Abstract:
Disclosed herein is a sample introducing apparatus which is designed such that the analytical flow path runs from the needle to the separation column without the flow path switching means placed at the downstream side of the needle. This design reduces dead volume, which in turn reduces the diffusion of the sample injected into the analytical flow path. Moreover, the absence of the flow path switching means at the downstream side of the needle to inject a sample into the analytical flow path eliminates connection of the pipe with the flow path switching means. This prevents the sample from remaining in the connecting part, thereby reducing sample carry-over and improving the accuracy of analysis.

Description:
RELATED APPLICATIONS 
     This application is a Continuation of U.S. application Ser. No. 12/235,145, filed on Sep. 22, 2008 now U.S. Pat. No. 8,191,404, claiming priority of Japanese Patent Application No. 2007-249613, filed on Sep. 26, 2007, the entire contents of each of which are hereby incorporated by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a liquid chromatograph and a sample introducing apparatus therefor. 
     2. Description of the Related Art 
     A liquid chromatograph, which is shown in  FIG. 1 , is equipped with an analytical flow path consisting of an eluent holder  1 , an eluent feeder  2 , a separator  4 , and a detector  5 . The eluent feeder  2  feeds an eluent from the eluent feeder  2  to the separator  4 . 
     The analytical flow path has a sample injector  3  placed between the eluent feeder  2  and the separator  4  (including a separation column). The sample injector  3  injects a sample into the flowing eluent, so that the sample is separated into components in the separator  4  and the separated components are detected by the detector  5 . 
     The liquid chromatograph is usually constructed of several units, such as a pump (as the eluent feeder  2 ), an autosampler (as the sample injector  3 ), a separation column (as the separator  4 ), and a detector (as the detector  5 ). 
     The autosampler as the sample injector  3  is recently dominated by that of direct injection type. The autosampler of direct injection type causes the inside of the needle to constitute a part of the analytical flow path while the eluent is being fed under a high pressure for analysis. 
     The autosampler of direct injection type injects a sample in the following manner. First, the needle is detached from the analytical flow path under high pressure, and the detached needle functions as a part of the sample introduction flow path in the autosampler. The sample introduction flow path, which is connected to a syringe to suck up and discharge a sample in the autosampler, sucks up a sample through the needle as the plunger of the syringe is pulled out. After sample sucking, the sample introduction flow path, which holds the sample sucked up through the needle, is switched such that the analytical flow path for the eluent to flow from the pump communicates with the analytical flow path connected to the separation column and the sample held in the sample introduction flow path is injected into the analytical flow path. The autosampler of direct injection type employs a valve to switch the analytical flow path and the sample introduction flow path. It is common practice to place one valve each in the flow paths upstream and downstream the needle. 
     An ordinary autosampler of direct injection type performs sample injection in a manner which is explained below with reference to  FIGS. 2 and 3 . 
     In an ordinary autosampler of direct injection type, the flow path takes the route shown in  FIG. 2  when a sample is discharged or sampling is not performed. The eluent delivered from the pump passes through the valve A  14  and then the needle  12 . The eluent passes further through the injection port  13  and the valve A  14  again and flows toward the separation column. 
     Also, in an ordinary autosampler of direct injection type, the flow path takes the route shown in  FIG. 3  when a sample is sucked up. At the time of sample suction, the valve A  14  acts to switch the flow path. The pump&#39;s action causes the eluent being delivered from the pump to flow directly to the separation column without passing through the needle  12  and the injection port  13 . After separation from the flow path to the separation column, the needle  12  and the sample loop  22  are connected to the syringe  20  which moves the liquid in the autosampler. With the needle  12  inserted into the vial  23 , the syringe  20  sucks up the sample so that the sample is held in the needle  12  and the sample loop  22 . 
     After sample sucking, the needle  12  is connected to the injection port  13 , and then the valve A  14  is switched again so that the flow path takes the previous route shown in  FIG. 2 . As the result, the eluent delivered from the pump enters the sample loop  22  and the needle  12 , and the sample held in the needle  12  and the sample loop  22  is pushed into the injection port  13 . Thus, the sample flows through the injection port  13  and the valve A  14  to reach the separation column. 
