Abstract:
A mobile phase supply device comprises an aqueous path including a first delivery pump for delivering an aqueous mobile phase, an organic solvent path including a second delivery pump for delivering an organic solvent mobile phase, and a mixer for mixing mobile phases from the aqueous path and the organic solvent path, and supplying the mixture to an analysis path of a liquid chromatograph. A flow resistance between the second delivery pump and the mixer is greater than a flow resistance between the first delivery pump and the mixer.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    1. Field of the Invention 
         [0002]    The present invention relates to a mobile phase delivery device for supplying a mixed solution of an aqueous mobile phase and an organic solvent mobile phase to an analysis path while changing the composition over time, and a liquid chromatograph provided with the mobile phase delivery device. 
         [0003]    2. Description of Background Technique 
         [0004]    As a liquid chromatograph, a gradient liquid chromatograph that is provided with an analytical column and a detector, and that performs separation analysis on a sample while changing over time the composition of a mobile phase flowing through an analysis path is known(see U.S. 2007-0144977 A1). A mobile phase that flows through an analysis path is generally a mixed solution of an aqueous mobile phase and an organic solvent mobile phase, and the mixing ratio is changed by adjusting the delivery flow rate of delivery pumps delivering the mobile phases. 
         [0005]    An example of a conventional gradient liquid chromatograph will be described with reference to  FIG. 5 . 
         [0006]    An upstream analysis path  2   a  and a downstream analysis path  2   b  are provided as analysis paths for performing separation analysis on a sample. An analytical column  8  for separating a sample, and a detector  10  for detecting a sample component separated by the analytical column  8  are provided along the downstream analysis path  2   b . One end of the upstream analysis path  2   a  is connected to a mixer  50 . An aqueous path  42  for delivering an aqueous mobile phase by a delivery pump  46 , and an organic solvent path  44  for delivering an organic solvent mobile phase by a delivery pump  48  are connected to the mixer  50 , the aqueous mobile phase and the organic solvent mobile phase are mixed by the mixer  50 , and the mixed solution is supplied to the upstream analysis path  2   a.    
         [0007]    The other end of the upstream analysis path  2   a  and one end of the downstream analysis path  2   b  are each connected to one port of a switching valve  30  of a sample introduction unit  6 . The sample introduction unit  6  includes a switching valve  30 , a sample delivery path  32 , a drain path  34 , and a trap path  36 . The sample delivery path  32  is a path for delivering a solution containing a sample by a delivery pump  33 . The trap path  36  includes a trap column  40 , and is capable of temporarily storing a sample delivered by the sample delivery path  32  in the trap column  40 . 
         [0008]    One end of the sample delivery path  32  and of an outlet path  34 , and both ends of the trap path  36  are connected to ports of the switching valve  30 . The switching valve  30  is for switching connection between adjacent ports, and switching between a state where the trap path  36  is connected between the sample delivery path  32  and the drain path  34  (a trap mode) and a state where the trap path  36  is connected between the upstream analysis path  2   a  and the downstream analysis path  2   b  (an injection mode) is enabled by the switching by the switching valve  30 . In the trap mode, the upstream analysis path  2   a  and the downstream analysis path  2   b  are directly connected, and in the injection mode, the sample delivery path  32  and the drain path  34  are directly connected. 
         [0009]    In the trap mode, a solution containing a sample is delivered from the sample delivery path  32 , and the sample is trapped in the trap column  40 . Then, switching to the injection mode is performed to thereby deliver a mobile phase solvent from the upstream analysis path  2   a , and the sample trapped in the trap column  40  is introduced into the downstream analysis path  2   b  together with the solvent. 
         [0010]    Pressure exerted on the delivery pumps  46  and  48  may suddenly and drastically change at the time of switching from the trap mode to the injection mode by the switching by the switching valve  30  in the manner described above. If pressure is changed suddenly and drastically during delivery of the aqueous mobile phase and the organic solvent mobile phase, the balance of delivery between the aqueous mobile phase and the organic solvent mobile phase may become lost, and the organic solvent mobile phase that is less viscous than the aqueous mobile phase and that flows more easily may be instantaneously delivered at a high flow rate. If the organic solvent mobile phase is delivered at a high flow rate when the trap mode is switched to the injection mode, a sample may pass through without being separated by the analytical column  8 . 
