Patent Publication Number: US-2019170782-A1

Title: Apparatus having function of diluting sample and method for diluting sample

Description:
FIELD 
     The present disclosure relates to an apparatus, such as an autosampler for liquid chromatography, having the function of diluting a sample and a method for diluting a sample. 
     BACKGROUND 
     An analysis system that has been suggested and implemented automatically performs pretreatment, such as a process for removing specific components unnecessary for analysis from a biological sample to extract required components as a sample and a drying and solidifying process for concentrating, and drying and solidifying, the extracted sample, and analysis such as liquid chromatographic analysis for the pretreated sample (see WO 2014/072979 A1). 
     In such an analysis system, a sample that undergoes predetermined pretreatment with a pretreatment apparatus is transferred to an autosampler of an analyzer. The autosampler collects the pretreated sample using a sampling needle, and injects the collected sample into a channel for analysis. In the autosampler of the analyzer, the sample transferred from the pretreatment apparatus may be prepared by being diluted with a diluent. 
     The sample is diluted in a sample preparation container inside the autosampler in the following manner specifically, a necessary amount of the sample transferred from the pretreatment apparatus and a necessary amount of the diluent are measured and collected using a syringe pump connected to the sampling needle. Then, the sample and the diluent are dispensed into the sample preparation container and then stirred. 
     If, to dilute the sample, the sampling needle is used to collect the sample and then to collect the diluent, the sample unfortunately enters a container storing the diluent, resulting in contamination of the diluent. In addition, the sample remains in the sampling needle, thus causing contamination and impairing the accuracy of the sample amount. 
     For these reasons, the diluent is preferably sucked into the sampling needle before the sample is sucked thereinto, and the diluent and the sample are preferably dispensed together into the sample preparation container. Sucking the diluent before the suction of the sample prevents the inside of the container for the diluent from being contaminated with the sample, and allows the sample in the sampling needle to be washed away with the diluent, thus preventing the sample from remaining in the sampling needle. 
     However, when the diluent is sucked into the sampling needle before the sample is sucked thereinto, and the diluent and the sample are dispensed together into the sample preparation container, the sample is dispensed into the sample preparation container before the diluent is dispensed thereinto. This results in formation of a phase of the sample at the bottom of the sample preparation container. The sample has a higher specific gravity than the diluent does, and thus the sample accumulates at the bottom of the sample preparation container. In such a state, even if the sample is repeatedly sucked into, and discharged from, the sampling needle in the sample preparation container, the sample remains accumulated at the bottom of the sample preparation container. This prevents the sample and the diluent from being properly mixed together. 
     In view of the foregoing background, it is therefore an object of the present disclosure to facilitate mixing the sample and the diluent together while preventing contamination and maintaining the accuracy of the sample amount. 
     SUMMARY 
     An apparatus according to the present disclosure includes: a sample part including a sample; a movable sampling needle configured to suck and discharge liquid through a tip end of the movable sampling needle; a sample preparation container configured to prepare the sample collected from the sample part with the sampling needle; a diluent part configured to store a diluent for diluting the sample; and a control unit configured to control operation of the sampling needle, the control unit including a dilution operation part configured to add the diluent collected from the diluent part to the sample collected from the sample part with the sampling needle to dilute the sample in the sample preparation container. The dilution operation part dispenses the diluent into the sample preparation container with the sampling needle before dispensing of the sample, dispenses, with the sampling needle, the sample into the sample preparation container into which the diluent has been dispensed, and thereafter dispenses, into the sample preparation container, the diluent sucked with the sampling needle before suction of the sample. 
     Specifically, in the apparatus according to the present disclosure, when the sample is diluted, the diluent is dispensed into the sample preparation container before dispensing of the sample, and thereafter the sample and the diluent are dispensed in this order. The diluent dispensed into the sample preparation container after dispensing of the sample is sucked with the sampling needle before suction of the sample. Dispensing the diluent prevents the sample from remaining in the sampling needle or a channel, and allows the sample amount to be accurate. Since the diluent is dispensed into the sample preparation container before dispensing of the sample, the sample is not accumulated at the bottom of the sample preparation container from immediately after dispensing of the sample. Consequently, the sample and the diluent are easily mixed together. 