     As mentioned above, an autosampler of direct injection type is constructed such that both the upstream side and the downstream side of the needle are connected to the valve. This construction is disclosed in Patent Documents 1 and 2 below. 
     Patent Document 1 
     Japanese Patent Laid-open No. 2007-121192 
     Patent Document 2 
     Japanese Patent Laid-open No. 2005-265805 
     The autosampler of direct injection type, which is used for the sample injector  3 , has a valve to switch the analytical flow path and the sample introduction flow path, the valve being placed at the upstream and downstream sides of the needle. 
     The disadvantage of the autosampler of direct injection type mentioned above is the necessity of more than one valve. Valves increase the dead volume which deteriorates the accuracy of analysis. 
     “Dead volume” means any space which exists in the flow from sample introduction to detection in the liquid chromatograph and which is useless, or rather harmful, to sample separation and hence is undesirable. 
     The dead volume has the following two adverse effects. 
     First, the dead volume diffuses the sample in the eluent, which results in the broadening of the detected peak. 
     The diffusion of the sample also reduces the theoretical plate number N (defined below) which is an index to represent the separation characteristics of the column.
 
 N= 5.54× t   R   2   /W   0.5h   2  
 
where, t R  retention time
 
     W 0.5h   2 : peak width at the middle of peak height 
     Moreover, the diffusion of the sample might mix again the zones of compounds purposely separated by the column. 
     Second, the dead volume increases the amount of carry-over or tends to cause carry-over. 
     “Carry-over” means a previously analyzed sample that remains in the liquid chromatograph to be detected in the subsequent analysis. 
     This is attributable to the dead space that exists at the joint between the pipe and the part or the valve. (There is no complete adhesion between the surface of the inserted pipe and the surface of the pipe receiver, and the gap between the surfaces forms the dead space.) The injected sample remains in the dead space and the remaining sample leaks out during subsequent analyses. 
     For these reasons, one of the important factors to improve the accuracy of analysis is how to reduce dead space in the analytical flow path from the autosampler to the separation column and from the separation column to the detector. 
     The conventional method of achieving the foregoing object is by using as thin a pipe as permissible (from the standpoint of flow rate and pressure) for the analytical flow path and also by using parts with a minimum area in contact with the liquid (thereby eliminating unnecessary spaces). 
     However, there is a limit to reducing the inside diameter and length of the pipe and changing the shape of parts to reduce dead volume. 
     OBJECT AND SUMMARY OF THE INVENTION 
     It is an object of the present invention to provide a liquid chromatograph and a sample introducing apparatus, which are capable of reducing carry-over easily by a simple structure without resorting to reducing the length and inside diameter of the pipe and contriving the shape of parts. 
     The gist of the present invention resides in a sample introducing apparatus intended to introduce a sample from a sample introduction flow path into a chromatograph equipped with a liquid feeding unit to deliver an eluent and an analytical flow path including a separator, which includes a sample sucking means for sucking up the sample, a flow path switching means for switching the separation and connection of the analytical flow path and the sample introduction flow path including a needle, and a control means for controlling the action of the sample sucking means and the flow path switching means, with the flow path switching means communicating with the upstream side of the needle and the separator communicating with the downstream side of the needle without the flow path switching means interposed between them. 
     The sample introducing apparatus according to the present invention is designed such that the analytical flow path runs from the needle to the separation column without the flow path switching means placed at the downstream side of the needle. This design reduces dead volume, which in turn reduces the diffusion of the sample injected into the analytical flow path. 
     Moreover, the absence of the flow path switching means at the downstream side of the needle to inject a sample into the analytical flow path eliminates connection of the pipe with the flow path switching means. This prevents the sample from remaining in the connecting part, thereby reducing sample carry-over. 