         [0011]    Further, with a nano-flow LC (liquid chromatograph) system where the flow rate of a mobile phase flowing through the downstream analysis path  2   b  is in units of nL, the mobile phases delivered by the delivery pumps  46  and  48  are split and delivered. According to such a nano-flow LC system, in the case of the pressure inside the trap path  36  in the trap mode being lower than the pressure on the delivery pumps  46  and  48 , if the switching valve  30  is switched from the trap mode to the injection mode, the pressure on the delivery pumps  46  and  48  is suddenly and drastically reduced, and the balance of delivery or the split ratio of the aqueous mobile phase and the organic solvent mobile phase is disturbed, and the delivery flow rate of mobile phase is greatly disturbed. 
         [0012]      FIGS. 4(A) and 4(B)  are graphs showing change over time of detected signals of a detector, and  FIG. 4(A)  shows a case where there is no reduction in the pressure on the delivery pump for delivering the organic solvent mobile phase at the time of switching from the trap mode to the injection mode (for example, trap column pressure of 6.5 MPa, and analytical column pressure of 5 MPa), and  FIG. 4(B)  shows a case where there is a reduction in the pressure on the delivery pump for delivering the organic solvent mobile phase at the time of switching from the trap mode to the injection mode (for example, trap column pressure of 2.0 MPa, and analytical column pressure of 5 MPa). The peak in FIG. (B) shown with a dotted-line circle is due to instantaneous delivery at a high flow rate of the organic solvent mobile phase caused by a change in the pressure at the time of switching from the trap mode to the injection mode which then results in a sample in the trap column passing through the analytical column without being trapped. In this manner, when a greater amount of organic solvent mobile phase than aqueous mobile phase is delivered at the start of analysis of a sample, the sample flows out without being separated by the analytical column. 
         [0013]    On another note, resistance tubes having approximately the same flow resistance are conventionally connected near mixers on the upstream side for each of the aqueous path and the organic solvent path. Mutual interference between a delivery pump for delivering the aqueous mobile phase and a delivery pump for delivering the organic solvent mobile phase may thereby be prevented, and the delivery flow rate of mobile phase may be stabilized. Stabilization of the delivery flow rate is based on a premise that the pressure on each delivery pump is not suddenly and drastically changed. If the pressure is changed gradually, the flow rate of the delivery pump is changed accordingly but the change is gradual, and the pressure on the delivery pump is at the end stabilized and the delivery flow rate is stabilized. However, if the pressure is suddenly and drastically changed by an external cause in the manner described above, the balance of delivery of the aqueous mobile phase and the organic solvent mobile phase is lost, and instantaneous flow at a high flow rate of a low-viscosity organic solvent mobile phase cannot be prevented. 
       SUMMARY OF THE INVENTION 
       [0014]    Accordingly, the present invention aims to suppress a change in the delivery flow rate of a mobile phase due to a change in the pressure at the time of switching the switching valve from the trap mode to the injection mode, and to prevent a problem of a sample passing through without being separated by the analytical column. 
         [0015]    A mobile phase supply device according to the present invention includes an aqueous path including a first delivery pump for delivering an aqueous mobile phase, an organic solvent path including a second delivery pump for delivering an organic solvent mobile phase, and a mixer for mixing mobile phases from the aqueous path and the organic solvent path and supplying the mixture to an analysis path of a liquid chromatograph wherein a flow resistance between the second delivery pump and the mixer is greater than a flow resistance between the first delivery pump and the mixer. 
         [0016]    At this time, it is conceivable to suppress a change in the delivery flow rate by making the flow resistances of both the aqueous path and the organic solvent path great. However, with a high-pressure liquid chromatograph, a high pressure has to be applied to an analytical column, and thus, it is difficult to increase the flow resistance of the mobile phase supply device from the viewpoint of relationship to the performance of a delivery pump. 
         [0017]    The sizes of the flow resistance of the aqueous path and the flow resistance of the organic solvent path are set to appropriate values based on the relationship between a delivery pressure necessary for the analytical column and the performance of the delivery pump. 