     In the apparatus of the present disclosure, the dilution operation part may dispense, into the sample preparation container, the diluent sucked with the sampling needle before the suction of the sample, and may thereafter suck, and discharge, liquid in the sample preparation container with the sampling needle once or more times, thereby stirring the sample and the diluent. 
     The sample dilution method according to the present disclosure is a method for diluting a sample by adding a diluent to the sample using a sampling needle in a sample preparation container. The method includes: dispensing the diluent into the sample preparation container with the sampling needle before dispensing of the sample; dispensing, with the sampling needle, the sample into the sample preparation container into which the diluent has been dispensed; and thereafter dispensing, into the sample preparation container, the diluent sucked with the sampling needle before suction of the sample. 
     Specifically, in the method according to the present disclosure, the diluent is dispensed into the sample preparation container before dispensing of the sample, the sample is then dispensed, and further thereafter the diluent sucked with the sampling needle before suction of the sample is dispensed. Dispensing the diluent after dispensing of the sample prevents the sample from remaining in the sampling needle or a channel, and allows the sample amount to be accurate. Since the diluent is dispensed into the sample preparation container before dispensing of the sample, the sample is not accumulated at the bottom of the sample preparation container from immediately after the sample is dispensed. Consequently, the sample and the diluent are easily mixed together. 
     In the method of the present disclosure, the diluent sucked with the sampling needle before the suction of the sample may be dispensed into the sample preparation container, and thereafter liquid in the sample preparation container may be sucked and discharged with the sampling needle once or more times, thereby stirring the sample and the diluent. 
     The apparatus of the present disclosure is configured to dispense the sample into the sample preparation container after dispensing of the diluent and thereafter to dispense the diluent sucked with the sampling needle before the suction of the sample. This prevents contamination, and allows the sample amount to be accurate, while the sample and the diluent are easily mixed together. 
     According to the method of the present disclosure, the sample is dispensed into the sample preparation container after dispensing of the diluent, and thereafter the diluent sucked with the sampling needle before the suction of the sample is dispensed. This prevents contamination, and allows the sample amount to be accurate, while the sample and the diluent are easily mixed together. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a block diagram schematically showing one example of the configuration of an analysis system. 
         FIG. 2  is a plan view illustrating one example of the configuration of a pretreatment apparatus. 
         FIG. 3  is a plan view showing one embodiment of an LC system. 
         FIG. 4A  is conceptual views for illustrating a dilution operation according to the embodiment step by step. 
         FIG. 4B  is conceptual views for illustrating a dilution operation according to the embodiment step by step. 
         FIG. 4C  is conceptual views for illustrating a dilution operation according to the embodiment step by step. 
         FIG. 4D  is conceptual views for illustrating a dilution operation according to the embodiment step by step. 
         FIG. 5A  is conceptual views for illustrating process steps following the dilution operation shown in  FIGS. 4A to 4D . 
         FIG. 5B  is conceptual views for illustrating process steps following the dilution operation shown in  FIGS. 4A to 4D . 
         FIG. 6  is a flowchart showing the dilution operation according to the embodiment. 
     
    
    
     DETAILED DESCRIPTION 
     One embodiment of an analysis system including an apparatus implemented as the apparatus of the present disclosure will be described with reference to  FIG. 1 . 
     An analysis system  1  includes a pretreatment apparatus  100 , a liquid chromatography system (hereinafter referred to as an “LC system”)  200 , a mass spectrometer (MS)  300 , and a system control apparatus  400 . The system control apparatus  400  controls operations of the pretreatment apparatus  100 , the LC system  200 , and the MS  300 . The system control apparatus  400  can be implemented as a dedicated computer or a general-purpose PC with software for controlling and managing the pretreatment apparatus  100 , the LC system  200 , and the MS  300 . 
     Details of the LC system  200  will be described below. The LC system  200  is configured to subject a sample pretreated by the pretreatment apparatus  10  to liquid chromatographic analysis, and includes an autosampler  202  for collecting the sample transferred from the pretreatment apparatus  100  and injecting the sample into an analysis channel for liquid chromatography. The autosampler  202  has the function of diluting the sample transferred from the pretreatment apparatus  100 , and is implemented as the apparatus according to the present disclosure. If the pretreatment apparatus  100  has the function of diluting the sample, the apparatus according to the present disclosure may be the pretreatment apparatus  100 . 