     In addition, the sample introducing apparatus according to the present invention does not need much modification of the conventional autosampler of direct injection type and hence it can be constructed and realized easily. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic diagram showing an example of the general structure and flow path of liquid chromatographs. 
         FIG. 2  is a schematic diagram showing an example of the flow path in an ordinary autosampler of direct injection type, the flow path working to discharge a sample from the sample introduction flow path or being in an idle state. 
         FIG. 3  is a schematic diagram showing an example of the flow path in an ordinary autosampler of direct injection type, the flow path sucking up a sample into the sample introduction flow path. 
         FIG. 4  is a schematic diagram showing an example of the liquid chromatograph according to one embodiment of the present invention. 
         FIG. 5  is a chromatogram of methylparaben (600 mg/L dissolved in 60% methanol) obtained by using an ordinary autosampler of direct injection type. 
         FIG. 6  is a chromatogram of methylparaben (600 mg/L dissolved in 60% methanol) obtained by using an autosampler according to one embodiment of the present invention. 
         FIG. 7  is a chromatogram showing carry-over induced by introduction of 60% methanol after the analysis of methylparaben (600 mg/L dissolved in 60% methanol) which was carried out by using an ordinary autosampler of direct injection type. 
         FIG. 8  is a chromatogram showing carry-over induced by introduction of 60% methanol after the analysis of methylparaben (600 mg/L dissolved in 60% methanol) which was carried out by using an autosampler according to one embodiment of the present invention. 
         FIG. 9  is a schematic diagram showing an example of another liquid chromatograph according to one embodiment of the present invention (having the switching means provided with a means for adjusting the flow rate resistance). 
         FIG. 10  is a schematic diagram showing the structure of the conventional liquid chromatograph which is referenced for comparison in the second embodiment of the present invention. 
         FIG. 11  is a schematic diagram showing the structure of the liquid chromatograph according to the second embodiment of the present invention. 
         FIG. 12  is a diagram showing the sample band in the flow path according to the conventional technology and one embodiment of the present invention. 
         FIG. 13  is a total ion chromatogram pertaining to one embodiment of the present invention. 
         FIG. 14  is a total ion chromatogram pertaining to another embodiment of the present invention. 
     
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The embodiments of the present invention will be described below with reference to the accompanying drawings. 
     The First Embodiment 
       FIG. 4  shows an example of the liquid chromatograph according to the present invention. 
     The eluent holder  1  is connected to the pump  7  through a pipe. The pump  7  sucks and delivers the eluent. The down stream side of the pump  7  is connected to the autosampler  8 . 
     The autosampler  8  consists of the needle to suck up and discharge samples, the injection port  13  communicating with the separation column, the valve A  14  to switch the flow path (as the flow path switching means), the valve B  15 , the syringe  20  to deliver the liquid in the autosampler, and the washing port  16  to clean the needle  12  of contaminants. (The outer wall of the needle is contaminated with a sample when the needle is inserted into the vial  23  to suck up a sample.) 
     With the tip of the needle  12  inserted into the vial  23  holding a sample, the syringe  20  sucks up the sample into the needle  12 . The sample which has been sucked up is held temporarily in the needle  12  and the sample loop  22  communicating with the needle  12 . Incidentally, the needle  12  and the sample loop  22  are collectively called the sample introduction flow path. 
     With the needle  12  moved to the injection port  13 , the valve A  14  (flow path switching means) switches so that the flow path from the pump communicates with the needle  12  and the injection port  13 . As the result, the eluent delivered from the pump enters the sample loop  22  and the needle  12  and the sample held in the sample loop  22  and the needle  12  enters the separation column  9  through the injection port  13 . 
     As mentioned in the section of prior art, the valve A  14  to switch the flow path connects and separates the sample introduction flow path (including the needle) to and from the analytical flow path. As the valve A  14  separates the sample introduction flow path from the analytical flow path, the sample introduction flow path communicates with the syringe  20 , so that the syringe  20  sucks up the sample. Except when the sample is sucked up, the valve A  14  keeps the sample introduction flow path connected to the analytical flow path, with the sample introduction flow path constituting a part of the analytical flow path. 