         [0018]    A liquid chromatograph according to the present invention includes an analysis path including an analytical column for separating a sample, and a detector for detecting a sample component separated by the analytical column, the mobile phase supply device according to the present invention, being connected to an upstream end of the analysis path and being, for supplying to the analysis path, a mobile phase solvent of a mixed solution of an aqueous mobile phase and an organic solvent mobile phase, and a sample introduction unit including a sample delivery path for delivering a solution containing a sample, a trap column for temporarily storing the sample, and a switching valve for switching a path to be connected, the sample introduction unit being capable of switching, by switching of the switching valve, to either of a trap mode where the trap column is connected to a downstream side of the sample delivery path and an injection mode where the trap column is connected between the mobile phase supply device and an analytical column. 
         [0019]    According to the mobile phase supply device of the present invention, the flow resistance between the second delivery pump of the organic solvent path and the mixer is greater than the flow resistance between the first delivery pump and the mixer, and even if the pressure on the first delivery pump and the second delivery pump is instantaneously changed according to an external cause, instantaneous flow at a high flow rate of the organic solvent mobile phase, which is less viscous than the aqueous mobile phase, may be prevented. 
         [0020]    According to the liquid chromatograph of the present invention, since the mobile phase delivery device of the present invention is provided, a sudden and drastic increase in the flow rate of the organic solvent mobile phase, which is less viscous than the aqueous mobile phase, due to an instantaneous change in the pressure at the time of the sample introduction unit being switched from the trap mode to the injection mode may be suppressed. Accordingly, a case where a sample passes through without being separated by the analytical column because of the organic solvent mobile phase being delivered at a high flow rate may be prevented. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0021]      FIG. 1  is a path diagram schematically showing an example of a liquid chromatograph. 
           [0022]      FIGS. 2A and 2B  are path diagrams for describing a structure of a sample introduction unit of the embodiment. 
           [0023]      FIG. 3  is a graph showing change over time of a flow rate of an organic solvent mobile phase for a case where a flow resistance of a second resistance tube is greater than a flow resistance of a first resistance tube, and a case where this is not so. 
           [0024]      FIGS. 4A and 4B  are graphs showing change over time of detected signals of a detector, and  FIG. 4A  shows a case where there is no reduction in the pressure on a delivery pump for delivering an organic solvent mobile phase at the time of switching from a trap mode to an injection mode, and  FIG. 4B  shows a case where there is a reduction in the pressure on the delivery pump for delivering the organic solvent mobile phase at the time of switching from the trap mode to the injection mode. 
           [0025]      FIG. 5  is a path diagram schematically showing an example of a conventional liquid chromatograph. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0026]    According to an embodiment of a mobile phase supply device of the present invention, by applying a split type where an aqueous path is split at a downstream side of a first delivery pump into a path joined to a mixer and a first split path different from the path, and where an organic solvent path is split at a downstream side of a second delivery pump into a path joined to the mixer and a second split path different from the path, instantaneous delivery at a high flow rate of an organic solvent mobile phase may be suppressed, and a sample may be prevented from passing through without being separated by an analytical column, even when the split ratio of each of the aqueous path and the organic solvent path is disturbed due to a change in the pressure at the time of switching from a trap mode to an injection mode. 
         [0027]    In another embodiment, the path, of the organic solvent path, joined to the mixer includes a path whose inner diameter is smaller than that of the path, of the aqueous path, joined to the mixer so that the flow resistance of the organic solvent path is greater than the flow resistance of the aqueous path. 
         [0028]    An example of a liquid chromatograph will be described with reference to  FIGS. 1 and 2 . 
         [0029]    As shown in  FIG. 1 , this liquid chromatograph has a mobile phase supply device  4  connected to an upstream end of an analysis path  2 , and a sample introduction unit  6 , an analytical column  8 , and a detector  10  are provided in this order along the analysis path  2  from the upstream. As shown in  FIGS. 2(A) and 2(B) , the analysis path  2  is formed from an upstream analysis path  2   a  and a downstream analysis path  2   b , and the downstream end of the upstream analysis path  2   a  and the upstream end of the downstream analysis path  2   b  are each connected to one port of a switching valve  30  of the sample introduction unit  6 . The analytical column  8  and the detector  10  are provided along the downstream analysis path  2   b.    