     One example of the configuration of the pretreatment apparatus  100  will be described with reference to  FIG. 2 . 
     The pretreatment apparatus  100  implements required processes for pretreatment using one pretreatment container  150  for each of samples. The pretreatment apparatus  100  includes a plurality of treatment ports for implementing respective pretreatment processes. The pretreatment container containing the sample is arranged in any one of the treatment ports, and thus the sample contained in the pretreatment container is subjected to one of the pretreatment processes corresponding to the one of the treatment ports. 
     The pretreatment container  150  is conveyed with a conveying arm  124 . The conveying arm  124  has a holding part  125  holding the pretreatment container  150  at its distal end, and rotates in a horizontal plane about a vertical shaft  129  holding the proximal end of the conveying arm  124  so that the holding part  125  draws an arc-shaped trajectory. The treatment ports and other ports that are destinations of the pretreatment container  150  conveyed are all provided along the arc-shaped trajectory drawn by the holding part  125 . 
     A sample setting part  102  is provided to set sample containers  106  containing the sample. A sampling arm  120  is provided near the sample setting part  102  to collect the sample from the sample containers  106  set in the sample setting part  102 . The sample setting part  102  is provided with sample racks  104 , which each hold some of the sample containers  106  and are arranged to form an arc shape. 
     The sampling arm  120  has a proximal end through which a vertical shaft  122  passes. The sampling arm  120  rotates in a horizontal plane about the shaft  122 , and moves up and down along the shaft  122  in a vertical direction. A sampling nozzle  120   a  is held near the distal end of the sampling arm  120  so that the tip end of the sampling nozzle  120   a  faces vertically downward. Using the sampling arm  120  allows the sampling nozzle  120   a  to move in a horizontal plane to draw an arc-shaped trajectory, and to move up and down in a vertical direction. 
     A dispensing port  132  is provided on the trajectory of the sampling nozzle  120   a  and on the trajectory of the holding part  125  of the conveying arm  124 . The dispensing port  132  is a port through which the sampling nozzle  120   a  dispenses the sample into the pretreatment container  150 . 
     Inside the sample setting part  102 , a plurality of reagent containers  108  are arranged to form an arc shape. A reagent arm  126  is provided to collect a reagent from the reagent containers  108 . The reagent arm  126  has a proximal end supported by the vertical shaft  129  shared with the conveying arm  124 , rotates in the horizontal plane, and moves up and down. A probe  127  is provided at the distal end of the reagent arm  126 . The probe  127  has a tip end facing vertically downward, moves in the horizontal plane to draw the same arc-shaped trajectory as that of the holding part  125  of the conveying arm  124 , and moves up and down. 
     The reagent containers  108  are conveyed to draw a circular trajectory in the horizontal plane independently of the sample setting part  102 , thereby arranging a desired reagent container  108  at a predetermined reagent collecting position. The reagent collecting position is a position along the trajectory of the probe  127  of the reagent arm  126 . At this position, a reagent is collected with the probe  127 . The probe  127  sucks a predetermined reagent, and then dispenses the sucked reagent into the pretreatment container  150  arranged in the dispensing port  132 , thereby adding the reagent to the sample. 
     A pretreatment container setting part  112  is provided at a position different from the position of the sample setting part  102 . The pretreatment container setting part  112  includes unused pretreatment containers  150  arranged to form an arc shape. The pretreatment container setting part  112  rotates in the horizontal plane to move the pretreatment containers  150  in a circumferential direction. As a result, an optional pretreatment container  150  is arranged at a position along the trajectory of the holding part  125  of the conveying arm  124 . The conveying arm  124  can hold the unused pretreatment container  150  arranged at the position along the trajectory of the holding part  125 . 
     Ports of a pretreatment part for containing the pretreatment containers  150  and implementing specific pretreatment processes include filtration ports  130 , stirring ports  136   a , and temperature control ports  138  and  140 . 