     The needle  12 , the syringe  20 , and the autosampler  8  are collectively referred to as the sample sucking means. 
     The valve A  14 , the valve B  15 , and the autosampler  8  work under control by the control means (not shown). 
     The valve A  14  has six connecting holes, with one closed by the plug  18  and one connected to the resistance coil  19  (which serves also as a drain pipe). (Closing with the plug  18  is not necessary if the valve A  14  has five connection holes.) The valve B  15  has a pipe connected thereto which passes the cleaning solution  17 . 
     The analytical flow path extending from the pump  7  passes through the valve A  14  (in its upstream side of the needle  12 ) and also passes through the injection port  13  (instead of passing through the valve A  14 ) to reach the separation column  9 , which is placed in the column oven  10 , (in its downstream side of the needle  12 ). The analytical flow path passing through the separation column  9  connects with the detector  11  and the data processing unit  6  sequentially. 
     An analysis was performed on methylparaben (600 mg/L dissolved in 60% methanol) by using an ordinary autosampler of direct injection type. The resulting chromatogram is shown in  FIG. 5 . The same analysis as above was performed by using the liquid chromatograph (mentioned above) in which the liquid flows from the needle  12  without passing through the valve. The resulting chromatogram is shown in  FIG. 6 . 
     The two chromatograms (shown in  FIGS. 5 and 6 ) differ in the theoretical plate number which denotes the sharpness of the measured peak. The former has a value of 3670, whereas the latter has a value of 4587 (both shown at the top). This result apparently shows that the chromatograph according to the present invention retains a higher value of theoretical plate number. 
     The amount of carry-over was determined by injection of 60% methanol after analysis of methylparaben (600 mg/L dissolved in 60% methanol). Experiments were carried out with an ordinary autosampler of direct injection type and the liquid chromatograph according to the present invention. The resulting chromatograms are shown in  FIGS. 7 and 8 , respectively. 
     The amount of carry-over was obtained from  FIGS. 5 and 7  in an experiment with an ordinary autosampler of direct injection type. The thus obtained amount of carry-over is 0.0050%. 
     By contrast, the amount of carry-over was obtained from  FIGS. 6 and 8  in an experiment with the liquid chromatograph according to the present invention. The thus obtained amount of carry-over is 0.0017%. The foregoing suggests that it is possible to reduce the amount of carry-over by using the liquid chromatograph according to the present invention. 
     In other words, the liquid chromatograph according to the present invention is simple in structure and yet is capable of reducing the amount of carry-over. 
     The above-mentioned embodiment of the present invention may be modified such that the valve A  14  is provided with the coil  12  to control the flow path resistance as shown in  FIG. 9 . The coil capable of changing the flow path resistance reduces the fluctuation of pressure that occurs when the flow path is switched, thereby stabilizing the base line. 
     The Second Embodiment 
     This embodiment demonstrates how the present invention produces its effect when applied to a conventional nanoflow liquid chromatograph disclosed in Japanese Patent No. 3823092. 
     This liquid chromatograph is that of trap column type, which is constructed as shown in  FIG. 10 . 
     It has the gradient pump  31 , the nanoflow pump  32 , and the loading pump  33 , whose flow rates are respectively tens to hundreds of μL/min, tens to hundreds of nL/min, and several to ten-odd μL/min. 
     It also has the 10-way valve  41  and the injection valve  44  (to switch the trap column), each having ten ports and taking two positions. It also has the backlash valve  46  and the sampler valve  51  (to switch the flow path), each having six ports and taking two positions. 
     It permits the sample  48  to be sucked up into the needle  49  by a metering device (not shown) in the autosampler  35 , in such a way that fluid-tightness is ensured between the tip of the needle  49  and the injection port  50 . Incidentally, pipes are numbered  52 ,  53 ,  54 ,  55 , and  56 . 