         [0030]    The sample introduction unit  6  is configured to be capable of switching, by the switching of the switching valve  30 , between a trap mode of trapping a sample in a trap column  40  (see  FIG. 2(A) ) and an injection mode of introducing a sample trapped in the trap column  40  into the downstream analysis path  2   b  (see  FIG. 2(B) ). The switching valve  30  has six ports, and is for switching connection between adjacent ports. In addition to the upstream analysis path  2   a  and the downstream analysis path  2   b , one end of a sample delivery path  32 , one end of a drain path  34 , and both ends of a trap path  36  are connected to the ports of the switching valve  30 . The sample delivery path  32  is a path for delivering a solution containing a sample by a delivery pump  33 , and the drain path  34  is a path for discharging liquid externally. The trap column  40  is arranged along the trap path  36 . 
         [0031]    As shown by a thick line in  FIG. 2(A) , in the trap mode, the trap path  36  is connected to the downstream side of the sample delivery path  32 , and the drain path  34  is connected to a further downstream side. When a solution containing a sample is delivered from the sample delivery path  32  in this state, only the sample component in the solution is trapped in the trap column  40 , and other solvents are passed through the trap column  40  to be discharged from the drain path  34 . At this time, the upstream analysis path  2   a  and the downstream analysis path  2   b  are directly connected. 
         [0032]    As shown by a thick line in  FIG. 2(B) , in the injection mode, the trap path  36  is connected to the downstream side of the upstream analysis path  2   a , and the downstream analysis path  2   b  is connected to a downstream side of the trap path  36 . When switching to the injection mode is performed after the sample is trapped in the trap column  40  in the trap mode, a mobile phase from the mobile phase supply device  4  flows to the trap column  40 , and the sample component trapped in the trap column  40  is eluted and is led to the analytical column  8  of the downstream analysis path  2   b . The sample led to the analytical column  8  is separated into each component, and is detected by the detector  10 . 
         [0033]    Returning to  FIG. 1 , the mobile phase supply device  4  includes an aqueous path  12   a  for delivering an aqueous mobile phase, and an organic solvent path  12   b  for delivering an organic solvent mobile phase, and the downstream end of the aqueous path  12   a  and the downstream end of the organic solvent path  12   b  are both connected to a mixer  27 . The upstream end of the analysis path  2  is connected to the mixer  27 , and a mixed solution of the aqueous mobile phase and the organic solvent mobile phase is supplied to the analysis path  2  as a mobile phase solvent. 
         [0034]    The upstream end of the aqueous path  12   a  is arranged in a container  14   a  for storing the aqueous mobile phase, and the aqueous mobile phase is pumped by a delivery pump  16   a  (a first delivery pump). One end of a split path  22   a  (a first split path) is connected to a downstream side of the delivery pump  16   a  along the aqueous path  12   a  by a joint  20   a . The other end of the split path  22   a  is arranged in the container  14   a , and a part of the aqueous mobile phase pumped by the delivery pump  16   a  is returned to the container  14   a . A flowmeter  18   a  is provided to a further downstream side of the joint  20   a , and the flow rate of the aqueous mobile phase being delivered to the mixer  27  is monitored. 
         [0035]    The upstream end of the organic solvent path  12   b  is arranged in a container  14   b  for storing the organic solvent mobile phase, and the organic solvent mobile phase is pumped by a delivery pump  16   b  (a second delivery pump). One end of a split path  22   b  (a second split path) is connected to a downstream side of the delivery pump  16   b  along the organic solvent path  12   b  by a joint  20   b . The other end of the split path  22   b  is arranged in the container  14   b , and a part of the organic solvent mobile phase pumped by the delivery pump  16   b  is returned to the container  14   b . A flowmeter  18   b  is provided to a further downstream side of the joint  20   b , and the flow rate of the organic solvent mobile phase being delivered to the mixer  27  is monitored. 
         [0036]    A flow rate control unit  50  for controlling the flow rate of the aqueous mobile phase and the organic solvent mobile phase delivered to the mixer  27  based on the measurement values of the flowmeters  18   a  and  18   b  is provided. The flow rate control unit  50  controls the driving of the delivery pumps  16   a  and  16   b  based on the measurement values of the flowmeters  18   a  and  18   b  such that the composition of the mobile phase solvent mixed by the mixer  27  becomes a predetermined composition. 
