and outlet ports  72  and  76 , respectively, of the injection valve  68  while limiting the amount of diluted sample exiting to the waste  80 . 
     The internal diameter of the tubing  64  is typically small (e.g., 6 inches of 0.005 inch tubing) and the pressure of the diluted sample flow is typically low (e.g., 20-30 psi). Thus sufficient time is allowed to lapse to ensure that the diluted sample fills the tubing  64  prior to loading the diluted sample into a sample loop  84  of the injection valve  68 . 
     During the period when the diluted sample flow is established, the mobile phase flows under high pressure from a pump system (not shown) through a high pressure inlet port  88 , sample loop  84  and high pressure outlet port  92  of the injection valve  68 . The mobile phase exiting the high pressure outlet port  92  flows to the chromatography column. 
       FIG. 4  shows an alternative configuration to  FIG. 3  where a sampling port  96  is coupled to a process line through a conduit or fluid channel  100  that may include one or more control valves  104 . This configuration enables sampling of a flowing stream in a process line. Samples can be drawn into the sample needle  34  at discrete times. Alternatively, samples can be taken from a slip stream (i.e., sample stream) in a process line. The sample reservoir  14  can be used to supply a standard for the chromatography measurements and can be used periodically between some or all of the sample stream measurements. Optionally, the sample or standard reservoir  14  is pressurized to at least partially compensate for pressure loss in the tubing. For example, the sample reservoir  14  can be a pressurized sample vial or pneumatic amplifiers  15  can be employed to increase the sample flowing from the reservoir  14 . 
     To prepare the diluted sample for injection into the mobile phase, the injection valve  68  is temporarily configured from the position shown in  FIG. 3  to a load position as shown in  FIG. 5  so that the sample loop  84  is removed from the high pressure path for the mobile phase. During the load stage, the diluted sample is received at the inlet port  72  and flows through the sample loop  84  before exiting the injection valve  68  at the outlet port  76 . Thus the diluted sample is loaded (step  230 ) into the sample loop  84  and is available for injection. During the load stage, at least one of the syringes  22  and  38  continues to push sample and/or diluent into the tee  60  and tubing  64 . As illustrated, the sample syringe  22  continues to push sample while the diluent syringe  38  is disabled. This mode of operation may be preferred if the stepper motor control of the sample syringe  22  provides more accurate volume control than would otherwise be possible using stepper motor control of the diluent syringe  38 , thereby enabling more accurate dispensing of the diluted sample to the injection valve  68 . It should be appreciated that other modes of syringe operation can also be used during the load stage, such as operating only the diluent syringe  38  or simultaneous operation of both syringes  22  and  38 . 
     The time during which the sample syringe  22  is active for loading the diluted sample into the sample loop  84  is carefully controlled to ensure that the entire volume of diluted sample in the tubing  64  is not passed to waste  80 . If the duration is too long, the sample loop  84  is loaded with the solvent (e.g., sample) in the tubing  30  that was used to push the volume of diluted sample. Thus knowledge of the volume of the tubing  64  between the tee  60  and injection valve  68 , and the flow rate of the diluted sample through the tubing  64  can be used to determine the proper duration and for synchronizing the activation times of the syringes  22  and  38  and the injection valve  68 . 
     After loading is complete, the injection valve  68  is configured for injection as shown in  FIG. 6  so that the high pressure mobile phase flows through the high pressure inlet port  88 , sample loop  84  and high pressure outlet port  92  before passing to the chromatography column. Thus the diluted sample previously loaded into the sample loop  84  is injected (step  240 ) into the continuously flowing mobile phase. 
       FIG. 7  illustrates a configuration in which a wash solvent is drawn from a wash container  108  into the sample syringe  22 .  FIG. 8  shows how the wash solvent is pushed through the tubing  30  and sample needle  34 . The particular wash solvent used is selected for its capability to dissolve any sample remaining in the sample needle  34 . This process ensures that no sample remains in the sample needle  34  to contaminate a subsequent sample. In some instances where the next sample to be drawn is the same as the previous sample, the wash process can be omitted; however, in other instances it may be preferable to perform the wash. 
     A series of separations may be performed by the chromatography system to obtain a measurement average and to reduce the effect of measurement noise. In this instance the preparation of a diluted sample for each measurement cycle preferably occurs while the immediately preceding separation is performed. 
     Table 1 lists the mean sample detected (peak area counts), sample standard deviation and the standard deviation normalized to the mean expressed as a percentage (i.e., % RSD) for measurements based on six injections for each of six different dilution ratios. The sample is caffeine and the diluent is 90/10 H 2 O/ACN (acetonitrile). The mean sample detected values exhibit a highly linear relationship with respect to the ratio of sample and diluent. A low value for normalized RSD is achieved for most dilution ratios and indicates that the dilution process is well controlled. At high dilution ratios, the relative flow rate for the sample is sufficiently small such that effects due to the discrete nature of the stepper motor driving the sample syringe  22  may be observed. Stated otherwise, the sample flow might not appear to be at a constant rate and can lead to greater variations from the desired dilution ratio. To reduce this effect, different system components can be utilized for different dilution ratio ranges. 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                   
                   
                 STANDARD 
                   
               
               
                   
                 DILUTIONRATIO 
                 MEAN 
                 DEVIATION 
                 % RSD 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                  2:1 
                 289310.2 
                 1458.0 
                 0.50 
               
               
                   
                  5:1 
                 112026.2 
                 618.3 
                 0.55 
               
               
                   
                  10:1 
                 56608.1 
                 352.9 
                 0.62 
               
               
                   
                  50:1 
                 10827.6 
                 74.9 
                 0.69 
               
               
                   
                 100:1 
                 6951.8 
                 127.7 
                 1.84 
               
               
                   
                 200:1 
                 3354.2 
                 31.4 
                 0.94 
               
               
                   
                   
               
             
          
         
       
     
     While the invention has been shown and described with reference to specific embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as recited in the accompanying claims.