     Operation proceeds as follows. First, the sample  48  is sucked up into the needle  49 . Then, the sample  48  is delivered to the trap column  45  by the loading pump  33  through the pipes  52 ,  53 ,  54 , and  55  and the flow path switching valves  51 ,  46 , and  44 . The eluents  36  and  37  are mixed together by the gradient pump  31 , and the mixture of eluents is delivered to the loop  42  and the loop  43  alternately by the 10-way valve  41  which is switched periodically. 
     The mixture of the eluents  36  and  37  and part of the water  40 , which have been delivered to the loops  42  and  43 , are forced into the port  1  of the injection valve  44  by the nanoflow pump  32 . This procedure realizes the nanoflow gradient liquid delivery at such low a flow rate as tens to hundreds of nL/min. 
     If desalting is necessary for the sample  48  held in the trap column  45 , the backlash valve  46  is switched so that the ports  5  and  6  are connected to each other (not shown) and the eluents  38  and  39  are delivered to the trap column  45 . 
     As the injection valve  44  is switched, so that the ports  1  and  10  are connected to each other and the ports  6  and  7  are connected to each other (not shown), the sample  48  which has been held in the trap column  45  is eluted into the separation column  47  by the nanoflow gradient liquid delivery mentioned above. The eluate is analyzed by the mass spectrometer  34 . 
     In the second embodiment, the conventional liquid chromatograph shown in  FIG. 10  is modified as shown in  FIG. 11  according to the present invention. 
     The modified chromatograph is constructed such that the injection port  50  of the autosampler  35  is connected to the port  8  of the injection valve  44  (to switch the trap column) directly through the pipe  56 . Incidentally, the numerals  57 ,  58 ,  59 ,  60 , and  61  denote closing plugs. 
     Eliminating the pipes used in the conventional analyzer greatly reduces the dead volume in the loading flow path from the injection port  50  to the trap column  45 . The result is that the spreading of the sample  48  in the flow path decreases and the loading time required for the sample  48  to be delivered to the trap column  45  decreases. 
     The conventional and modified liquid chromatographs shown in  FIGS. 10 and 11  were tested for the spreading of the sample  48  in the flow path and the loading time. The results are shown in  FIG. 12 . Incidentally, the modified one is constructed such that the piping to the port  8  of the injection valve  44  (to switch the trap column) is connected directly to a UV absorptiometer. 
     The foregoing test was carried out under the following conditions. 
     Flow rate of the loading pump  33 : 10 μL/min 
     Wavelength for detection by the UV absorptiometer: 214 nm 
     Sample: angiotensin 
     The numerals  62  and  63  in  FIG. 12  represent respectively the band of the sample measured by using the conventional chromatograph shown in  FIG. 10  and the modified chromatograph shown in  FIG. 11 . It is noted that the band  63  is taller and narrower than the band  62 . 
     The results shown in  FIG. 12  suggest that the foregoing modification greatly improves the separation behavior of liquid chromatography and also greatly reduces the loading time (and hence reduces time required for analysis). 
     The liquid chromatographs shown in  FIGS. 10 and 11  were tested for carry-over that occurs in analysis of enzymatic digests of human serum. The results are shown in  FIGS. 13 and 14 , respectively. 
       FIGS. 13 and 14  are total ion chromatograms obtained by the mass spectrometer  34 . The top represents the chromatogram obtained when the sample was injected. The middle represents the chromatogram of blank obtained for the first time. The bottom represents the chromatogram of blank obtained for the second time. 
     Carry-over in initial blank test was calculated for a component having a mass-to-charge ratio (m/z) of 825.78, which was selected as an example, by using the conventional and modified chromatographs mentioned above. The conventional one gave a carry-over as high as 34.8%, whereas the modified one gave a carry-over as low as 3.9%. 
     A probable reason for this is that the conventional chromatograph has the backlash valve  46  in the loading flow path and the connecting parts of the pipes hold the sample, resulting in a large amount of carry-over. 
     Thus, the embodiment of the present invention solves the problems with carry-over involved in the conventional liquid chromatograph.