     Each filtration port  130  is a port for applying negative pressure to the pretreatment container  150  arranged in the filtration port  130  to filtrate the sample. A stirring part  136  includes a mechanism to periodically operate the stirring ports  136   a  separately in the horizontal plane, and is configured to stir the sample solution in the pretreatment container  150  arranged in each stirring port  136   a.    
     The pretreatment apparatus  100  includes a sample transfer unit  142  for transferring the pretreated sample to the LC system  200  adjacent to the pretreatment apparatus  100 . The sample transfer unit  142  includes a moving part  144  moving in the horizontal plane in one direction (the direction indicated by the arrow shown in  FIG. 2 ) with a driving mechanism including a rack-and-pinion mechanism. The moving part  144  has an upper surface having a transfer port  143  in which a pretreatment container  150  containing the extracted sample is arranged. 
     The pretreatment apparatus  100  has a disposal port  134  for disposing a used pretreatment container  150 . The disposal port  134  is located near the dispensing port  132  and at a position along the trajectory of the holding part  125  of the conveying arm  124 . The pretreatment apparatus  100  further has a cleaning port  145  for cleaning the sampling nozzle  120   a . The cleaning port  145  is located at a position along the trajectory of the sampling nozzle  120   a . Although not illustrated, the pretreatment apparatus  100  further has another cleaning port for cleaning the outer and inner surfaces of the probe  127 . This cleaning port is located at a position along the trajectory of the probe  127 . 
     Subsequently, one embodiment of the LC system  200  will be described with reference to  FIG. 3 . 
     The LC system  200  includes a liquid feeding unit  204 , a column oven  206 , a detector  208 , and a control unit  236  in addition to the autosampler  202 . The liquid feeding unit  204  feeds, for example, two kinds of solvents to a mixer using a liquid feeding pump and feeds the resultant solution mixed with the mixer as a mobile phase. The autosampler  202  collects the sample from the pretreatment container  150  (sample part) transferred to the LC system  200  with the sample transfer unit  142  of the pretreatment apparatus  100  to inject the sample into the analysis channel for liquid chromatography. The column oven  206  includes an analysis column  207  separating the sample into components. The detector  208  detects the sample components into which the sample has been separated by the analysis column  207 . Examples of the detector  208  include an ultraviolet absorbance detector. The control unit  236  is configured at least to control operation of the autosampler  202 , and can be implemented as a computer provided inside the autosampler  202 , a system controller comprehensively managing the entire analysis system  200 , or any other suitable device. The liquid feeding unit  204  is positioned at the upstream end of an upstream side analysis channel  218 , and feeds the mobile phase through the upstream side analysis channel  218 . The analysis column  207  and the detector  208  are provided on a downstream side analysis channel  220 . The upstream side analysis channel  218  and the downstream side analysis channel  220  are each connected to an associated one of ports of a two-position valve  210  of the autosampler  202 , and are connected together through the two-position valve  210 . 
     The two-position valve  210  of the autosampler  202  includes six ports. A sample introduction channel  212 , a drain channel  214 , and the two ends of a sample loop  216  in addition to the upstream side analysis channel  218  and the downstream side analysis channel  220  are each connected to an associated one of the ports of the two-position valve  210 . Switching the two-position valve  210  allows selection of either of a state where the sample introduction channel  212 , the sample loop  216 , and the drain channel  214  are connected together in series while the downstream side analysis channel  220  is connected immediately downstream of the upstream side analysis channel  218  (the state shown in  FIG. 3 ) and a state where the upstream side analysis channel  218 , the sample loop  216 , and the downstream side analysis channel  220  are connected together in series. The sample introduction channel  212  communicates with an injection port  213 . 
     The autosampler  202  includes a sampling needle  222  sucking and discharging liquid from the tip end and a syringe pump  226  connected to the sampling needle  222  through a channel. The sampling needle  222  can move in a horizontal direction and a vertical direction using a driving mechanism (not shown). The sampling needle  222  collects the sample from the pretreatment container  150  transferred to the LC system  200  with the sample transfer unit  142  of the pretreatment apparatus  100 , and injects the sample into the analysis channel for liquid chromatography through the injection port  213 . 