         [0037]    A first resistance tube  24  is provided along the aqueous path  12   a , near the mixer  27 , and a second resistance tube  26  is provided along the organic solvent path  12   b , near the mixer  27 . Mutual interference between the delivery pumps  16   a  and  16   b  is prevented by the installation of the first resistance tube  24  and the second resistance tube  26 . 
         [0038]    The flow resistance of the second resistance tube  26  is greater than the flow resistance of the first resistance tube  24 . Accordingly, instantaneous delivery at a high flow rate of the organic solvent mobile phase, which is less viscous than the aqueous mobile phase, due to a change in the pressure at the time of switching from the trap mode to the injection mode is suppressed. 
         [0039]      FIG. 3  is a graph showing change over time of a flow rate of the organic solvent mobile phase for a case where the flow resistances of the second resistance tube  26  and the first resistance tube  24  were the same, and for a case where the flow resistance of the second resistance tube  26  was greater than the flow resistance of the first resistance tube  24 . 
         [0040]    In an example of the case where the flow resistances are the same for the second resistance tube  26  and the first resistance tube  24 , both resistance tubes were respectively a resistance tube whose inner diameter is 0.025 mm and whose length is 1000 mm. Here, to make the flow resistances the same is to make the sizes of the resistance tubes the same. However, even if the sizes of the resistance tubes are made the same, the resistance values are different depending on the type of mobile phase that is to flow through, and thus, the resistance value of the first resistance tube  24  through which the aqueous mobile phase flows is, in many cases, greater than the resistance value of the second resistance tube  26  through which the organic solvent mobile phase flows. 
         [0041]    In an example of the case where the flow resistance of the second resistance tube  26  is greater than the flow resistance of the first resistance tube  24 , the first resistance tube  24  was a resistance tube whose inner diameter is 0.025 mm and whose length is 1000 mm, and the second resistance tube  26  had a resistance tube whose inner diameter is 0.01 mm and whose length is 750 mm serially connected to a resistance tube whose inner diameter is 0.025 mm and whose length is 1000 mm. 
         [0042]    Results of delivering at a total flow rate of 600 nL/min, and delivering the aqueous mobile phase at 550 nL/min and a low-viscosity organic solvent mobile phase, such as acetonitrile, at 50 nL/min in the two cases described above are shown in  FIG. 3 . Here, a flow resistance of about 2 MPa may be reached in the first resistance tube  24 , and a flow resistance of about 4 MPa may be reached in the second resistance tube  26  with a greater flow resistance. However, these numerical values are not restrictive because an absolute pressure value is different depending on the type of column or the like to be installed. In the case an aqueous solvent passes through the first resistance tube  24 , and the solvent that passes through the second resistance tube  26  is the organic solvent mobile phase, which is less viscous than the aqueous mobile phase, an effect is achieved if the flow resistance of the second resistance tube  26  is greater than that of the first resistance tube  24 . 
         [0043]    In the graph in  FIG. 3 , the trap mode is switched to the injection mode after five minutes have passed from the start of acquisition of data. In the case when the flow resistances of the second resistance tube  26  and the first resistance tube  24  were approximately the same, the organic solvent mobile phase was instantaneously delivered at a high flow rate due to the change in the pressure according to the switching, and the disturbance in the flow rate was about 131.4 nL in terms of a flow rate value obtained from the area of the waveform. In contrast, in the case when the flow resistance of the second resistance tube  26  was greater than that of the first resistance tube  24 , the flow rate of the organic solvent mobile phase was not greatly disturbed, and the disturbance in the flow rate was about 4.3 nL in terms of a flow rate obtained from the area of the waveform. It can thereby be seen that a sudden and drastic change in the flow rate of the organic solvent mobile phase at the time of switching from the trap mode to the injection mode may be suppressed by making the flow resistance on the side of the organic solvent path  12   b  greater than the flow resistance on the side of the aqueous path  12   a.    
         [0044]    Additionally, a resistance tube  28  is provided to the split path  22   b  to make the split ratio of the organic solvent mobile phase a predetermined ratio. The size of the flow resistance of the resistance tube  28  is determined based on the size of the flow resistance of the resistance tube  26 .