     Switching a channel switching valve  230  allows the syringe pump  226  to be connected also to a cleaning fluid container  228  storing a cleaning fluid. If the cleaning fluid is fed from the syringe pump  226  while the syringe pump  226  that has sucked the cleaning fluid is connected to the sampling needle  222  connected to the injection port  213 , the inner surfaces of the sample loop  224 , the sampling needle  222 , and the sample introduction channel  212  can be cleaned. 
     The autosampler  202  includes a sample preparation container  232  for preparing the sample collected from the pretreatment container  150  with the sampling needle  222  and a diluent part  234  storing the diluent for diluting the sample. The sampling needle  222  has the function of dispensing the sample in the pretreatment container  150  transferred from the pretreatment apparatus  100  and the diluent in the diluent part  234  into the sample preparation container  232  to dilute the sample in the sample preparation container  232 . The sample preparation container  232  may be implemented as a well provided in the principal plane of a plate. 
     The control unit  236  controlling operation of the autosampler  202  includes a dilution operation part  238  configured to make the autosampler  202  dilute the sample. The dilution operation part  238  has a function obtained when an arithmetic element executes a predetermined program. 
     One example of the dilution operation achieved by the dilution operation part  238  will now be described with reference to the process charts shown in  FIGS. 4A  to.  5 B as well as the flowchart shown in  FIG. 6 . 
     To dilute the sample, first, the diluent is sucked from the diluent part  234  with the sampling needle  222  (see Step S 1  and  FIG. 4A ), and the sucked diluent is dispensed into the sample preparation container  232  (see Step S 2  and  FIG. 4B ). Subsequently, the diluent is sucked again from the diluent part  234  with the sampling needle  222  (see Step S 3  and  FIG. 4C ), and the sample is then sucked from the pretreatment container  150  with the sampling needle  222  (see Step S 4  and  FIG. 4D ). 
     Thereafter, the sample and the diluent sucked with the sampling needle  222  are dispensed into the sample preparation container  232  (see Step S 5  and  FIG. 5A ). As a result, phases of diluent are formed above and below a phase of the sample in the sample preparation container  232 . The total amount of the diluent dispensed into the sample preparation container  232  is an amount required to dilute the sample at a predetermined dilution rate. In this state, a tip end portion of the sampling needle  222  is moved into liquid in the sample preparation container  232 , and the sampling needle  222  sucks and discharges the liquid once or more times to stir the inside of the sample preparation container  232  and to mix the sample and the diluent together 
     The above dilution operation is merely an example, and the apparatus and the dilution method of the present disclosure should not be limited to this dilution operation. For example, in the dilution operation of the foregoing embodiment, the entire amount of the diluent sucked with the sampling needle  222  is dispensed into the sample preparation container  232 , and thereafter the diluent and the sample are sucked with the sampling needle  222  in this order. However, the diluent sucked with the sampling needle  222  may be only partially dispensed into the sample preparation container  232 , the sample alone may then be sucked with the sampling needle  222 , and the sample and the diluent may be dispensed into the sample preparation container  232 . 
     Returning to  FIG. 3 , after completion of the above dilution operation, the autosampler  200  sucks the diluted sample in the sample preparation container  232  with the sampling needle  222 , and injects the sample into the analysis channel through the injection port  213 . When the sample is injected through the injection port  213  into the analysis channel, the two-position valve  210  allows the sample introduction channel  212 , the sample loop  216 , and the drain channel  214  to be connected together in series. Thus, the sample injected from the injection port  213  is held in the sample loop  216 . Thereafter, switching the two-position valve  210  allows the upstream side analysis channel  218 , the sample loop  216 , and the downstream side analysis channel  220  to be connected together in series. Thus, the sample held in the sample loop  216  is introduced into the analysis column  207  by the mobile phase from the liquid feeding unit  204 . Components of the sample introduced into the analysis column  207  are separated from one another and detected with the detector  208 . Then, the detected components are further introduced into the MS  300 . 
     Signals fed from the detector  208  and the MS  300  are captured into the system control apparatus  400  (see  FIG. 1 ). Operations, such as quantitative determination of the components into which the sample is separated in the analysis column  207  and composition analysis of the components, are performed by software incorporated into the system control apparatus  400  and hardware such as a central processing unit (CPU) executing the software.